The Organic Matter of the Soil: A Study of

the Nitrogen Distribution in Different

Soil Types

A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE
SCHOOL OF THE UNIVERSITY OF, MINNESOTA

BY

CLARENCE AUSTIN , MORROW, B.S.,M.A.

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

JUNE, 1918

MINNEAPOLIS
UNIVERSITY PRINTING CO.

EXCHANGE

The Organic Matter of the Soil: A Study of

the Nitrogen Distribution in Different

Soil Types

A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE
SCHOOL OF THE UNIVERSITY OF MINNESOTA

BY

CLARENCE AUSTIN MORROW, B.S., M.A.

< i

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

JUNE, 1918

MINNEAPOLIS
UNIVERSITY PRINTING CO.

TO MY FATHER AND MOTHER

428979

ACKNOWLEDGMENT.

This investigation was carried out under the direction of Doc-
tor Ross Aiken Gortner. The author takes this opportunity to
express his appreciation and gratitude for the help extended during
the prosecution of this work, and for the unfailing kindness,
thoughtful advice and encouragement which was given.

C. A. M.

University of Minnesota.
Division of Soils.
January 1917.

TABLE OF CONTENTS.

Page
I. Introduction: Our present knowledge of the organic matter of

the soil 7

A. Early investigations ^ 7

B. The humus theory of Grandeau 12

C. The complexity of the ammonia soluble material 14

D. The presence of definite organic compounds in the soil.. 15

E. The origin of organic compounds in the soil 16

1. Nucleoprotein decomposition 17

2. Nucleic acid decomposition 17

3. Lecithin decomposition 18

F. Bacterial processes which influence the form of soil

nitrogen 19

1. Deamination or reduction , 20

2. Decarboxylation or amine formation 20

3. Hydrolysis 20

4. Oxidation 21

G. 'Nitrogen distribution in the soil . . . . 21

H. A summary of the nature of the organic matter of the

soil in the light of our present knowledge 29

II. Experimental: A study of the nitrogen distribution in different

soil types 31 -

A. The problem 31

B. The material : 31

1. Calcareous black grass-peat 31

2. Sphagnum-covered peat 31

3. Acid "muck" soil 31

4. Fargo clay loam 32

5. Fargo silt loam 32

6. Carrington silt loam 32

7. Hempstead silt loam 32

8. Prairie-covered loess 32

9. Forest-covered loess 32

10. Hempstead silt loam subsoil 32

C. The method 33

1. The method in detail for a peat soil 34

2. The method in detail for a mineral soil 36

3. The method for determination of "Jodidi num-

bers" ; 37

4. The determination of nitrogen 38

4. The analytical data 38

1. The analysis of "fibrin from blood" hydrolyzed in

the presence of 100 grams of ignited subsoil 38

2. Calcareous black grass-peat 41

3. Sphagnum-covered peat 42

4. Acid "muck" soil.. 42

5. Fargo clay loam 43

6. Fargo silt loam 44

7. Carrington silt loam 45

8. Hempstead silt loam 45

9. Prairie-covered loess 46

10. Forest-covered loess 47

11. Sphagnum-covered peat hydrolyzed in the pres-

ence of nine times its weight of a mineral
subsoil 47

12. Sphagnum-covered peat hydrolyzed in the pres-

ence of metallic tin 49

13. Analysis of a 1 per cent, hydrochloric acid extract

of sphagnum-covered peat and (in part) of
calcareous black grass-peat 50

14. Analysis of a portion of sphagnum-covered peat

soluble in 4 per cent, sodium hydroxide and
precipitated by hydrochloric acid and (in
part) of a similar solution from a calcareous
black grass-peat 52

15. Analysis of a portion of sphagnum-covered peat

soluble in 4 per cent sodium hydroxide and
not precipitated by hydrochloric acid and (in
part) of a similar solution from a calcareous
black grass-peat 54

16. "Jodidi numbers" ! 55

17. Summary tables 57

18. An attempt to isolate pure proteins from a soil 59

a. Extraction with 70 per cent ethyl alcohol 59

b. Extraction with absolute alcohol 6U

c. Extraction with 10 per cent sodium chlor-

ide 61

III. Discussion 62

A. Changes in nitrogen distribution in a protein when hy-
drolyzed in the presence of a mineral soil 62

B. The human nitrogen, its origin, and significance 63

C. The effect of the quantity of acid used for the hydrolysis
on the amount of nitrogen dissolved and the nitrogen
distribution in soils 66

D. The percentage of soil nitrogen extracted by acid hy-
drolysis 66

E. "Jodidi numbers" 67

F. Attempts to extract proteins from the soil 67

G. A consideration of the nitrogen distribution in different
extracts from the sphagnum-covered peat 67

H. General conclusions in regard to the distribution of soil

nitrogen in different soil types 68

IV. Summary 70

V. Literature cited 72

Biographical 80

I. INTRODUCTION: OUR PRESENT KNOWLEDGE OF
THE ORGANIC MATTER OF THE SOIL.

A. Early Investigations.

It has been recognized from the time of the alchemists that
manures are of fundamental importance for the growth of plants.
The alchemists believed that by a process of transmutation water
was converted into plant tissue. In an attempt to prove this an
interesting experiment was conducted by van Helmont (1648).
In a large earthen vessel he placed 200 pounds of dry earth, and
planted in it a small willow tree weighing five pounds, and for five
years he watered the plant either with rain or distilled water. .At
the end of that time he pulled up the willow and found that it
weighed 169 pounds and three ounces. The dry soil remaining after
the experiment was found to have lost only two ounces. He drew
the apparently justifiable conclusion that 164 pounds of roots, bark,
leaves, and branches had been produced by direct transmutation
of water.

It is evident that it was essential to establish the composition
of water and some of the components of the air before further
work could have real value.

Not until the discovery of oxygen by Scheele (1777) and
the proof of the composition of water by Cavendish (1784), as well
as the work of de Saussure (1804) regarding the role played by
carbon dioxide in plant and animal life, did we have any real
knowledge concerning the sources of matter stored up in plants.

During the first quarter of the nineteenth century organic com-
pounds were regarded as capable of being synthesized only in the
living cells of plants or animals. This idea that organic compounds
could be formed only through a special vital force was overthrown
by the classic work of Wohler (1828) when he prepared urea, a
purely animal product*, by evaporating ammonium cyanate to
dryness. This fact attracted the attention of chemists, and prac-
tically all work done from that time until 1840 was on some phase
of organic chemistry. This overthrow of the belief in a vital force
and the improved method of organic analysis by Berzelius (1808-
18) paved the way for a more thorough understanding of the part
taken by the organic matter in the soil, and consequently created
a renewed interest in scientific investigations relating to agricul-
ture.

Some important work had been accomplished previous to this
time. The most valuable was that of de Saussure's (1804) "Re-
cherches chimiques sur la vegetation" which was the first syste-
matic work showing the source of compounds stored up in the
plant. He pointed out that the quantitative increase in the carbon,
hydrogen, and oxygen, when plants were exposed to sunlight, was

*Fosse (1912) notes the presence of urea in certain plants.

8

due to the carbon dioxide of the air and water of the soil. He be-
lieved that the nitrogen of the soil was the chief source of the
nitro-gen found in plants. Unfortunately his conclusions were
not accepted at that time, and it was not until about fifty years
later, when other investigators had repeated his experiments, that
his results were finally accepted by botanists and chemists.

One of the first investigators to see the relation between chem-
istry and agriculture was that of Sir Humphry Davy (1813), who
published a book entitled, "Elements of Agricultural Chemistry."
This treated of the composition of air, soil, manures, plants, and
of the influence of heat and light upon the growth of plants.

Thaer (1809-10) contended that humus determined the fertility
of the soil, that plants obtained their food mainly from humus,
and that the carbon compounds of plants were produced from the
organic carbon compounds of the soil. These ideas gave rise to his
so-called humus theory, which was later shown to be inadequate.
His writings, however, did much to stimulate later investigation.

The French investigator Boussingault verified much of the
earlier work of de Saussure and secured many additional facts
concerning the chemistry of growth. His predecessors had sought
to solve the question as to whether plants assimilate the free or
uncombined nitrogen of the atmosphere. Boussingault (1838,
1838a) improved the methods for the determination of the point in
question, and showed that peas and clover could get their nitrogen
from the air while wheat could not. Unfortunately, he did not
make as much of this discovery as he might have done.

Boussingault was the first to have a chemical laboratory lo-
cated on a farm and to make investigations along a practical line
in connection with agriculture. His was the first agricultural ex-
periment station.

The investigations of de Saussure, Boussingault, Davy, Thaer,
and others paved the way for the work and writings of Liebig. He
published (1840) "Organic Chemistry in its Application to Agri-
culture and Physiology," which was an important factor in at-
tracting the attention of the public to> agricultural problems. Many
of his investigations and discoveries in the field of organic chem-
istry were applied directly to his interpretation of these problems.

He assailed the humus theory of Thaer and showed that humus
could not be an adequate source of the plant's carbon. By applying
the exact methods of chemistry to agriculture Liebig succeeded in
establishing that plants derive the carbon of their tissues from
the carbon dioxide of the air, and not from the carbon compounds
that may be present in the soil. He came to regard the ammonia
of the air as analogous with the carbon dioxide of the air, and
preached the doctrine that plants were able to derive their nitrogen-
ous food from the atmosphere. In the Farmer's Magazine, for in-
stance, he writes :

If the soil be suitable, if it contains a sufficient quantity of alkalis, phos-
phates, and sulphates, nothing will be wanting. The plants will derive their
ammonia from the atmosphere as they do carbonic acid. (Cited by Russell,
1912.)

Although the work of Liebig was not conducted in connection
with field experiments, it had a stimulating effect upon agricultural

investigation, and we are greatly indebted to him for summarizing
previous work and pointing out valuable lines of future research.
In his book (1840) he states, that—

a rational system of agriculture cannot be formed without the application
of scientific principles, for such a system must be based on an exact ac-
quaintance with the means of nutrition of vegetables, and with the influence
of soils, and actions of manures upon them. This knowledge we must seek
from chemistry, which teaches the mode of investigating- the composition and
study of the character of the different substances from which plants derive
their nourishment.

In one essential point, however, he fell into error. Lawes, the
pioneer experimenter on agriculture in England, flatly denied the
accuracy of Liebig's conclusions as regards nitrogen assimilation.
The results of the investigations at Rothamsted as conducted by
Lawes and Gilbert (1851) on the non-assimilation of atmospheric
nitrogen by crops, were accepted as conclusive evidence upon this
much discussed question.

The alkali soluble portion of the organic matter of the soil
has formed for many years the subject of keen interest and discus-
sion. This portion of the soil organic matter was called "humus"
by the earlier writers, but this name has in more recent times been
used by some American and most European investigators to desig-
nate the total organic matter of the soil. I have used the term
throughout this paper in its original meaning. In early days the
"humus" was regarded as being of very simple composition. De
Saussure (1804) for instance, described it as a "brown combustible
powder soluble in alkalies and ammonia compounds."

Klaproth* applied the name "ulmin" to dark colored amorphous
bodies such as those obtained by Vauquelin (1797) from the bark
of diseased elm trees. Sprengel (De Candolle** 1833, p. 280), who
obtained similar bodies from soils applied to these the name "humic
acid." Berzelius (1838) evidently had the general meaning of the
term "humus" in mind when he used the term "humin" in describ-
ing certain dark colored constituents of vegetable mold. Follow-
ing the use of the term "humin" as applied to what was considered
to be a definite organic body, a number of other workers took up
the study of similar substances, and a number of other terms more
or less related, soon appeared. The name of Mulder (1849) is asso-
ciated for the most part with the terms applied to humus-like sub-
stances which have appeared more or less in the literature from that
time to the present. For instance, he says :

At present seven different organic substances are known to exist in the
soil. They are crenic acid, apocrenic acid, geic acid, humic acid, and humin,
nlmic acid and ulmin.

These bodies were divided by him into two groups, one con-
sisting of crenic and apocrenic acids, and the other group embrac-
ing all the others. According to Mulder (1849) these seven or-
ganic bodies were intimately related, and five at least were five suc-
cessive steps in the decay of organic matter in the soil. The first
step in this decay he regarded as ulmic acid ; this on further
oxidation yielded humic acid ; and this in its turn, on still further
oxidation, geic acid. Continued oxidation produced apocrenic,
and finally crenic acid.

*(De Candolle 1833, p. 279) states "Das Ulmin ist von Klaproth entdeckt
ivorben."

** I" have been unable to verify the original citation, which, according to
Uoper was Kastner's Archiv. Bd. 7, p. 163; Bd. 8, p. 145. Presumably one of
these articles is that referred to by Russell (1912) entitled "Ueber Pflanzen-
Xaturlehre, Niirnberg, 1826.

10

A number of chemists have given the percentage composition
of the supposed acids, but no two agree. Nothing is known in re-
gard to their constitution. The lack of definite chemical character-
ization of these compounds is stated by Cameron and Bell (1905)
as follows :

The existence itself of these acids has never been satisfactorily demon-
strated. * * * * No satisfactory description of the physical or chemical
properties of these supposed acids, their salts, or characteristic derivatives,
have been recorded.

Nearly every writer on soils from the time of Mulder to Hil-
gard (1906, p. 126) spoke of these acids with the same assurance
as of oxalic or tartaric acids, or any other organic compound that
has well known derivatives.

The early investigators, including Mulder, soon found that
sugar, starch, carbohydrates generally, and even proteins, when
treated with strong acids or alkalies, gave rise to dark colored
compounds having the same general appearance and properties
as the humus substances arising in the soil through decay. How-
ever, a conspicuous feature of the work on these humus substances
is the discordant results for preparations bearing the same name
and often from the same source.

Robertson, Irvine, and Dobson (1907) reached conclusions that
although there were many strong resemblances between natural
and artificial humus preparations, in regard to properties and com-
position, yet there are important differences in constitution. Re-
cently Gortner (1916 c) has shown that in all probability the humin
formed from carbohydrates is actually formed by a polymerization
of furfural which is in turn formed from the carbohydrates by the
action of the acid. Gortner and Blish (1915), Gortner (1916 c), and
Gortner and Holm (1917) have likewise shown that the dark col-
ored products originating in an acid hydrolysis of protein sub-
stances have their origin in the tryptophane nucleus. Obviously,
if carbohydrate humin originates from furfural, and protein humins
originate in the tryptophane nucleus, mixtures of protein and
carbohydrate would produce a great variety of physically similar
but chemically different mixtures.

One of the important points at issue between the early in-
vestigators was whether humic acid and allied bodies contained
nitrogen as a constituent. Mulder (1849, 1862) held that nitrogen
was not a constituent of these substances, but was present as
ammonia, that is the acids were present in the soil as ammonium
salts, and in this connection he says :

In good arable soil — that is, one in which the organic constituents are as
far as possible decomposed — none of these substances contain nitrogen
as a constituent element; all their nitrogen exists in the state of ammonia.

Detmer (1871) came to the opposite conclusion, claiming that
nitrogen in humic acid as usually obtained, was present in organic
combination (not, however, bound in the humic acid molecule).
He obtained his humus by digesting with alkali. After precipitat-
ing with acid, he redissolved it in ammonia and precipitated the
mineral constituents with phosphoric and oxalic acids and am-
monium sulphide. After treating with potassium hydroxide and
precipitating with hydrochloric acid, he obtained a preparation

11

containing 1.5 per cent nitrogen. No ammonia was evolved on
making alkaline, but about 23 per cent of the total nitrogen was
evolved on treatment with sodium hypobromite. Detmer believed,
however, that humic acid did not contain nitrogen, but that the
nitrogen found was present in some organic compound occurring as
an impurity in his humic acid preparation. By a tedious process
he was able to lower the nitrogen content of his humic acid to
0.179 per cent.

Ritthausen (1877) attributed the high nitrogen content of peat
to the formation of complex, difficultly decomposable materials by
absorption of ammonia and pointed to the low ammonia content as
an indication of it, claiming it was not present as such after absorp-
tion. In an attempt to disprove Ritthausen's theory, Sivers (1880)
found that he could expel only very small amounts of ammonia by
heating with potassium hydroxide. He concluded that all the am-
monia taken in remained as such, and did not go to form complex
compounds. He maintained that most of the nitrogen was in the
form of protein but presented no conclusive evidence. Grouven
(1883) tried to show that the nitrogen of humus was due to the
absorption of ammonia by humic acids, but found frojn various
samples that only one-fiftieth of the total nitrogen was liberated
on heating with milk of lime, and only one-twentieth when heated
for two hours with potassium hydroxide.

It was found by Loges (1886) that the hydrochloric acid ex-
tract of the soil gave a precipitate with phosphotungstic acid, which
is recognized as being a precipitating agent for certain nitrogenous
compounds. Baumann (1887) found that certain black Russian
soils rich in humus, containing but small traces of ammonia in the
soil, gave a considerable amount of it on boiling the soil with dilute
hydrochloric acid. From this he suggested the presence of amino
and amide compounds in the soil. About the same time this sub-
ject was more thoroughly investigated by Berthelot and Andre
(1886). They found that the nitrogenous matter was split up pro-
ducing ammonia and soluble nitrogenous compounds, and that the
hydrolysis goes further the greater the strength of the acid, the
longer it is in contact with the soil and the higher the temperature.
A soil containing 0.174 per cent of nitrogen was heated on a water
bath for two hours with 7 per cent hydrochloric acid and 31.9 per
cent of its nitrogen was dissolved. Of this soluble nitrogen 17.8
per cent was ammonia. Similar experiments were conducted using
3.4 per cent hydrochloric acid, and 0.7 per cent hydrochloric acid
and distilled water.

Warington (1887) working with a sample of Rothamsted soil,
which had been heavily manured, showed the presence of a small
amount of amide nitrogen by using both hypobromite and nitrous
acid. It seems highly probable from these experiments that at
least a part of the nitrogen in the soil is present as amino com-
pounds. Eggertz (1888) found that the nitrogen content of thir-
teen samples of humus varied from 2.59 to 6.43 per cent and states
that the nitrogen was present in organic form and not as an am-
monium salt. Sestini (1899) also showed the presence of amino

12

nitrogen by the action of nitrous acid. Dojarenko (1902) working
with humus from seven Russian soils found appreciable quantities
of amino nitrogen. Unlike previous investigators he determined
the amount present quantitatively, and assumed all of the
amino nitrogen was present as amino acids. He also made deter-
minations of the ammonia by distillation with magnesium oxide,
and obtained the amide nitrogen by hydrolysis with dilute hydro-
chloric acid and subsequent distillation of the ammonia formed
with magnesium oxide.

B. The Humus Theory of Grandeau.

A tremendous impetus was given to the study of soils by the
work of Grandeau, because he believed that the ammonia extract
of soils contained the nutritive substances essential for the life
of the plant and for the fertility of the soil. The theory that the
humus extract was of such value had a great deal to do with re-
tarding the development of the study of the organic matter of the
soil.

The method of Grandeau (1872) is essentially the one in use
at the present time in America for the determination of humus. He
elaborated a method for the estimation of the "matiere noire" of
the soil by first leaching the soil with dilute acid in order
to set the humus free from its combination with the alkaline earths,
removing the excess of acid by washing with water, then moisten-
ing the soil with ammonia and allowing it to stand for a short
time (three to four hours, cf. Grandeau 1877, p. 149), after which
the humus solution was displaced by repeated washings with am-
moniacal water. The dark brown solution so obtained was evapor-
ated to dryness in platinum, weighed, ashed, and the amount of
"matiere noire" and of ash recorded. Grandeau regarded the humus
ash as an integral part of the humus. He believed that the organic
matter which dissolved was responsible for the fertility of the soil,
apparently not so much because of the carbon and nitrogen con-
tent, as for the high percentage of phosphoric acid and potash in
the humus ash.

The views of Grandeau wrere never generally adopted in Eu-
rope although often accepted by individual workers, but have been
more generally accredited in America, due to the sponsorship of
certain parts of Grandeau's humus theory by the late Professor
Hilgard. The American investigators, e. g., Hilgard (1906), Ladd
(1898), and Snyder (1895, 1897, and 1901), however, do not report
the humus ash as an integral part of the humus but call only the
volatile portion humus. They consider that the humus is, in part
at least, combined in the soil with inorganic substances ; these
compounds are called "humates" and to their abundance and pro-
duction has been ascribed an important part of the maintenance
of soil fertility.

Hilgard (1906) regarded the humus of the soil as a definite soil
product, formed from vegetable material in the soil under the in-
fluence of fungus and bacterial growths ; this conversion being

13

most efficiently carried out in the presence of only a moderate
amount of moisture, under the influence of a more or less rapid
circulation of air and in the presence of calcium carbonate to
neutralize any acids which may be formed. Under these conditions
the vegetable substance is converted into black, neutral, insoluble
humus compounds.

He believed that the nitrogen of plant debris which has become
an integral part of the soil must first be converted by humifying
bacteria and fungi into humus before the nitrogen can become avail-
able to the nitrifying bacteria and thus rendered available for the
use of the higher plants. His views of the persistence of plant ma-
terials in soils are contained in the following statement:

As a matter of course, the several organic compounds contained in plants
may continue to exist in soils for some time, varying according to conditions
of temperature and moisture. Thus dextrin, glucose, and even lecithin and
nuclein have been reported to be found. The activity of the numerous fungus
and bacterial ferments under favoring conditions, will of course, limit the
continued existence of such compounds somewhat narrowly so that they can
hardly be considered as active soil ingredients save in so far as they favor
the development of bacterial flora.

Suzuki (1906-08 a) made a study of the formation of humus by
treating oak leaves with a humus soil and various inorganic com-
pounds and concluded that not only calcium carbonate but also
magnesium carbonate promoted the decomposition of moist oak
leaves by fungi, judging from the amounts of carbon dioxide
evolved. He further states that the opinion of Hilgard corresponds
closely to the natural conditions of humification. Further studies
of Suzuki (1906-08 b) indicate that protein, starch, and pentosans
contribute to the formation of the black matter of humus, but
neither fat nor cellulose, and that protection from air is essential.

