Applications of The Acetylene-Ethylene Assay For Measurement of Nitrogen Fixation

Soil Bid. Biorhem. Vol. 5, pp. 47-81.
Pergamon
Press1973.
Printedin GreatBritain
APPLICATIONS OF THE ACETYLENE-ETHYLENE ASSAY

FOR MEASUREMENT OF NITROGEN FIXATION
R. W. F. HARDY, R. C. BURNS and R. D. HOLSTEN
Central Research Department, Experimental Station, E. I. du Pont de Nemours & Company,
Wilmington, Delaware 19898, U.S.A.
(Accepted 1 March 1972)
Summary-A comprehensive report of the acetylene reduction assay for measurement of Nz

fixation is presented. The objective is to facilitate the effective use and identify some potential
limitations of the method. The report is based on more than 200 accounts of the use of this tech-
nique in 15 countries during the last 5 years. Methods of measurement of N, fixation are
compared. Nomenclature, e.g., NJ&H,] fixed, is introduced to identify values of N2 fixation
determined by C,H,-C2H, assay. The biochemical basis of the assay is described along with
relevant characteristics including K,,,, CzH2/Nz conversion factor, and specific N2[C2&]-
fixing activities obtained with various systems. Effects of combined nitrogen, temperature,
light, pot, NZ, p&Hz and water on activity are summarized. Available methods for sample
preparation, assay chamber, gas phase, assay condition, termination of reaction, CIHQ
analysis and expression of results are compared. The many uses of the C2HZ-C2H4 assay for
investigations of the biochemistry of nitrogenase and physiology of N,-tiing organisms, defini-
tion of Na-fixing organisms and measurement of field Nz fixation by legume, non-legume, soil,
marine, rhizosphere, phylloplane and mammalian samples are tabulated.
INTRODUCTION
THE OBJECTIVE of this report is to provide a comprehensive summary of the use of the acety-
lene reduction (C,H,-C,H,) assay for the measurement of Nz fixation. The report is based
on more than 200 accounts from fifteen countries and four continents of the application of
this technique published during the past 5 years as well as experience from the authors’
laboratory where about 40,000 assays have been made. The exponential rate at which the
C,H,-C,H, literature has accumulated (Fig. 1) fulfills the predictions of Wilson and Fred
(1935) for Nz fixation and emphasizes the rapidly expanding application of this assay tech-
nique in enzymic, organism and field studies of N, fixation. The assay procedure which was
proposed by Hardy and Knight (1967), involves utilizing the nitrogenase (N,ase)-catalyzed
reduction of C2H, to C,H, coupled with sensitive gas chromatographic analyses and is based
on the inhibition of Nz fixation by C2H, (Schbllhorn and Burris, 1966) and the reduction of
C,H2 to C2H, (Dilworth, 1966). This interdisciplinary and comprehensive report is intended
to facilitate the use and identify some potential limitations of the method. In addition, pro-
posed terminology is introduced to clearly identify values for Nz fixation derived from
CzH,-CzH4 assays.
METHODS OF MEASUREMENT OF Nz FIXATION

Methods for the measurement of Nz fixation include growth and morphological deter-
minations, N-analysis (including isotopic methods) and reduction of alternate N,ase
substrates. No ideal method exists. Only ’ 5N, and C,H,-C,H, assays have been used at all
levels from purified N,ase to field samples. The preferred standard for Nz fixation is 15N-
enrichment from 15N2. However, the facility and sensitivity of the C,H,-C,H, assay make
it the assay of choice for many measurements of Nz fixation.
47
48 R. W. F. HARDY, R. C. BURNS AND R. D. HOLSTEN
NUMBER OF I
w,-v4
REPORTS
PER YEA?? 5. _
1965 66 67 68 69 70 71
YEAR
FIG. 1. The rate of appearance of C&-C&i4 literature emphasizing the rapid growth in the
utilization of this assajr te&nique.
