Author: Haskins, Arthur LaRue Title: The yield, composition and quality of Pennsylvania cigar-leaf tobacco as influenced by certain environmental conditions Place of Publication: Copyright Date: 1944 Master Negative Storage Number: MNS# PSt SNPaAg031 .1 1 <107109>*OCLC*Form:manuscriptitem2 lnput:BMM Edit:FMD 008 ENT: 980111 TYP: s DTI: 1944 DT2: LAN: eng 035 (OCoLC)38092054 037 PSt SNPaAg031 .1 1 $bPreservation Office, The Pennsylvania State University, Pattee Library, University Park, PA 16802-1805 090 20 Thesis 1944m $bHaski,AL $cst*221 68245 $cax+(Archival) 090 20 Microfiinn D344 reel 31.11 $cmc+(service copy, print master, archival master) 100 1 Haskins, Arthur LaRue. 245 14 The yield, composition and quality of Pennsylvania cigar-leaf tobacco as influenced by certain environmental conditions $ca thesis by Arthur LaRue Haskins. 260 $c1944. 300 56 leaves $bill. $c29 cm. 502 Thesis (M.S.)-Pennsylvania State College. 504 Bibliography: leaves 53-56. 533 Microfilm $bUniversity Park, Pa. : $cPennsylvania State University $d1997. $e1 microfilm reel ; 35 mm. $f(USAIN state and local literature preservation project. 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The Pennsylvania State College The Graduate School Departraent of Agricultural and Biological Chemistry THE YIT:LD, COMPOSITION AND QUALITY OF PENNSYLVANIA CIGAR-LIZAF TOBACCO AS INFLUENCED BY CERTAIN ENVIRONMENTAL CONDITIONS A Thesis by Arthur LaRue Raskins Submitted in partial fulfillment of the requirements for the degree of Master of Science February, 19^A Approved IJd^fJ^Ji^ A9f Mg^^^ > > ■ > > > > » » in » 111 » ) I 1 t > ) : ) ) • u ) ) t » 111 I > ) J ) ) » > ) » * > » •I 1 i » i 1 __ I > » » » J >. I I » 1 1 •> J » » > ■ I > 1 ) > . > > > > > • > ) > i t » I b I ) Professor of Soil and Phytochemistry. cA\jL^ Head of Department Table of _C pntent s. Page Introduction --• '1 The Experimental Field 2 Object of these Investi[::ations — ■ 3 Plan of the Experimental V/ork • 4 The Quantity and Distribution of Rainfall 6 The Influence of Season, Field Treatment and Curing Process on the Total Yield, Quality and Chemical Composition of Cigar-Leaf Tobacco ~ • • 10 The Com.position of Cigar-Leaf Tobacco as Influenced by the Season, Field Treatment and Curing 15 Methods of Analysis • 16 A Study o.' the Ash Content of the 194-2 and 194-3 Crops of Cigar-Leaf Tobacco 29 A Study of the Calcium and Nicotine Content of the 19A2 and 1943 Crops of Cigar-Leaf Tobacco and Loss of Organic I'Aatter as a Result of Curing 32 A Study of the iUtrogen Content of riature and Cured Samples of the 194-2 and 194-3 Crops of Cigar-Leaf Tobacco 35 A Study of the Potassium Content of the 194-2 and 1943 Crops of Tobacco as Related to Burning Quality 38 A Study of the Organic Acid Content of the 1942 and 1943 Crops of Cigar-Leaf Tobacco 39 Suirmiary and Conclusions -— - 4^ Acl Precipitation Sept> Precipitation Month 1941 1942 1943 1941 1942 1943 1941 1942 1943 1941 1942 1943~ in. m. in. xxi. in* m* in. in. m. m. m. m. 24 0.15 0.46 0.05 25 0.04 ♦ 26 1.31 27 1.33 1.66 0.06 0.49 0.23 0.82 28 0.3.0 1.18 0.72 0.01 0.04 2.27 29 . 0.02 0.69 0.07 0.07 30 2.48 0.29 0.11 0.42 0.02 31 3.21 0.10 3.71 0.71 7.15 0.13 Total 6.11 3.84 4.21 3.34 7.67 1.41 0, .74 3.52 0.05 0.19 I I - 9 - Table III The Dates of Seeding, Transplanting, Topping and Harvesting, Recorded for the Seasons of 194-1, 1942 and 1943 Seeding 1941 At)ril 14th 1942 May 20th Transplanting June 12th-17th June 10th 1943 May June 8th Topping Aug. 5th- 14th Aug. 11th- 14th Aug. 2nd Harvesting Aug. 29th-30th Sept. lst-2nd Aug. 30th Sept. 8th-13th - 10 - As may be noted in Table II, the Iv/^l crop was especially lacking in rainfall at a very important stage in the grov/th of the plants. No samples of this crop were taken for chemical analysis as the crop shov/ed profound symptoms of nutrient deficiencies* Hov/ever, samples of both mature and cured tobacco v/ere taken of both the 1942 and 1943 crops. The season of 1942 v^ras marked by a YJell distributed but excessive precipitation. Such conditions lead to a marked depletion of the mineral nutrients present in the soil which interfered v;ith normal production. Moreover, those conditions served to restrict normal root development and further restrict the inta^ke of mineral nutrients. The 1943 season v/as marked by a dearth of rainfall. The effect on the crop, however, did not appear to be as detrimental as the drought experienced in 1941, which could be accounted for by the stage of growth v/hen the drought occurred. The Influence of Season, Field Treatment and Curing Process on the Total Yield, Quality and Chemical Composition of Ciz-^ar-Leaf Tobacco The total yields, wrapper production and quality of the tobacco grov.Ti on the experiniental Dlots selected for these investigations and for the - !!■ seasons of 1941, 1942 and 1943 are presented in Table IV, ■ Table IV The Influence of Season and Field Treatment on the Yield and Quality of Cigar-Leaf Tobacco Produced on the Exnerimental Plots during the Seasons of 194-1, 194-2 and 194-3 Formula *v Plots of Fertilizer To ■tal Yield Totf 1941 -.1 VItrv 1942 — ■ners 1943 1941 Quality'' 1942 1943 Treatment 194-1 lb. /a 194-2 1943 lb. /A lb . /A /J A 1 0 2.096 1,288 1,566 78 53 69 G F F 16 Tl 1,965 1,260 1,534 75 A6 72 F F ;? 29 II 1,6U 1,408 1,212 69 42 65 F F F Ave. 1,892 1,319 1,437 74 69 B 6 3-9-0 1,931 1,389 1,542 75 46 75 F F F 20 It 1,908 1,350 1,685 82 35 71 F F to G F 22 « 1,621 1,305 1,502 74 29 69 F F to n D Ave. 1,820 1,348 1,576 77 37 72 C 3 3-9-6 1,887 1,357 1,830 75 49 73 G G P 19 tl 1,926 1,475 1,581 79 34 69 F F to G D 27 I! 1,784 1,487 1,430 74 52 72 P F to G P Ave. 1,866 1 , 440 1,614 76 45 71 D 9 3-9-12 1,817 "1 /roi 1,807 74 59 72 P F F 13 It 1,980 1,470 1,877 78 51 75 F F to G J 1 25 It 1,830 1,434 1,247 77 40 70 F F P 1 Ave. 1,876 1,475 1, 644 76 50 72 1 Table IV (continued) The Influence of Season and Field Treatment on the Yield and Quality of Cigar-Leaf Tobacco Produced on the Experimental Plots during the beasons of 194-1, 1942 and 1943 — - " — — - — " -- ■ — m- ^ - «..«■-•«—.- . Formula ■' ■■" ■■" '""" * "^'^ ^ " ^^~" " « Plots of Fertilizer To 19/.1 lb ./a tal Yie 1942 Id Tota 19/|.l /■■' .1 WraTi 1942 • ■ ■' -/" cers 1943 ■■ ;,-/■■ ■ 1941 Ou ali- ty Treatment 1943 lb. /A 942 1 943 I'd./A E 2 3- -9-18 »050 1,508 1, ,754 79 53 76 G G F 15 It 1 ,929 1,572 1, ,420 70 52 74 F F F 30 II -^ J ,909 1,394 }. , 4 479 75 34 76 G F P Ave. 1; ,963 1,491 J 551 75 46 75 F 8 3- -9-24 Ij ,982 1,429 1, ,519 75 53 75 F P F 11 !? 