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THE ROLE OF ANAEROBIOSIS IN ASYMBIOTIC
NITROGEN FIXATION1#
W. A. RICE AND E. A. PAUL
Department of Soil Science, University of Saskatchewan, Snskatoon, Saskatchewan
AND
L. R. WETTER
Prairie Regional Laboratory, National Research Coz~ncilof Canada, Suskatoon, Saskatchnua~z
Received January 30, 1967
niIicrobial fixation of atmospheric nitrogen was ~neasuredin soil amended
with ground wheat straw and incubated a t two moisture levels (field capacity
and \vaterlogged). Fixation equivalent t o 42-52 kg/ha in the soil a t held capacity
and 13-150 I;g/ha in t h e waterlogged soil was observed using 15N techniques
when the soil was amended with lyostraw or less. Using both I5Nand ICjeldahl
techniques, high rates of hsation (500-1000 kg/ha) were measured in soils
amended with 5520% straw and incubated under waterlogged conditions.
T h e high levels of nitrogen fixation in this study can be attributed t o a combination of ( a ) aerobic conditions required t o break down the plant residues
and ( b ) anaerobic sites which are essential for significant fixation rates. T h e
clostridia are strongly implicated in this process in nature. Upon incubation
in the laboratory, the nitrogen-hxing clostridia increased 1000-fold in the watersaturated soil and 100-fold in t h e field capacity soil. These organisms were also
active in field soils throughout the growing season. Identification studies indicated
t h a t these organisms were primarily Clostridiz~vnbutyriczlm.
Introduction
T h e transforination of atmospheric nitrogen to microbial proteiil by freeliving microorganisms was recorded early in the history of microbiology.
Microorganisms with the ability to fix N2 include species of azotobacter,
clostridium, and several blue-green algae. This classification has recently
been expanded to include a nluch wider variety of microorganisms (8, 10, 11,
14). The extent and significance of biological nitrogen-fixation by asymbiotic
bacteria in the soil has, however, never been completely established, although
many investigations have provided an excellent basis for postulations (6, 11,
19).
In general, significant nitrogen fixation, under both aerobic and anaerobic
conditions, occurs when soluble carbohydrates are added t o the soil (3, 4).
Fixation also has been shown to occur in the raw humus layer of forest soils
(3, 9). T h e importance of nitrogen fixatioil in nature, its role in soil fertility
and in geochemistry, however, requires further research.
Interesting claims of extremely high fixation rates (based on Kjeldahl
techniques) in waterlogged soils ainended with straw and incubated under
a normal atmosphere (containing oxygen) have recently been made (1, 2).
No detectable fixation occurred if oxygen was excluded from the atmosphere.
Soils which did not contain an excess of water failed to fix nitrogen when
incubated with straw in either the presence or absence of oxygen.
The study reported here was initiated ( a ) to use isotopic "Nn t o measure
the extent of nitrogen fixation in soils under varying moisture conditions in
lSasl<atchewan Institute of Pedology Publication R14.
2N.R.C. Contribution No. 9506.
Canadian Journal of
Microbiolosy. Volume
13 (1967)
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830
CANADIAN JOURNAL O F MICROBIOLOGY.
VOL. 13. 1967
the presence of crop residues, (b) to characterize and identify organisins
responsible for fixation under waterlogged conditions, and (c) to deterinine
the activity of anaerobic, asymbiotic, nitrogen-fixing organisms in soil.
Experimental Procedure
Nitrogen fixation was ineasured in a dark brown chernozemic soil by
determining the increase in atoll1 % 15N in soil-straw mixtures incubated in
a chamber containing 0.1 atin of Nz (8.22 atom % 15N),0.1 at111 Oz, and with
the remainder of helium. Replicates of 0.6 g of soil-straw mixture in small
polyethylene containers were incubated in a stainless steel chamber (inodified
Torbal BTL anaerobic jar) a t 27 "C for 28 days. For inass spectrometric
measurements of 'jN enrichments, incubated samples were digested for 18 h,
then distilled and the ammonia collected in 0.1 N HCl. I t was oxidized to
molecular nitrogen with alltaline hypobroinite and assayed with an AEI
MS3 mass spectroineter. The copper sulfate-selenium catalyst used in the
digestion mixture occasionally resulted in sinall losses of nitrogen during the
extended digestion period. Total nitrogen values, therefore, were determined
using a 2-h digestion on replicate samples.