(A) Growth and morphology

The simplest methods measnre growth as biomass or optical density increments in N-free
media and are used to select N2-fixing organisms, These methods have often led to erroneous
classification, however, particuIarIy with organisms which have a low N requirement and a
highly efficient capacity to scavenge traces of combined N (Postgate, 1971b). The presence
of specialized cells catled heterocysts has been correIated with N,-fixation by blue-green
algae; however, the correlation is not absolute. N2 fixation occurs in unicellular algae
(Wyatt and Silvey, 1969) and in a filamentous alga devoid of heterocysts (Haystead et al.,
1970; Stewart, 1971).
Methods based on N analysis are more definitive but require chemical manipulation and
lead to destruction of the sample. The Kjeldahl or Dumas methods assess total nitrogen in a
sample and do not distinguish N obtained from Nz from N obtained from other sources.
With legumes, uninoculated or non-nodulating legume or cereal controIs (Bell and Nutman,
1971; Brockwell, 1971; Sundara Rao, 1971) have been used to attempt to correct for N
from sources other than Nz. The use of lsNz avoids the need to correct. for N from other
than N2. Mass spectrometry is the routine detection system far j5N (Burris and Wilson,
1957), but optical emission has also recently been used (Akkermans, 1971). The former is
more accurate although the latter can be used with smaller samples. The ‘$N method is IO3
times as sensitive as the Kjeldahl method. Limitations of i SN-methods include expense of
15N2 and a mass s~~trome~er, relative ~l~sensi~~vity for field samples with low activity,
complexity of mass spectrometry, and a requirement for even more extensive chemical
manipulations than for the Kjeldahl procedure. Enrichment assays with a radioactive
isotope, 13NZ, are, at least thEoretically, more sensitive than lSNz; however, the short half
life of 13N and the sophisticated equipment for production and purification of the isotope
APPLICATIONS OF ACETYLENE REDUCTION 49
has limited this technique to a few laboratory tests (Campbell et al., 1967; Nicholas et al.,
1961). Another N-based method assesses Nz uptake by mass spectrometric determinations
of N, : Ar ratios in the atmosphere (Fay and Fogg, 1962; Sisler and ZoBell, 1951). Specific
N-based methods such as ammonia measurement by titration (Mortenson, 1961) or colori-
metry (Dilworth et al., 1965) are limited to in vitro analyses of relatively highly active
samples and require appropriate controls to correct for background NH, in the sample
and chemical treatment which destroys the sample to separate the NHJ.
(C) Alternate substrates

Alternate substrates of Nzase, including C2H,, nitriles, and isonitriles and evolution of
H, from H+ have been used in recent years to measure Nz fixation. Acetylene is the most
versatile in that it is applicable to N,ase, N,-fixing organisms and field samples. Other
substrates have been used for qualitative identification of N,-fixing activity. These include
isonitriles and nitriles; acrylonitrile is recommended for the latter since it is an excellent
substrate and is not highly toxic (Fuchsman and Hardy, 1970). Hydrogen evolution has
become a routine in vitro assay for N,ase (Bulen et al., 1965) and is measured by manometry,
gas chromatography and mass spectrometry. Measurement of Hz evolution by gas chroma-
tography (Fuchsman and Hardy, 1972) suggests the possibility of H, evolution as an assay
for Nz-fixing systems when insignificant H, uptake by H,ase occurs. The CzH,-CzH4
assay has been discussed by Bergersen (1970a), Dilworth (personal communication), Hardy
et al., (1968b, 1971a), Klucas (1969), Postgate (1970a), Silver (1967), and Stewart et al.
(196713) and Stewart (1969).
