Tisteieie ie yore ae reels, ee Ole «eles e hSET2COO TOEO O MOC 1OHM/18IN Cuiaupe E. ZoBELL was born in 1904, one of a pioneer family of eight living in upper Snake River Valley, Idaho. After receiving his credentials as a teacher, he was prin-~ cipal of an elementary school in Rigby, Idaho, for two years. He received the B.S. degree from the Utah State Agricultural College in 1927 and took the M.S. degree in 1929. As Thompson Scholar he obtained his Ph.D. degree at the University of California in 1931. He served as re- search associate at Hooper Foundation for Medical Re- search for a few months prior to taking charge of the re- search program in marine microbiology at the Scripps In- stitution of Oceanography. He studied the metabolism of lakes for a year as a special investigator at the University of Wisconsin in 1938-39. During the summer of 1939 he held a post-doctorate fellowship at Woods Hole Oceano- graphic Institution. Since 1942 he has been Associate Professor of Marine Microbiology and Assistant to the Director at Scripps Institution and Director of Research Project 43A of the American Petroleum Institute. His scientific publications number more than a hundred, in~ cluding ten papers on the metabolism of the Brucella, about thirty on bacterial physiology, and nearly fifty on the broader aspects of the bacteriology of the ocean and lakes. Many of the latter papers are listed in the bibliography of § this volume. Some of his more important contributions have been the relation of bacteria to solid surfaces, the role of microorganisms in the fouling of submerged surfaces, description of over sixty new species of marine bacteria, and the importance of bacteria in the formation and transforma- tion of petroleum hydrocarbons. He is an active member of the following professional societies; A.A.A.S., Amer. Biol. Soc., Amer. Assoc. Univ. Prof., Amer. Micro. Soc., Ecol. Soc. Amer. (Vice-president 1942), Limnological Soc. Amer., Oceanographic Soc. of the Pacific, Phi Kappa Phi, San Diego Soc. Nat. Hist. (President 1943), Sigma Xi, Soc. Amer. Bact. (President S. Calif. Section 1944- 45), Soc. Exper. Biol. & Med., and West. Soc. Natural- ists. He is a member of the editorial boards of the Bull. Scripps Inst. Oceanogr., Univ. of Calif. Publ. in Micro- biology, Jour. Ind. Biol., and Scripps Inst. Records of Observations. He has served asa board member of the San Diego Biological Research Institute, San Diego Clinical Lab. Association, the La Jolla Visiting Nurse Association, the San Diego Scientific Library Association, and the California Alumni Association (President S. Calif. Sec- tion 1942-43). He was an honorary member of the Byrd Antarctic Expedition II, sometime Distinguished Lec- } turer for the Amer. Assoc. Petrol. Geol., and Sigma Xi lecturer at several different institutions. A NEW SERIES OF PLANT SCIENCE BOOKS edited by Frans Verdoorn Volume XVII MARINE MICROBIOLOGY Ahr tet Dae WZ n : ! — —————— SS —_<== ———— ———— —— oo eS ee ee _—_— = ————— —— — a a __ a —— 200 meters 86.7 904.3 95.8 92.4 Saitoco,, |: * 79.6 gI.2 O2n7 88.1 5512000), (1). 74.3 87.3 89.3 83.9 >)2000)) 1) 65.5 82.1 81.9 a AE SF4000)) | 155 ATO 63.6 57-9 Bios > 50001) = 22 28.4 19.8 24.5 OOOO M Nas 0.6 1.8 0.4 T2 S17 C001 pee 0.2 ae O.I Topography of the sea floor:— The topography or profile of the ocean bottom is very irregular. There are submerged mountain-like structures of various dimensions which are termed ridges or rises, some of which rise precipitously for several thousand feet. Some, whose tops extend above the water, form islands. The term si/J is applied to a submerged elevation separating two basins. Ridges, rises, and sills influence the movement of water masses and thereby exert a pronounced effect upon the physical, chemical, and biological properties of the water. Certain elevations restrict the migrations of marine animals. For these and re- lated reasons the topography of the sea floor must be taken into account in the study of the ecology and activities of marine organisms. Large scale depressions are called troughs, trenches, or basins. A deep is the lowest part of a depression, being the antithesis of a mountain peak. Canyons are relatively long narrow depressions or furrows in the conti- nental shelf. Submarine canyons range in size from small gullies to vast depressions equalling the Grand Canyon of the Colorado River in all Chapter II —i1— The Marine Environment dimensions. The walls of submerged valleys are usually not nearly as steep as those of canyons. A shelf is a gently sloping seaward extension of the land around conti- nentsand continental islands from lowest tide level to the contour where the angle of descent increases rather sharply toward great depths. The water depth at the outer margin of the continental shelf ranges from 100 to 300 meters. Although there is considerable overlapping, the continental shelf marks the boundaries of a fairly distinctive biotic zone. The continental slope is beyond the outer edge of the shelf where the inclination downward is greater than 1°, or more than one meter in 60 meters. The declivity of the slope is usually much greater than 1°. The lower limit of the slope is even more arbitrarily defined than its upper limit but, in general, it is marked by the termination of the steep gradient. An abyss is the bottom of the deep sea. A bank is a fairly extensive, relatively flat-topped, submarine elevation approaching the surface. The water over it is usually sufficiently deep for navigation. The ecological significance of banks is indicated by the fact that some of the world’s best fishing grounds are over banks. A reef is a coral or rocky, elongated elevation, part of which may actually extend above the water level Biotic zones:— The marine environment is divided into biotic zones according to the nature of the plant and animal life inhabiting each. This is a function of such environmental factors as water temperature, salinity or osmotic pressure, depth, proximity to shore, etc. Marine organisms may be classified according to habitat into the categories of benthic or pelagic. Benthic or benthonic organisms, also called benthos, inhabit the sea floor. Those which live in the water and are not confined to the bot- tom are pelagic. The distinction between the neritic and oceanic zones is less sharply defined than that between the benthic and pelagic zones. Literally neritic denotes the zone of shallow water which is relatively near land, and oceanic refers to organisms which live in the open ocean. However, marine ecologists usually employ the term neritic to include the zone out- lined by the continental shelf, the oceanic zone embracing the open ocean beyond these boundaries. According to EKMAN (1935), the 200 meter depth contour roughly separates the neritic from the oceanic zone, this approximating the mean depth of water at the edge of the continental shelf. It is also the depth to which little or no sunlight penetrates. Some workers define the Jitéoral zone as that near the coast. Others define it as being synonymous with the iniertidal zone or the area between high- and low-tide water marks, but EKMAN (1935) considers the littoral zone as the floor of the continental shelf. The euphotic zone receives enough sunlight for the photosynthetic processes of plants. It is the zone of primary productivity in the sea. The depth of the euphotic zone varies with the factors which influence the penetration of light, but on the average it extends from the surface to a depth of around roo meters. Below the euphotic zone is the aphotic zone, a lightless region in which photosynthetic plants cannot grow. The ani- mal life in this zone, which comprises about 95 per cent of the sea, con- sists almost exclusively of carnivores and detritus feeders. Bacterial life abounds in the aphotic zone, particularly on the sea floor where organic matter, raining down from the euphotic zone, accumulates. Some work- ZoBell —12— Marine Microbiology ers recognize a dysphotic zone in the ocean between the euphotic and aphotic zones as a region 80 to 200 meters below the surface which receives some light but not enough for effective plant production. Penetration of sunlight:— There are several factors which influence the depth to which sunlight penetrates sea water. These include surface reflection, transparency of the water, the angle of incidence of light, and the intensity of the incident light. The latter two factors are largely functions of atmospheric conditions, the time of day, and latitude. Ordi- narily, light penetrates tropical water to greater depths than water at higher latitudes where the angle of incidence is greater. Data recorded by SVERDRUP ef al. (1942) indicate that an average of 65 per cent of the incident light (energy) in fairly transparent oceanic water is absorbed in one meter. Only 17 per cent of the incident light penetrates to a depth of 5 meters, 9.5 per cent penetrates to a depth of ro meters, 3.7 per cent to a depth of 20 meters, 0.31 per cent to a depth of 50 meters, and 0.0057 per cent penetrates to a depth of 100 meters. In less transparent coastal waters only 0.5 per cent of the incident light pen- etrates to a depth of 10 meters. Pure sea water, free of all suspended and coloring matter, permits the penetration of 22 per cent of the incident light to a depth of 10 meters, but only 3 per cent of the light penetrates pure sea water to a depth of 100 meters. Considerable difference is found in the transparency of water from dif- ferent parts of the ocean. According to CLARKE (1936), the water in the Sargasso Sea is more transparent than sea water in any other part of the world. It is 2 to 4 times more transparent than water in the Gulf of Maine which is characteristic of water on the continental shelf, and the latter is 3 to 4 times more transparent than water in Woods Hole Harbor. There is a marked difference in transparency of water for different wave lengths of light. For example, CLARKE (1936) reports that the transpar- ency of the Sargasso Sea is greater than that at Woods Hole Harbor by the following factors: 6-fold for green light, 7-fold for red, 11-fold for violet, and 16-fold for blue. In general, green light, or that having a wave length ranging from 4920 to 5350 A, penetrates sea water to greater depths than the shorter or longer wave lengths of the visible spectrum. Blue (4220 to 4920 A) and yellow (5350 to 5860 A) are more penetrative than orange (5860 to 6470 A) or violet (3900 to 4220 A). Red (6470 to 8100 A) is the least penetrative part of visible light. Sea water is relatively opaque to ultraviolet radia- tions. The decreasing intensity of orange, green, and blue light with increase in depth is given in Table II. TABLE II.— Per cent of transmitted light of three spectral bands in water at Dixon En- trance, Southern Alaska, taken from data by UTTERBACK (1936) :— DEPTH OF ORANGE GREEN BLUE WATER 6000 A 5300 A 4800 A meters per cent per cent per cent ° 100 100 100 5 18 35 26 Io 1.8 16 7.8 15 0.53 7-6 3-9 20 0.27 Reg 223 30 0.012 OnL2 0.082 Chapter II —13— The Marine Environment The amount of photosynthetic activity which occurs at different depths as indicated by oxygen production and carbon dioxide consump- tion, or other means, is one criterion of the penetration of light. At depths where there is much photosynthetic activity during the hours of daylight, more oxygen is liberated than the amount which is consumed by the respiration of organisms. With decreasing depth a point is reached where the amount of oxygen liberated is just equal to the amount con- sumed. It is termed the compensation point. CLARKE (1936) found the compensation point in the Sargasso Sea at a depth of 80 meters. In the Gulf of Maine the compensation point was at a depth of 24 to 30 meters and in Woods Hole Harbor it was only 7 meters. He observed measurable amounts of photosynthesis at 18 meters in Woods Hole Harbor, 40 meters in the Gulf of Maine, and 140 meters in the Sargasso Sea. Besides the effect of light on photosynthetic organisms which are the primary producers of the sea (CLARKE and OsTER, 1934), light is the most important factor controlling the diurnal migrations of plankton (CLARKE, 1936). Not only does light influence the migrations, vertical distribution, production, and physiology of zooplankton, but the attachment and rate of growth of sedentary animals are also influenced directly or indirectly by light (RUSSELL, 1936). The specific effect of radiations on the activities and distribution of marine microorganisms is outlined in Chapter V. The literature on the penetration of light through water has been re- viewed by ATKINS (1932). The reader is also referred to the symposium volume on the measurement of submarine light and its relation to biolog- ical phenomena by CLARKE (1936), RUSSELL (1936), UTTERBACK (1936), and others. Temperature of the marine environment:— Closely related to the penetration of light is the temperature of sea water, which is primarily a function of the intensity of solar radiation. The effects of volcanic dis- turbances and adiabatic heating or cooling are far less than the direct or indirect effects of solar radiation. The surface temperatures of sea water vary with season and latitude. Tropical waters in the open sea may have surface temperatures as high as 28° to 30° C., or 38° to 40° C. in localized regions near shore, while in polar seas water temperatures approximate the freezing point of the water. The freezing point is a function of the salinity. Water having a salinity of 35°/o begins to freeze at — 1.91° C. Because the salinity is never suff- ciently high, except in localized regions, to depress the freezing point lower than — 2.0° C., this approximates the lowest temperature found in the sea. The temperature range of the marine environment, — 2° to 40° C. is small contrasted with the range of air temperatures, — 65° to 65° C. The temperature of surface water usually fluctuates less than 1° C. throughout the day. Diurnal changes are barely perceptible below a depth of 10 meters and are probably too small to be of biological impor- tance. Annual changes may affect the temperature of sea water to a depth of 10 to 200 meters. Below this depth the water temperatures are fairly constant throughout the year, although fluctuations may be caused by the movements of water masses. The temperature of sea water usu- ally decreases with depth except in shallow turbulent seas or during the winter at high latitudes. The temperature of the water in the uppermost layers, which are ZoBell — 14 — Marine Microbiology mixed by the action of wind and waves, is fairly homogeneous. Below this homogeneous layer, which is ro to 100 meters deep, the temperature decreases rapidly with depth to about 5° C. or less at a depth of 200 to 1000 meters, depending upon the latitude. The zone of maximum temper- ature gradient is called the thermocline. Below the thermocline the tem- perature continues uniformly low to the bottom of the sea. Minor irregu- larities in the temperature curve may be caused by the intrusion of layers or tongues of colder or warmer water. The temperature range of water exceeding tooo meters in depth is 5° to — 1.5° C. Thus, about 90 per cent of the marine environment is per- petually colder than 5° C. However, SmirH (1940) points out that more than half of the ocean’s surface ranges from 15° to 30° C., and only 27 per cent of the surface water of the ocean has a mean annual temperature below 10° C. The effects of water temperatures on the activities and distribution of marine organisms have been summarized by HARVEY (1928), EKMAN (1935), SVERDRUP eé¢ al. (1942), and others. The temperature of the water also influences the density and movement of water masses. The movements of sea water:— Although the marine environment is fairly stable in most respects, in many places the water is in a state of continuous movement. In virtue of the mobility of its waters, the sea responds readily to all forces acting upon it. The principal movements are waves, tides, tidal streams, and currents. A wave is a ridge or swell of water normally having a forward motion. Waves may be propagated for long distances. Upon reaching shallow water, the wave steepness increases causing the waves to curl over and form breakers. In deep water the water particles within progressive as well as standing waves move in circles (SVERDRUP, ef al., 1942). Over deep water, surface waves sometimes attain a maximum height of nearly 100 feet, the height of a wave being the vertical distance from the bottom of the trough to the top of the crest. Besides tending to mix water, wind waves produce considerable agitation of bodies in the water, particularly at the surface and sometimes on the sea bottom. The action of waves and breakers along the coast and in shallow water is an important ecolog- ical factor. The attraction of the moon and sun causes fides, the alternate rising and falling of the surface of the ocean and of gulfs, bays, and other bodies of water connected with the ocean. The biological effects of tides are most noticeable along the coast, especially in the intertidal zone. The latter is alternately inundated and then left exposed to the air. In different parts of the world the vertical distance between the highest spring tide and the lowest neap tide ranges from less than one foot to 42 feet or more. Ac- cording to MARMER (1932), the largest observed tidal ranges occur in the Bay of Fundy near Nova Scotia, Port Gallegos at the southern end of Ar- gentina, and Frobisher Bay on Baffin Island where the maximum tidal ranges are 42, 36, and 35 feet respectively. Depending upon the bottom gradient, the intertidal zone may extend from a few feet to several miles from land. For further information on tides, tidal streams, ocean currents, and other movements of sea water, the reader is referred to JOHNSTONE (1928), MArMER (1932), PATTON and MARMER (1932), and SVERDRUP et al. (1942). The movements of sea water affect marine organisms in several ways Chapter II —15— The Marine Environment either directly or indirectly. In the latter category may be mentioned the effect of water movements upon the temperature of the marine environ- ment in various regions and upon the chemical composition of sea water. Vertical mixing or upwelling tends to bring nutrients to the euphotic zone. The greater abundance of microorganisms found in regions where warm and cold water meet or in regions of upwelling has already been mentioned. Salinity, chlorinity, and density:— The concentration of total solids dissolved in sea water is usually expressed as salinity in terms of parts per thousand, per mille, or grams of solids per kilogram of sea water, for which the symbol °/o) is used. For biological purposes it usually suffices to state the salinity as the nearest whole number or to the first decimal place, but because minute differences in salinity materially influence the dynamic stability of water masses, oceanographers give the salinity to the second decimal place. Unless diluted by heavy rainfall, melting ice, or rivers, the salinity of surface sea water generally ranges from 33 to 37°/o.. However, in regions of considerable fresh-water dilution, as in the Gulf of Bothnia, for exam- ple, the salinity may approach zero. In isolated regions where surface water evaporates rapidly, as in the Red Sea or in tide pools, the salinity may exceed 40°/ 9 for short periods of time. The salinity of the Dead Sea, like that of Great Salt Lake, sometimes reaches 320°/o9 while that in the Black Sea is only 16 to 23°/o, but these inland bodies of water are far re- moved from the great oceans of the world. The salinity of deep or bottom water of the oceans varies within narrow limits, approximately 34.6 to 35°/o0 (SVERDRUP et al., 1942). The chlorinity is the number of grams of halogens, calculated as chlo- rine, precipitable by silver nitrate, contained in a kilogram of sea water. Since analytical procedures for determining the chlorinity of sea water are simpler and more precise than those for determining salinity directly, and since there is a constant relationship between salinity and chlorinity, the salinity of sea water is generally calculated from its chlorinity: Salinity = 0.03 + 1.805 X chlorinity. The density or specific gravity of sea water can be calculated from the chlorinity if the temperature is known, At o° C. the density equals 0.999,931 + 0.001,470,8Cl — 0.000,001,57CI2 + 0.000,000,039,8Cl>. The density may also be obtained by reference to KNUDSEN’s (1901) Hydro- graphical Tables. Roughly the density of sea water ranges from 1.024 to 1.030. For hydrographical purposes the density of sea water may be deter- mined accurately to the fifth place. Since the first two figures are always the same, it is practical to express the density by omitting the first two figures and shifting the decimal point three places to the right so that a density of 1.02814, for example, is expressed simply as 28.14. Osmotic and hydrostatic pressure:— The osmotic pressure of sea water at any given temperature may be calculated from its salinity or chlorinity. According to THompson (1932), the freezing point of sea water, A, equals — 0.0966Cl — 0.0000052CI*. From the colligative properties of dilute solutions it is known that when A is — 1.86° C., the osmotic pressure is 22.4 atmospheres. Thus the osmotic pressure of sea water at o° C. approximates = x A or 12.04 X A. THOMPSON (1932) ZoBell — 16— Marine Microbiology points out that a small correction must be introduced for changes in sea water resulting from the heat of fusion and the heat of dilution, the cor- rected factor being 12.08. Therefore the osmotic pressure of sea water at o° C. equals 12.08 X A. If the osmotic pressure at 0° C., OPo, is known, the osmotic pressure at any other temperature, OP, can be calculated from the following formula: GEy Op 550 273 The osmotic pressure, density, and salinity of sea water of different chlorinities are summarized in Table III. The osmotic pressure of sea water of average salinity, 35.00°/o, is 23.07 atmospheres at o° C. or 24.69 atmospheres at 20° C. TaBLeE III.— The salinity, density, freezing point, and osmotic pressure of sea water of different chlorinities at o° C., calculated from KNUDSEN’s (1901) Hydrographical Tables:— CHLORINITY (°/0) 17.00 19.00 19.38 21.00 SALINITY (°/,0) 30.72 34-33 35-01 37-94 FREEZING POINT (°C.) —1.67 —1.87 —I.91 — 2.08 OSMOTIC PRESSURE (ATMOSPHERES) | 20.17 22.50 23.07 25053 DEwnsIty (AT 0° C. REFERRED TO DIST. WATER AT 4° C.) 1.02468 1.02758 1.02814 I.03049 Most marine organisms are stenohaline, or adaptable to only slight changes in salinity or osmotic pressure. Those which are able to live in water having a wide range of salinity or osmotic pressure are termed euryhaline. Certain crustaceans and the salmon are outstanding exam- ples of euryhaline organisms. Some bacteria thrive in fresh water and others live in the most concentrated brines, but most of the bacteria and allied microorganisms found in the sea at places which are remote from possibilities of terrigenous contamination tend to be stenohaline (see Chapter VIII). Contrary to popular conception, marine microorganisms tolerate hypertonic solutions no better than they tolerate hypotonic solutions. The hydrostatic pressure of sea water is primarily a function of depth and secondarily of temperature, chlorinity, compressibility, and latitude. For practical purposes the effect of atmospheric pressure on the hydro- static pressure is not generally considered. In fact, for biological purposes only the depth need be considered. Roughly, the hydrostatic pressure increases one atmosphere for each ten meters, an atmosphere being 15 lbs. per square inch or the weight of a 760 mm. column of mercury. At a depth of one mile the pressure approx- imates one ton per square inch. There are few land-dwelling organisms which can tolerate such pressures. Very little is known concerning the effect of pressure on marine organisms but certainly it does not exclude life from the abyssal regions of the sea. Living organisms have been re- covered from the greatest depths dredged. Bacteria appear to be more abundant in bottom deposits than elsewhere in the sea, regardless of the depth of overlying water or the pressure. The pressure influences the solubility of substances although the effect of pressure on the chemical or physical properties of sea water is consider- ably less than that of temperature or salinity. The effect of pressure on sampling apparatus must be taken into consideration because unless ade- quately protected, thermometers and other instruments will be broken by the pressure at great depths. Chapter II — 17 — The Marine Environment Chemical composition of sea water:— Sea water is a physiologically balanced salt solution containing more than half of the known elements. It is a dilute solution of several salts with some dissolved gases and traces of a vast number of organic compounds. Except for a few constituents which are produced or consumed by biological agencies, the composition of sea water is relatively constant. The concentration of the principal inorganic solutes in sea water having a salinity of 34.325°/00 is given in Table IV. TaBLe [V.— Concentration of elements, exclusive of gases, in sea water having a salinity of 34.325°/0 (from data recorded by SVERDRUP et al., 1942) :— oM./KG. PER CENT OF ELEMENT cM./KG. ELEMENT OR 9/09 TOTAL SOLIDS (cont.) OR 9/9 Chlorine 18.980 55-29 Tron* ©.000,02 Sodium 10.561 30.77 Manganese* 0©.000,01 Oxygen MEV QS 5.05 Copper ©.000,01 Magnesium Te 272 Sif Zine ©.000,005 Sulfur 0.884 2.57 Lead ©.000,004 Calcium ©.400 1.16 Selenium ©.000,004 Potassium 0. 380 septa Cesium ©.000,002 Bromine 0.065 0.189 Uranium ©.000,001,5 Carbon 0.028 0.081 Molybdenum ©.000,000,5 Strontium 0.013 0.038 Thorium <0.000,000,5 Boron ©.004,6 0.013 Cerium ©.000,000,4 Silicon* 0.004,0 0.012 Silver ©.000,000,3 Fluorine ©.001,4 0.004 Vanadium ©.000,000,3 Nitrogen* ©.000,7 Ya ©.002 Lanthanum ©.000,000,3 Aluminum ©.000,5 — Yttrium ©.000,000,3 Rubidium ©.000,2 a= Nickel ©.000,000,I Lithium ©.000,I = Scandium ©.000,000,04 Phosphorus* ©.000,I — Mercury ©.000,000,03 Barium ©.000,05 — Gold ©. 000,000,006 Iodine ©.000,05 — Radium* 3X 10 #8 Arsenic* ©.000,02 — * The quantity of the elements marked with an asterisk is highly variable in sea water primarily due to bio- logical activity. The concentration given is the maximum ordinarily found. The chlorine and bromine occur almost exclusively as chloride and bromide anions. Similarly sodium, magnesium, calcium, potassium, and strontium occur as cations. The oxygen reported in Table IV occurs mostly in sulfate ions, with smaller quantities in bicarbonate, borate, phosphate, nitrite, and nitrate ions. The nitrogen occurs as ammonium, nitrite, or nitrate ions and to a lesser extent in organic compounds. The ratios of the three principal anions of sea water, 7.e., carbonates, sulfates, and chlorides in sea water are the reverse of the ratios of these anions in river water: % CARBONATE % SULFATE % CHLORIDE River water 80 13 7 Sea water 2.0 Coarse sand 2 OLO) On 2) 347.6 Fine sand 0.2 to 0.02 3.476 Silt 0.02 to 0.002 0.03476 Clay < 0.002 0.00034 Terrigenous deposits can be characterized according to color which may be black, white, blue, red, yellow, green, or brown. Pelagic deposits consist of the remains of calcareous and siliceous organisms from the overlying water and the hydrous silicates of iron and aluminum from the disintegration of materials from submarine volcanic action. If the sediment consists primarily of the tests of pelagic foraminifera, it is termed globigerina ooze. In coccolith ooze the calcareous remains of Coccolithophoridae, minute flagellates, predominate. Pteropod ooze is characterized by a predominance of the calcareous shells of pteropods, small gastropod mollusks. Dzatom ooze and radiolarian ooze contain a large proportion of the siliceous skeletal material from diatoms or Radio- laria respectively. Pelagic deposits which contain less than 30 per cent of the calcareous or siliceous remains of organisms are known as red clay. For further information on the properties of marine bottom deposits the reader is referred to Murray and Hyjort (1912), TRASK (1939), and SVERDRUP ef al. (1942). Plant and animal population:— Over 8000 species of marine plants have been described, with species of red algae, diatoms, brown algae, dino- flagellates, green algae, and blue-green algae predominating in the order listed. The red and brown algae, which are known collectively as sea- weeds, are the most conspicuous plants along the coast and in shoal waters, but it is the microscopic diatoms and dinoflagellates that are most important in the economy of the sea. Diatoms and dinoflagellates are more or less universally and generally abundantly distributed through- out the photosynthetic zone. Though microscopic in size, their mass greatly exceeds the combined mass of all other primary producers includ- ing the more conspicuous seaweeds and higher plants. ‘The siliceous shells, or frustules, of diatoms are of considerable importance in sediments Chapter II — 23 — The Marine Environment and have formed extensive fossil deposits known as diatomaceous earth. Several hundred species of marine plankton diatoms have been described by Cupp (1943) who lists much of the relevant literature. Outstanding in importance among the higher plants is eel grass, Zos- tera marina, an angiosperm which is widely distributed along the protected coasts of Asia Minor, eastern Asia, Europe, and North America. Phyllo- spadix, a related genus, is confined to the open, wave-washed coasts of the Pacific. Six genera of Potamogetonaceae closely related to fresh- water Potamogeton species also inhabit the sea. In all there are only around 30 species of marine Spermatophytes (seed plants) and no known Pteridophytes (ferns) or Bryophytes (mosses). Besides providing food for animals and saprophytic microorganisms, aquatic Spermatophytes, algae, and certain diatoms provide anchorage for sessile microorganisms of various kinds including bacteria. The interrelationships of bacteria and plants are outlined in Chapter XIV. Vegetation is described by CoKER (1938) as “the broad base of the pyramid of life in the sea. The contrast to conditions on land is marked. None of the higher plants occur in the ocean remote from the shores. The great group of blue-green algae, abundant in lakes and rivers, are promi- nent in the ocean only in the waters near the mouths of large rivers or in tropical regions. The green algae, predominant in fresh waters, are sparsely represented in salt water and then chiefly where there is some admixture of fresh water. The off-shore plant life, barring the floating Sargassum, is of extreme simplicity of form. Even the relatively simple filamentous forms, so characteristic of all sorts of fresh water, are missing. Conditions in the sea have not favored cell aggregations and the associated specialization in form and development of larger bodies. .. . The basis of all the life in the modern ocean is to be sought in the microorganisms of the surface.”’ In comparing animal life in the sea with terrestrial and fresh-water faunas, COKER (1938) points out that ‘“‘the diversity in basic types of living animals is much greater in the ocean than in fresh water or on land. . . . There is in the ocean a predominance of the more primitive types of animals and of those types that constitute the possible links between the several phyla. In a way we may look upon the seas as representing a living museum of biological antiquities, or it might better be said, as com- prising the chief repository of the early archives of our family history.” This may also be said of the bacterial kingdom which is represented in the sea by a great diversity of species, including many primitive forms. The animal population of the sea has far-reaching effects on the marine environment and the microbial population. Some of the factors influ- encing the distribution of animals in the sea are discussed by EKMAN (1935). The sea is the home of over 60,000 species of mollusks, about half as many crustaceans, 15,000 fishes, and enough representatives of other classes to make a total of around 150,000 species of marine animals which have been described. Of the 48 major classes of animals only in- sects, reptiles, birds, myriopods, amphibia, and mammals are predomi- nantly non-marine. Most of the other classes are predominantly or al- most exclusively marine. In the latter category are coelenterates, cteno- phores, echinoderms, sponges, tunicates, and bryozoans, all of which are largely confined to the sea. Of special microbiologic and oceanographic interest are the Protozoa, of which there are many thigmotropic and planktonic types. In the ZoBell — JA — Marine Microbiology Woods Hole area, LACKEY (1936) found from 27,000 to 135,000 protozoans per liter of sea water. He observed 183 different species attached to sur- faces, 146 planktonic species, and 79 different species in bottom samples. Numerous representatives of all of the major groups as listed by CALKINS (1926) were observed in marine materials. LACKEY stressed the impor- tance of Protozoa as food for copepods and similar grazing animals (see also CLARKE and GELLIS, 1935). Protozoa also have an important func- tion in the turnover of organic matter. They ingest bacteria (Luck et al., 1931) and otherwise influence the bacterial population. More than a thousand species of Foraminifera, rhizopod Protozoa hav- ing calcareous shells, occur in the sea. ELtis and MESSINA (1940) catalog some 18,000 living and extinct species of Foraminifera. Species of the genera Globigerina and Biloculina are extraordinarily abundant, their dead shells making up a large part of the soft mud on many parts of the ocean floor. Similarly, the siliceous skeletons of Radiolaria give rise to radio- larian ooze. Tintinnids are protozoans belonging to the Class Ciliata, which occur in vast numbers, especially in coastal waters. At times tintinnids along with copepods and euphausiids make up a major part of the zooplankton. Copepods and euphausiids belong to the Class Crustacea. Copepods usu- ally constitute about 70 per cent of the zooplankton. Besides the afore- mentioned organisms, marine zooplankton also consists of ostracods, am- phipods, jellyfishes, siphonophores, worms, heteropods, pteropods, and the sperm, eggs, and larval stages of many animals. The term plankton is applied to the vast assemblage of feebly swim- ming or floating organisms, both plant (phytoplankton) and animal (zoo- plankton), which drift with the motion of the water. The chief compo- nents of phytoplankton are diatoms and dinoflagellates, with smaller num- bers of coccolithophores, silicoflagellates, blue-green algae, green algae, and the reproductive products of seaweeds. Being dependent upon radi- ant energy, active phytoplankton are confined to the euphotic zone. Zooplankton, like other animals, depend either directly or indirectly upon photosynthetic organisms for food. As is elaborated in Chapter V, the abundance of bacteria and allied microorganisms is closely linked with the abundance and activities of zooplankton and phytoplankton. Plankton are often classified on a basis of size as macroplankton, which are large enough to be seen with the naked eye or to be taken with a coarse net of No. oo to ooo bolting cloth; net plankton, which are smaller than I mm. yet large enough to be retained by a No. 20 or 25 silk bolting cloth net having a mesh of 0.06 to 0.08 mm.; and nannoplankion, minute organ- isms which are too small to be retained by a No. 25 net. Because nanno- plankton (from Greek xanos meaning dwarf), which range in size from 1 to 50m, are often removed from water by centrifuging (LOHMANN, 1922; WULFF, 1926), such organisms are sometimes termed centrifuge plankton. Nannoplankton can also be removed by filtering the water through hard filter paper (LEBOUR, 1917). Some workers apply the term microplankton to all planktonic organisms which are smaller than macroplankton as de- fined above, while others use the term microplankton as being synony- mous with nannoplankton as defined above. Free-floating or swimming organisms ranging in size roughly from o.2 to 5 uw have been designated ultraplankton. ‘The bacteria and yeasts which lead a planktonic existence belong to the ultraplankton, but most bacteria are associated with larger organisms or other particulate material where they lead a sessile existence. Chapter II — 25 — The Marine Environment Sessile organisms are termed epiplankton by HuBer-PEstatozzi (1938) in his treatise on phytoplankton. Many epiplankton are parasitic. The term neuston is applied to plants, animals, and bacteria which are associ- ated with the surface film of water. Under certain conditions the surface film of water serves as a mechanical support for neuston (WELCH, 1935). a — (, rs a lin Chapter IIT COLLECTION AND EXAMINATION OF SAMPLES ASE One of the most difficult of the peculiar problems of technic confront- ing the marine bacteriologist is the collection of samples of sea water or bottom deposits for analysis from any desired location or depth. There are very few places in the world where samples uncontaminated by land drainage can be obtained without the use of a boat, and it is often neces- sary to travel considerable distance from land. A small boat necessitates the special preservation and rapid transport of samples, while bacteri- ological work at sea even on a boat large enough to provide laboratory facilities is vicissitous to say the least. _ The collection of water samples:— Most of the hundred or more bacteriological water samplers which have been described are suitable only for collecting surface samples or samples from shallow depths. In the latter category are bottles from which the stopper can be removed by a string, spring, or messenger at the desired depth. JOHNSTON (1892) devised such an instrument with a glass-stoppered bottle fastened in a weighted metal frame which was lowered to the desired depth with a rope. A second line was attached to the stopper in such a way that pulling the line removed the stopper, thereby permitting the previously sterilized bottle to fill with water. When the tension on the line is released, the stopper drops back into position. Several modifications of this type of apparatus for removing the stop- per from a water bottle have been devised. HEYDENREICH (1899) at- tached the operating string in a different way. ESMARCH’s bottle for col- lecting samples with a rope and wire line is described by EyrE (1930). ZILLIG (1929) avoided the use of the troublesome second string, which has a tendency to become entangled with the supporting rope, by a mes- senger arrangement for activating the removal of the stopper. The rope was attached to both the bottle and the stopper in such a way that the messenger releases the rope from the bottle so that the bottle is suspended by the stopper which pulls the stopper from the bottle. A string connect- ing the stopper to the bottle provides for hauling the bottle to the surface. ABBOTT (1921) described a sampler in which a single line is connected directly to the stopper. Springs hold the stopper in the bottle until a sud- den jerk on the line by the operator removes the stopper long enough to permit the bottle to fill with water. A simplified version of such a mech- anism is described by WHIPPLE (1927), who pictures several other types of bacteriological sampling bottles which have been used by various workers. Although some most ingenious devices have been designed for remov- ing the stopper from bottles, all such bacteriological samplers have two inherent defects: They are useless at depths exceeding 5 to 40 meters where the hydrostatic pressure makes it impossible to remove the stopper, and the sample may be contaminated by bacteria from the outside of the sampler. MINERVINI (1900) took samples through a sea cock in the boiler room Chapter III — 27 — Collecting Samples at Sea of a steamer. This procedure is open to question because some of the sedentary organisms which grow in and around the external orifice of the sea cock will probably enter the sample. Most submerged surfaces, in- cluding ships’ bottoms and water conduits, are covered with a profuse growth of plant and animal life along with large numbers of bacteria. Metal cylinders for collecting samples:— Some of the pioneer work has been done on water samples collected with water bottles designed by hydrographers for collecting water for chemical analyses. Essentially Ekman, Nansen-Knudsen, Green-Bigelow, Sigsbee, and similar water bottles (Committee on Oceanography, 1932) are open metal cylinders through which the water flows while the device is being lowered to the desired depth. When a messenger is dropped down the hydrographic wire by which the bottles are lowered, the ends of the cylinders are closed by various kinds of valves thus entrapping a sample of water. In general, bacteriological results obtained with samples collected with such apparatus are of questionable validity. In the first place, they may contain contaminating organisms. Secondly, most of the metals used in the construction of hydrographic samplers have a bacteriostatic or bac- tericidal effect in sea water (page 31). Although glass bottles were used for collecting some of their surface samples, FISCHER (1894a), GAZERT (1906)), and many other workers used metal cylinders for sub-surface samples. This must be taken into consideration in the appraisal of their quantitative results. Otto and NEUMANN (1904) collected samples for bacteriological anal- ysis in a metal cylinder, the ends of which could be closed with rubber gaskets by dropping a messenger at the desired depth. A nickel-plated cylinder with cocks operated by a messenger was used by BERTEL (1911). None of the cylinders which go down open can be relied upon to bring up samples which are uncontaminated by adventitious bacteria. Even if the cylinders are first sterilized, which is not always practical, they may be contaminated by the water passing through them while being lowered. MATHEWS (1913) sought to obviate this difficulty by filling a glass- lined cylinder with 95 per cent alcohol to provide for the sterility of the apparatus as well as for the equalization of pressure inside and outside of the closed cylinder. One messenger was dropped to open the ends of the cylinder, which permitted the disinfectant to be flushed out by the water. Then, after allowing sufficient time for the disinfectant to diffuse away, a second messenger was dropped to entrap a water sample at the desired depth. Drew (1914) used this apparatus with expressed confidence for collecting samples for bacteriological analysis to depths of 800 meters. Others have used it or modifications with the cylinder filled with phenol solution or other disinfectants. Besides being somewhat complicated and inconvenient to use, there is always the possibility of the disinfectant diffusing out prematurely through a faulty valve. With a slightly compressible disinfectant such as 95 per cent alcohol, sea water would be forced into the cylinder through nearly any kind of valve feasible for use on such an apparatus. On the other hand, all of the disinfectant may not be washed out of the cylinder during the time the valves are opened and closed. YouncG et al. (1931) fitted a phosphor-bronze cylinder with a brass piston which aspirates a water sample when the piston is pulled out by a messenger-activated mechanism. A similar device made of a 1oo-ml. ZoBell — 28 — Marine Microbiology glass hypodermic syringe has been used by Dr. C. E. RENN at the Woods Hole Oceanographic Institution. It is difficult to exclude water from a cylinder fitted with a piston or plunger, especially at great depths, unless equipped with a valve near the orifice. If such a cylinder could be made absolutely leak-proof, the pressure of the water at great depths against the walls of the cylinder may bind the plunger unless the walls are very thick. REYNIERS (1932) used a glass cylinder fitted on each end with small- bore flexible rubber tubing to which cords are attached. When suspended by the rubber tubing, the latter is bent at a sharp angle thereby sealing the ends of the cylinder. After lowering the apparatus to the desired depth, a second line is used to take the tension off the rubber tubing, which permits the rubber tubing to straighten out, allowing the cylinder to fill with water. This ingenious sampler has the disadvantage of requir- ing two lines for its operation, and no provisions are made for excluding water from the ends of the inlet tubes, a possible source of contamination from the overlying water. The glass cylinder sampler described by BUTKEVICH (1932a) has rub- ber connections which are operated by a messenger-activated mechanism. Samplers having a capillary tube inlet:— RussELL (1892) perfected the evacuated glass bulb sampler of MassEa (Fig. 2A) by fitting a small flask with a glass tube, the end of which could be hermetically sealed. Provisions were made for breaking the end of the tube by dropping a mes- senger at the desired depth, thus permitting the flask to fill with water. It is not necessary to close such a sampling bottle because when left down until the water pressure on the inside is in equilibrium with that on the outside, the tendency will be for water to be forced out of the bottle as it is drawn toward the surface. Therefore there is no possibility of adven- titious organisms entering the sample while being hauled to the surface. The glass flasks are easy to clean, sterilize, and manipulate. Various modifications of this device have been used by PorTIER and RICHARD (1906), KRUSE (1908), PARSONS (1911), ISSATCHENKO (1914), and others (see Fig. 2). WILSON (1920) simplified the apparatus by using a large test tube fitted by means of a rubber stopper with a capillary glass tube, the end of which could be sheared off by a messenger-activated lever. GEE (1932q) attached it to an Ekman bottle. To provide for collecting samples at greater depths, he used a thick-walled test tube with the neck constricted to prevent the rubber stopper from being pushed into the tube by the hydrostatic pressure which increases with depth (Fig. 2D). Although GEE recommended it for “critical work at great depths,” ordinary too-ml. thick-walled test tubes are broken by pressures encountered at a depth of 600 to 1000 meters. ZOBELL and FELTHAM (1934) perfected a mechanism for breaking the capillary tube. Correcting a fault inherent in the construction of earlier models, the capillary tube was bent in such a way that the broken end of the tube through which the water enters the sampler was a few inches away from any part of the apparatus which might carry contaminating organisms (Fig. 2F). After nearly ten years of experimentation with various kinds of de- vices, ZOBELL (1941¢) described the J-Z bacteriological water sampling bottle. It consists of a universal metal frame carrying the messenger- Chapter III — 29 — Collecting Samples at Sea activated breaking mechanism which can be used for either citrate of magnesia bottles or collapsible rubber bottles. The former can be used to a depth of 200 meters without danger of breakage by the water pressure. Rubber bottles can be used to any depth in the sea. Both types of bot- tles are fitted with a short piece of heavy-wall rubber tubing which is closed by a piece of small-bore glass tubing hermetically sealed at the end (see Fig. 3). The assembled J-Z bottles are sterilized by steam pressure. They are sealed as soon as they are removed from the autoclave so that upon cooling they shall remain partly evacuated. The resulting reduced pres- sure facilitates the collection of samples. The rubber bottles are com- pletely collapsed prior to sealing. An important innovation in the J-Z sampler is the piece of rubber tubing, suggested by ScHACH (1938), which facilitates the construction and adjustment of the capillary inlet tube. An assembled bottle is con- Fic. 2. — Types of capillary-tube water samplers used by RussEtt (A), IsSATCHENKO (B), Witson (C), GEE (D), and ZoBrEtt (E). The arrows indicate the points where the tubes are broken by the breaking mechanisms to permit the entrance of water. nected to the carrier as illustrated by Figure 3. When the messenger en- gages the lever, the latter strikes the glass tube, causing it to break at a point of strain, FM, a file mark. When the glass tube is broken, the rub- ber tube straightens out so that the sample is taken from a position sev- eral inches away from any point of contamination on the carrier or cable to which it is attached. The carrier is adapted for ready connection to a standard hydrographic wire or cable. Several of the J-Z bacteriological water samplers can be connected seriatim on the cable as it descends into the water, thereby making it possible to collect samples concurrently from several different depths at the same station. When the messenger activates the uppermost sampler on the line, it releases a second messenger which activates a sec- ond sampler farther down the line. This releases the third messenger, the third releases the fourth, and so on. The J-Z samplers can be used on the same line with Nansen bottles, Allen bottles, reversing thermom- eters, and other standard hydrographic apparatus. C) rk = DONA (} 7® Ie Re Zs -- LEVER — SF FERS Less TUBE — aes i \-RUBBER TUBE BOTTLE 1 " 163 126 Cy | 10 240 228 184 19.3 20 203 188 165 12.2 35 241 216 198 8.7 It has already been pointed out on page 12 that the penetration of sunlight decreases rapidly with depth. Ultraviolet radiations are far less penetrative than visible radiations, the penetration of the former decreas- ing with wave length. This is graphically illustrated by Figure! 8 which PERCENT OF RADIATION IN METERS DEPTH Fic. 8. — Percentage of incident radiations of different wave lengths (expressed as Angstrom units) which pen- etrate clear sea to different depths (adapted from HUvLBurt, 1928). gives the absorption curves for ultraviolet radiations of different wave lengths in the abiotic range in clear sea water. According to the literature reviewed by ELtis e¢ al. (1925) and by BucHANAN and FULMER (1930), the bactericidal range of solar radiations is from 2100 to 2960 A, the max- imum being between 2500 and 2800 A. Wave lengths from 3000 to 3660 A are only feebly abiotic. From Figure 8 it will be observed that the penetrative power of wave ZoBell — 72 — Marine Microbiology lengths shorter than 2800 A is very small. Virtually all of the radiations which are most bactericidal are absorbed by the first meter of sea water. The intensity of the most lethal radiations is reduced nearly 50 per cent by passage through only 10 cm. of sea water. The lethal action of ultra- violet radiations decreases exponentially with decreasing intensity. If the intensity of sunlight is sufficiently intense at the surface of the water to kill a given bacterium in ten seconds, it would require more than 100 sec- onds to kill such a bacterium protected by a layer of sea water 40 cm. thick, and 1000 seconds to kill it in 70 cm. of water. The absorption curves in Figure 8 are based upon observations by Hutpurt (1928) in which the angle of incidence of the radiations is 90° and in perfectly quiet, clear sea water free of suspended matter. The transmission of radiations is reduced proportionately as the angle of inci- dence decreases. The transmission is also reduced when the surface of the water is ruffled and by the presence of particulate matter. So few as 100 bacteria per ml. of water greatly decrease the transmission of ultraviolet radiations. HUuLBurRtT (1928) found that sea water is much less transparent to ultra- violet radiations than is pure distilled water, the absorption coefficient for the wave length of 3030 A being o.or7 in sea water as compared with 0.005 in distilled water. The transmission of ultraviolet radiations by fresh water in lakes or rivers is primarily a function of its content of elec- trolytes and suspended materials, its turbulence, the angle of incidence, and the intensity of the incident radiations. In natural fresh water, BUCHNER (1893) noted only a feeble lethal action of sunlight on bacteria, the bactericidal power penetrating less than 3 meters. JORDAN (1900) found that in river water, sunlight is vir- tually without bactericidal action. Even in clear calm water it is doubtful whether abiotic radiations are active more than 5 feet from the surface, and due to the turbidity and constant movement of most bodies of water in nature, it is improbable that bacteria are subjected to radiations for sufficient time or in sufficient intensity to be killed. From his data on the seasonal and vertical distribution of bacteria in Lake Windermere, England, Taytor (1940) detected no bacteriostatic effect of the sun’s rays. During an eleven-week period he found almost as many bacteria in samples collected from the immediate surface as in samples from a depth of 1 meter, and there were generally more bacteria in surface water than in samples from a depth of 10 meters. It has been pointed out by PrEscotr and WINSLow (1931) that the tendency of bacteria to settle in standing water has been misinterpreted as a lethal action of sunlight. This criticism is applied to the work of CLEMESHA who attributed very great importance to the action of light in the self-purification of lakes and rivers in India after observing more bacteria in bottom water than in the superficial layers of water. The increasing abundance of bacteria from the surface downward as shown by the curve in Figure 7 on page 67 may appear at first sight to indicate a direct inhibitory effect of sunlight in surface waters, but there are other more cogent factors which account for the vertical distribution of bacteria. Unquestionably light does have an indirect effect upon the diurnal, seasonal, horizontal, and vertical distribution of bacteria in sea water or lake water through the intermediary of photosynthetic organ- isms and to a lesser extent of phototropic organisms. In the latter cate- gory are certain zooplankton which migrate towards the surface during Chapter V — 73 — Distribution in the Sea hours of darkness and sink to deeper water as the intensity of sunlight increases (SPOONER, 1933; CLARKE, 1933; RUSSELL, 1936). The abun- dance of diatoms, dinoflagellates, and other phytoplankton in water is directly associated with light intensities (STANBURY, 1931; PETTERSSON et al., 1934). Temperature as an ecological factor:— In a stable environment like the sea where the lack of organic matter or solid surfaces are known to limit the bacterial population, it is doubtful whether the temperature has much influence on the number of bacteria found there, although the tem- perature unquestionably influences the kinds of bacteria and their activ- ities. The total bacterial population is merely the dynamic balance be- tween the rate of reproduction and the death rate of bacteria in a given environment. Increasing the temperature may increase the rate of multi- plication of bacteria within certain limits, but it may also increase their rate of death. In all except surface waters, the temperature of the marine environ- ment is practically constant throughout the year. In surface waters the annual temperature range is usually only a few degrees Centigrade. If organic nutrients and other conditions essential for growth are present, minor increases in temperature may materially accelerate the rate of bac- terial multiplication and metabolic activity — but only temporarily. At the increased rate of assimilation of organic nutrients, a lack of the latter soon restricts further multiplication, in spite of the increased temperature, until more organic matter becomes available from some source. In the meantime, the increased temperature may have accelerated the death rate of the bacteria so that the increased temperature merely causes a temporary fluctuation in the bacterial population. Evidence for these views is forthcoming from data on the abundance of bacteria in sea water having different temperatures. The largest bac- terial populations in the sea occur on the sea floor which, in general, is the coldest part of the ocean. However, little difference has been found in the numbers of bacteria in shallow bottoms in tropical seas where temper- atures of from 20° to 25° C. prevail and in deep sea bottoms where the temperature is perpetually lower than 5° C. In warm shallow bottoms, bacteria multiply more rapidly, partly because the temperature is higher and partly because more organic matter is settling from the overlying water, but they are also dying off more rapidly. In cold deep bottoms which receive only a limited amount of organic matter, bacteria multiply more slowly but they live longer. When a dynamic balance is struck, it appears that the total bacterial population is independent of temperature except in so far as the temperature influences the availability of organic matter, the principal limiting factor. It has already been mentioned that an annual temperature change of about 8° C. in the surface water temperature at La Jolla has not caused any detectable fluctuations in the seasonal distribution of bacteria over a period of ten years. Similarly there are no differences in the number of bacteria in surface waters at different latitudes which can be correlated directly with temperature over a range of near o° C. in arctic waters to 25° C. in tropical waters. However, the temperature does influence the metabolic rate of bacteria and the kinds which are present. As will be elaborated elsewhere, there are marine bacteria which multiply at tem- peratures considerably lower than any temperature found in the sea and ZoBell a Marine Microbiology others which multiply at temperatures several degrees higher than those encountered in any marine environment. In environments less stable than the ocean or in regions where there are no other limiting factors, temperature is one of the most important ecological factors. In lakes and reservoirs, for example, there is often an indirect correlation between the vertical and seasonal distribution of bacteria with temperature. Moreover, seasonal changes in temperature are responsible for the annual overturn of the water which is character- istic of lakes in the temperate zones (WELCH, 1935). TAYLOR (1940) found no direct effect of temperature on the number of bacteria in water in English lakes, the annual temperature of which ranges from 2° to 20° C. The bacterial population of sea water stored in glass receptacles is largely a function of the temperature of incubation until a state of equi- librium is reached. Thereafter the bacterial population is independent of temperature within the range of from 0° to 25° C. The temperature range of growth of marine bacteria and the influence of temperature on their activities are discussed elsewhere in this volume. Seasonal distribution of marine bacteria:— Very few workers have been in a position to make sufficiently extensive or continuous observa- tions on the bacterial populations of oceans or lakes to show definite sea- sonal cycles of abundance. In Lake Ziirich, PFENNIGER (1902) observed a summer minimum, with spring and autumn maxima. He attributed the autumn maximum to the food supplied by dead plankton organisms. After making similar observations in Lake Ziirich, MINDER (1920) as- cribed the summer minimum to the lethal action of sunlight, but he failed to consider several other concomitant factors which are known to influ- ence the abundance of bacteria in bodies of natural water. RUTTNER (1932) correlated the spring and autumn maxima observed in Lake Lunz with the semiannual turnover of the lake. The highest counts occurred in the spring following the runoff of melted snow. Conversely FRED e¢ al. (1924) failed to find any definite seasonal cycles. These workers, who recognized the complexity of factors which influence the seasonal distribution of bacteria, observed the abundance of bacteria in Lake Mendota for three consecutive years. In 1920 the highest plate counts were observed during the summer, in 1921 the maximum occurred in the autumn, and during 1922 in the spring. In Lake Alexander in Minnesota, HENnrIcI (1938) found a gradual in- crease in the bacterial population as indicated by plate counts as well as by the submerged slide technic from the time that the ice went out in April until August when the maximum count was obtained. The bac- terial population decreased throughout September and October, at which time the survey was discontinued. Only minor fluctuations were noted. The fluctuations were probably less than could be accounted for by the sporadic distribution of microorganisms in water and variations directly attributable to sampling and counting errors. Significantly, the curve for the abundance of bacteria in Lake Alexander followed that for the abundance of plankton during the months when observations were made. Lioyp (1930) found the number of bacteria in the Clyde Sea to be remarkably constant throughout the year for all layers except the surface. The bacterial content of surface waters fluctuated widely, but there was no evidence of rhythmic seasonal variation. In interpreting her data, Lioyp says, ‘“‘Any variations outside the limits of the accuracy of the Chapter V — 715 — Distribution in the Sea experimental methods adopted appear to be erratic and therefore cannot be correlated with any factor varying seasonally.” Drainage from land was one factor which operated intermittently at irregular intervals to increase materially the bacterial population of surface water. The num- ber of bacteria in surface water was also observed to be unusually high during the time that herring fishing was in progress in the Clyde Sea. The range of temperature variation was not sufficiently wide during the year to affect the bacterial content of sea water. Samples of sea water have been collected from the end of the Scripps Institution pier five or six times a week for bacteriological analysis, using comparable procedures since January 1932. The only break in this series was during 1938. Besides giving information on the seasonal distribution of bacteria in sea water, the plates provided a source of cultures which have been used for studies on bacterial physiology and for other purposes. The monthly averages for the years are recorded in Table XX. TABLE XX.— Average plate counts on surface sea water at La Jolla, California, based upon about 24 daily plate counts for each month during a ten year period:— AVER- AGE 1932 | 1933 | 1934 | 1935 | 1936 | 1937 | 1939 | 1940 | 1941 | 1942 | FOR TEN YEARS January 324 787 107 650 517 624 209 85 578 359 | 424.0 February | 251 | 1001 570 | 685 | 1202 507 164 76 390 157 | 500.3 March 190 | 985 380 |1549 | 373 613 102 221 628 173.4) Sora April 186 | 26r | 650 | 468 | 639 | 450 T22) 320) ||| (O60 113 | 387.8 May 130 | 308 660 | 442 531 262 i187) 207 552 334 \'35453 June 156 366 560 941 450 565 168 196 506 332 | 424.0 July 433 | 492 540 | 720 | 861 | 306 165 168 | 636 | 661 | 498.2 August 696 | 1073 620 525 529 | 408 341 133 506 443 | 527.4 September| 423 |1738' | 650 879 699 168 264 212 579 596 | 620.8 October 108 | 587 7207 7s ee TGu Ns 2 0) | uke 239 | 504 7EO NE EO.O November go | 283 ASP wien. ||) Aer Ni yey Mabe | niche) || annals 719 | 518.4 December | 150 180 296 660 III 449 gr | 1018 769 52I | 424.5 The most striking feature of the monthly averages is their remarkable constancy throughout the year. From Table XX it will be observed that during only four months in ten years was the average plate count for the month less than 100 bacteria per ml. of sea water. The monthly average exceeded r1ooo bacteria per ml. only eight times. The lowest plate count obtained for any one day during the ten year period was on a sam- ple collected October 4, 1932, which contained only 7 bacteria per ml. of water. The count remained quite low during the first week in October 1932 to bring the average for the entire month down to 108 bacteria per ml. The highest count during the ten year period was on a sample of water collected on November 16, 1941. This high count of 30,700 viable bacteria per ml. and other high counts on two or three following days are directly attributable to a storm which stirred up the bottom and brought in large quantities of seaweed from the kelp beds a few miles away. Many of the high counts obtained during the winter months are at- tributable to the turbulence of water caused by storms. If it were permis- sible to disregard the plate counts obtained on the days during and im- mediately following such oceanic disturbances, the resulting averages for relatively calm days would show that the abundance of bacteria in the water parallels the abundance of phytoplankton more closely than any ZoBell — 76 - Marine Microbiology other observed property of the water. ALLEN’s (1937) data on the sea- sonal distribution of phytoplankton off the La Jolla pier from 1920 through 1939 show that phytoplankton organisms vary in abundance throughout the year much more than do bacteria. There is a definitely rhythmic seasonal cycle in the temperature of the surface water at this latitude. Year after year the water temperature has been observed to increase from a minimum of about 12° C. in the winter months to a maximum of about 23° C. reached during the summer months, but the abundance of bacteria in the surface water fails to parallel this seasonal periodicity, except for short periods of time. Likewise the annual distribution of bacteria in surface sea water can- not be correlated with the intensity of solar radiations. The latter has = 5 re) x Co /p) =) a 2 tJ a a W . P= | o < He 8) « = ra < O a - O a w ° J Fe cM) Aa MA Jd ANS O N D MONTH OF 1933 Fic. 9. — Seasonal distribution of bacteria (dotted line) in surface sea water at La Jolla, California, based upon the average of daily plate counts from January through December, 10933. The relative number of phytoplankton organisms per liter of sea water (solid line) and the intensity of solar radiation (dashed line) are also given. The latter was measured by the recording pyrheliometer at the Scripps Institution. been measured continuously by an automatic recording pyrheliometer which shows a definite seasonal cycle, the period of maximum insolation at this latitude being from May through July. Minimal plate counts are often observed during this period of maximum insolation as shown by the curves in Figure 9, but during other years there is no semblance of cor- relation between the abundance of bacteria in surface sea water and the intensity of sunlight. The studies stress the importance of continuous and extensive obser- vations preliminary to evaluating any single ecological factor. The com- plexity of the factors influencing the marine environment is also illus- trated by these studies on the seasonal distribution of bacteria. The com- plexity of the marine environment is emphasized by ALLEN (1941) in his “Twenty years’ statistical studies of marine plankton dinoflagellates of southern California.” Below the euphotic zone the marine environment appears to be highly uniform throughout the year. One would not expect to find seasonal Chapter V —W— Distribution in the Sea microbiological variations in such a constant environment. The frag- mentary observations which have been made show nothing to the con- trary. Effect of other organisms :— Prof. W. E. ALLEN of the Scripps Institu- tion has kept a daily record of the abundance of diatoms and dinoflagel- lates in samples of local sea water during the last 25 years. A comparison of these phytoplankton records with the bacterial plate counts during ‘the last ten years shows that, in general, the largest bacterial populations are found in water containing the most phytoplankton. This is shown better from an inspection of the daily or weekly averages than from the monthly averages, because the abundance of phytoplankton fluctuates within rather wide limits from day to day as does the bacterial population of sea water. It is for this reason that ALLEN (1941) stresses the necessity of continuous observations over a long period of time. Curves for the vertical distribution of bacteria follow very closely the curves for the vertical distribution of diatoms and other phytoplankton, as is illustrated by Figure 7 on page 67. Since phytoplankton constitute the principal source of food of bac- teria in the sea, as well as providing solid surfaces for attachment, it is not surprising that the abundance of bacteria is closely related to the abun- dance of phytoplankton. The total quantity of organic matter in sea water varies very little with depth until the bottom of the sea is reached. Actually there is often somewhat more organic matter in water below than in the euphotic zone in spite of the activities of photosynthetic organisms. Nevertheless, bacteria are usually most abundant where there is particulate organic matter. In studying the relation of marine bacteria to plankton diatoms in which bottles filled with sea water were suspended in the sea, GRAN (1933) observed that the abundance of bacteria increased and decreased with the diatoms. While there is nothing in GRAN’s experiments to indicate whether the bacteria were attached to the diatoms or whether the dia- toms provided the saprophytic bacteria with organic matter for growth, the observation provides supplementary evidence of the close relationship between bacteria and diatoms in the sea. WaksMan et al. (1933¢) analyzed sea water and plankton tows from the same water for the presence of bacteria. A number 20 silk net was used, which until the pores become clogged would not collect many nan- noplankton or bacteria. However, large numbers of bacteria were found in the plankton tows, presumably because the bacteria were attached to the plankton. From 500 to 2,270 times as many bacteria were found per unit volume of diatom plankton as were found in the unstrained water. These workers concluded that this indicates that a definite parallelism exists between the bacteria and the plankton content of the sea. The actual existence of such a relationship is substantiated by the fact that the bacteria from the diatom tow showed a considerable abundance of agar- liquefying organisms. While one encounters only infrequently such or- ganisms in the free water, they were found to make up 5.7 to 6.7 per cent of the total bacterial flora in plankton tows, as determined by the plate method. According to WAKSMAN et al. (1933c) bacteria occur only to a very limited extent floating free in water, most of them being attached to plankton organisms. Bacteria may live upon dead plankton or upon the ZoBell — 78 — Marine Microbiology excretion products of the cells, upon the cell membranes, and especially upon the mucilaginous substances which are excreted by certain algae. Since dead or decomposing plankton organisms serve as well or better for the attachment or food requirements of bacteria, the abundance of bac- teria may not always parallel the abundance of living plankton. The maximum bacterial counts may occur after the diatoms start to die. The data from an experiment conducted by WAKSMAN ef al. (1933¢) are sum- marized in Table XXI to illustrate this point. Not only do dead and decomposing plankton organisms furnish saprophytic bacteria with a ready source of food, thereby providing for the multiplication of bacteria, but when the particulate organic matter is decomposed, attached bacteria are liberated, thereby making it possible to detect more of them by plating procedures. TABLE XXI.