652
;5
•py 1
SOIL INOCULATION WITH
AZOTOBACTER
BY
PAUL EMERSON
DISSERTATION SUBMITTED TO THE GRADUATE FACULTY OF
THE IOWA STATE COLLEGE OF AGRICULTURE AND ME-
CHANIC ARTS IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY
NO. 3
REPRINTED FROM RESEARCH BULLETIN No. 45
IOWA AGRICULTURAL EXPERIMENT STATION
1918
Digitized by the Internet Archive
in 2010 with funding from
The Library of Congress
http://www.archive.org/details/soilinoculationwOOemer
February, 1918 Research Bulletin No. 45
Soil Inoculation with Azotobacter
<^ LSZ
By PAUL EMERSON
AGRICULTURAL EXPERIMENT STATION
IOWA STATE COLLEGE OF AGRICULTURE
AND MECHANIC ARTS
AGRONOMY SECTION
Soil Bacteriology
AMESs IOWA
IOWA AGRICULTURAL EXPERINENT STATION
OFFICERS AND STAFF
Raymond A. Pearson. M. S. A., LL. D.. President
C. P. Curtiss. M S. A., D. S.. Director
W. H. Stevenson. A. B.. B. S. A., Vice-Director
AGRICULTURAL, ENGINEERING
C. K. Shedd, B. S. A., B. S. in A. E., W. A. Foster, B. S. in Ed., B. Arch.,
Acting Chief Assistant
AGRONOMY
W. H. Stevenson. A. B., B. S. A., Cliief George E. Corson, B. S., M. S'., As-
H. D. Hiiglies. B. S., M. S. A., Cliief sistant in Soil Survey
in Farm Crops H. W. "Warner, B. S.. M. S., Soil Sur-
P. E. Brown, B. S.. A. M.. Ph. D., Chief veyor (ab.=-ent on leave)
in Soil Chemistry and Bacteriology -^ l, Rhodes, B. S., Soil Survey (ab-
L. C. Burnett. M. S. A., M. S., Chief ^ent on leave)
in Cereal Breeding .. , ^, . „ M. E. Olson, B. S., M. S'., Field Ex-
L. W. Forman. B. S. A., M. S., Chief periments
in Field Experiments . , ^ EI. Angell. Soil Surveyor
John Buchanan, B. S. A Supermtend- j p gjg-g. g g pie\& Exnerimenta
ent of Co-operative Experiment.^ q p j^^^^^,-, g g., m. S., Assistant
R. S. Potter A, B.. M. b.. Pn. u.. . ^^ rmno
Assista-t Ciiipf in Soil Chemistrv ^ ' Hnn=-on B q Eipli Exneri-
R g S'lvdpr. B. S., Assistant in Soil H. P. Hanf:on. B. ^-^ -l^ield iixpen
pviop^isfv ments (absent on leave)
H. "^'■. .Tobn^nn B S.. M. S . Assist-
ant in Soils (absent on le^-'^o'i
ANIMAL HUSBANDRY
W H Pew B S A Chief L. S. Gillette. B. S.. M. S., Assistant
J. M. Evvard, M, S., Assistant Chief Chief in Dairy Husbandry _
in Animal Husbandry and Chief in A. C. McCandlisb, M. S. A., Assistant
Swine Production in Dairv Husbandry
R. Dunn. B. S., Assistant in Animal Rodney Miller. B. S. A., Assistant in
Husbandry Poultry Husbandry
H. A. Bittenbender, B. S. A., Chief
in Poultry Husbandry
BACTERIOLOGY
R. E. Buchanan, B. S., iPh. D., Chief : Associate in Dairy and Soil Bacteriology
BOTANY
L. H. Pammel, B. Agr., M. S., Ph. D., I. E. Melhus, Ph. D., Chief in Plant
Chief Patbolo.gy
Charlotte M. Kin?, Assistant Chief in Botany
CHEMISTRY
A. W. Dox, B. S.. A. M., Ph. D., Chief S. B. Kuzirian, A. B., A. M., Ph. D.,
(absent on leave) Assistant
W. G. Gaessler, B. S., Acting Chief G. W. Roark. Jr., B. S., Assistant
A. R. Lamb. B. S., M. S'.. Assistant Lester Yoder, B. S., M. S'., Assistant
DAIRYING
M. Mortensen. B. S. A., Chief D. E. Bailey. B. S., Assistant Chief
B. W. Hammer, B. S. A., Chief in in Dairying
Dairv Bacteriology
ENTOMOLOGY
R. L. Webster, A. B., Acting Chief Wallace Park, B. S,, Assistant in Ag-
riculture
FARM MANAGEMENT
H B Munger, B. S., Chief O. G. Lloyd. B. S., M. S'., Assist. Chief
HORTICULTURE AND FORESTRY
S. A. Beach. B. S.. M. S., Chief J. B. Kendrick, B. A., Research As-
T. J. Maney, B. S., Chief in sistant in Pomology
Pomology ' A. T. Erwin, M. S., Chief in Truck
Harvey L. Lantz. B. S., Assistant in Crops
Fruit Breeding Rudolph A. Rudnick, B. S., Assistant
W. E. Whitehouse, B. S., Assistant in Truck Crops
in Pomology G. B. McDonald, B. S. F., M. F., Chief
Andrew Edward Murneek, B. A., Re- in Forestry
search Fellow in Pomology Frank H. CuUey. B. S. A., M. L. A.,
Chief in Landscape Architecture
RURAL SOCIOLOGY
G. H. Von Tungeln. Ph. B., M. .\., Chief
VETERINARY MEDICINE
C. H. Stange, D. V. M., Chief
GENERAL OFFICERS
F. W. Beckman, Ph. B., Bulletin Editor F. B. Colburn, Photographer
Gretta Smith. A. B., Assistant to Bulletin Editor
C, E. Brashear,_B'._S. A., Assistant to Director
""" JSaRYc QF CONGRESS j
. .RECEIVED
Soil Inoculation With Azotobacter*
BY PAUL EMERSON.
Following' tlie discovery of the nitrogen fixing' powers of the
symbiotic bacteria in the soil, early investigators found that the
power of utilizing the free atmospheric nitrogen was not confined
to the symbiotic bacteria alone. They noted increases in soils
which had borne no legumes and they found that fallow soils
in particular increased appreciably in nitrogen content. These
facts stimulated researches which led to the discovery of many
forms of bacteria which are able, when growing alone, to fix
nitrogen from the air. The chief of these is now known as the
azotobacter group.
It seems likely that the azotobacter will prove more effective in
fixing nitrogen than the symbiotic bacteria, although the general
requirements of the two classes of organisms are very similar.
The azotobacter are active in practically all soils regardless of
the kind of crop grown when conditions for their growth are
satisfactory. These conditions are probably much the same as
for the symbiotic bacteria except that these latter organisms
require the presence of a specific legume for fixing the greatest
amount of nitrogen. Azotobacter require a certain amount of
carbonaceous material in the soils and are usually stimulated by .
a small amount of nitrogen, but the exact optimum conditions
for their growth are as yet unknown. These organisms are active
in causing nitrogen increases in many soils, but the feasibility
of introducing them into the soil or of attempting to increase
their nitrogen-fixing powers by artificial means, and the effect
of the presence of growing plants on their efficiency are ques-
tions as yet unanswered, although Lipman has indicated that
under proper conditions successful inoculation may be accom-
plished in soils and Bottomley has successfully grown pure cul-
tures of these organisms in the presence of growing plants with
favorable results.
HISTOBIGAL
Beijerinck (2) isolated and described the first azotobacter (in
1901) . He found two species, one of which he named Azotohacter
chroococcum and the other Azotohacter agilis. The former was
isolated from the soil and the latter from a sample of water
taken from one of the canals in the city of Delft. Two years
later Lipman (36) added a third species, A. vinelandii, to the
list and the following j^ear isolated and described two more, giv-
ing them the names of A. deijerinckii and A. woodstownii. Of
the five organisms of this species, A. cliroococcum., A. deijerincMi
♦Thesis submitted in partial fulfillment of the requirements for the Degree
Df Doctor of Philosophy at the Iowa State College.
'■h ■'>«<•■ ...'
28
and A. vinelandii are considered the most important in soil in-
oculation studies.
The frequency with which investigators in all parts of Europe
and America have isolated azotobacter from various soils, indi-
cates that they are widely distributed. Christensen (10) found
that they were present throughout northwestern Europe, the
activity of the organism apparently depending on the basicity
of the soil. This view was later supported by the works of
Fisher (14), Lohnis and Pillai (45) and others. Ashby (1)
studied the soils of Mombasa, East Africa, Cairo, Egypt and
Rothamsted, England and found azotobacter forms present in
most eases. Lipman and Burgess (42) working with forty-six
samples of soil from variotis parts of the world, found that over
one-third of them contained azotobacter, the predominant form
being A. chroococcum. Many of the soils examined were museum
specimens and had been kept in tightly stoppered bottles for
long periods of time.
DESCRIPTION OF AZOTOBACTER.
Bei.jerinck characterizes the azotobacter r-s stout bacteria, 4-6
microns or less in length, sometimes longer, occurring as large
diplococci or short rods in young cultures, the hyaline cells often
containing a vacuole and the entire organism enclosed in a
mucilagenous wall of varying thiclaiess. They have a single
polar flagellum or bundles of 4-10 polar flagella of about the
same length as the organism itself. Beijerinck found no spores.
Vagler (65) writes that the older colonies produce involution
forms similar to those of yeasts while Heinz (22) and Fisher
(15) showed that the organisms can resist drying for six to nine
months. Later investigations by Mulvania (50) and Lohnis and
Smith (47) demonstrated that the organism produces spores and
completes a very complicated life cycle. Descriptions of azoto-
bacter and detailed cultural characteristics of the organism were
given by Lipman (35), Bei.jerinck (2), Prazmowski (54), "Warm-
bold (70), Bonazzi (6), Lohnis and Westerman (48), Lohnis and
Hanzawa (44), Jones (27) and others.
ACTIVITIES OF AZOTOBACTER.
Beijerinck first claimed that the isolated pure cultures of
azotobacter were able to fix the atmospheric nitrogen in appre-
ciable amounts; later, however, when working with Van Delden
(4), he retracted this statement, claiming that pure cultures did
not have this ability and that only in the presence of very small
celled organisms called radiobacter could the free nitrogen of
the air be fixed in the soil. Grcrlach and Vogel (18), Heinz
(23), Lipman (37) and Freudenreich (17) proved conclusively
that the earlier conclusions of Beijerinck were correct and that
29
the organism may fix considerable amounts of nitrogen in pure
cultures. Lipman accounts for the fact that Beijerinck did
not get a fixation of nitrogen in pure cultures by showing that
the organism will not fix nitrogen unless the reaction of the
medium is made neutral or slightly alkaline. When Beijerinck
later accepted this suggestion he found that his pure cultures
were able to fix atmospheric nitrogen.
STUDIES OF AZOTOBACTER.
Ver}' few investigators have attempted tO' inoculate soils with
azotobacter or other non-symbiotic nitrogen fixing bacteria
under conditions approximating those in the field. The influence
of various kinds of sugars, cellulose, inorganic salts, and various
organic compounds on the nitrogen-fixing power of the organ-
isms have been studied extensively. Gerlach and Vogel (19),
Pringsheim (55), Krainsky (33), Koch (30), Hoffman and Ham-
mer (25) and Stranak (61) have found that various sugars and
cellulose materially increase their nitrogen fixing powers while
Fisher (16), Christensen (10), Lohnis and Pillai (46), Wilfarth
and Wimmer (59) Kaserer (28), Rosing (59), Vogel (66),
Greaves and Anderson (20) and Pringsheim (56) have shown
that small amounts of lime, very small amounts of nitrogen,
various inorganic salts and even a very small amount of arsenic
will stimulate the nitrogen fixing power of the organisms in the
presence of certain carbon compounds. Stoklasa (60) studied
the products of the activities of the azotobacter organisms, con-
fining his researches largely to the amounts and kinds of gases
produced under different circumstances, under the influence of
various substances supposed to be energy sources, and under
varying temperature conditions. His results have been more or
less confirmed by the works of Thiele (64), Hoffman (24), Keller-
man and Smith (29) and Ehrenberg (13).
The activity of the azotobacter in soils in general, and partic-
ularly under laboratory conditions, was fully shown by the works
of Lipman (39), Voorhees and Lipman (68), Lohnis (43), Kuhn
(34), Freuclenreich (17), Dvorak (12), Remy (57), Remy and
Rosing (58), Jacobitz (26), Stranak (62), Headden (21), Peter-
son and Mohr (52), Koch and Seydel (31), Omeliansky and
Ssewerowa (51), Warmbold (71) and others who demonstrated
that under various conditions and in almost every type of known
soil these organisms are able to fix appreciable amounts of the
free atmospheric nitrogen. Only a few of these investigators,
however, have made any attempt to secure an active flora of
these organisms in the soil. Vogel (67) inoculated pure cultures
of azotobacter into soils that had been treated with grape sugar,
in some cases adding comparatively large amounts of nitrate of
soda. In pot experiments with oats and mustard, increases were
30
noted for the inoculated series, altho the pots receiving nitrate
of soda gave the greatest yields. When the experiment was re-
peated in the field the inoculated plots gave smaller yields than
the uninoculated, and the inoculation appeared to have an in-
jurious effect upon the crop.
A short time later Lipman and Brown (41) tried inoculation
experiments with A. vinelandii and A. beijerinekn. They sunk
four foot cylinders open at both ends into- the soil, filled the
cylinders with soil and inoculated the soil with the organisms.
The first summer the soils were left bare and then a rotation of
crops was followed and oats, corn and rye grown in succession.
While considerable variation Avas found in the nitrogen content
of the crops and in the dry weight, the general conclusion reached
was that the activities of the organisms did not increase the
nitrogen content of the soil. The results do not preclude the pos-
sibility that inoculation with the organisms in question may be
made of practical value, provided proper conditions for the best
growtli of the organisms are secured. Bottomley (7) and Bot-
tomley and Hall (9) experimented with oats, barley and some
root crops, and arrived at the same conclusions as did Lipman
and Brown. Stranak (63) also inoculated soils with azotobacter
and found a pronounced increase in the growth of potatoes,
grain and beets.