Advantages of the C2H,-C2H4 assay include the following features. The &Hz-CzH4
assay is 1Q3 to lo4 times as sensitive as “N methods. The facility of the method is
substantially greater than the other methods since the product is retrieved readily as a
sample of the gas phase which is directly analyzed without chemical treatment or manual
manipulation. The insignificant fraction of &Hz reduced in short term incubations
permits the use of C,H, as an internal standard or as a check for faulty technique. The
product, CzH4, is stable during storage. The gas chromatographic method gives specificity
by separation of C2H, from other hydrocarbons. The in situ assay process and chromato-
graphic equipment are relatively portable, simple and inexpensive. A high output is
readily obtainable with 30 assays per man hour in the field and 20 gas chromatographic
analyses per man hour in the laboratory. The biological sample is conserved and
techniques for continuous monitoring are possible. Disadvantages include the explosive
nature of C,H, and the need for quantitative handling of a gas substrate and product.
Biological parameters of the assay may produce possible limitations and various aspects
discussed below should be considered for all new systems or systems that produce unusual
results.
Two major precautions are common to estimation of Nz fixation by either C,H,-C,H,
assay or ’ 5N-enrichment from ’ 5N,. The environmental conditions, e.g., temperature,
pOz, light and moisture of the sample chamber must duplicate those of the sample in situ
to obtain a valid measure of in situ N2 fixation. Procurement of truly representative samples
is the major limitation of marine and soil samples, where considerable variability exists.
TERMINOLOGY
The increasing use of results from C,H,-C,H, assays for evaluation of Nz fixation makes
it desirable to develop a simple, definitive terminology that will clearly identify the analytical
origin of the data. We recommend use of the italicized, bracketed acetylene formula,
[C&J, to designate Nz fixation values which are derived from C2H,-C2H, assays, as in
N,[C,H,]-fixing activity and N,ase[C,H,]-activity. This terminology will be employed in
this paper.
N,ase-CATALYZED C,H, REDUCTION

(A) Biochemical basis of C, Hz-C, H4 assay
N,ase catalyzes ATP-dependent reductions of H + and six classes of molecules containing
triple-bonded carbon and/or nitrogen atoms (Fig. 2) (e.g., Burris, 1969; Hardy et aZ.,1971b).
Reactants include ATP, divalent cation (MZ+), reductant and electron acceptor; products
are ADP, Pi, oxidized reductant and reduction product. Electron acceptors may be endo-
genous protons which are reduced by 2e to Hz or they may be exogenous acceptors such as
Nz or &Hz which are reduced by 6 and 2e, respectively, to 2NH3 and CzH4.
THE Nzase REACTION
ATP ADP + Pi
M2+
REDUCTANT OXIDIZED REDUCTANT
ELECTRON _ e* _ REDUCTION
ACCEPTOR PRODUCT
(a)
2e*
ENDOGENOUS 2H+ l H, (H, EVOLUTION)
EXOGENOUS N, NH, (N2 FIXATION 1
C2H2 C&i., Qli2 REDUCTION 1
(b)
FIG. 2.The nitrogenase reaction subdivided into (a) electron activation (the limiting reaction)
and (b) substrate reduction. Substrate reduction may use endogenous electron acceptors, H+
or exogenous electron acceptors, NZ or C,H,. Use of the latter is the basis of the C&HZ-CZH4
assay.
The limiting reaction of N,ase is hydrolysis of ATP to ADP and P1 coupled with electron
transfer to form e*, a postulated activated electron or reduced species. Electron transfer
rate measured as a function of ATP consumption (Hardy and Knight, 1966) or total reduc-
tion product formation (Hardy et al., 1968b) is independent of exogenous electron acceptors
or the inhibitors of exogenous electron acceptors, H, and CO. Activation energy for ATP
consumption is also independent of exogenous substrate (Burns, 1969; Hardy et al., 1967,
1968b).