Clostridia were counted by the most probable number method using soilextract thioglycolate broth (15). For 30 days after incubation, the tubes
were observed for growth of clostridia, the criteria being gas production as
indicated by a t least 5 min of gas in the gas tube, and the production of
butyric acid. Aerobic organisms were counted on glucose mineral-salts agar
medium supplemented with amino acids and soil extract (KZHPOI 0.5 g,
glucose 5.0 g, yeast extract 5.0 g, soil extract 400 1111, agar 20 g, tapwater
600 1111).
Anaerobic nitrogen-fixing organisms were isolated by placing several small
soil aggregates on the surface of nitrogen-free agar and incubating in an
atmosphere of nitrogen. The agar plates were placed in large desiccators containing 100 g of inoist oats; the anaerobic atinosphere \ilas achieved by triplicate evacuation and refilling with iY2 passed over hot copper filings to reinove
oxygen. Pure cultures were obtained by subsequent streaking and growth on
a medium containing both soil extract and yeast extract. The purity of the
culture was checlted by inicroscopic examination and aerobic incubation.
When pure cultures were obtained, the organisms were transferred to a semisolid medium containing soil and yeast extract, and sub-cultured weeltly
throughout the period of investigation (18).
Results
The atom % 15X in the soil-straw inixtures after incubation in 15Nenriched
atmosphere is shown in Table I. There were no significant differences in
atom yo 15jN in the controls a t the four different residue levels. All incubated
treatments had a significantly higher atom yo 15N than that of the controls.
The amounts of nitrogen fixed by the soil are shown in Table 11. Fixation
a t the 5% straw level was tested by both 15N and Kjeldahl techniques. The
I5N technique indicated a soinewl~athigher fixation level. The total nitrogen
measurement (Kjeldahl), however, is affected t o a greater extent by soil
nitrogen losses than is the 15iYmethod. Significant fixation of nitrogen occurred
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R I C E E T AL.: ANAEROBIOSIS I N N I T R O G E N FIXATION
TABLE I
Atom % 15N in soil-straw mixtures after incubation in 15Nz-enriched atmosphere
for 28 days a t 27 OC
% straw added to soil
Treatment
0
Control
Field capacity
Waterlogged
1.O
0.5
0.3674~~
0.4411b
0.3858~
0.3677~~
0.4355b
0.3960~
5.0
0.3632a
0.4170
0.5571
0.3656a
0.7910
1.0367
NOTE: Any two means designated by the same letter are not significantly different a t the 5 %
level (5). Sx=0.0043. Shortest significant range: 0.0125-0.0147.
TABLE I 1
Nitrogen-fisation in dark brown chernozemic soil
pg N fixed
per gram of soil
straw
Straw, %
0
0.5
1 .O
5.0
5.0
20.0
Method of
nleasuring
+
Field capacity
Waterlogged
23
23
19
150
110
80
6
10
67
240
210
460
lsN*
15N
1%'
15N
I<**
I<
*LSN, measurement of '3N enrichment with mass spectrometer.
**K, total nitrogen by the Kjeldahl method.
p-----a/
/
-- - - - -
,
A e r o b e s , F.C.
C l o s t r i d i o , W.L.
A e r o b e s , W.L.
C l o s t r i d i o , F.C.
--
/
/
/
.---a
FIG. 1. Population changes in soil microorganisnls during incubation of soil plus 20%
straw at held capacity (F.C.) and under waterlogged (W.L.) conditions.
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CANADIAN JOURNAL OF
MICROBIOLOGY. VOL.
13. 1967
TABLE I11
The effect of nitrogen fertilization on the nu~nberof
N-fixing clostridia in soil
N-fixing clostridia per gram soil (X103)
Mg N applied
per kg soil
Summerfallow
Cropped
in all incubated samples, even in those to which no straw had been added.