Altho the experiments dealing with the inoculation of soils
with azotobacter have been inconclusive, it is believed that under
proper conditions such inoculation may be extremely profitable.
EXPERIMENTAL
The wdde distribution of non-symbiotic nitrogen fixing bacteria
in many types of soils is practically parallel with the distribution
of the symbiotic organisms, and since it is practical and profitable
to inoculate soil with the latter, even tho the particular organism
may be present, the following cjuestions quite naturally arise:
1. If the azotobacter are not present in the soil, can inocula-
tion be profitably accomplished?
2. What soil conditions are necessary for the greatest fixa-
tion of nitrogen by these organisms ?
These questions have an important bearing on the problem of
the maintenance of permanent fertility in soils from the nitrogen
standpoint and may govern the choice of the proper method of
farming. Some commercial concerns have placed cultures on the
market, claiming that they contain sufficient numbers of the non-
symbiotic nitrogen fixing bacteria to enable the farmer to solve
his nitrogen problem without growing legumes. However, results
of experiments showing that such cultures are capable of inoculat-
ing the soil were not found in the present investigation.
31
INCREASING THE NITROGEN FIXING POWER OF PURE
CULTURES.
Very little work has been done along the line of breeding pure
cultures of bacteria to an increased efficiency in their specific
actions, in fact, practically all the experiments have been carried
out with the idea of finding a method whereby the organism
could be kept alive for long periods without periodic transfers.
The earliest investigation along this line was that of Czaplewski
(11) who limited the amount of air in the tube by saturating the
plug with paraffine. Later Lunt (49) found that certain cultures
of water bacteria may be kept alive much longer in sterile water
than in ordinary culture media. In some cases he kept certain
organisms alive for two years by this method. BoUey (5) secured
good growths of B. amylovorus and Bad. dianthi in agar and
in bouillon by making transfers from cultures that had been
hermetically sealed for nine years. It is not stated whether or
not the organisms were tested for their pathogenicity and hence
their virulence is left in doubt. This work supports that of
Czaplewski in showing that cultures can be kept alive for long
periods of time if the transpiration is reduced to a minimum.
Some commercial concerns claim that they are able to increase
the efficiency of their particular cultures of legume bacteria by
alternate inoculations first on agar, then into sterile greenhouse
soil, growing the specific legume to which the organism in ques-
tion is adapted, and re-isolating the crsanism from the nodules
produced on the roots of the legume. If this is possible for the
symbiotic bacteria then it seems probable in the case of the non-
symbiotic organisms. The following cpiestions naturally suggest
themselves :
1. Can the nitrogen fixing power of azotobacter be increased
by periodic transfers on nitrogen free media?
2. Can the nitrogen fixing powers of azotobacter be increased
by growing the organism in the presence of growing plants ?
In outlining work to answer the above questions it was realized
that a large number of bacteria should be used. A number of
large celled nitrogen fixing organisms that had all the staining
reactions of the azotobacter type and closely resembled it in size
and shape, were isolated in pure cultures from soil secured from
the humus plots at the Iowa station and were designated with
laboratory numbers. At the same time pure cultures were se-
cured and their activities determined along with these of the
unnamed cultures. The pure cultures were kindly furnished by
Dr. J. G. Lipman of the New Jersey Agricultural Experiment
Station and also by the American Museum of Natural History of
New York,
32
MEDIA USED.
The nitrogen free medium used thruout the experimental
work was a modification of that proposed and used by Lipman
(35), and its composition was as follows:
Distilled water 1,000 cc
Di-potassium phosphate 0.2 grams
Magnesium sulphate 0.2 grams
Calcium chloride 0.02 grams
Dextrose 10.0 grams
10% Ferric chloride solution 2 drops
The solution was brought to boiling and made neutral to
phenolphthalein by the addition of N/10' NaOH. If a solid
medium was desired 1 % powdered agar w^as added. Sterilization
was accomplished by placing in the autoclave at ten pounds for
20 minutes.
Inoculation was secured by scraping off a two days' growth
from the agar slants with a sterile needle and transferring it to
flasks containing 50 cc. of the above solution. In onler to de-
termine w^hether the nitrogen fixing power of the organisms was
stimulated by the addition of nitrogen, the above solution with
the addition of 1 mg. of nitrogen as sodium nitrate was used.
PRELIMINARY STUDIES.
All of the organisms of the azotobaeter type including both
the pure cultures and the unnamed cultures, were inoculated
into 50 c. c. of both of the above solutions and tested for their
nitrogen fixing powers. The inoculated solutions were incubated
for tliree weeks at room temperature (22-25° C) and then
Kjeldahlized. The amount of nitrogen fixed by each organism
in the different solutions is shown in tal>le I. The same cultures
were transferred 12 times at three to four day intervals on nitro-
gen free media and their nitrogen fixing power tested in solu-
tioiis with and without nitrogen. The results appear in table II.
The laboratory organisms used in table I had been freshly
isolated and purified from the soil, the named cultures had been
kept on agar slants for varying periods of time. During the
time that the inoculated culture solutions were incubating the
transfers were being made in preparation for the inoculations
for table II.
Comparing the two tables we find that a ma.jority of the or-
ganisms decreased in their ability to fix atmospheric nitrogen,
altho a few showed a slight increase or at least retained their ef-
ficiency. From these the following eight were selected for fur-
ther study: No. 4, No. 22, No. 26, No 27, A. vinelandn, A. chroo-
cocciim., A. heijerincMi and A. cliroococcum (HCM) . These eight
organisms were studied under both laboratory and greenhouse
conditions,
33
TABLE I^NITEIOGIEN-TTKATTON OBT
PUBE ClUXiTUKES IN SQiLUTION
WITH AJNID WITHODT
MTKiOGEN.
N. Fixed in Mgs.
Organism
c " ?,
■sjl
Solution
with
Nitrogen
Lab. No. 1 . -
2.24
2.38
0.28
0.98
i.9e
3.22!
0.42
0.42
0.42
lost
1.12
2.80
7.141
0.2S
1.12
0.56
0.50
0.42
0.70
0.70
0.2S
0.56
a. 12
lost
4.20
0.84
0.70
0.84
1.96
V.U
Lab. No. 2-
1.54!
Lab. No. 3
1.99
Lab. No. 6
Lab. No. 7 -__
1.82
1.68
Lab. No. &
3.10
Lab. No. 10
0.84
Lab. No. 11
0.70
Lab. No. 12 -
1.40
Lab. No. 14
Lab. No. 15
lost
1.13
Lab. No. le - -
lost
Lab. No. IS
Lab. No. 19 - __
U.43
1.54
Lab. No. 20- -.
1.90
Lab. No. 21 __ ... _
2.10
Lab No. 22
2.53
Lab. No. 23- - . --
2.52
Lab. No'. 24-
1.82
Lab. No. 25- -
1.82
Lab No. 26-
2.10
Lab. No. 27
A. vinelandii
5.60
2.66
A. chroo'coecum (IHCIM)
A. chroocoecum
A. chi'Oococeum (Oolo).
A. beijerinekii
3.08
1.54
2.52
1.68
A. bsijerinekii No. 5
2.38
TAiBiLE n—NITBOOEN- FIXATION BT
FORE! COLTURES IN SOLUTION
WITH AND WITHOUT
NITRIOGEN.
After each org-anism has been transfer-
red twelve times on nitrogen- free
miedia at three to four day intervals.
N. Fixed in Mgs,
Organism
3 M
.2 S,
Lab. No. 1 --
0.84
0.14
0.14
1.82
0.00
0.28
O.OO
0.98
0.00
0.00
0.42
0.00
O.OO
0.00
0.00
0.98
0.42
3.'53
2.10
0.00
0.00
1.12
1.40
0.00
1.12
2.52
O.OO
0.42
1.12
1.12
Lab No'. 2
O.OO
Lab. No. S
0.99
Lab. No. 4
0.98
Lab. No. 6 _
0.84
Lab No. 7 -
0.28
Lab No. 9-
1.26
Lab. No. 10
0.00
Lab No. 11 - -
1.6S
0.28
Lab. No. 14-
1.121
Lab No. 15
1.40
Lab No. 16- -—
0.14
Lab No 18
0.42
Lab. Noi. 19 ---
0.42
Lab No 20
1.54
Lab. No. 21
0.84
Lab. No. 22
Lab No. 23
1.12
1.54)
Lab No 24
1.63
Lab No. 25
0.2S
Lab No. 26. - -
1.82
Lab. No. 27-
2.52
0.00
A. chroocoecum
A. chroocoecum (HICM)
A. chroocoecum (Colo).
1.82
1.12
1.26
2.92
A. beijerinekii No. 0.—
0.00
LABORATORT STUDIES.
The laboratory studies were arranged in a series of three ex-
periments as follows :
1. To determine the effect of transfers made every other day
on the nitrogen fixing- power of the organisms.
2. To determine the effect of transfers made once each week
in sand cultures variously modified.
3. To determine the effect of growing four of the organisms
on both agar and sand in large flasks with and without the
presence of growing plants.
34
Series 1. To Determine the Effect of Transfers Made Every
Other Day on the Nitrogen Fixing Power of
the Orga!msms.
Using the eight selected organisms transfers were made every
other day on the nitrogen free medium for a period of three
weeks. It was feared that such rapid transferring for so long
a period on a medium practically free from nitrogen would re-
duce the vitality of the organisms, accordingly each fifth trans-
fer was made on a modification of the medium consisting in the
addition of one milligram of nitrogen as sodium nitrate to each
liter of the regular dextrose agar. At the end of the transfer
period the organisms were inoculated into the nitrogen free and
nitrogen containing solutions incubated for the same periods of
time and the amount of nitrogen fixed determined by Kjeldah-
lizing. The results of the determinations are shown in table III.
TABIE nr— TIHiE EPFEiOT OF TIHuANiSIEEiHlS iMADE: EVElE;T OTHER DAT FOB FOUIB
WEEOBCS ON THE' NTTRlOGEiN MXINIG POWEIB OF THE OCROAJSfTSMS .
Organism
Nitrogen Fixed in Mgs.
Solution without
Nitrogen
Solution with
Nitrogen
Lab. No. 4
Lab. No. 25
Lab. No. 26
Lab. No. 27
A. vinelandii
A. chrooeoecum
A. ehrooeoccuni CHOM).
A. beijerinckii
(a)
(b)
(Av.)
(a)
(b)
0.14
0.42
0.28
0.70
0.42
0.00
O.OO
0.00
0.14
0.56
0.1+
0.14
0.14
0.28
0.98
0.2S
3.. 50'
1.98
0.70
0.98
0.28
0.42;
0.35
0.98
0.98
r^r.
0.2S
0.42
0.98
1.12
2.66
lost
2.66
1.40
1.40
0.S4.
2.66
1.79
0.28
0.28
(Av)
0.56
0.35
0.63
0.84
0.98
l.Oo
1.40
0.28
That these transfers should have been made at longer intervals
is evidenced by the fact that tables I and II showed that 12 of
the cultures had increased in efficiency after they had been
transferred every three days for 36 days. However, during the
latter work the organisms did not show any indications of a loss
of vitality and the growth at all times was vigorous and rapid.
Tabic III shows a decrease in the nitrogen fixing powers of all
the organisms except in the case of A. diroococcum (II CM)
which appears to have retained its efficiency thruout the ex-
periment.
Series 2. To Determine the Effect on the Nitrogen Fixing Power
of Transfers Made Each Week in Sand Cultures.
In the following experiment sand was used instead of agar as
the basis for the medium. Ground oats straw, ground red clover
hay and either the regular dextrose solution, or the dextrosf;
solution containing nitrogen were added. The tests were carried
out in tubes arranged as follows :
35
6.25 gr. sand+2.5 cc N. free dextrose solution.
6.25 gr. sand-i-2.5 cc dextrose solution containing 0.2 gr. NaNOs per
liter.
6.25 gr. sand + 3.5 cc N. free dextrose solution + 0.1 gr. clover nay.
6.25 gr. sand-|-3.5 cc N. free dextrose solution+0.1 gr. oats straw.
The organisms were transferred directly from the slants into
the tnbes and there allowed to incubate at room temperature for
seven days. A small portion of the sand was then transferred
to a fresh tube of the same medium as the original. As this par-
ticular experiment did not directly follow the others the efficiency
of the organisms was tested before they were inoculated into the
sand. Table IV shows the amount of nitrogen fixed by the pure
TAjB^LlE' IV-^TIBTE NITEiOOEN MXINO POWEIK OlF THE- PORIE CICPLiTUEElS
IMMEIDIAT'ElLY BiEIPIOBIE: the SIANID' CIU'LITHJIRE' EXPEIRIIlMElN'TlS.
Organi;
[Solution witnout
Nitrogen
Solution with
Nitrogen
Lab. No. 4
Lab. No. 22
Lab. Nc 26
Lab. No. 27
A. vinelandii
A. ehroococcum
A. chroococcum (HJQM)-
A. beijerinckii
(a)
0.14'
0.00
O.OO
0.00
O.OO
O.OO
0.00
0.98
(b)
0.70
0.00
0.14)
0.00
0.00
0.14
O.OO
0.84
(Av.)
0.42
O.OO
0.07
O.OO
0.00
0.07
0.00
0.91
(a)
(b)
0.28
1.54
3.36
3.0s
2.914
2.66
2.38
2.52
1.40
1.40
2.80
2.66
2.52
2.38
2.94
lost
(Av.
0.91
3.22
2.80
2.45
1.40
2.73
2.49
2.94
cultures at the beginning of this series of incubation, and the
same methods as in the previous experiments.
At the end of the fourth transfer period, i. e., four weeks
after the start of the experimental series, the organisms were
inoculated into dextrose solution and their nitrogen fixing powers
determined. After four more weeks of transferring or in all
eight weeks the final inoculation into dextrose solution was made.