The important conclusions from these characteristics for the C,H,-C,H, assay are (1)
the specific electron acceptor, e.g. Nz vs. C2H2, does not influence the rate of electron
transfer, (2) the rate of substrate reduction is proportional to the rate of electron transfer
and inversely proportional to the electron requirement per molecule, (3) when ATP or
reductant is limiting, as may occur at low carbohydrate levels, electron transfer by N,ase
decreases and this produces a corresponding decrease in either C,H, or Nz reduction, and
(4) N,ase activity, not N,ase concentration, is measured by either N, or C,H, reduction
Statements such as “the measurement of C&Hz reduction by nodules indicates the potential
for, rather than a measure of, Nz fixation” (Roughley and Dart, 1969), are misleading.
Various Nzase preparations show expected C2H, : N2 fixed ratios of about three when
C,H, reduced in the absence of N, is compared to Nz reduced in the absence of CzH2 (see
C,H,/NZ conversion factors). In our laboratory we have found a ratio with Azotobacter
N,ase preparations of 4. This elevated ratio is due to essentially complete elimination of H,
evolution by C2H, while about 25 per cent of the activated electrons are still used for
Hz evolution with Nz as the electron acceptor (Burns and Hardy, 1972; Hardy et al., 1971b).
Recent biochemical studies indicate differences between the N,ase sites for C2H, com-
plexation and for Nz complexation (Fig. 3). Hydrogen competitively inhibits only Nz
Substrate C,H,
N2
inhibitor CO
H2
FIG. 3. Proposed N,ase site and relationship of C2H2- and N2-reducing site.
reduction (Hwang and Burris, 1968), and CO inhibits both &Hz and N, reduction. Acety-
lene, but not N, showed a significantly different affinity in comparisons of vanadium-
N,ase (N,ase from bacteria grown on V in place of MO) and Mo-N,ase (McKenna et al.,
1970, 1971; Burns et al., 1971). These differences suggest that &Hz and CO complex with
MO while Nz and H, complex initially with another site and subsequently interact with
the MO-site for reduction. These mechanistic differences do not appear to be of significance
for the C,H,-C,H, assay.
(B) Characteristics of N,ase-catalyzed C,H, reduction

1. Product. Ethylene has been identified as the sole product of N,ase-catalyzed C&H2 re-
duction by mass spectrometry, infra-red spectrometry and gas chromatography. No further
reduction products have been found and, if present, are produced in amounts <O*Ol per
cent of that of CzH4. The reduction is stereospecific with cis-1,2_dideuteroethylene as the
major product and only possible traces of trans-1,2_dideuteroethylene formed in reactions
in D,O with Azotobacter and Clostridium N,ases. Ethylene does not inhibit N, fixation and
is not reduced by N,ase (Dilworth, 1966; Hardy et al., 1968b; Kelly, 1969).
2. Requirements and universality. Reduction of C,H, to C,H, appears to be uniquely
universal with respect to N,ase in vitro and in vivo. N,ase preparations from three different
aerobic bacteria, one anaerobic bacterium, two facultative anaerobic bacteria, three photo-
synthetic bacteria, three blue-green algae, and two nodulated legumes reduce C,H, to
52 R. W. F. HARDY. R. C. BURNS AND R. D. HOLSTEN
C,H, (Bergersen, 1970b; Biggins and Postgate, 1969; H. Bothe, personal communication;
Dilworth, 1966; Evans and Smith, 1971; Gallon et al., 1971; Hardy et al., 1968a, 1968b;
Hardy and Knight, 1967; Haystead et al., 1970; Kelly, 1968c, 1969; Kennedy, 1970a,
1970b; Koch et al., 1967a, 1967b, 1967c; Munson and Burris, 1969; Oppenheim et al.,
1970; Schiillhorn and Burris, 1967; Smith and Evans, 1970, 1971; Stewart, 1971a). All
N,ase preparations require ATP, reductant and anaerobic conditions. The requirement for
both MO-Fe and Fe protein fractions of Nzase for &I-I2 as for Nz reduction has been shown
with preparations from seven free-living bacteria (Biggins et al., 1971; Burns and Hardy,
1970; Burns et al., 1970; Hardy et al., 1968b; Kelly, 1968c, 1969; Kelly et al., 1967; Mous-
tafa, 1970; Moustafa and Mortenson, 1968; Vandecasteele and Burris, 1970) as well as from
GIycine max nodules (Bergersen and Turner, 1970; Evans, 1969; Klucas et al., 1968). A
report (Taylor, 1969) of the requirement of three fractions for Nz reduction and only two for
C,H, reduction has been refuted (Jeng et al., 1969).