— Comparative numbers of diatoms and bacteria per ml. of sea water afte different periods of incubation at 25° C.:— PERIOD OF INCUBATION ° 3 DAYS 7 DAYS 12 DAYS Diatoms 177 1,026 178 165 Bacteria 346 20,600 22,800 13,900 HENRICI (1938) observed a distinct parallelism between the abundance of bacteria and the abundance of phytoplankton in Lake Alexander. The view is expressed by PUTTER (1926) that the secretion of dissolved substances by phytoplankton is the most important process by which heterotrophic bacteria in the euphotic zone are nourished. According to BRAARUD and F6yN (1931), not less than 30 per cent of the anabolites of certain phytoplankton are secreted by the living cells. From their ex- perience with diatoms, MarsHALL and Orr (1930) doubt that phyto- plankton secrete dissolved organic matter. GRAN and RuupD (1926) and GAARDER and GRAN (1927) believe that certain phytoplankton secrete organic substances copiously. Various aspects of this controversial problem are discussed by ROBERG (1930) and KroGH (1931). WaKSMAN ef al. (1937) found no evidence that living diatoms nourish saprophytic bacteria, although dead diatoms are rapidly decomposed by bacteria and large bacterial populations are found associated with living diatoms. Quite likely the bacteria associated with living diatoms serve a useful purpose by producing ammonia, phosphate, carbon dioxide, and possibly other plant nutrients from the decomposition of dead organic matter. WAKSMAN et al. (1937) believe that amoebae, ciliates, copepods, and other grazing animals may be largely responsible for the destruction of living diatoms. Bacteria are also found associated with zooplankton and other ani- mals in the sea. However, in the euphotic zone, animals do not appear to exert as great an influence on the abundance of bacteria as do phyto- plankton and other plants. This is because phytoplankton organisms greatly outnumber zooplankton and nektonic animals in total numbers as well as in total weight. Apparently zooplankton have fewer bacteria associated with them than phytoplankton, the ratio of bacterial numbers per unit volume of zooplankton tow to numbers of bacteria in the un- strained water being 225:1 as compared with ratios of from 500:1 to Chapter V — 79 — Distribution in the Sea 2,270:1 for bacteria in phytoplankton tows (WAKSMAN et al., 1933¢). On the sea floor the direct influence of animals is more pronounced than that of plants. Dead animals, like dead plants, provide food for sapro- phytic bacteria. Large numbers of bacteria are also often associated with living marine animals, but it is doubtful whether this association materi- ally influences the distribution of bacteria in the sea except in localized regions. Many kinds of marine animals ingest and digest bacteria as a source of food (page 173). Such animals may tend to restrict the bac- terial population of the sea within certain limits. The interrelations of bottom fauna and bacteria have been investigated by MARE (1942). Animals which promote the precipitation and sedimentation of bac- teria probably play an important role in restricting the bacterial popula- tion. Lamellibranch mollusks, certain tunicates, sponges, coelenterates, and other ciliated mucus feeders filter large quantities of water, removing from it suspended material including bacteria. Sea mussels of average size studied by Fox et al. (1937) propelled from 2.2 to 2.9 liters of water per hour through their gill chambers removing all suspended matter. Some of the material removed from the water is swallowed by the mussel and the rest is rejected as strands of pseudofeces. Fox and CoE (1943) have demonstrated the ability of the California mussel to remove from sea water very fine, uncentrifugable, colloidal material such as Congo Red or boiled “‘soluble starch”’ particles which are considerably smaller than bacteria. In experiments conducted by ZOBELL and LANDON (1937), the California mussel reduced the bacterial population from 200 million bacteria per ml. of sea water to less than 10,000 per ml. in two hours. Dopcson (1928) has reviewed the earlier literature on the importance of mussels, oysters, and other animals in the purification of water. The early literature on the role of predatory protozoans and other bacteria-feeders upon the self-purification of natural water has been reviewed by MULLER (19126), PREscorr and WrnsLow (1931), and by BAIER (1935). There is clear-cut evidence that in polluted water which may contain millions to billions of bacteria per ml., protozoans and micro-crustaceans thrive at the expense of the bacterial population. This is illustrated by the experiments of Purpy and BUTTERFIELD (1918) in which the bacterial population of polluted water containing no Para- moecia remained fairly constant, but when Paramoecia were present the bacterial population decreased while the Paramoecia increased. A stage is reached when there are no longer enough bacteria present to provide a livelihood for the predatory animals. A survey of the literature fails to reveal any instance in which protozoans or other bacterivorous organisms have reduced the bacterial population below several thousand per ml. as determined by plating procedures. Since the bacterial popula- tion of sea water is generally in the order of magnitude of hundreds per ml., it is reasonable to conclude that bacterivorous animals are not a major factor in restricting the number of bacteria found in the open sea. In polluted water or bottom deposits, however, which support large bacterial populations, bacteria may constitute an important part of the dietary of small animals, and the predatory activities of such animals may be an important ecological factor. WAKSMAN (1937) relates that the significance attached to protozoans as a factor controlling the microbial population of the soil has been consid- erably modified in recent years. In many cases the protozoans are actu- ally beneficial, as shown by the observations of CuTLER and CRuMP (1929), ZoBell — 80 — Marine Microbiology MEIKLEJOHN (1930), and others. Although protozoans consume appre- ciable numbers of bacteria, CUTLER and Crump (1935) believe that the activities of protozoans in the soil keep the bacteria at a level of maxi- mum efficiency. The significance of the relationship between the fauna of lakes and the bacterial population is emphasized by Barer (1935) who has reviewed the rather extensive literature on the importance of bacteria as food for animals. BArER’s appraisal of the relative role played by bacteria as food for different classes of animals in different types of lakes is summarized in Table XXII. TABLE XXII.— Relative importance of bacteria as food for different classes of predacious animals in different kinds of lakes (from BatER, 1935):— CLASS OF PREDACIOUS ANIMALS TYPE OF LAKE OR ORES BIND TE SOUT Saya PROTOZOANS FILTERERS SEDIMENTATERS Eutrophic, rich in Predominant Predominant Considerable nutrients importance importance importance Dystrophic, rich in Slight Moderate Slight humus importance importance importance Oligotrophic, poor Considerable Moderate Slight in nutrients importance importance importance Further details on the relationship of marine bacteria to flora and fauna are given in Chapter XIV. The antagonistic effects of microorganisms:— Bacteria and allied microorganisms indigenous to the marine environment help to make the sea uninhabitable to exotic or adventitious species, and the indigenous species may have some antagonistic effect on each other. The chief way in which this may occur is by alteration of the food supply or by reduction of its concentration to levels below the minimal requirements of other organisms. Certain microorganisms produce specific toxic substances which inhibit the growth of other organisms in the immediate vicinity or destroy them. There are several other ways in which microorganisms are mutually antagonistic, a relationship which is designated as antibiosis in contradistinction to symbiosis. WAKSMAN (1937) writes: ‘‘When two organisms are capable of utiliz- ing the same nutrients, but are differently affected by environmental con- ditions (reaction, air supply, temperature), the one organism that finds conditions more suitable develops more rapidly and thus depresses the other.” This, together with the production of toxic substances, explains why certain fungi and bacteria are capable of growing in practically pure culture even in a non-sterile environment. Some workers regard the toxic or growth-inhibiting substances as “protective metabolic products pro- duced by microorganisms in their struggle for existence.” A classical example of a toxic or antagonistic substance is penicillin which is produced by a soil fungus, Penicillium notatum. Penicillin in ex- tremely dilute concentrations inhibits the growth of bacteria. The pro- duction by actinomycetes of toxic substances which limit the development of soil bacteria has been reported. A strain of Pseudomonas fluorescens isolated from water by Lewis (1929) produced in nutrient media a toxin Chapter V — 81 — Distribution in the Sea which inhibited the growth of bacteria, yeasts, and molds. Additional examples of specific and indirect antagonistic effects of microorganisms are given by WAKSMAN (19410). There are no quantitative data on the extent to which microbial an- tagonism may limit the bacterial population of the sea, but the presence of organic bacteriostatic substances in sea water has been definitely es- tablished. It has been known since the work of DE GrAxA (1889) that the profuse growth of bacteria in raw sea water eventually renders it unfit as a medium for the cultivation of bacteria, presumably due to the formation of toxic products. According to DE GIAxA (1889), Vibrio comma rapidly disappeared from raw sea water, the rate of disappearance being directly proportional to the abundance of other bacteria in the water, but in boiled or autoclaved sea water Vibrio comma survived for a long time. Similarly, KrrrpayAsHi and AIDA (1934) and KRASSILNIKOV (1938) ob- served that bacteria survived much longer in sterile than in raw sea water. Prescotr and WINSLOW (1931) give references to observations on the greater longevity of the typhoid bacillus in sterile sewage as contrasted with its rapid disappearance from raw sewage, which is attributed to bac- terial antagonism. In raw water freshly collected from the Black Sea, only 1,500 Staphylo- coccus aureus developed per ml. in experiments conducted by KrassIr- NIKOV (1938). Similar water previously sterilized by passage through a Seitz filter or by boiling supported the growth of 860,000 and 86,000,000 Staph. aureus respectively. Sea water is rendered more growth-promoting by boiling or autoclav- ing, not only because the heat treatment frees it from antagonistic micro- organisms, but because it also destroys certain thermolabile toxic prod- ucts. ZOBELL (1936) found that sea water sterilized by filtration was ap- proximately three times as toxic for coliform bacteria as was sea water sterilized by heating. When coliform bacteria, confined in all-porcelain Coors filter candles impregnated with collodion, were immersed directly in the sea they died approximately ten times as fast as when they were thus immersed in autoclaved sea water or in Berkefeld-filtered water. The average results from several such experiments are recorded in Table XXIII. These experiments prove that the toxic principle is thermolabile, water-soluble, and tends to be adsorbed by untreated Berkefeld bougies. TABLE XXIII.— Relative numbers of coliform bacteria which survived in stop pered, collodion- treated, Coors porous filter tubes when immersed in different kinds of water at 16° C. (from ZOBELL, 1936):— TIME IN MINUTES IMMERSED IN 30 60 fete) 120 ‘Formula C”’ control (see page 47) 100 gl 82 97 Natural sea water 47 25 I5 3 Autoclaved sea water 56 38 27 306 Berkefeld-filtered sea water 39 20 23 19 Further experiments have shown the toxic principle to be adsorbed by activated charcoal, diatomaceous earth, bentonite, and other surface- active substances. Also, it is precipitated from solution by iron and alu- minum salts in a slightly alkaline medium. There is a higher concentra- tion of the toxic principle in polluted sea water than in sea water collected from the open ocean. ZoBell — §2 — Marine Microbiology After noting that bacteria survived longer and were more active in heat-sterilized sea water or in artificial sea water, WAKSMAN and HotcH- KISS (1937) concluded that the antagonistic effects of microorganisms including nannoplankton help to explain the low numbers of bacteria usually found in natural sea water. Another manifestation of microbial antagonism is the progressive de- crease in the number of species of bacteria found in sea water during its incubation in the laboratory (ZOBELL and ANDERSON, 1936a). Asarule, the larger the bacterial population which develops in stored sea water, the fewer the species which survive. After several months storage, only two or three species of bacteria were found in samples of sea water which contained thirty to forty species when collected from the sea. TAYLOR (1940) directs attention to the commonly observed fact that the denser the bacterial population in natural waters, the smaller the num- ber of species. In Lake Windermere, which normally contains only a few thousand bacteria per ml., TAYLOR (1942) found scores of bacterial types, contrasted with only a few types found in polluted river water entering the lake. Besides contributing to the rapid destruction of adventitious species, the accumulation of hetero-antagonistic substances in the sea may help to explain the specificity of marine bacteria. In spite of the interchange of bacteria between the land and sea through the intermediaries of wind and water, most of the bacteria occurring in the sea at places remote from pos- sibilities of recent terrigenous contamination are quite unlike bacterial species occurring in soil or fresh-water environments. Bacteriophage in sea water :— Bacteriophage is a lytic, filter-passing, self-propagating principle which causes the destruction of susceptible bac- teria. It has been credited with contributing to the self-purification of polluted water. According to D’HERELLE (1926), the first record of bac- teriophage action was the observation of HANKIN in 1896 that unsterilized waters of certain rivers in India have a tendency to destroy bacteria. For example, just below Agra more than 100,000 bacteria per ml. were found in the Jumna River, while 5 kilometers farther down the river fewer than too bacteria were found in the water. The antiseptic property of the water was destroyed by boiling. Our knowledge of the occurrence and activity of bacteriophage in sea water is rather fragmentary. Bacteriophage is relatively resistant to ad- verse chemical and physical conditions, occupying a position in this re- spect intermediate between vegetative forms and spores of Bacillus sub- tilis (D’HERELLE, 1926). A high concentration of salt is not injurious to bacteriophage, BRUTSAERT (1924) having shown that the bacteriophage for Staphylococcus aureus and Staph. albus is active in broth containing as much as 14.5 per cent NaCl. After noting the occurrence of bacteriophage in rivers, soil, sewage, and in nearly all environments harboring large bacterial populations, pD’ Hf&RELLE (1926) remarked, ‘“‘It is everywhere present, one might say.” pb’ H&RELLE found bacteriophage active against dysentery bacilli in sea water at the mouths of rivers. He found the bacteriophage for Escherichia coli in the estuary of Mekong, French Indo-China. Although bacterio- phage is often present in sea water along the coast and particularly near the mouths of rivers, D HERELLE was unable to find it in water collected from the Indian Ocean at approximately 60° E. Long. and 10° N. Lat. Chapter V — 83 — Distribution in the Sea The presence of bacteriophage active against various bacteria has been reported by HaupuRoy (1923), FERNAND et al. (1925), FEJGIN (1926), ForTUNATO (1928), GILDEMEISTER and WATANABE (1931), and others who have examined coastal waters. FERNAND ef al. (1925) believed that the rapid disappearance of fresh-water bacteria observed in the sea is due primarily to the action of bacteriophage rather than to the unfavorable salt content of sea water or to other adverse physical conditions. GILDE- - MEISTER and WATANABE (1931) concluded that while bacteriophage may occur in sea water, the content is very small. GEE (1932d) wrote that, although the activity of bacteriophage in reducing the bacterial popula- tion of water has not been determined, it certainly may be expected in the intestinal contents of marine fishes. Davis (1933) was unable to demon- strate any bacteriophage in either sea water or in polluted sea-bath waters. Observations at the Scripps Institution indicate that bacteriophage for several different bacteria occurs in sea water along the coast, but it is rather dificult to demonstrate its presence. The infrequency with which positive results are obtained indicates that bacteriophage is not present in high concentrations or that it occurs only sporadically. The lytic prin- ciple has never been demonstrated in water collected beyond the littoral zone. Since bacteriophage is generally found associated with large numbers of rapidly multiplying bacteria, it is very doubtful if the sparse bacterial population characteristic of the open ocean is conducive to the develop- ment or activity of bacteriophage. The activity of bacteriophage may contribute to the destruction of bacteria in polluted water along the coast, but there are no experimental data or theoretical considerations to suggest that bacteriophage is a factor which limits the bacterial population of the open ocean. Effect of solid surfaces :— Unlike polluted waters, the bacterial popu- lation of which decreases from billions per ml. to millions per ml. during storage in small receptacles, the bacterial population of sea water which normally contains only a few hundred bacteria per ml. increases to thou- sands or millions of bacteria per ml. after being stored for a few days in glass receptacles. WAKSMAN and CarEy (1935a) attributed the increase in the bacterial population of stored sea water to the dying out of proto- zoans and other animals which devour bacteria and to the modification of “certain controlling factors (7m situ) injurious to free bacterial develop- ment.” Keys ef al. (1935), who observed similar changes in stored sea water, expressed the belief that the activity of bacteria in the sea is limited by the “extreme stability of the ocean as a chemical and physical factor.” This may be another way of saying that there are not more bacteria in the sea because they die off as fast as they multiply, but it fails to explain why the bacteria multiply faster than they die for a few days following storage of the water in glass receptacles. Temperature cannot be regarded as responsible for the increased bac- terial activity in stored sea water, because the bacterial population in- creases regardless of the temperature within the range of from 0° to 30° C. In fact, there is increased bacterial activity in sea water which is stored in bottles submerged in the sea under conditions which otherwise simulate very closely natural environmental conditions. Likewise the bacterial population increased a hundred- to a thousand-fold when the water was stored in the sea retained in glass bottles with open mouths which per- ZoBell — 84 — Marine Microbiology mitted the entrance of small predatory animals. When introduced from plankton tows into such bottles, protozoans and copepods survived with- out having any noticeable effect upon the bacterial population within the limits of hundreds to hundreds of thousands of bacteria per ml. of water. After observing that bacteria multiply proportionately faster in small receptacles than in larger ones, WHIPPLE (1901) concluded that the avail- ability of oxygen was the responsible factor. When the receptacles are only partly filled with water, proportionately more of the water is exposed to the atmosphere when it is stored in small receptacles than when it is stored in larger receptacles of similar shape. However, ZoBELL and STADLER (1940)) have shown that the multiplication and respiration of aquatic bacteria are independent of the oxygen tension throughout the range of from 0.30 to 36 mgm./liter. ZOBELL and ANDERSON (1936a) confirmed WHIPPLE’s observation that bacteria multiply in water stored in small receptacles appreciably faster than in identical water stored in larger receptacles of similar shape. After proving that neither temperature nor oxygen tension is responsible for the observed phenomenon, the increased bacterial activity was attributed to the beneficial effect of solid surfaces of the receptacles in which the water was stored. The beneficial effect of added glass beads, ignited sand, and other inert particulate materials substantiated this conclusion, which has also been confirmed by the observations of LLoyp (1937). In dilute nutrient solutions such as sea water which contain less than 10 mgm. of organic matter per liter, solid surfaces promote the multiplica- tion of bacteria. This is illustrated by the data in Table XXIV obtained by storing sea water in glass-stoppered Pyrex bottles for two weeks at 16° C., after which bacterial populations and oxygen consumption were measured. TABLE XXIV.— Number of bacteria found in sea water, which initially contained 276 bacteria per ml., after two weeks storage in the dark at 16° C. in glass-stoppered bottles of different capaci- ties. The area of glass exposed to the water and the ratio of volume in ml. of water to the area of glass surface are also given (from ZOBELL and ANDERSON, 1936a):— VOLUME OF SEA | SOLID SURFACE Ratio oF | AVERAGE NUMBER OF | OXYGEN CONSUMED WATER IN ML. IN CM.? ML.: CM.? BACTERIA PER ML. MGM./L. 14 27 1:2.64 1,863,000 3.42 120 148 Ty23 1,070,000 2ans 1,225 640 TO 52 553,000 TS 13,220 3174 I:0.24 261,000 0.97 Solid surfaces promote activities of bacteria in dilute nutrient solutions primarily by adsorbing organic matter, thereby making it more available to bacteria. ZOBELL (19430) has presented chemical and biological evi- dence that glass and other inert solids adsorb measurable quantities (2 to 27 per cent) of the organic matter in sea water. After observing the accumulation of organic matter on chemically cleaned glass slides, STarK et al. (1938) expressed the belief that adsorbed organic nutrients favor bacterial growth. Corroborative evidence is given by the studies of HEUKELEKIAN and HELLER (1940) on the relation between food concentration and solid surfaces. In their experiments, Escherichia coli failed to multiply when the concentration of food was less than 0.5 p.p.m. unless glass beads were added to provide adsorbing sur- faces. The beneficial effect of glass beads was noticed in solutions con- taining up to 25 p.p.m. of nutrients, above which concentration solid Chapter V — 85 — Distribution in the Sea surfaces were not particularly beneficial. By adsorptive concentration of nutrients, solid surfaces enable bacteria to develop in substrates otherwise too dilute for growth. Besides concentrating nutrients, solid surfaces in sea water provide surfaces for the attachment of sessile bacteria. The sessile habit of aquatic bacteria is the basis of the submerged slide technic employed by HENrIcI (1933, 1936), ZOBELL and ALLEN (1933, 1935), HorcHKiss and WAKSMAN _ (1936), SmitH and ZOBELL (1937), and others for the direct microscopic enumeration of bacteria. KUSNETZOWA (1937) believes that all water bac- teria are capable of attaching themselves to glass, and that many of them grow only when attached to solid surfaces. Several species of bacteria which grow only when attached to firm substrates have been described by HeEnrIcI and JOHNSON (1935), ZOBELL (19430), and ZOBELL and UPHAM (1944). Most of the bacteria in bottom deposits appear to be adsorbed on or attached to particles of sediment (WAKSMAN and VARTIOVAARA, 1938). Solid surfaces retard the diffusion of exoenzymes and partially digested food away from bacteria. Large molecules of organic matter must be con- verted into soluble substances by bacterial exoenzymes before the food can be assimilated by bacteria. Consequently in a dilute nutrient solution such as sea water, free-floating bacterial cells may not be able to digest and absorb enough nutrient to provide for their organic requirements. However, when the bacterial cells and organic nutrients are juxtaposed on solid surfaces, the bacteria may more effectively absorb the food which has been rendered soluble by their exoenzymes. Bacteria in an anchored position are less influenced by molecular bombardment (with the resultant Brownian movement and diffusion) which would tend to separate them from their exoenzymes and hydrolyzates. Also, solid surfaces probably facilitate the orientation of exoenzymes in the most advantageous posi- tion, thereby increasing their stability and activity. The beneficial effects of solid surfaces in dilute nutrient solutions help to explain why marine bacteria generally occur intimately associated with solid particles, as has been shown by the work of Ltoyp (1930), WAKSMAN et al. (1933¢), and others. In considering the factors which influence the distribution of bacteria in natural waters, PREScoTT and WINSLOw (1931) emphasized that in natural waters bacteria are to a great extent attached to large solid particles. Inert particles appreciably smaller than bacteria are not beneficial to bacteria; they may even be injurious to bacterial activity (ZOBELL, 1943b). Other evidence for the attachment propensi- ties of aquatic bacteria is reviewed on pages 56 and 193. Effect of sedimentation :— After stressing the importance of the low concentration of organic nutrients in sea water as a factor which limits the marine bacterial population, RENN (19370) declared that, ‘‘ Particulate substrates, necessary for the favorable development of large attached populations, tend to settle and carry large numbers of bacteria into the mud during sedimentation.”’ Thus, while the affinity of solids for bacteria tends to promote bacterial multiplication in dilute nutrient solutions, solid particles whose density exceeds that of sea water also tend to reduce the bacterial population. FRANKLAND and FRANKLAND (1894) concluded that sedimentation is of the utmost importance in the purification of water. JORDAN (1900) re- garded it as noteworthy that in all the instances recorded in the literature ZoBell — 86 — Marine Microbiology where a marked purification has been observed, the conditions are pre- cisely those which are most favorable for sedimentation. GAINEY (1939) emphasized the importance of sedimentation in the purification of water by declaring that “The decrease in bacterial numbers in water stored in reservoirs, while not entirely due to sedimentation, should probably be at- tributed largely to this factor.” He gives substantiating laboratory and field data. Applying Stokes’ law of falling bodies, Sprrra (1903) esti- mated that 20 to 50 per cent of the bacteria in canal water settle out due to their attachment to gross particles. RUSSELL (1891) recognized the importance of sedimentation in the dis- tribution of bacteria in the Gulf of Naples where many more bacteria were found in the bottom deposits than in the superficial strata of water. How- ever, the occurrence in bottom deposits of bacterial species never found in the overlying water convinced him that bacteria multiply in the bottom deposits and are not merely passive transients from the overlying water. HEnRICI (1939) ascribed the great preponderance of bacteria in lake bot- tom deposits as compared with the number found in the overlying water to several different factors, “the most important being the tendency of bacteria to be adsorbed by or otherwise attached to solid particles in the water, and to be carried by these particles to the bottom.” Sedimentation is most important in removing bacteria from sea water along the coast, particularly in localities where there is much land drain- age. In such places the precipitation and sedimentation of suspended matter are accelerated by flocculation which occurs when fresh water is mixed with sea water. Sedimentation is believed to be primarily respon- sible for the localization of the pollution of sea water by land drainage as manifested by the rapidity with which bacterial populations decrease with distance from sewage outfalls and from the mouths of rivers. In such places the bacterial population decreases much more rapidly than can be accounted for by dilution alone. From the average bacterial content of river water and that of sewage entering the Pacific Ocean, it is estimated that no fewer than 5 X 10” bac- terial cells enter the sea from the land along the coast of the North Pacific Ocean each day. Yet rarely does one find terrigenous bacteria in the sea at distances greater than a few miles from the mouths of rivers or sewage outfalls, in spite of water movements which are favorable for the wide- spread distribution of such organisms. The majority of the bacteria are carried to the sea bottom very near the point of entrance to the sea. The principal exceptions to this rule are caused by small quantities of floating solids and by fresh water flowing over the non-turbulent surface of sea water for considerable distances. Sedimentation also has a marked influence on the distribution of bac- teria in lakes, as shown by the quantitative studies of KLEIBER (1894) in Lake Ziirich. Bacteria introduced into the lake in large numbers by in- flowing streams are detectable only for short distances in the lake. The decrease in the bacterial population parallels the rapidly diminishing tur- bidity or cloudiness in the lake induced by the streams. An average of 10 bacteria per ml. was demonstrated in lake water a few meters from shore as compared with plate counts exceeding 10,000 per ml. of water in the mouth of the river. Since calculations indicated that this observation could not be attributed solely to dilution, KLEIBER considered sed’menta- tion to be the chief cause of the rapid diminution of bacteria where streams enter the lake. Chapter V — $7 — Distribution in the Sea In their ‘‘Elements of Water Bacteriology,” Prescott and WINSLow (1931) state that chief among the factors influencing the diminution of bacteria in surface waters appear to be sedimentation, the activity of other microorganisms, light, temperature, food supply, and perhaps more obscure conditions such as osmotic pressure. BAIER (1935), RUBENTSCHIK et al. (1936), TAYLOR (1940), and others stress the importance of sedimentation as a factor which limits the mi- crobial population of lakes, reservoirs, and bays. Sedimentation is not the prime cause of the paucity of bacteria in the open ocean, but it helps to explain the vertical distribution of bacteria. Here sedimentation occurs more slowly due to a dearth of suspended par- ticles. Most of the suspended particles in the open ocean are diatoms, dinoflagellates, and other plankton organisms equipped with special flota- tion adaptations which retard or prevent sinking. It is with such solid particles that the bacteria in the open ocean are associated. Following the death of the supporting plankton organisms, the organic tissues of the latter are gradually decomposed, leaving skeletal remains of greater den- sity. As the density increases and the flotation mechanisms disintegrate, the skeletal remains sink faster and faster towards the bottom. The rate of sinking of such organisms or their remains at different depths probably approximates the reciprocal of the curves which represent their abundance at different depths (see Figure 7 on page 67), assuming that the organ- isms are not completely decomposed. Of course, large numbers of plankton organisms are devoured by pred- atory animals and some may be completely decomposed by bacteria. However, the remains of numerous plankton organisms sink to the sea bottom, carrying with them attached bacteria. The numbers of bacteria found in the sea at different depths are directly proportional to the quan- tity of particulate material, regardless of whether the latter is living or dead. The paucity of particulate matter at depths exceeding 200 meters is believed to be ascribable primarily to the accelerated rate of sedimenta- tion when the remains of organisms reach this depth. A similar view is expressed by Lioyp (1930) who observed a progressive decrease in the bacterial population with depth in the Clyde Sea, paralleling the sinking of suspended matter. Effect of organic matter:— The concentration of organic nutrients in sea water is very low, being near or below the threshold of the require- ments for many kinds of bacteria. According to KRoGH (1934), sea water contains from 4 to 5 mgm. of total organic matter per liter, or, about 1/5000 as much as ordinary garden soil. An appreciable portion of the organic content of sea water is highly refractory to attack by bacteria. Keys ef al. (1935) estimated that only 10 to 15 per cent of the total or- ganic content of sea water is utilized by bacteria after storage for several days at 21° C. WaAKSMAN and Carey (19350) found that 50 per cent of the total organic content of sea water is readily subject to decomposition by bacteria, the rest being very resistant to bacterial attack. About 60 per cent of the organic matter decomposed by bacteria is completely oxidized to carbon dioxide and water, and 4o per cent is converted into bacterial cell substance or intermediate products of metabolism. S.milar results have been reported by WAKSMAN and RENN (1936). That organic matter is a factor which limits the multiplication of bac- teria in sea water has been established by the experiments of WAKSMAN ZoBell — 88 — Marine Microbiology and CAREY (19350). When they added 2.5 mgm. of glucose to a liter of natural sea water, they found 2,005,000 bacteria per ml. of water after 24 hours incubation as compared with only 625,000 in the control. This quantity of glucose increased the plate count to 3,850,000 when the sea water was also enriched with a little ammonium sulfate. During the same period the addition of 15 mgm. of glucose per liter of sea water increased the plate count to 24,050,000 bacteria per ml. These observations were confirmed by WAKSMAN and RENN (1936) who concluded that organic carbon is the principal factor which limits the bacterial population of sea water. When 1.5 mgm. or more of glucose is added per liter of sea water, com- bined nitrogen also becomes a limiting factor for the multiplication of bac- teria. The nitrogen requirements of most marine bacteria are satisfied by either ammonium salts or amino acids. All of the 15 ‘“‘representative aerobic bacteria of marine origin”’ studied by OstroFF and HENRY (1939) were found to utilize organic nitrogen compounds, but only 5 were able to utilize inorganic ammonium compounds as a source of nitrogen in a min- eral medium enriched with glucose. Most cultures grew luxuriantly on amino acids, which, as a class of compounds, were the best source of nitro- gen and carbon. The number of cultures which grew in glucose media utilizing different nitrogen compounds is shown in Table XXV. TABLE XXV.— Number of cultures of marine bacteria out of 15 tested which utilized dif- ferent nitrogen compounds in glucose media (from OstRoFr and HENRY, 1939):— NUMBER OF CULTURES ComMPOUND NUMBER OF CULTURES ComPouND WHICH GREW WELL CONT. WHICH GREW WELL Glutamic acid 9 Ammonium oxalate 4 Aspartic acid 8 Pyridine 3 Asparagine 8 Betaine 2 Propionamide 8 Uric acid 2 Urea 7 Tyrosine I Creatinine 7 Ammonium chloride I Acetamide 6 Ammonium formate ° Sodium hippurate 6 Ethylamine ° Di-ammonium phos phate 5 Aniline fo) dl-Alanine 5 Guanidine ° Cystine & Peptone 15 It is generally claimed that the minimum concentration of organic nutrients required for the multiplication of heterotrophs ranges from 0.001 to 0.01 per cent or 10 to 100 mgm. per liter. CURRAN (1931) reported that 250 mgm. of peptone per liter was the minimum quantity that supported the germination of bacterial spores. Bacteria which live in the ocean or in oligotrophic lakes must be peculiarly adapted to live in extremely dilute media because the organic content of sea water and many fresh-water lakes is less than 5 mgm. per liter. ZOBELL and GRANT (1943) found that most marine bacteria multiplied in mineral media containing only o.1 mgm. of peptone or glucose per liter. However, the bacteria multiplied very slowly in the presence of such low concentrations of organic nutri- ents. Probably the solid surfaces of the walls of the culture receptacles made it possible for the bacteria to utilize the small quantities of organic nutrients. Supplementing the solid surface by the addition of glass beads, glass tubes, or other inert solids promotes the growth of marine bacteria in media containing less than 10 mgm. of organic nutrients per liter. The optimum concentration of readily utilizable organic matter such Chapter V — 89 — Distribution in the Sea as peptone or glucose for the multiplication of marine bacteria is between 1000 and 10,000 mgm. per liter of sea water. This is 200 to 2000 times the concentration of organic matter in sea water, and only a small fraction of the latter is readily utilizable. This emphasizes that the concentration of organic matter in the sea is decidedly sub-minimal for bacterial activity. The addition of as little as o.1 mgm. of utilizable organic matter to sea water results in increased bacterial activity. Up to 100 mgm. per liter, doubling the concentration of organic nutrients approximately doubles the rate of bacterial multiplication. Although it is difficult to appraise quantitatively each of the numerous interrelated factors which influence the distribution of bacteria in the sea, the organic content of water is certainly one of the most important factors. Increases in the availability of organic nutrients in sea water are almost invariably accompanied by increased bacterial populations. The low con- centration of organic nutrients and the lack of solid surfaces for concen- trating organic nutrients are believed to be the principal factors which account for the small number of bacteria found in the sea. Wherever there is an influx of organic matter or an increase in the amount of sus- pended particles caused by land drainage, stirring up of the bottom, the activity of photosynthetic organisms, the appearance of seaweeds, fisher- ies activities in localized areas, upwelling, or convergence of cold and warm water which kills many organisms, bacteria are found in greater num- bers than in surrounding water. While the numbers of bacteria which may develop in sea water stored in the laboratory are directly proportional to content of utilizable organic matter, the bacterial population of sea water im situ is not necessarily in- dicative of the organic content of the water. A large bacterial population may more effectively utilize the organic content of sea water and reduce it to a lower level than a few bacteria, in which case there would be rela- tively little organic matter in the presence of large numbers of bacteria until the latter perish. The bacteria themselves contain very little or- ganic matter, it requiring a bacterial population of about 10,000,000 per ml. to be equivalent to 1.0 mgm. of organic matter per liter. On the other hand, in certain regions there may be enough organic matter to provide for the rapid multiplication of bacteria, but the population may never exceed more than a few thousand per ml. due to the activities of predators or other factors inimical to the prolonged survival of bacteria. Chapter VI MICROORGANISMS IN BOTTOM DEPOSITS It is in the sedimentary materials on the floor of the sea that the microbial population is most extensive and physiologically versatile. Large numbers of microorganisms of various kinds live in the mud or sand where they influence the activities of sedentary or burrowing animals, the diagenesis of bottom deposits, and certain properties of the overlying water. Viable microorganisms have been demonstrated in most samples of bottom deposits examined for their presence regardless of the depth of the overlying water, distance from land, latitude, or the composition of the bottom deposits. Numbers of bacteria in sediments :— CrErRTES (1884a) found appreci- able numbers of bacteria in all except four of t00 sediment samples, some of which were collected from water depths as great as 5,100 meters on the Talisman expedition, but his results have no quantitative significance be- cause he analyzed the samples only after prolonged storage. Employing plating methods, RUSSELL (1892) found from 25,000 to 300,000 bacteria per ml. of mud from the Bay of Naples as compared with 10,000 to 30,000 bacteria per ml. of mud in the vicinity of Woods Hole, Massachusetts. The bacteria from the Bay of Naples were generally different from the species isolated at Woods Hole, and at both stations the bottom flora dif- fered somewhat from the flora in the overlying water. Drew (1912) demonstrated an average of 160,000,000 bacteria per ml. of mud from the sea floor near Andros Island in the West Indies. Pre- dominating in the lime-rich mud was an organism which Drew described as Bacterium calcis. GEE (19320) estimated that the order of magnitude of the bacterial population of mud from the Florida Keys was hundreds of thousands per gram (wet basis). LLoyp (1931a@) found up to 300,000 bac teria per gram (dry basis) of mud from the Clyde Sea. Incidentally, the water content of recent marine sediments ranges roughly from 30 to 99 per cent, a factor which should be taken into ac- count in expressing quantitative results. Most mud quantities are ex- pressed on a wet or natural weight basis because this is more representa- tive of the spatial relations of the microorganisms. Unlike soil, in which there may be marked seasonal fluctuations in the water content, the water content of marine bottom deposits varies but little from time to time. In the calcareous deposits around the Bahama Islands, BAVENDAMM (1932) counted up to 16,800,000 bacteria per gram of wet mud. The bac- terial population decreased sharply with core depth. In this same region SMITH (1926) found an average of 565,000 bacteria per gram of calcareous mud. Unless otherwise stated all counts refer to aerobes. Sediments from the Channel Island region off the coast of southern California were found by ZOBELL and ANDERSON (19360) to contain up to several million viable bacteria per gram (wet basis). Like other workers who have made such studies, they found that the topmost layers of marine sediments generally contained many more bacteria per unit volume than did surface sea water. The bacterial content of the water of the mud- Chapter VI — 91 — Bottom Deposits water interface is comparable with that of the underlying mud. The bac- terial content of the water decreases sharply above this mud-water transi- tional zone. In bottom deposits of fresh-water lakes, HENRiIcrt and McCoy (1938) found from a few thousand to 500,000,000 bacteria per ml. Direct counts were even higher. RUBENTSCHIK ef al. (1936) demonstrated 3 billion bacteria per gram of mud from limans near Odessa. ISSATCHENKO (1937) found a maximum of 11 billion bacteria per gram of mud from the Kara Sea. These Russian workers employed the direct microscopic technic. Most of the bacteria were adsorbed upon sediment particles, a condition which tends to make plate counts lower than direct microscopic counts because large numbers of bacteria may be adsorbed to a single particle which may give rise to only one colony on a plate. The tendency for bac- teria to be adsorbed by marine bottom deposits has been emphasized by the studies of WAKSMAN and VARTIOVAARA (1938). Other general observations on the bacterial content of bottom depos- its have been made by Wirtt1Ams and McCoy (1935), DiGcELi (1936), BUTKEVICH (1938), HARTULARI (1939), and ELAZARI-VOLCANI (1943). Vertical distribution of bacteria in mud :— Wherever profile series have been examined, a progressive decrease in the bacterial population of bot- tom deposits from the surface downward has been observed (LiLoyp, 1931@; REUSZER, 1933; ZOBELL and ANDERSON, 1936); ZoBELL and FELTHAM, 1942). The decrease is most rapid in the topmost few centi- meters of sediment, below which the decrease is more gradual or even sporadic. This is illustrated by the data in Table XXVI. TABLE XXVI.— Number of bacteria per gram of mud (wet basis) in different core strata (from ZOBELL, 1942b):— CoRE NUMBER XIV-37 XIV-45 XIV-53 STATION LOCATION 32°26.4' N. 32°36.4' N. 33°03.3' N. 117°41.3' W. 117°27.8' W. 117°25.5' W. WATER DEPTH 3120 feet 3570 feet 1415 feet CoRE DEPTH BACTERIA BACTERIA BACTERIA IN INCHES PER GRAM PER GRAM PER GRAM o-I 38,000,000 7,500,000 840,000 I-2 940,000 250,000 102,000 4-5 88,000 160,000 63,000 g-10 36,000 23,000 19,000 I4-I5 2,400 8,700 1,500 19-20 400 2,100 2,200 29-30 180 600 370 39-40 330 200 190 59-60 250 300 210 79-80 130 b tele) 140 99-100 290 150 140 Significant numbers of viable bacteria have been found in marine sedi- ments at all depths sampled. WaxsMan et al. (1933c) reported the occur- rence of bacteria down to a depth of 90 cm. RITTENBERG (1940) demon- strated the presence of numerous bacteria throughout the length of several cores, some of which were longer than 350 cm. The numbers of bacteria found varied greatly from core to core. The abundance of bac- teria decreased from the surface downward, although in certain sediments, zones or strata of high bacterial population were found beneath strata of ZoBell — 92 — Marine Microbiology lower population. These discontinuous zones corresponded exactly with the loci of abrupt changes in the physical and chemical properties of the sediments, a striking example of the relation between bacterial population and environment. Representative data on the vertical distribution of bacteria in Minne- sota and Wisconsin lake bottoms are summarized in Table XX VII. Car- PENTER (1939) found a progressive decrease with depth in the bacterial population of mud from Crystal Lake, an oligotrophic lake in northern Wisconsin. Most of the organisms below the topmost layer of mud ap- peared to be either anaerobes or facultative aerobes. TABLE XXVII.— Bacteria per ml. of lake mud from different depths as determined from plate counts by HEnrici and McCoy (1938): CoRE DEPTH STATION 7 IN STATION I IN STATION 1 IN IN CM. LAKE ALEXANDER LAKE MENDOTA BRAZELLE LAKE o (surface) 123,400 148,000 147,000 2to4 51,060 II7,000 66,000 5 to8 EN 16,400 36,000 g to 12 14,980 19,400 22,900 13 to 20 8,425 12,400 2,750 21 to 30 1,860 12,800 710 31 to 4o 260 1,670 One of the longest cores on record was collected from the Gulf of Cali- fornia by Emery and Dietz (1941), it being nearly 17 feet in length. To obtain this core, the core barrel penetrated the bottom sediment to a depth exceeding 25 feet. Plate counts made by microbiologists showed that the bottom-most part of the core, as well as many other cores repre- senting penetrations ranging from 10 to 20 feet, contained a minimum of several hundred viable bacteria per gram (wet basis). Lower limits of the biosphere :— Finding significant numbers of living bacteria at such great depths, which represent geological ages of many centuries, provokes the question: what is the lower limit of the biosphere? From the extrapolation of the curves showing the abundance of bacteria in sediments with depth, one might expect to find some bacteria at depths of several hundred feet. In highmoor peat, which is a type of bottom deposit especially rich in organic matter, WAKSMAN and STEVENS (1929) found bacteria in almost undiminished numbers throughout the entire profile to the bottom of the greatest depth examined, 510 cm. In ancient sediments which are now above sea level, VON WOLZOGEN KUuR (1922) found sulfate-reducing bac- teria at depths of 10 to 37 meters by rigorously collecting samples for bac- teriological analysis from the walls of recent excavations. Sulfate reduc- ers have also been recovered from oil-well brines, which are presumably from ancient marine sediments, from depths exceeding a thousand feet by BASTIN (1926), GAHL and ANDERSON (1928), BASTIN and GREER (1930), GINTER (1930), GINSBURG-K ARAGITSCHEVA (1933), and others. Of course, it is questionable whether the sulfate reducers are indigenous to the oil- well brines or were adventitiously introduced in the process of drilling the wells. In their investigations to determine at what depths in the earth’s crust bacteria can live, LIESKE and HOFMANN (1929) found bacteria in coal at a depth of ro89 meters. Lipman (1931) reported the presence of living Chapter VI — 93 — Bottom Deposits bacteria in Pennsylvania anthracite at a depth of 1800 feet, but FARRELL and TURNER (1932) have questioned the significance of bacteria found in anthracite coal. ISSATCHENKO (1940) avers that bacteria, which he found in oil to a depth of 2000 meters, are indigenous species from near the lower limits of the biosphere. If his contention is substantiated, then we might expect temperature, organic matter, and water to be the chief factors which limit the depth to which living microorganisms may occur in sedi- ments. While there is no reason to believe that living bacteria are widely distributed in ancient sediments at great depths, accumulating field and laboratory data indicate that bacteria are alive and active in certain deeply buried deposits containing organic matter. The occurrence of viable bacteria at great depths in sediments leads one to wonder whether they have been reproducing throughout the thou- sands of years that they have been buried or if they are passively resting cells in a state of suspended animation. ZOBELL and ANDERSON (19360) have pointed out that conditions in the bottom deposits of the sea, namely low temperature and absence of free oxygen, have proven most conducive to great longevity of bacteria in a dormant state. Anaerobic bacteria may be physiologically active in such an environment, but some of the strict aerobes which have been found at a depth of several feet either have been carried there by burrowing organisms or other agencies or else they have been buried in a dormant state for many centuries. Since we have no reasons for believing that strict aerobes have been carried deep into the bottom deposits and since the aerobes in question are mostly sporogenous Bacillus species, these bacteria may have survived in a dor- mant state for many centuries. Of course, there is a possibility that the so-called “strict aerobes” are capable of reproducing in a highly reducing environment by physiological mechanisms still unknown to microbiolo- gists. While a few strict aerobes are found in bottom deposits at appreciable depths, their abundance decreases sharply from the surface downward (ZoBELL, 1938a). Curves depicting the vertical distribution of aerobes are reminiscent of logarithmic curves for the order of death of bacteria when the survivors are plotted against time. HEnrict and McCoy (1938) found that profile series of bacteria from lake bottom deposits give typical logarithmic death curves, suggesting that aerobes are slowly dying off. However, that many of the bacteria (probably anaerobes) in bottom deposits are physiologically active to great depths is indicated by the changes which they cause in the chemical composition and physico- chemical properties of the bottom deposits, as is elaborated in Chapter VII. Factors influencing abundance of bacteria in mud :— Neither distance from the mainland nor depth of the overlying water seems to have any direct influence upon the bacterial population of bottom deposits. This conclusion is based upon a study of several hundred mud samples, the bacterial content of which has been reported by various workers. In the Gulf of Naples, RussELL (1892) observed a decreasing abundance of bac- teria in mud samples with distance from shore, but in light of recent infor- mation this horizontal distribution appears to be more closely related to the organic content of the mud than to its distance from land or to the depth of the overlying water. This also applies to REUSzER’s (1933) ob- servation that the bacterial numbers on the continental shelf in the vicin- ity of Woods Hole decrease regularly with distance from land. ZoBeil — 94 — Marine Microbiology There is no evidence that either numbers or kinds of bacteria in bottom deposits are influenced by seasonal cycles except near shore or in very shallow water where bottom conditions are influenced by turbulence, storms, run-off, insolation, etc. In deep water, where environmental con- ditions are monotonously constant, one might expect no detectable sea- sonal fluctuations in the microbial population, but in shallow water, which is subject to cyclic changes caused by meteorological conditions and sea- sonal cycles in organic productivity, one might anticipate and does find seasonal changes in the bottom flora. This is especially true of lakes in the temperate zone which undergo a seasonal overturn. Even here, though, seasonal changes in the bottom flora are not as pronounced as are changes in the microbial population of the overlying water. Relatively small numbers of bacteria are found in coarse sand which occurs along the coast and in shallow water. However, the bacterial pop- ulation is more closely related to the character of the sediments than to their distance from land, because, as a rule, sand contains fewer bacteria than sediments consisting chiefly of smaller particles regardless of their topographical location. Table XXVIII shows the average number of bacteria found in different types of bottom deposits. The particle size and nitrogen content of the sediments is adapted from data by TRAsK (1932). The water content was determined by drying the samples for 24 hours at 105° C. TABLE XXVIII.— Average nitrogen, water, and bacteria content of sediment samples from the Channel Island region (from ZOBELL, 1938a):— MEDIAN DIAMETER BACTERIA PER Devon eae Scan wear Wesin NITROGEN CONTENT| WATER CONTENT pee pee IN PER CENT IN PER CENT (wer Basis) Sand 50 to 1,000 0.09 | 33 22,000 Silt 5 to 50 0.19 56 78,000 Clay 1to5 0.37 82 390,000 Colloid NH, 10,000 1,000,000 100,000 Urea fermentation — Urea — NH, 100 1,000 Proteolysis — Gelatin liquefaction 100,000 10,000,000 1,000,000 Proteolysis — Peptone — H2S 10,000 1,000,000 100,000 Denitrification — NO; —> Ne 100 10,000 10,000 Nitrate reduction — NO; — NO, 100,000 10,000,000 10,000 Nitrogen fixation ° ° ° Nitrification — NH, — NO, fo) ° fo) Sulfate reduction — SO, —> H2S 1,000 1,000 10,000 Dextrose fermentation 10,000 100,000 1,000 Xylose fermentation 10,000 -+- 10,000 Starch hydrolysis 10,000 100,000 10,000 Cellulose decomposition 1,000 + 1,000 Fat hydrolysis (lipoclastic) 1,000 a + Chitin digestion 100 -E ok Although their presence was not demonstrated in the analyses sum- marized in Table XXIX, nitrogen-fixing and nitrifying bacteria have been reported in marine bottom deposits (Cooper, 19370). Their significance is discussed in Chapter XI. Carrey and WAKSMAN (1934) reported the presence of nitrifying bacteria in mud samples collected from a water depth of 4,742 meters in the Atlantic Ocean just north of Bermuda. Microorganisms which decompose lignin and hemicellulose were Chapter VI — 99 — Bottom Deposits demonstrated in marine mud by BENSON and PARTANSKY (1934). Zo- BELL and STADLER (1940a) found bacteria in lake deposits which slowly oxidize various kinds of natural and purified lignin. According to ZOBELL and UPHAM (1944), agar-digesting colonies ap- pear on most plates of nutrient agar inoculated with marine mud, although they are relatively more abundant in sea water than in bottom deposits. BAVENDAMM (1932) was intrigued by the abundance of agar digesters found in marine mud. He also observed various kinds of cellulose fer- menters, urea bacteria, nitrogen fixers, sulfur bacteria, and sulfate reduc- ers in the mud. Species of Nocardia, Actinomyces, Pseudomonas, Micromonospora, and Mycobacterium which oxidize various kinds of petroleum hydrocarbons have been demonstrated in marine bottom deposits by ZOBELL et al. (1943). ELAzARI-VOLCANI (1943) found microorganisms in bottom sedi- ments of the Dead Sea which utilized kerosene and petroleum. Several species of Micromonospora which attacked paraffin wax, paraffin oil, and aromatic hydrocarbons as well as chitin, cellulose, and lignin were isolated from lake mud by ERIKSON (1941). Besides finding significant numbers of most of the physiological types of bacteria listed in Table XXIX, Wiritams and McCoy (1935) found both aerobic and anaerobic nitrogen fixers, thiosulfate oxidizers, and sulfur oxidizers in the bottom deposits of Wisconsin lakes. BAVENDAMM (1932) and BENECKE (1933) mention several kinds of sul- fur bacteria which have been found in marine mud. From marine mud near Kiel, BRENNER (1916) isolated Micrococcus selenicus, an anaerobe which allegedly reduces selenate and selenite. BuTKEVICH (1928) found certain iron-oxidizing bacteria in ferromanganese deposits on the sea bot- tom. THIEL (1925) noted the presence of bacteria and molds in marine muds and peat bogs which precipitate manganese. CARPENTER (1939) studied the relative abundance of urea fermenters, denitrifiers, cellulose decomposers, and pectin fermenters in lake mud. Proteolytic, pectin-fermenting, aerobic and anaerobic cellulose-decom- posing, denitrifying, anaerobic nitrogen-fixing, and fat-splitting bacteria were found in lake mud by OMELIANSKY (1917). ALLGEIER ef al. (1932) demonstrated in lake deposits the presence of bacteria which liberated methane, hydrogen, nitrogen, and carbon dioxide from the anaerobic fer- mentation of organic matter. From the mud of Red Lake (Rotsee) in Switzerland, DUGGELI (1936) isolated 72 species of bacteria including some which produce hydrogen and methane. Proteolytic organisms pre- dominated in the 530 samples of mud examined by DUGGELI. In reviewing the literature on the microbiology of muds, ISsSATCHENKO (1938) gives 171 references, most of which deal with the physiological types, distribution, and activity of microorganisms in Russian limans and lake deposits. Besides numerous representatives of the orders Eubacteriales, Ac- tinomycetales, Chlamydobacteriales, Myxobacteriales and Spirochaetales, which are generally regarded as bacteria, other microorganisms found in bottom deposits include yeasts, molds, and algae. From the Dovey salt marshes which are soaked with tidal sea water, ELtrorr (1930) isolated 48 fungi, most of them being common soil forms. Algae and diatoms are restricted to mud and water depths penetrated by sunlight. Protozoans and minute metazoan organisms are widely distributed in marine bottoms. Chapter VII ACTIVITIES OF (MICROORGANISMS vIn BOTTOM DEPOSITS Bacteria are important chemical and geological agents in bottom de- posits, not so much because they are indispensable to any one process, but rather because they help to accelerate many processes. The vast num- bers, physiological versatility, and almost universal distribution of bac- teria in recent marine sediments help to account for the long-range effectiveness of their work. In general, the geological activities of bacteria are neither specific nor confined to a limited number of species. The metabolic products of practically all bacteria have some effect upon their geological environ- ment. Evidence of their early creation (bacteria probably being one of the first forms of life on the earth) indicates that they have had an enormous span of time in which to exert their profound influences. Controlled lab- oratory experiments with samples of bottom deposits under environ- mental conditions simulating those on the sea floor demonstrate that bac- teria could be functional im situ, or in the environment in which they occur. This, together with the correlation of the numbers and kinds of microorganisms with chemical, physico-chemical, and geological condi- tions found in sediments, clearly shows that microorganisms are instru- mental in the modification and diagenesis of the sediments. Calcium carbonate precipitation:— According to estimates by VAUGHAN (1924), marine sediments contain an average of 20 per cent of calcium carbonate in the form of limestone, while the entire earth’s crust contains only about 6 per cent. Almost pure CaCO; is being deposited in certain parts of the sea, and in most bottom deposits precipitated CaCO; serves aS an important conglomerating or cementing material. While large quantities of CaCO; are deposited in the skeletal remains of animals and by calcareous algae (GLOCK, 1923), the activities of heterotrophic bacteria contribute to the process in various ways. Murray and [IRVINE (1889) attributed the precipitation of CaCO; to the reaction of ammonium carbonate with calcium sulfate: (NH,4)2CO3 + CaSO, = CaCO; + (NH4)2SO4 Ammonium carbonate originates from ammonia and CO, resulting from the microbiological disintegration of nitrogenous organic substances. In an obscure Russian publication which escaped attention until his observations were published in German (NApson, 1928), NADSON (1903) showed that the bacterial production of ammonia from proteinaceous materials promotes the precipitation of CaCO; in Lake Weissowo. Pro- teus vulgaris, Bacillus mycoides, Actinomyces albus, Bacterium albo-luteum, and Bacillus salinus were the principal organisms involved. Ammonia production tends to increase the pH of water and thus promotes the pre- cipitation of CaCO; (MoBErRG et al., 1934). Besides liberating ammonia from proteinaceous materials, certain organisms observed by NADSON re- Chapter VII — 101 — Activities in Deposits duced nitrate to free nitrogen or ammonia, thereby creating conditions sufficiently alkaline for the precipitation of both CaCO; and MgCO; with the formation of limestone and dolomite. Oolithic formations were also observed. NADSON (1928) believed that the bacterial reduction of sulfate also contributes to the precipitation of carbonates: CaSO, + CHsCOOH = CaCO; + H:S + H:0 + CO, Whether CaCO; will be precipitated depends primarily upon the pH. The destruction of the strongly acidic sulfate radicle tends to make conditions more alkaline, but the effect of sulfate-reducing bacteria on the hydrogen- ion concentration depends upon the nature of the organic matter used as a source of energy, as well as upon the concentration and form of calcium sulfate. RITTENBERG (1941) and others have found that under certain conditions the media in which sulfate reducers are growing become pro- gressively more acidic because the organic matter which is being utilized as a source of energy is oxidized to organic acids in sufficient concentra- tions to offset the effect of the sulfate which is reduced. DREW (r1o11, 1913) attributed the precipitation of CaCO; in the sea largely to the action of denitrifying bacteria according to the following reaction in which hydrogen and carbon are derived from the anaerobic oxidation of organic matter: Ca(NOs)2 + 3 He a C— CaCOx3 + 3 HO + No His conclusion was based primarily upon experiments in artificial media and the observation of large numbers of denitrifying bacteria in tropical seas where there was evidence of limestone formation. Believing that the process was fairly specific, DREW (1912) named the organism involved Bacterium calcis. This organism was more thoroughly described by KEL- LERMAN and SMITH (1914) who classified it as Pseudomonas calcis. It is closely related or identical to marine denitrifiers described by GRAN (1901), BAUR (1902), FEITEL (1903), and PARLANDT (1911). KELLERMAN and SMITH (1914) confirmed DREw’s observations on the precipitation of CaCO; in nitrate media by Pseudomonas calcis. They found further that ammonia production either from protein or nitrate decomposition resulted in CaCO; precipitation: Ca(HCOs)>2 + 2 NH,OH = CaCO3 -+- 2 H,.O + (NH4)2COs Other microorganisms may be instrumental in the process by oxidizing the calcium salts of organic acids, leaving calcium and CO, which, in the presence of water, unite to form CaCO;. These observations were con- firmed and extended by KELLERMAN (1915@). KELLERMAN and SMITH (1916) found halophilic lime-precipitating bacteria in Great Salt Lake which resembled those isolated from the sea. BERKELEY (1919) failed to find any evidence of denitrification (liber- ation of free nitrogen from nitrate) by bacteria in sea water collected off Vancouver Island. In artificial media, however, the bacterial reduction of nitrate resulted in the precipitation of CaCO;. BERKELEY attributed the precipitation of CaCO; in sea water primarily to the bacterial oxidation of organic calcium salts: Ca(COOCHs)2 + 4 O2 = CaCO; + 3 CO. + 3 H.O Lipman (1924) has questioned the ability of Pseudomonas calcis or other denitrifying bacteria to precipitate CaCO; from pure sea water ZoBell 102 — Marine Microbiology which has not been enriched with either calcium salts, organic matter, or nitrate. He regarded ammonia production from organic matter to be the most important factor. The utilization of CO, by plants, thereby increas- ing the pH of sea water, was also recognized as an important factor. Ina later publication, LrpMAN (1929) averred that under ordinary conditions bacteria do not possess the ability to precipitate CaCO; from sea water, a conclusion which was predicated primarily upon the paucity of bacteria in the open sea. This conclusion has been rendered untenable by the large numbers of bacteria found in marine bottom depos:ts, particularly cal- careous deposits (BAVENDAMM, 1932). Being guided by the work of Drew, who regarded CaCO; precipitation as being due to specific ‘‘calcium bacteria,’ Motiscu (1925) reported the discovery of aquatic bacteria which precipitate CaCO; by the formation of ammonia. He isolated Pseudomonas calcipraecipitans from sea water and Pseudomonas calciphilia from fresh water. Motiscu also described an Actinomyces sp. and a pink yeast, Saccharomyces olexudans, both of which allegedly precipitate CaCOs. SMITH (1926) grouped bacteria from “chalky mud” from the Bahama Banks into six categories according to their physiological activities and morphology. Only two groups, denitrifiers of the Pseudomonas calcis type and strong ammonifiers, formed calcite from calcium sulfate in sea water. Sterilized bottom mud was employed as a source of nutrients. The addi- tion of peptone and nitrate increased calcite formation. The production of calcite aggregations was enhanced by the presence of 0.2 per cent agar. From a review of the rather extensive literature on the subject and his own investigations, BAVENDAMM (1931, 1932) concluded that calcium pre- cipitation in tropical seas is primarily a microbiological process but that there are no specific “calcium bacteria.” He correctly emphasized that microbiological precipitation can be ascribed only to certain marine sec- tions. In other regions of the ocean, microorganisms may be dissolving calcareous deposits. Soil bacteria studied by Murray and LovE (1929) produced enough acids, including carbonic acid, from organic matter to make them much more effective than atmospheric carbon dioxide in dis- solving limestone. Whether the combined activities of microorganisms will tend to pre- cipitate or dissolve CaCO; depends upon the sum total of their effects on chemical and physico-chemical conditions, their effect on the pH of the environment being one of the most important considerations. While auto- trophic organisms are instrumental in the process, microbiological CaCO; precipitation is confined largely to sections where there is an abundance of organic matter. This condition is not fulfilled over most of the sea bot- tom, but it is certainly met in shallow, swampy areas where there is much CaCO; deposition. Although their preliminary experiments failed to prove the connection of the activity of microorganisms and the precipitation of CaCO; within the deposits of Lake Mendota, WiLLtaMs and McCoy (1934) isolated from the lake deposits many aerobic organisms which were capable of precipitat- ing CaCO; under artificial conditions. They also examined several com- monly known bacterial species which were endowed with this ability. Some of the bacteria precipitated CaCO; from solutions carrying the same amount of calcium as does lake water. Believing that nitrate reduction, the oxidation of organic calcium salts, or ammonia production from nitro- genous material accounted for the microbiological precipitation of CaCQOs, Chapter VII — 103 — Activities in Deposits Wiriiams and McCoy employed media which contained either peptone, calcium lactate, or potassium nitrate besides various other ingredients. KALANTARIAN and PETROSSIAN (1932) isolated Bacterium sewanense from Lake Sewan, Transcaucasia, Russia. This organism was believed to induce the precipitation of CaCO; by reducing nitrate and producing ammonia. From his extensive review of the literature, BArER (19372) lists the fol- lowing ways in which microorganisms may influence CaCO; equilibrium: (a) by their effect on the pH, (b) by producing or consuming COzs, (c) by oxidizing organic calcium salts and (d) by assimilating calcium. The di- rection and extent of the reactions depends upon the types of microorgan- isms present, the chemical composition of their surroundings, and other environmental conditions. ‘Deposition of iron and manganese:— There are several ways in which microorganisms are instrumental in the deposition and transforma- tion of iron and manganese. Autotrophic species such as Leptothrix ochracea and Gallionella ferruginea, which allegedly obtain their energy requirements by oxidizing ferrous iron, are generally associated with iron- bearing waters and bog-iron deposits. Similarly, species of Siderocapsa, Crenothrix, Cladothrix, and Clonothrix are often found in iron-bearing sur- face waters as revealed by the literature reviewed by HARDER (1919), ELLIS (1919), CHOLODNY (1926), DorFF (1935), and BAIER (19376). The taxonomic position and metabolism of many of the so-called ‘“‘iron- bacteria” are highly problematical but such organisms do promote the deposition of iron either directly or indirectly. HARDER (1919) reports that, ‘‘iron-depositing thread-bacteria have never been found in sea water and probably do not occur there on account of the high percentage of certain inorganic salts.”’ However, CHOLODNY (1926) has shown that iron bacteria can develop in chalybeate waters, the salt content of which is greater than that of sea water. THIEL (1925) re- ports finding in marine mud numerous heterotrophic bacteria and molds which precipitate iron and manganese, but he names no species. BUTKEVICH (1928) isolated Gallionella turtuosa from the Petschora Sea and Gallionella reticulosa from the White Sea. The organisms were found in ferromanganese concretions in bottom deposits. Leptothrix longissima was isolated from the sea by Moriscu (1910). In the black mud of the Sea of Azov and the Black Sea, ISSATCHENKO (1912) found Spirillum levocolelaenum, an organism which precipitates iron sulfide in the bacterial cell. ISSATCHENKO (1929) believed that bac- teria are the chief agents in the formation of pyrite. While ferrous sulfide is formed intracellularly by some bacteria and deposited as pyrite follow- ing their death, any bacteria which produce H2S may contribute to the deposition of iron as pyrite. Besides the autotrophic bacteria which oxidize iron or manganese to the corresponding hydroxides and the heterotrophs which precipitate the metals as sulfides by producing H,S, there are other ways in which micro- biological activities contribute to the deposition or transformation of iron. STARKEY and HALVORSON (1927) believe that heterotrophic organisms play a more important role in the deposition of iron than do autotrophs. This the heterotrophs do by decomposing organic compounds of iron, by changing the pH of the water or mud, and by altering the oxygen tension or Ex. Both the E, and pH of a system influence the state of iron. By ZoBell — 104 — Marine Microbiology producing oxygen, algae and diatoms may cause the precipitation of ferric iron. STARKEY and HALVORSON question the widespread occurrence and activity of autotrophic iron bacteria. THIEL (1928) is also of the opinion that the importance of true auto- trophic iron bacteria as geological agents has been overemphasized, while the effects of heterotrophs on the deposition and transformation of iron have been neglected. He reports that manganese as well as iron com- pounds are precipitated by heterotrophic bacteria and by certain fungi found in peat. HarDMAN and HEnrIcrI (1939) believe that Siderocapsa treubii, which has been regarded as an autotroph, probably is a heterotrophic organism, utilizing the organic radicle of organic iron compounds and merely depos- iting the iron as a waste product upon the cell capsules. This organism, together with the closely related Szderocapsa major, was found abun- dantly in alkaline, hard-water lakes of the drainage type and in certain rapidly flowing streams. Species of Siderocapsa were not found in neutral or acidic soft-water lakes of the seepage type. Leptothrix crassa and related iron-depositing bacteria were found in certain lakes having a high content of iron. Microbiological deposition of manganese is believed to occur in about the same way as iron deposition (DorFr, 1935; BAIER, 1937); ZAPFFE, 1931). In many environments iron and manganese are deposited together, and most of the bacteria which precipitate iron likewise precipitate man- ganese. According to JACKSON (1902), Crenothrix polyspora, Cr. man- ganifera, and Cr. ochracea are able to precipitate either iron, manganese, or aluminum from water. The precipitation of copper (as sulfide) in mud by bacterial activity has been reported by LOVERING (1927). Copper precipitation is due not to specific bacteria but rather to the action of reducing substances produced during bacterial metabolism. LOvERING believed that the precipitation of vanadium as vanadite and cornotite may be accounted for upon a basis of bacterial activity. In discussing the role of bacteria in halloysite for- mation, BUCHER (1921) stated that sulfate-reducing bacteria promote the precipitation of aluminum silicate. Effect on hydrogen-ion concentration :— As has been stated before, microorganisms are probably the principal dynamic agencies which alter the hydrogen-ion concentration, oxidation-reduction potential, gas ten- sion, and other physico-chemical conditions in bottom deposits. De- pending primarily upon the numbers and kinds of microorganisms present, the chemical composition of the substrate, and various environmental conditions, they may tend to increase or to decrease the hydrogen-ion concentration or pH. The following microbiologically activated processes tend to increase the hydrogen-ion concentration or to decrease the pH: (1) Production of CO, by respiring cells, (2) production of organic acids such as lactic, butyric, acetic, and formic acids, for example, from the decomposition of carbohydrates, lipids, proteins, etc., (3) oxidation of H2S or sulfur.to sulfuric acid or other acid sulfates, (4) reduction of sulfur to H2S, (5) formation of nitrite or nitrate, (6) assimilation of ammonium as a source of nitrogen or its oxidation as a source of energy, and (7) the liberation of phosphate from organic compounds. The extent to which these reactions may occur in the marine environment is discussed else- where in this volume. 2 Chapter VII — 105 — Activities in Deposits The hydrogen-ion concentration may be decreased or the pH increased by the following microbiological reactions: (1) Utilization of CO» by either chemosynthetic or photosynthetic autotrophs, chiefly diatoms and algae, (2) oxidation or decarboxylation of the salts of organic acids such as formate, acetate, propionate, lactate, etc., (3) reduction of sulfate to sul- fur or H.S, (4) reduction of nitrate or nitrite, and (5) the formation of ammonia from nitrogenous compounds such as amino acids, proteins, urea, purine bases, etc. Microorganisms capable of activating the aforementioned reactions have been demonstrated in sedimentary materials. The hydrogen-ion concentration of bottom deposits at depths to which CO, does not diffuse from the atmosphere is probably largely a function of the interrelated factors which influence the abundance, kinds, and activity of bacteria. An appraisal of these factors will require much additional quantitative in- formation. According to Mryapt (1934), bacterial activity has a pro- nounced effect upon the hydrogen-ion concentration of lake mud. KUSNETZOW (109352) reports that bacterial activity may affect the hydro- gen-ion concentration as well as the oxidation-reduction potential of an entire water basin. The influence of bacterial activities on the pH of water is discussed by ENEVOLDSEN (1927). Besides influencing the solubility and reactivity of calclum, magne- sium, iron, manganese, and other sedimentary constituents, the hydrogen- ion concentration also influences the oxidation-reduction potential of the sediments. Its effects on base exchange and on the properties of clay are discussed in the symposium volume edited by TRASK (1939). ZOBELL (19426) records that the hydrogen-ion concentration of the topmost layers of bottom deposits is usually nearly the same as that of immediately overlying water. The hydrogen-ion concentration of sea water ranges from pH 7.6 to 8.3, depending primarily upon the depth of the overlying water and its origin. In some cores from the Gulf of Cali- fornia and the Channel Island region off the coast of southern California it increases to as much as fH 8.9, and in other cores it decreases with depth to as low as pH 7.2. In fresh-water lakes the range is much greater, varying considerably from lake to lake (ALLGETER ef al., 1941). Effect on oxidation-reduction potential:— The oxidation-reduction (O/R or redox) potential of a system may be defined as its relative oxidiz- ing or reducing power or its electron-escaping tendency. The higher the concentration of free electrons in a solution the greater is its reducing power (or the lower its E;,), and the lower the concentration of free elec- trons the greater its oxidizing power (or the higher its E,). The reducing power, oxidizing power, or redox potential of a solution containing a re- versible oxidation-reduction system can be expressed conveniently in terms of Ey, measured in volts as E.M.F. compared with the E.M.F. of a normal solution of hydrogen ions (HEwIrT, 1936). BUCHANAN and FUL- MER (1928) point out that, in general, any system having an E, value less than another system will tend to reduce it, and the system having the higher value will tend to oxidize the lower. Most bacteria tend to create reducing conditions or to lower the Ey of culture media from an initial Ey, of + 0.2 or + 0.3 volt to Ey, —o0.1 or — 0.2 volt at pH 7.0 (HEwITT, 1936). Anaerobes generally require a lower Ey for the initiation of growth, and they create more reducing conditions or a lower E;, than do aerobes (REED and Orr, 1943). Not only do bac- ZoBell — 106 — Marine Microbiology teria influence the E;, of the medium in which they are growing; the Ey of the medium has a pronounced effect on the multiplication and metabolism of bacteria (see extensive reviews of literature by ELEmA, 1932; HEwirrT, 1936; and KANEL, 1941). For example, sulfate reduction, denitrification (ELEMA et al., 1934; KOROCHKINA, 1936), and the reductive deaminization of amino acids are enhanced by a relatively low Ex, whereas the oxida- tion of carbohydrates to CO, and water is favored by positive Ey values. Whether acids, alcohols, or other products result from the fermentation of carbohydrates has been shown by KuSNETzow (1931) to be partly a function of the E; of the medium. The redox potential of sea water is primarily a function of its oxygen content. COoopER (1937a) found the redox potential of normal surface sea water to be near Ey, + cay volt, increasing as the pH decreases. This value compares favorably with E;, + 0.46 to 0.50 volt found by HutcHIn- SON et al. (1939) in oxygenatéd lake water. Somewhat lower values were found in shallow inland seas by KusNetzow (19352). The redox potential of lake muds examined by KUSNETzOW (19350) ranged from E;, + 0.145 to — 0.142 volt. In Wisconsin lakes ALLGEIER et al. (1941) found Ep values ranging from + 0.512 volt in upper oxygenated water to — 0.140 volt in bottom deposits. Finding marked differences in the redox potentials on different dates led them to conclude that the poten- tials are controlled by dynamic factors that are in a state of flux, and not by static agents. Bacteria or their metabolic products are probably the responsible agents in the hypolimnion and in bottom deposits. In brown bottom deposits from the Barents and Kara Sea which con- tained appreciable quantities of iron and manganese, BRUJEVICZ (1937, 1938) found the E, to range from + 0.105 to 0.250 volt. In black mud rich in H2S, one might expect the conditions to be reducing. Several hundred potentiometric measurements made on profile sam- ples of bottom deposits from the Pacific Ocean show that, as a rule, the redox potential decreases with depth. Except under water containing HS, the redox potential of a sediments at the mud-water interface ranges from E, 0 or slightly positive values to Ey, — 0.2 volt. The redox potential decreases more or less regularly with depth to a depth of 25 to 50 cm. where the Ey is — 0.2 to — 0.4 volt. At greater depths the re- sults have been somewhat erratic, fluctuating from Ey, — 0.2 to — 0.5 volt at pH 7.0. One sample gave a reading of E, — 0.58 volt, this being the most reducing sediment examined. Bacterial cultures observed in the laboratory are able to create such reducing conditions, and unquestion- ably are at least partly responsible for the reducing conditions found in bottom deposits (ZOBELL, 1939a). The erratic results obtained at core depths exceeding 25 to 50 cm. are attributed partly to technical difficulties involved in making the measure- ments on freshly collected samples and partly to the decrease in the content of reversibly oxidizable or reducing constituents. The poising capacity, as indicated by the quantity of reducing substances in the sediments, usually decreases with core depth. This is shown by the data in Table XXX which gives the oxygen-absorbing capacity (spontaneous or abiogenic) of samples from different core depths. The redox potential has a marked influence upon the direction, velocity, and magnitude of chemical reactions which involve the exchange of elec- trons, or the oxidation or reduction of reactants. Consequently, the redox potential probably influences the diagenesis of bottom deposits in many Chapter VII — 107 — Activities in Deposits TABLE XXX.— Numbers of bacteria which developed aerobically and anaerobically in marine mud strata from different depths. The redox capacity in terms of spontaneous or abiogenic oxygen absorption and the redox potential (intensity) in terms of E corrected to pH 7.0 are also given (from ZOBELL and ANDERSON, 1936b):— CorE DEPTH | ANAEROBES AEROBES McmM. OXYGEN E}, IN pH or IN CM. PER GRAM PER GRAM | ABSORBED/GRAM VOLTS MUD 0 to 3 1,160,000 74,000,000 2.8 — 0.12 8.2 4 to 6 14,000 314,000 TE — 0.29 7.9 14 to 16 8,900 56,000 0.6 — 0.37 7.8 24 to 26 3,100 10,400 0.7 — 0.32 8.0 44 to 46 5,700 28,100 0.3 — 0.37 7.9 _ 66 to 68 2,300 4,200 0.4 — 0.34 7.8 ways. PEARSALL and MorTIMER (1939) find that the states of iron, sul- fur, and certain nitrogen compounds are functions of the redox potential of mud. Reducing conditions created by bacterial processes are believed by LovERING (1927) to promote the precipitation of copper, vanadium, and other minerals. Factors influencing oxygen consumption:— Most bacteria consume oxygen. The marine bacteria studied by JOHNSON (1936) consumed from 2.8 to 185 X 107! mgm. of oxygen per cell per hour as “‘resting cells” sus- pended in sea water at 25° C. About one-fifth this quantity of oxygen was consumed at 5° C., the Qu being 2.3 between 5° and 15° C. and 2.18 between 15° and 25° C. The addition of 0.04 per cent alginate increased the oxygen uptake by from 7.9 to 110 per cent, while the addition of 0.04 per cent glucose effected an increase of from 10 to 378 per cent. By determining the amount of oxygen consumed by bacteria in sea water and the number of bacteria present at any given time, ZOBELL (19406) estimated that marine bacteria multiplying in sea water consume an average of 20.9 X 10! mgm. of oxygen per cell per hour at 22° C. The rate of oxygen consumption was increased nearly four times when the sea water was enriched with 0.05 per cent of asparagine or glucose. The rate of oxygen consumption was found to be independent of the oxygen tension within the examined range of between 0.43 and 17.84 mgm. of oxygen per liter. The oxygen consumption of microorganisms in Lake Glubokoje, Rus- sia, studied by Liacina and KusNnEtzow (1937) ranged from 12.5 to 30.3 X 107! mgm. per cell per hour for bacilli to 128 to 184 X 1o- mgm. per cell per hour for torulae at 15° to 25° C. Their calculations clearly show that the respiration of bacteria could account for the depletion of oxygen from the bottoms of the lakes studied. Multiplying bacteria from Lake Mendota, Wisconsin, were found by ZOBELL and STADLER (1940b) to consume from 51 to 93 X 10 mgm. of oxygen per cell per hour in freshly collected lake water at 25° C. The rate decreased as oxidizable organic matter became a limiting factor, but the rate of oxygen consumption was independent of the oxygen tension within the examined ranges of 0.30 to 36 mgm. per liter. The rate of respiration is a function of temperature, the Qi being 2.1 between 8° and 25° C. (ZOBELL, 1940). The effect of the quality and quantity of or- ganic matter upon the respiration of marine bacteria has been studied by ZOBELL and Grant (1943) who found that concentrations of organic matter as low as 0.10 mgm. per liter are quantitatively utilized. ZoBell — 108 — Marine Microbiology The foregoing data on the rate of oxygen consumption by aquatic bacteria should suffice to indicate that the abundant bacterial flora of bottom deposits may play an important role in depleting dissolved oxygen in lake bottoms. Not only is all of the oxygen consumed in most bottom deposits faster than it can be replaced by diffusion from the atmosphere or photosynthetic processes, but there is generally an oxygen deficit in bot- tom deposits as shown by the spontaneous oxygen-absorbing capacity (see Table XXX). ZoBELL and FELTHAM (1942) estimated that there are enough bacteria in the topmost 1.0 cm. of each square meter of Mission Bay mud to consume from 8.4 to 25.2 mgm. of oxygen per day at 20° C. Bacteria in various lake deposits examined by Miyap1 (1934) con- sumed from 0.42 to 1.26 mgm. of oxygen per gram of mud in 60 hours at 15° C. In general, muds having the highest organic-carbon content and those under water containing the least dissolved oxygen exhibited the largest biochemical oxygen demand. WaxksMaNn and Horcukiss (1938) likewise found the oxygen consuming capacity of bottom deposits to be a function of the organic content. The biochemical oxygen demand of bot- tom deposits from the Woods Hole region was found to be from 0.08 to 2.83 mgm. of oxygen per gram in 14 days at 25° C. KUSNETzZOW and KARZINKIN (1931) attributed the zones of dimin- ished oxygen tension in Lake Glubokoje to bacterial activity, after ob- serving that the disappearance of oxygen corresponded with large bac- terial populations. This conclusion has been substantiated by more recent observations of KUSNETzOW (19350) who found that heterotrophic bacteria are the chief agents responsible for the depletion of oxygen from lake bottoms. Methane- and hydrogen-oxidizing bacteria were regarded by KUSNETZzOW as also being very important. Gases in bottom deposits :— Because of the relatively large bacterial populations and high content of organic matter in bottom deposits, it is primarily in or immediately above bottom deposits that the most oxygen is consumed. Likewise it is primarily in or near bottom deposits that other gases are produced or transformed by bacteria. Under anaerobic conditions appreciable quantities of methane and hydrogen may be produced from the decomposition of organic matter. According to WAKSMAN (19414), the anaerobic oxidation of organic mat- ter by aquatic bacteria results in the formation of methane, hydrogen, HS, and organic acids besides COs, phosphate, and ammonia, whereas under aerobic conditions organic matter is decomposed with the formation of CO:, phosphate, ammonia, and sulfate. The gaseous products resulting from the anaerobic decomposition of lake muds observed by ALLGEIER ef al. (1932) consisted of from 65 to 85 per cent methane, 3 to 30 per cent COs, 1 to 3 per cent He and from 3 to 22 per cent No. The catabolic H2S had probably combined with mineral constituents to give metallic sulfides. DUGGELI (1936) reported that the gas produced in deep muds from Red Lake in Switzerland consisted largely of methane and Ne with lesser quantities of H2, CO2, and H2S. Very little work has been done on methane production in marine muds but there is voluminous literature on the factors which influence the bac- terial production of methane in industrial wastes, soil, and under labor- atory conditions (BARKER, 1936a, b; BUSWELL, 1936). The general oc- currence of methane in natural gas which is usually associated with an- Chapter VII —= 109 — Activities in Deposits cient marine sediments, and the almost universal formation of methane in recent marine sediments rich in organic matter, suggest the importance of this gas as an end product of decomposing organic matter. The evolution of methane and hydrogen from Lake Glubokoje has been described by KusNETzow (19356) who discusses the importance of these so-called ‘‘mud gases.”” He believes that the oxidation of methane and hydrogen by autotrophic bacteria in the overlying oxygenated water plays an important role in depleting the oxygen content of lakes. The bacterial production of methane, hydrogen, CO, and HeS has been ob- served by BUTKEVICH (1938) in mud from the Caspian Sea. The anaerobic fermentation of organic matter usually results in the formation of a certain amount of hydrogen (BUSWELL, 1936). Several species of bacteria are endowed with this ability. Due to the chemical reactivity of nascent hydrogen and the tendency of various bacteria to activate molecular hydrogen, it is difficult to estimate how much hydrogen may be produced in bottom deposits. However, the fact that free hydro- gen is often detectable is evidence that it is formed, probably in very sig- nificant quantities. SUGAWARA et al. (1937) noted the presence of hydro- gen, along with methane and nitrogen, in lake deposits of high organic content. Large quantities of hydrogen may be consumed in the production of methane in mud. SOHNGEN (1910) postulated that methane is formed by the bacterial reduction of CO, in the presence of hydrogen: 4 He, + CO. = CH4 + 2 H:0 This mechanism of methane formation has been amply confirmed (Bar- KER, 1936a), but it is still problematical whether free hydrogen is always involved as an intermediate reactant in methane formation. STEPHENSON and STICKLAND (1931) observed that certain anaerobes use molecular hydrogen to reduce sulfate to H2S in the proportions indi- cated by the following equation: H.SO, oe 4 He = HS = 4 HO This observation has been confirmed by the work of STARKEY and WIGHT (1943) and others. Hydrogen may play a role also in the hydrogenation of organic matter with the formation of petroleum hydrocarbons. Nitrogen may be liberated from nitrogenous organic matter undergo- ing anaerobic decomposition, although the mechanism of the reaction is not well understood. Ammonia ordinarily is liberated from decomposing proteins but it will appear as a gas only when present in high concentra- tions or in an aJkaline environment. Nitrogen may be liberated from nitrates or nitrites by the process of denitrification, but since neither nitrates nor nitrites occur in bottom deposits having a low Ex, they are not likely sources of nitrogen. Often appreciable quantities of H2S are formed during the decomposi- tion of proteins and from the reduction of sulfate. The H2S may be either fixed in the sediments as metallic sulfides or may escape into the overlying water. Most black muds are rich in ferrous sulfide. If enough H.S is pro- duced to escape into the water, it tends to deplete the dissolved oxygen either abiogenically or through the activities of sulfur bacteria. HS pro- duced on the bottom of the Black Sea and in other stagnant bodies of water is primarily responsible for the prevailing anaerobic conditions in the overlying water. COPENHAGEN (1934) records that H2S generated in ZoBell mT ae Marine Microbiology the bottom of Walvis Bay on the South African Coast depleted the oxygen from the overlying water, and at times has been lethal to the fauna and flora. Mears (1943) relates that in certain polluted harbors (notably Calloa, Peru) so much HS is produced that metallic surfaces and surfaces coated with lead-base paints are blackened by the formation of metallic sulfides, giving rise to the phenomenon known in nautical parlance as the “‘Calloa Painter.” From his investigations on the “‘stinking putrefaction”’ in the Little Kiel, Germany, during summer, Baler (1935) concluded, that while the production of appreciable quantities of H2S corresponded with periods during which there was much organic matter undergoing decomposition, the H:S was derived primarily from the bacterial reduction of sulfate and only to a slight extent from proteins. Most if not all of the H2S was gen- erated in the bottom mud. : Autotrophic bacteria may be instrumental in the oxidation of HS, methane, carbon monoxide, and hydrogen at the mud-water interface or in the overlying water, provided that there is an adequate supply of dis- solved oxygen. Effect on organic content of bottom deposits :— Bacteria influence the quality and quantity of organic matter, both during the time that it is settling to the bottom and after it has been deposited and buried. The organic compounds which are most susceptible to attack, such as simple proteins and carbohydrates, are utilized first, leaving the more refractory compounds such as lignins, complex proteins, chitins, lipids, polyuronides, and other complexes known collectively as humus. This occurs in marine bottom deposits (WAKSMAN, 1933; TRASK, 1939) as well as in lakes (WAKS- MAN, 1941@). Illustrative data are provided by WaxKsMAN and Horcukxiss (1937) who found the biochemical oxygen demand (B.O.D.) of mud from differ- ent depths as follows: DEPTH OF NITROGEN CONTENT B.O.D. PER GRAM LAYER OF DRY MUD OF DRY MUD o to I cm. 0.095% 0.315 ml. Tptoys cm: 0.087% 0.265 ml. 5 to 20 cm. 0.070% 0.272 ml. The B.O.D. is the amount of oxygen consumed by bacteria in oxidizing organic matter in a given period of time. Thus the B.O.D. is partly a measure of the quantity of organic matter and partly a measure of its biological oxidizability. ANDERSON (1940) determined the organic content of a large number of marine muds and also their B.O.D. In general, the largest organic con- tent, as well as the highest 15-day B.O.D. per gram of organic carbon, was found in the topmost sections of the cores, although many unexplainable irregularities are reported. As a rule, the surface layers of mud contain the most organic matter, and that which is present is more readily decom- posed by bacteria than the organic matter at greater depths in the cores. In representative experiments conducted by ANDERSON (1940), the B.O.D. of samples was 1.39 ml. during the first 5 days, 0.27 ml. during the second five days, and 0.24 ml. during the third 5 days, thereby showing a progressive decrease in the oxidizability of the organic matter. Detailed analyses of cores from the southern California region by MoseErcG ef al. (1937) showed that the highest organic content is at the Chapter VII — i111 — Activities in Deposits surface of the cores. Within core samples 50 cm. long, the organic content near the lower end of the core fell to about two-thirds of that found near the surface. The amount of organic matter in the lower portion of the core was relatively constant. The vertical distribution of organic matter corresponded with the abundance of bacteria. From his extensive studies of the organic content of marine sediments, TRASK (1939) concluded that by the time the sediments have been buried to a depth of 30 cm., the quantity of organic matter seems to have de- creased about 15 per cent, and by the time that they have been lithified, some tens of millions of years after they have been deposited, the average decrease in organic content is about 40 per cent. He points out that these figures of 15 and 4o per cent are only rough approximations because the loss of organic matter differs greatly in different sediments. TRASK (1932) believes that anaerobic bacteria are the only living things which can alter organic matter after it has been buried to considerable depths. Further details on the transformation of organic matter are given in Chapter X. The petroleum problem :— Since the majority of the known petroleum deposits occur in ancient marine sediments, one is led to wonder whether organic matter is being converted into petroleum or protopetroleum in recent sediments. Assuming that petroleum is formed from plant or ani- mal organic matter, there are many ways in which the activities of bac- teria may contribute to the process. Field observations and laboratory experiments show that bacteria tend to convert organic matter into substances which are more petroleum- like. This they do by reducing the oxygen, nitrogen, phosphorus, and sulfur content of organic matter. The average proximate analyses of samples of organic matter representing different geological ages as com- piled from various sources is as follows: SOURCE OF CARBON HYDROGEN OXYGEN NITROGEN | PHOSPHORUS ORGANIC MATTER PER CENT PER CENT PER CENT PER CENT PER CENT Marine sapropel 52 6 30 II 0.8 Recent sediments 58 7 24 9 0.6 Ancient sediments 73 9 14 3 0.3 Petroleum 85 13 0.5 0.4 o.I Microbiologically produced hydrogen may be instrumental in the hydrogenation of certain kinds of organic matter, thereby converting it into substances which are more petroleum-like. Methane is known to be produced by the bacterially-activated hydrogenation of CO: (BARKER, 1936a), and it is not impossible that higher hydrocarbons may be formed by the hydrogenation of certain kinds of organic matter such as cellulose, for example. HS, which is a good reducing agent, may likewise con- tribute to the hydrogenation or reduction of organic matter. JANKOWSKI and ZoBELL (1944) have found that certain sulfate-reduc- ing bacteria are able to produce paraffin hydrocarbons ranging from Cy to C;; from various fatty acids and carbohydrates. The common occurrence of sulfate-reducing bacteria in oil-well brines (BAstTrIn and GREER, 1930; GAHL and ANDERSON, 1928) and the low content of sulfate in oil-well brines is very suggestive of the significance of sulfate reducers in petroleum formation. ZoBell — 112 — Marine Microbiolegy Besides their ability to produce and transform petroleum hydrocar- bons (Tausson and ALIOSCHINA, 1932), sulfate-reducing bacteria may help to liberate adsorbed oil by producing CO, which decreases the viscos- ity of oil. Likewise, sulfate reducers may dissolve limestone or dolomite, thereby liberating the adsorbed oil, or the latter may be set free by microbially produced surface-active or detergent substances. Other anaerobes besides sulfate reducers may also contribute to these processes. The low Ey, or highly reducing conditions, created by bacteria in bot- tom deposits tend to favor the formation and preservation of petroleum hydrocarbons. Trask and PATNODE (1942) point out that source beds of petroleum are usually highly reducing. In so far as is known, bacteria are the principal agents responsible for these reducing conditions. While there are several ways in which bacterial activities may con- tribute to petroleum formation, under certain conditions bydrocarbon- oxidizing bacteria may prevent the accumulation of oil. The literature on this subject has been reviewed by ZoBELL et al. (1943). Further in- formation on the relation of bacteria to petroleum formation is given by ZOBELL (1943a). Sulfur deposition:— There are two main types of elementary sulfur occurring in nature. The solfataric or volcanic type is formed from HeS and SO, in volcanic gases. The gypsum type is believed to result from the reduction of calcium sulfate. The gypsum type of sulfur deposits are as- sociated with marine sediments. There is evidence that gypsum or cal- cium sulfate is being reduced to sulfur at the expense of the buried organic matter which serves as a source of energy for the reaction. Although con- clusive proof is still lacking, it is generally believed by geologists and microbiologists that anaerobic bacteria are responsible for sulfur deposits of the gypsum type. Some of these sulfur deposits in Louisiana and Texas are a hundred or more feet thick. Unique sulfate-reducing bacteria, which appear to be indigenous species, have been isolated from sulfur-limestone- anhydrite formations from a depth of 1550 feet. Hunt (1915) attributed the origin of the sulfur deposits of Sicily to the bacterial reduction of sulfates in ancient shallow marine seas resembling present conditions in the Black Sea. Sulfur is also deposited either intracellularly or extracellularly by vari- ous autotrophic bacteria. The activities of autotrophic sulfur bacteria as well as those of heterotrophic sulfate-reducing bacteria are discussed in Chapter XII. As geological agents which deposit sulfur, the sulfate re- ducers are probably by far the more important of the two categories of microorganisms. Particle-binding action of microorganisms:— Besides their effects on the chemical and physico-chemical conditions of bottom deposits, there are several ways in which microorganisms may promote mechanically the diagenesis, lithification, or consolidation of particles of sand, silt, clay, and colloids which constitute marine sediments. The calcareous materials which are precipitated either directly or in- directly by bacteria in recent sediments tend to cement together solid particles such as clay, silt, or sand. CaCO; is one of the commonest cementing materials in sedimentary rocks. MgCO; is also important. Certain bacteria produce mucilaginous exudates or capsular materials which adhere to particles of sediments or which cause the particles to Chapter VII — 113 — Activities in Deposits adhere to the bacteria. The aggregation of particles of clay, silt, and sand hastens the rate of sedimentation and influences the structures of the resultant bottom deposits. The work of WAKSMAN and VARTIOVAARA (1938), RUBENTSCHIK ef al. (1936), and others has demonstrated that bacteria have a marked affinity for solids. Filamentous cells may weave particles together into intricate meshworks. The extent of the binding action depends primarily upon the types of microorganisms present and the quality of the organic matter (PEELE, 1936). The influence of micro- organisms on soil aggregations has been investigated by Martin and WAKSMAN (1940). Enzymes in bottom deposits :— In considering the evidence for vari- ous types of biochemical processes which occur in bottom deposits, KREPS (1934) points out that the absence of specific microorganisms does not prove the absence of the corresponding biochemical process because the latter may be effected by organic catalysts or enzymes. He believes that bacterial enzymes are concentrated upon the sea bottom where they con- tinue to function long after the bacteria have disappeared. There are no data available for appraising the part played by buried enzymes or the enzymes of resting cells in the transformation of bottom deposits, but it is possible that such enzymes may be very important catalytic agents. Seitz-filtered, germ-free sea water from Murman coast fjords was observed by Boxova et al. (1936) to catalyze changes in the oxygen, phosphate, nitrate, and ammonia content of water samples. Bacterial enzymes, including both oxidases and reductases, were found in greatest concentration in water collected just off the bottom. The observations suggest that marine bottom deposits may be especially rich in various enzymes from the remains of bacteria and other organisms. Certain bac- terial oxidases, in concentrations greater than can be accounted for by the presence of living bacteria, have been observed in bottom deposits by ZoBELL (19394). Chapter VIII CHARACTERISTICS OF MARINE BACTERIA Near shore the movements of water, wind, migratory birds, and other agencies provide for a continuous interchange of microorganisms between the land and the sea. Since many fresh-water and soil bacteria can sur- vive for prolonged periods of time in a salt concentration equal to or greater than that occurring in the sea, and since bays and estuaries pro- vide a gradual transition from fresh to salt water, one might expect to find soil and fresh-water bacteria in the sea freely intermingled with marine forms. Such a condition does prevail along the littoral zone, but only to a very limited extent in the open ocean. Although there are no infallible criteria for the differentiation of marine from non-marine bacteria, most of the bacteria which occur in the open ocean differ in certain respects from those found in non-marine habitats. This is probably because adventitious organisms either fail to perpetuate themselves in the marine environment or else lose their iden- tity in becoming acclimatized thereto. Unfortunately there have been no comprehensive studies of the general characteristics of marine bacteria as a group, although the observations of RUSSELL (1891), FISCHER (1894a), ISSATCHENKO (1914), BAVENDAMM (1932), BENECKE (1933), WAKSMAN and coworkers (1932-36), and BED- FORD (1933¢, b) are informative. The generalizations recorded below are based upon the published reports of these and other workers together with our own observations. Cell morphology :— About 80 per cent of the marine bacterial species catalogued by ZOBELL and UpHam (1944) are Gram-negative rods. From the random examination of colonies which developed on plates as well as from the direct microscopic examination of marine materials stained by the method of Gram, it is estimated that approximately 95 per cent of the bacteria occurring in the sea are Gram-negative. This compares favor- ably with the percentage of Gram-negative bacteria found in bodies of fresh water by TAYLor (1942), and is more than twice as many as TAYLOR and LocHHEAD (1938) found in soil (see Table XXX1I). Apparently there are proportionately more cocci in soil than in TABLE XXXI.— Comparison of main morphological groups of bacteria in soil, lake water, and marine materials:— MorPHOLOGICAL GROUP Sor} Sort? LAKE WATER® | MARINE MATERIALS‘ per cent per cent per cent per cent Gram-negative rods Bont 2007 95-5 94.6 Gram-positive rods 46.5 Hehael 3.8 Tie Gram-variable rods 9.4 °.9 Cocct 3.8 0.7 2.8 Others 4.2 0.2 0.5 1 625 soil cultures examined by TAYLor and LocHHEap (1938). 2 209 soil cultures examined by Toprr1nc (1937). 3 671 cultures from fresh-water lakes examined by TAYLOR (1942). 4 750 cultures from sea water and marine mud examined by ZOBELL et al. Chapter VIII — 115 — Marine Bacteria aquatic environments, but any generalizations must be preceded by more extensive investigations. Pleomorphism appears to be more common among marine bacteria than among the microflora of rivers, lakes, or soil. About one-fifth of the Gram-negative ‘‘rods’’ occurring in the sea are helicoidal, being properly classified as vibrio or spirilla. For the sake of simplicity, both straight and helicoidal rods are grouped together in Table XXXI since the investigators failed to mention vibrio or spirilla. This may have been an oversight, although there are perhaps more vibrio and spirilla in the sea than in soil or lakes. The majority of the bacteria found in the sea are actively motile. Flagella have been demonstrated on between 75 and 85 per cent of the pure cultures which have been examined, and a somewhat higher per- centage have been reported as “motile.’’ It is interesting to speculate upon the preponderance of motile forms as a possible aquatic adaptation. Spore-forming bacteria do not appear to be particularly abundant in the sea, although NEwrTon (1924) has described the characteristics of 80 different pure cultures of heat-tolerant bacteria which she isolated from the alimentary tracts of marine animals and from sea water. The ability of the organisms to tolerate a temperature of 80° C. for ten minutes was used as a criterion of spore formation. Twelve of her cultures survived boiling for three hours. Most marine bacteria are notoriously heat sensi- tive, as is discussed below. Marine spore-forming bacteria described by ZoBELL and UPHam (1944) include Bacillus abysseus, B. borborokoites, B. cirroflagellosus, B. fili- colonicus, B. imomarinus, B. submarinus, B. thalassokoites, and B. epi- phyticus. All of these were isolated from mud except B. epiphyticus. It was found associated with algae. On the average, marine bacteria are smaller than those which occur in milk, sewage, fresh water, or soil. Fresh-water bacteria are smaller on the average than soil forms. This generalization applies to representa- tives of the sub-order Eubacteriineae. Sulfur bacteria and representatives of the Chlamydobacteriales and Spirochaetales present many exceptions. Species of Pseudomonas, Vibrio, Flavobacterium, Achromobacter, and Bacterium predominate in the sea in the order named. Representatives of several other genera occur in smaller numbers (see Table XXXIV on p. 124). This may be contrasted with the preponderance of species of Bacillus and Actinomyces ordinarily found in soil. Due to their sanitary significance in fresh-water bodies, the importance of the enteric bacteria has been stressed by many investigators, but numerically they are out- numbered by species of the aforementioned genera. WAKSMAN (1934) writes, ‘‘The bacterial population of the sea is quite characteristic. It is distinct in nature from the population usually found on land, as shown by the more limited number of bacterial types found in the sea. Spore-forming bacteria, which comprise an important part of the bacterial population in soil, are practically absent in sea water, although they may be present in considerable abundance in the sea bottom. Cocci are also of limited occurrence in the sea. Motile rods and various types of vibrios, or comma-shaped organisms, usually make up the major part of the bacterial population thus far studied. The poverty of bacterial species in the sea depends largely upon the specific nature of sea water as a me- dium for the growth of these organisms.” For further information concerning the morphology of marine bac- teria the reader is referred to the literature summarized by ZOBELL and ZoBell 116. — Marine Microbiology Upuam (1944) who have prepared a list of marine bacteria and have described 60 new species. Of these, 49 are flagellated, 45 are Gram- negative, and 54 are rod-shaped. The average cell is 2 to 3 w in length by 0.4 to 0.6 w in width. Cultural characteristics:— In general, marine bacteria grow more slowly and the colonies are smaller than most microorganisms from soil, sewage, and milk. Whereas plates inoculated with terrestrial bacteria may be counted with good results after incubation from two to seven days at optimal temperatures, the numbers of macroscopically visible colonies of marine bacteria are still increasing significantly after ten days’ incu- bation (see Table IX on page 45). Diffuse, spreading colonies are en- countered less frequently on plates inoculated with marine materials than on plates inoculated with soil or fresh water. Depressed colonies occur on nearly all agar plates inoculated with sea water or marine mud, and occasionally an appreciable portion of the medium will be liquefied by agar digesters. Agar digesters are also found in fresh water and soil, but not nearly as frequently as in the sea. Because they are so conspicuous on agar plates, one may get the impression that agar digesters constitute a sizeable percentage of the marine microflora, but actually an average of only about one to two per cent of the colonies prove to be agar digesters. Over ten per cent of the pure cultures of marine bacteria described in the literature digest agar, but this is because agar digesters have been selected for special studies. Typical of water bacteria, more than half of those occurring in the sea are chromogenic. An examination by ZOBELL and FELTHAM (1934) of several thousand colonies developing on nutrient agar inoculated with sea water or marine mud showed that 69.4 per cent of them produced pig- ments. The distribution of colors was as follows: 31.3 per cent yellow, 15.2 per cent orange, 9.9 per cent brownish, 7.4 per cent fluorescent, 5.4 per cent red or pink, and 0.2 per cent green. The commonest type of fluorescence exhibited is greenish. Indigo, black, and silvery-sheen col- onies have been observed on special media. Twenty species of yellow chromogens were described by ZOBELL and UpuHam (1944), including Flavobacterium marinotypicum, Fl. marinovirosum, Fl. okeanokoites, Pseu- domonas neritica, Ps. obscura, Ps. oceanica, Ps. vadosa, Ps. xanthochrus, Vibrio adaptatus, V. marinoflavus, and V. marinovulgaris. In order to accentuate pigment production, the bacteria were grown in sea-water media enriched with Bacto-tryptone, neopeptone, and beef extract. The plates were incubated at 4° C. for three weeks following a preliminary incubation of 4 days at 25° C. Lower temperatures tend to favor pigment production. The unpublished observations of HARVEY Upuam working in the Scripps Institution laboratories indicate that in- fusions of fish, octopus, mussel, and other tissues promote the production of pigments by marine bacteria. Many marine bacteria gradually fail to produce pigment during prolonged laboratory cultivations. Yellow colonies predominated among those observed by GEE (19320) in sea water and mud around the Florida Keys. Of 855 colonies examined, 593 were yellow and 135 were red. According to PErRcE (1914), the red color of brine around marine salterns may be due to the abundant growth of chromogenic bacteria, although other organisms including Protococcus salinus and Dunaliella salina may be partly responsible. The discolora- tion of halibut has been shown by BEDFoRD (1933@) to be due to the Chapter VIII — 117 — Marine Bacteria growth of marine chromogenic bacteria which develop at o° C. From the skin of the discolored halibut he isolated various yellow, orange, red, and pink bacteria, some of which grew at temperatures as low as —5° C. Further reference is made to the extensive literature on chromogenic bac- teria which discolor marine fish on page 1go. SNow and FRED (1926) found that, except during the period of gross contamination, chromogenic bacteria constitute a high percentage of the microflora indigenous to Lake Mendota. An average of 52 per cent of the bacterial colonies were white or cream-colored, 35 per cent were yellow or orange, and 11 per cent were pink or red. Blue, violet, green, and black colonies were noted only infrequently. SNow and FRED contrast these findings with the percentage of chromogens of all of the Eubacteriales from various habitats described in the 1923 edition of BERGEy’s Manual of Determinative Bacteriology: 18 per cent white or cream, 18 per cent yel- low or orange, and g per cent pink or red. Only 3 per cent of the soil bac- teria listed in the Bergey Manual produced red or pink pigments as com- pared with rg per cent of the salt-water bacteria and 23 per cent of the fresh-water bacteria which were described as red or pink pigment pro- ducers. Considerably more work should be done on the factors which influence pigment production and the ecological significance of chromogenesis. However, the attractiveness of the more perspicuous chromogens should not influence one to overlook the less distinctive achromic varieties. Nearly all of the bacteria isolated from sea water or marine mud have proved to be facultative aerobes. They grow better in the presence than in the absence of atmospheric oxygen under ordinary conditions of labo- ratory cultivation. Neither strict aerobes nor strict anaerobes are com- mon in the sea, whereas both are fairly common in soil. The facultative aerobes tend to lose their ability to grow anaerobically after prolonged laboratory cultivation. These are provisional generalizations concerning the relationship of marine bacteria to oxygen, and may be subject to mod- ification when more information is available. Physiological characteristics:— Reference has already been made to the physiological versatility of marine bacteria. It is believed that there are bacteria in the sea which are capable of attacking nearly any kind of organic substrate, and many inorganic compounds are altered by the activities of marine microorganisms. The same may be said of soil and fresh-water microorganisms, although there are certain gross dissimilari- ties. As a group, marine bacteria are more weakly saccharolytic and prob- ably more strongly proteolytic than are either soil or fresh-water bacteria. Whether these properties are influenced by the salinity of the medium or whether they are peculiar adaptations of certain autochthonous species is problematical. Certainly the dissimilarities do not apply to individual species. Although ZoBELL and Grant (1943) present evidence which suggests that all heterotrophic marine bacteria are able to assimilate glucose, only 46 of the 60 cultures studied by ZoBELL and Upnam (1944) fermented glucose with the formation of acid, and none of them produced “gas” from glucose. This may be due to a general lack of fermentative ability, but more likely it is due to the efficiency of the organisms in assimilating utilizable organic matter. When cultivated in dilute solutions, the marine ZoBell — 118 — Marine Microbiology bacteria studied by ZOBELL and GRANT (1943) converted 30 to 35 per cent of the glucose into bacterial protoplasm or intermediate products of me- tabolism, the rest being oxidized to carbon dioxide and water as a source of energy. WAKSMAN and CAREY (19350) reported that around 60 per cent of the organic matter assimilated by marine bacteria is oxidized and 4o per cent is converted into bacterial protoplasm or intermediate products. It will be interesting to ascertain whether this economy of utilization is peculiar to marine bacteria which have become adapted to living in the extremely dilute organic nutrient concentrations existing in the sea. Rarely in the open ocean are bacteria encountered which produce acid from lactose. Gas producers are found only in contaminated water. Lac- tose fermenters are common in soil and most fresh-water bodies. Their sanitary significance is discussed in Chapter XVI. Very few marine bac- teria produce acid from sucrose, maltose, xylose, glycerol, mannite, or salicin, and gas producers have not been reported from the high seas. However, like glucose and lactose, most of these carbon compounds can be assimilated by marine bacteria. About one-third of the described species of marine bacteria metabolize starch, but little, if any, acid and no gas is produced from starch. The weak fermentative power of marine bacteria has been mentioned by CoupPIN (1915). As a group, marine bacteria are actively proteolytic, rapidly attacking most kinds of proteinaceous materials. Nearly all of them liberate am- monia from peptone, and approximately three-fourths of them liquefy gelatin. So many marine bacteria liquefy gelatin that it is not a satis- factory solidifying agent for plate-count media. Only a few marine bac- teria liberate detectable quantities of indol from tryptophane. Among the sixty new species described by ZOBELL and UpHam (1944), only Vibrio adaptatus and Vibrio marinofulvus formed indol in tryptophane broth. The observations of GRAN (1902), DREW (1912), KELLERMAN and SMITH (1914), MoiscuH (1925), and others on the abundance of denitri- fiers in the sea has focussed attention upon this group of bacteria. Some workers have concluded that most marine bacteria require nitrate, and that many reduce nitrate to nitrogen. The media employed by BERKELEY (1919), LIPMAN (1926), BAVENDAMM (1931), GEE (19320), and other ma- rine microbiologists all contain nitrate. However, ZoBELL (19412) failed to find that nitrate enhanced the growth of marine bacteria. A good many marine bacteria reduce nitrate to nitrite in appropriate media and a few liberate nitrogen from nitrate, but marine bacteria are not distinctive in this respect. Luminescence is not necessarily a physiological property of marine bacteria, although most described species of photogenic bacteria have been isolated from marine materials. When provisions are made for their detection, photogenic bacteria are found in most samples of sea water, and they are often associated with certain marine animals in considerable abundance. Several species of photogenic bacteria, some of which may prove to be identical, have been isolated from the sea and described. Besides the nine species of FISCHER (1894a) listed on page 3-4, FISCHER (1886) described Photobacterium indicum and Ph. phosphorescens (FISCHER, 1887). BEIJE- RINCK (1889) described Ph. duminosum for the first time and gave addi- tional information on Ph. phosphorescens, Ph. indicum, and Ph. fischert. The latter organism has been described by JoHNSON and SHUNK (1936) Chapter VIII — 119 — Marine Bacteria as Achromobacter fischeri. JOHNSON and SHuNK also described Achromo- bacter harveyi, which they isolated from marine materials. Katz (1891) isolated marine photogenic bacteria now known as Bacillus argenteo-phos- phorescens, Bacillus cyaneo-phos phorescens, Bacterium smaragdino phos phor- escens, Achromobacter phosphoricum, A. phosphoreum, and A. luminosum. Most of these are characterized by BERGEy ef al. (1939) who also list Pseudomonas pierantonti, Vibrio prerantonii, Bacterium giardi, Micrococcus phosphoreus, M. pfliigert, and Photobacterium pfliigeri. In a comprehen- sive résumé of the literature and compilation of species, DAHLGREN (1915) adds the following: Photobacteriwm balticum, Ph. cyaneum, Ph. javanense, Ph. plymouthii, and B. phosphorescens gelidus, all of which were found in sea water or associated with marine fish. DAHLGREN regards many of the afore-mentioned species as being synonymous. Bioluminescence is pre- ponderantly a property of marine organisms (HARVEY, 1940). The luminous marine bacteria studied by JoHNSON and Harvey (1938) required sea water for maximum efficiency. Luminescence, as well as respiration, fell off when sea water was diluted by more than 50 per cent with fresh water. Below a concentration of 10 per cent sea water, both respiration and luminescence were irreversibly destroyed, and only a small fraction of the cells remained viable. Doubling the salinity of sea water by the addition of salts did not markedly affect luminescence, but further increases in salinity were inimical to the respiration, luminescence, and viability of marine photogenic bacteria. Isotonic salt or sugar solutions were not so favorable as was natural sea water for the activity of the bacteria. The physiological characteristics of various groups of marine bacteria are discussed elsewhere in this volume. In the foregoing paragraphs only a few of the distinctive physiological characteristics of marine bacteria, as a Class, have been compared with bacteria from other habitats. Salinity requirements:— The preference of marine bacteria for sea water over either tap water or NaCl solution isotonic with sea water is illustrated by the data in Table VIII on page 44. Plate counts of either raw sea water or marine mud collected beyond the zone of land contam- ination are invariably higher when the nutrient media are prepared with sea water rather than with fresh water. Conversely, samples of sewage, soil, fresh water, human excreta, etc., show many more colonies when TABLE XXXII.— Relative numbers of bacteria from marine and terrestrial sources which developed on nutrient media prepared with different concentrations of sea water (from ZOBELL, 1941a):— Ss ee ee ee ee ae ee MEDIA NUMBER PER CENT OF SEA WATER IN MEDIUM INOCULATED OF ses WITH SAMPLES 100 75 | 50 | 25 | bdo) | ° Raw sea water 31 100 93 65 38 17 9 Marine mud 18 100 89 61 46 28 19 San Diego Bay mud 3 100 108 131 142 139 114 Mission Bay mud 4 100 107 117 142 156 106 Mission Bay water 5 100 103 115 108 118 97 Sewage 13 13 27 51 79 106 100 Tap water 8 4 be) 28 58 105 100 Inland soil 6 15 23 40 66 89 100 Mouth microflora 5 9 15 41 78 93 100 ZoBell — 120 — Marine Microbiology plated on media prepared with fresh water rather than with sea water. Diluting sea water by 50 per cent reduces both its growth-promoting properties for marine microflora and its toxicity for terrestrial bacteria, as shown by the data in Table XXXII on page 1109. Similar results have been reported by BERKELEY (1919), KORINEK (1926), LIPMAN (1926), and others. KoOrRINEK (1927) believes that marine bacteria can be distinguished from non-marine forms upon a basis of their salt tolerance. While this may not be true of every individual culture, it is statistically true of marine bacteria as a group. The effect of the dilution of sea water is shown by the following data which Lipman (1926) obtained by plating sea water collected one mile from land on sea-water media diluted with distilled water: PER CENT SEA WATER PER CENT DIST. WATER BACTERIA PER ML. 100 ° 270 70 30 200 50 50 150 30 70 ° ae) go ° As'shown below, Lipman obtained somewhat different results when he plated sea water collected from the wharf at the Tortugas Laboratory where there was much shore contamination: PER CENT SEA WATER PER CENT DIST. WATER BACTERIA PER ML. 100 ° 960,000 75 25 730,000 5° 50 640,000 25 75 680,000 The beneficial effects of sea water on marine bacteria and its inhib- itory effects on non-marine bacteria are most conspicuous when appro- priately diluted samples of marine or non-marine materials are plated on media containing different concentrations of sea water. This is illustrated by the experiments described above. Following laboratory cultivation, many bacteria seem to develop a tolerance for certain types of adverse conditions. Asa matter of fact, most old stock cultures of either marine or non-marine bacteria will grow almost equally well on either sea-water or fresh-water media when the media receive large inoculations. However, marked differences are noted in the salt tolerance of individual cells con- stituting the respective cultures if they are properly diluted and then plated on the two kinds of media. Under these conditions there emerge statistically determined preferences of marine bacteria for sea water and of the non-marine bacteria for fresh water. Within old stock cultures, though, some individual cells are found to be euryhaline over a wide range and others are relatively stenohaline. The salt requirements of the latter may be high, low, or intermediate, depending upon the origin and cultural history of the organism. According to KorInEk (1927), after a year’s cultivation original differ- ences in the salinity requirements between fresh-water and marine bac- teria were not eliminated. Conversely, 9 of 12 cultures isolated from the sea and maintained in the laboratory for five months by ZOBELL and MICHENER (1938) were able to grow in fresh-water media, although upon initial isolation they required sea-water media. The conditions under which the organisms are cultivated unquestionably influence the results. After laboratory cultivation on sea-water media for periods of from two to twelve years, 56 of the 60 new species of marine bacteria described Chapter VIII — 121 — Marine Bacteria by ZoBELL and UpHam (1944) have developed the ability to grow in fresh-water media. Upon initial isolation, all of them required sea water or isosmotic salt solution. Interestingly, the Bacillus and Micrococcus species isolated from the sea have proved to be much more euryhaline than Pseudomonas or Vibrio species. Only six out of 18 species of marine Pseudomonas and three out of 11 species of Vibrio became acclimatized to fresh water (and these only poorly), while all of the 8 species of Bacillus and all of the 6 species of Micrococcus became tolerant of fresh water. Micrococcus euryhalis, M. aquivivus, M. infimus, and M. maripuniceus grew equally well in either sea-water or fresh-water media after cultiva- tion for a few months in the laboratory. This was also true of Sarcina pelagia and Serratia marinorubra, both of which required sea water upon initial isolation from the sea. Neither isotonic salt solution nor artificial sea water is as good as is natural sea water for the cultivation of recently isolated marine bacteria. Moreover, marine bacteria are as sensitive to increases in salinity as they are to decreases. Our observations substantiate those of Kor1NEK (1926) that marine bacteria are even less resistant than are fresh-water bacteria to changes in osmotic pressure. Doubling the salinity of sea water either by the addition of NaCl or sea salt may reduce by 25 to 50 per cent the number of marine bacteria which will grow in it. Very few marine bacteria have been observed to grow in sea-water media to which 12 per cent NaCl has been added. Thus, while marine bacteria are often characterized as being halophilic or salt-requiring, they are far less so than are bacteria found in salted fish, salt-cured furs, pickle brines, salt lakes, and limans (see Chapter XVIII). Virtually no marine bacteria, exclusive of those found in salterns or asso- ciated with salted fish or similar marine products, grow in sea water to which 24 per cent NaCl has been added. Numerous bacteria which occur in strong brines flourish in media containing from 18 to 30 per cent NaCl (Hor, 1935). Studies on their salinity requirements indicate that, while bacteria from different environments differ in salt tolerance and in their ability to become acclimatized to changes in osmotic pressure, most of the bacteria found in the sea, exclusive of adventitious contaminants, are specifically marine. This conclusion is substantiated by the fact that very few com- monly known species of terrestrial bacteria such as members of the coli- form, subtilis-mesentericus, or Gram-positive cocci groups have been found in the sea except relatively near land. Temperature tolerance :— ForsTER (1892), DREw (1914), ANGST (1929), and others have emphasized the extreme thermal sensitivity of marine bacteria. BERKELEY (1919) believed that low plate counts obtained with sea water collected from depths of 20 to 100 fathoms may be due to the failure of some of the bacteria to tolerate the plating temperature of nutrient agar, 7.e., about 42° C. Lioyp (1930) suggested using plating temperatures ranging from 25° to 35° C. to avoid killing “ordinary micro- organisms’”’ indigenous to the sea. The data in Table X on page 46 show that if plating temperatures of the medium exceed 42° C. by a few degrees, plate counts on samples of sea water and marine mud are noticeably decreased. These studies were ex- tended by ZoBELL and Conn (1940) with the use of nutrient gelatin, which solidifies at lower temperatures than does agar. The data in Table ZoBell — 122 — Marine Microbiology XX XIII show that some marine bacteria are quickly injured at 35° C. However, it appears doubtful whether enough bacteria may be injured by the plating temperature of agar (40° to 45° C.) to contra-indicate the use of agar plates for estimating the abundance of viable bacteria. TABLE XXXIII.