2. ,092 1,304 1, 615 72 47 77 F F F 24 II 1, ,810 1,390 1> 531 70 47 79 F G P Ave. 1; ,961 1,374 _- y 555 72 49 77 10 3- -6-12 1, ,918 1,505 - 937 79 51 74 F P F 12 n Ij ,838 1,296 1, 837 74 46 74 F G F 28 !? Ij 1,609 1^ 405 74 53 74 P F P Ave. 1. ,861 1,470 , 726 76 50 74 H 4- 3- .1 2-1 - .1. Aw ,^/^ Ij ,818 1,476 - 775 73 57 77 G F to G F 18 !t 1; 1,255 1, 583 79 14 74 F F P • 26 t! ,911 1,364 1, 531 79 51 71 F i^' to G P H Ave. 1. ,871 1,365 1, 630 77 41 74 1 Table IV (contirmed) The Influence of Season and Field Treatment on tiie Yield and Quality of Cigar-Leaf Tobacco Produced on the Experimental Plots during the treasons of 1941^ 194^2 and 19.43 Formula of Treatment Plots Fertilizer Total Yield 1943 r^ li)./A lb. /A lb. /a rn otal ' 'ra'opers 1941 1942 1943 ST ^' Cuallty « 1941 1942 1943 64 79 Ave 3-9-12 11 1,338 1,331 1,334 1,240 1,174 1,207 1,515 60 1,432 58 1,473 59 52 67 60 66 59 62 P G F F F P = Poor, F = Fair, F to G = Fair to Good, G - Good, I H I The results on total yield, as indicated by Table IV, shoT/ that manure alone, fertilizers alone or manure in addition to six units of potash are not desirable fron this standpoint. Formulae of 3-6-12 or 3-9-18 v/ith manure gave better results. This Y:as true, also, in respect to quality. However, in all cases, the seasons had more influence on yield and quality than did the individual field treatments. The markedly poor yields recorded for 194-2 may be attributed to too hich a precipitation v/hen groY/ing and to too high a humidity in the curing shed. The curing season v/as featured by a very high humidity, Y/hich favored high enzymatic activity and loss in dry weight and in addition favored a disease knov.Ti as '^shed burn" where further decomposition was effected by means of microorganisms. k we The Com-Qosition of Cif^ar-Leaf Tobacco as Influenced b^^ the beason, Field Treatment and Curing. The leaves selected for the laboratory vfork re taken from the middle portion of the plants. The samples were dried and the midribs were removed, the remaining portions Y/ere then finely ground and kept in air-tight bottles until used. A niT-iber of determinations Y/ere made on both orr;anic and inorganic materials in the leaf samples. ~ 16 - Official Methods (15) v^ere used in certain instances, Y/hile methods formulated by Pucher, Vickery and Wakeman (16), (17) v/ere used in certain other determinations. Methods of Analysis Methods used in the chemical analyses v/ere those best adapted to plant materials, including tobacco. Moisture; - Moisture was determined on a two- gram sample by drying at 80^C. for three hours in a vacuum drying oven, maintained at a pressure of less than 50 mm. of mercury • Ash; - Y/elgh two grams of pov/dered tobacco in a tared porcelain crucible and place in electric muffle. Allow it to remain there for two hours at 500°C. Cool and weigh. Alkalinity of the water-soluble ash; - Ash 5 gms. in a platinum dish at 500^C. - cool. Moisten ash with 5f^ solution of ammonium carbonate, dry and heat to 200°C. to expel excess ammonium salts. Add hot water and disintegrate ash as much as possible with rubber policeman. Filter and v/ash thoroughly with boiling v;ater into a 600 ml. beaker. Titrate with 0.1 N acid using methyl orange as an indicator. ^ 17 > Report alkalinity in terms of number of m.e. per 100 gms . of sample • Calcium; - Ignite 10 gms. of the sample in a flat-bottom platinum dish in a muffle furnace. The temperature should be maintained at 500°C. over night* Moisten the ash with 5 ml* of hydrochloric acid and filter into a 250 ml* volumetric flask. Transfer a 50 ml. aliquot to a 250 ml. beaker, add ammonium hydroxide until the iron and aluminum hydroxides start to precipitate and immediately add 10 ml. of acetic acid. Heat to boiling and add 10 ml. of ammonium oxalate and boil until the precipitate is coarsely granular. Cool and allow to stand over night. Filter and wash with v/ater at room temperature until the filtrate is free from oxalates. Break the point of the filter with a platinum wire and vmsh the precipitate into the beaker in which the calcium was precipitated v/ith hot sulfuric acid (1 + 4-) and hot water. Add about 10 ml. of sulfuric acid (l + 4), heat to about 90^0* and titrate with 0.1 N potassium permanganate. Finally, add the filter paper to the solution and complete the titration. Potassium; - Potassium was determined on a neutral solution of the ash, using trisodium cobalti- ferf'S - 13 nitrite as the precipitating agent • A two gram sample of the groimd tobacco v/as ignited in a muffle furnace at 4.80-500^0. for 24 hours • The resultant ash was transferred to a 400 ml. bealcer using distilled water and one ml* of concentrated nitric acid* This solution was heated to boiling and cooled before filtering into a 200 ml* volumetric flask, washed and made up to volume v/ith distilled water. A 10 ml. aliquot was then measured into a 100 ml. beaker and neutralized by the dropv/ise addition of normal sodium hydroxide, using a drop of methyl red as an indicator. The solution was then acidified with one ml. of normal nitric acid and the potassium precipitated by the addition of five ml. trisodium cobaltinitrite solution, containing one gram of reagent per five ml. The beaker v/as placed in a v/ater bath maintained at 20^0. for two hours and then filtered through a tared porous-bottomed porcelain filtering crucible. The transfer v/as made with 0.01 N nitric acid from a wash bottle. The precipitate was washed ten times with two ml* portions of the dilute nitric acid and five times v/ith tv/o ml* TDortions of 95% alcohol. Suction was applied until the precipitate v/as quite dry, the crucible was then wiped with a clean cloth and placed in an electric drying oven for one - 19 - hour at llO^vC. After cooling In a desiccator. It was weighed. The weight of precipitate multiplied by the factor 0 #17216 gave the corresponding weight of potassium* The composition of this precipitate may be represented by the formula K^'^b.CoOhOz) e •^zO • I i 'if: -M Total nitro::en: - This was determined by the Kjeldahl method raodified to include the nitrogen of nitrates* A one-half gram sample of the ground tobacco was placed in an 800 ml* Kjeldahl digestion flask* To this 40 ml* of concentrated sulfuric acid, containing one gram of salicylic acid were