One of the latest additions to the idea of specific humificatio.il
of plant materials in the soil is that of. Trusov (1915). He inoculat-
ed various types of organic compounds with soil bacteria for vari-
ous lengths of time and concludes that humus has its origin in
lignin, albumen, starch, chlorophyll, and tannic substances; while
cellulose, hemicelluloses, mono- and disaccharides, glucosides, or-
ganic acids including amino acids, and wax forming substances
do not appear to have any part in its formation. He also finds that
the organic nitrogenous compounds used as nutrients for the micro-
organisms may serve as an indirect source of humus.

Weir (1915) has recently questioned the idea that the soluble
humus of the soil is an indication of the fertility of that soil and
that the humus nitrogen plays an important role in the nutrition
of plants. He removed 40 per cent of the nitrogen of the soil by
extracting the humus with sodium hydroxide and then used the
extracted soil for pot experiments. However, Gortner (1916 b)
has shown that in all probability a very considerable portion of the
humus nitrogen still remained in Weir's extracted soil, for he was
able to extract 90.3 per cent of the original nitrogen content of
the soil. This would indicate that nearly all of the soil nitrogen
could be extracted with sodium hydroxide.

Snyder (1897) prepared artificial humus by mixing a subsoil
with certain organic substances and allowing these to remain in a
moist condition for one year. At the end of the year humus was

14

determined on the resulting- mixture by extraction with ammonia,
folio-wing a previous leaching with dilute acid, an. I the humus so
found was regarded as having been formed in the soil during the
preceding year. Unfortunately Snyder did not correct for am-
monia soluble organic matter in the mixtures at the beginning of
the experiment. The results of Fraps and Hamner (1910) and
Gortner (1917) show that ammonia dissolves a considerable por-
tion of material from unchanged organic compounds, so that the
humus gain at the end of the experiment was in all probability
actually a loss when compared with -the amount of ammonia soluble
materials at the beginning of the experiment. Fraps and Hamner
(1910) as well as Gortner (1917) report a series of experiments af-
ter the general plan adopted by Snyder, with the exception that the
ammonia soluble materials were determined both at the beginning
and at the end of the experiments, and in each instance the am-
monia soluble material was found to decrease.

The experiments of Gortner (1917) furnish no evidence that a
specific "humification" of plant materials takes place in the soil
giving rise to an increased amount of "humus." He says:

On the contrary, all of the evidence is directly opposed to such a con-
clusion, and it appears altogether probable that the maximum amount of am-
monia soluble material is present in a soil immediately after a green manuring
crop has been plowed under and before the 'humifying' bacteria or fungi
begin their work.

Fraps and Hamner (1910) showed that the humus extract of
soil must contain substances from unchanged vegetable materials,
while Gortner (1916 a) pointed out that the extract must contain
substances from unchanged plant material, from bacteria and
protozoa.

C. The Complexity of the Ammonia Soluble Material.

With the development of chemistry the idea has been gradu-
ally abandoned, that the ammonia soluble compounds can contain
the whole of the organic matter which is responsible for the fer-
tility of the soil. The work of the U. S. Bureau of Soils in the
isolation of a large number of definite organic compounds from
the soil, has been a distinct contribution along this line.

As has been suggested by Gortner (1916 a) —

If one speculates on the nature of the soil organic matter, it becomes obvi-
ous that the variety of compounds which are present in a soil is limited
only by those compounds which were present in the plants growing upon
the soil, plus those compounds which compose the bodies of bacteria and
protozoa, plus the compounds contained in the soil fungi, plus all the various
compounds which may be formed from the above sources by decay, oxidation,
and all the intricate chemical reactions which take place in converting dead
organic material, either into living protoplasm on the one hand, or into
water, carbon dioxide, and nitrogen on the other. Undoubtedly these organic
compounds are not the product of 'humification' but are derived from un-
changed plant material, from protozoa, or from bacteria.

A part, or all, of these compounds would be found in the
"humus" extracted by Grandeau's method —

Inasmuch as the 'humus' extract of soils is undoubtedly a mixture of organic
compounds, many of which are colorless and in all probability are extracted
from unchanged plant or animal materials, and inasmuch as the soil pigment
present in this solution probably rarely exceeds 40 per cent of the 'humus', a
determination of 'humus', as ordinarily carried out, appears to be wholly
without scientific justification. (Gortner 1916 a.)

15
D. The Presence of Definite Organic Compounds in the Soil.

The following types of pure organic compounds have been
isolated from the soil. In view of Gortner's statement above it is
of interest to note that all of these compounds are colorless, and
that nearly all of them were isolated from an alkaline humus ex-
tract.

1. Paraffin hydrocarbons.

Hcntriacontane, C3iH«4. Schreiner and Shorey (1910 a, 1911 a).

2. Alcohols

Argosterol*, CseHUO. Schreiner and Shorey (1909, 1909 a)
Phytostcrol, C26H44O, H2O. Schreiner and Shorey (1910 a, 1911 b)

3. Esters

"Glycerides of fatty acids." Schreiner and Shorey (1910 a)
"Resin esters." Schreiner and Shorey (1910 a)

4. Acids

Oxalic acid, COOH-COOH. Shorey (1913)
Succinic acid, COOH-CH.-CHr-COOH. Shorey (1913)
Saccharic acid, COOH-(CHOH)4-COOH. Shorey (1913)
Acrylic acid, CH2=CH-COOH. Shorey (1913)
a-Crotonic acid, CH:1-CH = CH-COOH. Walters and Wise (1916).
a (l:ll)-Monohydroxystearic acid, CH,(CH,)tiCHOH (CH2)»COOH.

Schreiner and Shorey (1910 a, 1910 b)
Dihydroxystearic acid, CH3(CH2)7CHOH-CHOH(CH2)TCOOH.

Schreiner and Shorey (1908 a, 1909 a)
Benzoic acid, C6H5COOH. Shorey (1914)
Metaoxytoluic acid, OH-C6H3-CH3-COOH. Shorey .(1914)
Agroceric acid, C2iH42O3. Schreiner and Shorey (1909 a)
Paraffinic acid, C24H48O2. Schreiner and Shorey (1910 a)
Lignoccric acid, C:uH4*O2. Schreiner and Shorey (1910 a)
"Resin acids" (?). Schreiner and Shorey (1910 a)

5. Aldehydes

Salicylic aldehyde, C6H4-OH-CHO. Shorey (1913)

Vanillin, OH-C6H3(OCH3)-CHO. m-methoxy-o-hydroxy benzal-

dehyde. Shorey (1914)
Trithiobenzaldehyde, (C6H5CSH)3. Shorey (1913)

6. Carbohydrates

Mannitol, CH,OH-(CHOH)4-CH2OH. Shorey (1913)
Rhamnose, CH3-(CHOH)4-CHO, H2O. Shorey (1913)
Pentosan**, C5H8O4. Schreiner and Shorey (1910 a), Shorey and
Lathrop (1910)

7. Pyrimidine derivatives

Cytosine, C4H5N3O. 2-oxy, 6-amino pyrimidine. Schreiner and
Shorey (1910 a, 1910 c)

8. Purine bases

Xanthine, CsH4N4O2. 2, 6-dioxy purine. Schreiner and Shorey

(1910 a, 1910 c)
Hypoxanthine, C5H4N4O. 6-oxy purine. Schreiner and Shorey

(1910 a, 1910 c)

Adenine, C5H5N5. 6-amino purine. Shorey (1913)
Guanine***, CsHtNtO. 2-amino, 6-oxypurine. Lathrop (1912).

Schreiner and Lathrop (1912).
Tetracarbonimid****, C4H4N4O4. Shorey and Walters (1914)

9. Pyridine derivatives

a-Picoline. -y-carboxylic acid, CrHrNOa. Shorey (1906), Schreiner
and Shorey (1908 b)

*The composition of the compound was determined by a single analysis,
made with 0.1500 gram of the substance. It seems highly improbable that
the composition could be obtained accurately from this amount of material.

**Pentose sugars have been separated as hydrolysis products of substances
isolated from the soil (Schreiner and Shorey 1910 a).

**This compound was isolated from a steam heated soil.

**** This has been shown to be cyanuric acid (Wise and Walters 1917).

16

10. Amines

Trimethylamine, (CH3)3N. Shorey (1913)

Choline, HON(CH3)3-CH2-CH2OH. Trimethyl oxyethyl am-
monium hydroxide. Shorey (1913)

Creatinine, C^NaO. ^,-imino (n) methyl a-keto tetrahydro gly-
oxalin. Shorey (1911), Schreiner, Shorey, Sullivan and Skinner
(1911)

11. Organic phosphorus compounds

Nucleic acid. Shorey (1911 a, 1912, and 1913)
Lecithin, Aso (1904), Stoklasa (1911).

12. Amino acids

Arginine, CoHu&Os. a-amino, g-guanidine valerianic acid.

Schreiner and Shorey (1910 a, 1910 d)
Histidine, GiHflN3O2. a-amino, #-imidazole propionic acid.

Schreiner and Shorey (1910 a, 1910 d)
Lysine, CeHnN3O2. a £-di-amino, caproic acid. Shorey (1913)

Whether all of these compounds have actually been isolated
is perhaps an open question, in view of the minute quantities which
were obtained, insufficient in many cases for an, exact chemical an-
alysis as stated by Schreiner and Lathrop (1911) :

The amount of a substance obtained may be so small that extreme puri-'
fication is out of the question, and therefore) in such cases, where distinct
crystalline form or characteristic tests are not available the identification
becomes uncertain, as neither melting point nor analysis can be made.

E. The Origin of Organic Compounds in the Soil.

Chardet (1914) gives a discussion of the possible origin of
certain organic nitrogenous compounds that have been isolated
from the soil or might be expected to exist.

A very complete summary of our present knowledge of the or-
ganic matter of the soil is presented by Jodidi (1914) under these
headings: I. Introduction; II. The sulphur compounds of the
soil; III. The influence of certain factors on the quantity of nitro-
gen contained in the soil ; IV. The nature of humus substances ac-
cording to the older authors ; V. The observations of later authors
concerning the nature and behavior of humus to certain reagents ;
VI. Genetic relationship between the chemical compounds in the
soil and those in plants and animals ; VII. The nature of nitrogen
compounds in the soil; VIII. The organic nitrogenous compounds
of the soil ; IX. Separation of the nitrogenous compounds in a
sulfuric acid extract (i. e., hydrolysate) of the soil; X. Cleavage
products of nucleoproteins ; XL Lecithin products in the soil; XII.
Pyridine derivatives in the soil ; XIII. Ammonification of amino
acids and acid amides in the soil ; XIV. The occurrence of hydro-
carbons, alcohols, and aldehydes in the soil; and XV. The organic
acids occurring in the soil.

Different investigators have succeeded in isolating from soils
the following nitrogenous compounds which may be related to
or derived from the proteins: tetracarbonimid, a-picoline y-car-
boxylic acid, trimethylamine, nucleic acid, arginine, histidine,
lysine, proteoses, and peptones. Potter and Snyder (1915 b) have
shown that in some soils, at least, free amino acids and peptides
occur but the amounts are very small.

Since there have been so many nitrogenous compounds isolat-

17

ed from the soil that are related to proteins, it is well to discuss
some of the complex organic compounds such as nucleoproteins,
nucleic acids, and lecithins that find their way to the soil through
plant and animal remains. While the final decomposition products
are undoubtedly simple compounds or elements such as carbon
dioxide, methane, ammonia, nitrogen, and hydrogen, these products
are reached by fairly definite and well defined methods of cleav-
age. The process may be a rapid one, a slow one, or one entirely
arrested at certain stages, all depending on the factors present
in the soil.

1. Nucleoprotein decomposition. The nucleoproteins are with-
out doubt the most complex compounds that enter the soil. They
are common constituents of plants, animals, bacteria, and molds, and
hence occur wherever these live or die. The chemical changes
through which these compounds go during decomposition may be
rendered clear by the following scheme presented by Lilienfeld
(1892):

Nucleoprotein

Protein Nuclein

(Histone) I

! I

Protein Nucleic acid

The products are then protein and nucleic acid, the latter of
which has been isolated from the soil (Shorey 1911 a, 1912, and
1913).

2. Nucleic acid decomposition. Nucleic acids are constituents
of all nuclei and on decomposition yield a variety of compounds
composed of carbon, hydrogen, oxygen, nitrogen, and phosphorus.
The acids occur in both plant and animal cells.

Jones (1914) states that all plant nucleic acids contain a pen-
tose group, while on the other hand all animal nucleic acids yield
levulinic acid, which is formed from a hexose group in their mole-
cule.

The hydrolysis products may be classified, according to Forbes
and Keith (1914) as follows:

Nucleic acids

Phosphoric acid Carbohydrates Bases

Pentoses
Hexoses
Unidentified Purine Pyrimidine

Guanine . Cytosine
Adenine Thymine

Xanthine Uracil

Hypoxanthine

18

The purine and also the pyrimidine derivatives can be changed
one into the other by chemical means. In a parallel manner much
the same results can be obtained through the biochemical changes
brought about by bacteria and enzymes. By chemical agents Kos-
sel and Steudel ( 1903) transformed cytosine into uracil. In like
manner Fischer (1882) changed guanine into xanthine, and Kossel
(1886) changed adenine into hypoxanthine. It was shown by Schit-
tenhelm and Schroter (1904) that putrefactive bacteria, especially
those of the coli group, were able to convert guanine and adenine
into xanthine and hypoxanthine and that bacteria also have the
ability of breaking down nucleic acid itself. This change of nucleic
acid is also accomplished by certain enzymes, the nucleases (cf.
Jones 1914 and Euler 1912).

It will be clear from the above that the decomposition of
nucleic acid may take place in many steps and that the intermediate
as w'ell as the final products may be transformed one into the other.
This may be accomplished in the soil through the agency of micro-
organisms or enzymes. Schreiner and Lathrop (1912) working
with steam heated soils found that from the heated samples less
nucleic acid was obtained than from the unheated samples. On the
other hand the decomposition products of nucleic acid were present
in larger amounts in the heated soil, indicating that hydrolysis of
nucleic acid has been accomplished in this manner. That nucleic
acid decomposition does take place in the soil is evidenced by
the isolation of certain of its decomposition products, e. g., cytosine,
xanthine, hypoxanthine (Schreiner and Shorey 1910 a, 1910 c),
adenine (Shorey 1913), and guanine (Lathrop 1912, Schreiner and
Lathrop 1912).

3. Lecithin decomposition. A similar instance of a single
substance decomposing into several substances is that of the
lecithins. They are closely related to the fats in constitution and
are possible primary constituents of all plant and animal cells.
Lecithins are esters of glycerol with two molecules of higher fatty
acids (palmitic, stearic, oleic acids or other unidentified saturated
or unsaturated acids) with a molecule of phosphoric acid, which
is at the same time combined with the base choline. Mathews
(1915) states that in some cases choline can be replaced by neurine.
There are a number of different lecithins which are characterized
by the nature of the organic acid radicals present. The hydrolysis
may be indicated as follows :

L,ecithin

Acids Bases Glyceryl-phosphoric acid

I I

Palmitic Choline Glycerol

Stearic or Phosphoric acid

Oleic Neurine

or

Other higher
fatty acids.

19

The base choline is also widely distributed in both plant and
animal tissues as well as being a decomposition product of lecithins.
It has been shown to exist in the soil. Choline yields neurine by bac-
terial decomposition, and both of these compounds break up into
trimethylamine. This substance may also be added to the soil from
other sources, both animal and vegetable. As noted above Stoklasa
(1911) obtained evidences of lecithin in the soil and Aso (1904) re-
ports small quantities of lecithin present in soils rich in organic mat-
ter. Choline has been isolated from soil (Shorey 1913). The tri-
methylamine reported by Shorey (1913) possibly had its origin
in the lecithin molecule and it may be that the dihydroxy-stearic
acid of Schreiner and Shorey (1908 a, 1909 a) and the mono-
hydroxystearic acid (Schreiner and Shorey 1908 a, 1909 a) had
the same origin. (For a discussion of the organic phosphorus of
the soil sec (iortner and Shaw 1917).

F. Bacterial Processes Which Influence the Form of Soil Nitrogen.

We know that the decomposition of protein substances can be
brought about through bacterial activity or by the agency of en-
zymes widely distributed in the vegetable kingdom. We should
expect any protein materials present in the soil to be subject to
the action of the above agencies. Viewed in the light of the
researches of Emil Fischer (1899-06) protein hydrolysis leads to
disruption of the complex molecule and the formation of simple
molecules, as represented in the following scheme* —

i' Di-amino acids
Mon-amino acids
Acid amides

Fischer has shown that the amino acid combination in the pro-
tein molecule may be represented as follows:

HO H H O

I I! ! ! !J

H.N-C-C N-C-C-O H

I I

R R

O H

The group II ! being known as the "peptid group." The

— -C-N —

nitrogen in this group is in the form of the imino (-NH) radical.
Upon hydrolysis each "peptid group" takes up a molecule of water
forming a free carboxyl (-COOH) group changing the imino group
into an amino (— NH2) group.

As the protein hydrolysis continues, the proportion of nitrogen
in the amino form increases until it reaches a maximum at com-
plete hydrolysis. It has been shown by Van Slyke (1910, 1911)
that the amount of amino nitrogen formed is a measure rbf the
hydrolysis of the protein substance. The amino acids derived from
protein degradation may be acted upon by the bacteria in the soil
and bring about chemical changes which depend largely on the
character of the organisms present.

20

1. Deamination or reduction plays an important part in the
formation of ammonium salts in the soil. The amino group is
.split off as ammonia and non-nitrogenous organic acids remain.
It is not certain whether this process involves oxidation of the
amino acid to the ketonic acid first, or whether the deamination is
brought about by hydrolysis. If the hydroxy acids are first formed
they are subsequently reduced so that the fatty acids are formed
from the amino acids. This can be illustrated by the following
examples :

Aspartic acid will give succinic acid, and this by loss of carbon
dioxide gives propionic acid.

Tyrosine— >p-Hydroxy-phenyl-propionic acid— >p-Hydroxy-phenyl-acetic
acid— >p-Cresol— > Phenol

This deamination or reduction is in all probability what is
termed in soil chemistry ammonification.

2. Decarboxylation or amine formation involves the splitting
off of carbon dioxide by the action of so-called carboxylase bac-
teria. This may happen either before or after deamination. Their
formation is illustrated in the following reactions:

CH3-CH(NH,)-COOH- >CH:r-CH,.-NH,.+ CO2
Alanine Ethyl amine

C,iH4(OH)-CH2-CH(NH2)-COOH - ->CoH4(OH)-CH2-CHo-NH2 +CO2
Tyrosine p-Hydroxy-phenyl-ethyl amine

>NH2-CH2-CH:!-CH2-CH;-NH2+CO2
Lysine Cadaverine

NH2-C(NH)-NHCH2-(CH2)r-CH(NH2)-COOH-

Arginine NH2-C(NH)-NH-CH2-(CH2)2-CH2-NH2+CO2

Agmatine

Tryptophane gives rise to indole ethyl amine.
Mathews (1915) states—

If the splitting: off of carbon dioxide occurs after the deamidization an amine
cannot, of course, be formed, but the next lower carboxylic acid is produced
by way of the aldehyde.

Thus from tyrosine there may first be formed p-hydroxy-
phenyl-pyruvic acid, which may be reduced to p-hydroxy-phenyl-
lactic acid, reabsorbed and reexcreted in the urine ; or the p-hy-
droxy-phenyl-pyruvic acid may be split into p-hydroxy-phenyl
acetaldehyde and carbon dioxide, and the former be oxidized into
p-hydroxy-phenyl-acetic acid, which is excreted in the urine.

It is well to remember that any bacteria of the coli group will
split off carbon dioxide from an amino acid. It is evident that
mixed cultures of bacteria may be present in the soil and thus cause
more than one type of splitting to take place.

3. Hydrolysis takes place with liberation of carbon dioxide
Ehrlich (1911) has shown that yeasts can convert amino acids into
alcohols, liberating carbon dioxide and ammonia.

C6H4(OH)-CH2-CH(NH2)-COOH + H2O -- >

Tyrosine CoH4(OH)-CH2-C

Tyrosol

21

4. Oxidation results with liberation of carbon dioxide and
ammonia and the formation of a fatty acid containing one less car-
bon atom. A type reaction may be represented as follows :

R-CHr-CH(NH2)-COOH + Oa - ->R-CH2-COOH + CO2+NH3

That oxidation is a factor in the organic matter of the soil
is self-evident from the fact that carbon dioxide is constantly pres-
ent in the soil atmosphere in excess of the amount present in the
air, thus representing degradation of the organic matter to carbon
dioxide and water, arid also from the fact that ammonia is trans-
formed into nitrates, a process known in soil chemistry as nitrifica-
tion, a reaction which is carried out in the laboratory by the most
violent chemical oxidation, e. g., chromic acid. A further step in
this oxidation carries the nitrates through denitrification which re-
sults in the liberation of free nitrogen.

In carrying to completion these processes on protein material
one can easily postulate an almost unlimited number of organic
compounds, which are theoretically (and in all probability) pos-
sible. Very recently Robbins (1916) has produced some evidence
that the existence of certain of the organic compounds in the soil is
limited somewhat narrowly by specific bacteria, which either utilize
the nitrogen or the carbon of the compound as a source of energy.
Thus pyridine is destroyed by a specific bacterium which is able to
utilize the nitrogen, and the carbon of cumarin and vanillin is like-
wise a source of carbon for other specific bacteria.

G. Nitrogen Distribution in the Soil

The chemistry of soil nitrogen may to a large extent be con-
sidered as being the chemistry of protein undergoing hydrolysis.
The isolation of a number of amino acids indicates that proteins are
decomposed in the soil in much the same way as in acid hydrolysis
or animal digestion. Just how far the cleavages have already gone
in the soil previous to acid hydrolysis remains a matter of much
work before definite conclusions can be drawn.