— Relative numbers of colonies developing from sea water and marine mud plated with nutrient sea-water gelatin at different temperatures and cooled immediately to 12°C.:— POURING TEMPERATURE OF GELATIN ReGearA NUMBER OF SELES 30°C. 35°C 4orC. 45°C 50°C To % % % 7 Sea water 12 100 98.6 96.5 87.5 76.2 Marine mud 16 100 97-9 91.3 83-4 67.8 While brief warming during plating at temperatures ranging from 30° to 40° C. does not destroy the viability of large numbers of marine bac- teria, the majority of them are killed by exposure to this temperature for ten minutes, as shown by the following results obtained by ZOBELL and Conn (1940) who plated in duplicate ten samples of sea water and ten samples of marine mud: Samples held 10 minutes at aC.) /pgor Ch 4oiCuy | §or'C.,) -Se7-€.” (ereane. Per cent survival in sea water 100 81.3 21.9 6.8 3.0 Cellfalcicula + Thios pira + Spirillum +++4+-4+ Azotobacteriaceae Azotobacter +? Rhizobiaceae Rhizobium — Agrobacterium = Chromobacterium Gaffkya Micrococcaceae Micrococcus ++ Sarcina + Neisseriaceae Neisseria —_ Veillonella = Lactobacteriaceae Diplococcus + Streptococcus + Leuconostoc —_ Lactobacillus oa Microbacterium — Propionibacterium _ Butyribacterium = Corynebacteriaceae Corynebacterium — Listeria : = Erysipelothrix -_ Parvobacteriaceae Pasteurella — Malleomyces = Brucella = Hemophilus = Moraxella _ * Noguchia _ Dialister Bacteroides Aerobacter Klebsiella Erwinia _ Serratia ++ Proteus a+ Salmonella A Shigella - = Enterobacteriaceae Escherichia A A Achromobacteriaceae Achromobacter ++++ Flavobacterium +444 Alkaligenes A ZoBell — 126 — Marine Microbiology Se a Ee Table XXXIV cont. FAMILY GENUS OcCURRENCE IN THE SEA Bacteriaceae Kurthia + Actinobacillus _ Leptotrichia — Fusobacterium — Bacterium ++ Bacillaceae Bacillus ++4++4++4 Clostridium 4 Ain Sub-Order II. Caulobacteriineae Nevskiaceae Nevskia _ Gallionellaceae Gallionella a Siderocapsaceae Siderocapsa + Sideromonas + Caulobacteriaceae Caulobacter +? Sub-Order III. Rhodobacteriineae Chromotioidaceae Thiocystis Thios phaera Thios phaerion Thiocapsa Thiosarcina Lamprocystis Thiopedia Thioderma Lampropedia A moebobacter Thiodictyon T hiothece Thiopolycoccus Chromatium Rhabdomonas Thios pirillum Rhodacapsa Rhodothece ++++t++t+ 1 +444 04 Rhodobacteriaceae Rhodocystis Rhodonostoc Rhodorrhagus Rhodobacterium Rhodobacillus Rhodovibrio Rhodospirillum lists Ht Order II. Actinomycetales Mycobacteriaceae Mycobacterium a a: Actinomycetaceae Actinomyces Nocardia ++ Streptomycetaceae Streptomyces - Micromonos pora =F Order III. Chlamydobacteriales Chlamydobacteriaceae Sphaerotilus Clonothrix Leptothrix +++ Chapter VIII — 127 — Marine Bacteria Table XXXIV cont. FAMILY GENUS OCCURRENCE IN THE SEA Crenothricaceae Crenothrix + Beggiatoaceae T hiothrix Beggiatoa Thioploca Achromatiaceae Achromatium Thiophysa Hillhousia I++ +++ Order IV. Myxobacteriales Cytophagaceae Cytophaga ++ Archangiaceae Archangium _ Stelangium _ Sorangiaceae Sorangium - Polyangiaceae Polyangium = Synangium _ Melittangium _ Podangium — Chondromyces _ Myxococcaceae M yxococcus - Chondrococcus = Angiococcus _ Sporocytophaga + Order V. Spirochaetales Spirochaetaceae S pirochaeta — Saprospira +- ae Cristos pira Treponemaceae Borrelia ~ Treponema ate Leptospira + Through the courtesy of the Board of Editors of BErGEY’s Manual of Determinative Bacteriology, this Out- eo follows that used in the new (Sixth) Edition of this book now in press (see BREED é# al., 1944, The symbol A indicates that representatives of the genus have been found as adventitious organisms in the sea, z.e., in polluted water only or under other circumstances which strongly suggest that they are not indigenous species. It has been necessary to rely largely upon personal judgment in recording the frequency of occurrence of bacterial genera in the sea. The descriptions of many marine bacteria are so fragmentary that it is diffi- cult or impossible to ascertain the genus to which they belong. Early workers in the field indiscriminantly applied the generic name Bacillus or Bacterium to most rod-shaped organisms, many of which properly should be classified as Pseudomonas, Flavobacterium, Proteus, Achromobacter, Serratia, etc., in light of more recent developments in taxonomic bacteri- ology. This is true of the numerous species of Photobacterium and Hali- bacterium described by FiscHER (1894a@) and the Agarbacterium species described by ANGsT (1929), although certain species of these obsolete genera actually are Bacteriwm or Bacillus as defined by BERGEY ef al. (1946). ZoBell SS Marine Microbiology The number of plus signs in the table indicates the relative abundance of the genus as judged from reports in the literature and our own observa- tions. The plus signs do not indicate the relative numbers of species of each genus, since in many cases the species are not known. There may be only one marine species of Desulfovibrio, for example, but since it appears to be widely and abundantly distributed in the sea, particularly in bottom deposits, it is scored two-plus (++). On the other hand, there may be several marine species of Staphylococci or Actinomyces but since the pres- ence of representatives of these genera have been only infrequently re- ported, they are scored one-plus (+). Representatives of all orders have been found living in the sea. Attention is directed to the actinomycetes, which, because of the mold- like appearance of certain genera, are often not regarded as bacteria. Though not true bacteria, the actinomycetes belong to an order of Schiz- omycetes, the Actinomycetales. Species of Actinomyces have been found growing on dead marine algae, on submerged surfaces where organic mat- ter has accumulated, and in bottom deposits. ZoBELL and UPHAM (1944) described two new species, Actinomyces marinolimosus and Act. halotrichis. Species of Mycobacterium, Nocardia (Proactinomyces), and Micromonospora have been observed in sea-water enrichment cultures growing on petroleum hydrocarbons (ZOBELL ef al., 1943) and rubber products (ZoBELL and BECKWITH, 1944). They appear to be widely dis- tributed in the sea. There are nearly as many known marine species of the order Spiro- chaetales as those reported from non-marine habitats, notwithstanding the fact that the sea has not been extensively explored by bacteriologists. Spirochaeta plicatilis, which EHRENBERG (1838) found in sea water, has the distinction of being the first accurately described bacterium. Spiro- chaeta marina and probably Sp. eurystrepta live in the sea. Species of Saprospira and Cristospira have been found almost exclusively in shell- fish (see p. 187). The observations of ZUELZER (1928) suggest that Leptospira biflexa occurs in the sea. Chapter IX AQUATIC YEASTS AND, MOLDS Technically speaking, yeasts and molds as well as bacteria are Fungi. Fungi may be defined as achlorophyllous plants constituting a primary division of the phylum Thallophyta, co-ordinate with the Algae which contain chlorophyll. The division Fungi comprises the classes Myxo- mycetes (slime molds), Eumycetes (true fungi), and Schizomycetes (fission fungi or bacteria). The class Eumycetes consists of the sub-classes Phycomycetes, Ascomycetes, Fungi Imperfecti, and Basidiomycetes. Though primarily terrestrial, all sub-classes of Eumycetes except the Basidiomycetes have a number of aquatic representatives. No species of Basidiomycetes have been found living normally in bodies of water. Several hundred representatives of the Phycomycetes and smaller num- bers of Ascomycetes and Fungi Imperfecti live in aquatic environments. Organisms which in popular parlance are known as molds or mold fungz embrace certain orders of Phycomycetes, Fungi Imperfecti, and Ascomy- cetes. The latter also includes, besides other orders, the Saccharomy- cetales or yeasts. The true yeasts multiply by budding or by the forma- tion of ascospores, hence the sub-class name, Ascomycetes, to which they belong. The so-called wild yeasts or torulae belong to a family of Fungi Imperfecti, namely the Dematiaceae. Torulae, or wild yeasts, differ from the true yeasts in that they do not form spores. The asporogenous Mycoderma are also called wild yeasts. Occurrence of yeasts in the sea:— Yeasts are widely and commonly distributed in nature. Though probably not as well adapted, as a class, as bacteria to growing throughout a wide range of environmental condi- tions, there are yeasts which are capable of tolerating nearly any ex- tremes of osmotic pressure, hydrostatic pressure, temperature, pH, or oxygen tension found in the sea. Yeasts may be cultivated by the same methods as are used for bacteria, and they are more readily enumerated by direct microscopic counts than bacteria because, on the average, yeasts are larger than bacteria and have a more distinctive morphology. In spite of the fact that, for the most part, special nutrient media rich in carbohydrates or other utilizable organic matter have not been em- ployed, many bacteriologists report finding yeasts in the sea. Colonies of yeasts often appear along with bacteria on nutrient media designed primarily to detect bacteria. Most of the reports fail to describe the yeasts, so it is not known whether the organisms were true yeasts, torulae, or other yeast-like organisms. FiscHeR and BREBECK (1894) reported the presence of many Torula and Mycoderma and a few true yeasts in sea water. They found Blastoderma salmonicolor in a sample of sea water taken near the Azores Islands. It is problematical whether certain of these organisms are indigenous to the sea because they occur so commonly in the air. From the North Sea, NADSON and BurGwitz (1931) isolated 22 vari- eties of yeast-like organisms including 15 varieties of white Torula, 7 of ZoBell — 130 — Marine Microbiology red Torula, and one variety each of Dematium, Oidium, and Endomyces. The yeast-like organisms were found associated with Laminaria saccha- rina, Alaria esculenta, Fucus vesiculosus, and other seaweeds. The white Torula species ranged from 1.25 to 5.2 uw in width and from 2.5 to 9.4 win length. The red Torula species were somewhat larger, being from 1.75 to 5.0 w in width and from 3.75 to 15.0 win length. The yeast-like cultures developed readily in sea-water media enriched with mannite, sugars, or with Laminaria extract. They grew at temperatures as low as 2° C. Much better growth was observed at 12° C. Salt-tolerant varieties of the so-called “fat yeast,” Endomyces vernalis, along with Saccharomyces cere- visiae and Saccharomyces ellipsoideus, were also found. Endomyces ver- nalis as well as the Torula species were believed by NADSON and BurG- witz to be autochthonous marine species. FISCHER (1894a) noted the appearance of numerous yeast colonies along with molds and bacteria in sea water collected on the cruise of the S.M.S. Moltke. For example, his logbook records that in Sample rg col- lected 330 miles from the nearest land, the Azores, 242 colonies developed on nutrient gelatin inoculated with 0.25 ml. of sea water. Halibacterium pellucidum predominated, with nearly as many yeasts, most of which were believed to be Torula. Two of the latter were pink. Three colonies of mold fungi were noted. Of the 666 colonies which developed from o.5 ml. of sea water in Sample 26 collected one mile off Plymouth, England, only 13 were molds, but many were yeasts including 8 pink Torula. There were almost as many yeasts and molds as bacteria among the 262 colonies which developed from 0.1 ml. of Sample 29 collected in Wilhelmshaven Roads off the north coast of Germany. One Mycoderma species and 20 pink yeast were identified in this sample. Samples collected farther from land generally yielded fewer microorganisms of all types, particu- larly molds, but yeasts were generally found in all samples regardless of the distance from land. In some samples collected in the open ocean, actually more colonies of yeasts than bacteria developed on the gelatin plates. The nutrient composition, together with the relatively low pH (near 7.0) of the medium employed by FiscHER, may account for the prepon- derance of yeasts observed in some of the samples. Be this as it may, his extensive survey instructs us that yeasts are widely distributed in the sea. Besides the species of Torula which appeared to be fairly common in the sea, particularly in higher latitudes, FiscHER found species of Mycoderma and Saccharomyces at considerable distances from land. The widespread occurrence of yeasts in the sea, together with the fact that some of them grew better in nutrient sea-water media than in corresponding fresh-water media, led him to conclude that there are autochthonous species of marine yeasts. ISSATCHENKO (1914) reported the general occurrence of yeasts in the Arctic Ocean. Although ZOBELL and FELTHAM (1934) made no special efforts to estimate their abundance, yeasts were observed on most plates of sea-water agar inoculated with samples of marine materials collected near land as well as in the open ocean. A pink yeast, probably a Torula, was found by HUNTER (1920b) to be responsible for the spoilage of oysters. The yeast, which grows readily at low temperatures, produces a pink or reddish pigment. While exces- sive contamination of oysters with the yeast was attributed to careless handling, examinations of samples of surface and bottom water from the Chapter IX — 131 — Yeasts and Molds oyster-growing areas revealed the presence of the pink yeast. It could also be recovered from healthy oysters. There are frequent references in the literature to the occurrence of yeasts in lakes. Many of the numerous species of true yeasts, torulae, and yeast-like organisms found in the soil have been reported in lakes. Gen- erally they are most abundant near shore or associated with higher aquatic plants such as Sagitlaria, Myriophyllum, Naias, Zostera, Lemna, Elodea, Vallisneria, Potamogeton, Chara, Cladophora, etc. Marine molds:— Disregarding those obviously originating from air contamination, FISCHER (1894a) found molds in the sea far less frequently than either bacteria or yeasts. With very few exceptions, he found nu- merous molds in sea water only fairly close to land. The molds which FISCHER observed were common species of terrestrial fungi, primarily Penicillium and Aspergillus. WAKSMAN (1934) also observed that com- mon dust and wind-borne species of Penicillium and Aspergillus occupy a prominent place among the fungi reported in marine materials. In marine muds from the Woods Hole region, SPARROW (1937) found species of Penicillium, Aspergillus, Rhizopus, Alternaria, Cephalosporium, Trichoderma, Chaetomium, and Cladosporium associated with decaying phytoplankton. He doubts, however, that these are true marine fungi. Though definitely able to live in the sea, most of them were well-known terrestrial species. Owing to their small size and the difficulty of observing them under anything like natural conditions, the search for fungi associated with marine algae has been disappointing. The additions made by most my- cologists have been few and accidental. SPARROW (1934) remarked re- garding marine Phycomycetes that “‘one is at once confronted with a very real problem in endeavoring, first, to find sporangia which have not al- ready discharged their zoospores in the interval between collection and examination, and, secondly, to produce conditions in the laboratory favorable for the development and discharge of these sporangia. This, together with the very small size of zoospores produced by these fungi, has greatly retarded our knowledge.” PETERSEN (1905) made a systematic search for fungi along the coast of Denmark where he found several species of chytridiaceous Phycomy- cetes growing either parasitically or saprophytically on marine algae. He described the fungi now known as Petersenia lobata, P. pollagaster, Sirol- pidium bryopsidis, Olpidium laguncula, Ectrogella perforans, Rhizophydium discinctum, Pleotrachelus inhabilis, Pl. minutus, Pl. paradoxus, Pl. rosen- vingit, and Pontisma lagenidioides. SUTHERLAND (1915a) described five new species of Fungi Imperfecti, Mycosphaerella pelvetiae, Stigmatea pelvetiae, Pharcidia pelvetiae, Pleospora pelvetiae, and Macrosporium pelvetiae, which were found associated with species of the rockweed Pelvetia along the coast of Britain. He (19150) also described Orcadia pelvetiana, Didymosphaeria pelvetiana, Didymos- phaeria fucicola, and Hypoderma laminariae. These Pyrenomycetes were parasitic on Pelvetia, Fucus, and Laminaria respectively. From the green alga, Codium mucronatum, growing in the Puget Sound region, ZELLER (1918) isolated and described Chytridium codicola, Rhizophydium codicola, and Stemphylium codit. Twelve species of marine Phycomycetes were collected by SPARROW (1934) in the Kattegat near Frederikshavn, Jutland, including Pythium ZoBell — 132 — Marine Microbiology marinum, Eurychasma dicksonii, Sirolpidium bryopsidis, Pontisma lagent- dioides, Petersenia lobata, Petersenia pollagaster, Pleotrachelus olpidium, Pleotrachelus rosenvingit, Ectrogella perforans, Chytridium megastomum, and Chytridium polysitphoniae. ‘They were growing on either living or moribund marine algae. Near Woods Hole, Massachusetts, SPARROW (1936) isolated and described 15 marine species of Phycomycetes, two Myxomycetes, and a Protomyxa-like protozoan. SPARROW pointed out that marine fungi have been only very incompletely studied. Some of the fungi observed by him are true parasites which attacked healthy host plants unassisted by other organisms. Included in this category were Ectrogella perforans, Eurychas- midium tumefaciens, Olpidium sphacellarum, Rozella marina, and Chy- tridium magastomum, none of which was observed living saprophytically. Rhizophydium discinctum and Petersenia andreei were only doubtfully par- asitic. Labyrinthula chattont and Thraustochytrium proliferum were saprophytes found only on dead algal cells. Rhizophydium globosum, Pontisma lagenidioides, Sirolpidium bryopsidis, and Chytridium poly- siphoniae were true parasites on certain host plants and saprophytic on others. Besides noting the apparent obligate association of fungi with marine plants which strongly suggests that the fungi are true marine species, SPARROW noted the abundance in the sea of so-called chytridia- ceous fungi which possess biciliate zoospores. Most of the true chytrids inhabiting fresh-water algae are uniciliate, while in the sea the biciliate forms predominate. BARGHOORN and LINDER (1944), who observed along the coast of Maine, Massachusetts, and Connecticut many marine fungi which were quite unlike any known terrestrial organisms, were impressed by the diversity of fungi found in the sea. The following Fungi Imperfecti were isolated and described: Phialophorophoma litoralis, Diplodia orae-marts, Botryophialophora marina, Orbimyces spectabilis, Alternaria maritima, Helicoma maritimum, H. salinum, and Speira pelagica. Marine Pyre- nomycetes as follows were also described: Samarosporella pelagica, Cert- osporopsis halima, Remispora maritima, Amphisphaeria maritima, Lentes- cospora submarina, Halosphaeria appendiculata, Leptosphaeria orae-maris, Sphaerulina orae-maris, Peritrichospora integra, P. lacera, Halophiobolus cylindricus, H. opacus, H. longirostris, H. maritimus, H. medusa, H. hali- mus, and H. salinus. Most of these will be recognized as not only new species, but as new genera, since, with few exceptions, the organisms have no counterpart in terrestrial species or genera. The fungi described by BARGHOORN and LINDER (1944) were found in both brackish water and sea water of normal salinity. Most of them grew better in nutrient sea water than in corresponding fresh-water media, as illustrated by data on radial growth of mycelia, expressed in milli- meters per day: FRESH WATER SEA WATER pH 7.2 pH 7.4 Halophiobolus opacus 0.52 oe Xe) Halophiobolus salinus 2.74 3.16 Ceriosporopsis halima T2338 get / Peritrichos pora integra °.79 0.96 Helicoma salinum 0.68 I.14 Tolerance of relatively high salt concentrations was demonstrated by growth of the fungi in media containing three times as much salt as nor- Chapter IX — 133 — Yeasts and Molds mal sea water. With one exception the entire group developed best in media with an initial pH above 7.6, and, unlike most terrestrial molds, growth was definitely unfavorable in acid media. Their response to salin- ity and hydrogen-ion concentration was interpreted as a physiological modification to marine conditions. All species cultured made an appre- ciable growth at 5° C., although the most favorable temperature for growth on complex synthetic media was between 22.5° and 27.5° C. Special attention is directed to the slime mold Labyrinthula, a genus of Myxomycetes. According to WHIPPLE (1927), no aquatic species of Myxomycetes had been reported as late as 1927. This may be because labyrinthulae are regarded by some as rhizopods, since they possess pseudopodia and exhibit remarkable powers of amoeboid movement. SPARROW (1936) described two species of marine Labyrinthula, one of which is parasitic and the other saprophytic. The latter is not unlike L. macrocystis which CIENKOWSKY isolated from marine algae in 1867. It also resembles the Labyrinthula species which RENN (1936) found asso- ciated with diseased eel grass. JEPPS (1931) observed, in debris which collects in the bottom of aquaria containing marine algae, species of Labyrinthula, which infect and eventually destroy diatom cultures. In- jured Laminaria are also infected, but Miss JEpps found no evidence that uninjured Laminaria were parasitized by Labyrinthula. Two or more fungi may be involved. SPARROW (1943) relates that a Labyrinthula, along with Pyrrhosorus marinus and a Woronina-like fungus, has often been found in decaying marine algae. In his monograph on aquatic Phycomycetes, SPARROW (1943) states that most of the Phycomycetes discovered thus far in marine environ- ments are members of the orders Saprolegniales, the so-called water molds, and Lagenidiales. The Chytridiales, Plasmodiophorales, and Perono- sporales also have marine representatives. Most of these are chytrid-like forms living in or on algae. SPARROW points out that in many cases there are no hard and fast distinctions between aquatic, amphibious, and ter- restrial fungi. Representatives of all groups occur in both salt and fresh water. Significance of yeasts and molds in the sea:— Exclusive of parasitic varieties, the importance of yeasts and molds in modifying the marine environment is strictly secondary to that of the ubiquitous and more ver- satile bacteria. Most yeasts and molds require a medium rich in organic nutrients, particularly the simple carbohydrates, and the sea is notori- ously poor in organic matter. However, when associated with plants or animals or their products, aquatic fungi may be very active in the sea. As the causative agents of diseases of marine plants and animals, fungi may be extremely important. Fresh-water animals including fish are extensively parasitized by Saprolegnia and other aquatic fungi, and it may develop that many marine animals are also the victims of parasitic fungi. SPARROW (1936) described a fungus, Petersenia sp., which parasitizes rotifer eggs. Malformed sardine eggs collected and preserved for us by the Cali- fornia Fish and Game Commission were found to be filled with fungi. This research project was interrupted by the War before it could be estab- lished whether the fungi were responsible for the malformation of the sardine eggs or if the fungi attacked only moribund eggs. The preliminary observations suggest the possibility of fungus infections accounting for extensive failure of sardine crops. ZoBell — 134 — Marine Microbiology Parasitic and epiphytic fungi may occur on marine algae much more extensively than indicated by the fragmentary literature on the subject Such organisms are difficult to detect and identify even in properly col- lected specimens. The distinguishing morphological structures of the fungi are so minute and so intimately associated with the host tissue that they may escape detection. In some cases they have been mistaken for the fruit bodies of certain marine algae. For example, K1BBE (1916) described Chytridium alarium as a fungus parasitic on Alaria fistulosa, but SPARROW (1943) records that K1BBE mistook the cystidia of the alga for a fungus. According to the literature summarized by KIBBE (1916), LEMMER- MAN named a marine fungus, Dothidella laminariae, which is parasitic on Laminaria. ESTEE described Guignardia irritans, parasitic on Cystoseira and Halidrys. PATOUILLARD described Zignoella calospora, parasitic on Castagnea. PATOUILLARD and Hartot found Zignoella enormis on Stypo- caulon. REED described two ascomycetes, Guignardia ulvae on Ulva, and Guignardia alaskana on Prasiola. Coun described Chytridium poly- stphoniae on Polysiphonia, Olpidium plumulae on Antithamnion, and Olpidium entosphaericum on Hormiscia. FISCHER mentions Rhizo- phydium dicksonii on Ectocarpus, Olpidium sphacellarum on Sphacelaria, Olpidium tumefaciens on Ceramium, Olpidium bryopsidis on Bryopsis, and Olpidium aggregatum on Cladophora. Besides these parasitic marine fungi to which references are given by KiBBE (1916), SPARROW (1943) lists Rhizophydium gelatinosum, and Achlyogeton salinum, both of which are parasitic on Cladophora, Olpidium lauderiae parasitic on Lauderia, Rhizo- phydium marinum parasitic on Melosira, and Rozella marina parasitic in the sporangia of Chyiridium polysiphoniae. SPARROW (1936) suggests that the peculiar rhythms of blooming peri- ods and distribution of pelagic diatoms which cannot be attributed to hydrographic factors may sometimes be due to the activities of parasitic fungi. There is accumulating evidence, but no conclusive proof, that many of the diatoms and dinoflagellates which observers report are found in “poor condition” may have been parasitized. Diatoms parasitized by Ectrogelia perforans were observed by SPARROW (1934) to distort the frustules sometimes so much as to suggest that the fungus had dissolved the siliceous material and produced hypertrophy of the diatom cell. The possibilities of parasitism, coupled with the importance of diatoms and dinoflagellates as primary producers, invite early attention to this problem. The wasting disease of Zostera marina which has depleted the eel grass from hundreds of miles of coast in Europe and America may be due to a fungus infection. TurTrn (1934) believed Ophiobolus halimus to be the causative agent. RENN (1936) failed to find any Ophiobolus-like fungi associated with the rhizomes or leaves of diseased plants, but he did find a species of Labyrinthula in all of the diseased plants which he examined. The association of Ophiobolus (Halophiobolus) salinus, H. halimus, and H. maritimus with eel grass has been reported by BARGHOORN and LINDER (1944), who state that further research is required to ascertain whether these species are causative agents of the wasting disease, secondary in- vaders, or merely saprophytic. The brown alga, Macrocystis pyrifera, which is of considerable eco- nomic importance, may be subject to epidemics of fungus infections. Similarly, other commercially valuable marine algae may be affected by Chapter IX — 135 — Yeasts and Molds parasitic or saprophytic fungi, another problem which invites attention. Most of the fungi described by BARGHOORN and LINDER (1944) were isolated from wood or rope which had been submerged in the sea. His- tological examination of their natural substrata showed that the fungi penetrate and ramify the cell walls of wood and cordage fibers, inducing decay by enzymatic hydrolysis of the cellulose and other cell wall con- stituents. The fungi readily utilized cellulose, pectin, and starch under experimental conditions. Maltose, galactose, xylose, and asparagine were also utilized. Growth was vigorous and fairly rapid on wood flour agar. Many of the fungi attacked lignin. The evidence is convincing that fungi cause the deterioration of cordage fibers and wood under marine condi- tions. Importance of fungi in lakes :— In reviewing the literature on the sub- ject, WESTON (1941) states that there are between 700 and 800 species of Phycomycetes which are definitely aquatic, besides many others which, though normally terrestrial, are capable of living in water. In this latter category are several species of Penicillium and Fusarium. Relatively few species of Ascomycetes are adapted to aquatic life. Aquatic fungi are capable of activity and survival over a wide range of environmental conditions, being found in inland waters throughout the world. They grow equally well in direct sunlight or darkness. Certain ones are active at temperatures ranging from 1° to 33° C., and in water as acidic as pH 3.2 or as alkaline as pH 9.6. Most fungi require free oxygen. Aquatic fungi are both saprophytic and parasitic. Their ability to break down pectins, hemicelluloses, and cellulose is widespread. In at- tacking organic matter, fungi may influence the pH, oxygen content, and other chemical properties of the water. Most plants and animals living in lakes are susceptible to parasitism by aquatic fungi. Serious fungal epidemics among diatoms and desmids have been reported. The eggs of some animals are destroyed by fungi, and such organisms may also cause extensive infections of fresh-water fish. Fungi may be a limiting factor in aquatic biology. Many aquatic animals such as protozoans, rotifers, coelenterates, arthropods, etc., devour fungi spores from which they derive nourishment. According to WESTON (1941), water fungi are “‘ ubiquitous, abundant, versatile, hardy, and efficient, playing a significant part in the complexly interwoven pattern of biologic interaction in inland waters; as sapro- phytes in manifold capacities unceasingly active in the essential degra- dation of complex materials; and as parasites ever preying inconspicuously on the major groups of plants and animals important in hydrobiology, and occasionally so severely destructive as to reduce productivity.” Numerous species of Phycomycetes, which parasitize the eggs, em- bryos, and adult forms of various aquatic animals, are described by SPARROW (1943). Fresh-water algae, diatoms, and other aquatic plants are likewise infected. Some of the Phycomycetes are responsible for infections of epidemic proportions. For example, Rhizophydium agile has been reported to destroy up to 75 per cent of the Chroococcus turgidus cells in the field and in gross cultures. Attention is directed to the extensive bibliography on aquatic Phycomycetes compiled by SPARROW (1943). Chapter X TRANSFORMATION OF ORGANIC MATTER The chief function of bacteria in the carbon cycle is the decomposition of organic matter to CO:, water, ammonia, and certain minerals. The efficiency with which they perform this function is indicated by the low content of organic matter in sea water, which averages less than 5 mgm. per liter. It is because bacteria are able to mineralize virtually all kinds of organic matter that the sea has aptly been characterized as the world’s largest and most efficient septic tank. A second function of bacteria in the carbon cycle is that of converting waste and dissolved organic matter into bacterial cell substance which can be assimilated by filter-feeding and mud-eating animals (see page 173). The bacteria may convert as much as 30 to 4o per cent of the organic matter into bacterial cell substance while oxidizing the rest to CO, and water as a source of energy. Third, certain bacteria living symbiotically in the intestinal tracts of animals may aid the latter in the digestion of food. There is ample evi- dence that such bacteria are widely distributed, but there are no data for apraising quantitatively their importance in the carbon cycle. A fourth function is the primary production of organic matter by chemosynthetic and photosynthetic autotrophs. Below the photo- synthetic zone, chemosynthetic bacteria are the only primary producers which can synthesize organic matter from CO, and water. However, in spite of the academic interest centering around chemosynthetic and photosynthetic bacteria, they appear, when compared with green plants, to play a very minor role as primary producers in the sea. Quantity of organic matter decomposed :— Different workers, using various methods of approach, estimate that the primary production of organic matter in the sea ranges from 5 to 1000 grams of organic matter per square meter per year (see literature summarized by RILEY, 1941). Taking 10 gm./M7?/year as a conservative production figure, there is pro- duced in the oceans of the world a minimum of 3.7 X10” kilograms of organic carbon per year. Only a small fraction of this organic matter is TABLE XXXV.— Summer standing crop in Wisconsin lakes given as kilograms per hectare of lake surface on a wet-weight, ash-free basis (from JUDAY, 1942):— LAKE GREEN NEBISH WEBER MENDOTA LAKE LAKE LAKE Phytoplankton 1,875 2,767 608 1,069 Bottom flora 4,600 4,218 590 553 Total weight of plants 6,475 6,985 1,198 1,622 Zooplankton 120 177 42 74 Bottom fauna 414 138 122 147 Fish 35 23 Total weight of animals 534" Shih 199 244 Dissolved organic matter 15,201 27,901 3,820 2,866 * Excluding fish, the standing crop of which is not given. Chapter X — 137 — Organic Matter removed from the oceans per year in the form of fish, whales, commercial algae, and other products, and a similarly negligible amount is buried in bottom deposits. This means that more than 99.9 per cent of the total organic production in the sea must be decomposed. Plants oxidize some organic matter which they themselves have syn- thesized, but most of the plant tissue is ingested by animals or decom- posed by bacteria and allied microorganisms (see Figure 10). Grazing animals ingest a proportion of the diatoms, dinoflagellates, and other primary producers, converting some of the organic carbon into animal ,a\CARBON DIOXIDE Fad ait ye @ ‘ ia Pes +a PANTS === = pala Alen! enn yais: allel ts : \ \ BACTERIA \ ©) x \ \ DISSOLVED COLLOIDAL & PARTICULATE ORG. MATTER Fic. ro. — Carbon cycle in the sea. Solid lines represent processes in which bacteria exclusively participate, dot-dashed lines represent processes in which bacteria may participate, and dashed lines represent processes in which bacteria do not participate. Respiration (1), nutrition (2), decomposition (3), and COz fixa- tion (4). tissue, excreting varying quantities, and oxidizing most of it as a source of energy. Grazing animals in turn may be ingested by predatory animals. In small inland lakes where such problems are more susceptible to quantitative solution, JupAy (1942) estimated that the total weight of plants produced is about six to twenty times as great as the total weight of animals (see Table XX XV on opposite page). Insufficient data are avail- able to compare these estimates for lakes with conditions in the ocean. Certainly the production of bottom flora is far less in the ocean than in lakes because most of the ocean bottom lies beneath the levels that are ZoBell — 138 — Marine Microbiology penetrated by light. In oceans as well as in lakes there is much more “dissolved” organic matter than particulate (including plants and ani- mals). The so-called ‘‘dissolved”’ organic matter represents that which is not removed with a high speed centrifuge. It includes minute particles of de- composing organic material, some bacteria, colloids, and organic matter in true solution. Although some of the colloidal and minutely particulate organic matter can be assimilated by certain filter-feeding and mud- eating animals, most of it and all of that in true solution can be utilized only by bacteria and allied microorganisms. JUDAY (1942) points out that ‘“‘the dissolved organic matter is in a constant state of flux; it is con- tinually receiving both decomposing and excretory materials from the biota on the one hand and losing organic substances that change over to inorganic compounds on the other.”’ After noting marked increases in the bacterial population and oxygen consumption in filtered sea water incubated in the laboratory, WAKSMAN and CarEy (19352) concluded that sea water contains sufficient organic matter in true solution to support an extensive bacterial population under favorable conditions. In the decomposition of the organic matter in sea water by bacteria, a definite parallelism was obtained between bacterial multiplication, oxygen consumption, and liberation of nitrogen as ammonia. When sea water was placed in glass containers and kept under favor- able conditions, WAKSMAN and CArEy (19350) found that from 25 to 50 per cent of the organic matter was decomposed within ro or 12 days, as measured by the amount of oxygen consumed and ammonia liberated. About 60 per cent of the organic matter decomposed was completely oxidized, as shown by the amount of oxygen consumed, and about 40 per cent of it was converted into bacterial cell substance and other products of bacterial metabolism. The rapidity of the process was found to be influenced by the temperature, oxygen tension, abundance of or- ganic matter, and the chemical nature of the organic substrate itself. After five or six months storage in the dark the organic content of filtered sea water was found by ZoBELL and GRANT (1943) to be reduced to around 0.2 mgm./L. from an original 3 to4mgm./L. Glucose, glycerol, ethanol, lactate, succinate, starch, and asparagine in concentrations rang- ing from 0.25 to 5 mgm./L. were quantitatively utilized by bacteria in sea water in from 16 to 30 days at 22° C. From 60 to 70 per cent of the or- ganic substrate is oxidized and 30 to 4o per cent is converted into bac- terial protoplasm and intermediate products. Cultures attack concen- trations of glucose as low as 0.1 mgm./L. While the chief function of bacteria in the transformation of organic matter appears to be the utilization of “‘dissolved”’ organic matter, there is an extensive bacterial flora in the sea which is uniquely adapted to de- compose chitin, lignin, waxes, cellulose, and other complex organic sub- stances which are assimilated poorly, if at all, by other types of organisms. Decomposition of carbohydrates :— Simple sugars ranging from trioses to hexoses are readily assimilated by many species of marine bacteria. In a balanced mineral solution containing an available source of nitrogen, simple sugars are quantitatively utilized. Part of the carbon is oxidized to CO, as a source of energy and part is converted into bacterial proto- plasm. The quantitative oxidation of glucose by bacteria in sea water Chapter X — 139 — Organic Matter requires the addition of available nitrogen. This observation of WAKSMAN and CarEy (19350) suggests that the bacterial population of the sea and the decomposition of carbohydrates may be limited by the amount of available nitrogen present. Although widely and abundantly distributed in the sea, splitters of disaccharides such as sucrose, lactose, and maltose are fewer in numbers of species than are the bacteria which decompose simple sugars. About a third of the bacterial species isolated from marine materials hydrolyze starch. Achromobacter thalassius, Actinomyces halotrichis, Act. marino- limosus, Flavobacterium halohydrium, Fl. neptunium, Pseudomonas pleo- morpha, Vibrio marinopraesens, and V. ponticus are examples of amy- lolytic organisms described by ZOBELL and UpuHam (1944). Most of their Bacillus species from the sea, none of their Micrococcus species, and very few of their Pseudomonas or Vibrio species attacked starch. Fox (1934) demonstrated the ability of 69 different species of marine bacteria, 3 molds, and 2 yeasts to hydrolyze amygdalin with the gradual production of HCN. Several species of marine fungi studied by BARGHOORN and LINDER (1944) utilized maltose, galactose, xylose, starch, cellulose, and pectin. Growth of the fungi was most rapid in sea water agar enriched with either cellulose, pectin, or starch. Mannite was attacked by many species of marine bacteria observed by ENEVOLDSEN (1927). While on the steamer Dana, he noted an increased acidity in sea water samples treated with mannite, a carbohydrate-like hexatomic alcohol which occurs in certain marine algae. [ENEVOLDSEN believed that mannite could serve as a carbon source for marine bacteria in their natural habitats. Only a specialized few marine microorganisms are endowed with the ability to utilize cellulose, although such organisms may be demonstrated in most 1o- to 1oo-ml. samples of sea water and in nearly all one-gram samples of bottom deposits. Using the minimum dilution method, ZOBELL (1938a) demonstrated 1000 cellulose digesters per gram of mud, as compared with 100,000 glucose fermenters and 10,000 starch hy- drolyzers. Cellulose-decomposing bacteria were found to be generally present in sea water, and particularly abundant in bottom deposits and diatom tows, by WAKSMAN ef al. (1933a) in the Gulf of Maine and George’s Bank. Species of Cytophaga, Cellvibrio, and Cellfalcicula were identified. In crude cultures, the cellulose-decomposing bacteria were invariably accom- panied by numerous protozoans, including flagellates, ciliates, and amoebae, all of which feed upon bacteria. Most of the cellulose decom- posers were aerobic, but anaerobic forms were also demonstrated. Some of the latter produced gas. A variety of sugars were utilized by the cellu- lose decomposers. Some of them decomposed agar and other hemicellu- loses. BAVENDAMM (1932) found aerobic cellulose digesters in all marine mud samples, and anaerobic cellulose digesters in many samples which he examined. Many other workers refer to the occurrence of cellulose- splitting bacteria in the sea, but no noteworthy quantitative studies have been made on marine cellulose digesters. RUBENTSCHIK (1928)) reported that aerobic cellulose digesters are active in salt limans. He isolated Actinomyces melanogenes which decom- posed paper. Later he (1933) found that anaerobic cellulose digesters are ZoBell — 140 — Marine Microbiology also widely distributed in aquatic environments. The decomposition of cellulose by these organisms was unimpaired by a salt concentration as high as 15 per cent NaCl. RUBENTSCHIK (19280) related that from the salt lake Ssaky in Crimea he isolated Bacterium cellulosae album and Bac- terium cellulosae flavum which digest cellulose. Cellulose is decomposed primarily to CO; and water in the presence of oxygen. Under anaerobic conditions appreciable quantities of methane and hydrogen are produced from the decomposition of cellulose. Accord- ing to several theories of petroleum formation, cellulose may also be con- verted into higher hydrocarbons. ‘Theoretically, cellulose could be re- duced by either hydrogen or hydrogen sulfide to hydrocarbons. In view of the possible significance of the reaction, this question merits thorough investigation. From marine materials, STANIER (1941) isolated and described Vibrio fuscus, Pseudomonas iridescens, Cytophaga krzemieniewskae, and Cytophaga diffuens, all being new species which digest cellulose and agar. He also described Vibrio beijerinckit which digests agar but not cellulose. Accord- ing to STANIER (1941), Vibrio granii and Pseudomonas droebachense, two species previously named by LuNnDESTAD (1928), digest both agar and cellulose. About half of the agar digesters studied by STANIER utilized alginic acid, and most of them attacked starch and simpler carbohy- drates. Agar is the principal carbohydrate constituent of many marine algae, particularly certain Rhodophyceae. Although not attacked by most bac- teria, agar is digested by several marine species and a few terrestrial ones. Indicative of the abundance of agar digesters in the sea is the report of BAVENDAMM (1932) that marine muds from the Bahama Islands contain from 50,000 to 200,000 agar digesters per gram. It is estimated that be- tween one and two per cent of the bacteria occurring in the sea are able to digest agar. Agar-digesting bacteria were more commonly found in the sea than were cellulose decomposers by WAKSMAN ef al. (1933a@), especially in diatom tows and around larger marine algae or their residues. In 1.0 ml. of diatom tow, from 2,100 to 2,500 cells of bacteria capable of liquefying agar were found. Agar digesters made up 5.7 to 6.7 per cent of the total number of colonies developing on plates inoculated with the diatom tow. Starch, cellulose, inulin, galactan, and mannite were decomposed by most of the agar digesters. GRAN (1902) was the first to isolate a pure culture of agar-digesting bacteria from the sea. His Bacillus gelaticus is now known as Pseudo- monas gelatica. In a careful search for agar digesters in sea water off the Norwegian coast, LUNDESTAD (1928) found and described Achromobacter (Vibrio) granii, Flavobacterium rhodomelae, Fl. polysiphoniae, Fl. (Pseu- domonas) droebachense, Fl. delesseriae, F1. boreale, and Fl. ceramicola. Most of the cultures studied by LUNDEsTAD were able to grow at o° C., although their optimum temperatures ranged from 20° to 30° C. Some were killed by prolonged exposure at 31° C. VAN DER LEK (1929) demonstrated the occurrence in the sea of Vibrio agarliquefaciens, an organism which is capable of attacking cellulose and agar. ANGST (1929) found the following new species of agar digesters associ- ated with marine algae: A garbacterium aurantiacum, A. bufo, A. cyanoides, A. mesentericus, A. reducans, A. viscosum, and seven others which he described but failed to name. Chapter X — 141 — Organic Matter The decomposition of alginic acid, a polyuronide occurring abundantly in marine algae, was found by WAKSMAN et al. (1934) to be caused largely by certain specific bacteria. Other microorganisms such as fungi were responsible only to a very limited extent. Bacteria which decompose alginic acid occur abundantly in sea water, marine plankton, and in bot- tom deposits. Three new marine species which decompose alginic acid were described; namely, Bacterium alginicum, Bact. alginovorum, and Bact. fucicola. The last two species also digest agar. Pseudomonas hypothermis, Ps. marinopersica, Ps. perfectomarinus, Ps. periphytica, and Flavobacterium uliginosum are agar digesters described by ZoBELL and Upuam (1944). These organisms tend to lose their ability to digest agar after prolonged laboratory cultivation, even on agar slants. According to KINKEL (1936), cellulose-decomposing bacteria occur abundantly in lake mud. She also found in mud from Lake Mendota sev- eral species which fermented pectin. Cellulose-decomposing microorganisms, as well as those which attack lignin, are instrumental in the destruction of timbers, wooden pilings, ropes, fish nets, and other cellulose- or lignin-containing structures. Such microorganisms may work symbiotically with shipworms or other wood- borers. BARGHOORN (1942) reports the occurrence on the North Atlantic coast of marine fungi which cause the deterioration of both hard and soft woods as well as cordage fibres under marine conditions. Lignin decomposition :— Lignin is a complex, carbohydrate-like sub- stance which constitutes part of the woody structure of plants. Ac- cording to STEINER and MELOCHE (1935), from 10 to 20 per cent of the organic matter in phytoplankton and from 30 to 48 per cent of the organic residue in lake bottom deposits is lignin. WAKSMAN (1933) likewise found a much higher percentage of lignin in marine humus than in marine vegeta- tion. The difference between the lignin content of phytoplankton and bottom deposits is indicative of the relative insusceptibility of lignin to microbial decomposition. Lignin is slowly oxidized by certain bacteria. BENSON and PARTANSKY (1934) reported that the ligneous materials in sulfite waste liquors dis- charged by pulp mills are slowly decomposed by bacteria in sea water and marine mud. Sulfite waste liquor is primarily a calcium lignosulfate, or the residue of wood after the hemicelluloses are hydrolyzed by acid treat- ment and the cellulose is removed as pulp. The lignosulfate consists chiefly of lignin. When inoculated with marine mud, lignosulfate was found to be fermented with the production of CO2:, methane, and HbS. ZOBELL and STADLER (1940a) present evidence which indicates that, while lignin is oxidized less readily than are other major organic con- stituents occurring in lakes, it is slowly decomposed by bacteria found in water and bottom deposits. Several different purified lignins as well as various ligneous products were examined, using oxygen consumption and the disappearance of lignin as criteria of utilization. Most of the strains of Micromonospora found in lake mud by ERIKSON (1941) attacked lignin. Marine fungi which attack lignin and cellulose are believed by Barc- HOORN and LINDER (1944) to be dominantly responsible for the deteriora- tion of hemp, jute, and sisal cordage as well as pilings and other wooden structures in the sea. ZOBELL (1940) found that, provided some oxygen was present, puri- fied lignin and lignoprotein were slowly oxidized by marine bacteria. The ZoBell — 142 — Marine Microbiology rate of utilization was relatively independent of the oxygen tension. After the supply of dissolved oxygen was depleted or was reduced to such a low level that oxygen could not be replaced by diffusion as rapidly as it was consumed by respiring bacteria, the oxidation of lignin was retarded. The rate of oxidation of organic matter in sea water, as indicated by oxygen consumption, was found to be independent of the oxygen tension within the examined range of 0.43 to 17.8 mgm./L. Proteinaceous compounds :— As a class, marine bacteria are actively proteolytic. They rapidly decompose most simple proteinaceous com- pounds, and even the most complex compounds are slowly attacked with the liberation of ammonia and CO2. Every one of the 60 pure cultures studied by ZoBELL and UpHam (1944) liberated ammonia from peptone, 47 of them liquefied gelatin, and half of them hydrolyzed casein. Mixed cultures from mud or sea water break down peptone, gelatin, and casein with the production of CO: as well as ammonia. According to TRASK (1934), amino acids and simple proteins consti- tute a very minor part of the organic content of marine sediments. This is ascribable to the tendency of bacteria to decompose such compounds, which constitute an appreciable part of the protoplasm of plants and an- imals. Complex nitrogenous compounds are more abundant in sediments than are simple proteins, but neither simple nor complex proteins are proportionately as abundant in sediments as in the organic matter of plant and animal tissues. WaAKSMAN (1933) believes that lignoproteins and hemicellulose-protein complexes account for about 75 per cent of the organic nitrogen content of sediments. HEcuT (1934) reports that most simple proteins are completely de- composed in marine sediments even under anaerobic conditions. Decom- position of the bodies of marine invertebrates, birds, and mammals was observed to be most rapid in sea water containing dissolved oxygen. The presence of H2S and reducing conditions inhibited the decomposition of proteins. About 90 per cent of the combined nitrogen content of deeply buried sediments was present in chitin. The author is aware of no systematic investigations on marine proteo- lytic bacteria, although many investigators refer to the ability of marine bacteria to liquefy gelatin, utilize peptone, and to decompose fish muscle or the nitrogenous tissues of other marine animals. For example, ScHMIDT-NIELSEN (1901) was impressed by the rapidity with which bac- teria from Oslo Fjord decomposed proteins with the liberation of am- monia. He remarked that, although pouring sea water over boiled shrimps renders them temporarily more attractive in appearance, the shrimps are soon decomposed subsequently by bacteria from the sea water. Proteolytic bacteria are primarily responsible for the spoilage of fish, shellfish, crab meat, and other marine food products. Although not based upon quantitative studies, the opinion is rampant that marine fish are more susceptible to spoilage than fresh-water fish. Possibly marine bac- teria may be more actively proteolytic than corresponding fresh-water flora; there may be differences in the composition of fish from different environments; marine bacteria may be active at temperatures somewhat lower than fresh-water bacteria as a group, or there may be other explana- tions to account for this opinion. Fish and other marine food products soon show signs of spoilage if Chapter X — 143 — Organic Matter not properly refrigerated. The consistency and color of muscle tissue are altered by bacterial activity, and an odor of ammonia, indol, trimethyla- mine, or other protein-decomposition products is manifest. Quantitative tests for ammonia, indol, trimethylamine, histamine, tyrosine, and other protein-decomposition products have been proposed by various workers as a means of detecting the early stages of fish spoilage. A common property of the bacteria associated with spoiled fish is their ability to decompose proteins, peptones, peptides, and amino acids (BRADLEY and BAILEy, 1940; HUNTER, 1922; GEIGER ef al., 1944; GRIFFITHS, 1937; HARRISON and KENNEDY, 1922; SNOW and BeEarD, 1939; WooD, 1940). Ostrorr and HENRY (1939) investigated the ability of 15 represen- tative aerobic bacteria of marine origin to utilize 21 different nitrogen compounds. Asparagine, aspartic acid, glutamic acid, alanine, propiona- mide, acetamide, sodium hippurate, urea, and creatinine were commonly utilized either as a source of nitrogen or energy or both. Cystine, betaine, pyridine, and uric acid were utilized by some of the bacteria. Only one culture utilized tyrosine. Guanidine, aniline, and ethylamine were not utilized by any of the organisms. WaksMAN and RENN (1936) found that from 2 to 4 mgm./L. of glycine, alanine, phenylalanine, glutamic acid, tyrosine, and asparagine were almost quantitatively utilized by raw cultures of bacteria in sea water within 2 to 5 days at 20° C. From these results and others in which they noted the rate of carbohydrate decomposition to be dependent upon an available nitrogen source, they concluded that zooplankton in sea water were decomposed more rapidly than marine algae because of the greater proportion of available nitrogen in the former. WAxkSMAN eé¢ al. (1933@) noted that marine zooplankton were more susceptible than were marine algae to bacterial decomposition. The green alga, Ulva lactuca, which contains about 2 per cent nitrogen on a dry basis, was decomposed more rapidly than the brown alga, Fucus vesiculosis, which contains only half as much nitrogen. The bacterial decomposition of zooplankton pro- ceeded to completion with the liberation of ammonia and COs, whereas the complete decomposition of Fucus material required the addition of available nitrogen. Marine diatom plankton, which is relatively rich in proteinaceous ma- terial, was observed by WaKsMAN ef al. (1937) to undergo rapid oxidation and decomposition by bacteria in sea water. This was measured by oxy- gen consumption, nitrogen liberation, phosphate regeneration, and bac- terial multiplication. Only dead diatoms were attacked by the bacteria. Chitin decomposition:— Chitin is the chief constituent of the exo- skeleton of Crustacea and it occurs in some Mollusca, Coelenterata, and Protozoa. JOHNSTONE (1908) estimated that one sub-class of planktonic Crustacea, the Copepoda, produces several million tons of chitin annually, Most of this, as well as the chitin produced by other organisms in the sea, must be decomposed since relatively little accumulates in the marine sediments, and moreover, if it were not decomposed, it would become a serious drain upon carbon and nitrogen in the cycles of these elements. Chitin is generally believed to be a polymer of glucosamine in which each amino group is acetylated, the composition being C32Hs4Oo.N.. There are few if any animals which can assimilate chitin unaided by microorganisms. Although chitinase has been detected in the alimentary tracts of certain chitin-ingesting animals, it may have been produced by ZoBell — 144 — Marine Microbiology chitinoclastic bacteria which commonly occur in great numbers in the ali- mentary tracts of such animals. One of the best sources of chitinoclastic bacteria is the stomach contents of squid and other cephalopods which ingest chitinous food. Hock (1940) recovered chitinoclastic bacteria from the intestinal contents of seven different genera of marine animals. BENECKE (1905) isolated Bacillus chitinovorus from the polluted water of Kiel harbor. It is doubtful if this organism, which digests chitin, is a true marine species since it has been found in soil and it grows well in fresh-water media. WaksmaN et al. (1933¢), BERTEL (1935), and ZOBELL and ANDERSON (1936) have found chitinoclastic bacteria to be widely distributed in marine bottom deposits. From the lesions of live lobsters having a shell disease, HESS (1937) isolated a number of strains of chitinoclastic bac- teria. JOHNSON (1932) found chitinoclastic bacteria growing on crabs packed in ice. From the shells of crabs in an advanced stage of decom- position, she isolated several kinds of chitin-destroying bacteria. After finding chitinoclastic bacteria in 8 out of 27 samples of solar salt obtained from Africa, Spain, South America, California, and the West Indies, STUART (1936) concluded that such bacteria are probably widely distributed in the sea. Most of the bacteria were Gram-negative aerobes, morphologically resembling Serratia and Sarcina. STUART expressed the belief that halophilic chitinovors or chitinoclasts may be responsible for damage to skins and hides. Between o.1 and 1.0 per cent of the marine mud-dwelling bacteria studied by ZOBELL and RITTENBERG (1938) were able to attack chitin. Some of the bacteria could obtain both their energy and nitrogen require- ments from purified chitin, whereas others needed supplementary carbon or nitrogen compounds. Chitinoclastic bacteria were found in nearly all 5-gram samples of bottom deposits. They were most numerous at the mud surface, decreasing in abundance with core depth. Most of the 31 pure cultures of chitinoclastic bacteria isolated from marine materials by ZOBELL and RITTENBERG were small, Gram-negative rods. Many produced yellow, brown, orange, or pink pigments. One violet pigment producer resembling Chromobacter violaceum was observed. It, like some of the other chitinoclastic bacteria, had marked attachment propensities, growing almost exclusively tenaciously attached to strips of chitin. None of the chitinoclastic bacteria were able to digest cellulose, and as a class they were feebly saccharolytic. They were active at tem- peratures as low aso° C. Anaerobic as well as aerobic chitinoclastic bac- teria occur in the sea. CO, and ammonia are end products resulting from the bacterial decom- position of chitin. Acetic acid and reducing sugars have been detected in cultures as intermediate products. Hock (1940) isolated chitinoclastic bacteria from marine sands, mud, water, decomposing crabs, and the intestinal contents of animals which feed on crustaceans. Shells of Limulus, the horseshoe crab, were decom- posed relatively rapidly when buried in beach sand. Hock (1941) described two new species of marine chitinoclastic bacteria, Bacterium chitinophilum and Bacterium chitinochroma. Chitinoclastic bacteria were demonstrated in the mud and water of alpine lakes by STEINER (1931). Chitin was attacked both aerobically and anaerobically by raw cultures. From Lake Mendota mud, KINKEL (1936) isolated 13 types of chitinoclastic bacteria representing several different Chapter X — 145 — Organic Matter genera. Most of the strains of Micromonospora isolated from lake mud by ERIKSON (1941) readily decomposed chitin. Chitin is attacked more slowly than most other common types of organic matter. It is decomposed less readily anaerobically than aero- bically. In spite of the widespread distribution of chitinoclastic bacteria in the sea, chitin persists as one of the principal nitrogenous constituents of marine sediments (HECHT, 1934; WAKSMAN, 1933). Lipolytic bacteria:— Fats and oils of varying degrees of complexity are synthesized as part of the cell substance by most bacteria, yeasts, and molds. However, the amount synthesized is insignificant as compared with the amount which they decompose. The following mean values given by TRASK (1939) show the lipid content of the organic matter of sediments and that of the principal groups of organisms living in the water: ETHER CRUDE CARBOHY- EXTRACT PROTEIN DRATES per cent per cent per cent Diatoms 8 29 63 Copepods 8 65 22 Higher invertebrates Io 70 20 Marine sediments I 40 47 The ether extract includes oils, fats, certain pigments, sterols, and waxes. Further examination has revealed that not only is there a great decrease in the ether-extract content of organic matter deposited on the sea floor, but that fats and oils decrease proportionately faster than waxes. It is difficult to assess the relative importance of animals and bacteria in the decomposition of fats and oils, but it is known that lipolytic bac- teria are widely distributed in the sea. Nearly all types of fats and oils seem to be attacked by bacteria in sea water and marine mud. Certain pure cultures, notably anaerobes, hydrolyze tripalmitin, tristearin, and other pure fats with the liberation of fatty acids. Presumably the glycerol which results from the hydrolysis of triglycerides is oxidized as a source of energy. Some lipolytic bacteria utilize fatty acids under certain conditions. Whether the fatty acids are utilized or left apparently depends partly upon the organisms involved and partly upon the environmental or nu- tritional conditions. The hydrolysis of fats and the transformation of fatty acids are believed to be of considerable importance in the origin of petroleum. From the formulae of fatty acids, it is evident that the de- oxygenation or decarboxylation of fatty acids may result in the formation of petroleum hydrocarbons: CH;(CH:2),CH,COOH = CH3;(CH2),CHs3 + CO, Relationships of microorganisms to the generation of petroleum with particular reference to fats and oils are reviewed by Hammar (1934). In his doctorate dissertation work at the S.I.0., which is concerned with the transformation of lipids by marine anaerobes, W. D. ROSENFELD has found lipoclastic anaerobes to be widely and abundantly distributed in recent marine sediments, oil-well brines, tar sands, asphalt deposits, and in paraffin earth samples. Fatty acids are not readily oxidized under anaerobic conditions in the absence of glycerol, an observation which sug- gests that there is a concomitant oxidation of glycerol and reduction of fatty acid. Binary combinations of fatty acids appear to be assimilated ZoBell } — 146 — Marine Microbiology more readily by lipoclastic anaerobes than is either fatty acid alone. In long-term experiments with mixed cultures of lipoclastic anaerobes growing on lipid-rich algae from Mission Bay, a significant increase in the content of ether-soluble, unsaponifiable material was observed, thereby indicat- ing that lipids may be converted into hydrocarbons or hydrocarbon-like substances. The unsaponifiable material is a waxy substance which gives no colorimetric indications for the presence of sterols. Working on the hypothesis that petroleum is formed from the fats and oils of diatoms, THAYER (1931) found that, while marine bacteria may at- tack various kinds of fats, the only hydrocarbon resulting from the action of mixed cultures of marine anaerobes on fatty acids is methane. Acetic, propionic, butyric, valeric, caproic, heptylic, lauric, palmitic, margaric, and stearic acids were found to be decomposed quantitatively to CO. and methane by anaerobic organisms occurring in fresh-water and marine muds. The formation of methane from fatty acids has been reported by CooLHAAS (1928), Tarvin and BUSWELL (1934), and others. Most marine aerobes are able to assimilate some of the fatty acids, and all simple fatty acids are utilized by marine bacteria of one species or another. GINSBURG-KARAGITSCHEVA and RoDIONOWA (1935) noted an abun- dance of both aerobic and anaerobic lipolytic bacteria in mud from the Black Sea. They found one organism which reduced the iodine number of fats and produced unsaponifiable substances. STURM and ORLOVA (1937) isolated aerobic bacteria from Ala-Kule Lake in Russia which at- tacked fats and palmitic acid with the production of CO, and other inter- mediate products. ZoBELL and UpHAm (1944) described 13 species of marine lipolytic bacteria. Pseudomonas enalia, Ps. felthami, Sarcina pelagia, Vibrio algo- sus, Serratia marinorubra, and Bacillus submarinus were especially active in attacking triglycerides. The ability of the sulfate reducer to oxidize fats and olive oil was demonstrated by SELIBER (1928). More recent observations (BAARS, 1930) suggest that various strains of sulfate reducers can be differentiated upon a basis of their ability to utilize various fatty acids. The marine strain commonly known as Desulfovibrio aestuarii utilizes most of the fatty acids ranging from acetic to stearic. Reports from microbiologists working under the auspices of the American Petroleum Institute at the Scripps Institution of Oceanography indicate that certain strains of D. aestuarii produce ceresin wax and other hydrocarbon-like substances from fatty acids (JANKoWSKI and ZoBELL, 1944). Hecut (1934) buried the bodies of invertebrates, birds, and mammals in perforated celluloid boxes in different sedimentary environments and examined specimens for changes over a period of three years. Fats were found to be far more resistant to attack than proteins, and were still more slowly decomposed in a reducing environment. : Bacterial oxidation of hydrocarbons :— Petroleum consists primarily of hydrocarbons which are believed to have been formed in the sea, probably from the reduction of organic matter in anaerobic bottom de- posits. There are many ways in which bacteria may be instrumental in the formation and accumulation of petroleum hydrocarbons. Bacteria also destroy hydrocarbons under certain conditions. Besides their rela- tion to the petroleum problem, hydrocarbon-oxidizing bacteria play an important role in the carbon cycle. Chapter X — 147 — Organic Matter Many if not all plants including bacteria synthesize waxes and allied hydrocarbons to some extent. Baas BEcKING et al. (1927) found that 9.7 per cent of the organic matter of diatoms, mostly Awlacodiscus kittoni, was extractable by ether, and that 65.7 per cent of the ether extract was un- saponifiable. ‘The unsaponifiable material consisted of hydrocarbons, with lesser amounts of phytosterol and related alcohols. TRASK (1939) reports 8 per cent as the average ether-extractive content of the organic matter in marine diatoms. CLARKE and Mazur (1941) found that from 3 to 14 per cent of the ether extractives of marine diatoms consisted of hydrocarbons, part of which was identified as hentriacontane, C3,Hg,._ After six months incuba- tion in the presence of mud-dwelling microorganisms there was a marked decrease in the organic acid content of diatoms and an increase in hydro- carbons. Hentriacontane occurs commonly in plant tissues, often in con- siderable abundance as in the candelilla plant, Euphorbia cerifera, for ex- ample. Literature on the occurrence of hydrocarbons in the tissues of terrestrial plants has been reviewed by SEYER (1933), CHIBNALL ef al. (1934), and SANDERS (1937). Hydrocarbons also occur in animal tissues. Squalene, C3:.Hso, found in large amounts in the livers of sharks, is a no- table example. In certain environments unfavorable for the activity of hydrocarbon- oxidizing bacteria, hydrocarbons may accumulate in bottom deposits, but under other environmental conditions bacteria may oxidize hydrocar- bons. According to ZOBELL et al. (1943), species of Actinomyces, Micro- monospora, Mycobacterium, Pseudomonas, and other genera, which attack aliphatic, aromatic, naphthenic, and olefinic hydrocarbons in the presence of free oxygen, are widely distributed in sea water and marine mud. In general, long-chain hydrocarbons are attacked more readily than those of lower molecular weight, and aliphatic compounds are more susceptible to bacterial oxidation than are cyclic or aromatic compounds. Open-chain hydrocarbons having unsaturated bonds are attacked more readily than corresponding saturated compounds. Side-chains appear to be attacked preferentially. CO2, organic acids, bacterial protoplasm, and methane result from the action of bacteria upon complex hydrocarbons. There is some evidence that higher hydrocarbons are converted into simpler homo- logues besides methane. Anaerobic sulfate reducers found by Tausson and ALIoscHINa (1932) in lakes, rivers, limans, and the sea were able to utilize saturated aliphatic hydrocarbons containing ten or more carbon atoms per molecule. Naph- thenic hydrocarbons were not attacked by sulfate reducers. Upon a basis of thermodynamic considerations, these workers concluded that heavy hydrocarbons may be converted into polymethylene compounds by sul- fate reducers. * Desulfovibrio species of marine origin attack waxes and heavy oils with the formation of lighter hydrocarbons, according to NOVELLI and ZOBELL (1944). Neither aliphatic hydrocarbons simpler than decane nor aro- matic compounds were attacked. Decane was slowly utilized as a sole source of carbon. Tetradecane, cetane, and longer molecules were at- tacked anaerobically, progressively more readily as the chain length of the hydrocarbon increased. Micromonospora species isolated from Lake Mendota mud by Ertkson (1941) rapidly oxidized paraffin wax, paraffin oil, toluene, naphthalene, benzene, phenol, resorcinol, m-cresol, and 6-naphthol. From sediments ZoBell — 148 — Marine Microbiology of the Dead Sea, ELAZARI-VOLCANI (1943) obtained enrichment cultures in mineral media containing only kerosene or petroleum as the carbon source. Rubber, both natural and synthetic, is another type of hydrocarbon which is attacked by microorganisms (ZOBELL and BECKWITH, 1944). Although the decomposition of rubber in the sea is of little consequence in the carbon cycle, it is a problem of economic importance. Marine humus:— There is evidence that more than 99 per cent of the organic matter produced in the sea undergoes complete decomposition or mineralization. The remaining fraction of one per cent is buried to be- come an integral part of old marine sediments. This fraction, which is highly resistant to decomposition, is known as humus. It consists of the residues of plants, animals, and bacteria which have been subjected to the enzymes of animals and bacteria in the marine environment. Some marine humus originates from terrigenous deposition, but in the open ocean pelagic organisms, primarily plankton, contribute most of the organic residues. Nearer shore, Zostera and sessile algae may be impor- tant sources of humus in bottom deposits (JENSEN, 1915). The chemical composition of humus depends primarily upon the or- ganic matter from which it was derived and the transformation of the organic matter during and after its deposition. The relative roles played by autolytic enzymes, animal digestion, and bacterial activity constitute an unsolved problem, but it is generally agreed that bacterial activity is of prime importance. WAKSMAN (1933) states that only a small part of the organic matter built up by marine plants passes through the animal body, and that the major portion is destroyed post mortem through direct bacterial action. The quality and numbers of transformations caused by bacterial activ- ities depends to a great extent upon the kinds of microorganisms present and the environmental conditions. For example, certain constituents of buried humus in bottom deposits which are relatively stable in an anaer- obic environment may be subject to further decomposition by bacteria in the presence of free oxygen. The oxidation-reduction potential and the hydrogen-ion concentration are important factors which influence the state and composition of humus. WAKSMAN (1933) finds that marine humus consists predominantly of a lignoprotein complex and a hemicellulose-protein complex. Uronic acid, ether-soluble extractives, and alcohol-soluble extractives were also found. The chemical composition as well as relative amounts of the differ- ent fractions vary in different mud samples. Although humus is defined as being organic matter which is resistant to further decomposition by bacteria, there is evidence that humus is not absolutely resistant. Gradual decreases in the organic content of sedi- ments with core depth show that over a period of thousands of years of burial the organic matter is undergoing gradual decomposition, a process which appears to be progressively slower with age. WAKSMAN (1933) found that, in the presence of sufficient oxygen, marine humus undergoes a process of slow bacterial decomposition, as shown by the continuous liberation of CO, and ammonia. He points out that humus imparts certain characteristic properties to the marine bot- tom, making it a more favorable medium for the growth of bacteria and animals. In summary, the composition and amounts of humus both affect and are affected by the activities of marine microflora and fauna. Chapter X — 149 — Organic Matter By allowing the oxidation of organic matter to proceed for 15 to 30 days, WAKsMAN and Horcukiss (1937) found that 1.0 mgm. of organic matter in bottoms close to land consumed about o.1 ml. of oxygen under the most favorable conditions. The organic matter in bottom deposits was oxidized to a lesser extent at greater depths than in shallower bottoms nearer to land. Most of these observations were confirmed and extended by ANDERSON (1940). The activities of bacteria in producing and transforming humus in fresh-water lakes are outlined by WAKSMAN (19412). 