added and the flask rotated to insure a thorough mixing of reagent and sample* The m.ixture was allowed to stand at least thirty minutes, with frequent shaking. Five grams of sodium thioBulfate v/ere then added and the resultant mixture heated slowly until white fumes appeared* After cooling, ten grams of potassium sulfate, a few crystals of copper sulfate and a Hengar selenium pellet were added and the mixture heated in a moderate flame for one hour after clearing* The flask was allowed to cool and then diluted with 250 ml* of distilled water* A pre-determined volume of Green- bank alkali was added (to make the solution strongly alkaline), followed by a few pieces of mossy zinc. - 20 - then connected to the condenser and the contents mixed. The releaned ammonia was collected In -^bout 50 ml. of saturated boric acid solution, a total volume of about 200 ml. being distilled over. The distillate was then titrated v/ith 1/4 normal sulfuric acid using a mixed indicator of methyl red and methylene blue. A blank determination v/as run on the reagents employed. Protein nitro.p:en; - A one-half gram sample of ground tobacco v/as placed in a 400 ml. beaker, followed by 100 ml. of 0.5 per cent acetic acid solution. The suspension was then boiled for five minutes and allowed to cool. It was then passed through a filter paper of medium speed and the precipitate washed first with a hot dilute acetic acid solution and then v/ith a small volume of distilled water, the total wash water approximating a total of 100 ml* The filter paper and mass Y/as then transferred to an 800 ml. Kjeldahl digestion flask to ?/hich were added fifteen grams of Dotassium sulfate, a Hengar selenium pellet and 30 ml. of concentrated sulfuric acid. The contents were digested over a moderate flame for one hour after clear- .ng . The flask v/as allov/ed to cool and diluted with 250 ml. of distilled water. From this point the procedure is identical with that used for total nitrof^en. - 21 - ^^JL IJicotirie: - Place a 0.5 gm. sample (corresponding to 5-15 mg. nicotine) in a 50 ml. round bottom flask. Add 2-3 ml, vmter and an excess of 35fo sodium hydroxide, about 4 drops, use phenolphthalein indicator (solid barium hydroxide raay be used instead of sodium hydroxide) . Begin to heat with the micro-burner and at the same time connect to the steam still. Distil into 3 ml. hydro- chloric acid (1 + a) until the volume of the distillate is 100 ml. Add 1 ml. of 10,^ silicotungstic acid, A H2O . SiOs . 12 WO3 . 22 H2O, for each 10 mg. of nicotine. Stir, place on steam bath for an hour, cool and place in refrigerator over night. Filter on ashless paper, wash with hydrochloric acid (l + 99), bum off in Dlatinura crucible over low flame, then heat at full heat for 10 minutes. Cool and -/eigh. Weight of residue x O.II4.O - weight of nicotine. Volatile acids; - To five f.'rams of powdered tobacco in a 500 cc. Kjeldahl flask add 5 cc. of tartaric acid solution and 100 cc. of carbon dioxide free water. Connect the still; apply heat to the flask, regulating the flame so that boiling talces place in five to eight minutes. At the expiration of ten minutes from the time of applying flames, steam is TDassed in and 500 cc. of distillate collected at the rate of 10 cc* per minute. The original volume of solution in the reaction flask is marked with a red crayon and maintained during the distillation. Transfer the distillate to a 600 cc. beaker, heat to gS^'C^ add six drops of Phenol Red indicator, and titrate with standard 0.02 N sodium hydroxide. Reserve the solution for formic acid determination. Calculate total volatile acids as acetic on a moisture free basis. Blanlc is considered to be .00073 gram acetic acid. Recovery factor is 10^ 9 Determination pX the Non-volatile Organic Acids Preparation of the or^'anic acid extract; - The organic acids of the tobacco leaf were extracted accord- ing to the technique of Pucher, Vickery and V/akeman (16) . Ty70 grams of the dried and pov/dered tissue v;as acidified to pH 1.0 with 4 N sulfuric acid, mixed v/ith 3.5 grams of asbestos, and extracted v/ith specially prepared ethyl ether in a rubber analysis extraction apparatus for a period of 24 hours. At the end of the extraction period, 25 ml. of (carbon dioxide free) distilled v;ater and 2 ml. of (carbon dioxide free) 5 N sodium hydroxide was added to the ether extract, the system v/as thoroughly agitated to transfer the acids - 23 - to tlie aqueous phase, and the ether v/as carefully distilled off. The organic acid extract was made to volume at 100 ml. Determination of oxalic acid; - Oxalic acid was determined by the method of Pucher, Vickery and \7akeman (16)^ as folloY/s: A 25 ml* aliquot of the organic acid extract was acidified to congo red with O.S N hydro- chloric acid. The precipitate that formed was allovfed to settle, filtered off on asbestos in a Gooch crucible and washed with water. A drop of methyl red v;as added to the clear filtrate and amm.onium hydroxide v/as added to a faint alkaline reaction. Two to three ml. of glacial acetic acid 7/ere added, followed by 5 ml. of 10 per cent calcium chloride solution. After standing at least tv/o hours, the calcium oxalate was filtered on asbestos in a Gooch crucible and was v/ashed with a little very dilute ammoniiom hydroxide. Crucible and contents were then transferred to a 100 ml. beaker. Five ml. of 50 per cent sulfuric acid and 20 ml. of water v/ere added; and the solution v/as heated to boil- ing and titrated v/hile very hot with 0.02 N potassium Dormanganate solution. Determination of citric acid; - Citric and malic acids were determined by the simultaneous method 01 Puchor, Vickery and Wakeman (17) . In this method, ' citric and malic acids are oxidized simultaneously, and D entabromoacetone which is formed from the citric acid is separated from the malic acid oxidation product by extraction with petroleum ether, after which the acids are determined separately. The procedure that Y/as used is as folloY/s: A 5 ml* aliquot of the organic acid r solution ¥;as diluted to 20 ml. with water and 3 ml. of 50 per cent sulfuric acid were added. The mixture v;as boiled gently for eight to ten minutes to expel traces of ether, cooled, and 1 ml. of saturated bromine vmter v;as added. After five minutes the solution v-'as filtered 7/ith gentle suction through a Gooch crucible into a suction flask marked at 35 ml., and 2 ml. of 1 N