Walters (1915) has reported the presence of certain decompo-
sition products in the soil, presumably proteoses and peptones,
resulting from either a partial hydrolysis of proteins or by the syn-
thetic action of microorganisms. It has been recorded by Hoppe-
Seyler (1909, p. 413) that intermediate protein decomposition prod-
ucts may result from the action of water at high temperature, by
mineral acids, alkalies, oxidizing agents, enzymes and microorgan-
isms. There is little reason to suppose that the action of micro-
organisms is other than that of the enzymes which they produce.
Effront (1914) states that under the influence of the various tryp-
sins secreted by putrefactive bacteria, the protein molecule is split
into proteoses, peptones and amino acids. The proteoses and pep-
tones represent stages of decomposition between that of true pro-
teins and amino acids. Walters concludes —

that proteins undergo hydrolytic decomposition in the soil in much the same
way as in dig-estion by enzymes, acids, or alkalies, in the laboratory.

22

In an extensive examination of the nitrogen compounds of
processed fertilizers, Lathrop (1914) has reported the presence of
certain protein-like substances similar to those described above.

In his studies on the chemical nature of the organic nitrogen
in the soil, Jodidi (1911) thought water would be preferable to
either acids or alkalies for the purpose of extraction, since it would
not be so liable to alter the organic nitrogenous materials. He
found that the direct extraction of a soil by boiling with water for
ten hours removed only 2.92 per cent, and for twenty-four hours
the highest amount removed from any soil was 9.96 per cent of the
total soil nitrogen. Shmook (1914), however, reports 19.10 per
cent of the total nitrogen of a Laterite soil of Russia to be water
soluble.

The literature has been very thoroughly summarized by Potter
and Snyder (1914) in regard to the determination of ammonia in
soils. Both their work and that of Jodidi ( 1909) indicates that the
amount of ammonia is small. Kelley and Thompson (1914) in a
study of some Hawaiian soils reached the conclusion that ammonia
and nitrate nitrogen constitute but a small percentage of the total
nitrogen, and that the nitrogen is very largely in organic combina-
tion.

It is known that only a small part of the soil nitrogen is dis-
solved by dilute acids, yet it has been shown by Kelley and Thomp-
son (1914) that 1 per cent hydrochloric acid dissolves some organ-
ic nitrogen, for in every instance the soils contained only about
half as much ammonia nitrogen as was extracted by the acid.

In the soil studies of Potter and Snyder (1915 a) they find that
the nitrogen extracted by 1 per cent hydrochloric acid varied from
about -1.2 to 2.3 per cent of the total nitrogen, except in the case of
the peat it was only 0.67 per cent. This is contrary to the findings
of Gortncr (1916 a). Working with eight mineral soils he finds a
maximum of 4.18 per cent of the total nitrogen soluble in 1 per
cent hydrochloric acid with an average of 3.17 per cent. In three
peats he finds a maximum of 7.50 per cent with an average of 3.78
per cent, and in five samples of unchanged vegetable materials (oat
straw, alfalfa hay, oak leaves, sweet fern leaves, and grass from a
peat bog) he finds a maximum of 34.58 per cent with an average of
20.10 per cent. These findings would seem to indicate that in the
transformation of vegetable materials into the true organic mat-
ter of the soil there is a fall in the proportion of the total nitrogen
soluble in very dilute acids.

Shorey (1905) published results of his investigations which
gave the first definite knowledge of the individual amino acids
formed in the decomposition of soil organic matter. He worked on
a Hawaiian soil with a view to classifying the decomposition prod-
ucts of the nitrogenous substances in the soil. The method applied
was that proposed by Osborne and Harris (1903) for classifying the
decomposition products of proteins resulting from acid hydrolysis.
The method is a modification of that proposed by Hausmann (1899)
and is in short as follows : After hydrolysis the excess of the

23

mineral acid is removed by evaporation, and the nitrogen present
as ammonia determined by distilling with an excess of magnesium
oxide ; after separating the magnesia precipitate from the remain-
ing solution by filtration, the nitrogen was determined in the pre-
cipitate by the Kjcldahl method, the di-amino nitrogen in the fil-
trate was precipitated by phosphotungstic acid and determined by
the method of Kjeldahl and the mon-amino nitrogen determined
by difference.

He obtained in the acid solution 84.5 per cent of the total nitro-
gen in the soil, 52.3 per cent of which was found in the magnesia
precipitate. This result is in striking contrast to those obtained by
Osborne and Harris (1903) working on pure proteins, where they
found that the nitrogen contained in the magnesia precipitate does
not usually exceed 4 per cent of the total nitrogen and in most
cases is very much less. The amount of nitrogen insoluble in the
12 per cent acids used in the digestion may be designated as "hu-
min." The nitrogen in the magnesia precipitate has been desig-
nated by most investigators "humin" nitrogen. The total "humin"
nitrogen in the soil is then represented by the nitrogen in the
magnesia precipitate plus that retained by the soil. On -recalcula-
tion of his data it was found that the insoluble humin in the soil
after hydrolysis amounted to 15.3 per cent of the total nitrogen,
making a total humin nitrogen content of 59.1 per cent. This very
high result of total humin nitrogen was undoubtedly due to the soil
being hydrolyzed only seven hours with a relatively low concen-
tration of hydrochloric acid and the insoluble residue boiled the
same length of time with sulfuric acid. Complete decomposi-
tion of the proteins probably did not take place in the dilute acids
used in the short time that they were heated. As a result partially
hydrolyzed residues may have been precipitated by the magnesium
oxide, which would account for the high results.

Shorey (1906) concluded that even though we might know
much concerning the constitution of the compounds comprising
the various groups isolated from protein by this method of analysis,
we know nothing concerning their composition when isolated from
soil, inasmuch as we are not dealing with a pure protein (cf. also
Oiortner 1913, 1914, 1916 c).

The work of Suzuki (1906-08 c) gives us further knowledge of
the individual amino compounds formed in the decomposition of soil
organic matter. He worked with three samples of humic acid, o>ne
obtained from Merck (origin unknown to Suzuki), one prepared
from an unmanured soil, and one from a compost heap. After
boiling each preparation for ten hours with strong hydrochloric
acid, the undecomposed residue was filtered off, washed, and the
residue extracted twice in this manner with strong hydrochloric
acid. He determined the amounts dissolved as amide, di-amino, and
mon-amino acid nitrogen. From 65 to 75 per cent of the total
nitrogen was dissolved by the hydrochloric acid and in the extract
41 to 62 per cent of the nitrogen was not precipitated by phospho-
tungstic acid. A sample of humic acid was twice extracted, with

24

concentrated acid and the residue analyzed. His results calculated
on the ash free basis showed the residue to contain 64.11 per cent
carbon, 3.35 per cent hydrogen, and 0.80 per cent nitrogen. The
residue becomes lower in nitrogen*, hydrogen, and ash but richer
in carbon as the hydrolysis is continued.

Detmer (1871) pointed out that similar results were true in
peat beds where the deposits remained undisturbed for years. He
found that there is an increasing carbon and nitrogen content of
the humus for varying depths. This is shown by the following
table :

Carbon

Hydrogen

Oxygen

Nitrogen

Brown peat, near the surface...
Dark peat, 7 feet

57.75
62.02

5.43
521

36.02
3067

0.80
2 10

Black peat, 14 feet.

64.07

5.01

26.87

4.05

Likewise Gortner (1917) observed —

that there is a much greater wastage of carbon than nitrogen. Hilgard
(1906) calls attention to the increased nitrogen content of the humus over
that of the original vegetable materials. If we take the average carbon con-
tent of proteins as 51.15 per cent (average of 30 analyses given by Mathews
1915) a C:N ratio of 3.06 found in soil A-1916 would give a nitrogen content
of 16.71 per cent, which approaches very nearly to the average nitrogen content
of these 30 proteins, i. e., 17.66 per cent. It is evident that the materials re-
maining in the soils are rapidly increasing in nitrogen content.

Suzuki (1906-08 c) made further studies on a 500 gram sample
of the humic acid obtained from Merck. It was hydrolyzed with
concentrated acid and the solution obtained subjected to esterifica-
tion and fractional distillation according to the method of Fischer
(1901). He obtained:

Alanine • 2.39 gm.

Leucine , 2.16 gm.

Alanine + aminovalerianic acid • 0.11 gm.

Aminovalerianic acid 0.57 gm.

Proline (copper salt of active proline) . • 0.67 gm.

(copper salt of inactive proline) (?) 0.50 gm.

Aspartic acid • 0.06 gm.

Impure aspartic acid (?) • • 3.16 gm.

Glutaminic acid present

Tyrosine trace

Histidine trace

Ammonia 1.90 gm.

Copper salts of unknown acids • • • • • 30.30 gm.

As these compounds are typical protein decomposition prod-
ucts, his work proves that the humic acid examined by him was
either of a protein nature, a mixture of protein decomposition prod-
ucts, or probably both together with some as yet unknown com-
pounds. Unfortunately, the origin of the acid was unknown to
Suzuki, but he states that it was probably prepared from peat.

From a study on Michigan peat soils Jodidi (1909) has con-
cluded that the bulk of the organic nitrogen is made up of acid
amides, di-amino acids, and mon-amino acids. He used slightly
modified methods. The ammonia was determined as above by
distillation with magnesium oxide. The residue from distillation
with magnesia was dissolved in dilute sulfuric acid and the

*He stated that although the nitrogen content decreases, it is very difficult
to entirely remove all of the nitrogen.

25

di-amino acids precipitated by phosphotungstic acid. The nitrogen
in the precipitate of di-amino acids was determined by the method
of Kjeldahl. The filtrate from the di-amino acids containing the
mon-amino acids was oxidized by the Kjeldahl method and the
nitrogen determined. In some cases he secured the mon-amino
nitrogen by difference, stating that it was difficult to get a direct
determination of the mon-amino nitrogen by the Kjeldahl method.
He states that—

this percentage was usually higher than the one directly found by kjeldahl-
izing the filtrate from the phosphotung-istic acid precipitate.

In one experiment the percentage of mon-amino nitrogen by
direct determination was 62.83 per cent, while by difference the
result was 67.22, and in another case the results were 64.25 and
65.06 respectively.

It will be noted that this is a departure from the method used
by Shorey (1905) in that here the nitrogen is separated into three
fractions instead of the usual four.v The nitrogen in the magnesia
precipitate was distributed with the di-amino and mon-amino acid
nitrogen. This method of nitrogen distribution will be classed
as "Jodidi numbers" (in contrast to the Hausmann numbers) in
the subsequent portion of the paper.

Van Slyke's (1910) nitrous acid method was first applied by
Robinson (1911) to a study of peat soil, in order to determine the
amount of amino^nitrogen present. The ammonia nitrogen was re-
moved by previous distillation with magnesium oxide. The only
value of Robinson's work seems to be in the fact that his figures
for total and amino nitrogen increase to a maximum with in-
creasing time of hydrolysis, in much the same manner that pro-
teins react ; thus indicating that the amino groups were not ex-
isting free in the peat but in some form of combination which
did not react with nitrous acid. For example, after one hour's
hydrolysis the total nitrogen of the soil in solution amounted to
29.86 per cent, while the amino nitrogen was 4.62 per cent or a
ratio exceeding 6:1. After forty-two hours' hydrolysis the nitro-
gen of the soil in solution was 51.54 per cent of the total nitrogen
and the amino nitrogen was 25.07 or a ratio only slightly exceed-
ing 2:1. This ratio increases again with further hydrolysis so
that at the end of 138 hours the ratio is almost 3:1. However, the
amount of nitrogen in solution was so small that the experimental
error of measuring total and amino nitrogen must have been
quite large.

More recently Jodidi (1911) made a study of some Iowa soils
using a modification of the Osborne and Harris (1903) method.
The nitrogen removed from the solution by the magnesium oxide
was ignored by the author.* This contained a part of the so-called
"htimin" nitrogen. Subtracting the sum of the ammonia** and
di-amino nitrogen from 100 he found the per cent of the nitrogen in

^Experimental data presented later in this paper will show that this
fraction may exceed 9 per cent of the total nitrogen.

**He distinguishes the ammonia nitrogen originally present in the soil
as such, from that produced by acid hydrolysis.

26

solution as mon-amino nitrogen. It will be readily seen this con-
clusion is incorrect. The mon-amino acid nitrogen as deter-
mined represents the sum of the humin and mon-amino nitrogen.
It is very unfortunate that this mistake should have been made
since this gives us only the actual ammonia and di-amino acid
nitrogen for use in comparison with other investigations of the
organic soil nitrogen as distributed by acid hydrolysis. Kelley
(1914) following details as outlined by Jodidi (1911) has made
the same error and the criticisms above apply with equal force to
his data.

It is also extremely unfortunate that investigators should in
any case rely upon figures obtained "by difference" for any one of
their fractions. It is sometimes permissible to use figures obtained
in this manner, for example, in case a determination has been lost
and lack of time or other consideration prevents a repetition ;
but to constantly use figures obtained in this manner is unscientific,
especially, since by this method we have no means of determining
how great the experimental error of the method may have been.

Jodidi (1911) called attention to the fact that in the case of
protein substances the distillation of the hydrolyzed protein with
magnesium oxide gives pure ammonia. This, however, may not
hold true for the hydrolyzed portion of soils, since some protein
substances through decay yield organic bases. It has been shown
by Bocklisch (1885) that dimethyl amine is formed through putre-
faction of fish, and trimethylamine has been produced by the
putrefaction of wheat flour and fish. The bases putrescine and
cadaverinc result from the decay of organic substances under cer-
tain conditions. It is possible for certain di-amino acids to be-
come transformed into di-amines, as for example, arginine can be
decomposed into urea and ornithine through bacterial activity.
These processes can be expressed by the following equations :

NH2-C(NH)-NH-CH2-(CH2)2-CH(NH2)-COOH + H20-

Arginine

NH2

Hs-CHr-CH^CH (NH:)-COOH+C=O

l

Ornithine .NH,

Urea
NH,,-CH2-CH,-CHa-CH(NH2)-COOH -- >

Ornithine

CO2+NH^CH,-CH2-CH,-CH2-NH2
PUtrescine

Jodidi found that the ammonia obtained by distilling the
evaporated extract of the soil with magnesium oxide was actually
pure ammonia, thereby establishing the absence of any volatile
organic bases ; but that the phosphotungstic acid precipitate and the
filtrate from that precipitate did not represent di-amino and mon-
amino acids only.

In order to find out how much of the di-amino and mon-amino
nitrogen actually belonged to di-amino and mon-amino acids, the
solutions were subjected to analysis by the formaldehyde-titration

27

method of Schiff (1900, 1901, 1902) as modified by Sorensen (1908),
Henriques (1909), and Henriques and Sorensen (1910).

In a comparison of the amount of di-amino* acid nitrogen,
calculated as if histidine, arginine, and lysine were present in about
equal amounts, he finds that in plot E, 101.8 per cent; plot Q, 84.8
per cent; and plot U, 93.9 per cent of the nitrogen in the phos-
photungstate fraction was actually present as di-amino acids.

However, he obtains widely divergent results for mon-amino
acid nitrogen; in plot E, 91.64 per cent; plot Q, 52.63 per cent;
plot U, 40.12 per cent; plot H, 88.31 per cent; and plot J, 92.11
per cent of the total nitrogen in the "filtrate from the bases" was
actually present as mon-amino acid nitrogen whereas all should
have been present in this form if dealing with pure proteins only.
He, therefore, concludes that the di-amino and mon-amino acids,
or in other words, the bases and filtrate from the bases by hydro-
lyzing soils, contain other products- than are formed by hydrolysis
of pure proteins.

In a series of fertilized soils studied by Lathrop an.d Brown
(1911) they find that almost 98 per cent of the nitrogen in the soil
is of organic nature. The ammonia and nitrate nitrogen constitute
the remainder. Employing the same method to the distribution of
the soil organic nitrogen as Shorey (1905), they boiled 100 grams of
soil with 500 cc. of hydrochloric acid (sp. gr. 1.115) for three hours,
and used the filtrate after making to definite volume for the
analyses. The figures given for ammonia nitrogen represent the
actual amount of nitrogen as ammonia obtained by hydrolysis and
does not include the ammonia nitrogen already present in the
soil. They find that the plots which have received organic fer-
tilizers give the largest amount of ammonia on hydrolysis, the
amount being highest in the plot which has received manure alone
and lowest in the check plot.

Of the five soils studied, four contained over 25 per cent of
the "humin" nitrogen soluble in acid, while the other only showed
about half as much. Since the nitrogen of the soil not soluble in
acid may be considered "humin" nitrogen, the total amount in this
form in the above four soils was over 53 per cent, while in the
other soil, which received dried blood, it amounted to only 43 per
cent.

However, the fractions which they determined have actually
very little significance in a discussion of protein hydrolysis products,
inasmuch as a three-hour hydrolysis is far too short a time to com-
pletely decompose the protein molecule. This explains their high
figures for humin nitrogen and low ones for mon-amino acid
nitrogen. The di-amino and mon-amino acid nitrogen differ rather
widely but there seems to be no agreement between the form of
nitrogen and the plot treatment.

*The exact interpretation of his data is difficult to understand.

28

In conclusion they say —

these five samples of soil are really the same soil under long- continued
treatment of different kinds. It is not improbable that work on widely differ-
ent soils will show even much greater variations than those here noted.
The work shows, however, that even in such cases there is a difference in
the nitrogenous compounds in the soil, and that different decompositions of
the nitrogenous matter has taken place and probably will continue to take
place, under the different conditions imposed upon the soils in the field.

A very interesting study has been made by Shmook (1914)
of the nitrogen distribution in four Russian soils, one of the Podzol
type, two of the Chernozem type, and one of the Laterite type by
applying the method of Hausmann (1899). The water extract
from 100 grams of the Podzol soil showed a very high content of
soluble nitrogenous compounds. This amounted to 0.0452 gram
of nitrogen which constituted 19.10 per cent of the total soil nitro-
gen. This was distributed as follows : amide nitrogen 0.0034
gram, di-amino nitrogen traces, and mon-amino nitrog-eia 0.0408
gram. These results were deducted from the analyses of the
hydrolyzed soil. Thirty gram samples of soil were hydrolyzed
with 120 cc. of hydrochloric acid (sp. gr. 1.12) for 8 hours and
the analysis carried out as directed.

He finds, that the Chernozem and Podzol soils show a simi-
larity in the distribution of amide nitrogen, and that of the amino
acid nitrogen, but that the nitrogen distribution in the Laterite
soil is entirely different from the other types. He concludes that
the amount of protein in the soil is not in direct relation to the
amount of organic matter, and that the nitrogen insoluble in hydro-
chloric acid occurs in unknown form and composes only 1.50 to
1.90 per cent of the organic matter of the Chernozem and Podzol
soils, but 13.70 per cent of the total organic matter of the Laterite
soil, after subtraction of the protein nitrogen belonging to this
insoluble portion. He suggests that these results would indicate
that the organic nitrogen existed in the soil in large part as protein
material in the Chernozem and Podzol soils, but that a considerable
portion was of a non-protein origin in the Laterite soil, since
the amount of this insoluble "melanin" in pure proteins amounts
to from 0.60 to 1.80 per cent of the total protein nitrogen.*

Potter and Snyder (1915 a) made a study of some Iowa soils
using Van Slyke's (1911) method of protein analysis. Their soils
were the same type but had received different fertilizer treatment.
At the same time they also made a study of peat soil. The soils
were in all cases first extracted with 1 per cent hydrochloric acid
"in order to render the humus more soluble." They were then
hydrolyzed by boiling one part of the soil with two parts of 22
per cent hydrochloric acid for forty-eight hours. They also pre-
pared a 1 per cent sodium hydroxide extract of the acid leached
soils, and after precipitation with sulfuric and acetic acids the
resulting humic acid precipitate was subjected to the above method
of analysis.

The authors conclude: (1) that the humin nitrogen as deter-
mined by the Van Slyke method on soils extracted by dilute alkali

*Actually in some cas^s these results are much lower, and in others de-
cidedly Higher, e. gr., Van Slyke (1911) finds gelatin contains 0.07 per cent and
fibrin 3.17 per cent.

29

is very high when compared to the amounts in proteins ; (2) that
no typical class of organic compounds is extracted from the soil
by dilute alkali ; (3) that the amounts of amino acid and peptid
nitrogen in the soil are found to be very small compared to the
amounts of amino. acids formed by acid hydrolysis ; (4) (and this
is the most important for our purpose) that

nothing very significant can be deduced from the variations in the different
soils,

or in other words, the organic matter in the same soil type under
different fertilizer treatment, is essentially the same, and as I shall
show later in the experimental part of this paper, the organic mat-
ter (as distributed by Van Slyke's method) in different soil types is
essentially the same.

Lathrop (1916) recently made a study of protein decomposi-
tion in the soil. He added a high grade nitrogenous fertilizer to
the soil and allowed decomposition to proceed at laboratory tem-
perature, and at different periods topk samples and subjected them
to Van Slyke's method o.f protein analysis in order to determine
how the different fractions were affected by bacteria and other
agencies present in the soil.

From his \vork he concludes that the analysis obtained by the
Van Slyke method indicates that there is a formation of protein
taking place in the soil in the course of the decomposition of the
protein materials, and that apparently the new protein is somewhat
resistant to decomposition. He states, that —

this is indicated in (1) the unequal loss of mon-amino acids and hydroly/able
nitrogen from the soil during- the early stages, (2) by an increase in amide
nitrogen during the early stages, (3) by an increase in histidine nitrogen dur-
ing the early stages, (4) by an increase in the arginine nitrogen during the
later stages, and (5) by an increase in lysine nitrogen during the later stages.

This view that the protein nitrogen in the soil was largely con-
tained in the bodies of bacteria and protozoa had been previously
advanced by Shmook (1914).

It was stated by Loew and Aso (1906-08) that under favor-
able condition of growth protein material is excreted by yeast and
bacteria, and that soluble materials can pass through the cytoplasm
to the outside on death of the cell. They also state that the amount
of nitrogenous substances partly consisting of peptones excreted
by dead cells, is by no means inconsiderable.