The influence of the oats and clover in the presence of sand on
the nitrogen fixing power of the organisms used is shown in tables
V and VI, by the fact that both the large celled organisms of the
TAtBLE v.— NTTIBOiGEN FIXED BY THE PUBE COLTIPREIS APTE-B FOUR
TBAiNSFElBS IN ISLAND' AT PElRilODS! 'OF' SEVEN D'AYS EACH.
Nitrogen Fixed in Mgs.
Organism
dex. sol.
dex. sol.
+ N
dex. S0I.+
oats straw
dex. S0I.+
clover hay
Lab No. 4
0.28
0.07
0.77
0.42
0.14
0.21
O.07
0.14
0.35
0.2s
0.07
0.351
0.21
0.21
0.14
0.28
0.28
0.00
0.14
1.27
0.42:
0.07
1.19
0.35
0.98
Lab. No. '»
0.35
Lab. No. 26
Lab. No. 27
0.00
0.42
0.28
A. ehroococcum
A. chroococcum) (HCIM)
A. beijerinckii
0.21'
0.42
0.28
36
"HAiBLE' VI— NITRlOOEilSr iPIXED EiT THE FUBIE' OULTUElElS AFT'EIB EIGHT
THAaSHSE'EIRlS AT PiElRODOIDlSi OP SEiyEN DAYS- EACH.
Organism
Lab. No. 4
Lab. No. 23
Lab. No. 20
Lab. No. 27
A. vinelandii
A. chrooeoccum
A. chrooeoccum! (HCiM)
A. beijerinckii
Nitrogen Fixed in Mgs.
dex. sol.
0.30
0.20
0.70
0.20
0.20
0.30
0.10
0.00
dex. sol.
dex. sol.+
+ N
oats straw
0.20
0.30
0.20
0.20
0.40
o.oo
0.10
0.40
0.40
0.40
o.ao
0.20
0.40
0.40
0.00
0.20
dex. S0I.4-
clover hay
0.40
2.00
1.00
0.50
0.50
lost
1.40
O.b'O
azotobaeter type and the azotobaeter themselves, made gains in
their nitrogen fixing powers. There was no distinct gain due
to any one kind of carbonaceous material. Of the six organisms
showing gains A. cJiroococcum made the most notable, especially
in the presence of the oats straw. The nitrogen fixing power of
No. 4 appears to be rather constant thruout the series, with no
appreciable gain or loss. A. heijerinckii showed a decided loss in
its power to fix nitrogen in each of the four media, but gave a
slight indication that in the presence of the clover hay it might
be slowly regaining its power.
Series 3. To Determine the Effect of Growing the Organisms on
Both Agar and Sand With and WitJiont the
Presence of Growing Plants.
The main points considered in this experiment were: An
increase in the surface area over which the organism could
grow; an increase in the time between transfers and the grow-
ing of the organisms in the presence of an undetermined species
of alga3 and with growing oats and red clover plants. Two liter
Erlenmeyer flasks were used and arranged in the following man-
ner conforming to the outlines of the experiment:
Flask No. 1. 1000 cc N. free dex. agar+1 gr. CaCOj planted to oats.
Flask No. 2. 1000 cc N. free dex. agar-fl gr. CaCO 3 planted to
red clover.
Flask No. 3. 1000 cc N. free dex. agar-f 1 gr. CaCOa planted with
an undetermined species of algse.
Flask No. 4. 1000 gr. pure quartz sand + 180 cc N. free dex. solution
neutralized with CaCO:!, planted with oats.
Flask No. 5. 1000 gr. pure quartz sand + 180 cc N. free dex. solu-
tion neutralized with CaCOs, planted with red clover.
Flask No. 6. 1000 gr. pure| quartz sand+180 cc solution without
dex. neutralized with CaCOs, planted with an undetermined species
of algae.
Check flasks of sand and dextrose agar.
37
The flasks of agar were sterilized in the autoclave at ten pounds
for 30 minutes, but the flasks of sand were sterilized at 15 pounds
pressure for four hours once a day for three consecutive days.
Bacteriological tests on the sand at the end of that time showed
it to be sterile.
The culture of the algae used was so closely associated with a
bacterial growth that a separation would have required a long
time. For that reason it was not purified but was grown in sterile
distilled water, for about three months before inoculation. The
inoculation of the algae was made in the flasks about two weeks
before the inoculation vrith the azotobacter cultures in order that
the algae might make a sufficient gTowth to supply Vie bacterial
cultures with the proper amount of carbonaceous material. To
prevent contamination by the oat and red clover plants, the seeds
were soaked three minutes in a 1-500 solution of mercuric chlor-
ide, washed in sterile distilled water three times and then planted
in sterile agar plates. By this means the seeds were sprouted
and those which were contaminated were discovered and rejected.
The sprouted seeds were transferred from the plates to the flasks
by means of the platinum needle. A block of the agar containing
the sprouted seed was cut out and placed in the proper position
on the medium in the flask. The flasks were then carefully ob-
served for five days tO' insure the absence of contamination.
As all the flasks contained growing plants no attempt was made
to exclude the light, but neither were they placed in the direct
sunlight. They were kept on a table about eight feet from a
large window facing the west. All the flasks were plugged with
non-absorbent cotton and after inoculation a cap of paraffined
paper was placed over the mouth and held in place with a rubber
band. While the plants did not develop rapidly the oats grew
much faster than the clover for about three weeks, after which
time both began to lose chlorophyl and by the end of the five
weeks' experimental period, the majority of the plants had died.
The oats and clover in the flasks inoculated with A. chroococcum
(HCM) and the clover in the flasks inoculated with B. radicicola
showed a slight gain in growth and altho far from vigorous at
the end of the experiment were still alive and growing slowly.
ORGANISMS USET>.
The organisms used were A. chroococcum (HCM), A. vine-
landii, A. heijerinckii and for the purpose of comparison, B. radi-
cicola isolated from the nodules of sweet clover. The latter were
isolated and purifled especially for this series, and introduced to
compare the effects of symbiotic and non-symbiotic organisms on
the growth of the plant used. The results secured with it, how-
ever, were of no great significance.
38
After the bacteria had remained midisturbed in the flasks for
five weeks, transfers were made directly from the flasks into 50
CO. of the nitrogen-free dextrose solution, incubated for three
weeks, and the nitrogen fixed determined in the usual manner.
The total amount of nitrogen fixed by the bacteria themselves, as
well as the amount fixed by the bacteria but due to the stimulative
action of the plants on the bacterial activities, is shown in table
VII. There was a stimulation of the nitrogen fixing power of the
organisms due to the presence of a growing plant, especially no-
ticeable in the case of A. vinelandii and A. chroococcum (HCM)
and to some extent in the case of A. heijerinckii. A. vinelandii
was stimulated thruout the entire series except when grown in
sand in the presence of the algae. The oats and algae showed no
TAB'LE Vn-THE EFFElOT OF' GEIOWKSTG' PLlAW^T'S; ON THIE NITROGEN FIXING
FOWE'Ri OF PiU.RE' ICUXTUIRIES:
Inoculum
Medium
Plant Used
(a)
(b)
Nitrogen Fixed in Mgs.
(Av)
Z-5
Algae agar
AJgae
Vinelandii
Vinelandii
Vimlandii
Vinelandii
Vinelandii
Vinelandii
Vinelandii
Vinelandii
Olirooc'occmn (HCM)
Chroococcum (HCM)
Ohroococcum (HOM.) ._-
Chroococcum (HOM) ..-
Chroococcum (HCM) ..-
Cbroococcum (HCM) .—
Chroococcum (HOM) _.-
Chroococcum (HCM) .._
Beijerinckii
Beijerinckii
Beijerinckii
Beijerinckii
Beijerinckii
Beijennckii
Beijerinckii
Beijerinckii
B. rad
B. rad.
B:. rad
B. rad
rad..
check
sand check
agar.
sand-
agar-
agar-
clo.
clo.
ClO'.
ClO'.
clo.
rad., S. clo.
agar
sand
and
and
agar
sand
agar
agar
igar
sand
sand
sand
agar
sand
agar
agar
Tgar
sand
sand
sand
agar
agar
agar
sand
sand
Isand
check
check
oats
red clover-
oats
red clover-
check
check
oatsi
red clover. -
algae
oats
red clover--
algae
check
check
oats
red clover- -
oats
red clovsr-
oats'
red clover-
oats
red clover.
0.84
0.84
0.^
1.40
0.98
1.19
1.12
0.98
1.05
1.40
1.40
1.40
3.66
3. 53
3.59
2.10
2.38
2.24
4.20
lost
4.20
4.20
4.06
4.13
4.48
4.06
4.27
2.52
2.80
2.66
O.OO
O.OO
O.OO
0.28
0.14
0.21
0.28
0.44
0.30
1.82
3.22
3.52
S.92I
3.50
3.71
0.28
0.56
0.42
0.28
lost
0.28
3.22
5.18
4.20
0.00
0.00
O.OO
0.28
0.42
0.35
0.14
0.00
0.07
1.40
1.40
1.40
O.OO
O.OO
0.00
1.39
1.39
1.39
1.39
1.39
1.39
1.39
1.40
1.40
0.14
0.14
.014
0.14
0.a4
.014
o.m
0.56
0.56
0.42
O.OO
0.21
0.14
0.00
0.07
0.42
0.50
0.4191
0.84
0.84
1.19
1.19
3.5t
1.05
2.54
2.24
1.05
1.19
3.36
1.09
2.31
4.13
1.40
2.73
4.27
1.40
2i.8'7
1.47
1.40
0.07
0.36
0.00
01.35
2.52
0.00
2.52
2.S7
0.00
2.87
0.42
0.21
0.21
0.28
0.21
0.07
3.01
0.21
2.80
0.07
0.00
o.or
1.40
0.00
1.40
1.39
0.35
1.04
1.39
0.3S
1.04
0.21
0.35
0.14
0.14
'o'.n
0.07
39
difference when gTown on the agar and in the sand medium the
greatest stimulation was produced by the red clover. The activi-
ties of A. chroococcum were stimulated to the greatest extent by
the presence of algae in both sand and agar, the oats gave a poor
stimulation in both cases, and red clover gave good results in the
agar but not in the sand.
The nitrogen fixing power of A. 'beijerinckii was retarded by
the presence of algae, but was stimulated by red clover in both
the agar and sand. The oats stimulated this organism only when
grown on the agar. The nitrogen fixing power of B. radicicola
was so low thruout the experiment that the results are not con-
sidered.
CONCLUSIONS FROM LABORATORY STUDIES.
1. Transfers made on a nitrogen free dextrose agar more often
than once each week were detrimental to the nitrogen fixing power
of azotobacter and other large celled nitrogen fixing organisms
of the same type.
2. Transfers made once each week into a pure sand medium
containing some carbonaceous material were beneficial to the
nitrogen fixing power of the azotobacter in general, but the effect
on A. heijerincMi was detrimental.
3. The nitrogen fixing power of A. vinelandii was stimulated
to a marked extent when grown in large flasks for five weeks in
the presence of red clover and oats on both agar and sand. It
was stimulated by the presence of algae when grown on agar but
not when grown on sand.
The nitrogen fixing power of A. chroo'coccum was stimulated
markedly when grown on agar for five weeks in the presence of
growing oats and red clover, but to a less extent when grown with
the same plants in sand. The greatest stimulation for this organ-
ism was produced by growing it in the presence of algae in either
sand or agar for the same period of time.
5. The nitrogen fixing power of A. beijerinckii was stimulated
by the presence of red clover when grown on either sand or agar,
and by oats when grown in sand. Algae in either agar or sand
appeared to have a depressing effect on the nitrogen fixing power
of this organism.
GBEENHOUSE STUDIES.
At the conclusion of the first experiment the eight organisms
used in the laboratory series 1, 2 and 3 were also inoculated into
soils in pots in the greenhouse. Ground oats straw or ground
clover hay was added to these soils and the nitrogen fixing effi-
ciency of the organisms both in fallow soils and in the presence
of growing oats plants determined. Three experiments were car-
ried out in this test, as soon as the soil in which one crop had been
40
grown was sampled, it was immediately reseeded and another
crop grown. Strict account was kept of the amount of nitrogen
added in the seed and in the organic matter. The dry weight of
the crop and the N. content as well as the nitrogen content of the
soil was determined at the end of each experiment.
The soil used thruout the experiment was of the type classified
by the United States Bureau of Soils as Miami silt loam, and
according to tests in the laboratory did not contain azotobacter
or any similar organisms. A large amount of this soil was thor-
oly air dried^ sieved and mixed. Ten pounds were placed in each
of eighty glazed pots, seventy-two of which were given the fol-
lowing treatment : Half, or thirty-six pots received an applica-
tion of 22.68 grams ground oats straw, and the other half received
an equivalent amount of ground red clover hay. This application
(22.68 grams) was equivalent to a five-ton application of this ma-
terial per acre. The ground material was thoroly incorporated in
the soil, which was packed firmly in the pots. The pots used were
«lazed on the inside and made tight so there was no loss by
leaching, neither was there any drainage provided.
METHODS OP INOCULATION.
The inoculum used was the dextrose solution described above.
1500 c e were placed in each of six 2 L. flasks, inoculated with the
organism desired and incubated for seven days. Microscopic ex-
aminations were made at the end of the incubation period to in-
sure vigorous growth and the purity of the culture. 150 cc of
the solution was used as the inoculum for each pot. This was
poured over the surface of the dry soil and washed into it by the
addition of sufficient water to bring the moisture content up to
the optimum, in this case 25%. The pots were then weighed,
covered with a cloth, and allowed to remain undisturbed for three
days, in order to permit the moisture to become thoroly distrib'-
uted thruout the soil. The pots were then arranged in the follow-
ing manner and seeded to oats.
Thirty grains of Early Champion oats were planted in each
pot at each seeding. They were planted at five points. One in
the center of the pot and the other four were arranged between
the center and the edge at equal distances apart. Six seeds were
planted at each place and when the plants appeared they were
thinned out and but one plant left in each place. The discarded
plants were allowed to remain and decay on the soil in the pot
from which they were drawn.