potassium bromide were added. The temperature of the solution was then adjusted at 20 to 22''C. and 5 ml. of potassium permanganate, previously brought to the same temperature, v/ere added. The mixture was allowed to stand in a v/ater bath at 20"» to 22°C. for 10 minutes with occasional stirring, and was then chilled to 5^* to 10*^0. and decolorised with three per cent hydrogen Tjeroxide added dropwise with vigorous stirring. The oxidation mixture vas transferred to a 125 ml. pear-shaped separatory flask, and the suction 25 - flask vjas rinsed into the separatory flask ir/ith 25 ml* of petrolevun ether used in small portions* The flask was shaken vigorously and the aqueous layer v/as drawn off* The petroleum ether was transferred to a second flask. The aqueous solution was shaken again vrith 20 ml. of petroleuia ether and was then put aside for the determination of malic acid* The two petroleum ether extracts were combined and washed four times with 3 ml* of v/ater and the washings Y/ere added to the aqueous extract for the malic acid determination, A 3 nil • quantity of four per cent sodium sulfide solution vras added to the separatory flask, and the mixture v/as shaken vigorously* The aqueous layer, which turned reddish, was dra^m off into a 50 ml* Erlenmeyer flask. The Detroleum ether v/as then treated a second time w rith 3 ml. of sodium sulfide and washed three times with 3 ml. of v/ater, the aqueous solutions all being received in the same flask. To this solution was added * 2 ml. of 2 N sulfuric acid and a few quartz pebbles; and the solution xias boiled pently for three minutes to expel hydrogen sulfide. It was then cooled to room temperature and sufficient 1.5 N potassium permanganate added to produce a red color permanent for 20 seconds. This usually took 0.8 to 1.0 ml. of permanganate. The color was discharged by the addition of a small excess — 2o - of specially prepared halogen-free hydrogen peroxide, followed by the addition of 2 ml. of concentrated nitric acid, 3 nil» of standard silver nitrate, 1 ml. of ferric alum indicator solution, and 3 to 4 ^^1» of ethyl ether ♦ The flask v/as shaken vigorously to coagulate the silver bromide, and the solution was titrated with aminoniuin thiocyanate from a micro-burette to a faint salmon-pink color. Inasmuch as the conversion of citric acid to pentabromoacetone is not strictly quantitative, it was necessary to multiply the quality of citric acid determined by a factor of 1.12. Determination of malic acid: - Malic acid was determined by the method of Pucher, Vickery and. v;akeman (17) . The aqueous solution, together v;ith the v/ash fluid from the petroleum ether (obtained as described under the determination of citric acid), V/3E diluted to a 100 ml. A 25 ml. aliquot vras trans- ferred to a 300 ml. Kjeldahl flask to which 25 ml. of v/ater, 1 or 2 drops 18 N sulfuric acid and a few quartz pebbles were added. The flask was fitted with a bent distillation tube which extended into a 250 ml. wide-mouth Erlenmeyer flask charged with 10 ml. of a freshly filtered 2,4-dinitrophenylhydrazine solution and 20 ml. of vrater. The end of the distillation tube was dipped beneath the surface of the reagent in the - 27 - receiving flask, and the flame of a microburner v/as applied to the Kjeldahl flask. The receiving flask V7as inmiersed in a cold water bath and the rate of distillation v/as adjusted so that the receiving solution did not boil vigorously during the distillation. The distillation y/as continued until the volume of solution remaining in the Kjeldahl flask v^as reduced to somewhat less than 10 ml., usually taking from 12 to 15 minutes. The receiving' flask v/as then removed and the end of the distillation tube rinsed into it. The orange precipitate was transferred as completely as possible with water to a small fritted Gooch crucible and dried for a short time at 100^ to llO^C. Meanwhile the receiver v/as thoroughly drained, and the last traces of precipitate v/ere taken up in hot pyridine used in several successive portions but not more than 3 to 4' nil* in all. The pyridine Y/ashings v/ere transferred to a 25 m^l. volum^etric flask. The crucible was then fitted into a rubber stopper, carried on a cylindrical funnel — v/hich is attached to a test tube equipped with a side arm — and marked at 20 ml. Boiling pyridine w^as added in small portions to the crucible; and the contents w /ere gently triturated with a glass rod, after which - 2B - suction v^as applied and the pyridine was drav/n through each tirae* Three or four v;ashings usually were sufficient to dissolve and transfer all the precipitate to the 25 ml, flask used for the rinsing of the receiver. After being cooled to room temperature, the solution was made to volume with pyridine and mixed • Either a 2 ml. cr a 5 ml. alicuot of the pyridine solution Y/as transferred to a 100 ml. flask and 50 ml. of water were added, follov/ed "by 5 ml. of 5 N sodium hydroxide. The solution was then diluted to the mark and the intensity of the blue color vcas determined by the use of an Evelyn Photoelectric Colorimeter, using filter number 580, having trans- mission limits of 565 to 610 millimicrons. These readings v/ere converted into m.alic acid equivalents by comparing readings obtained against a standard reference curve v/hich had previously been prepared using solutions of pure malic acid treated in exactly the same manner as described for the aliquot from the organic acid fraction. - 29 - A_,StiKj^__jxL;_ the Ash Content of the 194-2 and 19A3 Crops of Ci>:ar~Leaf i'obc.cco For this study deteriiiinations of total ash and alkalinity of the soluble ash were made on mature and cured samples of "both the 1942 and 1943 crops. Samples for these and certain other studies were taken from the field at harvesting time and at the end of the curing season from the shed* The results obtained are presented in Table V. The ash content of the mature leaves of the 1942 tobacco, on a percentage basis, is somev/hat higher than that of similar samples of the 1943 tobacco, Hov;- ever, considerably more ash constituents v/ere removed by the latter crop as a greater total yield v/as obtained that season. This may be noted from the results presented in Table IV. The reason for determining the total ash of both ma ture and cured samples v/as to use this as a basis to determine the loss of diy matter in the shed as a result of the curing process. From the "apparent increase" in the i^ercenta^e of total ash this value may be computed. Hov/ever, using the calcium content as a criterion is supposed to be more accurate, ov.dng to the possible contamination of leaves by soil. The alkalinity of the water-soluble ash is im-Dortant as a measurement of the form of potassium Table V A Study of the Ash Coir:.ont of the 194-2 and 1943 Crops of Cigar-Leaf Tobacco. The alkalinity being expressed as ra.e. per 100 gram saaple. :~s. g^.' Treatment Plot KuBibers ^ Total Ash. 19/r3 A B 1942 1942 Mature Cured Mature 2 '^ D 13, 25 19.10 E 15, 30 19.16 F 11, 24 20.50 G 12, 28 18.60 H 18, 26 19.3-1 I 64-, 79 17.93 22.36 17.16 21.25 16.81 22.69 17.55 22.55 16.60 21.4-5 16.91 194-3 Cured _Alkr.ll:iity_ of the Soluble Ash . 