H. A Summary of the Nature of the Organic Matter of the Soil in
the Light of Our Present Knowledge.

It has been pointed out, that the organic matter of the soil
was at first considered to be a very simple thing, that the alkali
extract contained the essential plant nutrients, and that the process
of "humification" was the necessary step through which the or-
ganic matter of the oil must pass in order to be converted into food
materials for plant life ; but as the knowledge of chemistry de-
veloped it became evident that the problem was more complex.
A definite knowledge of the forms of organic matter in the soil
can only be secured when we have a thorough understanding of

30

the chemical products formed by the action of the bacteria, pro-
tozoa, and fungi on each other, and on the organic matter, both
animal and plant, that finds its way to the soil. The present method
is to study the soil with its complex mixture of organic and in-
organic compounds, and by the application of recognized methods
of chemical investigation unravel the mysteries tied up in it;
but unless we have the climatic and cultural conditions uniform,
we cannot hope to secure results that will be of general application.

There have been two methods of attacking the problem: first,
the isolation and study of the individual organic compounds ex-
isting in the soil, and, second, a study of the hydrolysis products.
It is evident that these methods are slow and tedious and unsatis-
factory, but on the other hand a study of all the possible combina-
tions of the organic substances existing in the soil appears to be
an endless task in the light of our present knowledge. In brief,
a complete and thorough knowledge of the organic matter of the
soil appears possible only after we have mastered all of the bio-
chemical processes which are characteristic of the fungi, protozoa,
and bacteria of the soil, and have a much deeper insight into
the chemical constitution of vegetable cells than we have at the
present time. Our present knowledge leads us to believe that
it is possible to isolate an almost infinite variety of chemical
compounds from a soil, the number and variety reaching a limit
only when we have isolated all of the compounds which are pres-
ent in the plants which grew upon the soil, plus those compounds
contained in the bodies of bacteria, protozoa, and fungi, plus all of
the compounds which may be derived from these compounds
under the peculiar soil conditions of decay, oxidation, bacterial ac-
tion, and the secretions of fungi and living plant roots.

31

II. EXPERIMENTAL: A STUDY OF THE NITROGEN
DISTRIBUTION IN DIFFERENT SOIL TYPES.

A. The Problem.

It has been shown in the historical study above that a number
of investigators have studied the organic nitrogen distribution in
the soil by applying either Hausmann's (1899) or Van Slyke's (1910,
1911) method of protein analysis. It has been demonstrated by
Potter and Snyder that various plots on a single soil type under
different fertilizer treatments gave, with Van Slyke's method, es-
sentially the same results.

I have taken up the problem at this point and made a similar
study of the organic nitrogen distribution in different soil types in
an attempt to see whether the forms in which nitrogen occur
differ from locality to locality and from soil type to soil type. 1
am concerned with the problem of distribution of the organic nitro-
gen in the soils and soil extracts studied.

B. The Material.

The study was made using two peats, one muck, seven mineral
surface soils, and one subsoil. All were from the State of Min-
nesota. The origin and type names are in accordance with the
surveys of the Bureau of Soils of the U. S. Department of Agri-
culture when such surveys were available. Almost all of the soils
used are portions of the identical samples employed by Gortner
(1916 a, and 1917) in his soil studies. The samples used in this
study were all air dry soils. The moisture was determined by
heating the soils to a temperature of 100° C. for 12 hours and then
weighing.

The descriptions of the soils follow :

1. Calcareous black grass-peat. This sample was selected
from a large bulk sample taken to a depth of 8 inches from a
grass bog near the Agricultural Experiment Station Farm, St. Paul.
The peat was black and well decomposed. The peat was ground
to a powder in a ball mill before using. The air dry material con-
tained 6.40 per cent moisture.

2. Sphagnum-covered peat. This sample of very strongly acid
peat was prepared from a large bulk sample taken from a bog on
the Experimental Farm near Grand Rapids. The superficial layer
of sphagnum and shrubs was first removed and a sample of the un-
derlying peat taken to a depth of 8 inches. The peat was poorly
decomposed. The sample was prepared by grinding to< a powder in
a ball mill. The air dry soil contained 5.90 per cent moisture.

3. Acid "muck" soil. A sample of this strongly acid soil was
obtained from a bog about two miles from the farm of the Agri-

32

cultural Experiment Station, St. Paul. The sample is a composite
of 10 samples taken to a depth of 8 inches lengthwise of the bog.
The vegetation of the bog was largely cattails (Typha spp.) and
rushes (Scirpus spp.) The sample contained 5.60 per cent moisture
in the air dry condition.

4. Fargo clay loam. The sample analyzed consisted of a
composite made from 144 borings taken to a depth of 8 inches from
a twenty-acre field on the Northwest Sub-station Farm, Crookston.
The sample was highly calcareous. This soil type has been de-
scribed by Mangum and Schroeder (1906). The moisture content
of the air dry soil was 3.92 per cent.

5. Fargo silt loam. This sample was a composite made from
100 borings to a depth of 6 inches, twenty borings being taken from
each of five virgin fields near Nerstrand, Rice County. The sam-
ple had a neutral -reaction. The soil type has been described by
Burke and Kolbe (1909). The air dry soil contained 14.89 per cent
moisture.

6. Carrington silt loam. This soil is represented by two sam-
ples. Each composite consisted of 100 borings to a depth o-f 6
inches, twenty borings being taken from each of five virgin fields
near Nerstrand, Rice County, for Sample number I, and twenty
borings being taken from each of five virgin fields near Morristown,
Rice County, for Sample number II. Sample 1 was strongly acid
while Sample II was but slightly acid. The soil type is described
by Burke and Kolbe (1909). The moisture content of sample I was
6.22 per cent.

7. Hempstead silt loam. A composite sample from 36 borings
to a depth of 6 inches was taken from 12 plots on the Agricultural
Experiment Station Farm, St. Paul. No commercial fertilizer had
been applied, but the land had long been under cultivation. The
soil type has been described by Smith and Kirk (1914). The sam-
ple was strongly acid. The air dry soil contained 3.07 per cent
moisture.

8. Prairie-covered loess. The sample consisted of 50 borings
taken to a depth of one foot, ten borings being taken from each of
five virgin fields near Luverne, Rock County. The sample was
somewhat calcareous. The area from which the sample was ob-
tained has not been subjected to a detailed soil survey. The air
dry soil contained 7.89 per cent moisture.

9. Forest-covered loess. This sample wras taken from five
virgin fields near Caledonia, Houston County, ten borings to a
depth of one foot being taken from each field and equal weights
from each boring being combined in the composite sample. The
sample was strongly acid. The air dry soil had a moisture content
of 1.87 per cent.

10. Hempstead silt loam subsoil. This was a third foot bulk
sample taken from a grove on the farm of the Agricultural Experi-
ment Station, St. Paul. The sample was strongly acid.

33
C. The Method.

The method of Van Slyke (1911) has been used throughout
this investigation because the nitrogen can be separated into a
larger number of fractions than when the earlier method of Haus-
mann (1899) is employed. The different fractions, however, are
not listed in the same manner as in the Van Slyke method, for
since we are not dealing- with pure protein material we cannot
correctly speak of arginine, histidine, cystine, and lysine nitrogen.

Van Slyke (1915) has called attention to the fact that his
method was devised for the analysis of pure protein material and
not for a heterogeneous mixture of nitrogen compounds. This
fact was not recognized by certain investigators. Grindley and
Slater (1915) applied this method to the analysis ot feeding stuffs
in exactly the same manner as though they were dealing with a
pure protein. Potter and Snydcr (1915 a) analyzed certain soils
and soil extracts and report their fractions as "arginine," "histi-
dirtfe," etc. Although they state (p. 2221) :

It is not thought that nitrogen as found by the Van Slyke method, work-
ing with such a complex as the soil, is in reality, all lysine, histidine, etc.,
nitrogen. It might be said that each group, as found, represents 'a class of
compounds having the particular reaction by which the lysine. histidine, etc.,
nitrogen respectively are determined.

It is obvious that there are other types of organic materials
which will interfere with the nitrogen distribution. It seems very
probable that in soils as well as in the material analyzed by Grind-
ley and Slater (1915) there must be many organic nitrogen com-
pounds which have no relation to the protein molecule, such as
purme bases, pyrimidine bases, nitrogenous lipins, nitrogenous pig-
ments, as well as other non-protein nitrogenous compounds (cf.
the list of non-protein nitrogenous compounds actually isolated
from the soil as given in the preceding part of this paper). Gortner
(1913) states that much valuable comparative data can be obtained
by the application of Van Slyke's method to the analysis of heter-
ogeneous materials ; but it is self evident that no analogy can be
drawn between the analysis of pure protein and the analysis of a
protein mixed with an unknown amount of foreign nitrogenous
compounds. The results of Potter and Snyder (1915 a) are of
little value in advancing our knowledge of soil proteins or for
comparison with analyses of pure proteins, but are extremely
valuable and interesting for comparison between themselves and
with other analyses of soils carried out under similar conditions.
It must be remembered that all data on similar material is strictly
comparable when the same method of analysis is followed.

It is possible that many of the non-protein nitrogenous com-
pounds may be split up during the hydrolysis of heterogeneous ma-
terial. Gortner (1913) has shown that uric acid nitrogen is dis-
tributed in all four of the major fractions after hydrolysis. The
ammonia nitrogen amounted to 15.27 per cent, humin nitrogen
35.98 per cent, basic nitrogen 12.97 per cent, and non-basic nitrogen
3S./8 per cent. "The humin nitrogen contained no trace of black
color and was probably calcium ureate." Probably all of the purines
and pyrimidines would behave in a similar manner.

34

The general method employed in this investigation will be dis-
cussed in detail for two soils, a peat and a mineral soil, inasmuch as
the experimental conditions vary in minor details with the two
types.

1. The method in detail for a peat soil. Duplicate samples
of 15 grams were hydrolyzed in the presence of hydrochloric acid
for 48 hours. The content of calcium oxide was taken into, account,
and corrections made so that the 100 cc. of hydrochloric acid used
was of sufficient concentration to neutralize the lime and at the
same time furnish a constant boiling acid. The hydrolysis was
carried out in 200 cc. long neck, round bottom Kjeldahl flasks, fitted
with modified Hopkins condensers made from a test tube which
fit rather loosely into the neck of the flask. By means of this device
any error due to products extracted from cork or rubber stoppers
was obviated. The flasks were heated to gentle boiling on the
same sand bath over an Argand burner, so that the rate of hydro-
lysis would be as near the same as possible.

After completion of the 48 hour hydrolysis the mixture was
evaporated in a Claissen distilling flask under diminished pressure
until all the hydrochloric acid possible was driven off. The residue
after this distillation was dissolved in 100-150 cc. of water, 100 cc.
of 95 per cent alcohol, and an excess of calcium hydroxide sus-
pended in water was added and the ammonia distilled off into
standard acid at a temperature of 40-50° C. under a pressure of less
than 30 mm., distillation being continued for at least a half hour.
The results are listed under "ammonia nitrogen."

The alkaline mixture in the distilling flask was filtered and
the precipitate well washed with hot water until free of chlorides.
A Kjeldahl determination was made of the filter and its contents,
and the results listed under "humin nitrogen."

The filtrate and washings from the humin were acidified with
hydrochloric acid and concentrated under diminished pressure to
less than 200 cc. To this solution was added 18 cc. of concen-
trated hydrochloric acid and the whole heated on the water bath
until hot. A solution containing 15 grams of phosphotungstic acid
was then added and the heating on the water bath continued for
an hour. The flask was then set aside in a cool place for 48 hours.
The precipitate of the bases was then filtered off and washed as
directed by Van Slyke (1911).

The basic phosphotungstates were suspended in 800 cc. of
water and brought into solution by the cautious addition of a 50
per cent solution of sodium hydroxide, a few drops of phenolph-
thalein being added to guard against too great an excess of alkali.
The phophotungstic acid was precipitated by the addition of a
slight excess of 20 per cent barium chloride, and the barium phos-
photungstate was filtered off and washed free of chlorides with
hot water.

The filtrate and washings were united, acidified with hydro-
chloric acid, and concentrated under diminished pressure to a very
small volume. After cooling the residue was filtered, washed and
made up to 50 cc. volume.

35

The washed precipitate of barium phosphotungstate and filter
were subjected to Kjeldahl determination for any nitrogen that
might be held by absorption, adsorption, or occlusion, as was also
the filter and contents remaining after the final filtration of the
solution containing the basic nitrogen. In all cases some nitrogen
was found. This nitrogen is probably derived from the "unad-
sorbed humin carried down with the !^asic phosphotungstates"
mentioned by Van Slyke (1915, p. 284). Inasmuch as my work was
done prior to this publication, I added this nitrogen to the total
nitrogen content of the bases instead of to the humin.

During the distillation after kjeldahling this precipitate the
cochineal indicator took on a color which made the acid solution
appear that complete neutralization might have occurred when in
fact it had not. This color change was noticed in every case with
the barium phosphotungstate distillate. This made titration diffi-
cult, since a new end point had to be established. Gortner and
Holm (private communication) have observed a similar color
change in the case of fibrin hydrolyzed in the presence of a large
excess of formaldehyde. They explain this finding on the assump-
tion that pyridine (or some similar base) is formed which is not
easily broken down in the Kjedahl process (cf. Dakin and
Dudley, 1914), and which greatly influences the color changes of
the indicator when the base is volatalized during the subsequent
distillation with alkali. Whether or not this is the cause of the
phenomenon observed in my materials cannot be ascertained, with-
out further investigation.

In no case did I attempt to separate the basic nitrogen into
the usual fractions of "arginine," "cystine," "histidine," and "lysine"
nitrogen, because I am not dealing with pure protein. Instead in
each case the total nitrogen liberated as ammonia was determined
on 25 cc. of the solution containing the bases. This was determined
in exactly the same manner that Van Slyke used for the determina-
tion of arginine nitrogen. The volume of standard acid neutralized
by the ammonia indicated the amount contained in the 25 cc. of
solution used. The nitrogen found is listed as "basic nitrogen set
free as ammonia by 50 per cent potassium hydroxide." The solution
remaining" from this determination was used in the estimation of
the total nitrogen of the bases. This was performed according to
Van Slyke's directions. The quantity of acid neutralized in this
determination was added to that neutralized in the basic-nitrogen-
set-free-as-ammonia-by-50-per-cent-potassium-hydroxide, thus se-
curing- the "total basic nitrogen."

The "amino nitrogen of the bases"" was determined in Van
Slyke's (1912) apparatus, using 10 cc. portions of the solution.

The filtrate from the bases was treated with sodium hydroxide
solution until a slight turbid precipitate -of lime was formed, and
then cleared immediately by the addition of acetic acid. This was
concentrated under diminished pressure and on cooling was made
to 200 cc. volume. The solutions were more or less violet in color.
"Total nitrogen in the filtrate from the bases" was determined on
duplicate portions of 25 cc. each by the method of Kjeldahl. The

36

digestion was continued for three hours after the solutions were
clear, so that the phosphotungstic acid would not interfere with
the accuracy of the determination. The "amino nitrogen in the
filtrate from the bases" was determined on duplicate portions of
10 cc. each by means of the Van Slyke (1912) apparatus.

2. The method in detail for a mineral soil. Duplicate portions
of 250 grams were hydrolyzed in 500 cc. round bottom Kjeldahl
flasks for 48 hours on different sand baths. Allowance was always
made for the lime content of the soil, and the requisite amount of
hydrochloric acid added to insure the presence of a constant boiling
acid (sp. gr. 1.115), and a volume of approximately 250 cc. The
solutions boiled smoothly and gave no trouble by bumping. Air
dry soil was used in all cases, but the moisture was determined on
a separate portion and all data calculated to the dry basis.

On completion of the hydrolysis each of the two samples was
diluted to a 1000 cc. in measuring flasks and allowed to settle for
at least 24 hours. The clear solution was then syphoned off and
an aliquot of 500 cc. analyzed according to the usual method of
Van Slyke. In nearly all cases this solution was straw color due
to the presence of ferric salts that had been formed during the
hydrolysis. No black color, the usual color of a protein hydrolysate,
was observed in any instance.

Another aliquot of 100 cc. was used for the determination of
total nitrogen in the solution by making duplicate Kjeldahl de-
terminations on 50 cc. portions. A second aliquot of 100 cc. was
used for the determination of ujodidi numbers" (cf. p. 25) when
they were determined.

The soil remaining in the measuring flask was washed free
from soluble nitrogen with a 1 per cent solution of potassium sul-
fate by decantation from tall soil beakers, the solution after set-
tling being syphoned off not oftener than twice a day. This meth-
od was employed in order to prevent the clay from forming a
colloidal suspension. Distilled water alone would remove all elec-
trolytes and allow a portion of the clay to remain in the solution
in colloidal suspension. It is known that suspensions of finely di-
vided clay carry a negative charge in pure water. Since it is
necessary for the complete precipitation of colloids to. have some
electrolyte present it was decided to use a 1 per cent solution of
potassium sulfate. The negatively charged colloid was thus pre-
cipitated by the positive ions of the potassium sulfate solution
and at the same time the salt would not interfere with the subse-
quent Kjeldahl determination.

A concrete example of the thoroughness of this washing may
well be given. It will be noted that 700 cc. of the original hydrol-
ysate was syphoned off for the different analyses. This left a total
volume of 300 cc. of residue and solution to be washed by decanta-
tion with 1 per cent potassium sulfate. By the methods of calcu-
lation given in the following paragraphs it was found that the re-
maining- solution contained 0.1089 gram of nitrogen. If three-
fourths of the wash solution is removed each time, there will remain

37

in the solution at the end of the fourth washing approximately 0.0004
gram of the original nitrogen. Actual Kjeldahl determinations
were made on 250 cc. portions from the fourth washing in the case
of duplicates from the same soil, and the results indicated that
0.0006 gram of nitrogen still remained in the solution. Since this
was within experimental error of the theoretical value, the method
of washing by decantation was followed in all the subsequent work
with mineral soils, or those which had mineral soils added previous
to the analysis.

The residue from the hydrolyzed soil was evaporated to dry-
ness on the steam bath in an evaporating dish, then further dried
at about 110° C. After cooling, this dry soil was passed through a
1 mm. sieve and after being thoroughly sampled, duplicate nitro-
gen determinations were made on 15 gram portions and the total
nitrogen remaining in the soil calculated. These results are listed
as ''insoluble humin nitrogen in the soil." The weight of the dry
soil divided by the average specific gravity (2.6) represented the
actual volume occupied by this soil residue. The total volume of
the hydrolysate minus the volume occupied by the insoluble resi-
due gives the actual volume of the soil solution.

Since the analysis was made on 500 cc. of the soil solution it
was necessary to recalculate the total "insoluble humin nitrogen in
the soil" in order to determine the amount of this humin nitrogen
actually belonging to the aliquot analyzed.

The total nitrogen belonging to the solution analyzed was
found by taking the sum of the total nitrogen in the solution and
the above insoluble humin nitrogen. Knowing the total nitrogen
content of the soil before hydrolysis and the total nitrogen in solu-
tion, the per cent of the total nitrogen in solution after hydrolysis
can be determined.

The 500 cc. aliquot was concentrated under reduced pressure
until the hydrochloric acid was practically removed and the am-
monia nitrogen determined in the manner outlined under the peat
analysis.

The "humin" fraction precipitated by the calcium hydroxide
was almost colorless or light yellow, due to the iron salts con-
tained in it. This bulky precipitate was always washed by de-
cantation after the method above described, except that distilled
water was used, the united washings being concentrated to 200
cc. or less for the precipitation of the basic nitrogen.

It was found necessary to use 35 grams of phosphotungstic
acid for the precipitation of the bases. The remainder of the
analysis was carried out as directed under peats.

3. The method for determination of "Jodidi numbers." A
100 cc. portion of the clear hydrolysate was concentrated under
reduced pressure and the ammonia nitrogen determined in the
usual manner. The residue remaining in the flask after this de-
termination was dissolved in concentrated hydrochloric acid and
phosphotungstic acid added.* After standing the usual length of
time the precipitate was filtered of! and the total nitrogen deter-

*lt will be noted that no "humin" fraction is separated. In that respect
the "Jodidi numbers" differ from Hausmann numbers.

38

mined on the filter and -contents by the Kjeldahl method and listed
as "basic N." The filtrate from the above precipitate was con-
centrated and made to 300 cc. volume. Duplicate determinations
were made on 100 cc. portions, and the total nitrogen listed as
nitrogen in the filtrate from the "bases."

4. The determination of nitrogen. Nitrogen was determined
on the soils and soil extracts in the usual manner, using 25-35 cc.
H.,SO4, 10 gm. K2SO4, and a crystal of CuSO4. All titrations were
made with N/14 acid and alkali so that the figures obtained repre-
sented milligrams of nitrogen without necessitating a calculation.

D. The Analytical Data.

The essential data which have already been published on the
soils studied are shown in Table I.

Table I. — Certain analytical data for the soils used in this paper.
Data of Gortncr (1$16 a).

3

'c "^

& ""

11

^ o

o tf "w

1^

|1

be

c G X

•** in

^ C _o

"1*

% Ss

o ^

o «

+> 0 X

C — • w

C cd
3 .O
.CJ ^

if

o
1

Calcareous black
grass-peat
Sphagnum-covered
peat

6.40
5.90

2.940
2.000

42.81
49.32

0.600
none

28.71
32.91

14.56
24.66

Acid ''muck" soil . .

5.60

1.340

14.58

none

7.14

10.88

Fargo clay loam. . . .
Fargo silt loam

3.92
14.89

0.250
0.823

2.678
10.02

2.360
0.200

2.66
9.91

10.72
12.17

Carrington silt loam.
Hempstead silt loam
Prairie-covered loess
Forest-covered loess

6.22
3.07
7.89
1.87

0.371
0.256
0.301
0.128

4.733
3.373
3.704
1.638

0.090
0.020
0.240
0.120

4.95
3.61
3.40
1.79

12.76
13.17
12.30
12.79

1. Analysis of "fibrin from blood" hydrolyzed in the presence
of 100 grams ignited subsoil. This analysis was conducted in order
to ascertain if possible the effect of soil minerals upon the hydro-
lysis of a pure protein. Fibrin wras selected because it was from
a sample already analyzed (Gortner 1916 c). The subsoil was first
ignited to redness in a muffle furnace for an hour, in order to drive
off all the organic matter, and subsequently cooled in a desiccator.