5. L Chapter XI THE ‘NITROGEN? CY CLE IN THE (SEA Considerable interest has centered around nitrogen compounds be- cause available nitrogen often limits the productivity of the sea. The origin, mode of formation, and fate of ammonia, nitrite, and nitrate is a time-honored problem, many aspects of which remain unsolved. Some workers believe that most of the fixed nitrogen available for plant nutri- tion enters the ocean from the atmosphere or from land drainage, whereas others contend that available nitrogen is derived primarily from the de- composition and transformation of nitrogenous compounds in the sea. Only the microbiological aspects of the problem can be reviewed here. Ammonia production:— Some ammonia is excreted by animals as a disintegration product of nitrogenous materials, but more ammonia is lib- erated from nitrogenous compounds undergoing bacterial decomposition. The ammonia may be utilized directly by phytoplankton (ZoBELL, 1935) or it may be oxidized to nitrite or nitrate. SCHREIBER (1927) found that there was little to choose between ammonia, nitrate, nitrite, and glycine as sources of nitrogen for bacteria-free cultures of Carteria. BRAARUD and F6yn (1931) noted that cultures of Chlamydomonas could use glycine, alanine, and asparagine, although amino acids were used less efficiently than either ammonia or nitrate. While amino acids may be utilized directly by certain plants, they are readily decomposed with the liberation of ammonia by bacteria. There is ample evidence from the investigations of WAKSMAN and | RENN (1936), OSTROFF and HENRY (1939), and others that most simple nitrogenous compounds and many complex ones are attacked by marine bacteria. If the nitrogen present exceeds the requirements of the bac- teria, ammonia is usually liberated. However, if carbohydrates or other oxidizable non-nitrogenous carbon compounds are present in excess, the nitrogen from decomposing nitrogenous material may be converted di- rectly into bacterial cell substance and thus not appear immediately as free ammonia (WAKSMAN and CarEY, 19350; WAKSMAN and RENN, 1936). In such circumstances, the nitrogen will be liberated as ammonia chiefly after the death and decomposition of the bacteria. Under ordinary conditions, ammonia production accompanies the bacterial decomposition of marine organic matter. The increased bac- terial activity which follows the storage of sea water in glass receptacles in the dark results in increased bacterial multiplication and ammonia production (Keys et al., 1935; WAKSMAN and CarEy, 1935a; ZOBELL and ANDERSON, 1936a). Mixed net plankton suspended in sea water was found by von BRAND ef al. (1937) to be decomposed by bacteria in the dark with the evolution of ammonia. The rapid liberation of ammonia from copepods being decomposed by bacteria in sea water was observed by WAKSMAN et al. (1938). The bacterial fermentation of urea is another important source of ammonia in the sea: (NH2)2CO -- HO = 2 NH3 a CO, Chapter XI — 151 — The Nitrogen Cycle Urea-decomposing bacteria were found by BAVENDAMM (1932) to be widely distributed in water and mud around the Bahama Islands. He be- lieved that the activities of such bacteria promote the precipitation of CaCO;: (NH2),CO + 2 H,0 ++ CaSO, = CaCOs -f (NH,4)2SO,4 BERTEL (1935) attributed the high pH values in water immediately over- lying the ooze to ammonia produced by urea-decomposing bacteria. RUBENTSCHIK (1925) found urea-decomposing bacteria in all samples taken from the Odessa limans. Throughout the euphotic zone, which is populated by urea-excreting animals, ZOBELL and FELTHAM (1935) found from 1 to 10 urea-decompos- ing bacteria per ml. of sea water. Surface mud, which is also populated by urea-excreting animals, was found to contain from 10 to 1000 urea- decomposing bacteria per gram. Some of these bacteria obtain their nitrogen requirements from urea without decomposing any beyond their needs and others ferment urea with the liberation of excess ammonia. Bacteria in the latter category were able to liberate enough ammonia in sea water enriched with urea to cause a reaction as alkaline as pH 9.7. Bacterial oxidation of ammonia:— Cooper (19370) points out that the oxidation of ammonia to nitrite is an exothermic reaction which is ac- companied by a decrease in thermodynamic potential or free energy of 59,400 gram calories at 25° C.: NH,t + OH- + 3/2 O.(gas) = H+ + NO, + 2 HO + 50,400 cal. This indicates that the reaction will proceed from left to right in the pres- ence of an appropriate catalyst or activator. The energy of activation may be provided by photic, chemical, or biological agents. A limited amount of photochemical oxidation of ammonia may occur in the topmost few centimeters of sea water but, owing to the rapid ab- sorption of ultraviolet radiations, will be of no importance below a depth of one meter. Purely chemical catalysis of the reaction has not been demonstrated under conditions which exist in the sea. Therefore, it is generally believed that bacteria are primarily responsible for the oxidation of ammonia to nitrite in the sea. Autotrophic organisms responsible for the oxidation of ammonia to nitrite, namely the Nitrosomonas, have been found in the sea by many investigators. However, failure to find them universally distributed in the sea, and failure to find specific marine Nitrosomonas species, leaves a large gap in our knowledge of the nitrogen cycle. Nitrification appears to be a much more common phenomenon in the sea than can be accounted for by the nitrifying bacteria which have been demonstrated. This dis- crepancy may be due to the inadequacy of the experimental methods which have been employed to demonstrate nitrifying bacteria. Most investigators have employed the conventional media employed by soil microbiologists to demonstrate nitrifiers in the soil. Essentially, such media consist of physiologically balanced mineral salts solutions en- riched with an ammonium salt and buffered with CaCO; or MgCO;. Sea water serves as the mineral solution for marine nitrifiers. VERNON (1898) demonstrated the presence of nitrifying bacteria in the Gulf of Naples. Branpt (1902) found them in two out of three samples of mud from the Kiel inlet, but he was unable to demonstrate such bac- ZoBell — 152 — Marine Microbiology teria in samples of sea water. Using similar methods, GRAN (1903) ob- tained only negative results with samples of water and mud from Nor- wegian fjords, except very near shore. Similarly, NATHANSOHN (1906) was unable to demonstrate nitrifiers in the Gulf of Naples beyond the zone which was obviously contaminated by land drainage. GaAzERT (19060) was rarely able to demonstrate nitrifiers in from 10- to 80-ml. samples of sea water collected off the southern coast of Africa or in the Antarctic on the South Polar Expedition of the research vessel Gauss. Negative re- sults were likewise obtained with sea weeds and diatom tows from the Sargasso Sea as well as off Kerguelen Island in the Indian Ocean. IsSATCHENKO (1914) demonstrated the presence of nitrifying bacteria in coastal water of polar Arctic seas and in bottom deposits from off the Murmansk coast. He was unable to find nitrifiers in surface waters of the open ocean. However, he believed that nitrifiers were indigenous to deeper waters and the sea floor. ISSATCHENKO (1926) detected no nitri- fiers where HS was present in the Black Sea. In shallow water such bac- teria were found in bottom deposits associated with sand and shells, but only infrequently in clay bottoms. KNrpowiTscH (1926) reported the presence of nitrifying bacteria in surface water of the Black Sea and the Sea of Azov. Nitrifying bacteria were demonstrated in all mud samples from Kiel harbor examined by THOMSEN (1910). He also found them in mud from the Helgoland channel and the Gulf of Naples. Negative results were ob- tained with samples of sea water collected at considerable distances from land. Likewise THOMSEN found no nitrifiers associated with plankton or sessile algae. The nitrifiers which he isolated from marine mud samples were morphologically and physiologically identical with terrestrial N7z- trosomonas. They grew well in sea water and could be acclimatized to higher or lower salt concentrations. They developed readily at 28° C. PrriE (1912) found no nitrifiers while on the Scottish Antarctic Ex- pedition. LIEBERT (1915) was unable to isolate nitrifying bacteria from water or bottom deposits of the open ocean. Mud from the North Sea also gave negative results except very near shore. Active nitrifiers were found in the Zuider Zee. BERKELEY’S (1919) attempts to demonstrate nitrifiers in sea water from off the coast of Vancouver Island were uni- formly negative. After finding no evidence of nitrification in 25- to 150-ml. samples of sea water collected from around American Samoa and Tortugas, Florida, LipMAN (1922) concluded that nitrifying bacteria were not present in the open sea. However, he obtained good nitrification in samples of bottom deposits from nearly all stations. Similar results were reported by Harvey (1928). From their rather extensive investigations, WAKSMAN et al. (19336) concluded that, beyond the zone of land drainage, surface sea water has either no nitrifying bacteria or only very few. On the other hand, active populations of nitrifying organisms were found in bottom deposits. Similar conclusions were reached by CarEy (1938) who has reviewed the literature on the occurrence of nitrifying bacteria in the sea. She found active nitrifiers in most samples of bottom deposits and in surface water samples collected near land. Water samples collected far from land, at depths ranging from 10 to 200 meters, gave negative results. Sam- ples of mud from great depths produced nitrite very slowly and in exceed- ingly small amounts. Concentrated diatom tows, copepod tows, and Chapter XI — 153 — The Nitrogen Cycle dinoflagellate plankton from Woods Hole and Vineyard Sound gave rise to nitrite when inoculated into ammoniacal sea-water solutions. Copepod tows which Carey collected off the Continental Shelf at a depth of 100 meters gave rise to ammonia and traces of nitrite. She concluded that nitrifiers occur primarily in bottom deposits, and that, by vertical mixing of water, they may be brought into the plankton layer. While there is hydrographic evidence that some ammonia and nitrite is produced on the sea floor, data accumulated in recent years suggest that most ammonification occurs in the surface layers of water. RAKESTRAW’S (1936) studies on the occurrence of nitrite in the sea indicate that nitrifi- cation likewise is primarily a surface phenomenon confined largely to the topmost 200 meters of water. Since nitrifying bacteria are seldom found NITROGEN MGM./L. 0.3 0.2 0.1 ce) 20.525, 30 735" 40,745 DAYS Fic. 11. — Changes in the ammonia, nitrite, and nitrate content of sea water enriched with mixed marine plankton (from VON BRAND et al., 1937). in the open ocean in the topmost 200 meters of water where most of the nitrite appears to be formed, the problem of nitrification in the sea must be regarded as unsolved in spite of the wealth of information on the sub- ject. It is the present author’s belief that, besides the soil-like nitrifier which has been commonly found in bottom deposits and near land, there are other marine nitrifying organisms which have escaped detection. In their experiments on the formation of nitrite and nitrate from ammonia liberated from decomposing plankton (Fig. 11), VON BRAND et al. (1937) were unable to isolate nitrifying bacteria. The nitrifiers found in marine materials by von BRAND et al. (1942) were inhibited by temperatures as low as 5° C. It will be recalled that over 80 per cent of the sea floor is colder than 5° C. Although there are many shallow, well- oxygenated marine bottoms where nitrifiers analogous to soil Nitrosomonas could be active according to modern concepts of their cultural require- ments, in many bottoms where nitrifiers have been demonstrated, the low temperature, low oxygen tension, low oxidation-reduction potential, and relatively high organic content would be expected to retard or inhibit nitrification. Nitrifying bacteria indistinguishable from the specialized nitrite- forming flora of soils are commonly found in fresh-water lakes. Such bacteria were found in mud from Lake Mendota by WiLL1AMs and McCoy (1935). Oxidation of nitrite to nitrate:— According to Cooper (19370), the oxidation of nitrite to nitrate is accompanied by a decrease of free energy, A F°o, of 18,000 gram calories. Like the oxidation of ammonia, there- fore, the reaction requires only activation: NO,- + 1/2 O2(gas) = NOs~ + 18,000 cal. ZoBell — 154 — Marine Microbiology Suitably equipped bacteria may oxidize nitrite to nitrate as a direct source of energy. Ndtrobacter species in the soil are endowed with this ability. Nitrobacter-like organisms have been demonstrated in the sea. Nitrate-forming bacteria are somewhat more difficult to demonstrate than nitrite-forming bacteria (WAKSMAN et al., 19330). Therefore it is not surprising that nitrate formers have been demonstrated in the sea only infrequently and that the status of our knowledge of them is very fragmentary. Most of the investigators, whose observations on nitri- fiers are summarized in the preceding section, looked for nitrate formers as well as nitrite formers, usually with little success. Nitrate-forming organisms have been found near shore and in shallow bottom deposits, but only rarely in the open sea. CAREY (1938) reports that nitrate production was noe than nitrite production in special media inoculated with various marine materials. In most deep-sea mud cores and plankton tows, no nitrate formers were found, although nitrite formers were usually present. Nitrate formation was observed to follow the formation of ammonia and nitrite in the exper- iments on the decomposition of marine plankton of VON BRAND é¢ al. (1937), as illustrated by Figure 11 on page 153. Attempts to isolate the organisms responsible for nitrate formation were unsuccessful. Although, in the light of circumstantial evidence, it is tacitly assumed by many oceanographers that nitrate is formed by bacteria at the bottom of the sea, and thence carried into the photosynthetic zone, there is no conclusive evidence bearing on either the mode or place of formation of nitrate in the sea. The problem is complicated by the dynamic nature of the marine environment. In the photosynthetic zone, nitrate may be utilized by plants as fast as it is produced, thereby precluding its accumu- lation. Elsewhere in the sea, nitrate may be reduced by bacteria. Reduction of nitrate and nitrite :— The reduction of nitrate or nitrite is an endothermic reaction and therefore is thermodynamically possible only when the required energy is forthcoming from an accompanying exothermic reaction. The sea abounds in bacteria which can obtain the necessary energy for the reduction of nitrate or nitrite from the oxidation of organic matter. However, inasmuch as there is relatively little readily available organic matter in the sea, the extent to which nitrate or nitrite may be reduced is highly problematical. Denitrifiers resembling Thio- bacillus denitrificans, which obtains energy for the reduction of nitrate by oxidizing sulfur, have been found in coastal waters by various workers, but evidence of their activity in marine environments is lacking. Most of the pioneer workers including RUSSELL (1893), FISCHER (1894a), VERNON (1808), GAZERT (19060), and GRAF (1909) demonstrated the presence in the sea of bacteria capable of reducing nitrate. Impetus was given to investigations by the hypothesis, advanced by BRANDT (1899), that the activity of denitrifying bacteria, or those which reduce nitrate to free nitrogen, destroy nitrate in tropical seas and hence prevent maximum phytoplankton growth. After finding that only near shore, where the organic and nitrate content of the water may be high, is there evidence of denitrification in the sea, GRAN (1901) concluded that Branvt’s hypothesis is untenable. The extensive literature on this con- troversial subject has been reviewed by WAKSMAN et al. (19330) and BENECKE (1933). Although he continued to defend his hypothesis for a quarter of a cen- Chapter XI — 155 — The Nitrogen Cycle tury, BRANDT (1929) finally retracted part of it by attributing the lack of nitrate in surface tropical waters to thermal stratification. However, he maintained that denitrifying bacteria destroy the surplus nitrogen com- pounds in the ocean, thereby providing the balance in Nature. From their own observations WAKSMAN ef al. (19330) concluded that, although denitrifying bacteria are present in the sea, the activities of such bacteria are so limited under marine conditions as to render them in most instances of little importance in limiting the nitrate supply of the sea. Lack of an available energy source was found to be the chief limiting factor. The problem of bacterial denitrification in the sea was attacked with renewed vigor after DREW (1911) credited marine denitrifiers with the ability to promote the precipitation of CaCO; in tropical seas (page 100). According to BAVENDAMM (1932), DREW’s (1912) Bacterium (Pseudo- monas) calcis is closely related to Bact. bauri, Bact. granii, and Bact. feiteli, denitrifiers which PARLANDT (1911) isolated from the Baltic Sea. It is also related to Bact. balticum and Bact. ornatum, marine denitrifiers studied by FEITEL (1903), and to Bact. russelli and Bact. brandti, marine denitrifiers described by IssatcHENKO and RostowzeEw (1911). Other marine denitrifiers named as new species include Bact. triviale, Bact. repens, and Bact. henseni isolated by GRAN (1901), Bact. actinopelte and Bact. lobatum isolated by BAuR (1902), and Bact. helgolanicum isolated by NADSON (1903). Unfortunately none of these denitrifiers is completely described in the available literature. Motiscu (1925) described Pseudomonas calciprecipitans and KALAN- TARIAN and PETROSSIAN (1932) described Flavobacterium sewanense, both of which were believed to be marine denitrifiers capable of promoting the precipitation of CaCO 3. Lioyp (19310) discussed the mechanism of denitrification and described B. (Vibrio) costatus which she isolated from the sea. About half of the bacteria found in the sea are capable of reducing nitrate to nitrite in sea water enriched with organic matter and nitrate. Thirty-four of the sixty species of marine bacteria studied by ZOBELL and UpuHAM (1944) reduced nitrate to nitrite in enrichment media. Most of their Bacillus species, Achromobacter stenohalis, Pseudomonas hypothermis, Ps. aestumarina, Ps. marinopersica, Serratia pelagia, Vibrio hyphalus, and V. marinagilis are examples of extremely active nitrate reducers. Pseu- domonas azotogena and Ps. perfectomarinus are the only organisms among the sixty species described by ZOBELL and UpHam which reduced nitrate to free nitrogen. Although denitrifiers are widely distributed in the sea, particularly in bottom deposits, it is estimated from the aforementioned pure-culture studies, as well as from ecological surveys, that fewer than five per cent of the bacterial species in the sea are endowed with the ability to liberate free nitrogen from nitrate or nitrite in the presence of an abundance of organic matter. Except in certain highly localized environments, there appears to be insufficient organic matter in the sea to provide for the activity of denitrifiers. GRAN (1901) classified marine bacteria in four categories according to their action on nitrate and nitrite: (z) Both nitrate and nitrite reduced to nitrogen, (2) Nitrate reduced to nitrite and ammonia, (3) Nitrite but not nitrate reduced, and (4) Neither nitrate nor nitrite reduced. Several other types of nitrate- and nitrite-reducing bacteria are now recognized. Throughout the foregoing discussion the term denitrification is applied exclusively to the liberation of free nitrogen. ZoBell — 156 — Marine Microbiology Nitrogen fixation :— BENECKE (1933) relates that KEUTNER was the first to make an extensive search for nitrogen-fixing bacteria in the sea, this being the subject of his doctorate dissertation at Kiel University. Species of the aerobic nitrogen fixer, Azotobacter chroococcum, and also the anaerobic Clostridium pastorianum were found (BENECKE and KEvt- NER, 1903). Azotobacter occurred primarily associated with the surface slime of algae, and Cl. pastorianum occurred chiefly in bottom water and ooze. After finding both types of nitrogen-fixers in the Baltic Sea, the North Sea, off the African coast, and in the Malay Archipelago, KEUTNER (1905) concluded that nitrogen-fixing bacteria are normal inhabitants of the sea. Azotobacter chroococcum was found to tolerate up to 8 per cent NaCl. REINKE (1903), believed Azotobacter to occur as an epiphyte on phytoplankton and larger marine algae, symbiotically obtaining utilizable organic matter therefrom while furnishing fixed nitrogen in return. Sev- eral other investigators have noted the occurrence of Azotobacter on marine as well as fresh-water algae, but a definite symbiotic relationship has never been established. FISCHER (1904) demonstrated that nitrogen fixers may obtain their energy from the hydrolytic products of agar liquefiers. This observation was confirmed by the PrincsHEIM brothers (1910) who inoculated nitro- gen-free agar media with cultures of Pseudomonas gelatica and Clostrid- ium pastorianum (also known as Bacillus amylobacter). Good growth of both organisms occurred. Presumably Ps. gelatica broke the agar down into substances which Cl. pastorianum could utilize. The strains of Azotobacter, which KEDING (1906) found on the surface slime of algae near shore, appeared to be identical with those found in soil. The strains of Azotobacter and Clostridium, which ISSATCHENKO (1914, 1926) found associated with marine algae, required enough salt for their development to lead him to believe that these nitrogen fixers were spe- cifically adapted to sea water. According to KorINEK (1932), there are species in the ocean which closely resemble Azotobacter morphologically, but they do not assimilate free nitrogen. He found that Azotobacter chroococcum grew only poorly in sea-water media. He admits that there may be marine strains of Asoto- bacter which fix nitrogen but he failed to find one. LLoyp (1930) likewise regarded the existence of active marine nitrogen-fixing bacteria as problematical. Azotobacter cells were only occasionally found by BAVENDAMM (1932) in enrichment cultures of calcareous mud from around the Bahama Islands. Cl. pastorianum was generally present. The occurrence in the sea of an abundant population of nitrogen- fixing Azotobacter and Clostridium is reported by WAKSMAN ef al. (19330). In the presence of a favorable source of energy, the bacteria were capable of fixing appreciable quantities of nitrogen. However, the extent to which such a process actually takes place in the sea remains to be deter- mined. vON BRAND et al. (1942) concluded that except for the sporadic presence of nitrogen-fixing bacteria, there is no evidence of nitrogen fixation in the sea. Nitrogen fixation is an endothermic reaction which requires consider- able energy. Nitrogen-fixing bacteria obtain this energy from the oxida- tion of organic matter. Only in the presence of readily available organic matter, therefore, can nitrogen fixers be expected to be functional. With- Chapter XI — 157 — The Nitrogen Cycle out enrichment with organic matter, filtered sea water does not support the growth of nitrogen-fixing bacteria. There are numerous accounts of the occurrence of both aerobic and anaerobic nitrogen-fixing bacteria in fresh-water lakes, but whether they are indigenous species which are functional in lakes or are adventitious forms from the soil is another unsolved problem. It has been definitely established that some species of blue-green algae fix atmospheric nitrogen. Pure cultures of Nostoc punctiforme and Ana- baena variabilis isolated by DREWES (1928) fixed 2 to 3 mgm. of nitrogen in 50 days in 250 ml. of medium. ALLISON et al. (1937) worked with pure cultures of Nostoc muscorum which fixed as much as 18 mgm. of nitrogen per roo ml. of medium in 85 days. In discussing the role of algae in the nitrogen cycle of soil, STOKES (1940) relates that DE demonstrated the ability of Anabaena variabilis, A. gelatinosa, and A. naviculoides to fix nitrogen. The ability of these blue-green algae from the soil to fix nitrogen suggests the possible importance of algae as nitrogen fixers in the sea. Chey XII BACTERIA WHICH TRANSFORM SULFUR COMPOUNDS Sulfur is essential for the growth of plants and is utilized partly as sulfate by most of them. Animals obtain their sulfur requirements largely from plants, other animals, or bacteria. Sulfur is liberated by bacteria, almost exclusively as hydrogen sulfide, from the remains of animals and plants. Specialized bacteria oxidize H,S to elementary sulfur, sulfate, or intermediate compounds of sulfur. Elementary sulfur, thiosulfate, tetrathionate, and other compounds intermediate between sulfur and sulfate may be oxidized by bacteria. Under certain conditions sulfate may be reduced to elementary sulfur or HeS by bacteria. Sulfite, thio- sulfate, and other sulfur compounds likewise may be reduced by bacteria and allied microorganisms. Besides their manifold effects on the sulfur cycle, bacteria which trans- form sulfur or its compounds may influence the pH, E,, color, carbonate content, oxygen tension, and other properties of water or bottom depos- its. Bacteria concerned with the sulfur cycle in the sea may render water or mud uninhabitable by other organisms or, on the other hand, they may be growth promoting. Sedimentary sulfur desposits have been ascribed to the activities of sulfate-reducing and sulfur-depositing bacteria. The importance of bacterial activities in the sulfur cycle in marine sediments is outlined by GALLIHER (1933). Liberation of sulfur from organic compounds :— Most organic sulfur occurs in cystine, cysteine, methionine, or other sulfhydryl amino acids. Lesser quantities are bound in the form of sulfatides, organic sulfates, sulfonates, sulfureted glucosides, etc. The liberation of sulfur from albu- minous material is a process which is more or less incidental to its decom- position. While all bacteria are not able to liberate HS from sulfur- containing organic matter, those which are endowed with this ability are so widespread in the sea that, except for special compounds, this process can be regarded as universal and non-specific. In bottom deposits from the Clyde Sea, Eris (1932) demonstrated from 10,000 to 3,000,000 saprophytes per gram, nine-tenths of which liberated H.S from albuminous material. ZOBELL (1938a) found from 10,000 to 1,000,000 H;S-producing bacteria per gram of bottom sediments from the Pacific Ocean off the coast of California. The bacterial decomposition of animal and plant remains generally results in the liberation of H,S. Abundant aeration seems to induce many bacteria, active in the decomposition of proteins, to transform sulfhydryl sulfur directly to sulfate instead of H2S. Such bacteria probably produce HS, but under favorable conditions the latter is abiogenically oxidized as fast as it is liberated from the decomposing protein. Under anaerobic conditions organic sulfur is converted almost quantitatively into HS by raw cultures of saprophytic bacteria. Traces of mercaptans may be pro- duced (BUNKER, 1936). Chapter XII — 159 — Sulfur Bacteria Little is known concerning the bacterial decomposition of organic sulfates, sulfonates, and related compounds. Under aerobic conditions the sulfur is probably liberated as sulfite or sulfate. Sulfate reduction:— Bacteria which reduce sulfate are abundantly and widely distributed in the sea. ZOBELL (1938a) demonstrated from 1000 to 10,000 sulfate reducers per gram of marine mud. RITTENBERG (x941) found sulfate reducers in each of several hundred samples collected at various stations off the coast of California and in the Gulf of California. They were found at all water depths and core depths from which samples were taken. Sulfate reducers were only rarely present in sea water except near the bottom. Although earlier workers had postulated that the bacterial reduction of sulfate is responsible for the accumulation of sulfide in marine bottoms, ZELINSKI (1893) was the first to claim the isolation of sulfate-reducing bacteria. From Black Sea mud he isolated organisms which he designated Vibrio hydrosulfureus and Bacterium hydrosulfureum ponticum which re- duced sulfate, sulfite, and thiosulfate to H,S. His descriptions of the or- ganisms suggest that he was working with mixed cultures. Actually it is rather difficult to obtain pure cultures of sulfate reducers. BEIJERINCK (1895) carefully described Spirillum desulfuricans, a sul- fate reducer which he isolated from Delft ditch water. He predicted the absence or paucity of sulfate in the deeper layers of soil in Holland due to the activity of the sulfate reducer. A similar organism named Spizrillum aestuarii was isolated from North Sea coast water and mud by vAN DEL- DEN (1904). Sp. aestuarii required sea water or 3 per cent NaCl for its growth, whereas Sp. desulfuricans was not active in 3 per cent NaCl media. After finding that these two organisms, as well as ELION’s Spirillum thermodesulfuricans, could be acclimatized to tolerate different salinities and temperatures, BAARS (1930) regarded all three organisms as strains of a single species which he called Vibrio desulfuricans. However, RITTEN- BERG (1941) was unable to adapt the strain which he isolated from the sea to grow in fresh water or to grow at temperatures exceeding 40° to 45° C. (The optimum temperature for growth of Sp. thermodesulfuricans is 55° C.). Likewise the marine sulfate reducer could not be induced to form spores, unlike the organism studied by STARKEY (1938), and no sporoge- nous marine sulfate reducers have been found. STARKEY (1938) isolated a sporogenous sulfate reducer from soil which was indistinguishable from Vibrio desulfuricans. He induced an asporo- genous strain of the latter to form spores and acclimatized it to grow at from 50° to 55° C., whereupon he proposed Sporovibrio as the generic name of the sulfate reducers. In the absence of adequate confirmation of STARKEY’s observation and since spore formation does not seem to be a primary or constant characteristic of sulfate reducers, the generic name Desulfovibrio is provisionally applied to previously described species of sulfate reducers in the sixth edition of the BERGEy (1946) Manual. Un- questionably there are sporogenous sulfate reducers, but it has not been established that all sulfate reducers form spores. The absence of free oxygen, the presence of sulfate, and the presence of organic matter are the chief requirements for the activity of sulfate-reduc- ing bacteria. Apparently there are strains which utilize nearly any kind of organic matter including proteins, sugars, starches, hydrocarbons, fats, ZoBell — 160 — Marine Microbiology and fatty acids. Besides playing the key role in the sulfur cycle in the sea, sulfate reducers play an important role in the transformation of organic matter. At least certain strains can utilize elementary hydrogen (see page 109). In marine bottoms the hydrogen for this reaction probably results from the anaerobic decomposition of organic matter. ISSATCHENKO (1914) noted the widespread occurrence of Desulfovibrio aestuarii in Arctic Sea bottoms where he believed it was an important biochemical agent. The ability of sulfate reducers to produce (JANKOWSKI and ZoBELL, 1944) and transform hydrocarbons (Tausson and ALIOSCHINA, 1932), the almost universal presence of such bacteria in marine sediments both recent and ancient (BASTIN, 1926), their ability to function over a wide range of environmental conditions, and the decreased sulfate content of oil-well brines strongly suggest that sulfate-reducing bacteria are intimately asso- ciated with petroleum genesis. Such bacteria are credited with being re- sponsible for the formation of sulfur of the gypsum type (page 112). The effect of sulfate reducers on iron corrosion is discussed by STARKEY and WIGHT (1943). BAVENDAMM (1932) noted the presence of large numbers of sulfate reducers in calcareous mud around the Bahama Islands where he believed they were instrumental in the precipitation of CaCO; under certain conditions: CaSO, + 8 H + CO, = CaCO; + 3 H20 + HS In this reaction, organic matter serves as the hydrogen donor. BAVEN- DAMM failed to take into account the acid that may result from the oxida- tion of the organic matter, and that HS itself is weakly acidic. The activities of sulfate-reducing bacteria are largely responsible for the occurrence of H2S in stagnant bodies of water. The Black Sea is a classical example of a large body of water in which few if any forms of life except bacterial are possible at depths exceeding a few hundred meters owing to the high concentration of HyS. Free oxygen is absent at depths exceeding 200 meters (SVERDRU?P et al., 1942). ISSATCHENKO (1924) found vigorous sulfate-reducing bacteria in bottom samples from the Black Sea, as did RAVICH-SHERBO (1930). Similar conditions occur in Norwegian fjords (StrR¢M, 1939), the Caspian Sea (BUTKEVICH, 1938), mud lakes or limans of Odessa (BUNKER, 1936), Lake Ritom in Switzerland (DUGGELI, 1924), and many other bodies of water, both salt and fresh, having poor vertical circulation. COPENHAGEN (1934) described an area approximately 25 by 200 miles in the Atlantic Ocean off Walvis Bay, South Africa, where HpS is periodi- cally liberated from bottom mud in quantities sufficient to be lethal to flora and fauna in the overlying water. Sulfate-reducing bacteria were isolated from the black mud. Incidentally, the mud contained no car- bonate, thereby suggesting that in this area sulfate reduction promotes the dissolution rather than the precipitation of CaCOs;. The black mud was rich in ferrous sulfide. BUNKER (1936) attributed the blackening of mud and sand in certain marine and lacustrine localities entirely to the action of sulfate-reducing bacteria. According to BUNKER, ISSATCHENKO has reported the seasonal production of HS in the Sea of Azov in sufficient intensity to destroy large numbers of fish and other aquatic animals. Whether sulfate reduction is a property of only one specific group of bacteria or whether there are several types of organisms endowed with Chapter XII — 161 — Sulfur Bacteria this ability is an unsolved problem. Certainly most microorganisms are capable of obtaining their sulfur requirements from sulfate by a process which BEIJERINCK (1895) called “non-specific” sulfate reduction. For such organisms, however, sulfate reduction is not an essential part of their oxidative metabolism as it appears to be for the Desulfovibrio. Desulfovibrio aestuarit can utilize not only sulfate, but also sulfite, thiosulfate, and sulfur as hydrogen acceptors. Ordinarily H:S is the chief end product, although there is evidence that sulfur may accumulate under certain conditions. Many marine microorganisms are able to re- duce sulfite and thiosulfate to HS. The oxidation of sulfur compounds:— Sulfur bacteria probably de- pend largely upon the activities of sulfate-reducing bacteria for H,S, thiosulfate, and other sulfur compounds which they oxidize. Several physiological types of bacteria, including both autotrophs and sapro- phytes, which oxidize sulfur or its compounds, occur in the sea. Some of the autotrophs obtain their energy chemosynthetically from the oxidation of H2S, sulfur, thiosulfate, or tetrathionate, and some use photic energy. The photosynthetic autotrophs can oxidize HeS in an anaerobic en- vironment, whereas most of the chemosynthetic autotrophs are strict aerobes or microaerophiles. This greatly restricts the localities in which sulfur bacteria can be functional in the sea. The penetration of sunlight is limited to the topmost hundred or so meters of water, and light pene- trates bottom deposits to a negligible extent. The presence of H2S in aerobic environments is limited largely to localities where it is being con- tinuously produced. Several physiological types of sulfur bacteria occur in shallow water where these conditions persist. Such conditions may also occur in growth zones or lamina at greater depths, where H2S from below diffuses upward into overlying oxygenated waters. Such lamina have been reported in the Black Sea, Caspian Sea, Lake Ritom, and elsewhere. The question of the bacterial activity in this thin layer or lamina in the Black Sea is elaborated by RAVICH-SHERBO (1930). The term “bacterial plate” or “bacterial fountain” has been applied to localized laminae (ELLIS, 1932). The phylogenetic position of the recognized genera of sulfur-oxidizing bacteria is outlined in Table XXXIV on page 124. They can be grouped for the convenience of this discussion into the following categories accord- ing to their pigmentation and sulfur metabolism: I. Achromic sulfur bacteria, which have neither bacteriopurpurin nor bacterioverdin and consist of (a) the Thiobacillus group, species of which oxidize various sulfur compounds, usually to sulfate, although sulfur may be deposited extracellularly and (b) Leucothiobacteria belonging to genera of Achromatiaceae and Beggiatoaceae, which deposit sulfur intracellu- larly. II. Purple sulfur bacteria, or Rhodothiobacteria, which contain bac- teriopurpurin and consist of (a) the Thiorhodaceae or Chromotioidaceae, the cells of which contain sulfur granules and (6) the Athiorhodaceae or Rhodobacterioidaceae, which do not deposit sulfur intracellularly. The green sulfur bacteria, or Chlorobacteria, are generally considered in this category, although they contain bacteriochlorin instead of bacterio- purpurin. The foregoing is not proposed as a new system of classification. It isa hybrid system designed only to expedite the discussion of bacteria which ZoBell Bia gaa Marine Microbiology oxidize sulfur or its compounds in the sea. In order to facilitate an under- standing of the confusing terminology in the literature, an attempt is made to combine the essential features of various classifications proposed by WINOGRADSKY, MOLIScH, ORLA-JENSEN, BUCHANAN, BAVENDAMM, ELLIs, VAN NIEL, and others. Achromic sulfur bacteria :— Bacteria of the Tzobacillus group oxidize sulfide, elementary sulfur, thiosulfate, or tetrathionate. Some of those which oxidize HS or other sulfides often deposit sulfur extracellularly, although sulfate is generally the end product of their oxidative metabo- lism. Several strains of Thiobacillus have been found in water. NATHANSOHN (1902) isolated Thiobacillus thioparus from sea water. It grew in sea water enriched with either potassium sulfide or sodium thiosulfate without a source of organic carbon. A little organic matter did not inhibit its development. It failed to grow in the absence of CO, and carbonate. In the presence of an abundance of sulfide, the latter was oxidized to sulfur and deposited extracellularly. Though able to oxidize elementary sulfur, it prefers sulfide, thiosulfate, or tetrathionate. The form of sulfur oxidized and the end products of oxidation depend upon the concentration of reactants and certain environmental conditions includ- ing the pH and Ey. Thiobacillus thioparus appears to be widely distrib- uted in coastal waters, marine mud, fresh water, and soil (BUNKER, 1936). BEIJERINCK (1904), who found Thiobacillus thioparus in the sea off the Dutch coast, reported that it oxidizes thiosulfate to sulfate and desposits sulfur: 2 S.03—— + O. = 280,77 + 258 Tetrathionate was oxidized less readily to sulfate and free sulfur: Sy(O)a +. Oz = 2 SO.an a 2 S RAVICH-SHERBO (1930) ascribed the absence of H2S in certain zones in the Black Sea to the activities of Thiobacillus thioparus: 2 HeS + 5 O2 = 2SO,-— + 2 HO Large numbers of Thiobacillus thioparus were noted in poorly oxygenated laminae or zones of the Black Sea which received HS produced by sul- fate-reducing bacteria. Thiobacillus denitrificans is often found in mud, water, and soil. Is- SATCHENKO (1914) noted its occurrence in Arctic Seas along the Murman coast of northern Russia. It is an anaerobe which utilizes nitrate as hy- drogen acceptor while oxidizing sulfide or elementary sulfur: 5 HS + 2HNO; = 5S+N2+6H,0 Thiobacillus thiooxidans oxidizes elementary sulfur to sulfate: S +20, = SO,-— Conditions in the sea do not seem to be conducive to the growth of this organism, although SasLAwsky (1927) isolated obligately halophilic strains from Russian mud lakes. According to ELtts (1932), Thiobacillus thiogenes (Molisch) and Thiobacillus bovista (Molisch) are exclusively marine species. The latter probably belongs to another genus, since its cells contain sulfur granules. The Achromatiaceae and Beggiatoaceae have been called Leucothio- Chapter XII — 163 — Sulfur Bacteria bacteria because they contain sulfur granules and no photosynthetic pig- ments. Mass cultures often appear white owing to the formation of sul- fur. According to ELLts (1932), the Leucothiobacteria includes all of the colorless sulfur bacteria. He does not regard the Thiobacillus group as “sulfur bacteria” because, unlike the Achromatiaceae and Beggiatoaceae, they do not store sulfur in their cells. Some workers, on the other hand, employ the term “sulfur bacteria” to embrace all classes of bacteria which either oxidize or reduce sulfur or its inorganic compounds, including sulfate reducers and all colorless forms which oxidize sulfur or its com- pounds, as well as the purple sulfur bacteria, regardless of whether the cells have sulfur granules or not. There are several marine representatives of the Achromatiaceae and Beggiatoaceae. In fact, most known species grow equally well in salt water and fresh water. They, along with purple sulfur bacteria, are abun- dant in shallow water where HS is being produced, particularly in pools among rocks where seaweed is undergoing decomposition. Achromatium oxaliferum occurs in sea water as well as in fresh water, according to BAVENDAMM (1924) and ELLIs (1932). It oxidizes H2S to sulfur which may be desposited intracellularly along with calcium oxalate granules: 2HS + 0O,;=2H0+25 BAVENDAMM also found Achromatium gigas in brackish water. Achro- matium miilleri was isolated from sea water off the Danish coast by WARMING (1876). Hinze (1903) discovered Thiophysa volutans in fine sand in the Gulf of Naples. It oxidizes H2S to free sulfur or sulfate. At low oxygen tensions, cells contain sulfur granules but in the presence of an excess of oxygen, the sulfide is oxidized to sulfate. EL ts (1932) claims that Thiophysa volutans is confined to marine waters. Motiscu (1912) found Thiospira bipunctata growing in the sea. It oxidizes H,S and deposits sulfur granules intracellularly. Mo.iscu also found Beggiatoa marina associated with rotting marine algae. The latter, like other species of Beggiatoa, is a filamentous form showing undulatory creeping. It oxidizes HS to sulfur which is deposited as intracellular granules. Beggiatoa mirabilis was isolated from sea water by CoHN (1865). Warminc (1875) found Beggiatoa minima in the sea. The last two named species, together with Beggiatoa arachnoides, Beggiatoa alba, and Beggiatoa leptomitiformis, were identified in calcareous mud off the Bahama Islands by BAVENDAMM (1932). The occurrence of Beggiatoa species in the sea has been reported by many workers (BAVENDAMM, 1924). Thiothrix annulata and Thiothrix marina, whose cells contain sulfur granules, were isolated from rotting algae in sea water by Motiscu (1912). He likewise found Thiothrix nivea and Thiothrix tenuis in Trieste harbor sea water and in the Baltic Sea. BAVENDAMM (1924) and ELLIS (1932) have reported the presence of T/iothrix species in the sea. According to BERGEY et al. (1939), the ocean floor is the habitat of Thioploca schmedlei, a colorless Beggiatoa-like organism which deposits sulfur granules in the cell. BAvENDAMM (1932) expressed surprise at not finding species of Thioploca in calcareous mud around the Bahama Is- lands. Exzis (1932) records that Thioploca schmedlei and Thioploca ingrica live in the sea. ZoBell — 164 — Marine Microbiology ':; Purple sulfur bacteria:— The purple sulfur bacteria, or Rhodothio- bacteria, contain bacteriopurpurin, a photosynthetic pigment. They can utilize radiant energy under suitable conditions, but not all species require sunlight for their activities. Most of them grow best in the presence of H2S. Some of the saprophytic and photosynthetic strains live anaero- bically, but a little free oxygen is required by the autotrophic strains which obtain their energy from the oxidation of H2S. The purple sulfur bacteria are classified into two categories upon a basis of the deposition of sulfur. The Thiorhodaceae embracing the genera of the family Chromotioidaceae deposit sulfur intracellularly. They are highly cosmopolitan. Representatives of most genera are aquatic. Some species have been found only in marine habitats. From a survey of the literature, BAVENDAMM (1924) listed the follow- ing Thiorhodaceae which have been observed in salt or brackish water: Thiocystis violacea, Thiocystis rufa, Thiocapsa roseopersicina, Thiosarcina rosea, Lamprocystis roseopersicina, Thiopedia rosea, Amoebobacter gran- ulae, Thiothece gelatinosa, Thiodictyon elegans, Thiopolycoccus ruber, Chro- matium warmingit, Thiospirillum jenense, Thiospirillum violaceum, Thio- spirillum rosenbergii, Rhodacapsa suspensa, and Rhodothece pendens. BAVENDAMM pointed out that these purple sulfur bacteria were found in sunlighted habitats containing H,S. The buffering capacity of sea water permits them to grow. Some of them can tolerate relatively wide temper- ature ranges. Their sulfur metabolism is dependent upon H2S produced by proteolytic saprophytes or sulfate reducers. In calcareous mud around the Bahama Islands, BAVENDAMM (1932) recognized Chromatium okenii, Chromatium weisii, Chromatium minus, Chromatium vinosum, and others. He described them as being actively motile and coming from the sea bottom generously filled with sulfur granules. They often appeared as red clouds in the sea water covering the mud in cultures. Deep_down into the mud they formed beautiful, characteristic, wine-red pellicles on the glass walls of culture receptacles. He remarked, regarding bottom biocoenoses, that besides numerous sulfur bacteria of all types, there was a large heterotrophic bacterial population, including many sulfate reducers. Closely associated with the sulfur bac- teria were many blue-green algae, especially species of Oscillatoria, a few sessile diatoms, and some protozoans. He regarded such an association as being ideal for the activity of the sulfur bacteria. Sulfate reducers pro- vided HS in the close proximity of algae which, during the hours of day- light, supplied free oxygen. In marine bottoms off the northern coast of Russia, IssaTCHENKO (1914) noted the occurrence of Amoebobacter granulae, Thiopolycoccus ruber, Thiosarcina rosea, Thiothece gelatinosa, Chromatium minus, Chr. minutissimum, Chr. rosea, Chr. vinosum, Chr. gobii, and Thiodictyon minus. The last two named sulfur bacteria were described by ISSATCHENKO as new species. In the Dreckee mud swamps along the Danish coast, UTERMOHL (1925) found a,maximum of 3000 Chromatium cells per ml. along with 2900 Thiopedia cells and several hundred cells of Thiocystis. The combined 6000 to 7000 purple bacteria per ml. of water caused a red coloration. It was from similar shallow “red water’ sounds that WARMING (1875) iso- lated achromic species of sulfur bacteria which were freely mingled with masses of purple bacteria. Further data on “red water” or so-called ‘‘bloody seas”’ are reported Chapter XII — 165 — Sulfur Bacteria by GIETZEN (1931). Extensive populations of purple sulfur bacteria along the Holstein coast, growing associated with decomposing plankton includ- ing algae, jellyfish, etc., imparted a distinctly red coloration to the sea. The presence of HS, microaerophilic or anaerobic conditions, and sun- light appeared to be requisite for maximum development of the purple bacteria. They grow throughout the entire temperature range of the sea. The Red Sea and the Vermilion Sea (Gulf of California) were so named because of the frequent red to brownish coloration of the water. Ac- cording to SVERDRUP ef al. (1942), certain algae, particularly Trichodes- mium erythraeum, or dinoflagellates are responsible for the color. ALLEN (1933) found Prorocentrum micans, a golden-yellow dinoflagellate, to be responsible for certain occurrences of ‘“‘red water”’ off the coast of south- ern California. Extensive patches of “red water’ caused by the ciliate protozoan, Mesodinium rubrum, and the shrimp, Munida cokeri, have been reported by CoKER (1938). While these or other organisms may sometimes cause “red water,” purple bacteria are often overlooked by oceanographers and hydrographers. It is significant that the hydro- graphic conditions in ‘‘bloody seas”’ are generally precisely those which would promote the growth of purple sulfur bacteria; namely, the presence of an abundance of decomposing plankton material which provides for HS production and reduced oxygen tension. Forti (1933) found large numbers of Thiopolycoccus ruber, Thiopedia rosea, and Beggiatoa alba in a Sicilian “lake of blood” or ‘‘bloody sea.” Sulfate reducers in the bottom water provided HS for the sulfur bacteria. ELLIS (1932) tells of Rhodothece pendens coloring sea water around Helgoland ‘‘as though with rose-red milk of sulfur.”” Rhabdomonas rosea, Rhodocapsa suspensa, and Amoebobacter roseum are other colored bacteria which live in the sea, according to ELLIs. Thiopedia rosea was the most abundant organism found in fresh-water plankton by UTERMOHL (1925). There may be as many as 10,000 of these purple bacteria per ml. of plankton tow. KnrpowrrscH (1926) found microaerophilic Thiopedia rosea down to a depth of 750 meters in the Cas- pian Sea. For further information on the ecology and physiology of the purple sulfur bacteria the reader is referred to the treatises of BAVENDAMM (1924), BAAS BECKING (1925), GIETZEN (1931), ELLIs (1932), and vAN NIEL (1931, 1936). In the second category of purple sulfur bacteria are the Rhodobac- terioides or Athiorhodaceae, which do not show intracellular sulfur gran- ules. They grow best in the presence of H2S and are often found associ- ated with Thiorhodaceae and achromic sulfur bacteria. Apparently the Athiorhodaceae are neither as abundant nor as widespread as other types of sulfur bacteria. Only one of the seven genera of Rhodobacterioides listed in the BERGEY (1939) Manual is described as having a marine habitat, namely, Rhodo- bacterium capsulatum which Mo tscu isolated from sea water. However, most of the Rhodobacterioides live in water. A more thorough search of the literature may reveal that most of the genera have marine represen- tatives. BAAS BECKING (1925) recovered a Rhodococcus or Rhodorrhagus from rotting Fucus near Moss Beach on the California coast. According to Baas BEckKInG, the sulfur bacteria in brine, brackish water, and fresh- ZoBell — 166 — Marine Microbiology water sulfureta are all very much alike. Sulfur bacteria appear to be independent of salinity over a range of 0.05 to 7.5 per cent NaCl. The purple forms prefer diffuse or subdued sunlight and a low oxygen tension. They require HS which, in sulfureta, is provided largely by sulfate re- ducers. Baas BECKING (1925) described a sulfuretum as a natural ecological community of bacteria operative in the sulfur cycle. It is a biocoenosis of sulfate-reducing bacteria and H2S-oxidizing bacteria of various kinds along with several other kinds of microorganisms. In the fresh water, brine, and brackish water which he studied, Chromatiwm and Beggiatoa were commonest, followed by Lamprocystis, Amoebobacter, Thiothrix, Thi- opedia, Thiopolycoccus, and Thiospirillum. Protozoans feeding on sulfur bacteria were common. Euglena and Phacus of various types, Oscilla- toria, Phormidium, and many diatoms were usually present. The green algae were represented by a Protococcus-like form. Nematodes and a large Spirochaete were observed. BAAS BEcKING found green bacteria in brackish water but not in brine. Chlorobacteria, or green sulfur bacteria, have been found in the sea, especially in brackish water. Although reference has been made to them for more than half a century, agreement concerning their physiology and taxonomic position is still lacking. According to vAN NIEL (1931), Chloro- bium limicola is the only strain which has been obtained in pure culture, although Chlorobacterium, Cyanodictyon, Pelodictyon, and possibly other genera have been named. NADSON (1912) described Chlorobium limicola which he found in brack- ish water and bottom mud in the Baltic, Black, and Caspian seas, as well as in the salt Lake Weissowo. He thought they were minute algae. BENECKE (1933), who found green bacteria in the Baltic Sea, relates that PASCHER designated them as small Protococcales. However, as VAN NIEL (1931) pointed out, bacteriochlorin, the green pigment of Chlorobium limicola, differs from the chlorophyll of algae. Moreover, these bacteria assimilate HeS in subdued sunlight and deposit sulfur extracellularly, therein resembling the purple Athiorhodaceae: 2 H.S'-+- CO; = (CELO) 438.0468 Transformation of selenium compounds :— The close analogy between the chemical behavior of sulfur and selenium provided the incentive for BRENNER (1916) to investigate the effect of bacteria upon selenium com- pounds. He isolated an organism, Micrococcus selenicus, from marine mud at Kiel which autotrophically oxidized selenide: 2 H2Se + 5 Op = 2 SeO,-—— + 2 HO It also oxidized selenite to selenate. When organic matter was added to the medium and oxygen excluded, Micrococcus selenicus reduced selenite and selenate as well as thiosulfate. Thiobacillus thioparus, which BREN- NER isolated from the mud, failed to utilize selenium compounds. Neither selenate nor tellurate was reduced by Desulfovibrio aestuarii studied by RITTENBERG (1941). Chapter XIII THE TEROUSPHORUS CYCLE The phosphorus cycle is relatively simple compared with cycles of nitrogen, sulfur, or carbon. Plants utilize phosphate phosphorus which they convert into organic phosphorus compounds. Animals depend upon plants for their phosphorus requirements. Bacteria fulfill a most impor- tant function in the regeneration of phosphate from the remains of ani- mals and plants. The storage of phosphorus in bacterial cells and the effect of bacterial activity on the solubility of phosphate are of secondary importance in the phosphorus cycle in the sea. Assimilation of phosphate by microorganisms:— Phosphate is es- sential for the growth of phytoplankton. At times a lack of phosphate may limit the primary productivity of the sea (REDFIELD, 1934). ATKINS (1926) discusses certain hydrographic factors which influence the replen- ishment and utilization of phosphate in the English Channel, where the vigorous growth of phytoplankton often leads to a depletion of phosphate. In such an event, the area has yielded its maximum production until phos- phates are regenerated by bacterial activity or replaced by the influx of water from nutrient-rich areas. The utilization of phosphate and nitrate in the synthesis of organic sub- stances proceeds at approximately parallel rates, according to SVERDRUP et al. (1942). Any deviation from the nitrate-phosphate ratio is believed to be dependent primarily upon factors which influence the rate of nitrifi- cation or the bacterial regeneration of phosphate. The ratio of nitrate-N to phosphate-P in offshore waters is approximately the same as the ratio of organic-N to organic-P in marine plankton, namely 7:1. This agree- ment suggests that the composition of the composite animal and plant population of the sea is determined by the rate of available nitrogen to phosphate-P in sea water. The optimum concentration of phosphate-P for the development of diatoms is about 50 mgm./M.?, according to HARVEY (1933). Water throughout the euphotic zone rarely contains more than 5 mgm./M.® during the photosynthetic season, and generally less than 1 mgm./M.* At such times a majority of the total phosphorus content of the water oc- curs in plant and animal tissues. Upon the death of the organisms, phos- phate is rapidly regenerated. As pointed out by RENN (19372), bacterial cell substance is notoriously rich in phosphorus. However, it is apparent from their minute size and small numbers that bacteria do not compete effectively for the sea’s lim- ited store of this element. There is no evidence that phosphate limits bacterial multiplication in sea water unless the latter is experimentally enriched with organic matter. RENN noted a slight decrease in the phos- phate content of sea water incubated in the dark, followed by an increase. He attributed the initial decrease to the assimilation of phosphate by bacteria, and the increase to the liberation of phosphate from organic com- pounds including autolyzing bacterial cell substance. ZoBell — 168 — Marine Microbiology Regeneration of phosphate:— Phosphorus occurs in organisms pri- marily in the form of phospholipids and nucleoproteins. Animal bones consist largely of tricalcium phosphate. Most organic phosphorus com- pounds seem to be very susceptible to decomposition by bacteria. Phos- phate is liberated very early in the process. In experiments conducted by SEIWELL and SEIWELL (1938), about half of the phosphorus content of freshly collected zooplankton was decom- posed in 24 hours after the death of the zooplankton, whereas oxygen con- sumption by bacteria in the experiments indicated that half of the total organic content of the zooplankton was not oxidized for four or five days. In similar experiments conducted by CoopER (1935), about one-fourth to one-third of the phosphorus content of marine zooplankton was liberated as phosphate during the first twelve hours of bacterial activity and nearly all of it in six days. Phosphate was liberated from phytoplankton some- what less rapidly. The rapid regeneration of phosphate from dead bacterial cells was ob- served by RENN (1937a@). He reported that after bacterial growth had passed its maximum in sea water enriched with phosphate and glucose, the regeneration of phosphate followed “almost at once — rapid at first, and then leveling off at 14 days toward completion.”’ Phosphate was not lib- erated from autolyzing, bacteria-free diatoms. There has been considerable speculation regarding the place of phos- phate regeneration. The occurrence of a far greater concentration of phosphate in deep water than near the surface has been interpreted by some oceanographers as indicating that phosphate is regenerated on the sea floor. While unquestionably a certain amount of phosphate is regen- erated in bottom deposits, the foregoing observations on the rapidity of phosphate regeneration, together with other considerations, strongly sug- gest that the majority of the phosphorus cycle is enacted in the euphotic zone. There large quantities of phosphate may be regenerated, but phos- phate may not accumulate because it is utilized by phytoplankton or bacteria almost as rapidly as it is regenerated. From their studies on the rate of sinking of decomposing plankton, SEIWELL and SEIWELL (1938) concluded that due to the low density of the great mass of organic material, most bacterial decomposition must occur in the upper levels of the ocean. Significantly, they found that the min- imum concentration of oxygen occurs in the western North Atlantic basin in water which has a density value somewhere near that of the great mass of organic matter, namely, 1.027,232+ 0.000,008. This suggests that most organic decomposition and hence phosphate regeneration takes place above the oxygen minimum layer, roughly 600 to 800 meters. Below this depth only denser, particulate organic matter, which constitutes only a small fraction of the total organic content of the sea, sinks. This is in agreement with the observations of RAKESTRAW (1936), who noted that the zone of maximum accumulation of phosphate roughly parallels that of nitrite. Similar conclusions were reached by REDFIELD (1942) who believes that the phosphate maximum and oxygen minimum in the North Atlantic are ascribable to the oxidation of organic matter within a few hundred meters of the surface. While part of the phosphate content of South Atlantic water at mid-depths may be derived from the isentropic movement of subsurface layers from higher latitudes, in equatorial and North Atlantic regions, phosphate may be added and oxygen removed by the decomposi- Chapter XIII — 169 — The Phosphorus Cycle tion of sinking organic matter. REDFIELD’s argument that some organic matter must sink to depths as great as 600 to 800 meters is a commentary on his belief that the vast majority of the organic matter must undergo complete decomposition nearer the surface. Whether a specific bacterial flora is involved in the liberation of phos- phate from phospholipids and phosphoproteins or whether all lipolytic and proteolytic bacteria are endowed with this ability is not known. Special investigations are needed to answer this question, as well as to elucidate the mechanism of phosphate regeneration. However, bacteria which decompose organic compounds with the liberation of phosphate appear to be abundantly and widely distributed in aquatic environments as well as in soil. Unless properly preserved, there is nearly always an increase in the phosphate content of either sea water or fresh water stored in the dark (ZoBELL and BRown, 1944). WAKSMAN et al. (1937) have noted a rapid regeneration of phosphate resulting from the bacterial decomposition of diatoms. WaAksMAN ef al. (1938) made similar observations on decom- posing copepods and other marine organic matter. Effect of bacteria on solubility of phosphate:— The solubility of calcium phosphate in sea water is primarily a function of the pH. The solubility is increased as a result of the activities of bacteria which pro- duce acidic substances and decreased by metabolites of alkaline character. In localized microspheres where the phosphate content of the water is relatively high, tricalcium phosphate may be precipitated from solution owing to an increased pH caused by bacteria. The reverse process, namely the dissolution of tricalcium phosphate, may be of considerable importance in the phosphorus cycle. It is of im- portance on land where bacterial activities promote the mineralization of rock phosphates. In the sea large quantities of phosphate are bound in the bones of animals in the form of tricalcium phosphate. Part of the lat- ter may be dissolved by the acidic digestive juices of carnivorous animals, and part by bacteria and allied microorganisms. This they do in four dif- ferent ways recorded in order of importance: (1) Generation of acids. (2) Decomposition of organic matter associated with the bones, thereby mechanically liberating some calcium phosphate, particularly from only partially ossified tissue. (3) Production of ammonium salts and other secondary reaction products which increase the solubility of tricalcium phosphate. To the extent that it increases the pH, microbiologically pro- duced ammonium may decrease the solubility of tricalcium phosphate, but ammonium chloride and ammonium sulfate have a slight solvent action. (4) Conversion of insoluble tricalcium phosphate into cell phosphoproteins or phospholipids by direct assimilation. Tricalcium phosphate, either in the form of bone or crystals, provides an excellent surface for the attachment of marine bacteria and for the con- centration of dissolved organic matter. This would promote increased bacterial activity in the immediate vicinity of the tricalcium phosphate where bacteria in microspheres may produce enough acid to have a solvent action, though surrounded by slightly alkaline water. Chapter XIV RELATION OF MARINE BACTERIA TO FLORA AND FAUNA It is almost axiomatic that the density of the plant and animal popu- lation of biotic zones is more or less proportional to the abundance of bac- teria and allied microorganisms. This is probably chiefly because bac- teria are predominantly heterotrophic, depending largely upon plants and animals for organic nutrients. However, there are other ways in which the flora and fauna are beneficial to bacteria. In turn, most bacteria are beneficial to plants and animals, although in localized regions bacteria may create antibiotic environmental conditions, and some bacteria are parasitic or pathogenic. Reciprocal relations of bacteria and plants:— It has been definitely established, as outlined in preceding chapters, that bacteria play an im- portant role in the production of plant nutrients including ammonia, nitrite, nitrate, sulfate, and phosphate. It is not known to what extent the ability of bacteria and allied microorganisms to produce these plant nutrients is unique, but certainly it is one of the most important functions of microorganisms. The plant nutrients are produced in considerable quantities and for the most part in the immediate proximity of plants. Bacteria also produce large quantities of CO, which is likewise essen- tial for plant growth. Though animals are generally credited with being the principal CO; producers in the sea, the composite bacterial population of the sea may produce more CQ, than the combined animal population. Based upon oxygen consumption data and considering that bacteria ap- proximate spheres having a mean diameter of 1.0 u and a density of 1.028, ZOBELL (19406) estimated that bacteria in the sea produce about 30 ml. of CO, per hour per gram of living cells at a mean temperature of 10° C. This may be contrasted with the value of 0.002 to 1.0 ml. of CO produced per hour per gram of marine animal tissue. Unfortunately, no data are available on the relative mass of bacteria and animals in the oceans of the world, but judging from the data of JupDAy (1942) on the standing crop of plants and animals in lakes, together with information on the bacterial population of these lakes, it is estimated that the ratio of the mass of the standing crop of bacteria to that of animals is about 1:200. The fact that bacteria produce CO: 30 to 15,000 times faster than animals per unit of mass indicates the relative importance of bacteria as CO, producers. Plants are the chief source of organic nutrients for saprophytic bac- teria. Plants are attacked immediately following their death. The secre- tions and excretions of living plants also provide food for bacteria asso- ciated with them. The secretion of organic matter by growing plants as well as the epiphytic association of bacteria with plants is discussed in the section beginning on page 77. There it is pointed out that bacteria occur primarily attached to phytoplankton or other particulate material which provides both food and solid surface. Symbiotic nitrogen fixation by bacteria associated with aquatic plants Chapter XIV —171— Relation of Marine Bacteria is an almost unexplored possibility. The fixation of nitrogen by Azoto- bacter growing epiphytically on algae (REINKE, 1903; KEDING, 1906) is hardly true symbiosis because, while the algae may provide the bacteria with a holdfast and organic nutrients, there is no evidence that the bac- teria contribute fixed nitrogen to the algae, at least not until after the death and decomposition of the bacteria. Marine plant pathogens:— Infections of marine plants by bacteria have been reported. An interesting example is the disease of kelp known as “black rot’? which was first noticed off the California coast by BRANDT (1923). It attacked the bladder kelp, Macrocystis pyrifera, the elk kelp, Pelagophycus porra, and the ribbon kelp, Egregia laevigata. The causative organism was kept in check by the colder water of winter and by proper cutting of the kelp beds. BILLET (1888a, b) isolated Bacterium laminariae and Bact. balbianiu from rotting kelp. ScHAUDINN (1903) described Bacillus sporonema, which he isolated from rotting Ulva. LAGERHEIM (1900) presented evi- dence that a fungus-like bacterium, which he named Sarcinastrum uro- sporae, is responsible for gall formation on red algae. CANTACUZENE (1930) found tumorous growths on Irish moss, Chrondus crispus, which he attributed to infectious bacteria. Tumors or galls on Saccorrhiza bulbosa were likewise found to be infected with bacteria. Healthy plants could be experimentally infected by inoculating them with the bacteria isolated from diseased plants. Large numbers of bacteria are associated with diseased eel grass, Zostera marina, but they have not been proved to be pathogenic. After finding Labyrinthula species in all specimens exhibiting symptoms, RENN (1936) concluded that these fungi were the etiological agents of the “wasting disease” of eel grass. Halophiobolus species (BARGHOORN and LINDER, 1944) may also be involved. Napson and Burewitz (1931) found several varieties of Torula and other yeast-like organisms parasitizing Laminaria, Alaria, Fucus, and other seaweeds. Several species of fungi parasitic on marine algae have been described by KipBE (1916), ZELLER (1918), SPARROW (1934, 1936), and others. Some of their observations are detailed in Chapter IX. Judging from the positive results that have been obtained from the few precursory or exploratory observations made to date, it is believed that pathogenic bacteria, yeasts, and mold fungi may extensively parasit- ize seaweeds, diatoms, dinoflagellates, and other marine plants. This is a most promising field for further investigation; a field in which the results may contribute to our knowledge of marine microbiology and general hydrobiology. Although he regards the study of aquatic fungi as still being a virgin field for research, WESTON (1941) writes, ‘‘On the plant life of fresh water, fungi are commonly, extensively and often destructively parasitic. Occa- sional serious epidemics have been reported, chiefly on algae such as diatoms, desmids or other plankton forms significant in aquatic ecology.” WESTON gives a résumé of the ever-growing literature describing aquatic fungi which have been found to be parasitic on fresh-water algae and higher aquatic plants. The latter appear to be less susceptible to parasit- ism than the simpler algae. He quotes Zopr to the effect that, ‘In the household of nature the infectious diseases of the lower organisms play a highly significant part.” ZoBell — 172 — Marine Microbiology Reciprocal relations of bacteria and animals:— There are several ways in which bacteria and animals are beneficial to each other either directly or indirectly. The bacterial production of plant nutrients, thereby providing for the growth of plants which animals may consume as food, is regarded as an indirect benefit. The part that bacteria play in aiding animals to digest their food is a direct benefit. The digestive tracts of most animals contain numerous bacteria which as a group are very versatile biochemically, being capable of attacking a wide range of materials. Collectively, these bacteria elaborate a formid- able equipment of enzymes which attack many of the substances swal- lowed by animals for which the consumer itself has no enzyme. The ~ power to break down pectins, hemicelluloses, cellulose, lignin, chitin, and other organic complexes is much more widespread among enteric bacteria than it is among animals which ingest these organic complexes. There are few data on the extent to which commensal bacteria may aid animals in the digestion of food, but with many animal species such bacteria may be virtually indispensable. Certain ship worms and wood borers, which are discussed elsewhere in this volume, are believed to depend upon com- mensal bacteria which help to digest cellulose and lignin. The function of bacteria in the pre-digestion or partial digestion of food is not confined to enteric forms. Grazing, filter-feeding, and mud- eating animals assimilate much finely particulate food which has been broken down by bacterial activity. Large numbers of bacteria, along with partially decomposed organic complexes, are ingested and digested by animals. Other bacteria find con- ditions in the intestinal tracts or on the integuments of the animals con- ducive to their growth and multiplication. Evidence is forthcoming from analyses which reveals that the intestinal contents of many marine ani- mals contain millions of bacteria per ml. Likewise the slimy integuments of aquatic animals are invested with an extensive bacterial population. From his observations on marine fish, SANBORN (1932) concluded that a more or less definite bacterial flora occurs on the surface of fresh fish. The integuments or cell walls of virtually all aquatic animals both large and small appear to be veritable bacterial gardens. _ Illustrative examples are the large bacterial populations associated with copepod tows, the difficulty experienced in attempting to obtain bacteria-free cultures of protozoa, and the bacterial content of fish slime. Predominating on the surface of halibut freshly taken at depths of 30 to 60 fathoms in the Atlantic Ocean by SANBORN (1932) were Achromo- bacter pellucidum and Rhodococcus agilis. These, together with Micro- coccus varians, M. citreus, M. candidus, Flavobacterium turcosum, and FI. fucatum, were observed by SANBORN on Atlantic halibut and by HARRISON (1929) on Pacific halibut. SANBORN (1932) also noted the presence of Micrococcus nitrificans, M. halophilus, Achromobacter geniculatum, A. am- biguum, and Flavobacterium annulatum on halibut. Unlike HARRISON’s findings on Pacific halibut, SANBORN noted on Atlantic halibut the pres- ence of Pseudomonas fluorescens, which was very actively proteolytic. SAN- BORN believed that Ps. fluorescens, along with two other equally active proteolytic marine species, Achromobacter geniculatum and Flavobacterium ucatum, have a direct economic bearing upon the keeping quality of fish. The formation of slimy or viscous growth, which is a common property of bacteria found on the integument of marine fish, is particularly pro- nounced with Ps. fluorescens, Achromobacter pellucidum, and A. viscidum. Chapter XIV —173— Relation of Marine Bacteria The latter is named by SANBORN as a new species which resembles A. Jito- ralis. Of the organisms listed above which were isolated from marine fish, only A. ambiguum, Micrococcus nitrificans, and M. halophilus proved to be salt-tolerant. They grew in media containing from 10 to 20 per cent NaCl. Species of Achromobacter predominated in the slime of the 22 haddock examined by STEWART (1932). She also observed species of Micrococcus, Flavobacterium, Pseudomonas, and many unidentified luminescent bac- teria in haddock slime. Nearly half of the cultures isolated from the slime of rr species of marine fish by GrpBons (1934a) were classified as Achromobacter, with smaller numbers of Flavobacterium, Micrococcus, Pseudomonas, Bacillus, Proteus, and Serratia occurring in abundance in the order listed. Luminescent bacteria have been commonly observed growing on fish and other marine animals. Such bacteria are often asso- ciated with the light organs of certain animals (HARVEY, 1940). Addi- tional data on the kinds of bacteria associated with marine fish are given in Chapter XVI on page 188. Their predilection for solid surfaces and their organic requirements, both of which are provided by animals, explain why certain bacteria are intimately associated with animals. The secretions and excretions of an- imals are a source of food for bacteria. The bacterial utilization of urea, uric acid, and other waste products of animals is mutually beneficial to both consumer and producer. Bacteria as food for animals :— Judging from their chemical composi- tion, bacteria should be highly nutritious and readily digestible. This supposition is substantiated by experimental and field observations which indicate that many kinds of animals ingest bacteria. Certain animals can live almost indefinitely on an exclusive diet of bacteria. Bacteria constitute an important part of the dietary of nearly all uni- cellular animals, according to Luck et al. (1931). They noted that Eu- plotes taylori grew better on mixed cultures of bacteria than on any one single strain. Heat-killed bacteria, filtrates, autolyzed bacteria, and phage-lysed bacteria failed to support Euplotes taylori. The failure of cer- tain bacteria to be assimilated by protozoans may be due to toxic meta- bolic products, unfavorable size or shape, capsular material, or other peculiarities. The importance of bacteria in the nutrition of pelagic copepods is stressed by the work of EstERLY (1916) who considered the question of the utilization of dissolved organic substances. He concluded that, al- though unable to utilize dissolved materials directly, copepods may sub- sist on bacteria which in turn utilize dissolved organic substances. Ac- cording to KrizENcKy and PoDHRADSKY (1927), one of the most impor- tant functions of aquatic bacteria is the conversion of dissolved organic matter into particulate (bacterial cell substance) organic matter which animals can utilize. The ratio of dissolved to particulate organic matter is estimated to range from 7:1 to 4000:1 in different parts of the ocean. It is the consensus of opinion (KRroGH, 1931; Fox and Cor, 1943) that, contrary to PUTTER’s theory, organic matter in true solution plays no significant role in the direct nutrition of aquatic animals, but that bac- teria serve as intermediary agents of vast importance. Given sufficient time and favorable conditions, including solid surfaces, bacteria utilize the organic content of sea water almost quantitatively (ZOBELL and GRANT, 1943), mineralizing roughly 70 per cent of it and converting 30 per cent into bacterial cell substance or intermediate products. - ZoBell — 174 — Marine Microbiology The experiments of CLARKE and GELLIS (1935) indicate that, while bacteria may be important as food for copepods, there seem to be insuffi- cient bacteria in the sea to provide for the complete nutritional require- ments of copepods. According to WAKSMAN and CarEy (19350), the bac- terial population of the sea is kept down to a certain minimum due to the consumption of bacteria by protozoans, copepods, and other marine ani- mals, notably those of the mucous-feeding or filtering classes. Several kinds of zooplankton organisms were found by VOROSCHILOVA and DIANOVA (1937) to ingest bacteria. These workers concluded that bacteria nourish zooplankton organisms and that the predatory activities of the latter tend thus to restrict the bacterial population of the Caspian Sea. Pack (1919) reported that ciliates in Great Salt Lake feed on bac- teria. Baas BECKING (192 5) noted that sulfur bacteria appeared to be the chief food of ciliates in sulfureta. Mud-dwelling detritus feeders studied by MacGinttte (1935) utilized bacteria as food. MARE (1942) stressed the importance of bacteria as food for bottom-dwelling animals in marine benthic communities. ZOBELL and FELTHAM (1942) estimated that around 1o grams (dry weight) of bac- teria are produced per day per cubic foot of mud in a shallow marine mud flat. The bacterial crop available for the nutrition of the animal popula- tion in deeper colder bottoms of the open ocean, where less organic matter reaches the sea floor, is considerably less than 10 grams per day. ZOBELL and. FELTHAM (1938) demonstrated that the sea mussel, Mytilus californianus, ingests and digests bacteria. Specimens were main- tained on an exclusive diet of bacteria for several months, during which time the mussels gained in size and weight. Few species of marine bac- teria are injurious unless they are present in such great numbers that their metabolic products vitiate the water. Bacteria were also found to sustain the growth of the sand-crab, Emerita analoga, to a limited extent. Emerita is more sensitive to large doses of bacteria than is the mussel and is less efficient in removing bacteria from suspension. The Gephyrean worms, Dendrostroma zostericola and Urechis caupo, were found to eat bacteria and to derive nourishment therefrom. BAIER (1935) listed several genera of rotifers, copepods, ciliates, and flagellates which may feed directly upon bacteria and small nannoplankton suspended in water. The larval stages of additional genera are planktonic bacteria feeders. A second group of animals ingest bacteria along with the mud and slime which they swallow promiscuously, digesting the usable portion and ejecting inert materials. Nematodes, mussels, tube worms, and the larvae of midges are in this category. A third group of bacteria feeders discussed by BAIER are animals which graze on solid objects like rocks, aquatic plants, and suspended solids of various kinds. Certain snails, ostracods, copepods, and amoebae are mentioned as examples of animals which feed in this manner. Concerning the latter group, BATER writes that it is not the decomposing plant substance but rather the bac- teria, the cause of the decomposition, which nourish the grazing animals. That bacteria are ingested and digested by highly diverse fauna is incontrovertible, but quantitative data on their relative importance in the food cycles in the sea are wanting. It is doubtful if bacteria are suf- ficiently abundant in sea water to constitute an appreciable item in the diet of marine animals, but cumulatively bacteria must play an important role in food cycles by synthesizing cell substances and by converting waste or dissolved organic matter into a particulate form which can be Chapter XIV —175— Relation of Marine Bacteria utilized as food by animals. In bottom deposits and as a constituent of the slime on solid surfaces, bacteria may be sufficiently abundant to provide for the more or less complete nutrition of certain animals. Accord- ing to Pearse et al. (1942), one of the important functions of bacteria in marine sand beaches is serving as food for nematodes, flatworms, proto- zoans, amphipods, and other small animals. Marine animal pathogens:— There is evidence that certain marine animals succumb to bacterial infections. However, conditions in the sea are not conducive to the propagation of microorganisms which cause acute infections of animals, although animal parasites are common. As soon as a pathogen incapacitates its host, the latter almost immediately falls prey to ever-present predators. There is no sanctuary in the sea for the ill or the old where only the fittest survive. Any diseased fish or other animal which can no longer swim quite as fast as his companions may soon be captured and eaten by a larger fish or other predator. Consequently, the pathogen which incapacitates its host may be destroyed also. Nevertheless, marine animals do have infectious diseases, perhaps much more extensively than indicated by the fragmentary literature on the subject. Animals kept in aquaria, experimental tanks, and elsewhere in captivity are quite susceptible to bacterial and fungus infections. PLEHN’s (1924) monograph on fish diseases describes many infections of fresh-water fish. Costly losses to the salmon industry and trout hatcheries have been caused by a generalized bacteremia or epizootic furunculosis, the etiologi- cal agent of which is Bacterium salmonicida (DUFF, 1932). Bacillus columnaris is a new species described by Davis (1922) which infects the epidermis, gills, and fins of several species of fresh-water fishes. ARONSON (1926) described Mycobacterium marinum which causes tuberculosis in certain salt-water fishes. Achromobacter ichthyodermis, the etiological agent of an infectious dermatitis of certain marine fishes, was described by WELLS and ZoBELL (1934). Since it has a polar flagellum, this organism should be designated Pseudomonas ichthyodermis according to the revised key. From the integument of diseased fishes, ZOBELL and Upxam (1944) isolated Bacterium marinopiscosus, but its pathogenicity was not established. A “soft shell” disease, which has killed large numbers of lobsters on the West Coast of North America and ruined the commercial value of others, is believed by HEss (1937) to be caused by chitinovorous bacteria. InNMAN (1927) described an infectious disease of sand fleas and other crus- tacea, which was caused by photogenic bacteria. A large number of aquatic animals are infected by photogenic bacteria, according to HARVEY (1940). The problem of the infection of fish and shellfish is discussed in Chapter XVI. WESTON (1941) relates that practically all the main groups of fresh- water animals, from the simplest protozoan to the most highly developed chordates are attacked, in some phase of development from egg to adult, by various aquatic fungi. He relates that an epizootic caused by the aquatic fungus, Aphanomyces astaci, killed millions of commercially valu- able crayfish in Europe. Species of Aphanomyces have caused destruc- tive epidemics among protozoans and copepods. ‘TIFFNEY (1939) discusses several species of the water mold, Saprolegnia, which exten- sively and destructively parasitize fish. Besides attacking a wide range ZoBell — — 176 — Marine Microbiology of fish species, Saprolegnia parasitica also infects various Amphibia and Reptilia. Saprolegnia ferax and Achlya flagellata are other common aquatic fungi which are pathogenic for animals. Eggs and larval stages of animals are usually more susceptible to fungus parasitism than are the adult forms. A species of Petersenia, a chytridiaceous fungus parasitic on marine rotifer eggs, was described by SPARROW (1936). Observations on malformed sardine eggs from off the coast of California suggest that fungus infections may contribute to the failure of the sagdine crop in certain years. Antibiotic conditions caused by bacteria:— In the sea as a whole the combined activities of bacteria and allied microorganisms tend to create environmental conditions which are conducive to the growth of plants and animals. However, in localized regions certain types of microbiological activities may be inimical to the well-being of other organisms. An outstanding example of the antibiotic activities of bacteria is the production of H,S in quantities which are toxic to both animals and plants. Some of the factors which influence H2S production are discussed in Chapter XII. Likewise in the presence of an abundance of utilizable organic matter, bacteria may reduce the oxygen tension of water below the threshold of tolerance for certain animals. This condition prevails in bottom deposits. The oxygen minimum layer found in the Atlantic and Pacific at a depth ranging from 600 to goo meters may be ascribable to the consumption of oxygen primarily by bacteria in water masses having a peculiar hydro- graphic history. Besides depleting the oxygen content of marine bottoms, bacteria re- duce the oxidation-reduction potential of bottom deposits to a level below that which is tolerated by most organisms. Most aquatic animals prefer or require a redox potential ranging from Ey 0 to + 0.2 volt. Bottom- dwelling bacteria sometimes create conditions in mud as reducing as Ei, — 0.2 to — 0.5 volt. Both the sea and its bottom are too well buffered for the pH to be greatly affected by microbiological activities, except in highly localized regions. There the acidic or basic substances produced by bacteria may have a profound influence on the residents of the small community, as in sulfureta, for example. Other toxic products besides HeS may be produced by bacteria includ- ing various nitrogenous catabolites. Such products can be expected to accumulate in significant quantities only in the immediate vicinity of a decomposing fish, for example, and there only temporarily. Additional information on the relation of marine bacteria to flora and fauna is given in the sections on Plant and Animal Population in Chapter II, Effect of Other Organisms and the Antagonistic Effects of Microorgan- isms in Chapter V, Biocoenosis and Bacteria in Bottom Deposits in Chap- ter VI, Bacteriology of Marine Fish in Chapter XVI, and Fouling of Sub- merged Surfaces and Bacteria Associated with Wood-borers in Chapter XVII, as well as elsewhere in the text. Chapter XV MICROORGANISMS IN MARINE AIR One of the first problems investigated by pioneer marine microbiolo- gists on oceanographic expeditions was the bacterial content of the atmos- phere. Prior to the observations of CERTES (18842), FISCHER (1886), and MINERVINI (1900), it was tacitly assumed that the air over the ocean at considerable distance from land was sterile. Dating from their investiga- tions, various kinds of microorganisms have been found in the air over the ocean at all stations and elevations where samples have been taken. Bacteria in marine air:— While on the Talisman Expedition, CERTES (18842) found bacteria in the air at considerable distances from shore. However, they were present in such small numbers that he felt certain that the aerial transportation of bacteria from land could not possibly account for the presence of bacteria in the sea. FISCHER (1886) made several hundred analyses of air over the ocean between Kiel and Trinidad while crossing the Atlantic on the S.M.S. Moltke. When the prevailing direction of the wind was onshore, terrige- nous bacteria were rarely found in the air at distances exceeding 10 to 20 miles from land. Only small numbers were carried more than 70 to 120 miles from land by strong offshore winds. However, he admitted the pos- sibility of bacteria being carried around the world with dust particles. He stressed the cleansing action of precipitation. Corroborative evidence of the paucity of bacteria in the air over the ocean has been recorded by LEVIN (1899), GAZERT (1902), EKELGF (1907), PIRIE (1912), and HESSE (1914). MINERVINI (1900) found air-borne bacteria in all collections taken over the Atlantic Ocean between Gibraltar and New York. Mold fungi and pink yeast were also noted. Rain water collected at sea contained small numbers of microorganisms. ‘There were far fewer bacteria in marine air than in air over the land. On various Pacific cruises of the Scripps, ZoBELL and MATHEWS (1936) found bacteria in the air over the ocean at all stations where tests were made. In general, the abundance of bacteria decreased progressively with distance from land. The numbers and kinds of microorganisms in the air appeared to depend upon meteorological conditions. At land stations terrigenous microorganisms predominated during offshore winds. During prolonged onshore winds, marine microorganisms predominated. The ability of the microorganisms to develop on fresh-water and sea-water media was considered indicative of their origin. Illustrative data are given in Table XXXVI on page 178. Besides their salinity requirements, other qualitative differences were noted in the microbial content of marine and continental air masses. In air masses known to have a marine history, Gram-negative, pigmented, asporogenous rods generally predominate, with relatively few cocci. GAZzERT (19060) and HEssE (1914) found virtually no cocci or spore-form- ing bacteria in marine air. By contrast, large numbers of cocci and Gram- ZoBell — 178 — Marine Microbiology TABLE XXXVI. — Average number of bacteria which developed on sea-water and fresh-water nutrient agar exposed to air under comparable conditions during a sustained onshore breeze (from . ZOBELL, 1942c): — DISTANCE INLAND SEA-WATER FRESH-WATER RATIO OF FROM SEA WALL MEDIUM MEDIUM SW:FW meters bacteria bacteria ° 461 43 10.70 100 548 74 7-40 200 263 stole) FAT 400 174 157 1.18 800 128 183 Ons 0.46 1600 49 106 positive, sporogenous rods generally characterize continental air. Only 15 of the roo different cultures of bacteria isolated at random by RITTEN- BERG (1939) from plates exposed to air over the ocean proved to be ter- restrial species. ReEITANO and MorsELtt (1938) found bacteria in the air at all stations where observations were made 2 to g kilometers from the coast over the Mediterranean Sea. Their analytical procedures precluded the possibility of differentiating marine from terrestrial bacteria. They failed to take into account meteorological conditions. This also applies to the observa- tions of GrUDICE (1939) who found from 12 to 280 bacteria per cubic meter of air in samples collected from 30 stations on the Red Sea and Mediter- ranean Sea. The California State Bureau of Sanitary Engineering (1943) presents evidence that Escherichia coli and other enteric organisms may be carried by the wind several miles from sewage outfalls. This observation further emphasizes the ecological and possible sanitary significance of the aerial transport of microorganisms. Proctor and PARKER (1942) reported the presence of a great variety of living microorganisms in the air at elevations exceeding 35,000 feet above the earth, which indicates the possibilities of a world-wide distribution by air currents. Yeasts and mold spores in air:— Pink yeasts, probably Torula spe- cies, have been observed on plates exposed to marine air by many investi- gators, including FISCHER (1894c). MCLEAN (1918) recovered two pink yeasts from marine air over Antarctica. The number of mold spores in air over the ocean generally decreases with distance from land. This is illustrated by the following data from RITTENBERG (1939): DISTANCE FROM LAND MOLDS DEVELOPING ON MOLDS DEVELOPING ON IN NAUTICAL MILES SEA-WATER MEDIUM FRESH-WATER MEDIUM o to 10 II5 200 to to 150 79 69 150 to 400 20 36 All of the mold fungi isolated from marine air by RITTENBERG proved to be common terrestrial species. The mold spores recovered from air over the ocean by F1IscHER (1886) were chiefly common varieties of Penicillium, Aspergillus, and Mucor. The fungus content of air is believed to be an indicator of its origin and history, since few if any mold spores enter the atmosphere from the ocean. Chapter XV — 179 — Microorganisms in Marine Air The comparative freedom of air from contaminating molds is most striking on research vessels and in seaside laboratories favored by prevailing on- shore winds. Additional information on air-borne fungus spores is given by Dur#amM (1942). Microbial content of precipitation:— There has been little additional work done on the microbial content of different forms of precipitation since FRANKLAND and FRANKLAND (1894) reported half a century ago that, ‘“‘Curiously but few determinations of the number of organisms in rain have been made.”” According to the FRANKLANDS, MIQUEL found an average of 4.3 bacteria per ml. of rain water collected at Montsouris Ob- servatory outside of Paris, and 19 per ml. in the middle of the city. The average number for three years was 4.3 bacteria and 4.0 molds per ml., which, with an annual rainfall of 60 cm., signified that about 5,000,000 organisms fall annually per square meter in that locality. Other workers have reported the presence of several thousand organisms per ml. of freshly fallen rain water. The FRANKLANDS stated that Buywip found as many as 21,000 organisms per ml. of water from hailstones and that Fou- TIN found 729 organisms per ml. of hailstone water. The number and kinds of microorganisms in rain water are influenced by the direction and velocity of the wind, the origin, course, and duration of the storm, and other meteorological conditions. Rain water collected at the Scripps Institution contained from 10 to 150 microorganisms per ml. Terrestrial forms, particularly mold spores, predominated in rain water containing the most microorganisms. The largest counts were usually obtained in the first rain. The rain water approached sterility as storms progressed, especially in the absence of local atmospheric turbu- lence. Rain water collected at considerable distances offshore contained an average of from 1 to 10 bacteria per ml., with few or no mold fungi. McLEANn (1918) recovered cocci, sporogenous rods, and yeasts from falling snow on Adelie Land, Antarctica. He believed that they were car- ried there from distant continents by air currents. Significant numbers of bacteria were found by SALIMOVSKAJA-RODINA (1936) in snow from mountains ranging in elevation from 2,050 to 2,800 meters and also in snow from polar regions. Predominating were colorless rods, although under certain conditions the snow was tinted by the presence of large numbers of pigmented microorganisms. Nineteen different species of yeast fungi colored white, pink, red, or black were isolated from the snow. Two species of red cocci and two yellow rods were found in freshly fallen snow. Species of Bacillus, Achromobacter, Flavobacterium, “EA Micrococcus were found by DARLING and SIPLE ( 1941) i in freshly fallen snow on Little America and Marie Byrd Land. Only one or two bacteria were found per pint of snow. The same general types of bacteria were found on plates of nutrient media exposed to air. Although they admit the possibility of migrating terns, gulls, or petrels carrying bacteria to this snow-covered region, DARLING and SIPLE were convinced that most of the bacteria were air-borne from the continents. Unfortunately, suitable media were not employed to ascertain if there were any marine bacteria in the air and snow of the Antarctic region. Pollens in marine air:— ERDTMAN (1938) made a quantitative study of pollens in the atmosphere while crossing the Atlantic Ocean from ZoBell — 180 — Marine Microbiology Sweden to New York. He found an average of from o.7 to 18 pollen grains per cubic meter of air at different stations. These figures for air over the ocean may be contrasted with the average of 18,000 pollen grains per cubic meter of air near Stockholm. In general, the number of pollen grains in marine air was related to the distance from land, the direction and velocity of the wind, and the flowering time of plants. Pollens of trees, shrubs, grains, and grasses were found several hundred miles from the nearest land. MEIER and LINDBERGH (1935) found pollens, as well as fungus spores, diatoms, and insect wings, in Arctic air several hundred miles from land and at an elevation exceeding 3000 feet. Pollen surveys over the ocean at different elevations made by air-lines and other agencies show that, while pollens are widely distributed, their abundance drops off sharply with distance from land and in the face of marine air currents. Aerial transport of marine bacteria:— There is ample evidence that bacteria, mold spores, and pollens from the land are carried around the _ world by the wind. Marine microorganisms likewise may be widely dis- seminated by the movements of the atmosphere, although there are few data on the transportation of marine bacteria inland. From an onshore wind having a velocity of 5 to 12 miles per hour fol- lowing a rainstorm, ZOBELL and MATHEWS (1936) recovered appreciable numbers of marine bacteria from air on a mountain 80 miles inland. Some marine bacteria probably fall with precipitation, but at inland stations their presence is masked by the preponderance of terrestrial forms. Microorganisms are introduced into the air over the land by atmos- pheric disturbances of various kinds, by the movements of animals, and by other agencies. Bacteria are being carried continually from the sea into the air, along with droplets of water. The transfer is most intense along rugged coasts when waves are dashed against rocks or along gently sloping shores where the incoming swell breaks into foam. The transfer is most extensive in the open sea where strong winds often blow spray from crests of waves. Large droplets fall back into the sea almost at once but small ones may be lifted to great elevations by advection and convection currents, and carried away by the wind to regions far re- moved from the place of origin. The almost universal distribution of sea Salt in the air makes this manifest. TABLE xxxvit. — Rate of fall of water droplets through air and the minimum distance they would be carried by a steady 10 mile per hour wind before they fell 100 meters in the absence of turbulence: — DIAMETER OF RATE OF FALL DISTANCE CARRIED DROPLET OF DROPLET BY 10 M.P.H. WIND microns cm./sec. miles I 0.003 9260.0 5 0.076 306.0 ie) 0.305 92.0 50 7.6 3.0) 100 30.5 0.9 500 760.0 0.036 According to evidence summarized by ZOBELL (1942c), droplets of water exceeding 1.0 mm. in diameter may be conveyed several thousand feet above sea level by convection currents, by orographic uplift, and by vertical motion caused by converging winds (see Fig. 12). In the absence Chapter XV — 181 — Microorganisms in Marine Air of any lifting force whatever, a water particle 0.5 mm. in diameter could be carried about a mile by a steady wind having a velocity of 10 miles per hour before it fell 100 meters. Larger droplets would not fall much faster because they break into small ones when they attain a velocity ex- ceeding 800 meters per second, unless frozen solid as in hailstones. The rate of fall of water droplets of different sizes through air as calculated from STOKE’s law of falling bodies is given in Table XX XVII on page 180. The calculations are based upon a steady state in which there is no turbu- lence. Since there is nearly always some degree of turbulence which may tend to retard the descent of falling bodies or actually carry them to higher elevations, and since the wind velocity often exceeds 1o miles per hour, the physical possibilities for the world-wide aerial transport of bacteria become apparent. Fic.12. — Principal ways in which bacteria in sea water are carried into the atmosphere: (A) orographic uplift, (B) spray from crests of waves some of which are 20 to 30 feet high, (C) convection, and (D) convergence of light warm air mass and heavy cold air front (taken from ZOBELL, 1942¢). The horizontal distance over which microorganisms can be transported is almost limitless and is largely determined by their ability to survive the atmospheric environment. The presence of microorganism in the atmos- phere at great elevations, far removed from their native habitats, shows that certain forms may survive the adversities of the atmospheric environ- ment for long periods of time. ; Chapter XVI SANITARY ASPECTS OF MARINE MICROBIOLOGY Treatises on ‘‘water bacteriology” are generally concerned primarily with the study of the sanitary properties of domestic water supplies, swim- ming pools, and sewage. For various reasons sea water is rarely consid- ered. Nevertheless, the sea does present certain problems of interest to sanitary engineers and students of public health. Outbreaks of oyster- borne typhoid and the development of beaches for recreational purposes have focussed attention upon some of these problems in recent years. The problems are of acute interest to those municipalities discharging raw or partially treated wastes into oceans, bays, or estuaries. Human pathogens in sea water :— It is the consensus of opinion that there are no autochthonous marine bacteria which infect man, but the lit- erature is replete with contradictory accounts of the viability of adventi- tious pathogens in sea water. Some workers hold that sea water is highly lethal for bacteria from land-dwelling animals while other workers present data which indicate that such bacteria can live almost indefinitely in the sea. A reconciliation of the divergent views requires that several factors which influence the survival of bacteria in sea water be taken into consid- eration. Artificial, synthetic, diluted, or autoclaved sea water, which has been used for many of the experiments, does not necessarily simulate natural sea water, and the biological properties of the latter may vary greatly. In his studies on the factors which influence the survival of pathogenic bacteria, DE GIAXA (1889) observed that enteric bacteria perish very soon in the sea. He found more than 100,000 bacteria per ml. of sea water 50 meters from a sewage outfall in the Gulf of Naples, 26,000 at a distance of 350 meters, and fewer than 100 per ml. 3000 meters from the sewage outfall. Controlled experiments showed that Bacillus anthracis and Vibrio comma were unable to compete with saprophytes in polluted sea water. The typhoid bacillus and pathogenic species of Staphylococcus | were even less resistant. Vzbrio comma lived for several days in heat- sterilized sea water, but in untreated sea water it soon disappeared. The period of survival was a function of the organic content and the bacterial population of the water. In grossly polluted water these organisms sur- vived for less than 24 hours. Experiments of various authors summarized by FRANKLAND and FRANKLAND (1894) show that, in general, human pathogens do not survive as long in sea water as in fresh water, although it is related that Vzbrio comma was observed by Nicati and RIEetTscH to remain viable in sterilized sea water for as long as 81 days. According to SOPER (1909), the virulence of Eberthella typhosa is not reduced by sea water in two or three weeks. The persistence of £b. typhosa in refrigerated oysters for 49 days is reported by KRUMWIEDE et al. (1926), who believed that a few typhoid bacilli may live for several months. Repeatedly washing the oysters with fresh sea water materially reduced the number of surviving typhoid bacilli. The occurrence of para- typhoid bacilli and other pathogens in sea water is discussed on page 191. Chapter XVI — 183 — Sanitary Aspects In experiments designed to simulate natural conditions in polluted sea water, BEarD and MEeapowcrorT (1935) noted a rapid diminution in numbers of both Eberthella typhosa and Escherichia coli, although some of each survived for more than a month. Representative findings are summarized in Table XXX VIII. The bacteria were suspended in freshly collected unfiltered water from San Francisco Bay in semipermeable mem- brane cells which were immersed in the bay. The rate of death of the bacteria was invariably higher in unfiltered water than in similar water which was sterilized by passage through an L-3 Chamberland candle. TABLE XXXVIII.— Number of enteric bacteria in filtered and unfiltered sea water in semipermeable cells immersed in the sea (from BEARD and MEADOWCROFT, 1035):— Tue Eberthella typhosa PER ML. IN Escherichia coli PER ML. IN pen FILTERED UNFILTERED FILTERED UNFILTERED SEA WATER SEA WATER SEA WATER SEA WATER ° 300,000,000 300,C00,000 500,000,000 500,000,000 I ,000,000 23,400,000 160,000,000 150,000,000 2 50,000,000 14,100,000 144,500,000 125,000,000 4 10,800,000 9,000,000 51,500,000 37,000,000 6 7,200,000 3,000,000 30,500,000 23,000,000 12 270,000 60,000 12,000,000 II,000,000 20 10,000 2,000 3,750,000 2,650,000 35 ° ° 1,200 975 Similar results were obtained by ZOBELL (1936), who suspended sew- age bacteria in different kinds of water in the sea in semipermeable tubes prepared by impregnating porous porcelain filter candles with collodion: SOLUTION IN WHICH BAC- PERCENTAGE SURVIVAL AFTER TIME IN MINUTES TERIA WERE SUSPENDED I 30 60 go 120 “Formula C”’ control 100 100 gl 82 07 Natural sea water 82 47 31 5 3 Autoclaved sea water 85 56 38 27 36 Filtered sea water 88 39 20 23 19 The ‘“‘natural” sea water was unfiltered and unheated. The “filtered” sea water was passed through a Berkefeld-W candle. A continuation of the experiment summarized above showed that, while 99.9 per cent of the sewage organisms were killed after two days suspension in sea water, a few survived for nearly a month. CARPENTER ef al. (1938) found that natural sea water killed 80 per cent of the organisms in sewage within half an hour. Porcelain or diatomaceous earth filters may remove marine sapro- phytes with which the terrestrial organisms cannot successfully compete, or may adsorb bactericidal substances from sea water. KirIBAYASHI and Ara (1934) found that Vibrio comma lived much longer in boiled or ster- ilized sea water than in comparable raw sea water. The average survival time of this organism in harbor water of Kellung, Formosa, was to days. GELARIE (1916) estimated that Vibrio comma could be eliminated by the natural flora and other adverse conditions in New York harbor water within 48 hours. Water from the Black Sea was found by KrRaAsSILNIKOV (1938) to be germicidal for terrestrial bacteria until boiled. Passing the water through ZoBell — 1384 — ‘Marine Microbiology a Seitz filter rendered Black Sea water less bactericidal for adventitious organisms. KRASSILNIKOV stressed the importance of the organic content of water as a factor which affects the survival of bacteria. Coliform bacteria surveys:— The sanitary significance of Escherichia coli, Aerobacter aerogenes, and other coliform bacteria in the sea is con- tingent upon two important considerations. First, do the intestinal tracts of marine animals normally harbor such organisms, and secondly, how long do coliform bacteria of fecal origin survive in the sea? Large numbers of coliform bacteria are introduced in the sea by land drainage, particularly in raw and partially treated sewage. It is estimated from data given by WARREN and RAwWNn (1938) that enough coliform bac- teria are discharged by sewage effluents along the west coast of the United States each day to give over a hundred for every liter of water in the North Pacific Ocean, if evenly distributed. However, such organisms are found only in tide water, harbors, and bays, which are often badly pol- luted. Comparable sanitary conditions were found by WESTON (1938) on the east coast of the United States. _ Extensive surveys around the Hyperion outfall shows the rapidity with which coliform bacteria succumb in the sea. This outfall carries raw sewage from nearly two million inhabitants of Los Angeles a mile into the ocean. As may be expected, millions of coliform bacteria per ml. are often found near the sewer outlet. The number decreases with distance from the outlet much more rapidly than can be accounted for by dilution. Surveys, summarized by KNOWLTON (1929), indicate that even during onshore winds and currents the count is often less than 1o coliform bac- teria per ml. in the surf. As many or more Escherichia coli may be intro- duced by bathers on the beach. Under no conditions were coliform bac- teria traced more than a mile or two from sewer outfalls in the open ocean. The effect of air currents, water movements, composition of the sewage, and other factors which influence the distribution of E. coli were consid- ered by a commission appointed by the California State Bureau of Sani- tary Engineering (1943). Only in solids or greases were coliform bacteria found to be able to survive for long periods of time in the sea. Similar observations were made in Europe by DIENERT and GUILLERD (1940) who concluded that, while sea water is neither antiseptic nor inimi- cal to E. coli, sewage discharged into the sea is rapidly purified by sedi- mentation, predatory organisms, and dilution. Lioyp (1930) was im- pressed by the virtual freedom of Clyde Sea water from coliform bacteria of fecal origin. ZOBELL (19410) failed to find coliform bacteria in any of 961 samples of sea water collected at stations remote from possibilities of terrigenous contamination, although large numbers were found in polluted bays and estuaries. Positive presumptive tests were obtained from the intestinal contents of 203 of the 387 marine fishes examined. The coliform bacteria isolated from the fishes were identified as follows using the ‘‘IMVIC” tests: Escherichia coli 6 per cent Aerobacter species 73 per cent Citrobacter species 2 per cent Imvic is a mnemonic which fixes in order the four tests commonly used in classifying coliform bacteria: (I) indol formation, (M) methyl-red reac- Chapter XVI — 185 — Sanitary Aspects tion, (V) Voges-Proskauer test, and (C) utilization of citrate as a sole carbon source (PARR, 1939). £. coli was found only in feedy fishes taken relatively near shore. Aerobacter species were more abundant than E. colt in the intestinal tracts of fishes. From the survey it was concluded that E. coli or other bacteria which ferment lactose with gas production do not constitute part of the normal intestinal flora of marine fishes, although, if ingested, such bacteria may survive for considerable periods of time. The absence of E. coli from Buzzards Bay and Vineyard Sound near Woods Hole was reported by Browne (1917). £. coli was found in the intestines of only 39.8 per cent of 93 scup which he examined. Clostridium welchii was found in 30.1 per cent of the fishes. The presence of these bac- teria in the fishes’ intestines seemed to be a function of the amount and type of food present. After noting that the bacteria in fewer than half of the 72 offshore fishes which he examined produced gas in lactose broth, GiBBons (19340) concluded that E. coli and Aerobacter aerogenes are present only in fish from contaminated water. He found E. coli in only 6.9 per cent of the positive lactose broth cultures. GRIFFITHS and FULLER (1936) detected only a few E. coli in commercial fish, from which they concluded that the E. coli con- tent of fish is largely due to handling. Additional literature reviewed by GRIFFITHS (1937) indicates that E. coli is not a normal intestinal inhabi- tant of marine fishes. The intestinal contents of sea fowls are generally reported to be free from coliform bacteria except in certain cases where there is evidence that the fowls have been feeding in polluted waters. Little is known regarding the intestinal flora of marine mammals. £. coli does not appear to be a normal inhabitant of the intestines of seals in captivity. The general absence of coliform bacteria in the sea except in areas known to be polluted with sewage confirms the validity of the test for coli- form bacteria as an indicator of sewage pollution. It follows that, wher- ever large numbers of E. coli are found in the sea, the possibility exists of typhoid, dysentery, and cholera organisms also being present. Al- though epidemics of these gastro-intestinal diseases have never been traced to bathing beaches, outbreaks of typhoid have been traced to infected oysters. Coliform bacteria are commonly present in small inland lakes which are more subject to pollution than the ocean. The coliform bacteria count of the hypolimnion of lakes was found by TayLor (1941) to be very low and relatively constant. More coliform bacteria were found in the epi- limnion where the numbers were greatest in summer and autumn, despite less terrestrial pollution during the summer. About 70 per cent of the coliform bacteria isolated from lakes by TayLor were E. coli types. Other sanitary problems of hydrobiological interest are reviewed by WutreLe (1927), PREscorr and WINSLOW (1931), NIKITINSKY (1938), GAINEY (1939), and SUCKLING (1943). Bacteriology of shellfish:— Oysters, clams, and mussels are often eaten raw or in a partially cooked condition. As a result, numerous cases of typhoid fever and Asiatic cholera have been traced to the ingestion of contaminated shellfish. Though normally free from dangerous bacteria in clean water, the pollution of bays and estuaries with unpurified sewage renders shellfish a potential source of infection. According to PRESCOTT and WinsLow (1931) who reviewed the literature on this subject, “In ZoBell — 186 — Marine Microbiology practically all of the American epidemics which have been traced to shell- fish, it was demonstrated that the oysters which caused the outbreak had been ‘floated’ or ‘fattened’ in brackish water near the mouths of polluted streams.” Rigorous control measures have done much to lessen the inci- dence of shellfish-borne diseases during the last two or three decades. The control of the sanitary conditions under which shellfish are raised and handled is a constant problem for health officials. Although typhoid fever is the disease most often discussed in this connection, such diseases as cholera, diarrhoea, and gastroenteritis also may be transmitted by shellfish (TANNER,1944). Botulism from shellfish is of very rare occur- rence. Obviously oysters that have been grown or floated in polluted water are undesirable as articles of human food, regardless of whether or not they contain specific organisms of disease. Several instances of the demonstration of typhoid bacilli in shellfish are related by HUNTER and HARRISON (1928). These workers record evidence for the survival of typhoid bacilli in oysters for from 9 to 42 days and of Escherichia colt for from 7 to 17 days. Shellfish poisoning is discussed on page 199. Dopcson (1928) gives experimental evidence that Eberthella typhosa survived in oysters, mussels, and other shellfish in sea water for more than three weeks. He cites several epidemiological instances of the prolonged survival of virulent typhoid bacilli in mussels and oysters. Large numbers of saprophytic bacteria are ordinarily associated with oysters. These are considered of little sanitary significance except that they promote the decomposition of shellfish. This can be retarded by proper refrigeration, although some marine bacteria slowly multiply at low temperatures. ‘TANIKAWA (1937) reported the slow multiplication of bacteria in oysters at o° C. There was little or no multiplication at — 5° C., as shown by the data in Table XX XIX. TABLE XXXIX.— Bacterial populations of oyster meats after different periods of storage at different temperatures (from TANIKAWA, 1937):— BACTERIA PER ML. OF OYSTER MEAT STORED AT STORAGE Sinica EO o7G: — 5°C. o days 1,600 1,600 1,600 6 days 6,600 3,600 3,400 17 days 66,500 4,100 2,700 24 days 1,600,000 8,900 1,800 TONNEY and WHITE (1926) noted a 458 per cent increase in the Es- cherichia coli content of shucked oysters held for 12 days at 5.8° C. In another lot of oysters the EZ. coli score increased 1490 per cent in 11 days at 5.8° C. The E. coli content of living oysters did not decrease from the eleventh to the 23rd day when stored at 5.8° C. After the 28th day a con- sistent decrease was noted, and after the 6oth day the EL. coli tests were negative. The Committee on Standard Methods for the Examination of Shell- fish of the American Public Health Association has outlined methods for collecting, handling, transporting, and examining oysters for their £. coli content. Oyster meats are scored by determining the highest dilution, in- creasing by powers of 10, in which the presence of £. coli is confirmed by the use of appropriate differential media. The results are recorded as the Chapter XVI — 187 — Sanitary Aspects numerical value of the greatest dilution in which £. coli is found. The sum of five such values is the “‘score.” An illustrative example follows: CONFIRMED PRESENCE OF E£, coli IN TEST NUMERICAL No. - VALUE I.O0 ML o.I ML 0.Or ML I + 5 i — 10 2 + = = I 3 ne a a r 4 f- + — 10 5 + i ae 10 Oyster score: 32 The necessity of confirming the presence of £. coli with dependable tests is emphasized by PERRY (1939) who points out that other types of coliform bacteria associated in large numbers with shellfish may have no sanitary significance. He recommends the Eijkman test in which the differential media are incubated at 46° C. in order to eliminate Aerogenes, Citrobacter, and other coliform types of bacteria which ferment lactose. Perry found that only o.1 to 10 per cent of the coliform organisms in oysters from Chesapeake Bay were E. coli. There was no constant rela- tionship between the two groups. Oftentimes no £. coli were found in too grams of freshly shucked or market oysters in which several thousand representatives of the coliform group were demonstrated. Experimental results of many investigators show convincingly that EF. coli is not an inhabitant of normal, unpolluted oysters (HUNTER and HARRISON, 1928). Likewise Clostridium welchit, Cl. aerogenes, Aerobacter aerogenes, A. cloacae, and Streptococcus faecalis are not found in normal, unpolluted oysters, although oysters are often contaminated with these organisms. Addi- tional data and many references are given by TANNER (1944) on the significance of oysters in the dissemination of disease, the purification of shellfish, microorganisms in market oysters, the bacteriological examina- tion of shellfish, and other aspects of the microbiology of shellfish. Oysters themselves may be susceptible to bacterial diseases. The death of a large number in English oyster beds in 1920 and 1921 was at- tributed by some investigators to bacterial infection, but doubt is ex- pressed by Eyre (1923). From diseased as well as from healthy oysters, Eyre isolated two new species, Vibrio fuscus and Spirillum ostreae. He also demonstrated the presence of Cladothrix dichotoma, five species of Bacillus, and two micrococci, which he believed to be variants of Micro- coccus cinnebareus. None of these organisms appeared to be pathogenic for oysters. Spirochaetes ostensibly parasitic in the gut of oysters were found in the digestive tract of 91 per cent of the Baltimore, Maryland, market oysters examined by DimitroFr (19260). He identified Saprospira grandis, S. lepta, S. puncta, Cristospira anodontae, Cr. speculifera, Cr. modi- alae, Cr. tenua, Cr. mina, Cr. balbianii, and Spirillum ostreae. Earlier, DimitrofF (1926a) described Spirillum virginianum which he found asso- ciated with oysters. BERGEy er al. (1939) report that Cristospira interro- gationis and Cr. pinnae were isolated from the intestinal canal by the scallop, Pecten jacobaeus. The spoilage of oysters takes place in three stages, according to EL1or (1926). First there is a period of rapid increase in acidity due to the bac- ZoBell — 188 — Marine Microbiology terial fermentation of glycogen, followed by a period of abundant gas production. Proteolytic bacteria subsequently complete the disintegra- tion of the oyster tissue. Bacteriology of marine fish:— Woop (1940) deduced, from the ab- sence of coliform bacteria in fish, that the likelihood of marine fish con- veying typhoid, dysentery, or other enteric infections to man is very re- mote except through careless handling. This deduction is in complete harmony with the findings of GRIFFITHS and FULLER (1936) and GIBBONS (19340). From his extensive survey, GIBBONS (19340) concluded that representatives of the genera Escherichia and Aerobacter seldom occur in fish taken from waters at considerable distances offshore. ‘The fecal forms are particularly rare, except in fish taken near shore or in contam- inated waters.” Only 11 of the 412 cultures isolated from the intestines of salmon by FELLERS (1926) proved to be E. coli. The kinds of bacteria which he found in fish slime were essentially the same as those in decomposing salmon flesh. Sarcina lutea, Micrococcus varians, and an acid-forming Streptococcus predominated. Fresh salmon slime contained 370 bacteria per ml. After two hours at 16.7° C. the count increased to 1,950 bacteria per ml., and after 24 hours there were 3,900,000 bacteria per ml. of slime. FELLERS concluded, as did HUNTER (1922), that the organisms respon- sible for decomposition of marine fish are those whose normal habitat is sea water or fish slime. HUNTER isolated 316 cultures, including 85 dif- ferent species, from sea water, decomposing salmon, and salmon canneries. All of them were asporogenous rods except 4 streptococci, 3 sporogenous rods, 1 actinomyces, and 1 pink yeast. Four coliform bacteria were identified. Species of Bacillus predominated among the organisms isolated from mackerel, haddock, halibut, sole, smelt, and butter-fish studied by Har- RISON et al. (1926). The tissues of fresh fish were found to be sterile. The gills act as an important source of infection. Puncturing or otherwise damaging fish introduces bacteria from the slime and intestinal contents. Fish decompose more rapidly when the alimentary canal is full of food than when empty. If fish are beheaded, eviscerated, and frozen soon after catching, they may be kept safely for several months. Spoiled fish may contain up to 400,000,000 bacteria per gram. Bacteria isolated from the surface slime of haddock were identified by REED and SPENCE (1929) as follows: 24 per cent Bacillus, 23 per cent Achromobacter, 22 per cent Pseudomonas, 18 per cent Proteus, 8 per cent Flavobacterium, and 4 per cent Micrococcus. In the intestinal contents of haddock 70 per cent of the bacteria were Proteus species. Members of the coli-aerogenes group were found in the intestinal contents of haddock only occasionally and never in the integumental slime. Achromobacter-like species predominated in the slime of haddock examined by STEWART (1932), followed in abundance by Micrococcus, Flavobacterium, and Pseudomonas. Escherichia coli was not found, and very few species of Aerobacter were iso- lated from haddock. From the slime and feces of 43 marine fish, representing 11 different species, GIBBONS (1934¢@) isolated 80 pure cultures of bacteria, including 31 Achromobacter, 18 Flavobacterium, 15 Micrococcus, 5 Pseudomonas, 5 Ba- cillus, 2 Proteus, 1 Eberthella, and 1 Serratia. Taken collectively, the bacte- rial flora of slime was found to be similar to that of fish feces. Most investi- Chapter XVI — 189 — Sanitary Aspects gators agree that the numbers and kinds of bacteria in the intestinal tract of fish are dependent primarily upon the food ingested recently. Accord- ing to HUNTER (1920), GRIFFITHS (1937), and others, the stomach and intestine of fasting fish are frequently bacteria-free. The flora, which Woop (1940) isolated from salmon, barracouta, whit- ing, mullet, and flathead taken from Australian waters, has a great deal in common with that isolated from marine fish in other parts of the world (see Table XL). He found that, if properly handled, fish may be kept for as long as 8 days at 7° to 8° C. without becoming too stale to be edible. After finding virtually no coliform bacteria of fecal origin, Woop con- cluded that there should be little danger of epidemics due to the consump- tion of fish, provided that a wide berth is given to sewer outfalls by fish- ermen. TaBLe XL.— The bacterial flora associated with fish and water, expressed as a percentage of the cultures examined (from Woop, 1940):— GENUS SEA WATER SLIME GuT GILLS TAP WATER % % % % % Achromobacter 26 19 30 31 30 Micrococcus 34 48 21 41 4 Pseudomonas 10 7 ate) 7 50 Flavobacterium 18 sy) I 12 8 Bacillus 12 9 35 9 8 From salmon of North Pacific waters, SNow and BEARD (1939) iso- lated 1838 cultures which they identified as follows: GENUS PER CENT GENUS PER CENT Achromobacter 53-7 Bacillus 1:5 Micrococcus 12.6 Serratia °.9 Pseudomonas 8.4 Rhodococcus 0.8 Kurthia 8.1 Yeasts 0.5 Flavobacterium 4.9 Aerobacter 0.4 Proteus yy Staphylococcus One Lactobacillus 245 Streptococcus 0.2 Sarcina 2.0 Escherichia o.1 The last four were found only in Columbia River salmon. Additional in- formation on the kinds of bacteria associated with marine fish is given on page 172. The controversial question of the bacterial content of fish flesh is re- viewed by GRIFFITHS (1937). It is generally agreed that the muscles of healthy fish are usually sterile, although some investigators have experi- enced difficulty in consistently obtaining sterile fish tissue. Infection of the muscle occurs immediately after death, and the bacterial population increases rapidly at a rate which is influenced primarily by the temper- ature. HUNTER (1920) observed an increase in the flesh of salmon from zero at the time of catching to as high as 155,000,000 per gram after 96 hours’ storage at from 10° to 21° C. During this period of storage the salmon may be so decomposed as to become unfit for human consumption. Kiser and BEcKwitTH (1944) found from none to 30,000 bacteria per gram of muscle tissue of freshly caught mackerel and from none to 64,000,000 per gram of intestinal contents. Predominating were species of Micrococcus, Achromobacter, Pseudomonas, Flavobacterium, Sarcina, Kurthia, Lactobacillus, and Streptococcus in the order named. ZoBell — 190 — Marine Microbiology After finding only from 20 to 625 aerobic and from none to 60 anaero- bic bacteria per gram of fish tissue, HIGHLANDS and WILLIAMS (1944) concluded that the chief hazard in the canning of sardines is in the hand- ling and processing and not in the bacterial flora normally associated with fish. The bacterial population increased rapidly during processing preparatory to canning. The increase was related more to the nature of the packing table surface than to any other single factor. Neither thermo- tolerant nor thermophilic varieties were encountered. Halophilic bac- teria were present only in low numbers. Eight per cent salt was required to inhibit bacterial growth for a commercially practicable period of time. The spoilage of fish may be retarded or prevented by proper handling to minimize infection of the muscle tissue and by refrigeration. While many bacteria associated with marine fish multiply slowly and are other- wise biochemically active at temperatures ranging from o° to — 5° C. (BEDFORD, 1933a, 6; GIBBONS, 1934c; HEss, 1934a, 0), their proteolytic activities are minimized by refrigeration at sub-zero temperatures. Fast- freezing and storage of mackerel at — 28° C. was found by KisER and BECKWITH (1942) to allay absolutely all bacterial activity. In fact, they observed a decrease in the bacterial flora, including species of Achromo- bacter and Micrococcus, in 15 days at — 20° C. Hess (1934a) reported - reductions of from 40 to 70 per cent in suspensions of marine bacteria frozen in sea water for eight minutes at — 16° C., but all organisms were not killed after 44 hours at this temperature. Some of the bacteria were actively proteolytic at — 3° C. GRIFFITHS (1937) reports a sharp rise in the bacterial population in haddock flesh stored in ice. After 7 to 11 days, counts of 100,000 or more bacteria per gram were found to indicate rapid subsequent decomposition. Fish having 1,000,000 or more bacteria per gram were not considered to be marketable. GRIFFITHS emphasized the desirability of additional research to establish bacteriological standards for acceptable quality of fish. Bacteria often affect the marketability of fish by causing discoloration. BECKWITH (1911) described Diplococcus gadidarum which caused the red- dening of cod and allied fishes. KELLERMAN (19150) who called it Micro- coccus gadidarum, isolated a similar red organism, Micrococcus litoralis, from salted codfish. KLEBAHN (1919) found Micrococcus morrhuae, Sar- cina morrhuae, and Bacterium halobium rubrum associated with reddened dry cod. GiBBoNs (1937) found 30 species of halophilic bacteria, includ- ing two species of Serratia, associated with the reddening of salted fish. A pink yeast, Torula wehmeri, along with Micrococcus albus-translucens, Micrococcus lutulentus, and Bacterium zopfii were isolated from reddened codfish by HanzAwa and TAKEDA (1931). Clathrocystis roseo-persicina, Oidium pulvunatum, Micrococcus roseus, Serratia salinaria, Vibrio halo- bicus desulfuricans, and Torula epizoa have been credited with causing the discoloration of codfish, according to TANNER (1944). The greenish-yellow discoloration of halibut was attributed primarily to Pseudomonas fluorescens by HARRISON (1929), who also isolated from halibut Flavobacterium marinum, Fl. balustinum, Fl. fucatum, Fl. mart, and Achromobacter pellucidum. BEDFORD (1933a) attributed the discolor- ation and subsequent souring of halibut to the activity of various pink, orange, and yellow marine bacteria, in addition to the fresh-water Ps. fluorescens. All of the bacteria were active at o° C., and some developed rb A= Genie Chapter XVI — 191 — Sanitary Aspects Sanitation of sea-water baths:— The dangers from bathing in pol- luted waters are well known. Gastro-intestinal infections and certain respiratory infections have been traced to infected swimming pools. Some health officers in coastal cities have mistakenly assumed that sea-water pools do not present the same sanitary problems as do fresh-water pools. Though summarized epidemiological data are not at hand, there are frag- mentary accounts of communicable diseases having been contracted in sea-water baths. The prolonged survival of pathogenic bacteria in modi- fied sea water is indicative of the public health hazards. Unless properly treated, sea-water swimming pools may be more menacing than fresh water. Municipal water supplies used as a source of fresh water for swimming pools are usually sanitary, whereas sea water may be pumped directly from polluted bays or beaches. Moreover, the chlorination or chemical treatment of sea water presents unique problems owing to its salt content. The control of algal growths in sea-water pools exposed to light pre- sents another problem. When enriched with nitrogenous wastes from bathers, both sessile and planktonic algae grow most profusely in sea water.