19T2 "194-2 194-3 194-3 Mature Cured Mature Cured ■7' 21.46 16.82 17.96 1, 16 6^ 26 20.84 16.70 20.41 19, 27 20.61 21.88 16.80 17 . 94 18.45 18.25 18.89 18.64 20.28 20.07 17.33 17.90 79 81 71 91 89 o u ^8 61 77 O 0 71 77 79 74 64 39 33 33 28 30 29 35 30 20 o 28 24 26 27 25 24 31 24 25 ■Wi ir3,JCrQi3s of Cl.^ar-Lcaf Tobacco and _Lp s s_pl\ Organic Llatter as a Result of Curins The calcium and nicotine content of both nature and cured samples- are listed in Table VI* The loss of dry weight during curing is based upon, the "apparent increase" in calciujn content as a result of this procer, s • Previous investigations (6) have indicated that more calcium is absorbed by tobacco plants during a dry rather than a v/et season, and the results based on the analysis of mature leaves of the 194-2 and 194-3 crops bears out this contention. Ov;ing to the fact that calcium appears to be more uniifornly distributed throughout the plant than elem.ents such as potassium, or organic compounds such as nicotine, the apparent increase in the percentage of calcium in cured leaves over that of mature leaves is usually em.ployed as a basis for calculating the loss of organic matter as a result of curing. These IS different plots of 9 different treatments as indicated in Table VI show an average loss of 22.08;:) and 2.63;,^ for 1942 and 1943 respectively. While this is an arbitrary mcasurem.ent, it is evident that m.ore loss Y/as incurred in 194-2 than in 194-3. Table VI The Cnlciun and nicotine Content of Mature and Cured Saiiiples of the 1942 and 194.3 Crops of Tobacco and Loss of Dry Matter on Curing . — -. — -. — - Plots Formula of Fertilizer Calc iuj.i I,'[a tur e 1942 Mature llicot Cured ine Mature ( I0 Aired LOSS Oj Mattel Curir -■ iJiy :* on Treat- ment 1942 Cured if^ Mature Cured 1942 1943 . ^ ' T i> 7" /'» >^. > A 1,16 1\ • 24- 3.80 3.84 — 2.23 /f • ^2 5.49 1.05 B 6,20 3-9-0 — . 4. 18 4.00 /f • 22 2.59 5.23 5.66 — — 5.21 C 19 , 27 3-9-6 3.55 4.21 3.81 4.00 2.00 2.47 4.64 5.52 15.68 4.75 D 13,25 3-9-12 3.43 4.41 3.96 4.01 1.35 2.18 5.11 6.22 22.23 1.25 E 15,30 3-9-18 3.40 4.20 3.61 3.62 JL. 9 / fC 2.49 5.05 5.98 19.02 0.28 F 11,24 3-9-24 3.24 4.33 3.63 3.66 1.69 2.29 5.20 5.82 25.17 0.82 G 12,28 3-6-12 3.17 4.34 3.91 4.00 1.77 9 ?Q 5.01 6.08 26.96 2.25 H 18,26 3-12-12 3.36 4.39 3.89 4.^4 1.82 2.55 4.93 5.67 23.47 14.38 I 64,79 3-9-12 3.18 4. 08 4.20 4.14 2.04 2.60 5.10 6.29 22.06 0 1 - 34 - The curing season of 1942 was marked by high- humidity. This favored a greater enzw.e nctivity^ but the p:reatest loss v/as no doubt due to the action of organisms v/hich degraded the leaves favoring a condition knovm as "shed-burn^^# It is aTroarent that the season overshadows the nroduction of nicotine over individual treatm.ents. During a moist season a relatively low nicotine content is noted, v/hile under dry conditions the nicotine content is much higher. Based upon the calcium content of mature and cured leaves of the 1942 crop, it appears that no loss of nicotine results during curing. This does not hold for the 19A3 season but if the loss of dry matter during curing is computed on the basis of total ash, little if any loss was apparent. These methods of calculation, hov;ever, appear to have certain limitations. A high nicotine content of cigar-leaf is usually indicative of a tobacco that will not ferment well. Some believe that this is on account of the nicotine, others believe that its presence signifies a metabolism leading to the formation of a substrate not adapted for the organisms involved in fermentation. A - 35 - -Study^pf^ of Mature and Cured bajiirle^ of th^JL9/,2 anCCl^A;^ Crops' of" Cii^ar-Leaf Tobacco'^ The results obtained on the nitroren content of mature and cured leaf samples of the 1942 and ±9 A3 crops are presented in Tables VII and VIII • The results presented in Table VII indicate a relatively high total nitrogen and protein content of the 1942 cro;n of tobacco. However, it is evident that considerable of the total and protein nitrogen v:as lost on curing. Such proioujid effects are abnormal, and may be attributed to the high humidity of the environi;ient, which favored the ixultiplicity of organisms deleterious to the integrity of the leaf* In 1943, according to Table VIII, the total n itrogen was somewhat lovver and the protein content markedly lov:er for mature leaves compared to those of the 1942 crop. However, the non-::rctein nitrogen content of the form.er leaves was very much higher* Furthermore, considerable of the protein nitrogen vras later converted to non-protein nitrogen as a result of curing* During a drought readily available carbohydrates are utilized in respiration. When these are more or less exhausted, proteins are hydrolyzed and otherwise i!mHPi^MBnir^^>»iw ,0 A B C D E F G H 1, 16 6, 20 19, 27 13, 25 15, 30 11, 24 12, 28 18, 26 64, 79 3-9-0 3-9-6 3-9-12 3-9-18 3-9-24 3-6-12 3-12-12 3-9-12 4.53 4.75 4.65 4.63 4.56 4.03 2.99 3.18 3.17 3.27 3.34 3.07 3.79 1.60 1.58 1.48 1.26 1.22 J. m/\.JL 1.25 7i> 3.19 > 1.77 3.50 1.97 3.50 1.98 3.50 1.92 3.83 1.90 3.84 1.94 3.32 1.81 3.45 1.75 3.22 1.75 7» 1.43 1.53 1.53 1.S9 1.93 1.91 1.52 1.65 1.47 I I Table VIII The Total, Protein and jlon-Protein llitrogen of Cigar-Leaf Tobacco as Influenced by Field Treatment, Season and Curing »~" '^ "^m- ' The Nitrogen Content of the 1943 Samples of Ci,r!:ar-Leaf Tobacco. , ■ I »—■■>■ n^ -*-— Formula Total Protein Mon-Proteln Total Protein Hon-Proteln Plot of IT N N M N li Treatment Nixmbers Fertilizer Mature Mature Mature Cured Cured Cured "'-'■' r ' ■ •f" % A 1, 16 4.28 2.00 0 OcJ; 4.09 1 ' ^ 2.54 B 6, 20 3-9-0 4.65 9 0 / 2.41 3.37 1*26 2.61 C 19, 27 3-9-6 4.32 1.9s 2»34 3.71 1 '^i J. • f-^X, 2.50 D 13, 25 3-9-12 4.33 2.12 2.26 4.57 1.54 3.03 E 15, 30 3-9-lQ 4.40 2.10 2.30 4.05 1.61 2.44 F 11, 24 3-9-24. 