A number of N,-fixing organisms, including 20 bacteria, 20 algae, 6 algal associations, I8
legumes and 12 non-legumes, have been shown to reduce CZH, to C,H,. No examples of
non-N,-fixing organisms including free-living ~~izu~i#rn, have ever been found to reduce
significant amounts of CzH2 to C,H+ {e.g., Hardy et al., 1968b; Yoshida and Ancajas,
1971). This absence indicates that the occurrence of non-N,ase catalysis of C2H, reduction
is very rare, if it exists at all.
3. Apparent Michaelis constant. Reported apparent X;n’sof C2H, are summarized in Table
1 for N,ase, N,-fixing organisms and some field and natural samples. In contrast to N,,
apparent Km’s of C2H, for in vivo and in vitro N,ase are similar. Most values are between
O+OOland 0.01 atm and their approximate similarity supports the concept that all Nzases
are similar. The k;, of C2Hz may be a useful test to document invoIvement of Nzase in
C,H, reduction by natural systems as has been done for soil (Brouzes et al., 1971b) and
mammalian intestinal contents (Elleway et al., 1971). A value of 0.09 atm reported for
rice rhizosphere is the sole major exception (Yoshida and Ancajas, 1971). The average for
all systems is 0.006 atm C,H,. This average is the basis for establishing the p&H,, which
will saturate N,ase with C&H, to the same extent that N,ase is saturated with N, when it is
exposed to ambient pN, (see p&H,).
4. C,H,/N, conversionfactor. The stoichiometric relationship between C&Hz reduced and
Nz fixed is of utmost importance where it is desired to convert measurements from the
&Hz-CzH4 assay to absolute values of Nz fixation, such as is required for N-balance stu-
dies. A theoretical conversion factor (&HZ reduced: Nz fixed) of three has been used in most
reported conversions. Experimentally determined conversion factors for N,ase as well as
for N,-fixing organisms and field samples are tabulated in Table 2. The average ratios,
excluding the unexpIained high values of up to 25 for anaerobic soil, are in the vicinity of
three with 3.6 for N,ase in tiitro, 4.3 for bacteria, 3 ‘2 for blue-green algae, 3 -9 for legumes,
2.4 for non-legumes and 4.3 for soil. The unusual soil ratios are attributed in one case to
the relatively low solubility of Nz vs. C,H, (Paul et al., 1971; Rice and Paul, 1971) in
waterlogged systems, and it was recommended that a normal pN2 of 0.8 atm be used. A
theoretical ratio of three was obtained when gas diffusion effects through the aqueous
phase were minimized. More ratio analyses are needed for the complex systems utilizing
short term incubations. In all cases specific assay systems should be calibrated with a
N-based method. On a biochemical basis it is difficult to accept ratios outside the area of
three to four in short term experiments unless interaction of CzHz and non-nitrogenase
factors are responsible, or unless there are unknown factors di~erentiaIly affecting the two
reductions.