T h e soil sainples used in this study were obtained from a field in which the
straw from the previous crop had been incorporated. Thus, there was a
reserve of energy in the system, and t h e population of nitrogen fixing organisms was active even in the absence of any additional crop residues.
Studies to determine the organisms responsible for the fixation implicate
the clostridia (Fig. 1). T h e population of these organisins increased 1000fold (10"107/g) in waterlogged soils during the early stages of incubation.
T h e total aerobic population increased 10-fold (from lo6-107/g). T h e clostridia
also increased in numbers in the soil incubated a t field-capacity moisture
levels.
The majority of the bacterial colonies growing aerobically on a low-nitrogensoil extract agar were punctiform. Less than ITo developed large colonies.
Microscopic examination revealed such organisms to be primarily pseudoinonads and bacilli. At no time were azotobacter observed.
T h e effect of nitrogen fertilizer ( N H 4 N 0 3a t planting time) on the number
of nitrogen-fixing clostridia in a soil supporting a crop of wheat was studied
ORTHIC
ELUVl ATED
80
5
Clostridio
Clostridio
Temp.
,
d
I
MAY
Moisture
\--..
-x-
- -f
I
I
JUNE
JULY
AUG.
I
SEPT.
I
OCT.
I
MAY
I
JUNE
I
JULY
I
AUG.
I
1
SEPT. OCT.
FIG.2. The effect of temperature and moisture on clostridia numbers in field soils.
10
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RICE ET
AL.: AX.4EROBIOSIS
833
I N NITROGEN F I X A T I O N
in a growth chamber experiment. Clostridia counts, conducted when the
crop was mature, are shown in Table 111. T h e clostridia showed a marlted
response to added nitrogen in cropped soils, but less response in sulnmerfallow
conditions. Plant growth was increased by the fertilizer-N but not t o the satne
extent as the organisms which increased threefold a t inoderate nitrogen
application levels.
T h e effect of environmental factors on clostridia in their natural habitat
was studied by following the changes in populatioll in relation t o soil inoisture
and temperature throughout the suininer months. iVIonthly clostridia counts,
soil temperature, and moisture content were recorded for the period hIay to
October, 1964 (Fig. 2). T h e eluviated soil profile norinally is fairly moist.
During the early part of the summer, clostridia numbers in this soil mere
correlated with temperature even though the soil was drying rapidly. Near
the end of August, rainfall increased the clostridia numbers despite the lower
soil temperatures.
In the drier orthic site, the high soil temperatures and low soil inoisture content reached in July may have caused a decrease in the number of clostridia.
T h e effects of the Ausust
- rainfall on numbers was also noticed a t this site.
Because of the variables involved, a study such as this has limited meaning.
However, it does give a measure of the number of organisms in the soil and
the response of these organisins to changes in the environment, indicating
that the pop~ilationwas active even when soil moisture was decreasing.
Soine of the chemical, physiological, and inorphological criteria used to
classify granulose-positive organisins are shown in Table IV. T h e organisins
capable of growth on nitrogen-free medium were reinarkably similar, with
the great majority falling in the sub-genus Clostridizim bzityricum a s described
TABLE IV
Physiological characteristics of anaerobic, butyricbutyl organisms in four soil members
of a chernoze~nicsoil
No. of organisms out of a total of eight
Physiological characteristics
Motile
Granulose
Catalase
Grarn
A M C t production
Illdol production
H?S production
Digestion of albumen
Digestion of corn mash
Gelatin liquefaction
Nitrate reduction
Fermentation :
glucose
sucrose
xylose
glycerol
soluble starch
ce!lulose
Pink D~rment
+
+
+
*I-IEG, humic eluviated gleysol.
'(AMC, acetylmethylcarbinol.
Calcareous
Orthic
Eluviated
II.E.G.*
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831
CANADIAhr JOURNAL OF MICROBIOLOGV.
VOL.