The length of the growing period was determined by the ap-
pearance of the seed-bearing spike. This period varied slightly
in each of the series, the first closed in sixty-three days, the second
x'n sixty-nine days, and the third in seventy days after planting.
41
PLAN OP EXPERIMENT
Pot. No.
Treatment
Inoculation
1— 3
2— i
5h- 7
6— 8
9—11
10—12
13—15
14—16
17—191
18—20
21—23
22—24
25—27
26—28--
29—31
30— .32L
33-33
34—36
37—39
38-^0
41—43
42—44
45-4T
46—48
49-51
50—52
5S—5S
54—50
57—59
£8—60
61—63
62—61
65-67
66—68
69—71
70--72
73^74
75—76
77—78
79—80
Fallow
iCropped
Fallow
Oropped
Fallow
Cl-opped
Fallow
lOlropped
Fallow
iCtopped
Fallow
Cropped
Fallow
(Oropped
Fallow
iCtropped
Fallow
'Cropped
Fallow
Cropped
FaUow
Cropped
Fallow
iCtopped
Fallow
lOtopped
Fallow
Ctopped
Fallow
Chopped
Fallow
Cropped
Fallow
Chopped
Fallow
(Ctopped
Fallow
Cropped
Fallow
iCtoppeQ
Oats straw
Oats straw
Olover hay
Clover hay
Oats straw
Oats straw
Clover hay
'Clover hay
Oats straw
Oats straw
'Clover hay
Clover hay
Oats straw
Oats straw
'Clover hay
Clover hay
Oats straw
Oats straw
Clover hay
Clover hay
Oats straw
Oats straw
Clover hay
Clover hay
Oats straw
Oats straw
Clover hay
Clover hay
Oats straw
Oats straw
iClover hay
Clover hay
Oats straw
Oats 'straw
Clover hay
Clover hay
Oats straw
Oats straw
Clover hay
Clover hay
A. chroococcum (HCIM)
A. chroorocciim (HOM)
A. cliriKirdcciini (HOM)
A. clii-c)<)c(.cciiiii eH'OM)
A. chroococcum
A. chroococcum
A. chroococcum
A. chroococcum
A. beijerinckii
A. beijerinckii
A. beijerinckii
A. beijerinckii
A. vinelandii
A. vinelandii
A. vinelandii
A. vinelandii
26 ID.
26 'D.
26 D.
26 r>.
27 D.
27 D.
27 D.
27 D.
23 D.
22 D.
22 D.
22 D.
4 D.
4 I>.
4 D.
4 D.
Mixed culture
Mixed culture
Mixed culture
Mixed culture
Cheek
Check
Check
Check
The pots were watered with tap water ahout every other day
and were weighed weekly. The loss in weight was replaced with
water in order to keep tlie moisture content at the optimum. The
growth of the plants was carefully noted and recorded by means
of photographs at different periods. The harvested plants were
dried, weighed, and tlie total nitrogen content determined by the
Kjeldahl method.
At the close of each series of experiments the soils were re-
moved from the pots, placed on a sterile oil cloth, thoroly mixed,
sampled and returned to the original pot. The sample taken at
this time approximated 500 grams dry weight. The pots were
seeded again as soon as possible and the experiment continued.
During the short period between sampling and reseeding the
moisture content was kept at the optimum.
42
The preliminary analyses, showing the nitrogen content of the
original air dried soil, and of the same soil mixed with the ground
oats or clover are as follows :
22.68 grs. ground oats straw contained 0.1416 grs. N. av. 6 dets.
22.68 grs. clover hay contained 0.4153 grs. N. av. 6 dets.
10 lbs. original soil contained 2.3494 grs. N. av. 6 dets.
10 lbs. original soil + oats straw contained. . 2.4910 grs. N. av. 6 dets.
10 lbs. original soil + clover hay contained. .2.7647 grs. N. av. 6 dets.
ACTION OF DENITRIFYING BACTERIA.
Some of the plants were very much stunted in their growth
and an experiment was conducted to determine whether this was
due to action by the denitrifying organisms. Samples weighing
about six or eight grams were drawn from near the center of each
pot by means of a sterile corkborer and placed immediately in
sterile tubes. Sterile water was added and a soil suspension made
from which inoculations were made into Giltay's denitrifying
solution. The solution was incubated three weeks and the amount
of nitrate nitrogen as well as the total nitrogen determined, the
first by the aluminum reduction method of Potter and Snyder,
and the second by the official method. The aluminum reduction
was carried out by aeration, thus leaving the original solution
available for analysis for total nitrogen. The results given in
table VIII show that the denitrifying organisms were not the
limiting factor in the growth of plants. Only the five soils in
pots Nos. 29, 45, 62, 64 and 66 show any great loss in nitrogen
and some of the pots show an actual gain in total nitrogen
content. This gain is particularly noticeable in the soils inoc-
ulated with A. heijerincMi, No. 26 and in the check pots.
FABLE Vni— THE ACTIVITIES OF THE DENITRIFYING BACTERIA IN THE SOILS
THREE WEEKS AFTER THE START OF THE EXPERIMENT
Pot
Nitrate N.
mgs.
N. mgs.
Total N. mgs.
Check
Amt. denitri-
fied
1
0.70
1.05
0.S8
0.91
0.90
0.56
0.70
0.791
0.70
0.42
0.70
0.84
0.86
0.65
0.56
lost
6.02
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
2
3
7.07
1.16
4 "
5 - _ __
5.60
4.20
8.12
7.84
8.14
7.07
7.07
8.96
6.16
7.56
6.51
51.10
8.68
8.54
8.93
7.77
7.49
9.80
7.02
8.21
1.72:
3.13
6
»:
7
8
9
10
0.46
0.74
11
12
13 .
'1.21
14
15
0.02
43
TABLE VIII— Continued.
Pot
N"itrate N.
mgs.
N. mgs.
Total N. mgs.
^, . Amt c
Check fj
enitri-
16 „ . —
1.71
0..56
0.90
0.42'
0.84
0.S4
0.56
0.70
0.78
0.S6
0.06
0.58
0.87
0.56
"1.54,
0.431
0.70
0.56
0.S8
0.79
0.S6
0.90
1.54
0.63
0.63
0.59
0.70
0.98
0.77
1.05
0.77
0..5S
O.W
0.91
0.81
O.EO
0.77
0.49
0.86
1.00
0.89
0.86
0.S6
1.33
0.91
0.78
1.26
1.31
i.v.e
0.9S
0.14
0.70
0.56
-0.84
0.49
0.67
0.87
1.03
0.65
0..51
6.10
7.50
7.70
8.61
8.40
7.91
9.38
7.87
8.12
8.66
9.08
9.24
8.751
9.94
8.23
8.23
8.23
8.23
8.23
8.2s
8.23
8 ''3
0.86
o.n
17
18 —
19
20 -
21 - ..
22
23
25 I"I"I~"III""I
26 -. -
7.21
6.44
7.42
8.04
7.28
5.04
6.80
5.32
9.10
7.98
7.30
7.48
8.62
8.15
5.60
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
0.25
0.93
0.75
27
28
0' 08
29 . . __
2 63
30
31 .-
6. 86
9.53
1.37
32
33
34 . .
8.54
7.56
8.68
7.00
0.44
6.58
9.10
8. ,54 •
9.47
7.86
7.34
8.12'
8.23
8.23
8.23
8.23
8.23
8.23
8.23
35
36 . -
37 -
37
38 .
89
39
40 -
0.11
41
43
4.90
8.19
5.46
8.23
8.23
8.23
0.04
2.77
44 -
6.86
2.80
6.30
6.16
6.02
7.28
7.S4
3.57
7.3E
7.. 35
6.5-8
8.32
8.23
8.23
8.23
8.23
8.23
8.23
8.23
0.39
45 - - -
4.66
46 - -. .
0.88
47
48 . .
0.8»
1.65
49
50
51
7.77
7.70
8.12
7.56
6.44
6.09
5.40
7.70
5.60
S.'S
8.20
8.89'
8.05
7.30
7.09
6.32
S.56
6.46
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23 . .
0.03
53
54
0.18
53 -.
0.83
56
l.U
57
58 —
59
60
1.81
1.77
61
8.23
63 .. .. .
1.96
5.60
4.76
7.28
1.54
7.28
6.16
6.72
4.6-2
8.40
7.98
6.02
7.70
9.80
8.12
2.74
6.86
6.07
8.. 54
2.52
7.42
6.86
7.28
5.46
8.89
8.65
6.89
8.75
10.43
8.63
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
8.23
5.49
63
1.37
64 —
2.10
65 . .
66 ._ .. . . ...
5.71
67
68
0.81
137
69 . -
0.95
70
n . . . - _
2.77
72 - .
73
1.34
74
75 . . ..
76
*'So denitrification is shown by :
44
PRELIMINARY TESTS FOR NITROGEN FIXATION.
To discover the action of the bacteria in the inoculated soils
samples were taken from the fallow pots four weeks after t': e start
of the experiment and their total nitrogen content determined.
Table IX shows a gain in the nitrogen content over the original
soil and the check soils but the actual gain due to the action of
the bacteria introduced was very slight. Organisms 22, 4 and
the mixed cultures showed no gain whatever, and the others
showed only a slight gain in those soils to which clover had been
TABLE IX — THE ACTIVITY OP THE BACTERIA IN THE INOCULATED
FALLOW SOILS AFTER FOUR WEEKS.
Grams of Nitrogen Per 10 Pounds of Soil.
Pot
Gi-ams N.
1
2
5
7
9
11
13
]5
17
19
21
23
25
27
29
SI
33
35
3Y
3&
41
43
45
47
49
51
5S
55
57
59
&1
63
65
67
60
n
Checks
73
74
77
78
M O'o
o
.Set.
. C3 O
0643
0643
8942
8304
0643
2281
3411
9581
2281
2281
2134
0219
2281
''239
7666
2772
2f281
0613
8942
8942
2281
1929
9581
7665
8409
9047
7027
5431
8409
8089
5431
.5750
9047
7451
2239
.5536
0043
93-66
0643
Average four checks-
lost
3.2281
3.8942
3.9581
3.2281
3.2281
4.0S96
4.0S57
3.2281
3.06i!3
4.0219
3.9'561
3.2558
3.2239
4. 0857
4.0219
3.2281
3.1920
3.8304
3.8304
e.25S8
3.228.1
3.9561
3.8304
2.8409
2.7679
3.8942
3.6069
2.9047
2.7132
3.5752
3.6069
2.8408
a. 7451
3.1320
3.443a
2.8089'
3.0324
2. 8089
3.2239
3.0643
3.1462
3.8942
3.8942
3.1462
3.2281
4.2203
4.0219
3.2281
3.1462
4.1176
3.9900
3.2419
3.2239
3.9261
4.1490
3.228]
3.1281
3.8623
3.862f
3.2419
3.210:'
3.9581
3.7984
2.8409
2.8393
3.798!
3.5750
2.872?
2.7610
3.5590
3.5909
2.8727
2.745]
3.2079
3.5250
2.6S12
3.0483
2.8727
3.H4]
2.4910
0.5533
2.4910
0.5552
2.7647
1.1295
2.7647
1.1295
2.4910
0.6552
2.4910
0.7371
2.7647
1.4556
2.7647
1.2572
2.4910
0.7371
2.49110
0.6552
2.7647
1.3529
2.7647
1.2258
2.4910
0.7509
2.4910
0.7329
2.7647
1.1614
2.7647
1.3843
2.4910
0.7371
2.4910
0.6871
2.7647
1.0976
2.7647
1.0976
2.4910
0.7509
2.4910
0.7109
2.7647
1.1934
2.7647
1.0337
2.4910
0.8499
2.4910
0.3453
2.7647
1.0837
2.764.7
O.8103
2.4910
0.S818
2.4910
0.2700
2,7647
0.7943
2.7647
0.S262
2.4910
0.3817
2.4910
0.2541
2.7647
0.4432
2.7647
0.7603
2.3494
0.3318
2.3404
0.6989
2.3494
0.5233
2.3494
0.7947
0.7288
0.7288
1.0025
1.0025
0.7288
0.7288
1.0025
1.O025
0.7288
0.T288
1.0025
1.O025
0.7288
0.7288
0.0025
1.002s
0.7288
0.7288
0.0025
1.0025
0.7288
0.7288
1.0025
1.0025
0.7288
0.7288
1.0025
1.0025
0.7288
0.7288
1.0025
1.0025
0.7288
0.7288
1.0025
1.0025
§0.
5 cS <u
o
0.1270
0.1270
0.0083
0.4531
0.2547
0.0083
0.3504
0.2228
0.0221
0.0041
0.1569
0.3818
O.00S3
0.0951
0.0961
0.0121
0.1909
0.0312
0.0312
.0.5672 + N in oats 0.1'. 16=--0. 7288
.0.5872' + N in clo. 0.4153^1. 0025
45
added. This difference may have been due to variation in the rate
of decomposition between the clover and the straw.
At the end of the three growing periods the soil in each pot
was sampled and the total nitrogen content of both the soil and
the entire crop determined. The amount of nitrogen found in
the determinations and its relation to the total amount due to
the bacterial activities is given in three separate tables, one for
each growing period. From these complete tables three condensed
tables have been made as follows : For the first growing period,
table X, for the second growing period, table XI, for the third
growing period, table XII, and a recapitulation table XIII.
TABLE X— THE NITROGEN FIXED BY BACTERIA-FIRST PERIOD
(Condensed from Appendix Table I.)
Treatment
Bact. Inoculum
Used
Grams N. per
10 Pound
s Soil
■p. o
3 a
P
o
2;
" ■« > ft
_c g o o
'" CS S Cj
2:
2;
1— 3—
P oats
C oats
F clover —
C clover
F oats
O oats
F clover
O clover .„
P oats
C oats
P clover ..
O clover
P oats
C oats — _
P clover .
O clover
P oats
O oats
P clover
O clover
F oats
O oats .