Duplicate portions of five grams fibrin and 100 grams ignited
subsoil were hydrolyzed in the presence of an excess of hydrochlo-
ric acid. Upon the application of heat, fumes of hydrogen chloride
were evolved for some time. The analysis was conducted like
that of the mineral soils, excepting that a 600 cc. aliquot was used
for the analysis, this amount of solution being equivalent to three
grams of fibrin. After making the ammonia determination the
humin precipitate was washed by decantation in the usual man-
ner and the filtrate made to 250 cc. volume. The first wash solu-

39

tion from the humin precipitate had a characteristic light blue
color in each case. Duplicate Kjeldahl determinations were made
on 25 cc. portions of this solution and the results listed as total-
nitrogen-in-the-filtrate-from-humin. The remaining 200 cc. solu-
tion was used for the precipitation of the bases and the subsequent
analysis, but of course the results were calculated on the basis of
the total volume. The total nitrogen belonging to the aliquot an-
alyzed was determined by adding the nitrogen obtained as am-
monia, humin, and total-nitrogen-in-the-filtrate-from-humin to the
insoluble-humin-nitrogen-in-the-soil. The filtrate-from-the-bases
was made to 250 cc. volume. Twenty-five grams of phosphotungstic
acid was used for the precipitation of the bases. The nitrogen
retained by the barium phosphotungstate was 0.0022 gram for
Sample I and 0.0020 gram for Sample II.

The experimental data showing the grams of nitrogen found
a-nd per cent of total nitrogen are given in Table II.

Table II. — Nitrogen distribution *in tJyree grams of Merck's "fibrin
from blood" hydrolyzed in the presence of 100 grams of ignited subsoil.

Grams nitrogen Per cent of total nitrogen

I

II

1 I

II 1

Av.

Total \

04577

04591

Ammonia N • •

0.0457

0.0455

9.98

9.91

.
9.95

Insoluble humin N in soil
Humin N pptd. by
CaCOH),

0.0125
00220

0.0130
0.0221

2.73
4.81

2.83
4.81

2.78
4.81

Basic N • •
\rgmine N • •

0.1219
0.0598

0.0995
0.0440

26.63
13.07

21.68
9.58

24.15
11.32

Histidine^T

none

none

none

Lysine N • •

Cystine N

0.0597

0.0531

13.04

11.57

12.30
0.51

Amino N in bases • •

00775

0.0746

16.93

16.25

16.59

N in filtrate from bases..
Amino N in filtrate from
bases

0.2725
02671

0.2909
02702

59.54
58.36

63.36
58.85

61.45
58.61

Non-amino N in filtrate
from bases • •

00054

00207

1 18

451

284

Total N regained ....-•••

0.4746

0.4710

103.69

102.59

103.14

iFrom data of Gortner (1916 c).

Table III shows a comparison of these analyses with other
analysis of the same sample of fibrin hydrolyzed alone, and in the
presence of three times its weight of cellulose (data of Gortner
1916 c).

Differences .between these analyses, together with differences
between duplicates in each series of analysis, and data showing
"maximum" and "average" experimental differences to be expected
are given in Table IV.

These comparisons will be considered in detail under "Dis-
cussion" in the latter part of this paper.

40

Table III. — Comparative analyses of three grains* of Merck's
'fibrin from blood" hydrolyzed alone and in the presence of carbo-

hydrate and of ignited subsoil.

c .M t;
I* c3

O
ti

1

0

<M

0

o

m

9

°1

*0

T3

c

•

3 ^

'O

5 -0

•** . ^

£

o

bjc

Q 55

a

9

s g

cC

5- 1 1

1

5

0,

. s

G

.C

1

x

•° "a! "2

£

c

"o

w *"

£2

c

"*""

0)

P

<C S-1

<C

o ^

G

?4-t ^

bJO

g-|o

g

-c

&
N

<v

0

c

5 ti

1

O ^j

§

1

CO

be

CO

r*

So

CO

-

O b£

C "^

O

5

H

I Per cent total jPer cent total] Per cent total
nitrogen I nitrogen I nitrogen

Ammonia N

1015

985

995

Humin N .. . .

283

7 72

7 59

Arginine N • .

1091

856

11 32

Histidine N

4 36

4 86

none

Lysine N

1205

11 04

1230

Cystine N . .

051

071

051

Amino N in filtrate from bases. . . .
Non-amino N in filtrate from bases
Total N regained

55.43
2.51
98.75

52.02
3.91
98.67

58.61
2.84
103.14

*The three gram portion contained approximately 0.4550 gm. nitrogen.

Table IV. — Difference between duplicate analyses (due to experi-
mental errors), the differences apparently due to the addition of carbo-
hydrate and of ignited subsoil, as well as Van Slyke's "maximum" and
<( average" differences to be expected.

V 0»

U O <D

•/. i

O>,Q

CM aJ

C 'U'O

"d c

"S C

g-Cd

8? 52

Van Slyke's

o ^

O r* E_,

o w

{^ ^ J-t

^ - S

(~\ Q1 1 ^
\ 11/1.1 1

'•£ o

^ bco

^ ^3

^n *** *^

•*•* ^ 5f

G-^- rK
^3 0>U

p, ^)

^ ^^

^2 ""'"

experimental

^•M

'C P

*o'£

'C O

• 'ct-1

differences

o

<M

— bu

C ^ ^ /*^v

S8°

s

^ ^ O

0"™*

fj :"2 O

c i

1-^

B^3

2g«S

0)Q

<D g

^ o °r~l

S^ 4J

•^^^

"^.S Q

•^ Bj

'O ^_) t-C

^o C

Is

rti O

irr

Si

S STstJ

ll-

I

c

0)

o

£•2

S ^ •*-*

£

°-2'b. °

o r 1 **^ ^

0) d

0) C ctf

<p 'S

o ^ r5

£O

M<

QJ

*

«K c^ *•<

^•* §

fe ^

rr-

-

P,

'S.S

^ d >>

9

ftr>-2'o

ft>-S '

s

.

s- ^A

•rn rj /-A.

M

•C""

^ C HH

•3

"<D

"t^ & O

"r^ O ^

^g*t-

a) w d

Q; m*O

be

O rH

(D^^CO

§^d

bJD fj 03 ^

c^«- o>ja

£

t

feo"^

®'o"

|SsS

||S2

d

C^

S

P-l

OH

<j

J

;

Ammonia N.. . .

0.01

0.48

0.07

—0.30

—0.20

0.37

0.12

Humin N

0.11

0.25

0.10

+4.89

+4.76

039

020

Arginine N. . . .

0.73

0.86

3.49

I ••**J''

. —2.35

+0.41

1.27

0.73

Histidine N. . . .

0.07

0.66

none

+0.50

—4.36

2.14

0.79

(0.93)1

Lysine N. ....

0.08

0.11

1.47

1.01

-f-0.25

1.23

0.61

Cystine N

0.10

0.00

+0.20

o'n

Amino N in fil-

trate from

bases

2.12

2.15

0.49

—3.41

+3.18

1.60

0.63

Non-amino N in

(0.60)1

filtrate from

bases ........

0.27

0.40

3.23

+1.40

+0.33

1.20

0.68

2The figure in parentheses represents the second greatest difference be-
tween duplicates observed by Van Slyke (1911).

41

2. Calcareous black grass-peat. Duplicate samples of 15
grams were hydrolyzed for 48 hours in the presence of 100 cc. con-
stant boiling hydrochloric acid, and the analysis conducted as de-
scribed under the method for a peat soil.

In the precipitation of the phosphotungstic acid with barium
chloride, it was difficult to determine when the precipitation was
complete. On addition of barium chloride a precipitate formed and
the pink color slowly disappeared. Both sodium hydroxide and
barium chloride were repeatedly added. The change in color oc-
curred several times during a half-hour. At all times test portions
gave immediate granular precipitates with both sodium sulfate
and barium chloride. This indicated that we could not depend
upon the test with sodium sulfate to determine when all the
phosphotungstic acid was precipitated. After the addition of a
large amount of barium chloride the solution was allowed to stand
several hours in order to allow equilibrium to be established. At
the end of this time test portions gave a heavy precipitate with
sodium sulfate, but no action with barium chloride. In all subse-
quent work, both with peat and mineral soils, the addition of barium
chloride to the solution of the phosphotungstates was extended
over an hour or more and the precipitate then allowecl to settle
over night.

This barium phosphotungstate precipitate was washed thor-
oughly by decantation with hot water until free of chlorides. This
operation was carried out in the original beaker, and in the case of
many mineral soils required from 500 to 1000 cc. to remove all
chlorides. The washed precipitate of barium phosphotungstate and
filter on kjeldahling gave 0.0021 gram nitrogen retained by Sample
I and 0.0055 gram by Sample II.

The experimental data showing the grams of nitrogen and
per cent of tgtal nitrogen are given in Table V.

Table V . — Nitrogen distribution in calcareous black grass-peat.

Grams nitrogen | Per cent of total nitrogen

I

II

I

II 1

Av.

Total N

04412

04412

Ammonia N

00833

00867

1888

1965

1926

Humin N

0.1145

0.1155

25.95

26.18

26.07

Basic N

00494

0 0441

11 20

1000

1060

Basic N set free as NH3 by
50% KOH .. .

0.0150

0.0139

3.40

3.15

3.27

Basic N not set free as NHa
by 50% KOH......
Amino N of bases

0.0344
0.0309

0.0302
0.0251

7.80
7.00

6.85
5.69

7.33
6.35

Non-amino N of bases • . . .

0.0185

0.0190

4.19

4.31

4.25

N in filtrate from bases
Amino N in filtrate from
bases
Non-amino N in filtrate from
bases ....

0.1932
0.1810
0 0122

0.1810
0.1672
00138

43.79
41.02
277

41.02
37.90
3 12

42.40
39.46
2.94

Total N regained

0.4404

0.4273

99.82

96.85

98.33

42

3. Sphagnum-covered peat. Duplicate 15 gram samples were
hydrolyzed in the usual manner. It was found in the ammonia de-
termination that all of the ammonia nitrogen could not be driven
off in a half-hour when the volume of solution was large and a
bulky precipitate of 'iron and aluminum hydroxides was present.
Continued distillation for a further half-hour in this case gave
additional ammonia nitrogen amounting to 0 0060 gram. In all sub-
sequent work with both peats and soils the volume was kept smaller
by the use of a more concentrated suspension of calcium hydroxide.

The calcium hydroxide precipitate containing the "humin"
nitrogen was difficult to digest in the subsequent Kjeldahl deter-
mination, due to the large amount of carbonaceous organic material
present. From 100-200 cc. of sulfuric acid were required for
the digestion. After digestion the material was transferred to a
1000 cc. flask and 250 cc. portions used for the distillation. It was
often necessary to heat the flask on a sand bath to prevent severe
bumping. This trouble was obviated in later work by adding zinc
dust instead of granulated zinc to the distilling flask. The nitrogen
retained by the barium phosphotungstate amounted to 0.0009 gram
in Sample I, and 0.0013 gram in Sample II.

The experimental data showing the grams of nitrogen and
per cent of total nitrogen are given in Table VI.

Table VI. — Nitrogen distribution in sphagnum-covered peat.

Grams nitrogen | Per cent of total nitrogen

1 I

1 II

I 1

II

Av.

Total N

03000

03000

Ammonia N

00659

00737

21 97

2457

2327

Humin N

0.0779

0.0804

25.96

26.80

26.38

Basic N

0.0281

0.0303

9.37

10.10

9.73

Basic N set free as NH3
by 50% KOH..........
Basic N not set free as

NH3 by 50% KOH. . . .

0.0091
00190

0.0088
00?15

3.03
632

2.93

7 17

2.98
675

Amino N of bases
Non-amino N of bases...
N in nitrate from bases..
Amino N in filtrate from
bases .

0.0171
0.0110
0.1396

0 1292

0.0145
0.0158
0.1184

01135

5.70
3.67
46.53

43.06

4.83
5.27
39.46

37.83

5.26
4.47
43.00

40.45

Non-amino N in filtrate
from bases

0.0104

0.0049

3.47

1.63

2.55

Total N regained

0.3115

0.3028

103.83

100.93

102.38

4. Acid "muck" soil One 25 gram sample was hydrolyzed
in the presence of 125 cc. concentrated hydrochloric acid for 48
hours. The -ammonia nitrogen was determined as usual, and the
precipitate of calcium hydroxide containing the '"humin" was
washed in a taM soil beaker in the manner described under the
method for a mineral soil. After digestion of this precipitate, the
material in the !£jeldahl flask was diluted to 500 cc. and duplicate
distillations made on 250 cc. portions. The bases were precipitated
with 15 grams phosphotungstic acid. The total nitrogen retained by
the barium phosphotungstate was 0.0022 gram. The solution of
the filtrate-from-the-bases was made to a volume of 250 cc.

43

The experimental data showing the grams of nitrogen and per

cent of total nitrogen are found in Table VII.

Table VII. — Nitrogen distribu tion in an acid "muck" soil.

Grams
nitrogen

Per cent
of total
nitrogen

Total N

Ammonia N

Humin N

Basic N

Basic N set free as NH3 by 50% KOH

Basic N not set free as NH3 by 50% KOH

Ammo N of bases

Non-amino N of bases

N in filtrate from bases

Ammo N in filtrate from bases

Non-amino N in filtrate from bases •

Total N regained

0.3350
0.0653
0.0925
0.0454
0.0104
0.0350
0.0306
0.0148
0.1300
0.1133
0.0167
0.3332

19.49
27.61
13.55

3.10
10.45

9.13

4.42
38.81
33.82

4.99
99.46

5. Fargo clay loam. Duplicate portions of 250 grams were
hydroh zed for 48 hours. The "hitinin" precipitate required 100 cc.
sulfuric acid for the digestion. After digestion the material was
transferred to a 500 cc. flask and 21>0 cc. portions used for the dis-
tillation. This method was followed subsequently with th'e "humin"
nitrogen determination of all the mineral soils.

The nitrate from "humin" was of a sirupy consistency in each
case. The first addition of 15 grams of phosphotungstic acid did
not entirely precipitate the bases. Five gram portions were added
from time to time until a total of 50 grams had been used. After
standing the usual length of time the precipitate of the bases was
filtered off, but even then the wash water caused the formation of
a small additional precipitate in the filtrate. After warming on the
steam bath this final solution was perfectly clear, and on standing
over night, only a trace of precipitate was formed so the precipita-
tion was considered complete. It appears probable that a portion
of this precipitate is due to the formation of inorganic phospho-
tungstates which consume a very large amount of the phospho-
tungstic acid, for if all of this precipitate had consisted o.f basic
nitrogen compounds the amount of nitrogen recovered should have
been greater than the amount which was actually found. In all
the subsequent work 35 grams of phosphotungstic acid was used
for the precipitation of the bases in the hydrolysates from mineral
soils.

The phosphotungstate precipitate dissolved very slowly in the
sodium hydroxide as did all other phosphotungstic acid precipitates
of the mineral soils studied. A Kjeldahl determination of the
barium phosphotungstate gave 0.0026 gram nitrogen. The solution
containing the nitrogen of the bases was made up to 100 cc. volume.

During the concentration of the filtrate from the bases so much
precipitate separated, that this was filtered off and the solution
made up to 300 cc. volume. The salt remaining was dissolved in
water and also made up to a volume of 300 cc. Aliquot portions
were taken from each solution and combined for the different de-
terminations.

The experimental data giving the grams of nitrogen and per
cent of total nitrogen are found in Table VIII.

Table VIII. — Nitrogen distribution in Fargo clay loam.

Grams nitrogen [ Per cent of total nitrogen

II

II Av.

Total N

03368

0 3338

Ammonia N

0 0788

"00821

23 40

24 60

24 (X)

Insoluble humin N in soil.
Humin N pptd. by
Ca(OH)2

0.0948
00352

0.0948
00266

28.15
1045

28.40
797

28.27
Q 21

Basic N .

i

00320

9 58

Basic N set free as NH3
by 50% KOH

0.0108

3?3

323

Basic N not set free as
NH3 by 50% KOH

00212

635

63=1

Amino N of bases • •

00215

6 44

Non-amino N of bases • •

00105

3 14

N in filtrate from bases. . .

0.1092

32.71

Amino N in filtrate from
bases

0 1027

3077

30 77

Non-amino N in filtrate

00065

1 95

1 9S

Total N regained

0.3447

103.26

103.77

1 Entire sample lost during precipitation of bases.

6. Fargo silt loam. Two 125 gram portions were hydrolyzed
for 48 hours with 500 cc. hydrochloric acid (sp. gr. 1.18). During
the first few hours large amounts of hydrogen chloride were
evolved.

The resulting hydrolysates from the two flasks were com-
bined and diluted to 2 liters. After settling, a 1 liter portion was
syphoned off and analyzed by the usual method. Two 100 cc. por-
tions were used for the determination of total nitrogen in solu-
tion, and another portion of 200 cc. was used for determination
of the "Jodidi numbers." The nitrogen retained by the barium
phosphotungstate was 0.0091 gram. The solution containing the
basic nitrogen was made to 100 cc. volume and that containing
the nitrogen in the filtrate from the bases to 300 cc. volume.

The experimental data showing grams of nitrogen and per
cent of total nitrogen are given in Table IX.

Table IX. — Nitrogen distribution in Fargo silt loam.

Total N

Ammonia N

Insoluble humin N in soil

Humin N pptd. by Ca(OH)2

Basic N

Basic N set free as NH3 by 50% KOH

Basic N not set free as NH3 by 50% KOH

Amino N of bases • • • •

Non-amino N of bases • •

N in filtrate from bases •

Amino N in filtrate from bases

Non-amino N in filtrate from bases

Total N regained

Grams
nitrogen

Per cent
of total
nitrogen

0.9238
0.2454
0.2118
0.03011
0.1119
0.0296
0.0823
0.0695
0.0424
0.3246
0.3015
0.0231

26.56

22.93

3.26

12.11

3.20

8.91

7.52

4.59

35.14

32.64

2.50

100.002

By difference.

Calculated.

45

7. Carrington silt loam. Sample I (Nerstrand origin). The
hydrolysis of 250 grams was carried out according to the method
described under Fargo silt loam. The "humin" nitrogen was de-
termined as outlined under Fargo clay loam. Sample II (Morris-
town origin) was hydrolyzed and the same methods of analysis
employed throughout as was applied to- Sample I. The nitrogen
retained by the barium phosphotungstate precipitate was 0.0028
gram in Sample I and 0.0025 gram in Sample II. In both samples
the solutions containing the bases and nitrate from the bases were
made to the same dilution as under Fargo silt loam.

The experimental data showing grams of nitrogen and per cent
of total nitrogen are given in Table X.

Table X. — Nitrogen distribution in Carrington silt loam.

I Sample No. I | Sample No. II

Grams
nitrogen

Per cent
of total
nitrogen

Grams
nitrogen

Per cent
of total
nitrogen

Total N ....

0 5124

04920

Ammonia N

0 1463

28 55

01381

2807

Insoluble humin N in soil
Humin N pptd. by Ca(OH)2. •
Basic N

0.1324
0.0304
00600

25.84
5.93
11 71

0.1210
0.03263
00725

24.59
6.63
1474

Music N set free as NPL by 50%
KOH

0.0172

3 36

00156

3 17

Masic N not set free as NH;t by 50%
KOH .. ...

004?8

835

00569

11 S7

Amino N of bases

0.0412

804

0 0393

799

Non-amino N of bases

00188

367

0033?

67S

N in filtrate from liases

0.1305

25.47

0 1390

28.25

Amino N in filtrate from bases
Non-amino N in filtrate from bases.
Total N regained •

i
0.4996

97.50

0.1272
0.0118
0.5032

25.85
2.40
102.28

Solution lost at this point.
2 Result calculated from the digestion of one-half the precipitate.

8. Hempstead silt loam. Duplicate 250 gram samples were
hydrolyzed for 48 hours. The "humin" was washed and the de-
termination carried out as described under mineral soils. The only
explanation which occurs for the higher ammonia nitrogen of one
hydrolysate is that Sample I must have been heated more strongly
during hydrolysis. From unpublished experiments conducted at
this Station it has been found that when a pure protein is hydro-
lyzed under vigorous boiling, a. greater amount of ammonia nitro-
g~en is invariably obtained than when hydrolyzed under slow boil-
ing conditions. This observation was made subsequently to my
own, so that the importance of the rate of boiling was not known
at the time of doing my work.

Only 25 grams of phosphotungstic acid were added for the
precipitation of the bases. After standing the solution gave a
further precipitate on addition of more phosphotungstic acid. It
was concluded, however, that the organic bases were entirely pre-
cipitated, for on washing the precipitate with the phosphotungstic
acid wash water no additional precipitate formed. The nitrogen
determination gave 0.0011 gram retained by the barium phospho-
tungstate. The solution containing the nitrate from the bases was
made to 300 cc. volume.

46

The experimental data giving the grams of nitrogen and pel
cent of total nitrogen will be found in Table XI.

Table XL — Nitrogen distribution in Hemp stead silt loam.

| Grams nitrogen | Per cent of total nitrogen
| I~ II " I II | Av.

Total "NT

0 3416

03485

01042

0.0989

30.50

28.38

29.44

Insoluble humin N in soil
Humin N pptd. by
Ca(OH)2

0.0981
0.0164

0.0920
i

28.72
4.80

26.40

27.56
4.80

Basic N

00389

11 39

Basic N set free as NH3
by 50% KOH

00078

2.28

2.28

Basic N not set free as
NH3 by 50% KOH

00311

9 10

9.10

00254

744

00135

395

N in filtrate from bases

00960

28.10

Amino N in filtrate from
bases

0.0839

24.56

24.56

Non-amino N in filtrate
from bases

00121

3.54

3.54

Total N regained ~.