13, 1967
by Prevot (16). I n general, these organisms were motile, Gram-positive rods
measuring 0.8-1.5 X 2.0-7.0 p. They occurred singly and in pairs. The sporangia were s~vollen,with ovoid or elliptical spores located centrally to subterminally. All organisms were catalase negative and did not grow aerobically on the surface of agar plates. When cultured anaerobically on agar
plates, these organisins produced large, viscid or butyrous, irregular colonies
with arborescent projections, soinetiines spreading over the entire plate
surface. Gas was produced abundantly and the presence of granulose was
demonstrated in most organisms. These organisms were saccharolytic and
non-proteolytic.
Two isolates were differentiated froin the C. butyriczlrn sub-genus by proteolytic activity and pink pigmentation. NIany of the organisms when first
isolated showed a faint pigmentation, but lost this characteristic after one or
two transfers, however, one of the 32 organisms tested in detail retained its
pigmentation and ~ v a sclassified as C. rubnun. T h e specific identity of the
proteolytic organism is uncertain.
T h e table expresses the numbers of organisms out of a total of 32 isolates
from the four soil profiles commonly occurring in glacial till topography. T h e
nitrate-reducing organisms were more predominant in warmer, drier soils
(calcareous and orthic) than in the eluviated and humic eluviated gleysol
soils which occupy the lower slope and depressional positions. Otherwise, all
the profiles contained organisins with a similar spectrum of physiological
activities.
Discussion
T h e iinportance of asymbiotic, nitrogen fixation in cultivated soils has
s o ~ n e t i ~ n ebeen
s
discounted because of the lack of direct evidence for its
occurrence, and because of the relative inefficiency and small population of
the organisn~sinvolved. T h e results presented here indicate that fixation
can occur in the soil. Fixation rates equivalent t o 13-150 kg/ha n7ereobserved
when the soil was amended with 1% straw or less. Even higher levels were
obtained ~ v h e nlarger amounts of straw were added. These levels of fixation
are substantially greater than those reported by other worlcers (3, 4) using
15N techniques, but compare favoral~lywith results obtained under similar
incubation conditions when analyses mere conducted using Kjeldahl techniques
(17 2).
A11axiinum fixation was obtained using a thin soil-straw layer (3 to 4 mn1)
saturated with water, but incubated in air. T h e high rates of fixation (equivalent to 500-1000 kg/ha) obtained when 5 and 20y0 straw were added t o
waterlogged soil suggest that this material supplied the energy required for
fixation. T h e soluble material in the crop residue could not have supplied the
required energy (4). Since typical nitrogen fixing organisms are not capable
of utilizing cellulose as a substrate, cellulolytic organisins inust have converted the straw residue to sinlpler intermediates nrliich were metabolized
by the nitrogen fixing organisms. T h e process of nitrogen fixation under
these conditions must, therefore, iilvolve a series of organisms under conditions
in which the upper portion of the soil-straw mixture mas aerobic but the
lower layers were oxygen deficient.
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R I C E ET 4L.: ANtZEROBIOSlS I N KITROGEN FIXATION
835
Nitrogen fixation in soil, and by individual organisms in vitro has been
shown to proceed more actively under conditions of poor rather than of good
aeration (1, 7, 13, 17). This was also found in this study when more than 1%
straw was added to the soil. T h e inhibition of nitrogen fixation by hydrogen
and the role of ferredoxin in electron transport to the nitrogenase system
suggest that nitrogen competes as an alterilative respiratory acceptor (11,
12) and that nitrogen fixation can be regarded as a for111 of respir>tion. I n
addition to converting nlolecular nitrogen to an available form, the biochemical process converting inolecular nitrogen to ammonia, and consequently
amino nitrogen,
- . under anaerobic conditions mav inalte feasible the oxidation
of energy yielding substrates which would otherwise remain unattaclted.
T h e substantial increase in nitrogen fixing clostridia implicates these organisms in the nitrogen fixation system. Although azotobacter were not present
in the soil, the possibility of fixation by organisms other than obligate anaerobes
cannot be ruled out (14). A number of nitrogen fixing soil organisms such as
the pseudomonads, facultative bacilli, and IClebsiella sp. are lcnown to occur
in the soils studied. T h e growth chamber and field studies, hotvever, showed
that clostridia are present in considerable numbers and that this segment of
soil microflora is not static. T h e response of the clostridia population to
e further evidence of an active
changes in soil moisture and t e ~ n ~ e r a t u rare
population and indicate that ainple micro-ecological habitats for the soil
anaerobes occur under natural conditions.