TABLE1. APPARENTK, OFC2H2 FORN*ASE, Nz-~~ti% ORGANISMS

AND NATURAL SYSTEMS
ApparentK,,,
System (atm x 103) References
Nitrogenase
Azotobacter chroococcum 2 Kelly, 1968a
A. vinelandii 2-9 Hardy et a/., 1968b
A. vinelandii 4 McKenna et al., 1970, 1971
A. vinelandii 10 Schollhorn and Burris, 1967
Clostridium pasteurianum 10 Dilworth, 1966
Glycine max 1 Bergersen, 1970a
Plectonema boryanum 2-6 Haystead er al., 1970, Stewart, 1971
Anabaena cylindrica >
Average 5
+fixing organisms
Bacteria
A. vinelandii 3-6 Hardy et al., 1968b
Clostridium asteurianum 3-8 Hardy et al., 19681,
Average 5
Algae ND
Legumes
G. max 7 Hardy et al., 1968b
G. max 22 Koch and Evans, 1966
Average 15
Non-legumes ND
N,-Fixing systems
Rhizosphere
Oryza sativa roots (90) ? Yoshida and Ancajas, 1971
Soil
Model soil 5 Brouzes, et al., 1971b
Mammals
Rumen l-54 Elieway, et al., 1971
Overall average* 6
* Rhizosphere is omitted.
ND-Not determined.
5. Specijic activities of N,ase and N,-jixing organismsandsystems. The specific N,[C,H,]-

fixing activity has been determined for some highly purified N,ase components and for a
variety of N,-fixing organisms and field and natural samples (Table 3). Activities of crystal-
line Azotobacter and highly purified Clostridium MO-Fe proteins are 1.2-l -5 pmoles
C,H, reduced/min/mg of MO-Fe protein (Burns and Hardy, 1972; Dalton et al., 1971),
while highly purified Fe proteins have activities of 2-l-2.7 (Moustafa, 1970; Moustafa
and Mortenson, 1969). Based on the activities of the individual components and a combin-
ing ratio of mg MO-Fe to Fe protein of 2-3, a calculated activity of 0 *5-l * 1 ,umoles C2H,
reduced/min/mg N,ase is suggested. Activities of Nz-fixing bacteria and algae range from
0’ 11 to 3 nmoles C,H, reduced/min/mg dry wt. with activities of the more active algae
approaching those of bacteria. Average algal activity is 0.9 nmoles C2H, reduced/min/mg
algal dry weight, while algal activities based on heterocysts are equal to or greater than
the average of 2.5 of bacteria. N&ixing lichens are about four-fold more active
than the free-living blue-green algal component, suggesting that the fungus may
enhance algal Nz fixation by lowering the p0, around the algae (Millbank, 1972).
TABLE2. CZH2/N2 CONVERSION

FACTORS
Conversion factor
Experimental system Range Average References
Nitrogenase
Azotobacter vinelandii 4.0-4.2 4.1 Burns and Hardy, 1972
A. vinelandii 3.74.2 4.0 Hardy et al., 1968b
A. vinelandii 3.2 Stewart et al., 1967b
A. vinelandii 2.7-3.0 2.9 Oppenheim et al., 1970
A. vinelandii 5.0-6.0 5.5 Fisher and Brill, 1969
Clostridium and Azotobacter 2.5 Schiillhorn and Burris, 1967
Clostridium 3.9-4.9 4.4 Dilworth, 1966
Clostridium 2.5 Jeng et al., 1969
Chromatium 3.4-3.5 3.5 Winter and Arnon, 1970
Chromatium 3.4 Yoch and Arnon, 1970
Glycine max 2.8-3.9 3.4 Klucas et a/., 1968
Rhodospirillum rubrum 2.84.0 3.4 Munson and Burris, 1969
Average 3.6
Nz-Fixing organisms
Bacteria
Azotobacter 6.0 Bergersen, 1970a
Azotobacter 3.04.5 3.8 Hardy et al., 1968b
Klebsiella 3.0 Bergersen, 1970a
Average 4.3
Algae
Anabaena cylindrica 2.8 Stewart et al., 1968
A. Jos-aquae 3.2 Stewart et al., 1968
Nostoc muscorum 3.6 Stewart et al., 1968
Average 3.2
Legumes