0.3536

103.51

101.29

1 Solution lost at this point.

9. Prairie-covered loess. In Sample I, 250 grams were hy-
drolyzed in the presence of 250 cc. of constant boiling hydrochloric
acid for 48 hours. The hydrolysate on cooling was diluted to 1000
cc. and a 500 cc. portion was syphoned off and used for the an-
alysis. In Sample II, two 125 gram samples were hydrolyzed in
the same manner as outlined under Fargo silt loam (500 cc. con-
stant boiling hydrochloric acid to 125 grams soil). The dilution
and aliquot used for analysis were also the same. The nitrogen
retained by the barium phosphotungstate amounted to 0.0020 gram
in Sample I, and 0.0072 gram in Sample II. The solution con-
taining the bases was diluted to 100 cc. in both samples.

The experimental data showing grams of nitrogen found and
per cent of total nitrogen are given in Table XII.

Table XII. — Nitrogen distribution in prairie-covered loess.

| Grams nitrogen | Per cent of total nitrogen

!

I

II

i

II

Av.

Total N

03907

04012

Ammonia N • •

0 1223

0 1194

31 30

29.76

30.53

Insoluble humin N in soil
Humin N pptd. by
Ca(OH)2

0.0922
00198

0.1007
0.0213

23.60
5.07

25.10
5.31

24.35
5.19

Basic N

0.0444

0.0562

11.36

14.01

12.68

Basic N set free as NH3
by 50% KOH
Basic N not set free as
NH3 by 50% KOH
Amino N of bases

0.0100

0.0344
0.0271

0.0132

0.0430
0.0322

2.56

8.80
6.93

3.29

10.72
8.03

2.92

9.76
7.48

Non-amino N of bases...
N in filtrate from bases..
Amino N in filtrate from
bases

0.0173
0.1152

0 1044

0.0240
0.1128

0 1033

4.43
29.49

26.72

5.98
28.12

25.75

5.20
28.80

26.24

Non-amino N in filtrate
from bases

0.0108

0.0095

2.76

2.37

2.56

Total N regained

0.3939

0.4104

100.82

102.30

101.56

47

10. Forest-covered loess. In Sample 1, 300 grams of soil
were hydrolyzed and diluted in the same manner as Sample I of
prairie-covered loess. In Sample II, 300 grams of soil were hy-
drolyzed under the same conditions as Sample II of prairie-covered
loess. The nitrogen retained by the barium phosphotungstate was
0.0024 gram in Sample I, and 0.0023 gram in Sample II. The
solution containing the nitrogen of the bases was Diluted to 100
cc. in both samples and the solutions containing the total nitrogen
of the filtrates were made to a volume of 300 cc.

It is observed that the volume of acid used in the hydrolysis
had little effect on the proportion of the different fractions. The
only observed difference is in the insoluble humin nitrogen retained
by the soil residue, and this is slightly larger in Sample II, which
was hydrolyzed in the presence of the greatest excess of acid. In
connection with this it must also be noted that there is a somewhat
larger quantity of nitrogen in solution in Sample II than in Sample
I. Much the same results are shown with the prairie-covered loess.
All increases or decreases in the various fractions due to the greater
excess of acid may well be considered to be within the experimental
error.

The experimental data showing grams of nitrogen found and
per cent of total nitrogen are given in Table XIII.

Table XIII. — Nitrogen distribution in forest-covered loess.

| Grams nitrogen | Per cent of total nitrogen

] I

II

I

II [ Av.

Total N

0.2224
0.0646
0.0574

0.0140
0.0316

0.00/4

0.0242
0.0175
0.0141
0.0621

0.0585

0.0036
0.2297

0.2362
0.0669
0.0662

0.00801
0.0325

0.0096

0.0229
0.0172
0.0153
0.0626

0.0568
0.0058

Ammonia N

29.05
25.81

6.29
14.21

3.33

10.88
7.87
6.34
27.92

26.30

1.62
103.28

28.32
28.03

3.30
13.76

4.06

9.70

7.28
6.48
26.50

24.05
2.45

U/O.UO2

28.69
26.92

4.84
13.98

3.69

10.29
7.57
, 6.41
27.21

25.17

2.04
101.64

Insoluble humin N in soil
Humin N pptd. by
Ca(OH)2

Basic N

Basic N set free as NH3
by 50% KOH

Basic N not set free as
NH3 by 50% KOH

Amino N of bases

Non-amino N of bases. . . .
N in filtrate from bases..
Amino N in filtrate from
bases

Non-amino N in filtrate
from bases

Total N regained

By difference

Calculated.

1 1 . Sphagnum-covered peat hydrolyzed in the presence of nine
times its weight of a mineral subsoil. Duplicate 10 gram samples
were hydrolyzed in the presence of 90 grams subsoil with constant
boiling hydrochloric acid for 48 hours. The hydrolysate was con-
centrated as much as possible and ammonia nitrogen determined
on the entire mixture by distillation with an excess of calcium hy-
droxide for one hour.*

*This was the first attempt to determine the nitrogen fractions in the
presence of a mineral soil. For certain reasons later analyses have already
been reported in this paper. The analyses as reported in this paper are
by no means in chronological order, which may explain seeming1 inconsist-
encies.

48

The residue remaining after hydrolysis was washed free of
chlorides on ordinary funnels. It was then digested in three
Kjeldahl flasks using a total of 30 grams potassium sulfate and
360 cc. sulfuric acid. After digestion the material was transferred to
a 1000 cc. flask, made to volume, and the nitrogen content deter-
mined as described under sphagnum-covered peat. It will be ob-
served that the total "humin" nitrogen is determined here instead
of being reported in two separate fractions as was done in the
case of all other analyses containing mineral soil.

The combined filtrate and washings from the "humin" precipi-
tate were concentrated in the usual manner and made to 200 cc.
volume. Duplicate Kjeldahl determinations were made on 25 cc.
portions of this solution and the results listed, as total nitrogen-
in-the-filtrate-from-humin. The remaining 150 cc. portion was
used for the precipitation of the bases, the subsequent procedure
being completed as described in the "fibrin from blood" analysis.
The barium phosphotungstate precipitate retained 0.0016 gram of
nitrogen in Sample I, and 0.0053 gram in Sample II.

In the second sample the combined filtrate and washings
from the phosphotungstic acid precipitate of the bases were brought
to near the neutral point with 50 per cent sodium hydroxide and a
small amount of acetic acid added at once. After the addition of
the acid it seemed possible that the neutral point had not been
reached the first time, so more sodium hydroxide was added until
the neutral point was just passed and acetic acid again added. The
resulting solution was placed in a double-necked distilling flask
and an attempt made to concentrate the solution under diminished
pressure. Frothing was so intense that it was impossible to effect
any concentration. When alcohol was added the distillation con-
tinued quietly as long as any alcohol was present, but after that the
frothing continued. The mixture behaved like a concentrated soap
solution. The solution was finally concentrated in an evaporating
dish over a water-bath and the resulting solution made to 300 cc.
volume. On shaking the solution frothed very badly.

The experimental data showing the grams of nitrogen found
and the per cent of total nitrogen are given in Table XIV.

A comparison between these analyses and those of the peat
hydrolyzed alone is made in Table XV, the data of the peat hy-
drolyzed alone being taken from Table VI, and recalculated from
a 15 gram basis to a 10 gram basis.

49

Table XI]7. — Nitrogen distribution in sphagnum-covered peat
livdrolyscd in the presence of nine times its weight of a mineral subsoil.

Grams nitrogen
I II"

I Per cent of total nitrogen
" I | "II | Av.

Total N

02441

02441

Ammon'a N

0.0625

0.0621

25.60

25.44

25.52

Humin N

0.0658

0.07561

26.96

j

26.96

Basic N • •

00268

0 0374'

1098

]

10.98

Basic N set free as NH3
by 50% KOH
Basic N not set free as
NH3 by 50% KOH

0.0095
00173

0.0105

3.89
709

4.30

4.09

\mino N of bases • •

00177

00179

725

733

7.29

00091

373

N in filtrate from bases..
Amino N in nitrate from
bases • •

0.0954
0.0836

0.0948
0.0815

39.08
34.25

38.84
33.39

38.96
33.82

Non-amino N in filtrate
from bases
Total N regained

0.0118
0.2505

0.0133
^_..._:

4.83
102.62

5.45

5.14
102.42

1 These results are evidently incorrect and in the "average" column the
figures from the first column only are used.

Table XV. — Comparative analyses of sphagnum- cohered peat
hydrolyzed alone and in the presence of nine times its zveight of a min-
eral subsoil.

Grams Nitrogen

Apparent
distribution

of Nin
subsoil in
per cent of
total
nitrogen

Peat

Peat
Subsoil1

fncrease ( + )
or
Decrease(— )

Total N

0.2000
0.0466
0.0527
0.0195

0.0060

0.0135
0.0105
0.0090
0.0860

0.0776

0.0084
0.2048

0.2441
0.0623
0.0658
0.0268

0.0100

0.0168
0.0178
0.0090
0.0951

0.0825

0.0126
0.2500

+0.0441
-J-0.0157
+0.0131
+0.0073

+0.0040

+0.0033
+0.0073

\mrr>.onia N . .

35.60
29.71
16.55

9.07

7.48
16.55

Humin N

Basic N

Basic N set free as NH3 by
50% KOH

Basic N not set free as NH3
by 50% KOH
Amino N of bases
\on-amino N of bases.
N in filtrate from bases....
AmJ.no N in filtrate from
bases • •

+0.0091
+0.0049

+0.0042
+0.0452

20.64
11.11

9.52
102.50

Non-amino N in filtrate from
bases

Total N regained ..........

1 Ninety gm. of subsoil contained 0.0441 gm. of soil nitrogen.

12. Sphagnum-covered peat hydrolyzed in the presence of
metallic tin. This peat was hydrolyzed in the presence of a reduc-
ing agent because it was thought that the amount of "humin" nit-
rogen would be reduced, for according to Samuely (1902) the
formation of this dark colored product is due to an oxidative proc-
ess. Hlasiwetz and Habermann (1871 and 1873) hydrolyzed pro-
tein with hydrochloric acid in the presence of stannous chloride in
order that the solution should remain colorless. Cohn (1896-97

50

and 1898-99) believed that the use of »a reducing agent was not
essential, but according to Otori (1904) this is a mistake.

It is perhaps significant that the "humin" nitrogen was reduced
to 3.90 per cent by the presence of a reducing solution. It is not
known whether there was sufficient tin present to maintain a reduc-
ing solution throughout the hydrolysis inasmuch as the ferric iron
in the peat would have an oxidizing action on the stannous salt.
The sample was known to contain iron but the amount was not
determined.

Duplicate 15 gram samples were hydrolyzed with 100 cc. hydro-
chloric acid (sp. gr. 1.115) for 48 hours in the presence of five and
ten grams of tin respectively. The tin was first partially dissolved
in the acid before the samples of peat were added. The deter-
mination of "humin" nitrogen was carried out as directed under
sphagnum-covered peat excepting that the digested material was
diluted to 500 cc. instead of to 1 liter. The nitrogen retained by
the barium phosphotungstate was 0.0023 gram in Sample I; and
0.0047 gram in Sample II. The solution containing the filtrate from
the bases was diluted to a volume of 300 cc. The experimental data
giving the grams of nitrogen found and the per cent of total
nitrogen are given in Table XVI.

Table XV L — Nitrogen distribution in
hydrolyzed in the presence of metallic tin.

sphagnum- covered peat

Grams nitrogen | Per cent of total nitrogen

1 I .
5 gm. tin

II

10 gm. tin

I

II

Av.

Total N

0.3000
0.0633
0.0699
0.0320

0.0099

0.0221
0.0193
0.0127
0.1 4041

0.1297

0.0107
0.3056

0.3000
0.0578
0.0650
0.0417

0.0100

0.0317
0.0219
0.0198
0.1380

0.1307

0.0073
0.3025

Ammonia N
Humin N

21.10
23.30
10.67

3.30

7.37
6.43
4.23
46.80

43.23

3.56
101.86

19.27
21.67
13.90

3.33

10.57
7.30
6.60
46.00

43.57

2.43
100.83

20.18
22.48
12.28

3.31

8.97
6.86
5.41
46.40

43.40

2.99
101.34

Basic N ....

Basic N set free as NH3
by 50% KOH
Basic N not set free as
NH3 by 50% KOH....
Amino N of bases ' • •

Non-amino N of bases. . . .
N in filtrate from bases..
Amino N in filtrate from
bases

Non-amino N in filtrate
from bases

Total N regained

1 This result is from a single determination of nitrogen.

13. Analysis of a 1 per cent hydrochloric acid extract of sphag-
num-covered peat and (in part) of calcareous black grass-peat.
Acid extraction was made of the two peats in direct contact with
1 per cent hydrochloric acid. For the extraction 125 gram por-
tions were placed in 2.5 liter acid bottles and two liters of 1 per
cent acid added. In the case of calcareous black grass-peat, how-
ever, the calculated amount of hydrochloric acid necessary to neu-
tralize the calcium oxide was first added and then sufficient dilute
acid to make two liters of a 1 per cent solution. Five hundred

51

grams were taken in the case of sphagnum-covered peat, while 750
grams were taken in the case of calcareous black grass-peat. The
bottles were shaken at intervals during five days and then the con-
tents filtered through two thicknesses of a good grade of cheese
cloth and squeezed in the hands. The resulting solution was then j
filtered through two thicknesses of filter paper on a Buchner funnel.

This extract was colored in each case but the calcareous black
grass-peat gave a deeper straw colored solution than did the sphag-
num-covered peat. This was probably due to the presence of a
larger amount of iron in the one case than in the other. The cal-
careous black grass-peat is known to contain a very considerable
quantity of iron. The wash water in both instances was also straw
colored.

It has been shown by a number of investigators, e. g., Jodidi
(1909), Kelley and Thompson (1914), and Gortner (1916 a) 'that
considerable amounts of nitrogen are dissolved from certain soils
by this preliminary treatment. Xhe acid solution thus obtained
should contain the ammonia, acid amides, amines, amino acids, and
all other organic nitrogenous compounds soluble in water or very
dilute acid. The 1 per cent hydrochloric acid extracted 8.57 per cent
of the total nitrogen from sphagnum-covered peat, and 5.09 per
cent from the calcareous black grass-peat.

Duplicate nitrogen determinations were made on 250 cc. por-
tions of the acid extract and from these results the total nitrogen
in the bulk solution determined. The 5500 cc. solution from sphag-
num-covered peat, containing 0.6468 gram nitrogen, and the 5000
cc. from the calcareous black grass-peat, containing 0.4690 grain
nitrogen, were used for analysis. These solutions were concen-
trated under reduced pressure to about 200 cc. and then hydrolyzed
for 48 hours, after first adding 75 cc. concentrated hydrochloric
acid to the solution from sphagnum-covered peat, and 100 cc. to
the solution from the calcareous black grass-peat. During evap-
oration under reduced pressure considerable hydrolysis took place
for the solutions turned dark brown in color. During hydrolysis
of calcareous black grass-peat silicic acid separated in the con-
denser.*

The analysis of sphagnum-covered peat shows that over 65 per
cent of the nitrogen is in the form of ammonia. Potter and Snyder
(1915 a) have shown that a very small amount of the nitrogen
in the 1 per cent hydrochloric acid extract of soils exists in the soil
as ammonia nitrogen. It seemed probable that if an extract of the
peat contained so much ammonia nitrogen after hydrolysis, the air
dry peat must contain an appreciable amount in the ordinary con-
dition. The ammonia nitrogen was determined directly on a 5 gram
sample of the air dry material. An excess of calcium hydroxide
solution was added and the mixture distilled under reduced pressure
for forty-five minutes. It was found that 5.40 per cent of the total

*This was also true with all of the mineral soils studied, and is probably
due to the presence of inorganic fluorides.

52

nitrogen of the soil existed in the form of ammonia nitrogen.*

The precipitates containing the "humin" nitrogen were
washed by decantation until practically all the dissolved nitrogen
was removed. After digestion the material was diluted to 500 cc.
and 250 cc. portions used for distillation. Before concentration the
filtrate from the "humin" precipitate of sphagnum-covered peat
was reddish in color and after concentration this color changed to a
cherry red. Twenty-five grams of phosphotungstic acid was used
for precipitation of the bases in sphagnum-covered peat. The
barmm phosphotungstate precipitate retained 0.0022 gram nitrogen.
The solution containing the basic nitrogen was diluted to 50 cc.
and the one containing total nitrogen-in-filtrate-from-bases to 250
cc.

The experimental data showing the grams of nitrogen found
and per cent of total nitrogen are given in Table XVII.

Table XVII. — Nitrogen distribution of a 1 per cent h\drochloric
acid extract of sphagnum-covered peat and (in part) of calcareous
black grass-peat.

Sphagnum-covered
peat

Calcareous
black grass-peat

Grams
nitrogen

Per cent
of total
nitrogen

Grams
nitrogen

Per cent
of total
nitrogen

Total N

06468

04680

Ammonia. N • •

04230

. 55 40

03013

64 38

Humin N

00657

10 16

00582

1244

Basic N

00389

601

i

Basic N set free as NH3 by 50%
KOH

00181

280

Basic N not set free as NH3 by 50%
KOH

00208

321

Amino N of bases

0.0266

4.11

Non-amino N of bases

0.0123

1 90

N in filtrate from bases

0.1335

20.64

Amino N in filtrate from bases

0.1107

17.11

Non-amino N in filtrate from bases.

0.0228

3.53

Total N regained

0.6611

102.21

1 Distribution of remaining' nitrogen not determined.

14. Analysis of a portion of sphagnum-covered peat soluble
in 4 per cent sodium hydroxide and precipitated by hydrochloric
acid and (in part) of a similar solution from a calcareous black
grass-peat. The organic material soluble in 4 per cent sodium
hydroxide was next extracted from new portions of the two differ-
ent peats. Twelve 5 gram portions were leached with 1 per cent
of hydrochloric acid to the absence of calcium and the excess of
acid removed by washing with distilled water, until the filtrate
indicated only a faint trace of free acid when tested with Squibb's
litmus paper. After leaching and washing, each 5 gram portion
was washed into tall glass-stoppered cylinders of 500 cc. .capacity

*This was further indicated by greenhouse experiments. The peat was
treated with calcium carbonate at the rate of 4000 pounds per acre and planted
to barley. The plants made a very rapid growth during the early stages of
development and finally lodged. Next to this was a plot of calcareous black
grass-peat which contained only 0.88 per cent of its total nitrogen in the
form of ammonia nitrogen. When limed and sown to barley, it did not show
any abnormal growth.

53

with 4 per cent sodium hydroxide and filled to the mark. These
were thoroughly shaken and placed on their sides, thus allowing
the peat to settle on the sides of the cylinder, thereby exposing a
very large surface to the action of the hydroxide. The shaking was
repeated at intervals for nine days. The cylinders were then thor-
oughly shaken, placed in a vertical position and allowed to settle
for four days before the supernatant liquid was syphoned off and
filtered. The samples of calcareous black grass-peat were almost
completely dissolved by the hydroxide solution.

These filtered solutions were neutralized with hydrochloric
acid (solution tested faintly acid) when a brown flocculent precipi-
tate separated. This was allowed to settle for several hours and
the cider colored solution syphoned off. The brownish black pre-
cipitates were filtered and after draining over night were thoroughly
mixed with a large volume of water and again filtered and drained.
The resulting precipitates were hydrolyzed with 200 cc. of hydro-
chloric acid for 48 hours. This amount of concentrated acid was
added and the flask brought to boiling until hydrogen chloride
fumes were evolved showing the presence of constant boiling acid.
Silicic acid separated on the condenser during the hydrolysis of
calcareous black grass-peat. Even after the hydrolysis there still
remained some small lumps of the "humus" precipitate.

The entire hydrolysate was used for the ammonia determina-
tion. After this determination the "humin" precipitate was thor-
oughly ground in a mortar to insure complete disintegration, al-
though this seemed hardly necessary as the solid was already in
a fairly fine state of division. This precipitate was washed in the
usual manner by decantation, the filtrate concentrated by evapora-
tion and made to 250 cc. volume. Duplicate portions of 25 cc.
were used for the determination of total nitrogen in the solution
and the result listed as total nitrogen-in-the-filtrate-from-humin.
The remaining 200 cc. portion was used for precipitation of the
bases and subsequent analysis.

The high content of carbonaceous organic matter made the
"humin" precipitate very difficult to digest. The sulfuric acid
required was 120 cc. and the digestion extended over 10 days be-
fore complete decoloration was effected. Of course precautions
were taken to prevent the absorption of ammonia from outside
sources, The material was diluted to 500 cc. and 250 cc. portions
used in the distillation. The basic nitrogen was precipitated with
25 grams of phosphotungstic acid. The nitrogen retained by the
barium phosphotungstate was 0.0043 gram. The solution contain-
ing the basic nitrogen was made to 50 cc. volume, and that con-
taining the total nitrogcn-in-the-filtrate-from-the-bases was made
to a volume of 250 cc.

The experimental data showing the grams of nitrogen and per
cent of total nitrogen are given in Table XVIII.

54

Table XVIII. — Nitrogen distribution in that portion of a sphag-
num-covered peat soluble in 4 per cent sodium hydroxide and precipi-
tated by hydrochloric acid and (in part) of a similar solution from a
calcareous black grass-peat.

Sphagnum-covered
peat

Calcareous
black grass-peat

Grams
nitrogen

Per cent
of total
nitrogen

Grams
nitrogen

Per cent
of total
nitrogen

Total N

0.2860
0.0464
0.0950
0.0309

0.0070

0.0239
0.0174
0.0135
0.1175
0.0965
0.0210
0.2898

0.6344
0.0781

0.2093

i

Ammonia N . .