P clover
O clover
P oats
O oats
P clover _-
C clover
F oats
lO oats
P clovsr
O clover
P oats
C oats
P clover
C clover _—
P. nothing _— -
O. nothing
P nothing
C nothing
A. chrooe. (HClM) —
-4. chrooe. (HCM) —
A. chrooe. (HCM) —
A. chrooe. (HCM)— .
A. chrooe.
3.3499
2.9S83
4.3058
4.1808
3.2210
3.5380
4.5726
4.4928
5.1701
5.2867
4.4S94
4.5801
3.9267
5.1120
2.4572
4.2388
3.4887
3.5170
4.4823
4.5722
3.6458
3.5720
4.5023
4.3114
3.4101
3.5823
4.4001
4.3114
3.6065
3.50C6
4.4630
4.30-23
3.3865
3.60S8
4.3787
4.4720
3.5942
3.5085
3.3002
3.5403
3.3833
2.9670
4.3488
4.-2684
3. -2582
3.5808
4.6183
4.5787
5.2218
5.28-24
4.4827
4.6613
3.95159
5.1794
2.4817
4.3457
3.5235
0.5133
4.5-271
4.6833
3.6822
3.6217
4.5473
^4196
3.4442
3.5519
4.4441
4.3866
3.6423
3.. 5323
4.5076
4.3965
3.4203
3.60-21
4.4123
4.5794
3.6301
3.. 5689'
3.3332
3.6097
3.6232
3.7309
3.8963
4.0046
3.6232
3.7309
3.8963
4.O046
3.6232
3.7309
3.8963
4.0046
3.6232
3.7309
3.8063
4.0046
3.6232
3.7309
3.8963
4.0016
3.6232
3.7309
3.8963
4.0046
3.6232
3.7-209
3.8963
4.0046
3.62.32
3.7309
3.8963
4.0046
3.6232
3.7309
3.8963
4.0046
2— 4.
5— 7-
6— S-.
&-11.-
I(t— 1'^
0.4525
0.-2638
13-15--
14—16-
17—19 -
A. chrooe.
A. chrooe.
A. beyer. .. -. -
0.7220
0.5741
1..5966
18—20—
A. beyer. ..
1.5515
21—23—
')9 — Oi ,
A. teyer.
A. beyer.^ .._
0.5884
0.6567
25—27-
A. vine. - -
0.34-27
26—28-
A. vine. _.
1.4485
29^—31
A. vine.
30—32-
A. vine. -,
0.3411
33—35 -
No. 26 .
34—36 -
No. 26
37— 3&-
No. 26 -
0.6808
3S^}0..
41—43-
42-44--
No. 26
No 27
No. 27
0.6787
O.O590
45-^7-
46—48 -
No. 27
No. 27
0.6510
0.4149
49—51-
No. 22 .-
50—52—
53—55-
No. 22
No. 22
0.5478
.S4-56-
57— 59-
58—60
I.o. 22
No' 4-IZ-II-II-I."
No. 4
0.3820
0.0193
61-63-
6113
62-61
No 4 -
3919
65—67-
66-68-
Mixed culture
Mixed culturs
Mixed culture
Mixed culturs
Check -
6»-7l-
70—72—
73—74
0.5160
0.5748
7.5—76-
Cheek
77— 7S-
79^80-
Check
Check
46
FIRST GROWING PERIOD.
The determinations for this period are shown in appendix
table I and in condensed table X. As indicated by table IX,
there was a steady increase in the total amount of nitrogen fixed
in all the soils. This increase is still more marked if the last
columns of tables IX and X are compared. The bacteria were
increasing-ly active in fixing- the free atmospheric nitrogen and in
practically every case the total amount fixed due to the bacterial
solution was more than doubled during- the latter five weeks of
this series.
These activities may be divided into two classes, as the bacteria
were more markedly affected by the presence of clover hay or
of oats straw. In the first class A. ckroococcmn, A. chroococcum
{HCM), No. 26, No. 22 and the Mixed Culture stood out prom-
inently. None of these four organisms showed any fixation due
to the presence of the decaying oats straw, but they did show ap-
preciable g'ains due to the presence of the clover hay. The pres-
ence of the oats straw had apparently either inhibited the activi-
ties of the organisms or increased the activities of the other forms
that are incapable of fixing nitrogen for their own use and have
utilized that fixed by the inoculating organisms. Organisms 4
and 27 showed a decided stimulation due to the clover hay and
were able to utilize the oats straw as a source of energy,
A heijerincki and A. vinelmidii were more markedly affected by
the presence of oats straw. The stimulation of the activities of
the former due to the presence of the decaying clover was parallel
to that of the other organisms, and in addition the presence of the
decaying oats straw stimulated its nitrogen fixing powers to over
250% of that of any other organism in the series with the single
exception of A. vinelandii. On the other hand, A. vinelandii,
while showing a marked stimulation due to the presence of the
oats, also showed that the clover hay affected its activities much
the same as the oats straw affected the other organisms, that is,
the presence of the decaying clover hay in the cropped soils, de-
creased its nitrogen-fixing power, and in the fallow soils, com-
pletely inhibited it.
SECOND GROWING PERIOD.
The results for this period are shown in appendix table II, the
more important parts of which are repeated in condensed table
XI. In comparison with the first gTowing- period the results for
the second period are decidedly lower thruout the second series.
Not only are the total amounts of nitrogen found lower, but also
the total amount of dry matter produced in the crop, indicating
a possible direct relation between bacterial action and crop yields.
These low results are explained by the fact that this series as
47
grown during' the hottest part of the summer, the pots being
planted in the latter part of June and harvested during the earl-
ier part of August. The results confirm those given in Table X
except that in this series the only organism stimulated by the
presence of the decaying oats straw was organism 27 in the
cropped pots. Each of the inoculated organisms showed a direct
stimulation due to the presence of the clover hay.
The organisms may be divided into two classes according as
their activities are stimulated or retarded by the presence of
TIAJBCLE XI— THE NinHiOO'EJST MXEID BY BiAlCITIElRlLA— iSECIOiND' PERIOD.
('Oondensed from appendix Table 2.)
Dupli-
cate
Pots
Tieatment
Bacterial inoculum
used.
Grams Nitrogen per 10 lbs. soil.
g > 2
sou
£ Z't
tic 2
2g^
•^2
1— 3—
2— 4._
&— 7...
&- 8...
9—11..
10-412...
13—15..
14^16...
17—19...
IS— 20..
21—23..
22—24..
25—27.-
2&— 28„
291—31...
iO— 32..
33—35..
34—36..
87—39..
38—40..
41—13..
42—44-.
45—47-.
46^48..
49^51-
50^52..
53—55..
54—50-
57—59..
5S— 60.-
61—68-.
62^64-.
eo'— 67-.
66— 68-.
69—71-
70—72-
73—74-.
75-76-.
77—78-.
79—80-
oats
oats -.
clover
clover
oats
oats
clover
clover
oats
oats
clover
clover
oats
oats
clover
clover -....
oats
oats
clover
clover
oats
oats
clover
clover
oats
oats
clover
clover
oats
oats
clover
clover
oats
oats
clover
clover
nothing- ...
nothing
nothing
A. chroocoecum (HCIM).
A. chroocoecum (HiOM).
A. chrooeoceum (HICM).
.4.. chroocoecum (HIOM).
A. chroocoecum
A. chrooeoceum
A. chroocoecum
A. chroocoecum
A. Beijerinckii
A. Beijerinckii
A. Beijerinckii
.4. Beijerinckii
A. vinelandii
A. vinelandii
A. vinelandii
A. vinelandii
No. 26
No. 26
No. 26
No. 26
No. 27
No. 27
No. 27
No. 27
No. 22
No. 22
No. 22
No. 22
No. 4
No. 4
No. 4
No. 4
Mixed culture
Mixed culture
Mixed culture
Mixed culture
Check
Cheek
Check
nothing ... Oheck
2.7340
3.0128
2.8218
3.0916
3.3205
3.6592
3.3123
3.6402
3.6867
2.9607
e. 54791
2.7947
3.5242!
3.8836
3.1813
3.4901
2.7155
2.9924
2.6134
2.8626
3.3343
3.6743
3.21681
3.5857
2.8239
3.1115
2.6936
2.9763
3.3205
3.6591
3.4143
3.6822
2.7547
3.0350
2.8912
3.1730
3.4921
3.8492
3.0648
3.3747
2.7015
2.9770
8.O508
3.3429
3. 4487
3.8004
3.5089
3.8613
2.7083
2.9845
2.8804
3.170O
3.3977
3.7442
3.2667
3.5925'
2.6146
2.8812
2.8246
3.1262
3.3998
3.7465
3.1740
3.4913
2.5210
2.7781
2.6935
2.9S10
3.21396
3.6031
3.38521
3.7159
2.8835
3.1115
2.7518
3.0.537
2.9758
3.2798
2.7736
3.0364
3.3370
3.1866
3.6107
3.4603
3.3370
O.0485
0.1799
3.1866
3.6107
3.4603
3.3370
'"0^2729
0.0298
3.1866
3.6107
3.4603
3.3370
3.1866
3.6107
3.4603
3.3370
O.O630
0.1254
"o.0384
0.1719
3.1866
3.6107
3.4603
0.2385
3.3370
3.1860
3.6107
3.4603
3.3370
3.1866
0.1503
0.1897
0.4010
3.6107
3.4603
3.3370'
3 I860
0.1335
0.1322
3.6107
3.4003
3.3370
3.1866
0.1358
0.0310
3.6107
3.4603
0.2550
48
growing plants such as clover. In the first class are included A.
chroococcum {RCM) , A. heijerinckii, A. vinelandii, No. 27 and
the mixed cultures. The first three organisms have had their
nitrogen-fixing powers stimulated by the presence of the plants
in practically the same ratio and have fixed similar amounts in
both the fallow and cropped soil. A. h&ijerincJdi showed the
highest fixation of any of the eight for this series. The mixed
cultures showed no fixation whatever in the fallow soils, but
quite an appreciable amount in the cropped soils. No. 26, on
the contrary, fixed an appreciable amount of nitrogen in the
fallow soils but none at all in the presence of the growing oats
plants. A. chroococcum. and No. 4 showed the same stimulation
under practically the same conditions, namely, that they possess
a greater nitrogen-fixing power in the presence of decaying clover
if no crop is grown upon the soil, while No. 22 was apparently
neither stimulated nor retarded by either fallow or cropped con-
ditions, but was affected by the presence of the decaying oats
straw.
THIRD GROWING PERIOD.
The results for this period are shown in appendix table III
and condensed table XII. The total nitrogen content of the soil
according to tables IX, X and XI, increased steadily through-
TABLE XII— THE NITROGEN FIXATION BY BACTERIA— THIRD PERIOD
(Condensed from Appendix Table III).
Bacterial Inoculum
Grams Nitrogen per 10 Pounds Soil
+ ,^
-Sll
>>
Treatment
Used
a
l£-^
.^ O'S
"S 53
Ǥ
3
o
in s
amt.
mov
crop
.s-^l
■^
^•^
fi;
^
1 ^
;z;
^
<
1
F oats
A.
chrooc. (HCM)
2.56-2:6
2.8618
2.8502
0.0146
R
F oats
A.
chrooc. (HCM)
2.7342
3.3166
2.8502
0.4063
0.2405
'O oats
O oats
i
chrooc. (HCM)
2. 3643
2,9647
3.3284
4
A.
chrooc. (HCM)
2.1930
2.6754
3.32S4
5
F clover _
A.
chrooc. (HCM)
2.0255
3.5486
3.1239
0.4247
7
F clover
A.
chrooc. (HCM)
2.8274
3.4296
3.1239
0.3057
0.3652
6
O clover .
A.
chrooc. (HCM)
S.2852
4.0691
3.6021
0.4670
S
O clover
A.
chrooc. (HCM)
2.S688
3.16142
3.6021
0.0121
0.2395
9
F oats — -
A.
Chroococcum..
21.7489
3.3344
2.8502
0.4842
n
F oats _
A.
Chroococcum..
2.3103
2.8024
2.8502
0.2421
10
oats
A.
Chroococcum..
2.3652
2.9149
3.3284
12
O oats
A.
Chroococcum..
2.S7S5
2.9537
3.3284
13
F clover
A.
Chroococcum...
2.8928
3.5089
3.1289
0.3850
13
F clover
A.
Chroococcum..
3.1540
3.8265
3.1239
0.7026
0.5438
14
O clover
A
Chroococcum..
2.8489
3.5649
3.6021
Ifi
'O Clover
A
Chroococcum.
2.7501
3.4326
3.6021
17
F oats
A.
beijerinckii
2.5789
3.1279
2.8502
0.2777
19
F oats
A.
beijerinckii
2.4S05
3.0088
2.8502
0.1586
0.218B
18
4
heijerinckii
beijerinckii
2.2943
2.8469
3.3284
20
O oats
A.
2.07317
2.5764
3.3284
21
F clover
A.
beijerinckii
3.5208
4.2705'
3.1239
1.1466
2i3'
F clover ..
A.
beijerinckii
S.0S68
3.6836
3.1239
0.5597
0.8532
49
TABLE XII— Continued
Treatment
Bacterial Inoculum
Used
Grams Nitrogen Per 10 Pounds Soil
-0
-a 1 -°
5i ^"S--
w
3
■" tj > a.
■-Ill
.5 c c
^t
O
a.