Glycine max 5.4-a.4 6.6 Bergersen, 1970a
G. max (pOZ=0.7 atm) 3.0 Bergersen, 1970a
G. max (pOZ=0.2 atm) 6.2 Bergersen, 1970a
G. max (complete growth cycle) 2.3 Hardy et al., 1971a)
Pisum arvense 1.7-3.5 2.6 Oghroghorie and Pate, 1971
Trifolium subterraneum 1.5-3.7 2.6 Roughley and Dart, 1969
Average 3.9
Non-legumes
Alnus rubra 2.0-2.8 2.4 Russell and Evans, 1970
A. glutinosa 2.3 Akkermans, 1971
Average 2.4
Nz-Fixing systems
Soil
Fallow soil 3.8-8.6 6.9 Steyn and Delwiche, 1970
Irrigated lawn 3.1-6.3 4.2 Steyn and Delwiche, 1970
Native vegetation 4.1 Steyn and Delwiche, 1970
Wheat field 3.1-5.5 4.3 Steyn and Delwiche, 1970
Aerobic soil 3.0 Paul and Rice, 1970; Paul et al., 1971
Waterlogged soil >3.0 Paul and Rice, 1970; Paul et al., 1971
Model soil (aerobic) -3.0 Brouzes et al., 1971a, 1971b
Model soil (anaerobic) up to 25 Brouzes et al., 1971a, 1971b
Legumes are more active than non-legumes; average reported activities are 0.20 and
0.045 nmoles/min/mg fresh weight of legume and non-legume nodules, respectively.
Rhizosphere and mammalian samples are only 10e3 to low5 as active as N,-fixing
organisms or nodules; these low activities reflect the high dilution of N,-fixing organisms
or unfavorable conditions in these natural samples.
6. Inhibitors. Carbon monoxide is a potent inhibitor of N, fixation and also inhibits

C,H, reduction by N,ase, N,-fixing algae and soybean nodules (Burns et al., 1971; Dil-
worth, 1966; Hardy et al., 1968b; Hwang and Burris, 1968; Sprent, 1969a; Stewart, 1971;
Stewart et al., 1968; Stewart and Lex, 1970; Tiepkima and Yocum, 1970). The reported
K, for CO inhibition of C,H, reduction by Nzase is O-8 to 3 x 10e4 atm CO and is similar
to that for NZ. The kinetics of inhibition of C,H, reduction by CO may be a useful test to
document involvement of N,ase in C,H, reduction by new systems. Hydrogen does not
inhibit C,H, reduction by N,ase (Hwang and Burris, 1968); the reported inhibition of C,H2
reduction by H, in the case of the Gunnera-Nostoc symbiosis (Silvester and Smith, 1969)
should be re-examined with different samples of HZ.
(C) Factors afleeting C,H, reduction

An understanding of the factors which affect C2H, reduction is fundamental for the
development of optimal methodology and evaluation of assay results.
1. Combined nitrogen. Ammonia, the normal product of N,ase, is not formed during
C2H, reduction, so repression-derepression effects of NH3 on N,ase, as well as possible
effects of biosynthetic reactions of NH3 on N,ase, are not encountered. This interruption in
ammonia formation increases in importance with length of incubation time and is a major
reason for utilization of only short term incubations.
No evidence has been found for feedback inhibition of N,ase activity by combined forms
of nitrogen which is, of course, a desirable characteristic for the C2H,--C,H4 assay. Ammon-
ium chloride inhibits Azotobacter N,ase[C,H,] only at concentrations > 50 mM and the
inhibition parallels that produced by KC1 (Hardy et al., 1968b). Similarly, glutamate or
aspartate at < 50 mM do not inhibit bacteroid N,ase[C,H,] (Kennedy, 1970a, 1970b).
Combined forms of nitrogen do appear to control N,ase activity in living organisms.