4.49 2.14 2.35 3.93 1.23 2.70 G 12, 28 3-6-12 4.3s 2.16 p 00 4.15 1.41 2.74 H 13, 26 3-12-12 4.12 1.8S 2.24 3.47 n 1 s 2.32 I 6>4, 79 3-9-12 4.36 2.09 ft m f^ ( 4.0s 1 ' -^ J- »/+0 2.62 1 .^ '"1 o broken dov.Ti to rorm energy-producing materials; oxalic acid being one of the stable by-products of these reactions. SorneY;hat similar reactions appear to result on curing which is ordinarily a slov drying process, somewhat analogous to that of a drought. The relatively high non-protein nitrogen foujid in the plants previous to harvesting indicates that the TDrotein content vras sonevrhat higher during the ripening process. This contention is further strengthened by the high o"alic acid of the nature leaves. Non-protein nitrogen is found to be an excellent nitrogen carrier for the v^ildfire oroanism. During its netabolism on such a substrate considerable ouantities of free anmonia are liberated. If siich reactions ^a.:e place v.dthin the leaf, a "break do\Tn" of the tissues take Place. The abrupt breaking of a drought follov/ed bv continued v/et leather usually favors the penetration V of the leaf by these or.'^anisms, vith disastrous results, especially if the crop is approaching maturity. This condition has been referred to by some investi- gators as "a:nmonia toxicity". A Gtudv of the Potassiu|n_Cpiitent_of_.the.J.2iL2_ax^^^^ For these studies, saaples of cured tobacco T/ere taken for analysis and burning tests. - 39 - The results or tne burning tests for the I94I crop are likevdse listed. It is of interest to note that the ' growins season of this particular crop closely approached that of 194,3. It is evident frora Table IX that a :n oist season rather than a dry season favors tVio intake of potassiui^. The burn of all sanples of the 19^2 crop was classed as good. The potassim content of these sanples averaged 3.37;.'. On the other hand, the burn- ing quality of the 194.3 crop ^7as quite poor. It is interesting to note that the -otassiurn content of these samples averaged but 2.12;j. A Study of the ,.Oj-anic,_jicid intent of tie 19/ p and 1943_Craos _oi_ Ci/;ar- L e a f To b a c c o In these studies it v/as doc iced t o aeternine the volatile acid content of both nature and cured leaves of the 19^2 crop of tobacco. It v/as thought that possibly the volatile acid content v/ould sliov/ an increase as a result of curing. Iiov;evor, the results were by no means highly ri-nificant in this respect, so that it v;as decided not to re-ieat then on the 194-3 samples. Considerable attention v;as given as to the oxalic, citric and malic acid content of mature and Table IX The Potash Content and the Burn of Cigar-Leaf Tobacco as Affected by Field Treatment, Reason and Curing TreF.t"ient Plot Numbers Field Treatment Fertilizer T/A Manure Formula Quantity lb ./A Potassium Cured Cured 1942 1943 -/„ r1 Burn 1941 1942 1943 1, 16 10 B 6, 20 10 3-9-0 C 19, 27 10 3-9-6 D 13, 25 10 3-9-12 E 15, 30 10 3-9-18 F 11, 10 3-9-24 G 1 ^ 28 10 3-6-12 H 18, 26 10 3-12-12 I 64, 79 0 3-9-12 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 3.19 2.32 F to G 3.74 2.43 P to F 4 . 13 3.96 1.97 2.26 1.75 /j. • O/C /^ m J I 6 G P to F P to F P to F P to F 4.05 A. • <<1 / P to F G P to F 3.53 2.25 P to F G P 3.53 1.43 P G P to F 1 o Ps, Poor, P to F — Poor to Fair, F= Fair, P to G - Fair to Good, G = Good - 41 cured tobacco oi" both cro-os. The results obtained are presented in Tables <^ X and }'I . The o::alic acid content of both matured and cured samples of the 194-2 tobacco v;as loY/er than that obtained for 1943 . At this point it may be stated that a low oxalic acid content usually is associated with good burning tobacco and one rhich ferments readily. On the other hand, a high oxalic acid content is evidence of poor cuality tobacco, both from the stand- rffc •-• " 1^ oint of burn and ease of fermentation. It may be noted that the malic acid content is higher than that for citric acid in mature leaves, but such a condition is som.ewhat reversed as a result of curin g. This is in agreement v.dth the findings of Garner, Bacon and Foubert (5), and Vickery and Pucher (27). Hov;cvcr, the citric acid content of the 19/;.3 tobacco v/as sonev/hat higher and the malic acid some\:hat lov/er than that of 1942. During the curing of the 1942 tobacco a greater decrease of malic acid v/as noted than held true for the 1943 tobacco. It is readily apparent, therefore, that the effect of the gro\7ing and curing season tends to overshadow the effects of a given field treatment. The term "curing", according to Garner (3), (4) J Garner, Bacon and Foubert (5); Loov/ (11), and others is £omev;hat indefinite in meaning, being used to include Table X . . The Volatile Acid, Oxalic, Citric and Malic Acid Content of the 1942 Crop of Tobacco as Affected by Season, Field Treatment and Curing Plot Volatile Acids Oxalic Iiature ■i Acid Cured Citric rlature Acid Cured Ifelic Acid Trcr.traent IluuVoers ..ature Cured rigm./S gras. mgra./5 [^ms. nature Cured '^3 A 1, 16 9-42 3*00 6.76 3.41 B 6, 20 10,25 — .i.-* -» 2.97 mmmm.^.^ 6.62 mmmm,mm^^ 1.75 19, 27 8.01 10.10 1.S8 3.07 2.95 7.25 7.27 3 • 04 E H 13, 25 8.95 7.93 2.25 2.39 2.97 0 • ^/C 5.38 3.36 15, 30 9.02 8.03 2.23 2.31 2.33 7.57 4.39 2.93 11, 24 8.75 10.33 2.20 3.04 0 <<> 6.96 7.65 2.71 12, 28 8.01 10.96 0 ,'-^ Q 3.05 1.93 6.98 8.00 /i.40 18, 26 6.38 10.99 2.09 3.17 2.20 7.30 2.78 64, 79 6.65 9.39 2.31 3.11 2.56 4.72 7.21 3.40 1 4>- ■■ Table XI The Oxalic, Citric and Ilalic Acid Content of the 1943 Crop of Cigar as Affected by Field Treatiaent, treason ana Curing -Leaf Tobacco Treatment A B n D E F U H I Plo ■»- Ilurabers 1, 16 6, 20 __Oxalic Acid luature Cured 3.26 3.20 19, 27 2.94 13, 25 3.76 15, 30 3. 38 11, 24 3.49 12, 28 3.01 18, 26 3.24 6/+, 79 3.54 4.05 3.75 3.91 3.67 3.92 4.19 3.65 3.31 4.25 Citric Acid Mature Cured' $ 3.93 3.38 2.64 2.77 ::> .10 3.71 3.02 2.52 3.16 '/O 4 . 16 6.25 4.38 4. SO 3.99 4.39 5.08 5.01 4.73 :.iallC Mature p 6 1 ^ 7.15 6.96 7.02 6.29 5.37 7.54 6.59 7.19 Acid, ,. Cured 4.66 4.73 3.94 3.94 4.63 5.31 4 . 