. Jl
>-°
12
2i
^
z
<
22
clover
A. beijerinckii
2.7429'
3.4256
3.6021
24
clover
A. beijerinckii
2.7694
3.4584
3.6021
Wi
F oats
A. vinelandii
A. vinelandii
A. vinchindii
2.5431
2.447S
2.3917
2 3491
3.0847
2.9601
2.9781
2.9494
2.8502
2.8502
3.3284
3.3284
0.2315
0.1189
97
F oata -
0.1767
26
oats
28
oats
F clover
29
A. rinrliiniHi
S.0172
3.6598
3.1289
0.5359
31
F clover
A. r'nuUmiUi
3.0711
3.7252
3.1239
0.6013
0.5686
80
clover
A. rhir'KiniHi
2.7429
3.4284
3.6021
32
clover
A. rhicUindii
2.7363
3.3959
3.6021
33
F oats
No. 26
2.4609
2.9850
2.80O2
0.1348
S3
F oats
No. 26
2.3823
2.8887
2.8502
0.0385
0.0862
34
oats
oats
No. 26
]So. 20 ,
^*b. 25
2.4848
2.3321
2.9S95
3.0673
2.9746
3.6162
3.3284
3.3284
3.1239
.%
' 0^4923
m
F clover
,
39
F clover
No. 26
2.0164
2.4459
3.1239
0.2463
38
lO clover
No. 26
2.7749
S.4477
3.6021
40
'O clover
No. 26
2.8877
3.5&S8
8.6021
41
F oats
No. 27
No. 27
2.4478
2.4871
2.9691
3.0169
2.8502
2.8502
0.1189
0.1667
43
F Oatsi
0.1428
4f1
oats _,
oats
No. 27 ..
2.4583
2.4917
2.8143
3.0736
3.1595
S.4137
3.3284
3.3284
3.1289
44
No. 27 .
No. 27
45
F clover
0.2898
47"
F clover
No. 27
3.0564
3.7074
3.1239
0.5835
0.4366
46
clover „„
No. 27 .
3.0181
3.7421
3.6021
0.1400
48
iO clover
No. 27
2.8621
3.5807
3.6021
0.0700
49
F oats
No. 22
2.4961
3.0278
2.8502
0.1776
51
F oats
No. -221
2.2186
2.6911
2.8502
0.0888
50
oats
oats .
No 221 .
2.5580
2.4451
3.1751
3.0758
3.32S4
3.3284
5?;
No. 22
53
F clover
No. 22!
2.7230
3.3029
3.1239
0.1790
55
F clover
No. 23
2.8667
3.4773
3.1239
0.3584
0.2662
54
clover
clover _.
F oats
No. 23
No. 23
2.7893
2.6236
2.3430
3.5060
S.29S3
2.8321
3.6021
3.6021
2.8502
5fl
57-
No. 4
59
F oats
'O oats
No. 4 .
2.4094
2.3170
2.60S7
2.9126
2.8086
3.3108
2.8502
3.3284
3.3284
0.0624
0.0312
58
No. 4
No. 4
60
lO oats
_.
fil
F clover
F clover
clover
No. 4
3.0173
2.8208
2.8877
3.6599
3.4216
3.6172
3.1239
3.1239
3.6021
0.5860
0.2977
0.0151
m
No. 4.^
0.416S
62
No. i
M
a Clover
F oats
No. i_
2.8224
2.3496
3.5098
2.8501
3.6021
0.0076
65
Mixed culture
2.8502'
67
F oats
Mixed culture
2.3234
2.8183
2.8502
m
oats -
Mixed ciiltiire
2.4381
3.0435
3.3284
fiS
lO oatsi - .
Mixed culture
2.3586
2.89S7
3.3284
69
F clover
Mixed culture
3.0761
3.7313
3.1239
0.6074
71
F clover
Mixed culture
2.7831
3.3759
3.1239
0.2520
0.4297
70
2.8366
3.5867
3.6021
72
C clover .
Mixed ci.lture
3.0306
3.7664
3.6021
0.1643
0.0822
73
F nothing-
Check
2.3216
2.8161
*
*
*
74
F. nothing
Check
2.0944
2.5405
*
*
*
77
F nothing
Check
2.1074
2.5568
*
*
78
F nothing
Check
2.4085
2.9215
*
75
nothing
Check
2.7760
3.3649
!t
t
t
7fi
nothing
Cheek
2.5375
3.1041
+
+
+
79
nothing
Check
2.4911
3.1522
t
t
t
80
nothing
Cfteek
2.4901
3.1362 t ' t t
•Average four fallow checks 2.7086.
■fiAverage four cropped checks 3.1868.
50
out the first growing period, declined somewhat during the
second, and according to table XII, there was a pronounced ten-
dency to increase again during the third period. The crop re-
sponse of this last period of growth confirms the results of the
determinations, the amount of dry matter produced being prac-
tically midway between the production of the first and second
growing periods. Figs. 1-6, which show the growth of oats in rep-
resentative pots for the three periods, show that the first crop
when ready to harvest was in the majority of cases leafy and
heavy and showed a decided tendency to lodge ; the second crop
in the same stage of growth was somewhat dwarfed in appearance
and with no indication of leafiness or weakness of stem ; the third
crop, while not as heavy as the first, showed all of its characteris-
tics except that as a whole the production was more uniform and
did not show the variation in the total dry weight of the harvested
crop. The bacterial activities, which are plotted in the tables
shown in fig. 7, varied in the same proportion as the crop re-
sponse of the treated soils, being practically parallel with the
production of the dried weight cf the crop. The activities in-
creased during the first growing period, declined thruout
the second, but increased again during the third. The discus-
sion of the third and last period of growth will be a combina-
tion of the activities of the inoculated bacteria as discussed in
the first and second growing periods.
The last column in table XII indicates that each inoculated
bacterial culture acted without exception in the same general
manner instead of showing the expected variations. All of the
inoculated bacteria fixed greater amounts of nitrogen in the
soils to w^hich clover hay was added as organic matter than in
soils that were treated with the same amount of oats straw, and
the growing crop on these soils reduced the nitrogen-fixing power
of each and every one of these organisms. The activities of any
one of the eight organisms used during the third period of growth
would be an accurate measure for the activities of any of the
others, a fact not even indicated in the other periods of growth.
Conclusion: Table XIII, recapitulating tables X, XI and
XII, shows that inoculation, especially in fallow soils to which
clover hay or oats straw was added, is not only possible but
practical. The amounts of nitrogen shown in these tables are
the actual amounts fixed by the organisms in ten pounds of soil
and if these amounts are calculated on a 2,000,000 pound acre
basis, the result is distinctly profitable. With proper soil condi-
tions the greenhouse experiments can be duplicated in the field.
All of the organisms have shown an appreciable fixation of
nitrogen but A. beijerinckii and A. vinelandii have been de-
cidedly the most active. This finding confirms the suggestion of
51
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52
Fio. 1. Oats at end of first growing period, immediately before harvest; in pots 2, 6, 10. 14, IS, 22
26, 30, 24, 38
\U
Fig. 2. Oats at end of first growing period, mmediately before harvest; pots 44, 48, 52, 56, 60, 64,
68, 72, 76, 80
Fig 3. Oats at end of second growing period, immediately before harvest; pots 2, 6, 10, 14, 18, 22 ,
26 30, 34, 38
Fig 4 Oats at end of second growing period, immediately before Harvest; pots 42, 46, 50, 54, 58,
62, 66, 70, 75, 79
\ -v.
1/7' \
\\\
r--^r
IG. 5. Oats at end of third growing period, mmediatelv before harvest; pots 4, 8, 12, 16, 20 24.
28, 32. 36, 40
Fig. 6. Oats at end of third growing period, immediately before harvest; pots 44 48, 52, 56 60
64, 68, 72, 76, 80
54
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LEGEND.
Oata added as monure
yjote HepffaHooJ
OqH adaea as manure
pais kept cmpped-- «-
Oovei-Qddsd as manure
oofs kept fallow
C/over added os monure
;OoM HepfcmppecJ
tr
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Fig. 7. This graph shows the variation in bacterial activity in the different growing periods
55
Lipman and Brown (41) by proving definitely that these organ-
isms are capable of being profitably inoculated into field soils,
provided that organic matter, carrying a sufficient amount of
nitrogen as a stimulus, is supplied.
Simimary.
1. When three crops of oats were grown continuously on this
soil the nitrogen content of the soil increased during the first
cropping period, decreased during the second, and increased
slightly again during the third.
2. The nitrogen-fixing powers of the bacteria and the crop
response were parallel with the total nitrogen content of the soil.
3. The nitrogen-fixing powers of some types of azotobacter
and other large celled organisms of the same general character,
were stimulated to a greater extent by the presence of decaying
clover hay than of decaying oats straw.
4. The nitrogen-fixing powers of A. heijerinckii and A.
vinelandii were stimulated to a greater extent by decaying oats
straw than by clover hay, especially during the earlier stages of
decomposition. ,
5. The nitrogen-fixing powers of the azotobacter and other
large celled organisms of the same general type eventually be-
came greater in fallow than in cropped soils.
6. The non-symbiotic nitrogen-fixing organisms of the azoto-
bacter group were all eventually influenced in their activities in
the same manner and by the same materials.
7. Soils may be profitably inoculated by azotobacter and
other large celled organisms of the same type, the best effects
being secured in this work by an inoculation with A. heijerinckii
or A. vinelandii.
8. The conditions necessary for the greatest fixation are :
Good environmental factors such as tillage, drainage, etc. ; the
presence of a rapidly decaying organic matter carrying a small
nitrogen content, and freedom from growing plants.
ACID EXTRACT, AMINO, NON-PROTEIN AND POLYPEP-
TID NITROGEN CONTENT OF THE POT SOILS.
Introduction : The nitrogen of the soil is found in many com-
plex combinations, in the determination of which the Bureau of
Soils has isolated a large number of nitrogenous compounds and
many different forms have been discovered. In investigating
methods for the determination of amino acids and nitrates in a
limed and unlimed soil, both with and -without heavy applications
of manures. Potter and Snyder (53) have found that they could
accurately measure the 'amino nitrogen by a modification of the
method devised by Kober and Sugiura (32). They discovered
56
no tendency for the amino acid to accumulate under the condi-
tions of the experiment. Accordingly in the present investiga-
tion determinations were made of the acid extract, non-protein,
amino, and polypeptid nitrog^en of some of the soils inoculated
with the azotobacter cultures used in the g:reenhouse experi-
ments, in order to prove this point and also to discover if the
bacterial action had any effect on the accumulation or disap-
pearance of these nitrogenous forms.
Soils used: Only the three soils inoculated with A. chroo-
coccum, A. heijerinckii and A. vinelandii were analyzed.
METHODS.
Acid extract : Place 166 gr. of air dried soil on a wetted
double filter paper in a Buchner funnel and extract with 600 e. c.
of a 1% HCl solution using g-entle suction. Keep the soil barely
covered with the solution and when extracted, wash with 200 to
300 c. c. of pure distilled water. Dry as quickly as possible,
and determine the nitrogen content of the filtrate jjy the official
salicylic acid method.
Alkali extract: The non-protein, amino, and polypeptid ni-
trogen determinations are based on the amounts extracted by a
1.5% NaOH solution. Shake 150 gr. of the air dried acid ex-
tracted soil with 600 c. c. of the NaOH solution and centrifuge to
a clear solution. At least 210 c. c. cf the clear solution must be
obtained.
Non-protein nitrogen : Pipette off 25 c. c. of tbe alkali extract,
neutralize Avith a sulphuric acid solution and add sufficient tri-
chloracetic acid to make a 2.5% solution. To do this use 4.3 c. e.,
of a 1 3/10 N. H2SO4 solution and 0.75 c. c. of a saturated tri-
chloracetic solution. This method precipitates the proteins which
are removed by filtering. Pipette 10 c. c. of the clear filtrate
into large test tubes, add a couple glass beads, 2 drops of a 5%
CuSO^ solution, 1 c. c. C. P. HoSO^, and approximately 1 gr.
C P. potassium sulfate. Digest and distil as in the regular
Kjeldahl method determining the ammonia colorimetrically.
Amino acid nitrogen : Pipette 80 c. c. of the alkali extract into
100 €. c. measuring flasks, neutralize with strong HCl until
neutral to litmus, add 7 c. c. saturated lead acetate solution, fill
the flask to the mark with concentrated NH^OH and shake vig-
orouly. Allow to settle for a few minutes then pass through
double filter, using gentle suction and obtain at least 80 c. e. of
the filtrate. Measure oft' 75 c. c. of this filtrate, add 25 c. c.
saturated Ba(0H)2 and phenolphthalein as indicator and distill
over steam bath under reduced pressure until there remains a
volume of about 25 or 30 c. c. It is important that the reaction
of the solution throughout this distillation should be at all times
57
alkaline. Discard the distillate, wash residue into 100 c. c. gradu-
ate, cool, make up to 75 c. c, filter quickly to remove all car-
bonates, pipette 50 c. c. into 100 c. c. measuring flasks, make ap-
proximately neutral with N/10 HCl and add 40 c. c. of buffer
solution, stopper tig'htly and keep in cool place, if possible, on
ice. (The buffer is made by dissolving- 0.2 gr. molecules of boric
acid in water, adding 100 c. c. of COo free N/10 NaOH solution
and making up to 1000 c. c. with pure COo free water. Three
volumes of this mixed with one volume of 0/1/N HCl makes the
desired solution.)
Use pure water as cold as possible to prepare fresh the fol-
lowing solution: Place 10-20% copper chloride solution in 20-30
volumes cold Avater, add a few drops phenolphthalein and a sat-
urated solution Ba(0H)2 until the purple color just forms.
Centrifuge, decant oft' the clear liquid, wash with cold water
and recentrifuge, repeating until there is no pink color formed
by the addition of phenolphthalein in the wash water. Suspend
the copper hydroxide in about 100 c. c. cold water and add ap-
proximately 1 c. c. to the cool flasks, shake vigorously, make up
to the mark, and allow to warm up to the room temperature. Fil-
ter through No. 589 blue ribbon filter, pipette oft' 50 c. c. of the
filtrate and determine the copper complex present as shown be-
low as a measure of the amino nitrogen. Pipette off 40 c. c. of
the filtrate for the determination of the polypeptid nitrogen.
Polypeptid nitrogen: Hydrolize the polypeptids into amino
acids by adding approximately 5 c. c. concentrated HaSO;^ to
the 40 c. e. and placing under a steam pressure of 8-10 pounds
for 10-12 hours. Remove the excess acid with a saturated solu-
tion Ba(OH)o keeping the solution slightly alkaline to phenol-
phthalein, filter and wash with carbonated water at least three
times. Evaporate the filtrate to about 35 or 40 c. c, place in
100 c. c. measuring flasks, neutralize with N/10 HCl, add 40 c. c.
buffer solution, 1 c. c. of the copper hydroxide solution in the
cold water as for the amino determinations and determine the
copper present in the same manner.