16.22
33.22
10.80

2.45

8.35
6.08
4.72
41.08
33.74
7.34
101.32

12.31
32.99

Humin N

Basic N

Basic N set free as NH3 by 50%
KOH

Basic N not set free as NH3 by 50%
KOH

Amino N of bases

Non-amino N of bases

N in filtrate from bases

Amino N in filtrate from bases

Non-amino N in filtrate from bases.
Total N regained

1 Distribution of the remaining- nitrogen not determined.

15. Ana'ysis of a portion of sphagnum-covered peat soluble
in 4 per cent sodium hydroxide and not precipitated by hydro-
chloric acid and (in part) of a similar solution from a calcareous
black grass-peat. The filtrate remaining from the brownish black
precipitate formed by acidifying the sodium hydroxide extracts
of the soil with hydrochloric acid (cf. section 13) were concentrated
in the usual manner to about 700 cc. when a heavy precipitate of
sodium chloride separated. On standing over night there also
separated a heavy flocculent brown precipitate. This may have
been due to the salting out effect of the sodium chloride on some
of the organic substances in the solution. The solution was sat-
urated with hydrogen chloride in the cold and the mixture then
divided into two portions and hydrolyzed for 48 hours. The cold
material after hydrolysis was united and filtered through glass
wool and the precipitate washed with concentrated hydrochloric
acid. The filtrate was allowed to stand in a tall soil beaker when
more salt separated, due to the increased concentration of the hydro-
chloric acid. The salt that separated was freed from the mother
liquid by packing in a centrifuge and washing with acid a number
of times. The salt washed as free of the solution as possible was
dried on the steam bath. It was nearly white in color. The glass
wool was dried and ground with the salt. After being sampled, 15
gram portions were used for Kjeldahl determinations. The results
were listed as nitrogen-retained-by-the-salt.

The combined nitrates were concentrated and analyzed. After
the digestion of the humin nitrogen, the material in the Kjeldahl
flask was made to 500 cc. volume and the distillations carried out
on 250 cc. portions. The nitrate and washings from the humin
were acidified and evaporated under diminished pressure to less
than 200 cc. and then made to 250 cc. volume. Duplicate nitrogen
determinations were made on 25 cc. portions of the solution and

55

the results listed as total nitrogen-in-the-nltrate-from-humin.

The total nitrogen in the hydrolysate was determined by add-
ing the nitrogen obtained as ammonia, and humin (in salt and that
precipitated by calcium hydroxide) to the total nitrogen-in-the-
filtrate-from-humin.

Fifteen grams of phosphotungstic acid were used for the pre-
cipitation of the bases. The barium phosphotungstate from the
sphagnum-covered peat retained 0.0055 gram nitrogen. The solu-
tion containing- the basic nitrogen was made to. 50 cc. vUim" '•»* '
that containing the total nitrogen-in-the-filtrate-from-the-bases was
made to 250 cc. volume.

The experimental data showing the grams of nitrogen and per
cent of total nitrogen are given in Table XIX.

Table XIX. — Nitrogen distribution in that portion of a sphagnum-
cohered peat soluble in 4 per cent sodium hydroxide and not precipi-
tated by hydrochloric acid and (in part) of a similar solution from a
calcareous black grass-peat.

Sphagnum-covered
peat

Cateareous
black grass-peat

Grams
nitrogen

Per cent
of total
nitrogen

Grams
nitrogen

Per cent
of totat
nitrogen

Total N

03736

06204

Ammonia N ; . . .

0.0993

26.58

0.2003

32.29

Humin N pptd. by Ca(OH)2
Humin W retained in NaCl

0.0335
00102

8.97
273

0.0652
0 0169

10.51
2.72

Basic N

00324

867

2

Basic N set free as NH3 by 50%
KOH

00070

1.87

Basic N not set free as NH3 by 50%
KOH

00254

680

\mino N of bases . .

0.0160

4.28

Non-amino N of bases

00164

4.39

"V in filtrate from bases

0 20061

5369

•\mino N in filtrate from bases

0 1721

4607

\on-amino N in filtrate from bases..

0.0285

7.62

Total N regained

0.3760

100.64

1 Calculated from one determination. Duplicate lost in digestion.
8 Distribution of the remaining nitrogen not determined.

16. "Jodidi numbers." These determinations were carried
out as directed previously on Fargo clay loam, Fargo silt loam,
Hempstead silt loam, prairie-covered loess, and forest-covered
loess.

The resulting data in grams and in per cent of total nitrogen
are shown in Tables XX, XXI, XXII, XXIII, and XXIV. The
figures for similar fracions from the complete Van Slyke analyses
are added for comparison. These results will be discussed later.

56

Table XX. — "fodidi numbers" determined on 100 cc. of hydrol-
ysate of Fargo clay loam, together with a comparison of similar frac-
tions taken from the Van Slyke analysis.

Grams nitrogen

Per cent of total
nitrogen

Average
data of
Van Slyke
analysis

TablfVlIl

Total N
Ammonia N
Residue from above acidi-
fied with HC1 and bases
pptd. direct, "Basic N".
N in filtrate from "bases"
Total N regained

I

0.0484
0.0160

0.0033
0.0315
0.0508

II

0.0478
0.0160

0.0055
0.0277
0.0492

I

II

Av.

33.06

6.82
65.08
104.96

33.47

11.51
57.95
102.93

33.26

9.16

61.52
103.94

24.00

9.58
32.71

Table XXI. — "Jodidi numbers" determined on 200 cc. of hydrol-
ysate of Fargo silt loam, together with a comparison of similar frac-
tions taken from the Van Slyke analysis.

Grams
nitrogen

Per cent of
total
nitrogen

Average data
of Van Slyke
analysis.
Table IX.

Total N

0 1424

Ammonia N

0.0487

34.20

26.56

Residue from above acidified
with HC1 and bases pptd. di-
rect, "Basic N" ; . . .
N in filtrate from "bases"
Total N regained

0.0193
0.0777
0.1457

13.55

54.56
102.31

12.11
35.14

Table XXII. — "Jodidi numbers" determined on 100 cc. of hydrol-
ysate of Hempstead silt loam, together with a comparison of similar
fractions taken from the Van Slyke analysis.

Average

Grams nitrogen

Per cent of total
nitrogen

Van Slyke
ana ysis

s

Table XI

Total N

I
00487

II 1

0.0513

I

11

Av.

Ammonia N

00207

00197

4251

3840

4045

2944

Residue from above acidi-
fied with HC1 and bases
pptd. direct, "Basic N".
N in filtrate from "bases"
Total N regained

0.0068
0.0243
0.0518

0.0067
0.0255
0.0519

13.96
49.90
106.36

13.06
49.71
101.17

13.51
49.81

103.77

11.39
28.10

57

Table XXIII. — "Jodidi numbers" determined on 200 cc. of hydrol-
ysate of prairie-covered loess, together with a comparison of similar
fractions taken from the Van Slyke analysis.

Grams
nitrogen

Per cent of
total nitrogen

Average data
of Van Slyke
analysis.
Table XII.

Total N

0.0601

Ammonia N . .

00244

4060

3053

Residue from above acidified
with HC1 and bases pptd. di-
rect "Basic N"

0.00361

5.99

12.68

N in filtrate from "bases"

0.0321

53.41

28.80

Total N regained

100.002

1 By difference. 2 Calculated.

Table XXIV. — "Jodidi numbers" determined on 200 cc. of hydrol-
ysate of forest-covered loess, together with a comparison of similar
fractions taken from the Van Slyke analysis.

Grams
nitrogen

Per cent
of total
nitrogen

Average data
of Van Slyke
analy sis.
Table XIII.

Total N

0.0340

Ammonia N . . . . . ...

0.0130

3824

2869

Residue from above acidified
with HC1 and bases pptd. di-
rect, "Basic N"

0.0059

1735

1398

N in filtrate from "bases"

00175

51 47

2721

Total N regained

0.0364

107.06

17. Summary Tables. Certain of the preceding analyses have
been summarized in Tables XXV, XXVI. In Table XXV are
shown the amounts and percentages of soil dissolved by the acid
during hydrolysis as well as the amount of nitrogen and percentage
of the total nitrogen dissolved. Table XXVI summarizes average
nitrogen distribution of Tables V, VI, VII, VIII, IX, X, XI,
XII, XIII, XIV, XVI, XVII, XVIII, and XIX.

Table XXV. — Percentages of soil and of soil nitrogen dissolved
by hydrolysuig the different soil types.

Soil type

&>
a

a

C/3

Grams
soil
taken
(dry basis)

Grams
soil
dis-
solved

Per cent
soil
dis-
solved

Grams
nitrogen
in
soil

Grams
nitrogen
dis-
solved'

Per cent
of dis-
solved
nitrogen

Fargo clay loam

I

TT

240.2
2402

43.2
432

17.99

0.6005

0.4252

70.79

Kargo silt loam

T

222.8

54.8

24.60

1.8336

1.4237

77.65

Carrington silt loam . .
Hempstead silt loam .

Prairie-covered loess .
Forest-covered loess .

I
I
II
I
II
I
II

235.5
242.3
242.3
230.3
230.3
294.4
294.4

36.5
32.3
. 33.3
42.3
45.3
29.4
32.4

16.61
13.33
13.74
18.37
19.64
9.98
11.11

0.8738
0.6201

0.6933
0.3768

0.6191
0.4395
0.4508
0.5260
0.4991
0.2735
0.2511

70.91
70.87
72.69
75.91
72.02
72.19
66.65

aThe figures in this column were obtained by subtracting the "insoluble
humin nitrogen" remaining in the residual soil from the nitrogen figures ob-
tained by multiplying the original weight of soil taken (dry basis) by the
nitrogen content of the soil. These figures may or may not agree with the
figures obtained in kjeldahling a portion of the solution, due to experimental
errors, and perhaps to errors introduced in using a uniform factor (2.6) for
specific gravity. The figures in this column are free from any error of this
sort.

paadAOO

•jl opiums

*I

tUBOl .

xio}Suu.n!0 H

•UIBO|

•ureoi

PPV

in

•TJH A'q
.HO«X %t

•TOH

(! jou
in

°^3X

S ^*JU OS r-H

laaudg H X

paa^.voo -u i uuS «

HX

H.X

<u —

58

ON CM ^T O 00 ON

ro 10 ON -^ OO CvJ VO OO O ° ^ \O ^O
10 co >— i LO SO Ox t^ rj- (M 00 CM u-j 10
CM

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CM co VO C^i co CXJ t~v! -^ LO ^j CM

CM " CM — i

CM CM

ON T-H LO O LO co CM •— ' CM ON vC

Tf ^ ""> *- ' -3- T-H TJ- 00 GO ON ^J- ;

ON * * t< co co

l-H CM T-H

\OT-H O
T-H o CO
OvO CM

r_<r_OT»-T-HCOT-i
CM T-H ON vO T-H LO CM
'T-'orx'coCM
CM -H O

OO COCO T^

T-H Tj-CM co

O**CMCM co
CM CM T-.

T-H o O O\ Tf
CO
T-H
O

CM VO CO ON ON ON ON VO CM ^ CM

LO ON ON O 00 CM vO ON 00 T-I ^t

LO'**VO'O ^ vbt^coco'coLOCM

CM CM ^H co co O

oo co oc

CM cot^ ON

'**VO'ON ^
CM

vo r^ o

CNlTfO

''Jfl' '

^
pq

59

18. An attempt to isolate pure proteins from a soil. From
all the positive evidence it would seem that a very considerable
portion of the nitrogen in the soil exists in other forms than pro-
tein. For example. Potter and Snyder (1916) found an average of
20.46 per cent of the alkali soluble nitrogen of 'a soil to be non-
protein in nature. Bacteria and fungus spores all contain chitiri
which on hydrolysis yields glucosamine which will give amino
(-NH,) nitrogen of non-protein origin.

The average C/N ratio that exists in this series of six mineral
soils studied has been shown by Gortner (1916 a) to be 12.23. The
average nitrogen content of the six soils is 0.355 per cent which
would indicate 2.22 per cent protein (NX6.25) if all of the nitrogen
existed in this form. However, the total organic matter in these
soils averaged 7.48 per cent (carbonX 1.724), showing that less
than 30 per cent of the soil organic matter could be of protein na-
ture. This is shown equally well by a comparison of the C/N
ratios. The average analyses of sixteen plant proteins as recorded
by Mathews (1915) gives carbon 52.08 per cent and nitrogen 17.73
per cent. The average C/N in these vegetable proteins is 2.94.
The high C/N ratio in soils indicates that the organic matter does
not consist essentially of protein material (cf. however, Gortner
1917, when a ratio of 3.06 was found indicating that the organic
matter in this instance was essentially protein).

In view of the desirability of demonstrating the presence or
absence of proteins in soils an attempt was made to isolate from
a soil either alcohol soluble or salt soluble proteins. A 6 inch
bulk sample of Fargo silt loam from Morristown, Rice County,
containing 0.397 per cent of nitrogen in the air dry soil, was used.

a. Extraction with 70 per cent ethyl alcohol. The soil was
first leached with 1 per cent hydrochloric acid to the absence of
calcium and washed with distilled water until practically all chlo-
rfdes were removed. For this purpose sixty-one 100 gram por-
tions were taken. It required about 150 liters of acid to remove
all traces of calcium.

After leaching, the soil was allowed to air dry in the green-
house and there remained 5700 grams air dry soil. Five hundred
gram portions were placed in twelve 2.5 liter acid bottles and ex-
tracted successively with 70 per cent ethyl alcohol. A fresh por-
tion of alcohol was added to the first bottle of the series, shaken,
allowed to stand over night, syphoned off, and placed in the bottle
next ahead in the series. This was continued until six successive
portions of fresh alcohol had been added. The alcohol was ab-
sorbed by the dry soil to such an extent that a 500 gc. portion of
fresh alcohol had to be added to the bottles towards the end of
the series. Almost 15 liters of alcohol were used, but only a little
over 10 liters were regained at the end, since so much was retained
by the soil.

The combined extracts were filtered until practically free of
clay and then concentrated under diminished pressure at a tem-
perature of 55° C. The extracts were in all cases straw colored.

60

A heavy yellow precipitate separated on concentration but dis-
solved very largely in the fresh portions of the alcohol extract
The greater portion of water was finally removed by evaporating
four times with 93 per cent alcohol. More water, however, could
have been removed by evaporating with absolute alcohol. The
trace of clay, remaining after concentration, was filtered off on a
dry filter paper and the solution diluted to 500 cc. with 93 per
cent alcohol. The total nitrogen was determined on duplicate 25
cc. portions of the solution by the Kjeldahl method. The results
gave 0.1320 gram nitrogen in the total alcohol extract.

Tests were made on portions of this alcohol extract for the
presence of proteins or protein-like substances. Precipitates were
obtained with phosphotungstic acid and lead acetate. A heavy
yellow precipitate formed on dilution with water. This yellow
precipitate was readily soluble in sodium hydroxide (suggests
presence of acids or phenols), and in concentrated hydrochloric
acid. The biuret test and Liebermann's reaction both gave nega-
tive tests, and Millon's reaction gave an extremely faint test (mere
trace).

One 25 cc. portion of the alcohol solution was diluted with
water and extracted four successive times with chloroform, fol-
lowed by a single extraction with ether. The chloroform and ether
extracts were combined and evaporated to dryness in a platinum
dish on the steam bath and later in a hot air oven at 102° C. and
the residue weighed. The results indicated the presence of 0.3108
gram of organic matter, making a total of 6.2160 grams in the en-
tire alcohol extract.

After the chloroform-ether extraction there remained a dark
brown granular insoluble substance. This was filtered off on a 5
cm. Buchner funnel and the filter and precipitate used for the
determination of nitrogen. The results indicated the presence of
0.0010 gram maximum protein nitrogen in this solution, leaving
0.1310 gram of non-protein nitrogen in the same extract.

The negative color tests and the small amount of nitrogen
indicate that no protein is present in this soil that is soluble in
alcohol.

Another 25 cc. portion of the alcohol extract was evaporated
to dryness on the water-bath in .1 platinum dish and subsequently
dried'in an oven at 96° C. with a short final heating at 103° C. The
organic matter amounted to 0.6126 gram, making a total content
of organic matter in the alcohol solution 12.2520 grams. From the
data given it was found that only 1.08 per cent of the organic mat-
ter in the 70 per cent alcohol ext -act of the soil was nitrogen.

b. Extraction with absolute alcohol. A 100 gram portion of
the unleached soil was extracted with absolute alcohol for 60
hours in a Soxhlet extraction apparatus. The extraction flask had
a capacity of 500 cc. and all the joints of the apparatus were ground
glass. Several pieces of broken porcelain were placed in the flask
to prevent bumping-, as the amount of dissolved organic matter
increased. An alundum extraction thimble was used to hold the

61

soil. The alcohol syphoning back became colorless several hours
before the extraction was stopped. At the end of the extraction
the solution in the flask was deep straw color.

The total alcohol extract was evaporated to small volume and
then the total nitrogen determined, which was found to be 0.0011
gram. This .was equivalent to 0.0660 gram of nitrogen in 6 kilo
of soil before leaching with acid. It will be recalled that 0.1320
gram nitrogen dissolved in the 70 per cent alcohol after first leach-
ing with 1 per cent hydrochloric acid.

This would indicate that many of the organic nitrogenous
compounds are in combination with the lime or other bases present
in the soil, and that this combination is broken up when leached
with 1 per cent hydrochloric acid.

c. Extraction with 10 per cent sodium chloride. After com-
pletion of the alcoholic extraction, the soil residue was dried in an
air o.ven at about 65° C. and placed in a 20 liter bottle. To this
was added 2000 cc. of 10 per cent sodium chloride solution for
each kilo of dry soil. The bottle was shaken repeatedly and allowed
to settle over night. The presence of an electrolyte caused the
clay to settle rapidly, so that a clear solution could be withdrawn.
After the soil had been in contact with the salt solution 20 hours,
two 100 cc. portions were withdrawn and analyzed for nitrogen.
The results showed that 0.1350 gram nitrogen was contained in .5
liters of the supernatant liquid. After shaking and standing for
three days duplicate determinations were again made on 100 cc
portions with the result that 0.1600 gram nitrogen was present in
the corresponding solution. This indicates that the longer the soil
is in contact with the sodium chloride solution the greater the
amount of organic nitrogen extracted.

Two 500 cc. portions of the salt extract were used for precipi-
tation with phosphotungstic acid. After the addition of 45 cc. of
concentrated hydrochloric acid, the phosphotungstic acid was
added. The gelatinous precipitates formed were filtered on an
ordinary funnel and washed with the filtrate, and then used for
the determination of nitrogen by the usual method. The duplicates
averaged 0.0028 gram nitrogen, thus making the total nitrogen
precipitated by phosphotungstic acid 0.0280 gram in the 5 liters of
supernatant liquid. This shows that 17.50 per cent of the nitrogen
extracted from the residual soil was precipitated by phosphotung-
stic acid. This represented the maximum protein nitrogen in the
10 per cent sodium chloride solution.

62

III. DISCUSSION.

A. Changes in nitrogen distribution in a protein when hydro-
lyzed in the presence of a mineral soil. From a study of Table
III it is seen that the histidine nitrogen formed when fibrin is
hydrolyzed alone is 4.36 per cent and when hydrolyzed in the pres-
ence of cellulose it is 4.86 per cent of the total nitrogen. By re-
ferring to Table II where fibrin is hydrolyzed in the presence of
ignited subsoil we see the histidine nitrogen is entirely lacking a.nd
that the nitrogen precipitated by calcium hydroxide amounts to
4.81 per cent. This corresponds very closely to the amount of
histidine found in the other two cases. In other points the three
analyses agree within experimental error.

It appeared possible that the histidine nitrogen might have
been converted into the nitrogen fraction precipitated by calcium
hydroxide. It is a well known fact that histidine can be precipi-
tated by silver nitrate in slightly alkaline solutions. Since .there
are a large number of mineral constituents in the soil it may be
possible that the histidine could be precipitated by some of these
and thus be found with the calcium hydroxide precipitate.

With this idea in mind an analysis of histidine was made in
the presence of an ignited subsoil, only three fractions being de-
termined. One 0.5000 gram sample of histidine di-hydrochloride*
and 50 grams ignited subsoil were boiled in the presence of 100
cc. of hydrochloric acid (sp. gr. 1.18) for 48 hours. The solution
was diluted to 500 cc. in a graduated flask and two 200 cc. portions
syphoned ofif and analyzed. The solution was deep straw color.
The determinations for ammonia nitrogen gave negative results.
The precipitate formed by calcium hydroxide was washed by de-
cantation until free of dissolved nitrogen compounds, and the total
nitrogen determined. This precipitate was bulky due to the pres-
ence of large amounts of ferric and aluminum hydroxides. The
average humin nitrogen in the two samples was only 0.0006 gram.

The filtrate from the calcium hydroxide precipitate was con-
centrated to a small volume and the entire solution used for the
nitrogen determination. Sample I, contained 0.0371 gram in the
filtrate, and Sample II, 0.0365 gram, making an average of 0.0368
gram.

The residual soil was practically colorless, and a determina-
tion indicated that it was nitrogen free. The volume occupied by
the soil residue was 17.3 cc. By calculation it was found that the
total nitrogen regained in the original solution was 0.0903 gram,
theoretical 0.0921 gram, or a recovery of 98.01 per cent.

Thus practically all of the histidine was recovered in the fil-
trate from the calcium hydroxide precipitate, indicating that the
hypothesis was incorrect.

*The histidine di-hydrochloride was prepared from dried blood as out-
lined by Abderhalden (1910). Total nitrogen found 18.42 per cent; calculated
18.42 per cent.

63

It is possible that the specific character of the histidine nitro-
gen is destroyed by certain oxidizing agents present during the
hydrolysis, with the result that histidine nitrogen is not precipi-
tated in the basic fraction. A study is being conducted at the pres-
ent time on the hydrolysis. of pure protein in the presence of certain
inorganic oxidizing agents. It is hoped that some light will be
thrown upon the disappearance of histidine as well as the formation
of "humin" and of the ''nitrogen precipated by calcium hydroxide."