~ mvery closely related species comprise
The observation that C. b z ~ t y r i c ~or
the majority of the clostridia isolated from cultivated field soils cannot be
attributed to isolation techniques selective for C. bz~tyricum.In a preliminary
study, two of nine organisms isolated from lawn and greenhouse soils had
In addition, control cultures of
properties characteristic of C. pnstez~rianz~m.
C. bz~tyricz~m,
C. ncetobz~tylicz~nz,
and C. pastezlrinnz~mwere used with all test
media. T h e significance of a uniform type of nitrogen fixing clostridia in the
soil may be related to the relatively high degree of aero-tolerance of C.
butyrictrm (7).
-
References
1. BARROW,N.J. and JENICINSOX,
D. S. 1962. The effect of waterlogging on fixation of
nitrogen by soil incubated with straw. Plant Soil, 16, 258-262.
2. BREMNER,
J. M. and SAAIV,I<. 1958. Denitrification in soils. I. Methods of investigation.
1. Am. Sci. 51. 22-39.
3. CHANG,P. and ~CNOWLES,
R. 1965. Non-symbiotic nitrogen fixation in some Quebec
soils. Can. J. Microbial. 11, 29-38.
4. DELWICHE,C. C. and WIJI,ER,
. -1. 1956. Non-symbiotic nitrogen
- fixation in soil. Plant
Soil, 7, 113-129.
5. DUNCAN,
D. B. 1955. Multiple range and multiple F-tests. Biometrics, 11, 1-42.
6. I-IALL.A. 0. 1905. On the accumulation of fertilitv bv land allo\ved t o run \vild. 1. Asr.
--0
~
7. HART,&I. G. R. 1955. A study of spore-forming bacteria in soil. Ph.D. Thesis, University
of London, England.
8. HINO,S. and WILSON,P. W. 1958. Nitrogen fixation by a facultative bacillus. J. Bacteriol.
7.-,
6 4nziinx
-"" -"".
9. HUSER,R. 1963. Proble~nezur biologischen Luftsticlrstoff-bindung in Waldboden. Z.
Pflanzenernaehr. Dueng. Bodenlr. 103, 220-226.
10. MAIIL, M. C., WILSON,P. W., FIFE, M. A., and EWING,W. H. 1965. Nitrogen fixation
by members of the tribe Klebsiellene. J. Bacteriol. 89, 1482-1487.
11. MOORE,A. W. 1966. Non-symbiotic nitrogen fixation in soil and soil plant systems.
Soils Fertilizers, 29, 113-128.
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836
CANADIAN JOURNAL O F MICROBIOLOGY.
VOL. 13, 1967
12. MORTENSON,
L. E. 1963. Nitrogen fixation: role of ferredoxin in anaerobic metabolism.
Ann. Rev. Microbiol. 17, 115-135.
13. PARKER,C. A. and SCUTT,P. B. 1960. T h e effect of oxygen on nitrogen fixation by Azotobacter. Biochim. Biophys. Acta, 38, 230-238.
14. PAUL,E. A. and NEWTONJ. D. 1960. Studies of aerobic non-symbiotic nitrogen-fixing
bacteria. Can. T. Microbiol. 7. 7-13.
15. POCHON,
J. a n d T A R ~ I E U X
P., 1962: Techniques d'analyse en microbiologie du sol. Editions
de la Touvelle. Sainte ~Mande.
16. PREVOT,A. 1966. ~ 1 a n u a for
l the classification and determination of the anaerobic bacteria. Lea and Febiger, Philadelphia, Pennsylvania.
17. SHUNK,I. V. 1929. Microbiological activities in the soil of a n upland bog in Eastern
North Carolina. Soil Sci. 27, 283-303.
1957. Manual of microbiological methods.
18. SOCIETYOF AMERICANBACTERIOLOGISTS,
McGraw Hill Book Co.. Inc.. New York.
19. WINTERS,N. W. 1924. ~ o i conditions
i
which promote nitrogen fixation. J. -4m. Soc.
Agron. 16, 701-716.