37 VjJ I - 44 - the separate operations of barn curing, feraentation and aging, vrhile the tobacco r,roY:er restricts the term to the process of drying the ripe leaf in a soecially constructed shed and under such conditions as will develop the desired properties or '-'ualities. In these investigations the term ''curing" has been considered as It --roceeds in the curing shed. Curing is essentially a life process, as is shovni by the fact that hilling the proto-olasri bv - ' V excessively lov; or high temperatures or by means of poisons such as chloroform effectively prevents normal curing. According to Valleau and Jolinson (2$), tlie relative humidity is a very important factor in curing. As the leaves slowly die and become yellov; in color a humidity of Z% is desirable. If it exceeds 90,;o shed- burn may result. On the other hand, if the humiditv is much less than S5/o, the tobacco nay dry out too raioidly and result in an imperfect cure. The temperature of the curing shed is another important factor to be taken into consideration. Leaf activity practically ceases at 40^F. but becomes more active as the temperature increases. The best temperature for the first stable of curing^ or -.he yell stage, according to Valleau and Jorinson (25)^ lies ov/ - 45 - betv/een 60^F. and 100°F» The chemical changes taking place during the curing process are numerous. As a result of thorough curing all the starch and reducing sug' rs are said to disappear, and there is a decrease in pentosans and malic acid. On the other hand, there is an increase in citric acid, vhile the cellulose remain unaffected. At the same time there is said to be a large decrease in nicotine and total nitrogen (5) • The physiological processes characteristic of cigar-leaf tobacco indicate the presence of amylases, proteinases, dcamidases, o::idases and other enzymes. The process of starvation to v;hich the leaves are subjected appears to lead to an Increased secretion of amylase. That the chemical changes which take place during curing are enzymatic in nature appear to be V;'ell established. Furthermore, these enzj'-mes must of necessity be leaf enzymes, as normally there does not appear to be micro-organisms on the leaf surfaces in any great number. As to the role played by leaf enzymes during curing, this has yet to be established It is apparent, hov/ever, that bacterial enzymes play a decided role during fermentation. - 46 - The investi.'^ations of LIcKinstry, Haley and Reid (12); Reid, McKinstry and Haley (21); Reid, Haley, iicKinstry and Surraatis (18)^ have established the fact that under the proper conditions of moisture, temperature and aeration, there is a raT)id increase in the nunber of organisms present on or v/ithin the tobacco leaves during fermentation. The nLimber and activities of these or^'^anisms de^jend in large measure on the chemical composition of the leaf. Other things being equal, vrell cured tobacco V7ill ferment more satisfactorily than poorly cured tobacco. The investi::ations of LIcKinstry (13) indicate that malic and citric acids furnish a great deal of the energy requirements for the fermentation organism. Furthermore, a relatively high citric acid content is usually indicative of a tobacco of good cuality and one which ferm.ents readily. However, it appears that for best results a considerable part of this acid should be in combination with potassium. Active potassium, liberated during fermentation, suffices to increase the alkalinity of the medium, a condition Y:hich further favors the y^rocess. Attention las "oeen caj,led to the fact that ordinarily the curing process is somewhat analogous to the condition of plants undergoing a drought in - 47 - the rielci^ Txs both represent a condition of slow starvation. In this connection it r:iay be stated that the production of oxalic acid in the field and in the curing shed v/arrants this concluision* - 4-S ~ Suimnary and Conclusions This Investigation had for its purpose a study of the yield, composition and quality of Pennsylvaxila cigar-leaf tobacco as influenced by ce:-:_.ain environ- mental conditions including field treatnont, crowing season and curing. Saiaples choren for this study were taken from mature ^jlants in the field from selected ex.oerinental plots and aaditional samples -vere ta!:en of the same plants at the end of the curing season. Determinations of individual leaf constituents were conducted in both mature and cured samples for the seasons of 1942 and 1943. The results obtained v/ere correlated -..ith the yield and ; uality of tobacco from the various plots studied and included the seasons of 194.1, 1942 and 1943. Kotv:ithstanding the abnormal-ly dry seasons of 1941 and 1943, and the abnormally wet season of 1942, better yields and wrapper production were obtained wlien ma anurial additions wore supplemented v/ith fertilizer mixtures of well-balanced nutrients than with manure alo ne, fertilizer alone or \'hen the manure was su-oT;lemented with an unbalanced fertilizer mixture. In this connection it may be stated that fertilizers having the formula of 3-6-12 or 3-9-12 gave better 4-9 - n** G suits than aiiy other individual treatraent. From the standr/oint of chemical coinpocition the effect of the growing and curing seasons overshadowed the effects of a ,i;iven field treatment, and this held for both inorganic and organic constituents. Studies of ash and ash constituents shor.'ed the percentage of total ash to be practically the sarae for both seasons regardless of troatr.ient. Hovjever, nore ash Tias renoved by the 194-3 tobacco since the total yield was much greater. The alkalinity of the soluble ash showed a much higher value for 194?- than for 1943. This was due to the fact that there vras a greater concentration of organically combined potassium in these particular samples. Such a condition favors the "burn" of tobacco and is usually correlated with the total quantity of potassiiJin absorbed by the plants. The calcium content and total ash showed an "apparent increase" as a result of curing. This was due to a loss of organic matter during curing, a loss that was far greater in 194-2 than in 194-3 ♦ Previous investigations showed that under dry conditions the calcium content v:as greater and the potassium content was considerably lower than under moist conditions. The results submitted show this to - 50 - be true for 194-2 and 1%3* The nitro,':;en content of the 1942 and 194-3 samples of mature tobacco analyzed practically the same. However, in 194-2 the protein content v/as much higher than the non-protein content. During curing there v/as a noticeable loss of total nitrogen and