It is of interest to note that the average total nitrogen in the
two samples in Table II is 0.4584 gram (cf. Gortner 1916 c, where
total nitrogen on four determinations of three grams averaged
0.4551), showing that all of the nitrogen present is accounted for
in the method of analyses used.

Table IV shows the difference between the duplicate deter-
minations of the analysis of fibrin alone and in the presence of car-
bohydrate and of subsoil, and the differences apparently due to the
addition of 100 grams ignited subsoil to the 3 grams of fibrin. Van
Slyke's (1911) "maximum" and "average" differences to be ex-
pected between duplicate determinations are also given in the table
for reference.

From a study of Table IV it will be seen that the difference
between the analyses of fibrin hydrolyzed alone and in the presence
of ignited subsoil are, in the case of most of the fractions, within
the maximum allowed by Van Slyke for experimental error. The
only differences which are certainly greater than experimental
error are those of humin nitrogen, histidine1 nitrogen, and amino
nitrogen in the filtrate from the bases. It is observed that prac-
tically the same error occurred with the amino nitrogen in the fil-
trate from the bases when hydrolysis was carried out in the pres-
ence of carbohydrate.

From this analysis one can only draw the conclusion that even
if the organic matter of the soil consisted entirely of pure protein,
one would not obtain the same nitrogen distribution by the Van
Slyke analysis in the presence of soil that one would obtain in the
cibsence of the soil, or in other words, the presence of ignitied min-
eral subsoil intefferes with the Van Slyke analysis in much the
same manner as carbohydrates (Gortner 1916 c, and Hart and Sure
1916).

B. The humin nitrogen, its origin and significance. In such
a discussion one must first consider the source of humin nitrogen
in pure proteins.

Osborne and Jones (1910) suggest that perhaps tryptophane
and histidine are responsible for the humin formation, basing their
pbsttilation on the fact that zein, which contains no tryptophane
and but little histidine, gives only small amounts of humin on
hydrolysis.

Gortner and Blish (1915) hydrolyzed zein in the presence of
both tryptophane and of histidine and found that a large part of
the tryptophane was converted into humin nitrogen, whereas none

64

of the histidine was converted into humin but was all recoverable
in the bases. I have shown that histidine is practically all recov-
ered in the nitrate from the humin when it is hydrolyzed in the
presence of an ignited mineral subsoil. Histidine, therefore, can
be eliminated as a factor in the formation of humin nitrogen in the
soil. Gortner and Blish conclude that —

in all probability the humin nitrogen or protein hydrolysis has its origin in
the tryptophane nucleus.

Gortner (1916 c) has shown that the humin nitrogen is in-
creased by the addition of carbohydrate material to protein, and
suggests that this increase may be due to both physical and chem-
ical causes.* He presents evidence to show that the action of car-
bohydrate is probably due to the furfural produced from the car-
bohydrate and shows that increasing quantities of furfural cause
the humin nitrogen to steadily increase, and the work of Gortner
and Holm (1917) shows that the presence of formaldehyde during
hydrolysis causes a gradual increase in the amount of humin nitro-
gen up to a maximum very much larger than the amount of normal
humin nitrogen, and then a decrease with increased amounts of
aldehyde.

Shmook (1914) states that during the hydrolysis of his soils
there separated on the walls of the condenser a substance violet blue
in color, and that this appears during the hydrolysis of pure pro-
tein an$ is recognized as Liebermann's reaction for protein sub-
stances. The above conclusion in regard to the hydrolysis of a
pure protein is incorrect, since no color appears on the neck of the
flask or condenser in such an analysis. When furfural is heated
alone with hydrochloric acid a characteristic colored substance is
deposited on the condenser. It has been shown by Gortner (1916
c) that at the same time a polymerization ( ?) of furfural to humin
takes place very rapidly. He found that when, 1.165 grams of
furfural were heated with 100 cc. of hydrochloric acid (sp. gr.
1.115) for 18 hours that 76.40 per cent of the original furfural was
converted into insoluble "humin." Our mineral soils on hydrolysis
gave a deposit on the condenser similar to that described by
Shmook. The reaction indicates the presence of furfural, which
is in turn formed from the carbohydrates in the soil. This must
be considered to be a distinctive furfural reaction.

The humin nitrogen actually present in the soil may easily be
a very small part o-f the nitrogen found. It is evident that there
must be many nitrogenous organic compounds present in the soil
which have no relation to protein material, such as pu'rine, pyrimi-
dine bases, nitrogenous lipins, and nitrogenous pigments besides
a number of other non-protein substances. It is certain that the
humin nitrogen will be greatly changed by the presence of many
of these compounds. The calcium hydroxide here drags down all
the organic nitrogenous compounds which are soluble in dilute
acids, but insoluble in hot water and dilute calcium hydroxide, to-

*Practically the same increase in humin nitrogen occured when fibrin
was hydrolyzed in the presence of a mineral subsoil. The humin in this
case was not due to the presence of carbohydrate since the soil had lost all of
its organic matter by ignition.

65

gether with the calcium salts of nitrogenous organic acids, the
calcium salts of the purine and pyrimidine bases in addition to
the humin formed from the protein material, and other organic
compounds that are adsorbed, absorbed, occluded, or combined with
the iron and aluminum hydroxides present.

From Table XXVI we find that from 4.84 to 9.21 per cent of
the total nitrogen is precipitated by calcium hydroxide. It can
readily be seen that this does not represent true humin nitrogen,
since the calcium hydroxide does not contain any black colored sub-
stances formed by hydrolysis. The solution from which it is pre-
cipitated is colored only by ferric compounds, therefore, the or-
ganic material in this precipitate must consist of colorless organic
compounds adsorbed by or combined with the lime. This por- .
tion of the nitrogen consists almost certainly of non-protein mate-
rial. In all pure proteins the nitrogen retained in the calcium hy-
droxide precipitate is supposed to consist entirely of deeply colored
compounds. This study of the. distribution of organic nitrogen
in the soil has led to a new fraction, not previously reported. Cer-
tain of the analyses were carried out before the importance
of this fraction was realized, but in most of the analyses I have
reported this fraction as "nitrogen precipitated by calcium hy-
droxide," because of its unknown nature. Further investigations of
this fraction are highly desirable.

Another point of interest is observed in the humin nitrogen of
the sphagnum-covered peat hydrolyzed in the presence of metallic
tin. There is a decided decrease in this fraction as compared with
the peat hydrolyzed alone. As noted earlier, Samuely (1902), sug-
gested that humin formation might be due to an oxidation process
and certain of the earlier workers (cf. Hlasiwetz and Habermann
1871 and 1873) hydrolyzed protein in the presence of stannous chlo-
ride in order to obtain a colorless solution instead of one deeply
colored by the presence of humin.

I, therefore, hydrolyzed some gliadin in the presence of tin and
found that while the solution remained colorless, nevertheless small
balls of black material were formed. The humin nitrogen (insolu-
ble in acid + that pptd. by Ca(OH)2) was 1.17 per cent of the total
nitrogen, while gliadin hydrolyzed alone gave a dark colored hy-
drolysate and a humin nitrogen content of only 0.67 per cent.

Recently Spriestersbach (private communication) has hydro-
lyzed fibrin (from a different sample than mine) alone and in the
presence of stannous chloride and finds in the fibrin hydrolyzed
alone 1.67 per cent of total humin nitrogen, of which 1.06 per cent
is "acid insoluble" (cf. Gortner 1916 c) and 0.61 per cent precipi-
tated by calcium hydroxide. In the sample of fibrin hydrolyzed
in the presence of tin he finds 0.91 per cent of total humin nitrogen,
of which 0.25 per cent is "acid insoluble" and 0.66 per cent pre-
cipitated by calcium hydroxide. The nature of his hydrolysate
agreed in all respects with mine, i.e., was colorless or faint straw
color, with tiny black balls of humin floating on the surface or at-

66

tached to the sides of the flask in which the hydrolysis was carried
out.

These experiments suggest interesting possibilities, but dis-
cussion must be deferred until we know more of the exact reac-
tions taking place.

The true humin nitrogen remains in the residual soil after
hydrolysis. The amount of nitrogen in this fraction varies from
22.93 per cent to 28.27 per cent of the total nitrogen for the mineral
soils studied. This represents more nearly the true humin nitrogen,
in that the black coloring matter formed by acid hydrolysis remains
in this portion, but in addition we should also find here all organic
nitrogenous compounds insoluble in fairly strong solution of hy-
drochloric acid, all of the nitrogen adsorbed by the carbohydrate
humins, etc. Potter an'd Snyder (1915 a) express surprise at the
large proportion of nitrogen in this fraction but when one considers
the heterogeneous nature of the soil organic matter it is perhaps
more surprising to find that over 60 per cent of the nitrogenous
compounds are soluble in strong hydrochloric acid. Further study
is necessary before the full significance and origin of this humin
nitrogen can be thoroughly understood.

C. The effect of the quantity of acid used for the hydrolysis
on the amount of nitrogen dissolved and the nitrogen distribution
in soils. Throughout this investigation acid at least as strong as
constant boiling hydrochloric acid was used for the hydrolysis
since that is the strength recommended for the analysis of pure
proteins.

In the case of two soils, however, one of the duplicates was
hydrolyzed in the presence of 1000 cc. concentrated acid to 250
grams of soil, the other being hydrolyzed in the presence of 500
cc. of constant boiling acid to 250 grams of soil, in order to see if
any noticeable differences would be observed on the resulting analy-
ses. The two soils thus hydrolyzed were the prairie-covered loess
and forest-covered loess.

The results show little difference between the duplicates.
Table XXV shows that the larger volume of the stronger acid dis-
solved a greater per cent of the soil, due to the fact that more of
the mineral constituents were soluble in acid of this concentration.
At the same time, however, the amount of nitrogen extracted was
less.

D. The percentage of soil nitrogen extracted by acid hydro-
lysis. Shorey (1905) working with a single Hawaiian soil ex-
tracted 84.68 'per cent of the total soil- nitrogen by acid hydrolysis.
Jodidi (1911) working with eleven Iowa soils found from his
'studies a minimum of 68.90 per cent, a maximum' of 83.94 per cent,
and an average of 75.77 per cent; Lathrop and Brown (1911) in
five Pennsylvania soils found a minimum of 70.60 per cent, a maxi-
mum of 73.71 per cent, and an average of 71.78 per cent; Shmook
(1914), working with four Russian soils, found a minimum of
60.60 per cent in the Laterite soil, a maximum of 87.67 per cent in
the Podzol soil, and an average of 68.33 per cent; Kelley (1914),

67

working- with nine soils of the Laterite class common to the Ha-
waiian Islands, found a minimum of 67.51 per cent, a maximum
of 91.80 per cent, and an average of 82.17 per cent; and Potter and
Snyder (1915 a) in seven Iowa soils, found a minimum of 68.68
per cent, a maximum of 76.47 per cent, and an average of 74.41
per cent.

The grand average of all of these thirty-seven soils from wide-
ly different origin gives 75.91 per cent of the soil nitrogen in solu-
tion in the hydrochloric acid extract. In my studies I found a
minimum of 66.63 per cent, a maximum of 77.65 per cent, and an
average of 72.19 per cent extracted by the acid.

These results indicate that the nitrogen of practically all soils,
in so far as investigated, dissolves to about the same extent during
the acid hydrolysis.

E. "Jodidi numbers." A study of Tables XX, XXI, XXII,
XXIII, and XXIV shows that the nitrogen distribution by this
method does not give accurate results when compared with similar
fractions of the Van Slyke analyses. The ammonia nitrogen and
nitrogen in the filtrate from "bases" are all much too high, while
"basic N" corresponds fairly well with the true basic nitrogen.
If one desires accurate data in regard to the distribution of the
organic nitrogen in the soil he should not use "Jodidi numbers."

F. Attempts to extract proteins from the soil. The attempt
to isolate alcohol or salt soluble proteins from the soil was not suc-
cessful. The maximum protein nitrogen in the 70 per cent alcohol
extract from 6 kilo of soil amounted to. only 0.0010 gram, while
the maximum protein nitrogen extracted by the 10 per cent sodium
chloride amounted to 0.0280 gram or 17.50 per cent of the total
nitrogen in solution. A larger amount of organic nitrogen was
extracted by alcohol when the soil was first leached with 1 per cent
hydrochloric acid.

The amounts of possible protein were so small that it seems
safe to conclude that no appreciable quantities of alcohol soluble
or salt soluble proteins are found in the soil.

G. A consideration of the nitrogen distribution in different
extracts from the sphagnum-covered peat. Nitrogen distribution
was determined on extracts of a sphagnum-covered peat soluble
in (a) 1 per cent hydrochloric acid, (b) 4 per cent sodium hydroxide
and not precipitated by acidification, and (c) 4 per cent sodium
hydroxide and precipitated by acidification with hydrochloric acid.
Of the three extracts only the second approximates the distribution
of nitrogen in a pure protein. The figures for the ammonia nitro-
gen are abnormally high in the hydrochloric acid extract.

The humin nitrogen is high in all the extracts, but is excessive
in fraction (c). It is clear that carbohydrates from the soil must
be present in all three fractions used, and must have some share
in bringing the humin nitrogen up to such high figures. The
nucleic acids (Shorey 191 la, 1912) would be found in the hydro-
chloric acid precipitate from the sodium hydroxide solution, and

68

the purine and pyrimidine compounds of these nucleic acids, as
well as the lecithins (Aso 1904, Stoklasa 1911) and nitrogenous
lipins and nitrogenous acids would be precipitated with the true
humin by the calcium hydroxide.

The basic nitrogen figures are not widely divergent although
there may be some significant differences. The differences be-
tween the nitrogen in the filtrate from the bases is perhaps the most
significant of all. An amino nitrogen of only 17.11 per cent in the
filtrate from the bases such as is found in the hydrochloric acid
extract is far lower than has ever been obtained in an analysis
of a pure protein and indicates that the nitrogen of this extract is
essentially non-protein.

Unfortunately it was impossible to complete the corresponding
analyses on the calcareous black grass-peat, but the fractions ob-
tained would indicate a distribution similar to that of the sphagnum-
covered peat.

H. General conclusions in regard to the distribution of soil
nitrogen in different soil types. From a study of Table XXVI we
observe a great similarity between the different fractions. This is
practically the same deduction made by Potter and Snyder (1915 a)
in regard to their study made on a single soil type under different
fertilizer treatment.

• I find that the nitrogen distribution in a soil is very uniform
whether in the same soil type under different fertilizer treatment,
or in different soil types. This is to be expected, for if one were
to take fifty Van Slyke analyses of protein at random and compare
the average analyses with that of another fifty analyses, one should
expect to find results agreeing closely with each other. This
expectation should also hold true for the hydrolysate of soils, since
in each soil are to be found many of the nitrogenous compounds
contained in the plant and animal products that find their way
to the soil together with their decomposition products. Since
there is such a great variety of different nitrogenous substances
in the soil, it stands to reason that the nitrogen distribution in
soils is an average distribution, and as such should not be ex-
pected to vary widely from soil to soil.

It has been shown in the earlier part of this discussion that
when fibrin was hydrolyzed in the presence of ignited subsoil, no
histidine fraction was obtained.

For reasons which were stated previously, I have not tabu-
lated the nitrogen distribution under the different headings used
for the analysis of pure proteins. However, in view of the results
obtained on the fibrin hydrolyzed in the presence of ignited sub-
soil, it is perhaps worth while to consider what values the histidine
fraction would have had. The Fargo clay loam, Fargo silt loam, and
Sample I of the Carrington silt loam, gave results indicating that
this fraction was absent, while Sample II of Carrington silt loam
gave 2.97 per cent, Hempstead silt loam 0.78 per cent, prairie-cov-
ered lo.ess 1.21 per cent, and forest-covered loess 1.25 per cent of
histidine nitrogen.

69

Potter and Snyder (1915 a) find this fraction to be present in
all of their eight soils, with a minimum of 1.99 per cent and a maxi-
mum of 6.30 per cent. They do not give sufficient analytical data
to permit a recalculation of their figures in order to ascertain if
any errors in calculation were made. However, it is possible that
their rinding is due to the fact that all of their mineral soils repre-
sented a single soil type. It is possible that this form of nitrogen
may have been especially abundant in their particular soil. It is
of particular interest to note that their soil was from the Wisconsin
drift area, as was my sample of Carrington silt loam from Morris-
town, which gave my maximum amount of nitrogen in this frac-
tion. The sample of Carrington silt loam from Nerstrand was sit-
uated on the Kansan drift and gave no "histidine" nitrogen.

70

IV. SUMMARY.

This paper deals with a study of the nitrogen distribution in
different soil types, by applying Van Slyke's method. Tables have
been presented showing such distribution for the following mate-
rials :

a. Fibrin hydrolyzed in the presence of an ignited mineral

subsoil (together with data of fibrin hydrolyzed alone
and in the presence of carbohydrates).

b. A calcareous black grass-peat.

c. An acid, sphagnum-covered peat, hydrolyzed alone, in the

presence of a mineral subsoil, and in the presence of
stannous chloride.

d. An acid "muck" soil

e. Seven samples of mineral surface soil representing the fol-

lowing soil types : Fargo clay loam, Fargo silt loam,
Carrington silt loam (two samples from different glacial
drifts), Hempstead silt loam, prairie-covered loess, and
forest-covered loess.

f . Extracts of a sphagnum-covered peat and of a calcareous
black grass-peat soluble in (a) 1 per cent hydrochloric
acid, (b) 4 per cent sodium hydroxide but precipitated
by acid, and (c) 4 per cent sodium hydroxide and not
precipitated by hydrochloric acid.

The following conclusions are evident :

1. The figures for the ammonia nitrogen in a protein analysis
are not appreciably changed when the hydrolysis is carried out in
the presence of an ignited mineral soil equal to twenty times the
weight of the protein material.

2. The "humin" nitrogen is greatly increased by the addition
of ignited mineral soil. It was shown that histidine nitrogen cannot
account for this increase, neither is it due to the presence of car-
bohydrates, since the soil lost all its organic matter on ignition.

3. Attention has been called to the fact that the analysis of
a pure protein in the presence of even an ignited mineral soil docs
not give reliable results for the different fractions, and that such
determinations are of value only when used for purposes of com-
parison. Such data should not be compared with analyses ot pure
proteins.

4. Since practically all mineral soils give furfural on treat-
ment with acid it is very likely that a very considerable amount
of the total humin nitrogen found is due to the presence of carbohy-
drates in the soil, which give rise to furfural during hydrolysis.
This may combine with certain of the nitrogenous compounds and
cause an increase in the "humin" nitrogen, as well as adsorb or
occlude nitrogenous compounds in the "humin" formed from fur-
fural by polymerization.

71

5. This investigation of the distribution of organic nitrogen
in the soil indicates a new fraction, the nature of which has not
been previously recognized. This is the fraction of nitrogen re-
moved from a colorless solution by calcium, iron, and aluminum
hydroxides on the addition of calcium hydroxide. The nitrogen
retained in this fraction must consist almost entirely of non-pro-
tein material, since the organic substances in "this precipitate have
been shown to be colorless organic compounds adsorbed by or
combined with the metallic hydroxides. This fraction has been
reported as nitrogen precipitated by calcium hydroxide.

6. The true humin nitrogen remains in the residual soil after
hydrolysis, but in addition non-humin nitrogenous compounds
must also be retained in this fraction.

7. The strength and volume of the hydrochloric acid used in
hydrolysis has little effect On the nitrogen distribution of the hy-
drolysate provided acid as strong as constant boiling acid is used,
in the proportion of at least two parts of acid to one of soil.

8. Results gained from a study of different soils -indicate that
the organic nitrogen dissolves, during hydrolysis, to almost the
same extent regardless of the origin and nature of the soil.

9. Some very interesting figures are found in the comparison
of the different extracts from sphagnum-covered peat (Table
XXVI). The portion soluble in sodium hydroxide and not pre-
cipitated by hydrochloric acid gives a nitrogen distribution ap-
proximating very closely that of a normal plant protein. The nitro-
gen dissolving in the preliminary hydrochloric acid leaching shows
a nitrogen distribution which is certainly not due exclusively to
protein materials, e. g., an ammonia nitrogen percentage of 65.40
and amino-nitrogeii-in-filtrate-from-bases of 17.11 per cent.

10. When an attempt was made to isolate alcohol soluble
and salt soluble proteins from the soil the amounts obtained were
so small that, it seems safe to conclude that no appreciable quan-
tities of these types of proteins are present.

11. The most significant fact brought out by this study is
that the organic nitrogen distribution in different soil types is very
uniform. This is to be expected since it has been pointed out that
the nitrogen distribution in soils is an average distribution of all
the plant and animal nitrogenous products that find their way to
the soil.

72

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BIOGRAPHICAL.

Clarence Austin Morrow was born near Morrow, Warren
County, Ohio. He graduated from the Hillsboro, Ohio, high school
in June, 1901. He entered the Ohio Wesleyan University in the
Jail of 1902, and received the degree of Bachelor of Science in June,
1906. During 1906-08 he held the position of Assistant in Chem-
istry at Oberlin College. He received the degree of Master of Arts
from the same institution in June, 1909. In 1909-10 he was acting-
head of the Departments of Chemistry and Physics at Doane Col-
lege. Having been appointed the John Harrison Scholar in Chem-
istry at the University of Pennsylvania he entered that institution
in the fall of 1910 and continued his graduate work. In the spring
of 1911 he was appointed John Harrison Fellow in Chemistry, but
later resigned this to take the position of Professor of Chemistry
at Nebraska Wesleyan University, which position he has held to
date (January, 1917). During 1914-15 he was granted leave of
absence for study in the Division of Soils, University of Minnesota,
where he held the position of Assistant in Soil Chemistry. Here
he studied for the degree of Doctor of Philosophy.

Major subject, soil chemistry.

Minor subject, organic chemistry.

Member of the American Chemical Society.

Member of the American Association for the Advancement of
Science.

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