Copper determination: Place the beakers containing the 50
c. c. on the hot plate, heat to boiling and neutralize with dilute
HNO3. Boil down to about one-half and add bromine water
until a decided bromine color appears, evaporate to about 10-15
c. c, add 20-30 c. c. pure water and a little more bromine water
and evaporate down again to 10-15 c. c. Cool, add 2-3 c. c.
glacial acetic acid, a few crystals potassium iodide, a few drops
of starch solution and titrate immediately with .001/N sodium
thio-sulfate until the blue color disappears. Each c. c. of the
.001/N thio-sulfate solution is the equivalent of 0.000028 gr,
amino acid nitrogen.
58
Preliminary determinations : In addition to the work on the
soils, an unsuccessful attempt was made to determine tlie amount
of non-protein and amino acid nitrogen fixed by the ba-ter:a in-
oculated into the dextrose solution used in the other experiments.
250 e. c. of the dextrose solution was inoculated with the organ-
isms indicated in Table 14 and incubated three weeks at room
temperature. Enough c. p. sodium hydroxide w^as added di-
rectly to the solution to make 1.5% and the determination car-
ried out in the above manner. A slight trace was the greatest
amount found.
This table shows a decided increase in the soils under field
conditions over the same soils in the dry state, the greatest in-
crease taking place during the earlier periods of growth. The
results of these determinations are grouped in three tables, each
showing the amount of the different nitrogenous forms found at
the end of each growing period.
Discussion of results : A comparison of the results given in
Tables XVI, XVII and XVIII, shows that there was a definite
variation of the nitrogenous forms with the length of the time
of cropping. In almost every case the amount extracted by the
acid varied Avith the length of time that the soil had been cropped,
growing smaller and smaller, and the amino and polypeptid
nitrogen gave similar results. The amount of the^e nitrogenous
'MB/LlE XIV— lAlMINO' AlCID AND' NON-PiRlOT'EIN" NITKIO'GEN FIXED' BY THE PUlBE
OULa'UIREiS' IN vSO'I/UTION.
-id
E
Inoculum
Non-protein N.
Amino Acid N.
1
A. chroococcum CBCIMI- _-_
trace
tracs
s?
A. chroococcum CHCIM)
«
A. chroococcuin (DfflCIM) -
4
No. 26
5
No. 26
fi
No. 26
7
A. chroocoecum C.HCM) and
A. ehroocoecum CHCIM) and
A. ehroocoeeum (HCM) and
No. 26
8
9
No. 20
No. 28—
trace
TAKLE' XV— THE' AMOHINT O'V iDIFFlEKENT NITKlOGENOTJiSi PORlMS IN THE SOIL
AT THE' ■BElGiINNKNO OE' THE' 'EXIPEIRIIlM'ENTS, ALISO THE SAMIE SOIL PLtJiS
the: EQUIVALEINT' OE' EH'K TOMS 01ROX.TND' OATS', STKAW OK GlROI]NI>
OLOVEiR HAY ADIDEB' TO' 'J'll i: s.\.\I l"LE'. DEiTE'BMINATIONS BASED ON THE
AiMOiUNT' IN 2ft GlR;. 'OiF' 'TH'i': S.\:iIJ''lvl<7 AND' iRIEIS'UlJT'Si ,KXPIRES!SED IN (MG.
NTTROG'EflSr AND' IN PEK OENT' O'P THE TO'TALi NTTIBOGEN.
Soil
OZ8
c
1 '^
o c
o.S ti
Zi ii E
< « E
£
"o-O M
ix-n E
c
Original . _ _ _ -
12.95
IS. 75
15.40
1.1243
1.1666
1.4424
8.7
8.5
9.3
2.22T5
2.339'0'
2.3475
17.2
17.0
15.2
O.CS40
0.1025
0.1050
0.7
0.7
0.7
0. 210O
0.2550
0.2625
1.6
Original + oatS— _
Original + red
clover hay
1.8
1.7
59
TABLE SVT— (AIMOTHSTTS OF T!H'E iDIFFEIRENT FOKlMiSi OP NffTElOOEJST IN THE
CRIOOPP'BID ANJy FALLOW Ii:N OiOULAT'EID SOILiS AT TCtlEi END OP THE PIBlST
PEIBTOID OP GBOWTIH. (BEISUlLrPS EXPIRESBEID IN M'G. MT'KOiGEN FOUND
AND' IN PEiB CENT OP T'HEI T'O'TIAIj NITfilOGlEIN' CIONTENIT, BASED ON 25 GR.
SAMIPLE.
Pots
5K.
2 2 M
1
2 ti
C r- C
0.~ 3
i
° 6^3
c
Ih
c
H S^
< t:^
c^
k, ii3
Ph
< ^3
Oh
£i.°
IX
9— ll-_.
14.33
2.0242
14.1+
3.1675
22.0
0.1260
0.8+
0.4725
3.2+
10—12—
15.60
1.9181
12.3—
2.6650
17.0+
0.1505
0.9+
0.2975
1.9
13—15—
20.39
2.2151
10.8 +
2.3690
11.6+
0.1400
0.7—
0.4aS0
2.2 +
14—16—
19.81
1.9S18
10.0+
3.0650
15.4+
0.1085
0.5+
O.2O0O
1.0+
17—19—
23.03
2.2939
9.91+
3.5000
15.2+
0.1015
0.4+
0'.2625
1.1 +
IS— 20l__
23.09
1.1121
4.7 +
2.8500
a2.3i+
0.1680
0.7 +
0.2100
0.9+
21— 23-._
19.77
2.3424
11.8+
2.7175
13.8—
0.2810
1.1 +
0.4200
2.1 +
22—24—
20.19
0.S648
4.8+
21.7325
13.5+
0.1400
0.7—
0.3500
1.7+
25—27
17.50
2.4030
13.7+
2.6950
15.4
0.1820
1.0 +
0.4375
2.5
26—28-..
22.54
1.9878
8.S+
2.7425
12. a+
0.2110
0.9+
0.31.50
1.4—
29^31...
20.94
2. 3848
11.4—
2.5150
12.0+
0.1750
0.8+
0.2100
1.0
30^-S2...
118.69
0.7515
4.1—
2.5875
13.8+
0.09SO
0.5+
0.3200
1.7+
TABLE XVn— AM'O'UNTISi OP THE DIPPEBENT PIOIRMISI OP NTTEOGEN IN THE
PALLO'W AND ORiOiPPED' IXOOULATE© SOIL® AT THE: END OP THE SECOND
G'RiOiWING PERIOD. RESIULTS EXPRES'^'ED' IN M!G. N. POUND AND' IN PEK
CENT OP THE TOTAIL N. CONTENT EASED O'N 25 -GEiAM SAiMP'LflB.
Pots
„ c .
2 g M
1 bi
'G re 3
c
2 M
g.S§
a
0-T3 <^
*-'
H SS
< i:3
Oh
2^ 2^
o.
< « S
CU
£-Z B
0-
9-11...
16.45
1.6515
10.0
1.8675
11.1+
O.O70O
0.4+
0.2800
1.7+
10—12...
15.44
lost
3.41S0
22.1 +
0.1110
0.T+
0.3675
2.4^
13-15...
21.48
2.1896
10.2
2.0700
9.0+
0.1260
0.6—
O.160O
0.7+
14—16
19.32'
0.7257
3.V+
3.99'2S
15.5—
O.140O
0.7+
0.1225
0.6+
17—19
16.62
0.9212
5.5+
2.6825
16.1 +
0.4750
2.8+
0.1750
1.1—
18^20
15.84
0.7257
4.6—
3.4075
21.5+
O.02O1
0.1 +
0.3300
2.2+
21—23
19.74
1.7S95
9.1—
3.5000
17.8—
0.1190
0.6—
0.2100
1.0+
22—24
19.78
0.7500
. 3.8—
3.33OT
16.8+
O.063O
0.3 +
0.2800
1.4 +
25—27-..
17.28
1.4727
8.5+
2.6370
15.3—
0.2660
1.5+
0.2100
1.2+
26—28
16.32
0.6030
3.7—
2.6000
15.9+
0.0630
0.4—
0.29'76
1.8+
29—31...
20.31
2.1363
10.5+
2.8330
13.9+
O.091O
0.4+
O.280O
1.3+
30—32
20. 6®
1.1000
5.4 +
3.4900
16.9—
0.0'700
0.3+
0.2275
1.1 +
TABLE XVIIT— AMIO'UNTB OP THE DrPPEiBENT POIRMS OP' NITROGEN IN THE
PALL'OW ANiD CiROiPPED INOCiULA,T'EID' iSiOTLlSi AT THEi EINlD OP THE THIBD
AND LAST GR'O'WTNIG PEIRiTOD. iREISUODTS IBABED' ON 23 GIEAM S'AIMPLE,
EXPRESSED IN IMG. N. POUND' u-m-D IN PEiR CENT O'P TOTAL N. CONTENT.
Pots
Total N.
content
2Sgr.
c
u
1 TJ
o c
2^ £ E
c
'Ev. 'i
< « E
c
a
0-T3 5
c^-s3
c
£.
9^11...
16.93
1.2833
7.5+
2.1475
12.7—
0.0420
0.2+
0.2275
1.3+
10—12...
15.65
0.5773
3.7 —
3.2850
20.9+
0.0490
0.5+
O.210O
1.3 +
13—15...
20.23
0.7485
S.7—
2.3675
11.2+
O.042O
0.2+
0.2450
1.2+
14—16...
18.19
0.6306
3.4+
3.3005
18.1 +
O.091O
0.4t
O.2100
1.1 +
17-^19...
16.90
1.1773
e.9+
2.7025
15.9+
0.0210
0.1 +
0.2625
1.6—
18—20..-
14.94
0.5560
3.7+
3.6823
24.7—
0.06.30
0.4+
O.140O
0.9+
21—23
22.03
1.7727
8.1—
2.8560
VZ.9+
0.0420
0.2'—
0.2800
1.3—
2r2-24-..
18.21
0.7000'
3.8+
3. 03 GO
16.5+
0.3240
1.8—
0.1750
0.9+
25—27
a6.7l
1.506O
9.0+
2.6925
16.1 +
0.0350'
0.2+
0.5950
3.6—
26^28
15.66
0.7212
4.C +
3.6350
23.2+
0.0420
0.31—
0.3150
2.0+
29-31—
20.36
2.2060
10.8+
2.740O
13.4+
O.056O
0.4 +
0.7075
3.4+
3D--32
18.091
0.5939
3.3—
3.4500
11.3—
0.0770
0.4+
0.2800
1.5 +
60
compounds became smaller, as decomposition of the oro:anic mat-
ter proceeded, at a sligiitly faster rate than the total nitrogen
content of the soil became depleted. The non-protein nitrog'eu
also varied considerably, altho' not in the marked degree shown
by the other forms. Neither the oats straw or the red clover hay,
added as manures to the pots, showed any effect on the forms of
nitrogen determined, further than the small amount shown in the
preliminary determinations. If there was a difference in the
soil under field conditions it evidently was too small to he
measured by these methods. It is entirely possible that the
amounts of these complex nitrogenous compounds are rapidly
changing into other forms and that the per cent they bear to the
total nitrogen content remains somewhat constant, varying only
with the amount of organic matter present in the beginning, then
as decomposition proceeds and the more complex combinations
are broken up, this percentage relation becomes smaller and
smaller until it reaches a constant.
Once decomposition had begun in the soil there was absolutely
no tendency for the more complex nitrogenous forms to accumu-
late under conditions approximating those in the field. Instead of
an accumulation there was a steady reduction. How closely this
reduction is coupled with the decay of the organic matter and
what would be the final equilibrium between the total nitrogen
content and the nitrogenous compounds are questions for further
study.
Summary.
1. The acid extracted, non-protein, amino and polypeptid
nitrogen changed into other forms with the advance of decompo-
sition much faster than the total nitrogen contents of the soils
in question decreased.
2. Oats straw and clover hay added as manures at the rate
of five tons per acre had little effect in influencing this change.
3. The amounts of non-protein and amino acid nitrogen fixed
by bacterial cultures in solution were negligible.
4. Bacterial inoculation had apparently no effect on the
amounts of non-protein, amino or polypeptid nitrogen in the soil.
5. There was no tendency for the above forms of nitrogen to
accumulate in the soil under conditions approximating those in
the field.
Acknowledgments: I wish to express my thanks to Dr. P. E.
Brown for his help and suggestions thruout this work and to Dr.
H. S. Potter for his suggestions in the determinations of the com-
plex forms of nitrogen.
APPENDIX TABLE 1.
=====
==
= ^
Found in Tot Soils, Cniculated
1
==
—^
on B
osia of 4491 vj. 'n Fallow and 4536 gr.
1
S = °
1
1
liind of Crop
Grown
in
cropred.—
P..t
Ji
m
izi
2
-"lias
„ % c 0.^
n-c
Inoculum Used and
Pot No.