* Bacteriology of ice:— The bacterial content of sea ice presents no special sanitary problems because there is little likelihood of human patho- gens being in the sea, and little sea ice is used for the preservation of foods or for the preparation of drinks. These generalizations, however, do not apply to the large quantities of ice which are harvested from lakes and rivers. From his review of the literature and his own investigations, JENSEN (1943) concluded that, if ice harvested from lakes is handled properly, it is not an important factor in the spread of typhoid or other enteric fevers. It is always a potential source of danger, though, because bacteria, such as the typhoid bacillus, may remain viable for several months in ice. FRANKLAND and FRANKLAND (1894) described experiments of PrupD- DEN in which there were 1 million typhoid organisms per ml. of ice held at from o° to — 10° C. for 11 days, 72,000 after 77 days, and 7,000 after 103 days. The typhoid bacillus tolerated continuous low temperature in ice better than alternate freezing and thawing. According to the FRANK- LANDS, ice from various lakes in the vicinity of Berlin contained from a few to as many as 14,400 bacteria per ml. JENSEN (1943) found coliform bacteria in only a small percentage of the samples of ice recently harvested from lakes of the upper Mississippi River valley. An average of 22 coliform bacteria per liter were detected * According to an editorial in Public Health News, of New York State Department of Health, Volume 12, No. 3, page 402, polluted harbor waters may be responsible for outbreaks of conjunctivitis, otitis, tonsilitis, rhinitis, sinusitis, sore throat, furuncles, laryngitis, and ring- worm, besides various enteric infections. The Second Quarterly Report of the New York State Department ot Health for 1932 states ‘from all of the data on hand it is very probable that most of the increase (in typhoid fever) may be charged to bathing in polluted harbor waters condemned by the Department of Health.” A higher incidence of typhoid fever was observed in the city blocks bordering waterfronts and tide flats. The California Bureau of Sanitary Engineering (1943) details the case of a lifeguard who presumably contracted para- typhoid fever from gulping several swallows of surf water while m: aking a rescue near E] Se- gundo, California. In this region both Paratyphoid A and B organisms were isolated from water samples taken 200 or 300 feet of the Hyperion outfall. As a result of a sanitary survey of certain beaches around Los Angeles, the Bureau has caused to be posted signs which read, “WARNING: This beach from high tide line seaward, including adjacent shore waters, is polluted with sewage and is dangerous to health. The public is excluded from these areas under order of the California State Board of Public Health.’ Schistosomiasis or swimmer’s itch and fungus infections are not uncommonly contracted by sea bathers. ZoBell — 192 — Marine Microbiology in 41 cakes of ice examined in 1943. The average aerobic plate count was 4 per ml. at 37° C. and 7 per ml. at 20° C. Fifteen samples were sterile or showed fewer than two bacteria per ml. JENSEN asserted that Escherichia coli died off in ice more rapidly at 0° to — 5° C., the temperature of natural ice on lakes, than at — 20° C. or lower. £. coli remained viable for long periods when frozen in distilled water at — 16°, — 4o°, and — 79° C. Species of Achromobacter, Aerobacter, Bacillus, Cellulomonas, Chromobac- terium, Flavobactertum, Micrococcus, Proteus, Pseudomonas, Serratia, and Spirillum were found in newly harvested lake and river ice. Bacteria were found in polar ice and snow by GazErT (1902), MCLEAN (1918), and DARLING and SIPLE (1941). It was directed to the attention of the author by Dr. E. B. FREp of the University of Wisconsin that more bacteria have been found in water im- mediately under the ice on Lake Mendota than at greater depths. This observation was confirmed by demonstrating the presence of from two to three times as many bacteria in the water adjacent to a 4o-cm. layer of ice covering Lake Mendota as in water samples collected at depths of from two to five meters beneath the ice. The ice itself generally contained fewer than a hundred bacteria per ml. compared with several thousand bacteria per ml. found in the underlying water. There are several pos- sible explanations, none of which has been established as the cause of the concentration of bacteria next to the ice: (a) Nutrients may be somewhat more concentrated in the water adjacent to ice because as water solidifies, most of the dissolved solids remain in the liquid fraction. (6) Ice may pro- vide a beneficial solid surface for increased bacterial activity or for adsorp- tion of nutrients (see page 84). (c) Trihydrol, a form of water which pre- dominates in ice, may stimulate the growth and activity of bacteria, as suggested by the work of BARNES and JAHN (1934). Harvey (1933) noted that recently melted ice water seemed to stimulate the growth of diatoms. While ice cannot be relied upon to prevent the spoilage of fish and other flesh foods, it has a preservative value. According to TARR and SUNDER- LAND (1940), ice prepared from water treated with o.1 per cent of either benzoic acid or sodium nitrite is relatively effective for preserving fish fillets and other flesh foods. JENSEN (1943) reports that chlorine-water ice, azochloramide ice, katadyn silver ice, sodium propionate ice, and other kinds of germicidal ice show promise for various purposes. Ice presents special problems where dilution and sedimentation are depended upon to minimize the danger from sewage discharged into rivers or lakes. Unless proper precautions are taken, the warmer, less dense water from sewage effluents may flow over the surface of colder, more dense water, and under certain conditions even over the surface of ice for considerable distance. Such a condition is very noticeable in Great Salt Lake where fresh water flowing over the surface of the denser saline water in mid-winter freezes to form a sheet of ice, over which more fresh water flows farther and farther from land, until higher temperatures or turbu- lence breaks up the ice sheet. Bacteria and allied microorganisms preserved in glacial ice may be transported hundreds of miles to sea in icebergs. Icebergs play an impor- tant role in the transportation of sedimentary materials at high latitudes. Chapter XVII ECONOMIC IMPORTANCE OF MARINE MICROORGANISMS Marine microorganisms are of economic importance in many ways besides causing diseases and bringing about the decomposition of marine animals and commercial algae. Their indirect effects upon the primary productivity of the sea and their possible role in the origin of oil have already been mentioned. A few additional ways in which bacteria and allied microorganisms are of direct economic concern to man are outlined on the following pages. Fouling of submerged surfaces:— In nautical parlance fouling is the attachment and growth of a heterogeneous assemblage of plant and animal organisms on ships’ bottoms, piles, water conduits, and other submerged structures. Virtually all types of structural materials, whether they be of wood, metal, concrete, glass, or plastic, sooner or later become fouled when submerged in the biotic zone. The assorted organisms are popularly known as “‘ barnacles,”’ “‘shells,”’ “seaweeds,” or ‘‘moss.”” While barnacles or algae are usually the principal offenders, not infrequently a hundred or more different species of organisms have been identified in fouled surfaces, exclusive of bacteria and allied microorganisms. Sometimes nearly a hundred tons of fouling organisms are found on the bottom of a large ves- sel after it has been in the water for a year. Several pounds of fouling or- ganisms may accumulate on the bottom of a flying boat within a few days. By increasing the resistance of ships in water, fouling organisms dimin- ish the speed of a vessel, prolong the voyage, increase fuel consumption, and augment wear and tear on the machinery. Fouling organisms necessi- tate the drydocking of vessels at frequent intervals for cleaning, scraping, and re-painting, costly processes which take the average vessel out of commission for three or four weeks each year. The fouling problem is of gravest concern to the Navy, particularly when operating far from home bases. Microorganisms are the primary film formers on submerged surfaces. On badly fouled surfaces bacteria may constitute as much as 8 or 9 per cent by volume of the total cumulation. Bacterial attachment to previ- ously cleaned glass surfaces may be detected in less than an hour following immersion in the sea. The numbers increase more or less geometrically with time until their abundance, together with the simultaneous attach- ment and growth of diatoms, algae, protozoans, suctorians, and various larvae, defeats census attempts. This may be illustrated by data summa- rized in Table XLI on page 194. Laboratory as well as field observations (ZoBELL and ALLEN, 1935) suggest that bacteria may play an important role in the fouling of sub- merged surfaces. This they may do in a variety of ways: (z) By affording the planktonic larval stages of fouling organisms a foothold or otherwise mechanically facilitating their attachment. (2) By discoloring glazed or bright surfaces. VISSCHER (1927) and others have shown that bright, ZoBell — 194 — Marine Microbiology light-reflecting surfaces are fouled less readily than dark or discolored ones. (3) By serving as a source of food. Barnacles, mussels, tunicates, and other fouling organisms are nourished by bacteria (see page 173). (4) By promoting the deposition of the calcareous cements of sessile organ- isms. (5) By increasing the concentration of plant nutrients, including CO, and ammonia, which result from the bacterial decomposition of organic matter. TaBLe XLI.— Numbers of different types of fouling organisms found per square inch of glass slide after different periods of submergence in the sea at La Jolla, California (from ZOBELL, 1939b):— ORGANISMS 24 HOURS 48 HOURS 96 HOURS Bacteria 1,876,000 13,240,000 78,100,000 Diatoms 940 3,750 8,200 Protozoans 72 290 1,100 Barnacle larvae Out On3 Ih Other organisms 389 1,360 9,700 Under certain conditions the primary film formers may form a pro- tective layer over antifouling paints designed to discourage barnacles, or they may otherwise effectively reduce the toxicity of antifouling paints. Bacteria attack and slowly decompose certain protective surface ma- terials. On the other hand, under certain conditions bacterial films may help to keep surfaces free from larger fouling organisms by the production of antibiotic substances. The interrelationships among the organisms in fouling cumulations are very complex and not well understood. The available information stresses the desirability of taking microorganisms into account in the scientific approach to the preparation of antifouling surfaces (ZOBELL, 19380). Algae, particularly species of Ectocarpus and Herposiphonia, diatoms, hydroids, barnacles, oysters, bryozoans, and serpulids were the most abundant sedentary organisms observed on submerged surfaces by COE and ALLEN (1937) during their nine years’ study at La Jolla, California. Sixty species of diatoms, 15 species of algae, and 8 species of foraminifera were commonly recognized on submerged plates, along with a diversity of sponges, hydroids, nemerteans, annelids, bryozoans, mollusks, crusta- ceans, and ascidians. No record was kept of the presence of bacteria and allied microorganisms. Bacteria associated with wood-borers:— Wooden structures exposed in the sea are subject to the depredations of several kinds of destructive animals of which the drilling mollusk or “ship-worm,”’ Teredo navalis, and the boring gribble, Limnoria lignorum, are the commonest. Details re- garding these and other boring animals and the extent to which they dam- age pilings, wooden boats, timbers, etc., are given in the literature sum- marized by CROSTHWAITE and REDGRAVE (1920). Additional informa- tion is given by Kororp (1923) who believes that Limnoria digests the wood which it swallows. Doubt is expressed that Teredo utilizes wood as food. It is an inadequately explored possibility that these and other wood- boring animals depend partly or largely upon bacteria for their nutrition. Plankton organisms have been regarded as the principal food of Teredo navalis, although Dore and MILLER (1923) found that wood loses about 8o per cent of its cellulose and 15 to 56 per cent of its hemicelluloses during Chapter XVII — 195 — Economic Importance passage through the digestive tract of Teredo. The lignin content was de- creased only slightly. However, it is still indeterminate whether the cellu- lases and lignases are elaborated by Teredo or by commensal bacteria. The gut of engorged Teredo as well as its burrows contain large numbers of cellulose- and lignin-digesting bacteria. Conceivably the bacteria convert the cellulose and lignin into products which are more readily assimilated by the wood-borers. The bacteria themselves are nutritious. The number present at any one time on the wood or in the gut of wood-borers is not enough to provide for the complete requirement of the wood-borers, but the “standing crop”’ of bacteria is not an adequate criterion. Considering that bacteria repro- duce every hour or two under favorable conditions, the standing crop multiplied by 10 to 20 would be more representative of the amount of bacteria available per day as food. Cellulose- and lignin-decomposing microorganisms may help to condi- tion the wood for initial attack by wood-borers. The extensive degrada- tion of wooden structures by fungi in the sea has been reported by BARG- HOORN and LINDER (1944), who observed a conspicuous softening in the outer parts of pilings and submerged wood. The activities of such organ- isms may help to explain why wood becomes increasingly more suscep- tible to attack by wood-borers as its period of immersion is prolonged. Given a sufficiently extended time, either bacteria or fungi could inde- pendently effect the complete deterioration of wooden structures. The rate, though, is far from being commensurate with the rapid destruction of wood by Teredo or Limnoria. Destruction of cordage and fish nets:— After periods of from three to five weeks’ immersion in the sea, textile fibers were found by DorEE (1920) to be completely rotten. Fabrics had become coated with a bac- terial or diatomaceous film within a week after immersion. Cotton was found to be less durable than linen or silk. Wool was the most resistant. The deterioration was shown to be caused by microorganisms and not by abiogenic oxidation, light, or salts. The deterioration of fish nets in Lake Erie was found by ROBERTSON and Wricut (1930) to be due to cellulose-digesting bacteria. Both linen and cotton lines, seines, and nets were attacked. According to RoBERT- SON (1931), the value of fiber seines, nets, traps, and lines used by com- mercial fishermen in the United States in 1930 was about $15,000,000. Such equipment lasts an average of less than two years, its durability be- ing affected primarily by the activities of cellulose-decomposing bacteria. The usefulness of the nets, seines, and lines can be extended by applying copper resinate and other preservatives. The tendency of certain pre- servatives to decrease the flexibility, impart undesirable colors, or other- wise adversely affect the properties of the fiber equipment complicates the problem. Manila ropes and cotton nets were found by ATKins and WARREN (1941) to be destroyed after 14 months alternate wetting in sea water and drying, as in ordinary use. Preliminary treatment of the fibers with cop- per naphthenate was found to prolong the useful life of the rope by 40 per cent. The occurrence of fungi on the North Atlantic coast which infect cord- age fibers, including hemp, jute, and sisal, causing deterioration under marine conditions has been reported by BARGHOORN (1942). BARGHOORN ZoBell — 196 — Marine Microbiology and LINDER (1944) have investigated the physiology of several such fungi. According to IMSHENETSKY and Koxurina (1941), microorganisms cause the destruction of jute coverings on ships. Bacterial deterioration of cork and rubber :— Cork is a ligno-cellulose- suberin complex filled with air spaces which are responsible for its buoy- ancy. Large quantities of cork are used for floats on fish nets, fish lines, life preservers, etc. Although relatively resistant to bacterial attack, cork is decomposed by marine microorganisms which slowly destroy its buoyancy by rupturing the cell walls of the cork. Eventually, pieces of cork continuously or periodically exposed to sea water break to pieces. Rubber is generally regarded as being biologically inert, but highly purified rubber, both natural and synthetic, as well as various rubber products, are susceptible to bacterial oxidation in the presence of minerals and moisture. Even the small areas of rubber gasket exposed to water in citrate of magnesia bottles filled with sea water causes the consumption of oxygen as the rubber is oxidized by bacteria. According to ZOBELL and BECKWITH (1944), rubber is attacked by many marine bacteria, including species of Actinomyces, Mycobacterium, Micrococcus, Micromonospora, Nocardia, Pseudomonas, and Bacillus. This is not particularly surprising when it is recalled that rubber is an unsatu- rated hydrocarbon having the composition (C;Hs),. Unsaturated hydro- carbons are quite susceptible to bacterial oxidation. Synthetic rubbers are closely related chemically to natural rubber. Some of the synthetic rubbers are more readily oxidized by bacteria than is natural rubber. Rubber products are prepared by compounding vulcanizing agents, accelerators of vulcanization, antioxidants, softeners, fillers, etc., with rubber hydrocarbon. Manufacturers have devoted little attention to the development of antimicrobial agents for rubber products, for the obvious reason that they have been more concerned with wearing qualities, heat- resistance, tensile strength, elasticity, vulcanizability, and other proper- ties than with biological inertness. Only when it is continuously in con- tact with moisture does the bacterial deterioration of rubber become a practical problem. Most rubber products, including hoses, bumpers, rub- berized products, certain chlorinated rubber paints, bearings, etc., used at sea are either submerged or subject to frequent wetting with sea water. For such rubber products the development of antimicrobial qualities may be desirable. Halophilic bacteria in solar salt:— Typical marine bacteria are hardly halophilic since very few of them grow well in media containing more than 5 per cent salt. However, a small percentage of the bacteria in the sea are able to grow in saturated salt solutions. Such organisms are particularly abundant in marine salterns where halophilic chromogens often impart a red color to the brine and crude salt. PErRcE (1914) regarded red chromo- genic bacteria as being the principal cause of the pink to red coloration of San Francisco Bay salterns, although the algae, Protococcus salinus and Dunaliella salina, may be partly responsible. PErIRcE did not identify the bacteria. They were obligate halophiles, growing in concentrated brine but not in diluted brines. The bacteria grew on salt codfish, giving it a red color. Unless measures are taken to sterilize re-crystallized and purified solar salt, it may carry halophilic bacteria which are instrumental in the Chapter XVII — 197 — Economic Importance spoilage of raw furs, fish, caviar, meat, pickles, and other products on which salt is used as a preservative. According to RAHN (1934), natural rock salt as well as crystallized salt manufactured from rock salt is prac- tically free from bacteria, except such as it may gather up in the process of shipment, storage, and handling. However, salt from marine or solar salterns often carries halophilic bacteria (CLAYTON, 1931). CLAYTON and GrBBs (1927) traced pink halophiles to sea salt which stained salted hides and produced pink blotches on salt fish. BROWNE (1922) traced the discoloration of fish to Bacterium halo- philicum, which was isolated from sea salt along with Spirochaeta halo- philica. BAUMGARTNER (1937) discovered Bacteroides halosmophilus in salted anchovies. The red, brownish, and other spoilage organisms associated with salted food products were traced by PETROWA (1936) to saline lakes from which the salt was reclaimed. Other problems attending the preservation of food with solar salt are discussed by TANNER (1944). The cause of the discoloration of salt codfish was shown by HARRISON and KENNEDY (1922) to be Pseudomonas salinaria that had been intro- duced with solar evaporated salt used in curing the codfish. It, like Sarcina litoralis, Bacterium trapanicum, and several other species of halo- philes isolated from salt samples and spoiling salt fish by GIBBONS (1937), grew in media containing 20 per cent NaCl. Hor (1935) gives additional information and literature on bacteria which cause the spoilage of various foods in concentrated salt solutions. He points out that halophilic bac- teria are widely distributed in salt lakes, limans, salt gardens, soil, and elsewhere in nature. STUART (1936) detected the presence of proteolytic and chitinoclastic bacteria in nearly all of the 27 samples of solar salt which were examined from different parts of the world. He likewise noted the association of chromogenic halophiles with the reddening of salted fish and hides. StTu- ART believes that halophilic bacteria introduced with salt used for curing may be responsible for damage to skins and hides. Using silica gel media containing 14.5 per cent salt, MoorE (1940) demonstrated the presence of halophilic bacteria in several samples of packing house salt, crude solar evaporated sea salt, tainted steer hides, and tainted sealskins. Before effective control measures were taken, microorganisms damaged large numbers of Alaska sealskins which had been packed with salt as a pre- servative on the Pribilof Islands preparatory to shipping to the processing plant in the States. Spoilage of marine food products :— Reference has already been made in preceding chapters to the microorganisms which cause spoilage of oys- ters and fish. Such microorganisms present special problems to food technologists, because many of the microorganisms are active at the tem- perature of ice, some grow in concentrated salt solutions, others are both psychrophilic and halophilic, and fish tissues are very susceptible to de- composition. Much progress has been made in recent years in improving the quality of “fresh” marine food products by fast-freezing processes, rapid transit, and more careful handling, but the marine microbiologist is still confronted by numerous unsolved problems in this field. Typical of the problems confronting the section of the canning indus- try specializing in sea foods is the blackening of lobsters. The thorough investigations of REED and MacLeEop (1924), revealed that species of Pseudomonas, Flavobacterium, and Bacillus, along with other natural bac- ZoBell — 198 — Marine Microbiology terial flora of lobsters, rapidly produced ammonia and HS after the lob- sters were scalded. The H2S reacted with iron in the lobster flesh, result- ing in undesirable blackening. Prompt acidification of the prepared meat, followed by immediate sterilization, proved to be remedial. Canneries are confronted by the additional difficulty that the raw material is usually not delivered in as fresh condition as that in which meat, fruit, or vege- tables can be procured. No conceivable canning process can correct prior decomposition. In an effort to determine the cause of the rapid spoilage of fish and its attendant large economic loss to man, LUCKE and SCHWARTz (1937) made a bacteriological study at various times during the process of catching and marketing fish. The bacterial content of the high seas, about 50 bacteria per ml., was not regarded as the serious source of infection encountered in polluted near-shore water, which in many places contained millions of bacteria per ml. Fishes taken from water having a high bacterial content did not keep well and had an offensive taste. The fish seemed to become infected by handling on board ship, particularly when handled in such a way that the intestinal contents were voided or the skin broken. The bacteria of polluted sea water, ice, and fish holds were regarded by LucKE and ScHWwaArvTz as being secondary sources of infection. They em- phasized the importance of proper killing, cleaning, and washing proce- dures to minimize bacterial spoilage. Bacteria were found to multiply rapidly in infected fish, even in iced holds, resulting in relatively high bac- terial counts by the time that the fish were unloaded in port. Conse- quently the fish were subject to rapid spoilage on shore unless frozen. A mixture of salt and ice instead of ice alone was recommended in order to obtain a temperature sufficiently low to retard effectively the multipli- cation of spoilage bacteria. Similar observations were made by Woop (1939) who also stressed the importance of proper handling and refriger- ation. The value of benzoates, nitrites, and other preservatives used in conjunction with ice to allay spoilage is discussed by TARR and SUNDER- LAND (1940) and by TANNER (1944). ' Frozen fish were observed by STEWART (1935) to spoil more rapidly when permitted to thaw than when held at the same temperature without previous freezing. She recommends refrigeration temperatures of from — 12° to — 21° C. for prolonged preservation of marine fish. REAy (1935) confirmed STEWART’s observations on the effects of alternate freezing and thawing, but he found that fish could not be kept indefinitely even at — 21° C. without undergoing undesirable changes in quality. An appalling quantity of obviously spoiled fish is dumped or processed for fertilizer owing to inadequate control of microbial processes prior to the delivery of the fish to the retailer or cannery. There is a division of opinion among authorities regarding how much decomposition is allow- able before the food is no longer fit for human consumption. Fish and other marine foods which are partially pre-digested by certain non- pathogenic bacteria may be entirely wholesome, but those which have been properly preserved are generally more palatable. The solution of this problem presents a challenge to the marine microbiologist and food technologist. Food inspectors have been guided by one or more of the following properties of commercial fish in passing upon its freshness or eatability: Discoloration, physical consistency, odor, bacterial population, ammonia content, trimethylamine value (BEATTY and GIBBONS, 1937), and tyrosine Chapter XVII — 199 — Economic Importance content. Further details of this problem are given by Dyer et al. (1944) who have developed what they believe to be a practicable method for determining the index of spoilage by measuring the pH at the surface of fish or fillets with a glass electrode. The range of fresh fish flesh was found to be from pH 6.2 to 6.8. Between pH 6.8 and 7.5 the fish was found to be in an incipient stage of spoilage, and above pH 7.5 the fish is in an advanced stage of decomposition. The test was applied during a two year period to cod, haddock, and flounder with equally satisfactory results. Histamine produced post mortem in fish muscle as a result of bacterial activity is believed by GEIGER et al. (1944) to be a good criterion of the keeping quality or stage of decomposition of fish. Histamine results from the decarboxylation of histadine, one of the amino acids composing fish flesh. Many theories have been advanced to account for the occurrence of poison in mussels and other shellfish. Ptomaine or toxin production by putrefactive microorganisms has been regarded as a possible cause. How- ever, it has now been established by the observations of SOMMER and as- sociates at the University of California that the food of shellfish is the source of the poison. The poisonous food has been shown to be a dino- flagellate called Gonyaulax catenella. Gonyaulax digitale, G. polygramma, G. spinifera, and G. triacontha may be involved also, although most out- breaks of mussel poisoning on the California and Oregon coasts have been associated with an abundance of G. catenella in coastal waters. There is some evidence that species of Ceratium, Prorocentrum, Gymnodinium, Noctiluca, and possibly other genera of diatoms or dinoflagellates may contain the toxic principle. The identical poison has been demonstrated in sand crabs. Further information and additional references on shellfish poison are given by SoMMER e¢ al. in Archives of Pathology, Volume 24, pages 537-559 (1937). Seaweeds which are used for food, agar, alginates, fibers, insulation, paper pulp, and other purposes are susceptible to decomposition by bac- teria during transportation and storage. Unfortunately little is known regarding the microorganisms responsible for the spoilage of seaweeds. Control measures are largely lacking in scientific principles and effective- ness. r ‘ i Ambergris is a solid, fatty substance produced by the sperm whale. Pure ambergris has a peculiar sweet, earthy odor which makes it invalu- able as an ingredient of perfumes, but that found floating in the sea and in the intestines of whales has a disagreeable smell. It has been suggested that the activities of bacteria which slowly decompose crude ambergris are responsible for its foul odor. On the other hand, BEAUREGARD (1897) stated that bacteria tend to purify ambergris by decomposing extraneous material. From concretions of ambergris he isolated an organism which he called Spirillum recti physeteris. Chapter XVIII MICROBIOLOGY OF INLAND WATERS Frequent reference has been made to conditions in inland seas, lakes, and limans in the preceding chapters. The general relationships of organ- isms to each other and to the environment are much the same in inland bodies of water as in the oceans, although there are certain qualitative and quantitative differences. The latter are probably no greater in magni- tude, however, than the differences which exist between different parts of the ocean. Though each environment may have unique features, fun- damental observations of conditions in one body of water often help to explain conditions in another. A few of the outstanding microbiological features of certain unique bodies of inland water are outlined on the fol- lowing pages. A key to the periodical literature is also given. The Black Sea:— Peculiar hydrographic conditions, coupled with microbiological activities, render nine-tenths of the Black Sea virtually uninhabitable by any form of life except bacteria. Since the influx of fresh water from precipitation and land drainage exceeds evaporation, the density of the surface waters (salinity about 16°/ 9) is sufficiently less than that of bottom water (salinity up to 23°/o) that there is little vertical mixing. As a result, the oxygen content of the water below the photo- synthetic zone is renewed only very slowly. The oxygen which does penetrate the water is effectively consumed by bacteria either directly or indirectly, so that there is little or no oxygen present at depths exceeding 200 meters. The lower limit of animal life appears to be from 130 to 190 meters. At greater depths, only anaerobic bacteria are active. In the oxygen-poor waters immediately below the photosynthetic zone, bacteria consume oxygen while oxidizing the organic matter which is raining down from the productive surface layers. Sulfur bacteria are also believed to be instrumental in the utilization of oxygen in the zone wherein the overlying oxygenated water merges with deeper H,S-containing water. The abiogenic oxidation of H2S likewise helps to deplete the oxygen content of the water. The H:S results partly from the anaerobic decomposition of albumi- nous compounds. More is believed to result from the bacterial reduction of sulfate. According to DANILTCHENKO and TSCHIGIRINE (1926), sulfate reduction is most intense on the bottom of the Black Sea where the con- centration of H:S sometimes reaches 6000 ml./L., calculated at 760 mm. pressure and o° C. It was in the Black Sea where sulfate-reducing bac- teria were first demonstrated by ZELINSKI (1893). Since that time sulfate reducers have been observed in the Black Sea by numerous investigators. IsSATCHENKO (1924) found vigorous sulfate reducers, presumably Desulfo- vibrio desulfuricans or D. aestuarii, in all samples examined. Some of the samples were collected from depths as great as 2,118 meters. The cultures produced from 0.3 to 0.5 gm. of HeS per liter of media in the laboratory. ISSATCHENKO (1929) asserted that H,S-producing bacteria are respon- sible for the formation of ferrous sulfide in the Black Sea. According to Chapter XVIII — 201 — Microbiology of Inland Waters him, H2S reacts with iron intracellularly to give black inclusion bodies o.5 # in diameter or larger: H2S + Fe(HCOs). = FeS + 2 H:CO; After the death of the bacteria, crystals of ferrous sulfide are liberated. The Black Sea gets its name from the color caused by ferrous sulfide. ISSATCHENKO and EGoROVA (1939) were unable to confirm EGounov’s celebrated hypothesis that there is a ‘bacterial plate”’ (see p. 161) or zone of sulfur bacteria in the Black Sea where oxygenated surface water merges with H,S-containing bottom water. After failing to find sulfur bacteria in samples taken at one meter intervals throughout the boundary layers, it was concluded that H2S must be oxidized abiogenically as it diffuses up- ward into oxygenated water. However, RAVICH-SHERBO (1930) found large numbers of Thiobacillus thioparus in the so-called “thin layer”’ or “bacterial plate.”” KNnrpowitscu (1926) reported the presence of T/io0- pedia rosea and other sulfur bacteria in the Black Sea as well as in the Caspian Sea and the Sea of Azov. In the Caspian Sea, where hydrographic and microbiological condi- tions are akin to those in the Black Sea, BuTKEvicH (1938) found large numbers of sulfate reducers. Near the mouth of the Volga River he found from one to two million bacteria per ml. of water, or the equivalent of a bacterial biomass of about 1 gram per cubic meter. This compared favor- ably with the biomass of plankton algae. The bacteria were very active biochemically. Denitrifiers were widely distributed. Both denitrifying and nitrifying bacteria were reported in the Black Sea by KnrpowITscH (1926). As pointed out by ISSATCHENKO (1926), the presence of H.S pre- cludes the possibility of nitrification in deeper water, but nitrifying organ- isms were abundant in shallow, sandy, or shelly bottoms. The investigations of PoTERIAYEV (1936) on sanitary problems attend- ing the disposal of sewage in the Black Sea are noteworthy. Except in closed basins such as bays or firths, there is generally an intermixing of sea water with sewage, which results in a rapid coagulation and biochem- ical oxidation of the latter. RAVICH-SHERBO (1936) diagnosed a fatal disease of Amphioxus lan- ceolatum in the Black Sea as being caused by a red chromogenic bacterium which he described briefly, but did not identify. Bacteriology of Russian limans:— The shallow salt lakes bordering the Black Sea have been the subject of extensive and intensive study by microbiologists. Unique conditions exist here where the salinity ranges from a few parts per thousand (when flooding rivers overflow into the mud lakes or limans) to concentrated salt solutions following prolonged periods of drought and evaporation. Especially noteworthy are the voluminous contributions of RUBENTSCHIK and associates on the limans or mud lakes around Odessa. Representative of the work of RUBENTSCHIK (1925) are his observa- tions on urea-splitting and proteolytic bacteria which are active in saline media at o° to — 2° C. He isolated and described Sarcina psychrocarteria and Bacillus psychrocartericus, both of which attacked urea at — 2.5° C. From the Kilyalnizki Liman, RUBENTSCHIK (1928a) isolated sulfate reducers which used the decomposition products of cellulose as an energy source but not cellulose itself. The sulfate reducers were able to grow in media containing from o to 20 per cent NaCl. In nature, the bacterial ZoBell ee Marine Microbiology destruction of cellulose probably provides the energy source for sulfate re- duction. Both aerobic and anaerobic cellulose decomposers are widely distributed in the limans, according to RUBENTSCHIK (1933). He (19280) isolated Actinomyces melanogenes and several other aerobic cellulose de- composers from liman mud and water. A good review of the microbiology of liman mud is given by RUBENTSCHTIK and GOICHERMAN (1935), who show that nitrifiers which oxidize ammonia to nitrite are active in concen- trated salt solutions. Azotobacter-like organisms were found in the liman mud but there was no evidence reported that they fixed nitrogen in the limans. The freshening of certain limans in the Odessa region from 1932 to 1934 owing to floods from increased precipitation was found by RUBENT- SCHIK and GOICHERMAN (1936) to be responsible for an appreciable in- crease in the bacterial content. In terms of separate physiological groups the increases were as follows: Sulfur bacteria 100 to 10,000 times Sulfate reducers Io to 1,000 times Putrefying bacteria 10 to 1,000 times Nitrifying bacteria 1o to. ~— Ioo times Denitrifying bacteria 100 to 10,000 times Urea splitters 100 to 10,000 times Cellulose anaerobes 1o to —Ioo times Cellulose aerobes 10 to 1,000 times The salt optimum of halophilic sulfate reducers isolated from the limans was observed to decrease from an average value of from 5 to 8 per cent NaCl in 1932 to an average of from 2 to 4 per cent in 1934 after the freshening of the limans. Most of the bacteria occurring in the mud were reported by RUBENT- SCHIK et al. (1936) to be adsorbed on the sediment particles. Adsorbed bacteria may survive in mud for long periods without undergoing detect- able changes. Nitrification is diminished and sulfate reduction is in- creased when the bacteria responsible for these processes are adsorbed on mud particles. In limans near Odessa containing 256°/o) of salt, BARANIK-PIKOWSKY (1927) demonstrated the presence of denitrifiers, H»S-producers, and other physiological types of bacteria. He described several new species to which he applied numbers but no names. BERGEY et al. (1939) have assigned names to his numbered species as follows: No. 19 Micrococcus halophilus No. 27 Achromobacter galophilum No. 22 Micrococcus pikowskyi No. 30 Flavobacterium halophilum No. 25 Achromobacter pikowskyi No. 36 Achromobacter halophilum The extensive literature on the microbiology of liman mud has been summarized by ISSATCHENKO (1938). Great Salt Lake :— This extremely saline body of water, now covering an area of 1,120 square miles in northern Utah, is the remnant of Lake Bonneville, an old fresh-water lake 19,000 square miles in area. At the present time it is saturated with NaCl, NasSO,, and probably CaCOs, the water containing about 335 grams of salt per liter, or about ten times as much as normal sea water. Pack (1919) reported that one crustacean, four protozoans, nine algae, two fly larvae, and several bacterial species live in Great Salt Lake. Two ciliates inhabiting the lake were found to Chapter XVIII — 203 — Microbiology of Inland Waters feed on bacteria, small plants, and small protozoans. The brine shrimp, Artemia gracilis, which abounds in the lake, was shown by JENSEN (1918) to be an obligate halophile. Lake water diluted to a specific gravity of 1.044 to 1.027 was found to be most favorable for the development of Artemia. The specific gravity of undiluted lake water is about 1.15. In his review of the literature on life in Great Salt Lake, EARDLEY (1938) lists the brine shrimp, 3 flies, 5 protozoans, and 13 species of algae. He relates that at times the brine shrimps ‘“‘congregate in such numbers as to tint the water over wide areas.” During certain years the puparia of the fly Ephydra were reported to drift upon the shore in long windrows, and countless swarms of the adult flies were observed over the water, in which they drop their eggs freely. The extensive calcareous bioherms along the exposed lake shores were believed by EARDLEY to consist princi- pally of deposits of the alga, A phanothece packardii, although the possible importance of bacterial activity in the precipitation of the carbonate deposits was not overlooked. The occurrence of small algae in the lake has'been reported by various investigators. However, the extent to which they are growing in the un- diluted brine is problematical. According to ParricK (1936), who identi- fied 24 genera and 62 species of diatoms from lake bottom samples, no diatom flora is found living in the lake proper today. Patrick avers that the living forms reported from the lake have been found, no doubt, in the surrounding brackish marshes and river estuaries which have a much lower salt concentration. FLOWERS (1934) stated that the lake water proper harbors six species of algae, including four Myxophyceae and two Chlorophyceae. Numerous additional species were found in surrounding brackish water. An abundant bacterial flora representing several genera was observed on glass slides submerged in the lake by Smiru and ZOBELL (1937). The development of micro-colonies on the submerged slides established that the bacteria multiplied in the brine and were not merely passive inhabi- tants. Datnes (1917) demonstrated the presence of from 200 to 625 bacteria per ml. of lake water by plate count procedures. Pigmented rods predominated. The decomposition of organic matter in the lake was regarded as evi- dence of bacterial activity. _ZoBELL et al. (1937) found an average num- ber of 167 bacteria per ml. which formed colonies on nutrient lake-water agar. Most of the organisms proved to be obligate halophiles whose growth required 6 to 15 per cent NaCl. Sea water was found to be little better than fresh water for the cultivation of lake bacteria. Conversely, very few marine, soil, and sewage bacteria were able to grow in media pre- pared with undiluted water from Great Salt Lake. This is illustrated by the data in Table XLII. TaBie XLII.— Relative numbers of bacteria from different sources which developed on nutrient agar prepared with various dilutions of Great Salt Lake water (L.W.), fresh water, and sea water:— SOURCE OF SAMPLE 75% 50% 25% 10% FRESH SEA L.W. L.W. L.W. L.W. WATER WATER Sewage 0.0 0.0 6.7 18.1 100.0 9-5 Soil 0.0 0.8 12.6 29.8 100.0 13.7 Pacific Ocean 0.2 0.6 10.4 64.3 6.2 100.0 Great Salt Lake 100.0 96.4 51.2 To 3.8 Aor ZoBell — 204 — Marine Microbiology When suspended in brine from the lake, most sewage or fresh-water bac- teria were killed within a few minutes. The Dead Sea:— The salt composition of Dead Sea water is distin- guished from that of other naturally occurring brines by the predominance of magnesium chloride and the relative abundance of bromides. The total salt content ranges from 227 to 330 grams per liter. Although this body of water has the popular reputation of being lifeless, ELAZARI- VotcanlI (1940) found numerous bacteria in water and mud samples. He reports the presence of several species of algae, including genera of Chloro- phyceae, Diatomeae, and Cyanophyceae. ELAZARI-VOLCANI isolated organisms similar to Micrococcus morrhuae, Bacterium trapanicum, Bacterium halobium, Pseudomonas indigofera, and Sarcina morrhuae from the Dead Sea. Flavobacterium maris-moriu., Chromobacterium maris-mortui, Pseudomonas halestorgus, and Flavobac- tertum halmephilum were described as new species. The organisms thrived in media containing from 3 to 30 per cent salt. Organisms carried into the Dead Sea by inflowing streams failed to grow in the saline waters, but certain terrigenous spore formers exhibited remarkable salt resistance. In bottom sediments of the Dead Sea, ELAZARI-VOLCANI (1943) dem- onstrated the presence of cellulose decomposers, hydrocarbon oxidizers, denitrifiers, and sulfur bacteria. He found both aerobes and anaerobes in stratified cores. Some of the latter were 170 cm. long and were collected from water depths of 70 to 330 meters. The bacteria developed in enrich- ment media containing 25 per cent salt. Fresh-water lakes:— The factors which influence the distribution and activities of bacteria in fresh-water lakes have been discussed in the preceding chapters. The parts played by bacteria and allied microorgan- isms in lakes as producers of plant nutrients, in the cycle of elements, as geological agents, in the food cycles of animals, as agents which influence physico-chemical conditions, as parasites, and in their relations to higher organisms are analogous to those features of marine bacteria, to which frequent references have already been made. Although most hydrobiologists and limnologists recognize that bac- teria play an important part in the economy of lakes; hydrobacteriology of lakes, like that of the sea, has lagged considerably behind other biolog- ical sciences in participating in the study of lake metabolism and ecology. Whereas zoologists and botanists have classified their organisms as to species, habitat, and activity, bacteriologists have only the most general ideas regarding the characteristics, distribution, and activities of bacteria which live normally in lake water and bottom deposits. Until a decade ago the emphasis had been placed upon the occurrence of bacteria of sani- tary significance in lakes (MINDER, 1920). The fragmentary literature on the subject suggests that the micro- flora of lakes differs qualitatively from that of the surrounding soil and inflowing streams. For example, SNow and FRED (1926) noted that nearly half of the bacteria isolated from Lake Mendota, Wisconsin, were brilliantly pigmented, whereas fewer than ten per cent of the known spe- cies of soil bacteria are chromogenic. Regarding the characteristics of bacteria in Lake Mendota, they write: “The bacteria of lake water, taken far out from shore, show certain well defined characteristics. The kind of microorganisms present in the Chapter XVIII — 205 — Microbiology of Inland Waters water is more or less constant. These indigenous forms are present at all seasons and at all depths. As compared with other organisms commonly studied, the majority of them grow slowly on ordinary culture media, and on plate cultures appear as punctiform colonies. About 10° to 25° C. is the optimum for their growth. In general they do not form acid or gas from sugars. They do not curdle milk and the majority of the true lake forms liquefy gelatin slowly. A considerable proportion are chromogenic, but long incubation at comparatively low temperatures is necessary to bring out the deep color. It is in respect to pigment production that the typical water flora of Lake Mendota is most easily recognized. A large number of pure cultures were selected from well isolated colonies on plates poured during the winter, when the true water bacteria were more prevalent.” Data summarized in Table XXXI on page 114 show that TAYLOR (1942) found nearly three times as many Gram-negative rods in English lakes as in soil, and twelve times as many Gram-positive rods in soil as in lake water. There were five times as many cocci in soil as in lake water. The chances for transplantation are so great that virtually any soil microorganism may be found in lakes, but apparently only certain types find conditions suitable for their multiplication or prolonged survival in lakes. BaIER (1935) compared the bacterial species found in three shallow lakes around Kiel, Germany, where each body of water was found to have a more or less distinctive microflora. No attempt was made to isolate new species, but he recognized many common soil forms and several sulfur bacteria. BArer was especially impressed by the differences in the fun- gous flora of soil, lake water, and bottom deposits. He concluded that the failure of soil fungi spores to germinate in aquatic environments is due to their inability to compete with bacteria for the limited supply of oxygen and organic matter. The total number of bacteria found in any given lake depends upon the distance from land, depth, season, type of lake, and other factors, many of which are only poorly understood. After reviewing the literature on the subject, HENRICI (1939) declared that data are as yet too incom- plete to warrant any general conclusions regarding the distribution of bacteria in different types of lakes. The largest bacterial populations usually occur in eutrophic lakes, which are richer in organic nutrients than are oligotrophic lakes. Dystrophic lakes, which are rich in humus, occupy an intermediate position, although the microflora in many dystrophic lakes differs more in quality than in quantity from that of other types of lakes. The eutrophication or dystrophication of lakes (WELCH, 1935) is influenced by bacterial activities and the latter are in turn modified by other factors. This chain of causes and effects further emphasizes the necessity of having detailed information on microbiological activities when attempting to explain the metabolism of lakes. One of the distinguishing characteristics of oligotrophic lakes is the disappearance of oxygen in the hypolimnion during the period of stagna- tion. Kusnerzow and KarziInKIN (1931), Miyapi (1934), KUSNETZOW (19356), and others have shown that bacterial activity in deeper water or in lake bottoms is primarily responsible for the depletion of oxygen. Com- bining field and laboratory observations, LracinA and KUSNETZOW (1937) calculated that the decrease in the oxygen content of the waters of Lake Glubokoje in Russia could be accounted for by the respiration of bacteria. Similar conclusions were reached by ZOBELL (1940a), who investigated ZoBell — 206 — Marine Microbiology the factors which influence oxygen consumption by bacteria in Lake Mendota. Other things being equal, bacteria are generally more abundant in the epilimnion than in the hypolimnion of fairly deep, well stratified lakes. The differences are not marked. Differences in the abundance of bacteria at different depths disappear entirely during overturns and are non- existent in shallow lakes. Only slight or no differences in the vertical distribution of bacteria were observed by FRED et al. (1924) in Lake Mendota and by GrAHAM and YOUNG (1934) in Flathead Lake, Montana. Progressive decreases were found by KusNETzow and KaArZINKIN (1931) in Lake Glubokoje and in various lakes in northeastern Wisconsin by BERE (1933). Additional observations by KLEIBER (1894), MINDER (1920, 1927), PFENNIGER (1902), DUGGELI (1924), FRED ef al. (1924), RUTTNER (1932), Zi (1932), and others on the factors influencing the distribution of bacteria in lakes are recorded in Chapter V. The densest bacterial populations are almost invariably found in bot- tom deposits. HENric1 and McCoy (1938) counted from a few thousand to 500,000,000 aerobes per ml. of lake mud (see Table XXVII on page 92). According to HENRICcr (1939), the bacterial content of the overlying water is no criterion of the number or kinds of bacteria in bottom deposits. The observations of WILL1aMs and McCoy (1935), KusNETzow (1935), RUBENTSCHIK and GOICHERMAN (1935), DUGGELI (1936), and Car- PENTER (1939) on the numbers, kinds, and activities of bacteria in lake deposits are noteworthy (see Chapter VI). The seasonal distribution of bacteria in lakes is influenced by changing water temperatures, plankton pulses, overturn, runoff, etc. As in the sea, the abundance of bacteria in lakes responds more quickly to changes in organic content than to any other environmental factor. Therefore most seasonal fluctuations can be traced either directly or indirectly to factors which influence the quantity and quality of organic matter. In about half of the lakes examined by BERE (1933) the bacterial content was quan- titatively proportional to the organic and inorganic nutrients in the water, and in about one-third of the lakes the bacterial content was proportional to the organic content alone. The relation of bacteria to the cycle of organic matter in lakes has been reviewed by WAKSMAN (19414). Some of the ways in which aquatic fungi influence the transformation of organic matter are outlined by WESTON (1941). The activities of para- sitic and pathogenic fungi are of considerable importance in aquatic biology. Bacteria are also known to infect fresh-water organisms, some of which are of economic significance. The studies of KLEIN and STEINER (1929) and DUGGELI (1924, 1934) on Swiss lakes, those of FRED e¢ al. (1924) on Wisconsin lakes, those of KUSNETzOW (19350, 1939) on Russian lakes, and those of BAIER (1935) on German lakes are illustrative examples of the results which may be ex- pected to accrue from intensive hydrobacteriological investigations of particular bodies of water. After making a survey of the numbers of various physiological types of bacteria throughout the lake in question, these workers undertook to estimate bacterial activity in situ. This was achieved either by controlled experiments in which concentrations, tem- perature, and other environmental conditions were designed to simulate the natural environment, by comparing the abundance of various physi- ological types of bacteria present from time to time with the chemical composition of the water, or by a combination of both methods. Chapter XVIII — 207 — Microbiology of Inland Waters DUcGGELt (1924) was one of the first to make extensive use of selective media specifically designed to estimate the relative abundance of various physiological types of bacteria in Lake Ritom in Switzerland. He was particularly concerned with the factors which influence the removal of oxygen and the occurrence of HS. The formation of the latter was shown to be due to the activities of Desulfovibrio desulfuricans. The H2S was oxidized either abiogenically or by certain sulfur bacteria, thereby deplet- ing the oxygen. Chromatium species occurred in sufficient abundance in the boundary zone between the overlying oxygenated water and the deeper, H»S-containing water to cause a rosy red color. In the bottom water of Lake Ritom, DiGGELI found up to 30 mgm./L. of H:S, a concen- tration which was toxic for nearly all forms of life, including most bacteria except those that produced H2S by reducing sulfates. KLEIN and STEINER (1929) made quantitative studies on ammonifica- tion, nitrification, nitrogen fixation, nitrate reduction, and sulfate reduc- tion in Lake Lunz. Their comprehensive activity determinations gave new impetus to the study of hydrobacteriology. Seasonal fluctuations in the ammonia, nitrate, and oxygen content of Lake Mendota were found by Domocattra e¢ al. (1926) to be due almost entirely to bacterial activity. Increases in bacterial numbers were gener- ally accompanied by increases in ammonia. An increase in nitrification followed the production of ammonia except in waters deficient in oxygen. From 800 to 3800 bacteria per ml. of water were found, with little evidence of seasonal or vertical variations. BAIER (1935) noted a close correlation between chemical, physico- chemical, biological, and bacteriological conditions in five shallow lakes around Kiel. The foul odor emanating from the Little Kiel, a black, brackish backwater within the city limits of Kiel, was shown to be due to the activities of sulfate reducers. Several factors which influence the numbers, kinds, and activities of microorganisms in inland waters were examined with the help of much relevant literature and original research, BAIER stressed the importance of microorganisms as biochemical agents and as a food source for aquatic animals. More intensive and extensive studies on the activities, distribution, and characteristics of aquatic microorganisms are needed in order to gain a complete understanding of the productivity, metabolism, and ecology of bodies of water. While hydrobacteriological research can contribute much to the pure science of bacteriology, its chief objective should be an elucidation of general hydrobiology, biochemical and geological processes in bottom deposits, and other practical problems. The importance of hydrobacteriological research was emphasized by THIENEMANN (1927) in a ten-year progress report of the Hydrobiological Institute Plén of the Kaiser Wilhelm Society. A literal translation of his statement follows: “The greatest need of limnology, the satisfaction of which would be of benefit to many associated departments, is bacteriological information. It may be momentarily surprising if I insist that hydrobacteriology as such is virtually non-existent. It is true that there have been investiga- tions regarding bacteria living in water, but for the most part they have had reference primarily to practical hygienic problems. It is well known that bacteria play an extremely important part in the cycle of life- materials; it may even be the most important part, if we are justified in assigning degrees of importance. No matter how detailed may be our methods in water chemistry, even the most intensive delving into purely ZoBell — 208 — Marine Microbiology chemical processes is not going to solve for us the mystery of the meta- morphosis of matter, if we neglect bacterial action.... What has been said here about the importance of bacteriological research to hydrobiology and limnology applies also to fisheries and biology in general. Wherever we encounter the difficult problems of the cycle of substances on this earth, we also encounter the necessity of considering bacteria.” Sufficient progress has been made by hydrobacteriologists during the last decade to substantiate the views of THIENEMANN expressed above, but the solution of most of the problems to which he refers is still woefully wanting. For the microbiologist with adequate training in chemistry, biochemistry, physical chemistry, general biology, and hydrography, the field of hydrobacteriology or marine microbiology is most promising, being almost virgin, for research in pure and applied science. BIBLIOGRAPHY Aspsott, A. C., 1921: The Principles of Bacteriology (Lea & Febiger, New York, roth Ed., 686 pp.). ALLEN, W. E., 1921: A brief study of the range of error in micro-enumeration (Trans. Amer. Micro. Soc., 40:14-25). , 1933: “Red water” in La Jolla Bay in 1933 (Science, 78:12—13). , 1937: Plankton diatoms of the Gulf of California obtained by the G. Allan Hancock Expedition of 1936 (Hancock Pacific Expeditions, Univ. South. Calif. Press, Los Angeles, Calif., 3:47-59). , 1941: Twenty years’ statistical studies of marine plankton dinoflagellates of South- ern California (Amer. Midland Naturalist, 26:603-635; 25 ref.). ALLGEIER, R. J., Harrorp, B. C., and Jupay, C., 1941: Oxidation-reduction potentials and pH of lake waters and of lake sediments (Trans. Wisconsin Acad. Sci., 33:115-133; 11 ref.). ALLGEIER, R. J., PETERSON, W. H., Jupay, C., and Brrcg, E. A., 1932: The anaerobic fer- mentation of lake deposits (Internat. Rev. d. ges. Hydrobiol. u. Hydrogr., 26:444-461; 21 ref.). ALLISON, F. E., Hoover, S. R., and Morris, H. J., 1937: Physiological studies with the nitro- gen-fixing alga, Nostoc muscorum (Botan. Gaz., 98:433-463; 35 ref.). AMANN, J., 1911: Die direkte Zihlung der Wasserbakterien mittels des Ultramikroskops (Centralbl. f. Bakt., IT Abt., 29:381—-384). 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W., 1934: Ueber die Beziehungen zwischen den Stoffwechselvorgiingen der Mikroorganismen und dem Oxydo-reduktionspotential im Medium, I. Versuche mit denitrifizierenden Bakterien (Biochem. Zeitschr., 270:317-340; x2 Iref.): Error, C., 1926: Bacterial flora of the market oyster (Amer. Jour. Hyg., 6:755—776; 31 ref.). ELLroTT, JESSIE S., 1930: The soil fungi of the Dovey Salt Marshes (Ann. Appl. Biol., 17:284- 305; 21 ref.). Ets, B. F., and Messina, A. R., 1940: A Catalogue of Foraminifera (Amer. Mus. Nat. Hist., New York, 30,000 pp. in 30 volumes). Exits, C., WELLS, A. A., and BorHMER, N., 1925: The Chemical Action of Ultraviolet Rays (Chem. Cat. Co., New York, 362 pp.; many ref.). Es, D., 1919: Iron Bacteria (Frederick A. Stokes Co., New York, 179 pp.; 53 ref.). , 1932: Sulphur Bacteria. A Monograph (Longmans, Green and Co., New York, 261 pp.; 335 ref.). Emery, K. O., and Dietz, R. S., 1941: Gravity coring instrument and mechanics of sediment coring (Bull. Geol. Soc. Amer., 52:1685-1714; 10 ref.). " ENEVOLDSEN, V., 1927: Ueber vielleicht durch Bakterienwachtum verursachte Anderungen in der Wasserstoffonenkonzentration natiirlicher Wasser (Biochem. Zeitschr., 181:251-266; 4 ref.). ErpTMAN, G., 1938: Pollen grains recovered from the atmosphere over the Atlantic (Medd. Géteborgs Botan. Tradgard, 12:185—106; 8 ref.). ErIkson, D., 1941: Studies on some lake-mud strains of Micromonospora (Jour. Bact., 41:277- 300; 18 ref.). EstTerLy, C. O., 1916: The feeding habits and food of pelagic copepods and the question of nutrition by organic substances in solution in water (Univ. Calif. Publ. in Zool., 16:171-184; 18 ref.). Eyre, J. W. H., 1923: Some notes on the bacteriology of the oyster (including description of two new species) (Jour. Roy. Micro. Soc., 43:385-304). , 1930: Bacteriological Technique (Wm. Wood & Co., New York, 3rd ed., 617 pp.). FarrELL, M. A., and Turner, H. G., 1932: Bacteria in anthracite coal (Jour. Bact., 23:155- 162; 12 ref.). FEITEL, R., 1903: Beitrige zur Kenntnis denitrifizirender Meeresbakterien (Wiss. Meeresun- ters., Abt. Kiel, N.F., 7:89-110; 7 ref.). FEJcIN, B., 1926: Etudes sur les microbes marins, II. Etude sur la forme imperceptible des bactéries dans l’eau de mer (Bull. Inst. Océanogr., Monaco, No. 484:1-7; 20 ref.). FELLERS, C. R., 1926: Bacteriological investigations on raw salmon spoilage (Univ. Wash. Publ. Fish., 1:157—-188; 54 ref.). FERNAND, A., Semp£, and CHAVANNE, 1925: Propriétés antimicrobiennes de diverses eaux fluviales ou marines. Pouvoir bactériophagique (Bull. Acad. de méd., 93:184-187). Marine Microbiology — 215 — Bibliography Fiscuer, B., 1886: Bacteriologische Untersuchungen auf einer Reise nach Westindien (Zeitschr. f. Hyg., 1:421-464; 3 ref.). , 1887: Bacteriologische Untersuchungen auf einer Reise nach Westindien, IT. Ueber einen lichtentwickelnden, im Meerswasser gefundenen Spaltpilz (Zeitschr. f. Hyg., 2:54-05; 7 ref.). 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Bacteria associated with the reddening of salt fish (Jour. Biol. Bd. Canada, 3:70-76; 17 ref.). GIETZEN, JOHANNA, 1931: Untersuchungen iiber marine Thiorhodaceen (Centralbl. f. Bakt., II Abt., 83:183-218; 97 ref.). GILDEMEISTER, E., and WATANABE, H., 1931: Untersuchungen iiber das Vorkommen von Bakteriophagen in Oberflichenwasser (Centralbl. f. Bakt., I Abt., 122:556-575; 10 ref.). GINSBURG-KARAGITSCHEVA, T. L., 1933: Microflora of oil waters and oil-bearing formations and biochemical processes caused by it (Bull. Amer. Assoc. Petrol. Geol., 17:52—65; 18 ref.). GINSBURG-KARAGITSCHEVA, T., and Ropronowa, K., 1935: Beitrag zur Kenntnis der im Tiefseeschlamm stattfindenden biochemischen Prozesse (Biochem. Zeitschr., 275:396-404; 8 ref.). GINTER, R. L., 1930: Causative agents of sulphate reduction in oil-well waters (Bull. Amer. Assoc. Petrol. Geol., 14:139-152; 23 ref.). Grupice, A., 1939: Esame batteriologico dell’aria sul Mediterraneo e sul Mar Rosso (Ann. di med. navale e coloniale, 45:520-523). Gtock, W. S., 1923: Algae as limestone makers and climatic indicators (Amer. Jour. Sci., 6:377-408; 48 ref.). Grdr, Dr., 1909: Forschungsreise S. M. S. ‘“‘Planet’”’ 1906/07 (Herausgegeben vom Reichs- Marine-Amt. Berlin, Verlag von Karl Siegismund, 4:1—198; 18 ref.). GrauaM, V. E., and Younc, R. T., 1934: A bacteriological study of Flathead Lake, Montana (Ecology, 15:101-109; 9 ref.). Gran, H. H., 1901: Studien iiber Meeresbakterien, I. Reduction von Nitraten und Nitriten (Bergens Museums Aarbog, No. 10:1-23; 8 ref.). , 1902: Studien iiber Meeresbakterien, II. Ueber die Hydrolyse des Agar-Agars durch ein neues Enzym, die Gelase (Bergens Museums Aarbog, No. 2:1—-16; 7 ref.). , 1903: Havets Bakterier og deres Stofskifte (Naturen, Bergen, 27:33-40; 72-84). , 1933: Studies on the biology and chemistry of the Gulf of Maine, II. Distribution of phytoplankton in August, 1932 (Biol. Bull., 64:159-182; 13 ref.). Gran, H. H., and Ruup, B., 1926: Untersuchungen iiber die im Meerwasser gelésten orga- nischen Stoffe und ihr Verhiltnis zur Planktonproduktion (Avhandl. Norske Videnskaps- Akad. i Oslo, 1. Matem.-Naturv. Klasse, No. 6:1-14; 16 ref.). Grirritus, F. P., 1937: A review of the bacteriology of fresh marine-fishery products (Food Res., 2:121-134; 62 ref.). Grirritus, F. P., and Futter, J. E., 1936: Detection and significance of Escherichia coli in commercial fish and fillets (Amer. Jour. Pub. Health, 26:259-264; 13 ref.). Hatt, I. C., 1929: A review of the development and application of physical and chemical principles in the cultivation of obligately anaerobic bacteria (Jour. Bact., 17:255-301; 247 ref.). Hatvorson, H. O., and ZIEcteER, N. R., 19334: Application of statistics to problems in bac- teriology, I. A means of determining bacterial population by the dilution method (Jour. Bact., 25:101-121; 30 ref.). Marine Microbiology — 217 — Bibliography —- — , 19330: Application of statistics to problems in bacteriology, III. A con- sideration of the accuracy of dilution data obtained by using several dilutions (Jour. Bact., 26:559-567; 5 ref.). Hammar, H. E., 1934: Relation of micro-organisms to generation of petroleum (Problems of Petroleum Geology, Amer. Assoc. Petrol. Geol., Tulsa, Okla., pp. 35-49; 81 ref.). Hanks, J. H., and WerntTrAvB, R. L., 1936: The preparation of silicic acid jellies for bacteri- ological media (Jour. Bact., 32:639-670; 10 ref.). HAanzawa, J., and TAKEDA, S., 1931: On the reddening of boned codfish (Arch. f. Mikrobiol., 2:1-22; 48 ref.). Harper, E. C., 1919: Iron-depositing bacteria and their geologic relations (U.S. Geol. Survey, Prof. Paper 113:1-89; 65 ref.). HarpMAN, YVETTE, and Henrict, A. T., 1939: Studies of freshwater bacteria, V. The dis- tribution of Siderocapsa treubii in some lakes and streams (Jour. 