a decrease in protein nitrogen as well* The non-protein nitrogen was quite high in the leaves of the 1943 crop of mature tobacco. Such a condition indicates an hydrolysis of leaf proteins as a result of drought. This condition w-as aggravated on c ur mg • The nicotine content was noticeably higher in 1943 than in 1942* It v/as considered that the ^^apparent increase^' in the nicotine content on curing v/as due to a loss of organic matter. The nicotine content of all samples of the 1942 tobacco v/as ouite lov:. In respect to rii-ied acids it _iay be stated that the degradation of :)roteins as a result of drought is followed by an accumulation of oxalic acid. To a certain extent this holds true on curing as t-iir Is a "drying out" process to a considerable degree. It is not sur^orising, therefore, to find "chat tiie oxalic acid content of the 1943 samples was much higher than those of 1942. The malic acid content of mature tobacco is :iuch - 51 - higlier than its citric acid content, a condition v/hich tends to become revised as a result of curing* Such conditions were found to hold true for all saLiples of both the 1942 and 1943 crops of tobacco. The decrease in malic acid and the increase of citric acid during curing, v;hich v/as so apparent in the case of the sainples of the 194-2 crop, is considered desirable. Both of these acids serve as enercy material for the orcanisms of f ernientation. It is apparent, hov/- ever, that for sone reason not yet understood that tlie 1 -1 +-- hiPiher the citric acid content the higher the nualiu \r The study of the tobacco produced in 1942 and 1943 indicates an overshadowing influence of the grow- .ng season. These ex^oeriiients serve to substantiate the sratei-ient that for best results the rainfall should be both anple and v/el]. aistributed throughout the grov/- mg season m a "^ ( r-, ition to auple supplies of oreanic and inorganic Dlant nutrients as soi .* 1 idditions • - 52 - A c knov/1 e d .foment s The author v.dshes to express his appreciation for the many helpfiil suggestions of Dr# D. E. Haley during the progress of this investigation and in the preparation of this manuscript, and the cooperation of Dr. 0. E* Street, U. S. Department of Agriculture, and Mr. A. Vincent Hurst, Bayulc Cigars, Inc., is gratefully acl:nor:ledged and deeply appreciated^ - 53 - Bibliography !• Behrens, J. 189^» V/eitere Beitrage zur Kenntnis der Tabakpflanze. V, Der Anatomische Bau imd die Bestandteile des Tabakblatts in ihrer Beziehung zur Brennbarkeit* Landw. Ver* Sta. ^3: 271-301. 2. 3. K. Garner, W. ¥• 1907 • The relation of the composition of the burning qualities of tobacco* Agr* Bur. Plant Ind. Bull. 105. the leaf to U. S. Dept. Garner, W. W. 1909. Principles and practical methods of curing tobacco. U. S. Dept. Agr. Bur. Plant Ind. Bull. 143* Garner, V/. W. 1913. Tobacco curing. Bull. 523* U. S* Dept. /Igr. Farmers^ 5. Garner, V/. W., Bacon, C. W., and Foubert, C. L. 19U* Research studies on the curing of leaf tobacco* U. S. Dept. Agr. Bur. Plant Ind. Prof. Paper 79. 6. Haley, D. E., Nasset, E. S., and Olson, Otto. 1928. A study of certain constituents of the leaf and their relation to the burning Gualities of tobacco. Plant Physiol., Vol. 3: 185-197. 7. Haley, D. E. 1929. On the relation of potassium to iron in the combustion of cigar-leaf tobacco. Science, Vol. 70: 17-18. S. Haley, D. E., Street, 0. E., Farrell, M. A., and Re id, J. J. 1943* The yield and composition of cigar-leaf tobacco as influenced by fertilizer and preceding crop. Penna. Agr. Expt. Sta. Bull. MO. - 54 - 9. Haley, D. E., and Reld, J. J. 1943. The bearing of potassium on the quality of tobacco. Soil Science, Vol. 55: 79-86. 10. 11. 12. 13. 1 / 1 ^ 1, J • 16. 17. Johnson, E. M., and Valleau, \V. D. 1940. Control of Blackfire of tobacco in v/estern Kentucky. Kentuclcy Agr. Expt. Sta. Bull. 399. Loew, 0. 1899. Curing and fermentation of cigar-leaf tobacco. U.S. Dept. Agr. Rpt. 59. J. -J. McKinstry, D. W., Haley, D. E., and Reid, 1938. A bacteriological study of the bulk fermentation of cigar-leaf tobacco. Jour. Bact., Vol. 35: 71. McKinstry, D. v;. 1938. The relation of bacterial activity to the disaDDearance of citric and raalic acids in the bulk fermentation of tobacco. Doctor^ s Dissertation. The Pennsylvania , State College, State College, Pa. Nessler, J. 1867. Der Tabaks seine Bestandteile unci seine Behandlung. (Mannheim.) Official and Tentative Methods of Analysis of the Association of Official Agri- cultural Chemists. 5th Ed. 1940. Pucher, G. V/., Vickery, H. B., and VJakeman, A. J. 1934* Determination of the acids of plant tissues. II. Total organic acids of tobacco leaf. Ind. and Eng. Chem., Anal. Ed., Vol. 6: 140-143. Pucher, G. V/., Vickery, H. B., and Wakeman, A. J. 1934* Determination of malic acid in plant tissue. Ind. and Eng. Chem., Anal. Ed., Vol. 6: 238-291. i - 55 -- 18. Reid, J. J., Haley, D. E., McKinstry, D* \N., and Surmatis, J. D. 1937* The relation of catalase activity to the microflora of cured and fermenting tobacco* Jour# Bact* 34- • 4-60. 19* Reid, J. J*, McKinstry, D* ¥•, and Haley, D. E* 1937. The fermentation of cigar-leaf tobacco. Science, Vol. 86: AOA* 20. Reid, J. J., McKinstry, D. V/., and Haley, D. E. 1938. Studies on the fermentation of tobacco. I. The microflora of cured and fermenting cigar-leaf tobacco. Pa. Agr. Expt. Sta. Bull. 356. 21. Reid, J. J., McKinstry, D. W., and Haley, D. E. 1938. Studies on the fermentation of toDacco. II. Microorganisms isolated from cigar-leaf tobacco. Pa* Agr. Expt. Sta. Bull. 363. 22. Schlosing, Th. 1860. Uber die Verbrennlichkeit des Tabaks. Landw. Ver. Sta. 3: 93-100. o 'i 3. Shear, G. M. 1941. Factors affecting physiolosical breaMovm of maturing tobacco. Va. Agr. Expt. Sta. Tech. Bull. 74. 24. Thomas, J. J., Haley, D. F., and Olson, Otto. 1936. The absorption of plant nutrients in relation to the production of Pennsylvania cigar-leaf tobacco. Pa. Agr. Exp. Sta. Bull. 331. 25. 26. Valleau, W. D., and Johnson, E. LI. Tobacco diseases in Kentucky, Agr. Exp. Sta. Bull. 32S, 1932. Kentucky Van geraaielen, J. M. 1899. Uber die Zusammensetzung der Asche der Tabaksblatter in Beziehung zu ihrer futen Oder schlechten Oualitat insbesondere zu . 56 - ihrer Brennbarkeit. 409-436 • Landw. Ver. Sta. 37 21. Vickery, H. B^, and Pucher, G, W* 1933* The effect of the curing process on the organic acids of tobacco leaves • Conn. Agr. Exp, Sta. Bull. 352.