Determinations
1
fi
2
[2
11
1
Q
>
11.1
ill
i,chrooc. (HCM)
F
P
3.0309
2.7668
4.2744
4.H171
3.3630
3.2079
4.3373
4.2547
3.3499
2.9SS3
4.305S
4.1809
"oroiio"
3.54SD
2.9747
4.3056
4.1073
U.835«"
7.1150"
6.0123"
o.irai"
3.S499
2.9870
4.3058
4-2684
3.3833
2.9S70
4-3488
4.2684
3.6232
3.7309
3.8963
4.0046
Clover
dover
e'. {.'.'.'.'— —
0.2638
A, c/l">-toi.i""'
F
F
■'.SCOl
3.4450
4.6663
■1.4770
3.6S20
3.0310
4.57S)S
4.50S7
3.2210
S.53S0
4.5726
4.492S
""o'oisis"
0.0136
3.2210
3.6244
4.5726
4.1702
"i.noo"'
""eSoo""
1.0564"
l^roS"
3.2210
3.5808
4.5726
4.5787
3.2532
3.5S0S
4.6183
4.57S7
3.6232
3.7309
3.8963
4.0046
11
ID-IZ
13-lS
14-lC
0at3
Clover
Clover
0.7220
0.5741
4. iictjcwckii
[7-19—
IS-M- - -
21-2S
F
F
C
F
P
c
F
Oats _
Oats
UKiVcr
Clover
Oats ,--
Oats
CiOvcr --
Clover -.-
Oats
5.1230
5.1392
4.5102
4.b8Sl
S.OOOl
5.17.55
2. iOol
4.2S05
3.4tSV
5.2173
6.3343
4.3CS7
4.6722
3.8973
6.0-185
2.44V4
4.1912
3.4SS7
5.1701
5.28617
4.4394
4.E8C1
3.9267
5.1120
2.4572
4.23SS
3.4SSr
"i'.mii'
"o.oise"
"aoiss"
"oroise"'
5.1701
5.2731
4.4394
4-5666
3.0267
5.0984
2.45r2
4-2252
3.48S7
""o^Sso""
"7?oi6o""
"i'.'dim"
S.2050"'
"6.6093"
o.oSs"
0.0810
"6.1205
5.1701
5.2S24
4.4394
4.6613
3.9267
6.1791
2.4S72
4.3467
5.2218
6.as24
4.4S27
4.6613
3.9659
5.1794
2.4817
4.3457
3.6232
3.7309
3.8963
4.00-16
3.ia32
3.731-.'
3.'S«i
4-l«16
1.5086
1.5515
0.5SC4
25-27-
20-28 — -
20-31 — -
0.3427
1.44S5
Az. seD
,\3-»5
Oats
0.01-36
3.S034
0.4900
•n 19'
1'
Glover
4.4823
SS-40
Olovcr
6.0136
4.55S6
■t.VXili
6.1247
4-6833
4.0SS3
4.0046
0.6/87
F
Oats ---
Onts -.
3.5S30
3.556-2
3.70S7
3.5S79
3.6458
3.5/20
"oToiseT"
3.6458
.1.5584
i'.im'
3.6158
3.6^22
3.0232
F
Clover _ -
4.5023
l6-(8 —
Clover
4.3114
0.0136
4-2973
9.(H60
0.1217
4.4195
4.41i;5
4.0046
0.4149
F
Oats
3.3«30
3.4573
3-41(1
3.4101
3.4442
Oats
3.6323
0.0136
3.5187
2. 1850
3.5519
3.5519
3.7309
F
Clover -
4-4001
4.40.11
51-56
C
aovcr -
4.272SI
4.S0OO
4.3114
0.0136
4.2978
6.7850
0.08S8
4.3866
4,3Mi6
4.0040
0.3t20
3.6M4
3.59S7
3.6065
3.5323
0.0:33
Oats
3.4'.i27
3.5085
3.5006
0.01S6
S.4S10
3.8650
3. 5323
3.730')
F
Clover ..-
4.^.310
4.4C44
4.4630
._.
4.4630
4.4630
4..i076
3.8903
V.6113
62-(H
Wis".d cultures
00-07
Clover
4.2230
4.3817
4.3023
O-0136
4.2fS7
7.1400
0.107S
4.3905
4.3905
4.O0W
H.3919
Oats
3.5563
3.6514
3.603S
0.0136
3.5902
1-0450
3.1:021
3.6021
3.7109
F
Clover
4.3087
4.3687
4.3ia7
4.4123
3.SJ63
0.516(1
C
Clover .—
4.4135
4.5105
4.4720
0-0136
4.4684
9-530O
i.l210
4.57114
4.5794
4.0046
0.5748
Check
Average
P
Nothing _.- —
3.6369
5.5515
3.5912
3.594'.
3.5942
3.6301
3.4816
75-76 —
Nothing
3.5562
3-4609
3.5CS5
0-0136
3.4949
5.5650
0.0740
3.5689
3.56S9
3.5S93
V
Nothing
3.S002
3.3002
3.3002
3.3002
3.3002
3.3332
JS-90 - —
Nothing
3.4609
3.6197
3.5103
0.0136
3.6267
C.7700
0.0830
3.6097
3.6097
APPENDIX TABLE II.
Actual Grams N. Found in Pot Soils, Calculated
on Basis 41J6 gr. in Fallow, 4101 gr. in
Cropi ed. — Pots
Determination
,
c
S.5
SZ°^
Hi
!|i
ttoS.g
g-ss
oP°S6
•3.3-S
o
-^1
2.7340
3.0128
3.3370
2.8S64
3.0910
3.1866
3.3205
3.6592
3.6107
3.3397
3.6102
3.4603
2.6867
2.9607
3.3370
2.5610
2.7947
3.1866
3.5242
3.8S36
3.6107
3.2020
3.4901
3.4003
2.7165
2.9924
3.3370
2.6263
2.86-26
3.1866
3.3343
3.6743
3.6107
3.2897
3.5857
3.4003
2.8235
3.1115
3.3370
2.7306
2.9763
3.1866
3.3205
3.6591
3.6107
3.463(5
3.6322
3.4603
2.7517
3.0356
3.3370
2.9120
3.1730
3.1866
3.4921
3.8492
3.6107
3.0901
3.S747
3.4603
2.7015
2.9770
3.3370
3.0069
3.3J"9
3.1806
3.4487
3.S0O4
3.6107
3.5425
3-8613
3.4603
2.70S3
2.9845
3.3370
2.9083
3.17(0
3.1866
3.3977
3.7442
3.0107
3.2959
3.69-25
3.4603
2.6146
2.S812
.1.3370
2.8681
3.1-202
S.lwd
3.3998
3.7465
3.6107
3.2031
3.4913
3.4003
2.5210
2.7781
3.3370
2.7221
2.9310
3.186'j
3.2696
3.0031
3.01/7
3.4091
3.7159
3.4 .(3
Aver!i:--e
2.8235
3.1115
3.0537
2.9768
3.2793
t
2.7857
3.0364
II
Onts ...
C?lovt.r .
Oats ..
Oats ...
Clover .
Olovcr .
Oats . . .
Clover ...
Clover . .
Oats ... .
Oats ....
Clover .-
Clover ..
Oats ...
Oats ....
Clover .-
Clover _.
Oats ....
Oats ....
Clover ..
Clover ..
Oata ....
Onts ....
caover ..
Clover ..
Nothing .
Nothing
2.7517
2.85.37
3.6443
2.7227
2.SCS3
3.3977
3.3051
2-8001
2.75IS
2.9696
2.fl»5
2.7900
3.2557
3.2177
2.7083
2.5910
3.4318
3.3(ftl
2.7577
2.!I-2S6
3.4400
3.04.<10
2.6790
3.2096
3.3183
2.8S79
2.7618
2.9S'20
2.7340
2.S21S
3.3-205
3.3123
2.6807
2.5479
3.5242
3.1813
2.6134
3.3343
3.2681
2.8235
2.0936
3.3-205
3.4143
2.7.547
2.S9I2
3.4921
8.0618
2.7015
3.0503
3.44S7
3.50S9
2.6146
2.8-246
3.3908
3.1740
2.8235
2.7518
2.9758
2.T7S6
0.0136
0.0130
0.0136
0.0136
O.OISO
6.0136
0.0136
'6.6l36
0.0136
6.0130
"o.6i36
"616136"
"6.61S6
0.0130
6.0136
0.0136
3.2445
2.8235
2.7547
2.8776
8.4921
3.0512
2.8748
3.3977
3.2531
2.8235
2.7382
2.9758
2.7600
"'l?4995 "
' 016282'"
"2?i746"
"6"64i6""
"i.7S7""
"T6297"
"ir6253"
""6!m43"
1.8895
0.0205
i?S346"
"016452"
"2"ol7s'"
6.6566"
"s^nss""
""6^629""
"2?2545"
6?6S4"'
2.2800
0.0149
"i"6s66'"
"'6S362"
"I'sffls"
"6^6472"
"'i'so-Is'"
"6'6336""
2.6103
0.0428
""s^oior"
li.mi
""i^iooo"
6^0127
2.2315 "
0.0422
1.6933
0.0376
'T.m&'
""6;6ie9"
"ilm'
'"o"657""
0.1663
0.1897
0.4010
IIJ.O4B0 -t- clover N. -|- 3.4603
APPENDIX TABLE III.
Az. 27D
41
42
43
64 -.
05
Ai-iual Grams N. Found in Pot Soils, Calculated
on Basis of 3741 er. in Fallow and 3786
gr. in Cropped Pots
Oats ...
Oats -.
Oats ...
Clover .
Clover -
Clover .
Clover .
Onts
Oats
Oats
Clover ..
Olover _-
Clover ..
Olover — .
Oats . ,
Oats
Oats
Oats
Olov(!r ...
Clover ...
Clover ...
Olover ...
Onts
Oats
Oats
Oats
Clover ...
Olover 11'
Clover ...
Oats
Oats
Oats
Oats
Clover ...
Olover ...
Olover ._
Clover ..
Oats
Oats
Oati
Oats
Olover ..
Clover ..
Clover _-
Olover ..
Oats ._-
Oats
Oats
Oats
Clover ..
Olover .-
Cnover ..
(/lover .-
Oata
Oats
Oats ....
Oats
Clover ..
Olover .-
Clover ..
(plover .-
Oats
Oats
Oata -.
Oats „-.
(Jlovor .^.
Clover ..
Olover _.
Olover ..
Nothing
Nothing
Nothing
Nothing
Nothing
Nothing
Nothing
Nothing
2.S30O
2.8131
2.7619
2.1864
2.3321
2.3038
2.3586
2.9059
2.S751
3.1808
2.5971
2.5018
2.81S9
3.0499
2.8773
2.6574
2.1074
2.4216
2.9583
3.2ni
2.8405
2.5656
2.32-27
2. 4478
3.0070
3.5203
2.7129
3.0761
2.7694
2.5731
2.3983
2.4478
2.3719
3.0-237
2.7429
2.4740
2.5044
2.3954
2.3050
2.9714
2.7969
2.0027
2.4478
2.4663
2.4871
2.4640
2.8405
2.9548
3.063O
2.4478
2.5713
2.1991
2.3321
2.4497
2.6971
3.0237
2.9152
2.^71
2.7959
2.8274
3. 0666
2.1074
2.3954
2.4616
3.-2852
2.8274
2.8688
2.7189
2.36-52
2.3103
2.3785
2.89-28
2.8489
3.1646
2.7501
2.57S7
2.2913
3.4805
2.5131
2.3917
2.4478
2.3491
3.0172
2.7429
s.oni
2.7363
2.4009
2.4848
2.3823
2.4478
2.4583
2.4871
2.4917
2.8143
3.0181
3.056-1
2.4451
2.7230
2.7893
2.S067
2.3160
2.4094
2.6037
3.0173
2.8877
2.8208
2.3496
2.4381
2.3234
2.3210
2.0944
2.7700
2.6375
2.1071
2.4085
2.4911
2.4901
0.0136
0.6136
0.0136
0.0136
"o'oiso
0.0130
"6?6i3«"
"6?6i36'
0.0136
"o'orn"
0.0136
0.6136
"6^136"
0.0136
oToise"
0.0136
0.0136
6.6136
'6"6i3o'
O.0136
"6?6ii6'
"616136"
0.6136
"6^6136"
"6'oi36"
"6'6i36"
0.6136
2.3507
2.7S12
2.17t«
2.9-255
3.2710
2.8274
2.8562
2.57S7
2.2807
2.4S(B
2.0601
2.6431
2.3761
2.4478
2.3351
3-0172
2.7293
3.0711
2.7227
2.7113
2.0161
2.8741
2.4478
2.4447
2.4871
2.4781
2.S14S
3.0015
2.4961
2.5444
2.2180
2.4315
2.7230
2.7757
2.6607
2.6100
2.4085
2.4775
2.4765
3.0050
3. mo"
2.5500
2.7790
3.7675
3.1550
"3"iS6"
3.8030
1.1575
3.4015
2.2166'
3.9045
S.7480
"i'.imi
3.4770
2.9195
0.0715
0.0904
O.0S68
o.oiio
0.0975
'6ru68'
0.1002
0.0785
"6.6512'
0.1296'
'6'i69i'
0.1103
"o'iisj'
0.1280
"6^940'
6'i263'
o'issT
6.1293'
0.0420
'6ri6i6'
'6?i3-27'
0.10S7
0.1055
"0.6664"
4.3356 j 0.15S5
2.9196 I 0.113.8
2.7489
2.4231
2.3103
2.4653
2.!,l)-28
2.y(;34
3.1540
2.863-1
2.6787
2.4178
2.4617
3.0172
2.4478
2.6650
2.4871
2.6261
2.S143
3.1106
3.0564
2.9705
2.4901
2.0393
2.21S0
2.6508
2.9047
3.3165
3.0817
2.9781
2.9091
2.9-191
3.0598
3.4381
3.7252
3.3V59
2.9091
3.0730
3.0109
3.1695
:'..4137
3.7431
3.027S
3.1751
2.0911
3.0758
2.8502
3.S-2&1
2.8502
1.3860
J. 7020
0.16S0
i.iioo"
2.8502
0.2345
3.3281
2.8502
0.11S9
3.3284
3.1239
0.6369
3.0021
3.1239
0.0013
3.00-21
2.8602
0.1348
3.3281
2.S0O2
0.0385
3.S-2S4
3.1239
0.40-23
3.3284
2.8602
3.3-28.1
3.1239
3.6U21
3.1239
3.C021
S.82S1
3.1239
3-6021
-'\.v. ck. fallow 2.70S6 -f N. c
Av. ck. fa.llow 2.7086 + N. c
jVv. cU. cropped 3.1.SfiS -t- N.
Av. ck. cropped 3.1868 + N.
ntent of oat.s ground 0.1410=2.8502
nlcnt ground clover 0.4163 = 3.1239.
•ontent ground oats 0.1416=3.3284.
■ontent ground clover 0.4163=3.0021
*•
61
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