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Press, London, 194 pp.; 131 ref.). , 1933: On the rate of diatom growth (Jour. Mar. Biol. Assoc., 19:253-276; 15 ref.). Havupuroy, P., 1923: I. Recherches sur le bactériophage de d’Hérelle (Présence du principe dans |’eau de mer) (Bull. Inst. Océanogr., Monaco, No. 433:1-12). Hecut, F., 1934: Die chemische Zersetzung der tierischen Substanz wahrend der Einbettung in marine Sedimente (Kali, 28:209-215). Henrict, A. T., 1933: Studies of freshwater bacteria, I. A direct microscopic technique (Jour. Bact., 25:277—-287; 12 ref.). , 1936: Studies of freshwater bacteria, III. Quantitative aspects of the direct micro- scopic method (Jour. Bact., 32:265—280; 7 ref.). , 1938: Studies of freshwater bacteria, IV. Seasonal fluctuations of lake bacteria in relation to plankton production (Jour. Bact., 35:129-139; 10 ref.). , 1939: The distribution of bacteria in lakes (Problems of Lake Biology, Amer. Assoc. Adv. Sci., Washington, D. C., Publ. No. 10:39-64; 60 ref.). Henrict, A. T., and Jonson, DELIA E., 1935: Studies of freshwater bacteria, II. Stalked bacteria, a new order of Schizomycetes (Jour: Bact., 30:61-93; 25 ref.). Henricl, A. T., and McCoy, ELizaBETH, 1938: The distribution of heterotrophic bacteria in the bottom deposits of some lakes (Trans. Wisconsin Acad. Sci., 31:323-361; 34 ref.). Hess, E., 1932: The influence of low temperatures above freezing upon the rate of autolytic and bacterial decomposition of haddock muscle (Contr. Canad. Biol. Fish., N.S., 7:147-16 ae 20 ref.). , 1934a: Cultural characteristics of marine bacteria in relation to low temperatures and freezing (Contr. Canad. Biol. Fish., N.S., 8:459-474; 40 ref.). , 1934): Effects of low temperatures on the growth of marine bacteria (Contr Canad. Biol. Fish., N.S., 8:489-505; 33 ref.). , 1937: A shell disease in lobsters (Homarus americanus) caused by chitinovorous bacteria (Jour. Biol. Bd. Canada, 3:358-362; 12 ref.). 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Hinze, G., 1903: Thiophysa volutans, eine neue Schwefelbakterie (Ber. deut. botan. Ges., 21 :309-316; 7 ref.). HitcuHens, A. P., 1921: Advantages of culture mediums containing smal] percentages of agar (Jour. Infect. Dis., 29:390-407; 26 ref.). Hire, H., Grppines, N. J.,and WEAKLEy, C. E., 1914: The effect of pressure on certain micro- organisms encountered in the preservation of fruits and vegetables (West Virginia Univ. Agric. Exper. Sta., Bull. 146:1-67). Hyort, J., and Ruup, J. T., 1938: I. Deep-sea prawn fisheries and their problems. II. A bottom-sampler for the mud-line (Norske Videnskaps-Akad. i Oslo, Hvalradets Skrifter, No. 17:1-151; 37 ref.). Hock, C. W., 1940: Decomposition of chitin by marine bacteria (Biol. Bull., 79:199-206; 17 ref.). , 1941: Marine chitin-decomposing bacteria (Jour. Mar. Res., 4:99—106; 11 ref.). Hor, T., 1935: Investigations concerning bacterial life in strong brines (Rec. d. Trav. botan. néerlandais, 32:92—-173; 101 ref.). Horowitz-Wiassowa, L. M., and GrinBERG, L. D., 1933: Zur Frage iiber psychrophile it» Mikroben (Centralbl. f. Bakt., II Abt., 89:54-62; 20 ref.). Horcuxiss, M., and WaAxksMaAN, S. A., 1936: Correlative studies of microscopic and plate |; methods for evaluating the bacterial population of the sea (Jour. Bact., 32:423-432; 9 ref.). Houston, A. C., 1904: Results of a number of separate bacteriological observations bearing ;, on the general question of the pollution of estuarial waters and shell-fish (Roy. Comm. hk Sewage Disposal Report No. 4, 3:191-300). HusBer-PEsTAtozzi1, G., 1938: Das Phytoplankton des Siisswassers (Die Binnengewidsser, 16(1) :1-342; 63 ref.). Huxpurt, E. O., 1928: The penetration of ultra-violet light into pure water and sea water (Jour. Opt. Soc. Amer., 17:15-22). Hont, W. F., 1915: The origin of the sulphur deposits of Sicily (Econ. Geol., 10:543-5793 54 ref.). Hunter, A. C., 1920a: Bacterial groups in decomposing salmon (Jour. Bact., 5:543-552; 11 ref.). Hunter, A. 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ZoBell — 224 — Marine Microbiology REDFIELD, A. C., and Keys, A. B., 1938: The distribution of ammonia in the waters of the Gulf of Maine (Biol. Bull., 74:83-92; 10 ref.). REED, G. B., and MacLeop, D. J., 1924: A bacteriological and chemical study of certain problems in lobster canning (Contr. Canad. Biol. Fish., N.S., 2:1-30; ro ref.). Reep, G. B., and Orr, J. H., 1943: Cultivation of anaerobes and oxidation-reduction poten- tials (Jour. Bact., 45:309-320; 11 ref.). REED, G. B., and SPENCE, C. M., 1929: The intestinal and slime flora of the haddock. A pre- liminary report (Contr. Canad. Biol. Fish., N.S., 4:257-264; 7 ref.). REINKE, J., 1903: Symbiose von Volvox und Azotobacter (Ber. deut. botan. Ges., 21:481-483; 3 ref.). Rerrano, U., and MorsE tt, G., 1938: Ricerche sui germi dell’aria marina a diversa distanza dalla costa (Ann. di med. navale e coloniale, 44:530-534). Renn, C. E., 1936: The wasting disease of Zostera marina, I. A phytological investigation of the diseased plant (Biol. Bull., 70:148-158; 10 ref.). ,1937a: Bacteria and the phosphorus cycle in the sea (Biol. Bull., 72:190—195; 8 ref.). , 19370: Conditions controlling the marine bacterial population and its activity in the sea (Jour. Bact., 33:86-87). : RevuszeEr, H. W., 1933: Marine bacteria and their réle in the cycle of life in the sea, III. The distribution of bacteria in the ocean waters and muds about Cape Cod (Biol. Bull., 65:480- 497; 33 ref). Reyniers, J. A., 1932: Apparatus for taking water samples from different levels (Science, 75 :83-84). Ritey, G. A., 1941: Plankton studies, III. Long Island Sound (Bull. Bingham Oceanogr. Coll., 731-93; 32 ref.). RITTENBERG, S. C., 1939: Investigations on the microbiology of marine air (Jour. Mar. Res., 2:208-217; 15 ref.). , 1940: Bacteriological analysis of some long cores of marine sediments (Jour. Mar. Res., 3:191-201; 20 ref.). , 1941: Studies on marine sulphate-reducing bacteria (Dissertation, Univ. Calif., II5 pp.; 93 ref.). RITTENBERG, S. C., ANDERSON, D. Q., and ZoBEL1, C. E., 1937: Studies on the enumeration of marine anaérobic bacteria (Proc. Soc. Exper. Biol. Med., 35:652-653; 6 ref.). RoserG, M., 1930: Ein Beitrag zur Stoffwechselphysiologie der Griinalgen (Jahrb. f. wiss. Botan., 72:369-384; 13 ref.). Rospertson, A. C., 1931: Preservation of textile fish nets (Ind. Eng. Chem., 23:1093—1098; rr Tek.) RosBerTson, A. C., and Wricut, W. H., 1930: Investigations upon the deterioration of nets in Lake Erie (U. S. Bur. Fish., Document 1083:149-176). Rott, H., 1939: Zur Terminologie des Periphytons (Arch. f. Hydrobiol., 35:59—-69; 27 ref.). RosENTHAL, L., 1937: ‘‘Chromium-sulphuric acid’”’ method for anaerobic cultures (Jour. Bact., 34:317-320). Rossi, G., 1936: Direct microscopic and bacteriological examination of the soil (Soil Sci., 41:53-66; 14 ref.). RUBENTSCHIK, L., 1925: Ueber die Lebenstiatigkeit der Urobakterien bei einer Temperature unter o° C. (Centralbl. f. Bakt., II Abt., 64:166-174; 26 ref.). , 1928a: Ueber Sulfatreduktion durch Bakterien bei Zellulosegarungs-produkten als Energiequelle (Centralbl. f. Bakt., II Abt., 73:483-496; 13 ref.). , 19286: Zur Frage der aeroben Zelluloserzertsung bei hohen Salzkonzentration (Centralb). f. Bakt., II Abt., 76:305-314; 8 ref.). , 1933: Zur anaeroben Zellulosezersetzung in Salzeen (Centralbl. f. Bakt., II Abt., 88:182-186; 6 ref.). RuBENTSCcHIK, L., and Cuarr, S. S., 1937: Etude sur la vitalité des microbes (Ann. Inst. Pas- teur, 58:446-458; 33 ref.). RUBENTSCHIK, L., and GOICHERMAN, D. G., 1935: On the microbiology of mud salt lakes, I. Investigation of the Kujalnizki liman (Mikrobiologiia, 4:403-420; Russian with English summary; 23 ref.). , 1936: The influence of a decrease in salt-content in limans on the micro- flora oF eel muds (Arkh. biol. nauk, 43(ser.2-3):217-227; Russian with English summary; 17 ref.). RUBENTSCHIK, L., Rorstn, M. B., and BreLyansxy, F. M., 1936: Adsorption of bacteria in salt lakes (Jour. Bact., 32:11-31; 25 ref.). Russet, F. S., 1936: Submarine illumination in relation to animal life (Rapp. et Proc.-Verb. Conseil Perm. Internat. p. l’explor. de la Mer, 101:1-8; 34 ref.). Russe Lt, H. L., 1891: Untersuchungen iiber im Golf von Neapel lebende Bakterien (Zeitschr. f. Hyg., 11:165-206; 20 ref.). Marine Microbiology — 225 — Bibliography Russet, H.L., 1892: Bacterial investigation of the sea and its floor (Botan. Gaz., 17:312—32 4) )- , 1893: The bacterial flora of the Atlantic Ocean in the vicinity of Woods Hole, } Mass. (Botan. Gaz., 18:383-3953 411-417; 439-447). Rutter, F., 1932: Bericht iiber altere, bisher unveréffentlichte bakteriologische Unter- suchungen an den Lunzer Seen (Internat. Rev. d. ges. Hydrobiol. u. Hydrogr., 26 :438-443; 9 ref.). SaLimovskajA-Roprna, A. G., 1936: Ueber die Mikroflora des farbigen Schnees (Arkh. biol. nauk, 43(ser. 2-3):229-238; Russian with German summary; 14 ref.). SaNnBorN, J. R., 1930: Certain relationships of marine bacteria to the decomposition of fish (Jour. Bact., 19:375-382; 5 ref.). , 1932: Marine bacteria commonly found on fresh fish (Jour. Bact., 23 :349-351). SANDERS, J. M., 1937: The microscopical examination of crude petroleum (Jour. Inst. Petrol. Tech., 23:525-573; 51 ref.). SANFELICE, F., 1889: Ricerche batteriologische delle acque del mare in vicanza dello sbocco delle fognature ed in lontananza da questa (Boll. Soc. nat. Napoli, 3:32-37)- SasLawsky, A. S., 1927: Ueber eine obligat halophile Thionsdurebakterie (Centralbl. f. Bakt., II Abt., 72:236-242; 15 ref.). 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J., 1939: Studies on bacterial flora of North Pacific salmon (Food Res., 4:563-585; 29 ref.). Snow, Lartitra M., and Frep, E. B., 1926: Some characteristics of the bacteria of Lake Mendota (Trans. Wisconsin Acad. Sci., 22:143-154} 16 ref.). SéHNGEN, N. L., 1910: Sur le réle du méthane dans la vie organique (Rec. de trav. chim.. 29 :238-274; 8 ref.). Soper, G. A., 1909: The discharge of sewage into tidal waters (Jour. Amer. Med. Assoc., 52:122I-1224). Sparrow, F. K., 1934: Observations on marine Phycomycetes collected in Denmark (Dansk botan. Ark., 8(6):1-33; 15 ref.). — , 1936: Biological observations on the marine fungi of Woods Hole waters (Biol. Bull., 70:236-263; 12 ref.). , 1937: The occurrence of saprophytic fungi in marine muds (Biol. Bull., 73:242-248; 4 ref.). , 1943: Aquatic Phycomycetes (Exclusive of the Saprolegniaceae and Pythium) (Univ. Mich. Press, Ann Arbor, 785 pp.; 736 ref.). Spirta, O., 1903: Weitere Untersuchungen iiber Flussverunreingung (Arch. f. 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D., 1938: A paradox in the adaptation of marine bacteria to hypotonic solutions (Science, 87:328-320; 5 ref.). ZOBELL, C. E., and RITTENBERG, S. C., 1938: The occurrence and characteristics of chitino- clastic bacteria in the sea (Jour. Bact., 35:275-287; 18 ref.). ZOBELL, C. E., and STADLER, JANICE, 1940a: The oxidation of lignin by lake bacteria (Arch. f. Hydrobiol., 37:163-171; 24 ref.). , 1940): The effect of oxygen tension on the oxygen uptake of Jake bac- teria (Jour. Bact., 39:307-322; 29 ref.). ZOBELL, C. E. and Upuam, H. C., 1944: A list of marine bacteria including descriptions of sixty new species (Bull. Scripps Tnst. Oceanogr., 5:239-292; 160 ref.). ZUELZER, MARGARETE, 1928: Zur Hydrobiologie der Spirochaeta icterogenes syn. biflexa in den Tropen, IV. Mitteilung (Centralbl. f. Bakt., I Abt., 105:384-393). ABBOTT, 26, 200 Aida, 47, 81, 183, 2190 Alioschina, 112, 147, 160, 226 Allen, Esther C., 56, 57, 230 —— (in Moberg et al.), 19, 20, 100, 222 Allen, E. J., 21, 215 Allen; M: C. (im Waksman et al.), 141, 228 Allen, W. E., 60, 76, 77, 85, 165, 193, 209, 212 Allgeier, 95, 99, 105, 106, 108, 209 Allison, 157, 209 Amann, 52, 209 Anderson, B., 2, III, 215 Anderson, D. Q., 37, 82, 84, 90, OI, 93, 95, 107, 110, 144, 149, 159, 209, 230 (in Rittenberg et al.), 50, 224 (in ZoBell et al.), 203, 230 Angst, 121, 127, 140, 209 Aronson, 175, 200 Atkins, 13, 19, 167, 195, 209 Baars, 146, 159, 209 Baas Becking, 147, 165, 166, 174, 209 Baier, 9, 79, 80, 87, 96, 103, 104, 110, 174, 205, 207, 209 Bailey, 143, 211 Ball, 40, 211 Bancel, 3, 209 Baranik-Pikowsky, 202, 209 Barghoorn, 132, 134, 135, 139, 141, I7I, 195, 209 Barker, 108, 109, III, 200 Barnes, 192, 210 Basset, 69, 210 Bastin, 92, 111, 160, 210 Baumgartner, 197, 210 Baur, 101, 155, 210 Bavendamm, 42, 90, 99, 102, I14, 118, 139, 140, I5I, 155, 156, 160, 162, 163, 164, 165, 210 Beard, 143, 183, 189, 210, 225 Beatty, 198, 210 Beauregard, 199, 210 Becking (see Baas Becking) Beckwith, J. D., 128, 148, 196, 230 Beckwith, T. D., 124, 189, 190, 210, 219 Bedford, 9, 114, 116, 122, 190, 210 —— (in Young et al.), 27, 31, 229 Beijerinck, 118, 159, 161, 162, 210 Benecke, 9, 54, 56, 99, 114, 144, 154, 156, 166, 210 Benson, 99, 141, 210 Bere, 51, 53, 206, 210 Bergey, 2, 5, 117, 119, 124, 127, 159, 163, 165, 187, 202, 210 Berkeley, 42, 44, I01, 118, 120, 121, 152, 210 Bertel, L., 44, 210 Bertel, R., 7, 27, 31, 64, 66, 144, I51, 210 Bieljansky (in Rubentschik et al.), 87, 91, 113, 202, 224 Bye OW, 9, 19, 211 Billet, 171, 211 Birge (in Allgeier et al.), 95, 99, 108, 209 Boehmer (in Ellis et al.), 71, 214 Bokova, 113, 211 Borsook (in Bokova et al.), 113, 211 Braarud, 78, 150, 211 Bradley, 143, 211 Brandt, K., 151, 154, 155, 211 Brandt, R. P., 171, 211 Brebeck, 120, 215 Breed, 52, 127, 211 Brenner, 99, 166, 211 Brew, 52, 211 Brown, B. F., 169, 230 Brown, C. J. D., 40, 211 Browne, 185, 197, 211 Brujevicz, 106, 211 Brutsaert, 82, 211 Buchanan, 71, 105, 162, 211 AUTHOR INDEX Bucher, ro4, 211 Buchner, 72, 211 Bunker, 158, 160, 162, 211 Burgess, 5, 215 Burgwitz, 129, 171, 222 Buswell, 108, ro9, 146, 211, 226 Butkevich, 9, 28, 40, 91, 97, 98, 99, 103, 109, 160, 201, 211 Butler (in Waksman et al.), 78, 143, 1609, 228 Butterfield, 36, 47, 48, 79, 212, 223 CALKINS, 24, 212 Cantacuzene, 171, 212 Carey, 9, 19, 36, 83, 87, 88, 98, 118, 138, 139, 150, 152, 153, 154, 174, 212 — (in Waksman et al.), 51, 52,77; 78, 79, 85, 91, 140, 141, 143, 144, 155, 156, 169, 228 Carpenter, L. V., 183, 212 Carpenter, P. L., 92, 99, 206, 212 Cassedebat, 60, 65, 212 Cattell, 69, 212 Certes, 2, 3, 60, 90, 177, 212 Chait, 08, 224 Chavanne (in Fernand et al.), 83, 214 Chibnall, 147, 212 Chlopin, 69, 212 Cholodny, 54, 55, 103, 212 Christensen (in Keys et al.), 83, 87 150, 219 Clarke, F. W., 15, 17, 212 Clarke, G. L., 12, 13, 24, 65, 73, 174, 212 Clarke, H. T., 147, 212 Clayton, 197, 212 Coe, 79, 173, 212, 215 Cohn, 2, 163, 212 Coker, 23, 165, 212 Conn; H: J., 52, 55, 212 Conn, J. E., 38, 45, 46, 12%, 122, 123, 230 Coolhaas, 146, 212 Cooper, 9, 98, 106, 151, 153, 168, 213 Copenhagen, 109, 160, 213 Coupin, 118, 213 Coumary (in Geiger et al.), 143, 199, 21 Crosthwaite, 194, 213 Crump, 79, 80, 213 Cunningham (in Fox et al.), 79, 215 Cupp, 23, 213 Curran, 88, 213 Cutler, 79, 80, 213 DAHLGREN, IIQ, 213 Daines, 203, 213 Daniltchenko, 200, 213 Darling, 179, 192, 213 Davenport (in Fred et al.), 36, 63, 70, 74, 206, 215 Davis, B. L., 83, 213 Davis, H. S., 175, 213 Day, 45, 228 Deevey (in Hutchinson et al.), 106, 218 d’Herelle, 82, 213 Dianova, 174, 228 Diehl (i Larsen et al.), 33, 60, 220 Dienert, 184, 213 Dietz, 34, 35, 92, 214 Dimitroff, 187, 213 Dodgson, 79, 186, 213 Domogalla, 207, 213 Dore, 194, 213 Dorée, 195, 213 Dorff, 103, 104, 213 Drew, 7, 27, 31, 42, 67, 90, 101, 118, I2I, 155, 213 Drewes, 157, 213 Duff, 175, 213 Diiggeli, 43, 68, 70, 91, 99, 108, 160, 206, 207, 214 Durham, 179, 214 Dyer, 199 ,214 EARDLEY, 203, 214 Egorova, 201, 218 Ehrenberg, 2, 128, 214 Ekeldf, 6, 177, 214 Ekman, 11, 14, 23, 214 Elazari-Volcani, 9, 91, 99, 148, 204, 214 Elema, 106, 214 Eliot, 187, 214 Elliott, 99, 214 Ellis, B. F., 24, 214 Ellis, C., 71, 214 Ellis, D., 103, 158, 161, 162, 163, 165, 214 Emery, 34, 35, 92, 214 Enevoldsen, 105, 139, 214 Erdtman, 179, 214 Erikson, 99, 141, 145, 147, 214 Esterly, 173, 214 Eyre, 26, 187, 214 FARRELL, 93, 214 Feitel, ror, 155, 214 Fejgin, 83, 214 Fellers, 188, 214 Feltham, 28, 37, 55, 59, 61, 62, 63, 64, 70, 91, 95, 90, 108, 116, 130, I5I, 174, 230 Fernand, 83, 214 Field (in Baas Becking et al.), 147, 209 Finn (in Young et al.), 27, 31, 229 Fischer, B., 2, 3, 4, 6, 7, 27) 31, 42s 60, 64, 65, 70, 114, 118, 123, 127, 129, 130, 131, 154, 177, 178, 215 Fischer, H., 156, 215 Fleming (in Moberg et al.), 110, 222 —— (in Sverdrup et al.), 9, 10, 12, 14, 15, 17, 21, 22, 33, 160, 165, 167, 221, 226 Flowers, 203, 215 Forster, 4, 121, 123, 215 Forti, 165, 215 Fortunato, 83, 215 Fowler, 21, 215 Fox, 79, 139, 173, 215 Féyn, 66, 78, 150, 211, 215 Frankland, E., 5, 215 Frankland, P., 70, 85, 122, 179, 182, 192, 215 Fred, 9, 36, 43, 53, 54s 93, 79 74s I17, 204, 206, 215, 225 (in Domogalla et al.), 207, 213 Fuller, 185, 188, 216 Fulmer, 71, 78, 105, 211 GAARDER, 70, 78, 215 Gahl, 92, 111, 215 Gainey, 86, 185, 215 Galliher, 158, 215 Gazert, 6, 27, 31, 38, 43, 00, 61, 152, 154, 177, 192, 215, 216 e, 8, 9, 28, 20, 32, 371 39) 43 54s 62, 83, 90, 116, 118, 216 Geiger, 143, 199, 216 Gelarie, 183, 216 Gellis, 24, 174, 212 de Giaxa, 81, 182, 216 Gibbons, 9, 123, 173, 185, 188, 190, 197, 198, 210, 216 Gibbs, 197, 212 Giddings (in Hite et al.), 69, 218 Gietzen, 165, 216 Gildemeister, 83, 216 Ginsburg-Karagitscheva, 92, 146, 216 Ginter, 92, 216 Giudice, 178, 216 Glock, 100, 216 Goicherman, 202, 206, 224 Grif, 7, 61, 154, 216 Graham, 206, 216 Gran, 10, 66, 77, 78, 101, 118, 140, 152, 154, 155, 215, 216 Grant, 36, 88, 107, 117, 118, 138, 173,230 — (in ZoBell et al.), 99, 112, 128, 147, 230 ZoBell See Marine Microbiology ee ee a eee eee ee ee eee eee SS eee Gratia (in Basset et al.), 69, 210 Gray, 55, 227 Greenberg (in Moberg et al.), 19, 20, 100, 222 Greer, 92, III, 210 Griffiths, 143, 185, 188, 189, 190, 216 Grinberg, 124, 218 Guggenheim, 49, 50, 220 Guillerd, 184, 213 Haas (in ZoBell et al.), 909, 112, 128, 147, 239 Hafiord (in Allgeier et al.), 105, 106, 200 Hall, 49, 216 Halvorson, 48, 103, 104, 216, 226 Hammar, 145, 217 Hanks, 45, 217 Hanzawa, 190, 217 Harder, 103, 217 Hardman, 104, 217, 228 sen Waksman ef al.), 150, 169, 22 Harrison, C. W., 186, 187, 218 Harrison, F. C., 9, 143, 172, 188, 190, 197, 217 Hartulari, 91, 217 Hartzell (im Larsen et al.), 33, 69, 220 Harvey, E. N., 119, 217, 219 Harvey, H. W., 14, 18, 19, 152, 167, 173, 175, 192, 217 Hashimoto (im Baas Becking et al.), 147, 209 Hauduroy, 83, 217 Hecht, 142, 145, 146, 217 Heller, 84, 217 Henrici, 9, 35, 40, 43, 56, 57, 58, 68, 74, 78, 85, 86, 91, 92, 93, 96, 104, 205, 200, 217 Henry, 88, 143, 150, 222 Hess, 9, 123, 144, 175, 190, 217 Hesse, 8, 177, 217 Heukelekian, 36, 84, 217 Heusner (in Moberg et al.), 110, 221 Hewitt, 49, 105, 106, 217 Heydenreich, 26, 217 Highlands, 190, 217 Hilen, 56, 218 Hinze, 163, 218 Hitchens, 51, 218 (in Breed et al.), 127, 211 Hite, 69, 218 Hjort, 22, 34, 218, 222 Hock, 144, 218 Hof, 121, 197, 218 Hofmann, 92, 221 Hoéglund (in Pettersson et al.), 73, 223 Hoover (in Allison ef al.), 157, 209 Horowitz-Wlassowa, 124, 218 Hotchkiss, 9, 57, 82, 85, 94, 108, I10, 149, 218, 228 (in Waksman et al.), 51, 52,775 78, 79, 85, 91, 144, 150, 152, 154, 155, 150, 169 Houston, A. C., 218 Houston, C. W., 48, 226 Huber-Pestalozzi, 25, 218 Hulburt, 71, 72, 218 Humm (in Pearse et al.), 95, 175, 223 Hunt, 112, 218 Hunter, 130, 143, 186, 187, 188, 189, 218 Husson, 3, 2090 Hutchinson, 106, 218 IMSHENETSKY, 196, 218 Inman, 175, 218 Irvin, 100, 222 Issatschenko, 8, 9, 28, 29, 91, 93, 99; 103, 114, 130, 152, 154, 155, 150, 160, 162, 164, 200, 201, 202, 218 JACKSON, 104, 219 Jahn, 192, 210 Jankowski, 111, 146, 160, 219 Jensen, A. C., 203, 219 Jensen, L. B., 192, 219 Jensen, P. B., 148, 219 Jepps, 133, 219 Johnson, D. E., 85, 144, 217, 219 Johnson, F. H., 107, 118, 119, 160, 219 Johnson, M. W. (im Sverdrup et al.), 9, 10, 12, 14, 15, 17, 21, 22, 33, 160, 165, 167, 226 Johnston, 26, 219 Johnstone, 14, 143, 219 Jordan, 72, 85, 219 Juday, 136, 137, 138, 170, 219 (in Allgeier et al.), 95, 99, 105, 106, 108, 209 KALANTARIAN, 103, 155, 219 Kanel, 106, 219 Karzinkin, 53, 57, 108, 205, 206, 210, 220 Katz, 119, 2190 Keding, 156, 171, 219 Kellerman, ror, 118, 190 219 Kennedy, 143, 197, 217 Keutner, 156, 210, 217 Keys, 18, 83, 87, 150, 217, 224 Kibbe, 134, 171, 219 Kinkel, 141, 144, 210 Kiribayashi, 47, 81, 183, 219 Kiser, 123, 124, 189, 190, 219 Klebahn, 190, 220 Kleiber, 63, 68, 86, 206, 220 Klein, 206, 207, 220 Kligler, 49, 50, 220 Kluyver (im Elema et al.), 106, 214 Knipowitsch, 152, 165, 201, 220 Knowlton, 184, 220 Knudsen, 15, 16, 220 Kofoid, 194, 220 Kokurina, 196, 218 Korinek, 44, 48, 120, 121, 156, 220 Korochkina, 106, 220 Krassilnikov, 81, 183, 184, 220 Kreps, 113, 220 (in Bokova et al.), 113, 21 Krizencky, 173, 220 Krogh, 19, 33, 78, 87, 173, 220 (in Keys et al.), 83, 87, 150, 219 Krumwiede, 182, 220 Kruse, 28, 220 Kullmann, 52, 220 Kusnetzow, S. L., 9, 53, 105, 106, 107, 108, 109, 205, 206, 220, 221 Kusnetzowa, Z I., 85, 220 LACKEY, 24, 220 Lagerheim, 171, 220 Landberg (in Pettersson et al.), 73, 223 Landon, 79, 230 Larsen, 33, 69, 220 Lebour, 24, 53, 220 Levin, 5, 6, 177, 221 Lewis, 80, 221 Liagina, 107, 205, 221 Liebert, 152, 221 Lieske, 92, 221 Lindberg, 180, 221 Linder, 132, 134, 135, 139, 141, 171, _195, 209 Lipman, 41, 42, 44, 61, 92, IOI, 102, 118, 120, 152, 221 Lloyd, 9, 36, 42, 52, 63, 64, 67, 68, 70, 74, 84, 85, 87, 90, 91, 97, 121, 155, 156, 184, 221 Lochhead, 114, 227 Lohmann, 24, 53, 221 Love, 102, 222 Lovering, 104, 107, 221 Luck, 24, 173, 221 Lucke, 198, 221 Lukyanowa (in Bokova et al.), 113, 211 Lundestad, 140, 221 Lyman, 21, 221 MAcGINITIE, 95, 96, 174, 221 Macheboeuf, 609, 210 (in Basset et al.), 69, 210 Maclay (in McCready et al.), 45, 221 MacLeod, 197, 224 Manil (in Basset ef al.), 69, 210 Mare, 79, 95, 96, 97, 174, 221 Marmer, 14, 221, 223 Marshall, 78, 221 Martin, 113, 221 Mathews, D. J., 27, 221 Mathews, H. M., 177, 180, 221, 230 Matudaira, 44, 221 Mazur, 147, 212 McCoy, 9, 35, 49 43, 91; 92, 93, 96, 99, 102, 103, 153, 206, 217, 220 —— (in Stark et al.), 84, 226 McCready, 45, 221 McEwen, 66, 70, 230 McLean, 178, 179, 192, 221 McMillin (in Baas Becking et al.), 147, 209 Meadowcroft, 183, 210 Mears, IIo, 221 Meier, 180, 221 Meiklejohn, 80, 221 Meloche, 141, 226 Messina, 24, 214 Michener, 120, 230 Miller, 194, 213 Minder, 68, 70, 74, 204, 206, 221, 222 Minervini, 6, 26, 177, 222 Miyadi, 105, 108, 205, 222 Moberg, 19, 20, 100, I10, 222 Molisch, 102, 103, 118, 155, 162, 163, 222 Moore, 45, 197, 222 Morris (in Allison et al.), 157, 209 Morselli, 177, 224 Mortimer, 107, 223 Miiller, 53, 79, 222 Murray, A. N., 102, 222 Murray, E. G. D. (in Breed et al.), 127, 211 Murray, J., 22, 100, 222 Napson, G., 100, ror, 129, 166, 171, 222 Nadson, S., 100, 155, 222 Nathansohn, 152, 162, 222 Naumann, 33, 56, 222 Neumann, 7, 27, 31, 61, 65, 67, 222 Newton, 115, 122, 222 Nikitinsky, 185, 222 Novelli, 147, 222 OMELIANSKY, 90, 222 Orlova, 146, 226 Orr, A. P., 18, 78, 221, 222 Orr, J. H., 49, 50, 51, 105, 224 Oster, 13, 212 Ostroff, 88, 143, 150, 222 Otto, 7, 27, 31, 61, 65, 67, 222 Owens (in McCready et al.), 45, 221 Oyama (in Sugawara et al.), 109, 226 PACK, 174, 202, 222 Parker, 178, 223 Parlandt, ror, 155, 222 Parr, 185, 223 Parsons, 28, 223 Partansky, 99, 141, 210 Patnode, 112, 227 Patrick, 203, 223 Patton, 14, 223 Pearsall, 107, 223 Pearse, 95, 175, 223 Peele, 113, 223 Peirce, 116, 196, 223 Perry, C. A., 187, 223 Perry, H. M. (im Harrison eé¢ al.), 188, 217 Petersen, 131, 223 Peterson (in Domogalla et al.), 207, 213 (in Allgeier et al.),95, 99, 108, 209 Petrossian, 103, 155, 219 Petrowa, 197, 223 Pettersson, 73, 223 Pfenniger, 70, 74, 206, 223 Pfliiger, 3, 223 Pirie, 7, 152, 177, 223 Plehn, 175, 223 Podhradsky, 173, 220 Portier, 28, 223 Poteriayev, 201, 223 Prescott, 72, 79, 81, 85, 87, 185, 223 Pringsheim, 156, 223 Proctor, 178, 223 Purdy, 79, 223 Piitter, 78, 173, 223 RAHN, 197, 223 Rakestraw, 18, 153, 168, 223 (in von Brand et al.), 150, 154, 156, 227, 228 Ravich-Sherbo, 160, 161, 162, 201, 223 Rawn, 184, 228 Reay, 198, 223 Redfield, 18, 167, 168, 223, 224 Redgrave, 104, 213 Reed, 49, 50, 51, 105, 188, 197, 224 Reinke, 156, 171, 224 Reitano, 178, 224 Renn, 9, 19, 28, 85, 87, 88, 133, 134, 143, 150, 167, 168, 171, 224 Marine Microbiology — 233 — Author Index —— 9 von Brand ¢ al.), 150, 153, 154 ay —— (im Waksman et al.), 51, 52, 77 78, 79, 85, 91, 144, 228 Reuszer, 9, 42, 59, 61, 7°, OF, 93, 94) 96, rg 228 —— (in Waksman ¢é al.), 51, 52, 77, 78, 791 85s OTs 139s 140, 143) 144s 22 Revelle (in Moberg et al.), 19, 20, 100, II0, 222 Reyniers, 28, 224 Richard, 28, 223 Riley, 136, 224 Rittenberg, 50, 91, ror, 144, 159, 166, 178, 224, 230 Roberg, 78, 224 Robertson, 195, 224 Robinson, 18, 227 Rodionowa, 146, 21 Roisin (in at techik et al.), 87, QI, 113, 202, 224 Roll; 57, 224 Rosenfeld, 145 Rosenthal, 51, 224 Rossi, 55, 224 Rostowzew, 155, 218 Rubentschik, 9, 87, 91, 98, 113, 139, 140, I51, 201, 202, 206, 224 Russell, F. S., 13, 73, 224 Russell; H. L., 2, 4, 5, 28, 29, 60, 65, 86, 00, 93, 97, 114, 154, 224, 225 Ruttner, 74, 206, 225 Ruud, 19, 34, 78, 216, 218 SALIMOVSKAJA-RODINA, 179, 225 Sanborn, 123, 172, 225 Sanders, 147, 225 Sanfelice, 60, 225 Saslawsky, 162, 225 ach, 29, 225 Schaudinn, 171, 2 a Ni ielsen, = 65, 66, 70, 142, Beir eeibers (in Geiger et al.), 143, 199, 216 Schneider, 37, 226 Schreiber, 150, 225 Schwartz, 198, 221 Sears, 19, 211 Seiwell, 18, 19, 168, 225 Seliber, 146, 225 Sempé (in Fernand et al.), 83, 214 Setter (in Carpenter et al. ; 183, 212 Seyer, 147, 225 Sheets (1 ack et al.), 24, 173, 221 ona (in Sugawara et al.), 109, 22 Shunk, 118, 119, 219 Sigurdsson (in Dyer et al.), 199, 214 Siple, 179, 192, 213 Smith, B. W., 14, 225 Smith, N. R., 90, 101, 102, 118, 279, 22 Smith, P. W. P. (in Harrison et al.), 188, 217 Smith, W. W., 58, 85, 203, 225 —— (in ZoBell et al.), 203, 230 Snow, | oe E., 143, 189, 225 Snow, Laetitia M., 43, 53, 54, I17, 204, 225 Séhngen, 109, 225 Sommer, 199 Soper, 182, 225 Sparck, 70, 215 Sparrow, 131, 132, 171, 176, 225 133, 134, 135, Spence, 188, 224 Spitta, 86, 225 Spooner, 73, 225 Spray, 51, 225 Stadler, 36, 99, 107, 141, 230 —— (in Stark ef al.), 84, 226 Stanbury, 73, 226 Stanier, 140, 226 Stark, 84, 226 Starkey, 103, 104, 109, 159, 160, 226 Steiner, 1 *., 141, 226 Steiner, M., 144, 206, 207, 226 Stephenson, 109, 226 Stevens, 92, 228 Stewart, 123, 173, 188, 198, 226 Stickland, 109, 226 Stokes, 157, 226 — (in aksman et al.), 78, 143, 169, 228 Stone, 50, 226 Strgm, 95, 160, 226 Stuart, 144, 197, 226 Sturm, 146, 22 Suckling, 185, 226 Sugawara, 109, 226 Sunderland, 192, 198, 226 Sutherland, 131, 226 Sverdrup, 9, 10, 12, 14, I5, 17, 21, 22, 33, 100, 165, 167, 226 —— (1m Fox et al.), 79, 215 TAKEDA, I90, 217 Tammann, 69, 212 Tanikawa, 186, 226 ores 37, 48, 186, 187, 190, 197, 19 Tarr, 9, 52, to, 198, 226 Tarvin, 146, 226 Tausson, 112, 147, 160, 226 Taylor, 9, 60, 63, 72, 74, 82, 87, 114, 185, 205, 227 Thayer, 146, 227 Thiel, 99, 103, 104, 227 Thienemann, 207, 208, 227 Thomas (in Luck et al.), 24, 173, 221 Thompson, 15, 18, 227 Thomsen, 152, 227 Thornton, 55, 227 Tiffney, 175, 227 Tolman (in Baas Becking et al.), 147, 209 Tonney, 186, 227 Topping, 114, 227 Trask, 9, 10, 22, 33- 34, 94, 105, 110 III, I12, 142, 145, 147, 227 Tschigirine, 200, 213 Turner, 93, 214 Tutin, 134, 227 UrnHam, 5, 85, 99, 114, 115, 116, 117, 118, 121, 124, 128, 139, 141, 142, 146, 155, 175, 230 UtermGhl, 53, 54, 164, 165, 227 Utterback, 12, 13, 227 VAN DALFSEN (in Elema ef al.), 106, 214 van Delden, 159, 227 Van der Lek, 140, 227 van Niel, 162, 165, 166, 227 Vartiovaara, 55, 85, 91, 113, 228 Vaughan, 100, 227 Verjbinskaya (in heeya etal.), 113, 211 Vernon, 151, 154, 227 Visscher, 193, 227 von Brand, 9, 150, 153, 154, 156, 227, 228 von Volzogen Kihr, 92, 228 Voroschilova, 174, 228 WAKSMAN, 9, 10, 36, SI, 52, 55, 77, 78, 79, 80, 81, 82, 83, 85, 87, 88, 91, 92, 94, 98, 108, 110, 113, 114, 115, 118, 131, 138, 139, 140, I4I, 142, 143, 144, 145, 148, 140, 150, 152, 154, 155, 156, 169, 174, 206, 218, 221, 228 Walker, 45, 228 Warming, 2, epee 164, 228 Warren, A. K., 184, 228 Warren, F. J., 195, 209 Watanabe, 83, 216 Weakley (in Hite et al. ), 69, 218 Weinberg (in Carpenter et al.), 183, 212 Weintraub, 45, 217 Welch, 9, 25, 66, 74, 205, 228 Wells, On (in "Ellis et al.), 71, 214 Wells, N. A., 175, 228 Weston, A. D., 184, 228 Weston, W. H, 135, I71, 175, 206, 220 Wharton (in Pearse et al.), 95, 175, 223 ee 2 36, 84, 133, 185, 220 White, 186, 227 Wight, 109, 160, 226 Williams, ‘7 T., 9, 91, 99, 102, 103 153, 206, 229 Williams, O. B., 190, 217 Wilson, F. C., 28, 29, 229 hrs (in Fred et al.), 36, 63, 7°, 74, 206, 215 Wilson, P. W., 52, 229 Winogradsky, 55, 162, 229 Winslow, 72, 79, 81, 8s, 87, 185, 223 Wollack (in "Hutchinson ef al. ), 106, Wood, A. J. (in Dyer et al.), 199, 214 . J. F., 143, 188, 1809, 198, Wrig right, 195, 224 Wulff, 24, 229 Youne, O. C., Young, R. T., 27, 31, 220 206, 216 ZABOR (in von Brand et al.), 153, 156, 228 Zapfie, 104, 229 Zelinski, 159, 200, 229 Zeller, 131, 171, 229 Ziegler, 48, 216 Zih, 70, 206, 229 Zillig, 26, 229 ZoBell, 5, 9, 18, 28, 20, 30, 31, 36, 37,39) 41, 42, 43) 45, 46, 48, ST, 55 56, 57, 58, 59, OL, 62, 63, 64, 66, 67, 70, 75, 79, 81, 82, 84, 85, 88, 90, OI, 93, 94, 95, 96, 98, 90, Tos, 106, 107, 108, III, I12, II3, I14, IIS, 116, 117, 118, 119, 120, 121, 122, 123, 124, 128, 130, 138, 130, 141, 142, 144, 146, 147, 148, 150, I51, 155, 158, 159, 160, 169, 170, 173, 174, 175, 177, 178, 180, 181, 183, 184, 193, 194, 196, 203, 205, _219 222, 224, 225, 228, 220, 230 in Rittenberg et al.), 50, 224 Zuelser, 128, 230 Abyss, 11 Achlyogeton salinum, 134 Achromatiaceae, 162, 163 Achromatium, 127 — gigas, 163 — mulleri, 2, 163 — oxaliferum, 163 Achromobacter, 97, 115, 123, 125, 173, 188, 189, 190 — ambiguum, 172, 173 — aquamarinus, 124 — fischeri, 119 — galophilum, 202 — geniculatum, 172 — granii, 140 — halophilum, 202 — harveyi, 119 — ichthyodermis, 175 — litorale, 5 — litoralis, 173 — luminosum, 119 — pellucidum, 172, 190 — phosphoreum, 119 — phosphoricum, 119 — pikowskyi, 202 — stationis, 124 — stenohalis, 155 — viscidum, 172 Achyla flagellata, 176 Actinomyces, 57, 99, 102, 115, 126, 128, 147 _ albus, 100 — halotrichis, 128, 139 — marinolimosus, 128, 139 — melanogenes, 139, 202 Actinomycetales i in sea, 126, 128 Adsorption of bacteria, 83-87, ol, 202 Aerial transport of microbes, 178, 180, 181 Aerobacter, 125, 185, 188, 189 — aerogenes, 184, 185, 187 — cloacae, 187 Aerobes, 68 —and En, 490 —in mud, 90-99, 107, 117 Agar, as solidifying agent, 44 — plating temperature, 121, 122 — substitutes, 45 A garbacterium, 127 — aurantiacum, 140 — bufo, 140 — cyanoides, 140 — mesentericus, 140 — reducans, 140 — viscosum, 140 Agar-digesting bacteria, 44, 77, 110, 140 — in muds, 9 — relation ‘ie Ne fixers, 156 — species of, 140, 141 Aged sea water, 44, 58 Aggregation of particles, 112, 113 Air, bacteria in, 3, 5, 7, 177-181 — pollens in, 179, 180 — yeasts and molds in, 178, 179 Ala-Kule Lake, 146 Algae, blue-green, 23, 164 — brown, 134 — cause of red water, 165 — decomposition, 143 — film-forming, 193, 194 — green, 23, 143 —in Dead Sea, 204 — in Great Salt Lake, 202, 203 — in mud, 99 — in sea, 22, 23 — molds associated with, 131-135 — N-fixation by, 157 — nitrogen content, 143 — parasitism of, 131-135, 171 —red, 140, 171 — relation to N-fixers, 156, 157 Alginic acid, 140, 141 Alternaria, 131 — maritima, 132 Aluminum ppt., 104 Ambergris, 199 GENERAL INDEX American Petroleum Institute, 146 Amino acid utilization, 88, 143 — by plants, 150 — effect on CaCO; ppt., 100-103 Ammonia, oxidation, 151-153 — production, 102, 142, 150, I51 — utilization by plants, 150 Ammonia-N, in sea water, 18 — microbial utilization, 88, 150 Ammonifiers, 8, 98, 102, 142, 150, I5I, 207 Amoebobacter, 124, 166 — granulae, 164 Amphisphaeria maritima, 132 Amygdalin decomposition, 139 Amyloclastic bacteria, 98, 118, 139 Anaerobes, and En, 49 — enumeration, 49-51 — in mud, 93, 97, 98, 107 — isolation of, 51 — jars, 50, 51 Analysis of samples, at sea, 39, 40 — direct counts, 51-58 — plate count media, 41-44 — Pai dilution method, 48, Andeos Island, 7, 90 Animals, bacterial flora, 5, 6, 7, 172, 173 — bacterial nutrition, 79, 80, 96, 137 072-E 75), — effect on bacteria, 78-80, 96, 97, 172 — injured by H,S, 160 — in sea, 23-25, 77-80 — light organs, 173 Antagonism, microbial, 80-82, 176 Antarctic Ocean, bacteria i in, 7, 60 Antibiosis, 80, 176 A phanomyces ‘astaci, 175 A phanothece packardii, 203 Aphotic zone, 11 Aquatic yeasts and molds, 129-135, 171 Arctic Ocean, 8, 130 — seas, 5, 6, 8, 152, 160, 162 Artemia gracilis, 203 Artificial sea water, 21, 47, 48, 121 Ascomycetes, 129, 135 Ascorbic acid for anaerobes, 50 Aspergillus, 131, 178 Atlantic Ocean, 3-8, 60, 61, 64, 67 Attachment, count, 57, 58 —organisms, 56-58, 85, 124, 193, 194 _ eponeiaiiies of chitinoclasts, 144 Autotrophic bacteria, 95, 103, 104, 161-166 Azores Islands, 129, 130 Azotobacter, 125, 156, 202 — chroococcum, 156 Bacillus, 5, 93, 115, 121, 126, 155, 173, 187, 188, 189 — abysseus, 115 — amylobacter, 156 — anthracis, 182 — argenteo-phosphorescens, 119 — borborokoites, 115 — cereus, § — chitinovorus, 144 — cirroflagellosus, 115 — columnaris, 175 — cyaneo-phosphorescens, 119 — epiphyticus, 115, 124 — filicolonicus, 115 — gelaticus, 140 — granulosis, 5 — halophilus, 5 — immomarinus, 115 — kildini, 8 _ limicola, 5 —limosus, § — litoralis, 5 — maritimus, 5 — mycoides, 5, 100 — pelagicus, 5 — phosphorescens gelidus, 119 — psychrocartericus, 201 — salinus, 100 — sphaericus, § — sporonema, 171 — submarinus, 115, 146 — subtilis, 69, 82 — thalassokoites, 115 — thalassophilus, 5 — tumescens, 5 Bacteria, as "chemical agents, 100- 113 — as food, 24, 79, 80, 96, 172-175, 203 — as geological agents, 100-113 — importance of, 1, 8, 9, I0O-I13, 150-176, 193-199 — in sea, 2-8, 38, 59-68, 75, 90-09, 124-127 Bacterial, genera in sea, 124-127 — growth near o° C., 3-8, 45, 98, 122, 123, 144, 190, 201 — periphyton, 57, 85, 124, 193 — “plate,” 161, 201 — stratification, 68, 161, 201 Bactericidal action, of metal, 31, 32 — of sea water, 47, 81, 182, 183 Bacteriochlorin, 166 Bacteriological samplers, 26-33 Bacteriophage, 82, 83 Bacteriopurpurin, 164 Bacteriostatic substance in water, 47, 81 Bacterium, 8, 115, 126 — actinopelie, 155 — albo-luteum, 100 — alginicum, 141 — alginovorum, 141 — amforeti, 8 — arcticum, 8 — balbiani, 171 — balticum, 155 — barentsianum, 8 — bauri, 155 — beijerincki, 8 — brandti, 155 — breitfussi, 8 — calcis, 101, 155 — cellulosae album, 140 — cellulosae flavum, 140 — chitinochroma, 144 — chitinophilum, 144 — fausseki, 8 — feiteli, 155 — flavum, 8 — fucicola, 141 — giardi, 119 — granit, 155 — halobium, 204 — halobium rubrum, 190 — halophilicum, 197 — halophilum, 5 Ea see 155 — henseni, 155 _ hydrosulfureum ponticum, 159 — immotum, 124 — knipowittchi, 8 — laminariae, 171 — linkoe, 8 — lobatum, 155 — marino piscosus, 175 — marinum, 8 — ornalum, 155 — papillare, 8 — repens, 155 — russelli, 155 — salmonicida, 175 — septentrionale, 8 — sewanense, 103 — siccum, 8 — smaragdinophosphorescens, 119 — sociovivum, 124 — spirale, 8 — trapanicum, 197, 204 — triviale, 155 — 2z0pfii, 190 Bacteroides, 125 — halosmophilus, 197 sea Marine Microbiology <= 200 General Index Bahama Islands, 7, 90, 140, 151, 156, 160, 163, 164 Baltic Sea, 3, 155, 156, 166 Bank, 11 Barents Sea, 8, 106 Barnacles, 193, 194 Basin, 10 Beaches, ecology of, 175 — pollution of, ror Beggiatoa, 127, 163, 166 — alba, 163, 165 — arachnoides, 163 — leplomitiformis, 163 — marina, 163 — minima, 2, 163 — mirabilis, 2, 163 Beggiatoaceae, 127, 162, 163 Benthic organisms, 11 Benthos, 11 Biloculina, 24 Binding action of microbes, 112, 113 Biocoenosis, 95, 164, 166 Bioluminescence, 3, 4, 118, 119, 173 Biomass, 7 201 Biosphere, lower limits of, 92, 93 Biotic zones, 11 Black rot of kelp, 171 Black Sea, 81, 95, 200, 201 — anaerobic conditions, 68, 109, 200 — bacterial stratification, 68, 161, 201 — iron bacteria, 103 — lipoclasts, 146 — nitrifiers, 152 —salinity, 15, 200 — sulfate reducers, 159, 200 — sulfur bacteria, 162, 166, 201 — water germicidal, 81, 183 Blastoderma salmonicolor, 129 “Bloody seas,” 164, 165 Boat laboratory, 39 B.O.D. of mud, 108, 110, 149 Boring gribble, 194 Boiryophialophora marina, 132 Bottom deposits, bacteria in, 2, 3, 6, 7, 67, 86, 90-909 — bacterial activity in, 100-113 — biocoenosis in, 95-97, 164, 166 —chitinoclasts in, 144 — collection of, 33-36 — Enh of, 105, 106 . — enumerating organisms in, 54-56 — enzymes in, 113 — factors influencing microbes, 93- Oe =—TIETIUIC, 22 — oceanic, 22 — of lakes, 91, 206 — organic content, 19 — organisms in, 21, 22, 93-97, 100- 113 — oxygen consumption in, 107, 108, IIO, 149 — pelagic, 22 — pH of, 104, 105 — terrigenous, 22 Brandt’s hypothesis, 154, 155 Breed and Brew method, 52 Brine, discoloration, 116, 196 — shrimp, 203 Buffers in sea water, 19 ‘‘Calcium bacteria,” 102 ; Calcium carbonate ppt., 100-103, 151i — effect of sulfate reducers, 160 — role of denitrifiers, 155 Calcium phosphate, 169 “Calloa Painter,” 110 Canyon, submarine, 1o Capacity, poising, 106 Cape Cod region, 61, 65, 70 Capillary tube water samplers, 28- 31 Carbohydrate, assimilation, 117, 118, 138-141 — content of organisms, 145 — relation to nitrogen, 138, 139 Carbonates, in sea water, 19, 20 — precipitation, 100-103, I5I, 155, 160 Carbon cycle in sea, 136-149 Carbon dioxide, in sea water, 19, 20 — production, 108, 137, 170 Caribbean Sea, 65 Carrier for submerged slides, 57 Caseinate, for plate counts, 43 ar ter Sea, bacterial stratification, 68, 161 — bacteria in mud, 98 — biomass, 97 — gas produced in, 109 — H,S in, 160 _ sulfate reducers, 201 — sulfur bacteria, 165, 166 Caulobacter, 124, 126 Cellfalcicula, 125, 139 Cell morphology, 114, 115 Cellulose, decomposers, 98, 99, 139, 140, 195 , — decomposed by fungi, 135 Cellvibrio, 125, 139 Centrifuge plankton, 24 Cephalosporium, 131 Ceratium, 199 Ceratophyllum, 96 Ceriosporopsis halima, 132 Chaetomium, 131 Chalky mud, 102 Channel Island region, 90, 105 Chara, 96 Characteristics of marine bacteria, 114-128 —attachment propensities, 56-58, 124, 193 — cultural, 116, 117 — physiological, 117-119 — salinity requirements, 119-121 — temperature tolerance, 121-124 Chemical composition of sea water, 17-21 Chitin decomposition, 98, 143-145 Chitinoclastic bacteria, in muds, 144, — as parasites, 144, 175 Chlamydobacteriales, 126, 127 Chlorinity of sea water, 15, 16 Chlorobacterium, 161, 166 Chlorobium limicola, 166 Cholodny, direct counts, 55 — ultrafiltration method, 54 Chromatium, 126, 164, 165, 207 — gobii, 8, 164 — minus, 164 — minulissimum, 165 — okenit, 164 — rosea, 164 — vinosum, 164 — warmingii, 164 Chromobacterium, 97, 125 — maris-mortui, 204 Chromogens, 116, 117, 164, 166, 190, 196 — in lakes, 205 Chromous sulfate for anaerobes, 50 Chroococcus turgidis, 135 Chytridiaceous fungi, 132, 133 Chytridiales, 133 Chytridium, 131-134 — alarium, 134 — codicola, 131 — megastomum, 132 — polysiphoniae, 132, 134 Citrate of magnesia bottles, 29, 30 — effect of pressure on, 32 Citrobacter, 184, 187 Cladosporium, 131 Cladothrix, 103 — dichotoma, 187 — intricata, 5 Clathrocystis roseo-persicina, 190 Clay particle size, 22, 94 Clonothrix, 103, 126 Clostridium, 126, 156 — aerogenes, 187 — pastorianum, 156 — welchit, 185, 187 Clyde Sea, bacteria in mud, go, 158 — coliform bacteria, 184 —distribution of bacteria, 67, 68, 72, 74, 75, 87 — HS producers in, 158 _ tides and bacteria, 63 — “water bacteria” in, 97 Coal, bacteria in, 92, 93 Coccolith ooze, 22 Codfish, 190, 107 Coliform bacteria in, fish, 184, 185 — ice, 191 — sea water, 184, 185 — shellfish, 186, 187 Collection of, mud samples, 33-36 — water samples, 26-33 Compensation point, 13 Concentrating water for counts, 53 Conn direct count, 55 Copepods, bacteria as food for, 173, be oie — decomposition, 150 — in sea, 24 — lipid and protein content, 145 Copper precipitation, 104 Cordage fiber decomposition, 135, 141, 195 | Coring devices for mud, 33-36 Cork decomposition, 196 Counts, dilution method, 48, 49 — direct microscopic, 51-58 — media for, 41-45 — water blanks for, 46, 48 Crabs, chitinoclasts on, 144 Crenothrix, 103, 127 — manganifera, 104 — ochracea, 104 — polyspora, 104 dds fairy 127, 128 — anodontae, 187 — balbianii, 187 — interrogationts, 187 — mina, 187 — modialae, 187 — pinnae, 187 — speculifera, 187 — tenua, 187 Crystal Lake, 92 Cultural characteristics, 116, 117 Cyanodictyon, 166 Cyltophaga, 127, 139 — diffuens, 140 — krzemieniewskae, 140 Dead Sea, 9, 148, 204 Decomposition of, carbohydrates, 138-141 — chitin, 98, 143-145 —cordage fibers, 135, 141, 195 — cork, 196 — fish, 116, 123, 142, 190, 197, 198 — fish nets, 141, 195 — hydrocarbons, 99, 146-148, 196 — lignin, 99, 135, 141, 142, 195 Be ee 98, 99, 145, 146 — plankton, 143, 150, 153 — rubber, 148, 196 — wood, 135, 141, 194, 195 Deep, 10 Tie Deep sea, bacteria in, 2-4, 7 Dematium, 130 Denitrification, 101, 154, 155 Denitrifiers, 154, 155 — effect on CaCO; ppt., ror — in sea, 7, 8, 98, 118, 155, 20% Density of sea water, 15, 16 — of marine bacteria, 53 Depth of sea, 10 — effect on bacteria, 3, 4, 7, 65-70, 206 Desulfovibrio, 125, 147, 159, 161 — aestuarii, 146, 160, 161, 166, 200 — desulfuricans, 200, 207 Dextrose fermentation, 98 Diagenesis of sediments, 100-113 Diatomaceous earth, 22 Diatom ooze, 22 Diatoms, decomposition, 143 — ether extract, 145, 147 — film-forming, 193, 104 — fungus infections, 133, 134, 135 —in Dead Sea, 204 — in Great Salt Lake, 203 — in mud, 97, 99 — in sea, 22, 67, 73 , — relation to bacteria, 77, 78, 140 — shellfish poison, 199 Didymosphaeria, Jucicola, 13% _ ens, 131 Dilution, method counts, 48, 49 — water blanks, 40, 46-48, 58 Dinoflagellates, 22 — cause of red water, 165 — parasitized by fungi, 134 — shellfish poison, 199 Diplococcus, 125 — gadidarum, 190 Diplodia orae-maris, 132 Direct microscopic counts, 51-58, 91 — compared with plate counts, 51, $3.08," a, —on bacteria in mud, 54-56 Discoloration, of fish, 116, 117, 190 — submerged surfaces, 193 Disease, amphioxus, 201 — contracted in sea water, I9Qr — kelp, 171 ZoBell ao Marine Microbiology — lobsters, 144, 175 — oysters, 187 — typhoid bacillus, 182, 183, 186, IQI Disinfectants for water samplers, 27 Distance from land, 60-63, 86, 93 Distilled water for blanks, 46-48 Teeee Gr of bacteria in sea, 3-7, 9-89 ot of depth, 3, 4, 7, 65-60 — factors influencing, 59-89 — fluctuations in, 59, 60, 74 — seasonal, 74-77 — solid surfaces and, 36, 56-58, 78, 83-85 Diurnal variations, 64 Dothidella laminariae, 134 Dunaliella salina, 116, 196 Dysphotic zone, 12 Eberthella typhosa, 182, 183, 186 Economic problems, 9, 193-199 Ectrogella perforans, 131, 132, 134 Eel grass, 23 — disease of, 133, 134, 171 Egounov’s hypothesis, 201 En, anaerobiosis and, 49 — effect on iron ppt., 103 — effect on microbial activity, 106 — of bottom deposits, 105, 106 — of sea water, 106 Elodea, 96 Emerita analoga, 174 Emery-Dietz coring device, 34, 35 Endomyces vernalis, 130 Enumerating marine bacteria, 41-58 Environment, marine, 10-25 Enzymes in mud, 113 Epilimnion, 68, 206 Epiphytic fungi, 134 Epiplankton, 25 Escherichia, 125, 188 — coli, 5, 82, 178, 183-188 Esmarch cultures, 39 Ether extract, from ipoclasts, 146 —in marine humus 148 — of decomposing diatoms, 147 — of organisms, 145 Eubacteriales in sea, 124-126 Euglena, 166 Euphausiids, 24 Euphotic zone, 11, 66 Euplotes taylori, 173 Eurychasma dicksonii, 132 Eurychasmidium tumefaciens, 132 Euryhaline organisms, 16, 120 Examination of samples at sea, 39,40 Expedition, Moltke, 3, 130, 177 — Nathorst, 5 — Planet, 7 — Plankton, 3 — Scottish Antarctic, 7 — Solar Eclipse, 5 — South Polar, 6 — Talisman, 2 Extracts for nutrient media, 42 Fabric deterioration, 195 Fat hydrolysis, 98, 909, 145, 146 Fatty acid utilization, 143, 146 Fauna, effect on bacteria, 78-80, 96, 97, 172, 173 Fermentative power, 118 Ferric salts for media, 42 Film-formers, 56-58, 85, 193, 194 Filtration, effect on sea water, 47, 183 Fish, bacteriology of, 4, 172, 188-190 — coliform bacteria, 184, 185 — decomposition, 123, 142, 197, 198 — discoloration, 116, 117, 190, 197 — diseases, 133, 175 — effect on bacteria, 60, 75 — extract media, 42 — net deterioration, 141, 195 — parasitized by fungi, 133 — spoilage, 143, 190, ‘107, 198 Flathead Lake, 206 Flavobacterium, US, £23; X25, 2735 188, 189 — amocontactum, 124 — annulatum, 172 — balustinum, 190 — boreale, r40 — ceramicola, 140 — delesseriae, 140 — droebachense, 140 173, — fucatum, 172, 190 — halmephilum, 204 — halohydrium, 139 — halophilum, 202 — marinotypicum, 116 — marinovirosum, 116 — marinum, 190 — maris, 190 — maris_mortui, 204 — neptunium, 139 — okenokoites, 116 — rhodomelae, 140 — sewanense, 155 — turcosum, 172 — uliginosum, 141 Fluctuations in bacteria, 59, 74 Food spoilage, 142, 144, 197-109 Foraminifera, 22, 24, 194 Formula C dilution water, 47, 81 Fouling organisms, 56, 193, 104 Freezing point of sea water, 13, 16, 123 Fresh water bacteria, 114, 115, 204 — on submerged slides, 56 — salt requirements, 43, 48, II9-121 — thermal sensitivity, 122 eo water media, for bacteria, 43, oe fungi, 132 Fungi, classification, 120 — imperfecti, 120, I3I-135 —in air, 178 _— infect kelp, 134 — in lakes, 135, 171 — in sea, 131-135, IOI — lignoclastic, IAI — parasitic, 131-135, 171, 175, 176 Fur spoilage, 197 Fusarium, 135 Gallionella, 124, 126 — ferruginea, 103 — reticulosa, 103 — turtuosa, 103 Gases, in mud, 108-110 — in sea water, ae) Gas producers, 118 Gee-Esmarch tubes, 39 Gee water sampler, 29 Gelatin, liquefaction, 118 — media, 44, 122 — plating temperature, 122 Genera of bacteria in sea, 124-128 Geological activities of bacteria, 100-104 German ¢ Polar Expedition, 6 Glass-bulb water sampler, 28-30 Glass-disk anaerobe plate, 50 Globigerina, 24 Globigerina 00ze, 22 Gonyaulax, catenella, 199 — digitale, 199 — polyeramma, 199 — spinifera, 199 — triacontha, 190 Gram character, of marine bacteria, II4 — of soil bacteria, 114 Grazing animals, 137 Great Salt Lake, 15, 58, 174 102, 202, 203 — water bacteriostatic, 203 Green Lake, 136 Green sulfur bacteria, 166 Guignardia, alaskana, 134 — irritans, 134. — ulvae, 134 Gulf, of Bothnia, rs — of California, 64, 105, 159, 165 — of Maine, 130 — of Naples, 4, 60, 65, 86, 90, 93, 97, I51I, 152, 163, 182 Gymnodinium, 199 Haddock, bacteria of, 173, 188 Halibacterium, 4, 127 — aurantiacum, 4 — liquefaciens, 4 — pellucidum, 4, 180 — polymorphum, 4 — purpureum, 4. — roseum, 4 — rubrofuscum, 4 Halibut, bacteria on, 172, 188, 190 Halophiles, r2r, 106, 197 —_— chitinoclastic, 144 — in Great Salt Lake, 202, 203 —in limans, 201, 202 Halophiobolus, 134, 171 — cylindricus, 132 — halimus, 132, 134 — marilimus, 132, 134 — medusa, 132 —_ longirostris, 132 — opacus, 132 — salinus, 132, 134 Halosphaeria appendicula a, 132 Harbor water, bacteria in, 60, 61 Heat sensitivity of marine bacteria, 38, 46 Helgoland, 152, 165 Helicoma, maritimum, 132 — salinum, 132 Hemicellulose decomposition, 139 Henrici-McCoy mud sampler, 35 Henrici submerged slides, 56 Hentriacontane, 147 Hides spoilage, 144, 197 H-ion conc., effect of organisms on, IQ, IOI, 104, 105 — effect on CaCOs ppt., ror — effect on plate counts, 42, 43 — effect on PO, solubility, 169 — of sea water, 2: Histamine, 143, 1 Humboldt’ Piet Expedition, 4 Humus, lignin in, 141 — marine, 94, 148, 149 Hydrobacteriology, importance of, I, 2, 207, 208 Hydrocarbons, 146-148 — bacterial oxidation, 146-148, 196 — fats as progenitor, 145 — from cellulose, 140 — synthesis, 147 Hydrogen, effect on SO, reduction, 109 — for anaerobes, 49-51 — from cellulose, 140 — HC formation, 109, 111 — produced in mud, 108, 109 Hydrogen sulfide, effect on iron ppt., 103 — effect on oxygen tension, 109, 207 — from organic matter, 158 — from sulfate, 160, 161, 207 —in “bacterial plate,”’ 20r — oxidation, 162-166 — produced in mud, 108-110 — toxic to animals, 200, 207 Hydrostatic pressure of sea water, 3, 16 — effect on organisms, 16, 32, 33, 69 — effect on water samplers, 29, 32 assimilation, 99, 33 Hyperion sewage outfall, 184, 191 Hypoderma laminariae, 131 Hypolimnion, 68, 206 Ice, bacteria in, 191, 192 Impression smears, 55 IMVIC test, 184 Incubation temperature, 45, 46 Indian Ocean, 82, 152 Indol, indicator of fish spoilage, 143 — production, 118, 184 Infections from sea water, I9Ir Inland waters, (see Lakes), 200-208 — coliform bacteria i in, 185 — fungi in, 135 Intertidal zone, 11, 14, 63 Inverted microscope, 53, 54 Iron, bacteria, 56, 99, 103, 104 — effect on plate counts, 42 — precipitation, 103, 104 J-Z water sampler, 29-31 Kara Sea, 91, 106 Kelp disease, 134, 171 Kerosene oxidation, 148 Kiel Harbor, 3, 144, 151, 152, 166 Kurthia, 126, 189 Laboratory work at sea, 39 Labyrinthula, 132-134, 171 — chattoni, 132 — macrocystis, 133 Lactobacitlus, 125, 189 Lactose fermenters, 118 Lagenidiales, 133 Lake, Ala-Kule, 146 — Alexander, 74, 78, 96 — Bonneville, 202 Marine Microbiology — 237 — General Index ' a — Glubokoje, 107-109, 205, 206 — Lunz, 70, 207 _ Mendota, 63, 70, 74, 102, 107, 117, 136, 141, 144, 147, 153, 192, 204, 205, 207 — of “blood,” 165 — Ritom, 68, 160, 161, 207 — Sewan, 103 — Ssaky, 140 — Weissowo, 100, 166 — Windermere, 62, 72, 82 — Ziirich, 63, 68, 70, 74, 86 Lakes, aquatic plants in, 96, 131 — bacteria in mud, 91, 99, 201, 202, 206 — bacterial population, 60, 62, 63, 68, 86, 114 — cellulose decomposers in, 141 —chitinoclasts in, 144 — En of mud, 106 — fluctuations in bacteria, 60 — nitrifiers in, 153 — organic matter in, 136, 137, 141 — oxygen consumed in, 108 — pH of mud, 105 — yeasts in, 131 Lamprocystis, 126, 166 — roseopersicina, 164 Land drainage, 61-63, 74, 75, 86, 150, 152 Land, effect on bacteria in sea, 7, 3 Lentescospora submarina, 132 Leptosphaeria orae-maris, 132 Leptospira, 127 — biflexa, 128 Leptothrix, 57, 126 — crassa, 104 — longissima, 103 — ochracea, 103 Leucothiobacteria, 161, 163 Light, as ecological factor, 13, 64, 60, 76 — germicidal action, 70-72 — penetration, 12, 13, 67 — producing bacteria, 3, 4, 118, 119, 173 Lignin digestion, 99, 141, 142, 195 — by fungi, 135 Limans, bacteria in mud, 91, 99, 202 — bacteriology of, 201, 202 —cellulose digesters in, 139, 201, 202 — sulfate reducers in, 147, 160, 201 — urea decomposers 1n, 151, 201 Limnoria, 194, 195 — lignorum, 104 decomposition, 98, 99, 145, 14 — in organic matter, 145 Lipoclastic organisms, 98, 145, 146 Little Kiel, 207 Littoral zone, 11 Lobsters, bacteria on, 3, 144 — blackening of, 197, 198 — shell disease, 144, 175 Luminescent bacteria, 3, 4, 118, 119, 173 Mackerel, bacteria of, 188 Macrobenthos, 96, 97 Macroplankton, 24 Macrosporium pelvetiae, 131 Manganese ppt., 99, 103, 104 Mannite utilization, 139 Marine bacteria, characteristics of, 114-128 — genera of in sea, 124-127 — media for, 41-45 — physiological characteristics, 117- 119 — sessile habits of, 51, 52, 56-58, 85, sid Oe — specific gravity of, 53 — temperature optima, 45, 46 — thermal sensitivity, 121-124 Marine Biological Association, 9 Marine environment, 10-25 Media, for anaerobes, 49-51 — for plate counts, 41-45 —sea-water vs. fresh-water, 43, 44 — semi-solid, 51 — solidifying agents for, 44, 45 Mediterranean Sea, 7, 64, 178 Medium 2216, 41, 42 Meiobenthos, 96, 97 Mesodinium rubrum, 165 Metal cylinders, bactericidal effect, 31, 32 — for samples, 27, 28 Methane, from cellulose, 140 — from fatty acids, 145 — produced in mud, 108, 109 be a niagaee ss ethod, anaerobiosis, 49-51 — Breed and Brew, 52 : — collecting mud, 33-36 — collecting water, 26-31 — concentrating water, 53 — direct microscopic, 51-58 — of enumerating bacteria, 41-58 — Petrofi-Hausser, 52 — successive dilution, 48, 49 Methylene blue, 50 Microaerophiles, 49 Microbenthos, 96, 97 Microbial antagonism, 80-82 Micrococcus, 121, 123, 125, 173, 188, 189 — albus-translucens, 190 — aquivivus, 121 — boreus, 8 — candidus, 172 — catharinensts, 8 — centropunctalus, 8 — cinnebareus, 187 — citreus, 172 — euryhalis, 121 — gadidarum, 190 — gelatinosus, 8 — halophilus, 172, 173, 202 — infimus, 121 — litoralis, 190 — lutulentus, 190 — marinus, 8 — maripuniceus, 121 — minutissimus, 8 — morrhuae, 190, 204 — nitrificans, 172, 173 — pfliigeri, 119 — phosphoreus, 119 — pikowskyi, 202 — roseus, 190 — sedimentarius, 124 — sedimenteus, 124 — selenicus, 99, 166 — varians, 172, 188 Micro-colonies, 58 Micromonospora, 99, 126, 128, 145, 147 Microplankton, 24 Microspira murmanensis, 8 Mindanoa Deep, ro Mission Bay, 62, 64, 108, rr9, 149 Molds, importance of, 133, 134 — in air, 178, 179 — in lakes, 135 — in mud, 990 — in sea, 4, 130-135 — iron ppt., 103 — marine, 131, 133 — on submerged surfaces, 56 Moltke Expedition, 3, 130, 177 Morphology of marine bacteria, 114, IIs Mouth microflora, 119 Movements of sea water, 14, 15 Mucor, 178 Mud, agar digesters in, 140 — bacterial activity in, 100-113 — biocoenosis, 95-07, 164, 166 — B.O.D. of, 108, rr0 —chitinoclasts in, 144 — direct microscopic counts, 54-56 — effect of storing samples, 37, 38 — enzymes in, 113 —factors influencing bacteria in, 93-07 . — gases in, 108, 110 — kinds of microbes in, 97-09 — lake, 91, 99, 201, 202, 206 — microorganisms in, 4, 90-99 — mold fungi in, 131 — nitrifiers in, 152, 153 — nitrogen fixers in, 156 — oxygen consumption in, 108 — profile series, 91-93 — sample collection, 33-36 — water content, 90 Mundi cokeri, 165 Murman coast, 8, 113, 152, 162 Mussels, 79, 174, 185 — poisonous, 199 Mycobacterium, 99, 126, 128, 147 — marinum, 175 M ycoderma, 120, 130 Mycosphaerella pelvetiae, 131 M yriophyllum, 96 M ylilus californianus, 174 M yxomyceles, 132, 133 Nahrstoff Heyden media, 43 Naias, 96 Nannoplankton, 24, 82 Nansen bottles, 31 Nathorst Expedition, 5 Nebish Lake, 136 Neritic, bottom deposits, 22 — zone, II Net plankton, 24 Nets, deterioration of, 141, 195 Neuston, 25, 66 Nitrate, in media, 43 — in sea water, 18 — reduction, 98, 154, 155 Nitrification, 151-154 Nitrifiers, 8, 98, 151-154, 201 Nitrite, in sea water, 18 — oxidation, 153, 154 Nitrobacter, 124, 154 Nitrogen, content of mud, 94 — content of sea water, 18, 20, 21 — cycle in the sea, 150-157 — fixers in sea, 8, 08, 99. 156, 157 — limits carbon utilization, 138, 139 — produced in mud, 108 — requirements of bacteria, 88, 143 Nitrosomonas, 24, I51, 152, 153 Nocardia, 99, 126, 128 Noctiluca, 199 North Sea, 3, 4, 65, 129, 152, 156, 159 Norwegian coast, 140, 152, 160 Ocean, depth and size, ro Oceanic, deposits, 22 — organisms, Ir — zones, II Oidium, 130 — pulvunatum, 190 Oil, bacteria in, 93 Oil-well brines, bacteria in, 92 — lipoclasts in, 145 Olpidium, 131, 134 — aggregatum, 134 — bryopsidis, 134 — entosphaericum, 134 — laguncula, 131 — lauderiae, 134 — plumulae, 134 — sphacellarum, 132, 134 — tumefaciens, 134 Ophiobolus, halimus, 134 — salinus, 134 Orbimyces spectabilis, 132 Orcadia pelvetiana, 131 Organic matter, 19, 137, 168, 173 — anaerobic fermentation, 99 - —as Ss Sao III — attacked by fungi, 135 — content of sea water, 18, 87, 88 — content of sediments, 19, 94, I10, III — density of, 168 — dissolved, 19, 138, 168, 173 aa on bacteria, 36, 77, 87-89, 20 — in aged sea water, 58 — in lakes, 136, 137, 206 — humus formation, 148, 149 —low concentration, 84, 85, 87-89, 138 — microbial utilization, 87, 135, 138 — oxygen consumed by, 107, 108,138 — phosphorus from, 168 — required by bacteria, 88 — secreted by plankton, 78 — sulfur liberated from, 158, 159 O/R potential, for anaerobes, 49 — of sediments, 105-107 — petroleum, 112 Oscillatoria, 164 Oslo Fjord, 142 Osmotic pressure of sea water, 15 — effect on bacteria, 48 Oval tubes for anaerobes, 50, 5 Oxygen, absorbed by muds, 107 — consumption, 84, 107, 108, 149, 205 — content of sea water, 20 N — effect on bacteria, 36, 84, 95, 107, 142 118, ZoBell ota Marine Microbiology — minimum layer, a 168 —relation to En, 4 Oysters, bacteriology of, 185-188 ‘score,’ 187 _ spoilage by yeasts, 130 — typhoid bacillus in, 182, 186 Pacific Ocean, 61, 62, 64, 98, 184 Parasitic fungi, 131-135 Particle-binding action, 112, 113 Particle size of sediments, 04 feta material, 19, 84-87, 168, 17 Peharena, animal, 175, 176 — cholera vibrio in sea, 81, 182, 183 — for lobsters, 144, 175 — in sea water, 182, 183, I91 _ paratyphoids in ee IQ — plant, 131-135, 1 — survival in shellfich, 185 — typhoid bacillus in sea, 182, 183, 186, 191 Peat, bacteria in, 92 Pectin fermenters, 99, 141 Pelagic, zone, rz — bottom deposits, 22 Pelodictyon, 166 Penicillium, 131, 135, 178 — notatum, 80 Periphytic ’bacteria, 57, 84, 193 Peritrichospora, integra, 132 — lacera, 132 Peronosporales, 133 Petersenia, 131-133, 176 — andreet, 132 _— lobata, 131, 132 — pollagaster, 131, 132 Petroff-Hausser counter, 52 Petroleum, assimilation, 99 — bacterial oxidation, 99, 146-148 — effect of hydrogen on, 109 — fats as progenitor, 145 — from cellulose, 140 —relation to sulfate reducers, 160 Petschora Sea, 103 PH (see H-ion conc.) — indicative of fish spoilage, 199 — optima for marine molds, 133 Phacus, 166 Pharcidia pelvetiae, 131 Phialophorophoma litoralis, 132 Phormidium, 166 Phosphate, assimilation, 167 — in sea water, 18, 167, 169 — regeneration, 168 Phosphorus, cycle, 167-169 — in sediments, 111 Photobacterium, 127 — annulare, 4 — balticum, 110 — caraibicum, 4 — coronatum, 4 — cyaneum, 119 — degenerans, 4 _ delagadense, 4 — fischeri, 118 — glutinosum, 4 — hirsutum, 4 — indicum, 3, 118 —javanense, 119 _ luminosum, 118 — papillare, 4 — pfliigeri, 119 — phos phorescens, 118 — plymouthii, 119 — tuberosum, 4 Photogenic bacteria, 3, 4, 118, 119, 173 Phycomycetes, 129, 131-135 Phyllospadix, 23 Sens, characteristics, 117- Py toplankton! 24, 73 — effect on bacteria, 60, 67, 75, 77; 78, 96 — in lakes, 136 — molds associated with, 131 — nitrogen requirements, 88, 150 — vertical distribution, 67 Riemer production, 116, 117, 164, I _ by sulfur bacteria, 164, 166 —in fish, 116, 117, 190 Pink yeasts, 102, 130, 178, 188, 190 Planet Expedition, 7 Plankton, 24, 25, 77 — bacteria associated with, 52, 67, 77, 78, 87 — decomposition, 150, 153 Plants, effect on bacteria, 77, 78, 95, 96, 170 — marine, 22, 2 Plaamacieohorales: 133 Plate counts, deviation, 59, 60 — effect of temperature, I2I-123 — media for, 41-45 — vs. dilution method counts, 48, 49 Plating temperature, 121, 122 Pleospora pelvetiae, 131 Pleotrachelus, 131 — inhabilis, 131 = minutus, 131 — olpidium, 132 — paradoxus, I3I — rosenvingit, 131, 132 Poising capacity of mud, 106 Poison of shellfish, 199 Pollens in air, 179, 180 Pollution, of sea water, 184, 191 = terrigenous, 61 Pontisma lagenidioides, I31I, 132 Pools, swimming, 191 Potamogeton, 23, 96 Precipitation, microbes in, 179 Prescription bottles, for counts, 39, 40 — for water blanks, 40 Pressure-cooker anaerobe jar, 50 Pressure, hydrostatic, 16, 32, 33, 60 — osmotic, 15, 16, 48 Primary productivity, 136, 137, 167 Prince Rupert water sampler, 31 Proactinomyces, 128 Prorocentrum, 199 — micans, 164 Proteus, 125, 173, 188, 189 — vulgaris, 100 Protococcales, 166 Protococcus, 166 — salinus, 116, 196 Protozoans, effect on bacteria, 78-80 — importance of, 24 — ingest bacteria, 24, 36, 79, 173 — in Great Salt Lake, 202, 203 — in sea, 23, 24, 132 — in sediments, 97, 90, 164 Pseudomonas, 99, 115, 121, 125, 147, 173, 188, 189 — aestumarina, 155 — azotogena, 155 — calciphilia, 102 — calcipraecipitans, 102, 155 — calcis, 101, 102, 155 — coenobios, 124 — droebachense, 140 —enalia, 146 — felthami, 146 — fluorescens, 80, 172, 190 — gelatica, 140, 156 — halestorgus, 204 — hypothermis, 141, 151 — ichthyodermts, 175 — indigofera, 204 — iridescens, 139 —_ marinoglutinosus, 124 — marinopersica, 141, 155 — membranoformis, 124 — membranula, 124 — neritica, 116 — obscura, 116 — oceanica, 116 — perfectomarinus, 141, 155 — periphyta, 124, 141 — phosphorescens, 3 — pierantonit, 119 — pleomorpha, 139 — salinaria, 197 — sessilis, 124 — stereotropis, 124 — vadosa, 116 — xanthochrus, 116 Psychrophiles, 3-8, 45, 98, 122, 123, 144, 190, 201 Pteropod ooze, 22 Public health’ problems, 182, 183, 185, 188, 191, 197, 199 Purple bacteria, 161, 164-166 — cause of red water, 1605 Piitter’s theory, 173 Pyrenomycetes, 131 Pyrrhosorus marinus, 133 Pythium marinum 132 plate Radiations, 12, 13, 64, 67, 69-73, 76 Radiolaria, 22, 24 Radiolarian ooze, 22, 24 Rain, microbes in, 179 Rainfall, effect on bacteria, 60, 62 coe method of counting ‘bacteria, Red clay, 22 Red Lake, 99, 108 Redox potential of muds, 106 Red Sea, 15, 165, 178 Red water, 164, 165, 207 Reef, 11 Remispora maritima, 132 Renn mud sampler, 33 Reuszer’s medium, 42 Reynier’s water sampler, 28 Rhabdomonas, 126 — rosea, 165 Rhizophydium, 131 — agile, 135 — codicola, 131 — dicksontui, 134 — discinclum, 131, 132 — gelatinosum, 134 — globosum, 132 — marinum, 134 Rhizopus, 131 Rhodobacterioides, 165, 166 Rhodobacterium capsulatum, 165, Rhodocapsa, 126 — suspensa, 164, 165 Rhodococcus, 165, 189 — agilis, 172 Rhodorrhagus, 126, 165 Rhodothece, 126 — pendens, 164, 165 Rhodothiobacterium, 161, 164-166 Ridge, 10 Rise, 10 River water, bacteriophage in, 82 — chemical composition, 17 — thermal sensitivity of bacteria ; 122 Rope deterioration, 135, 141, 195 Rossi direct counts, 55 Rotifer eggs parasitized, 133, 176 Rotted sea water, 58 Rowboat crane, 40 Rozella marina, 132, 134 Rubber-bottle water sampler, 29-31 Rubber deterioration, 148, 196 Saccharolytic activities, 117, 118, 138, 139 Saccharomyces, 130 — cerevisiae, 130 — ellipsoidus, 130 — olexudans, 102 Salinity, of sea water, 15, 16 — requirements of bacteria, 42-44, IIQ-I2I — requirements of fungi, 132, 133 Salmon, bacterial flora of, 188, 189 Salt, bacteria i in, 196, 197 — chitinoclasts in, 144 — lakes, 15, 58, 140 — tolerance, 42-44, IIQ-121 Salterns, 116 Samarosporella pelagica, 132 Samplers, mud, 33-36 — water, 3, 6, 26-33 Samples, "effect of corps 36.37, = examination at ee 2, 39, 40 — temperature of, 38 San Diego Bay, 62, 63, 119 Sand, bacterial content, 94 — crabs, 174, 199 — particle size, 22, 94 San Francisco Bay, 196 Sanitary problems, 182-1092, I9Q, 201 Saprolegnia, 133, 175 — ferax, 175 — parasitica, 176 Sapropel, marine, 111 Saprospira, 127, 128 — grandis, 187 — lepta, 187 — puncta, 187 Sarcina, 125, 189 — litoralis, 197 — lutea, 188 — morrhuae, 190, 204 — pelagia, 121, 146 —— psychrocarteria, 201 Sarcinastrum urosporae, 17% Sardines, bacteria of, 133, 190 — eggs parasitized, 133 Sargasso Sea, 12, 13, 60, 152 Schistosomiasis, 191 197, Marine Microbiology ba” eae General Index Schizomycetes in sea, 124-126 Scottish Antarctic Expedition, % 152 Scripps Institution, 9, 41, 61, 64, 75, 83, 116, 146, 170 Sea-cock water sampler, 26, 27 Sea, depth and extent, 10 Sea’ fowls, 185 Sealskins, spoilage, 197 Sea of Azov, 103, 152, 160, 201% Seasonal distribution, 74 Sea water, aged, 58 — bacteria i in, 2-9, 62 — chemical composition, 17-10 — dilution water blanks, 46-48 — for media, 4, 5, 8, 42-44, 119-121 — organic content, 19, 87, 88 — properties of, 13-20 — required by yeasts, 130 — synthetic or artificial, 21, 47, 48, 121 Seaweeds, 6, 22, 60, 130, 152, I71, Sadireiitation, 85-87 eh (see mud or bottom de- sits Selenium oxidation, 166 Semi-solid media, 51 Serratia, 125, 173, 188, 190 — marinorubra, 121, 146 — pelagia, 155 — salinaria, 190 Sessile organisms, 56-58, 83-85, 124, 193, 194 Sewage, bacteria, 119, 183 — outfalls, 182, 184, IQI, 201 Shelf, rz Shell disease of lobsters, 144, 175 Shellfish, bacteriology of, 185-188 — poison, 199 Ship laboratory, 39 Pag fouling of, 193, 194 worms, 194 Sie spoilage, 142 Siderocapsa, 104, 124, 126 — major, 104 — treubit, 104 Sideromonas, 124, 126 Silica gel media, 45 1, 10 Silt, bacterial content, 94 — particle size, 22, 94 Sirolpidium bryopsidis, 131, 132 Skins, damaged by bacteria, 144 Slime, molds, 133, 134 —on “submerged surfaces, 56 Slope, 11 Snow, microbes in, 179 Soil bacteria, 44, 114, 119 we, surface, 35, 56-58, 78, 83-85, South Seas, 61 Specific gravity, of bacteria, 53 — of sea water, 15, 1 Specificity of marine bacteria, 82 Speira pelagica, 132 Spermatophytes in sea, 23 Sphaerotilus, 126 Sphaerulina orae-maris, 132 Spirillum, 125 — aestuarii, 159 — desulfuricans, 159 _ levocolelaenum, 103 — marinum, 5 — ostreae, 187 — recli physeteris, 199 — thermodesulfuricans, 159 — virginianum, 187 Spirochaeta, 127 — eurystrepla, 128 — halophilicum, 197 — marina, 128 — plicatilis, 2 Spirochaetales in sea, 127, 128 Spoilage of fish, 143, 190 — criteria of, 108, 199 2 ay 5 of oysters, 187 Spore formers, 3, 4, 115 Sporocytophaga, 127 Sporovibrio, 159 Squalene, 146 Standing crop in lakes, 136, 137 Staphylococcus, 182, 189 — albus, 82 — aureus, 81, 82 Starch hydrolysis, 98, 118, 139 Stemphylium codit, 131 Stenohaline organisms, 16 Stigmatea pelvetiae, 131 Stinking putrefaction, 110 Storage, of fish, 189, 190 — of oysters, 136 — of samples, 36-38, 82-85, 150 Stratification, bacterial, 6, 65-68, 70 — in mud, 91-93 Streptococcus, 125, 188, 189 — faecalis, 187 Successive dilution method, 48, 49 cay a utilization, 117, 118, 138, 139 — limited by nitrogen, 138, 130 Sulfate reducers, deposit sulfur, 112 — effect on CaCO; ppt., ror — effect on hydrogen, ro9 — from limans, 201, 202 — in oil-well brines, 92 210) sea, 6, 68, 159, 160, 200 —in sediments, 92, 98, 90, 160, 166 — liberate adsorbed oil, 112 — oxidize hydrocarbons, 147 — produce hydrocarbons, 111 — utilize lipids, 146 Sulfate reduction, 159-161 — in Black Sea, 200 Sulfite waste liquor, 141 Sulfur, bacteria, 68, 99, 161-166 — “bacterial plate,” 161, 201 — cycle in sea, 158-166 — deposition, 112 Sulfureta, 166 Sulfur- oxidizing bacteria, 161-166 — cause of red water, 165 Sunlight, effect on bacteria, 3, 7, 64, 69-74, 76 — penetration of water by, 12 Surface, solid, 36, 56-58, 78, 83-85, Suriice water, bacteria in, 6, 66 nwa of bacteria in sea, 182-184, IQI Swimming pools, ror Symbiosis, 80 Synthetic sea water, 21, 121 — for water blanks, 47, 48 Talisman Expedition, 2, 90, 177 Tap water, bacteria in, 119 — for water blanks, 47 Temperature, as ecological factor, 13, 14, 73, 74 — effect on bacterial respiration, 107 — for marine molds, 133 — growth at low, Bec 45, 98, 122, 123, 144, 190, 201 — incubation, 45, 46, aoa — of ocean, 13, 14, 6 — of storing samples, 37-39 — range of growth, 3-8, 38, 45, 122- 12 _ isaac of bacteria, ete 24 — vertical] distribution of, Teredo, 194, 195 — navalis, 194 Thermal sensitivity, 38, 46, 121-124 Thermocline, 14, 68 Thiobacillus, 124, 161, 162, 163 — bovista, 162 — denitrificans, 154, 162 — thiogenes, 162 — thiooxidans, 162 — thioparus, 162, 166, 200 Thiocapsa, 126 — roseopersicina, 164 Thiocystis, 126, 164 — rufa, 164 — violacea, 164 Thiodictyon, 126 — elegans, 164 — minus, 8, 164 Thiopedia, 126, 166 — rosea, 164, 165, 200 Thiophysa, 127 — volutans, 163 Thioploca, 127, 163 — ingrica, 163 — schmedlei, 163 Thiopolycoccus, 126, 166 — ruber, 164, 165 Thiorhodaceae, 164 Thiosarcina, 126 — rosea, 164 pes Se, 125 bipunctata, 163 Thiospirillum, 126, 166 — jenense, 164 — rosenbergii, 2, 164 — violaceum, 2, 164 Thiosu fate, oxidation, 162 _ reduction, 159, 161 Thiothece, 126 — gelatinosa, 164 Thiothrix, 127, 163, 166 — annulata, 163 — marina, 163 — nivea, 163 — tenuis, 163 Thraustochytrium proliferum, 132 Tides, 14 — effect on bacteria, 63, 64 Tintinnids, 24 Topography of sea floor, 10, 11 Tortugas, 37, 120, 152 Torula, 129, 130, 171, 178 — epizoa, 190 — wehmeri, 190. Toxic products in water, 81 Choo ape of sea water, 12 Trenc Treponema, 127 Tricalcium phosphate, 169 Tricoderma, 131 Trimethylamine, 143, 198 Trough, 1o Turnover in lakes, 68 Typhoid bacillus, 182, 183, 186, 19x — in ice, 191 Tyrosine, 143, 198 Ultrafiltration method, 54 Ultraplankton, 24 Ultraviolet radiations, bacteria, 70 — penetration in water, 12, 64, 71, effect on 72 Urea fermenters, in mud, 98, 99, 151 Valley, submarine, 11 Vanadium ppt., 104 Vancouver Island, 152 Vegetation in sea, 23, 96 Vermillion Sea, 165 Vertical distribution, in lakes, 206 — in mud, 91-93 — of bacteria in water, 3, 6, 65-68 — of phytoplankton, 67 Vibrio, 115, 121, 125 _ ada ptatus 116, 118 — agarlique ‘aciens, 140 — algosus, 146 — beijerinckit, 140 — comma, 81, 182, 183 — costatus, 155 — desulfuricans, 159 — fuscus, 139, 187 — granii, 140 — halobicus desulfuricans, 190 — haloplanktis, 124 — hydrosulfureus, 159 — hyphalus, 155 — marinagilis, 155 — marinoflavus, 116 — marinofulvus, 118 — marinopraesens, 139 — marinovulgaris, 116 — marinus, § — phytoplanktis, 124 — pierantonii, II9 Pa bn” 139 Volume of water and bacteria, 36 Walvis Bay, r10, 160 Wasting disease, 131, 134, 171 Water, blanks, 40, 46-48 — molds, 133 —samplers, 26-33 — samples storage, 36-38 Waves, 14 Weber Lake, 136 West Indies, 3, 7, 67 White Sea, &, 103 Wilson water sampler, 29 Wind transport of microbes, 178, 180, 181 Wood, borers, 194, 105 — destruction by fungi, 794s 195 Woods Hole, 5 131, 132, 185 Worms, ingest bacteria, 174 —ship, 194 135, 141, 5,9, 28, 42, 90, 93, 108, Yeasts, aquatic, 129-131 _ CaCO, ppt. by, 102 — importance in sea, 133, 134 —in air, 178 ZoBell Oa Marine Microbiology — in lakes, 131 — in sediments, 99 — on fish, 123, 189 — on submerged surfaces, 56 — pink, 102, 130, 178, 188, 190 Zignoella, calospora, 134 | | BS, “i { ¥ — enormis, 134 ZoBell Medium 2216, 41, 42 ZoBell water sampler, 29-31 Zones, biotic, 11 Zooplankton, 24 — decomposition of, 143 == = AK — ingest bacteria, 174 — in lakes, 136 — phosphorus content, 168 — relation to bacteria, 78-80, 174 Zostera marina, 23,134 148, 171 Zuider Zee, 4, 152 aaa A AA ih Vs WA ty ¢ } Tce) he a Ae . 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