This activity is of potentially great concern for the &Hz-CzH4 assay. Nitrogenase synthesis
and N,-fixing and CzH,-reducing activities are repressed in organisms grown on combined
N. Acetylene reduction is absent from Azotobacter vinelandii, Clostridium pasteurianum,
Klebsiella pneumoniae and Gloeocapsa grown on combined nitrogen. Glutamine and am-
monia but not glutamate or casein amino acids repressed N,ase[C,H,] activity in K.
pneumoniae. Addition of combined N has been shown to inhibit development of N,ase
[C,H,] activity in other experiments with bacteria and algae. Thus, 20 mM NH3 abruptly
stopped N,ase[C,H,] synthesis in C. pasteuriunum (Daesch and Mortenson, 1968a, 1968b).
N,ase[CzH,] activity of A. chroococcum and A. cylindrica (Dalton and Postgate, 1969;
Neilson et al., 1971; Smith and Evans, 1970) is higher in cells incubated under Ar vs. NZ.
The absence of NH3 synthesis under Ar presumably produces derepression with a resultant
increased synthesis of N,ase. This effect could occur in long term C,H,-C,H, assays and
may be one of the factors causing the rate of C2H, reduction to increase as the time of
incubation increases, as observed in some assays (Rinaudo et al., 1971). It is clear from these
studies of effects of fixed nitrogen that long term assays should be avoided. This should
seldom cause difficulty because the sensitivity of the C,H,-C,H4 assay eliminates the need
for long term assays in almost all cases.
2. Temperature. The effect of temperature of &HZ-reducing activity has been examined
with Nzase and N,-fixing bacteria, legumes and non-legumes. (Akkermans, 1971; Anon.,
1971; Burns et al., 1971; Dart and Day, 1971; Gibson, 1971; Hardy et al., 1968b; Moustafa,
1969a; Roughley and Dart, 1969; Wheeler, 1971). Activation energies have been calculated
for Azotobacter N,ase. The Arrhenius plots for both C,H, reduction and Nz fixation are
TABLE 3. SPECIFIC ACETYLENE-REDUCING ACTIVITIES OF NITROGENASE AND ~~~~~~~~ ORGANISMS AND FIELD
AND NATURALSAMPLES
experimental system nmoles C2H2 ~du~d~rnin References
Nitrogenase
Purified Clostridium MO-Fe nrotein 1200/mg protein Dalton et a/., 1971
Purified Clostridium Fe protein 2700jmg protein Moustafa and Mortenson 1969
Crystalline Azotobacter MO-Fe protein 144O/mg protein Burns and Hardy, 1972
PurifiedAzotoburter Fe protein 2100/mg protein Moustafa. 1969a
N,-Fixing Organisms
Bacteria
Clostridium pasteurianum 3/mg dry wt Daesch and Mortenson, 1968a
Rhodospirillum rubrum 2/mg dry wt Munson and Burris, 1969
Algae
Anabaena cyli~r~ca 1 +O*/mg dry wt Stewart ef af., 1968
A. ffos-aquae 2.4*/mg dry wt Stewart er al.. 1968
A. flos-aquae 3*7*/mg dry wt Kurz and La&e, 1971
Mastigocladus iaminosus 0.5*/mg dry wt Stewart et al,, 1968
Nostoc entophytum 05*/mg dry wt Stewart et al.. 1968
N. muscorum 1.9*/mg dry wt Stewart et al.; 1968
Nostoc sp. 0.2-I -8*/mg dry wt Stewart et al., 1967b, 1968
Tolypothrix tenuis O.P*/mg dry wt Stewart et al., 1968
Ap~~ani~ome~n _f?os-aquae 0_4*img dry wt Stewart et al., 1967b
Gloeotrichia echinulata 0.2*img dry wt Stewart et al., 1967b
Gloeocapsa O*ll/mg dry wt Wyatt and Silvey, 1969
Heterocysts of variety of algae 1’ l-8.4/mg dry wt Jewel1 and Kulasooriya, 1970
Lichens
Peltigera canina 5/mg algal dry wt Millbank, 1972
Nostoc component N 1.25jmg algal dry wt Millbank, 1972
Legume nodules
Glycine max 0. 15jmg fr. wt Hardy et al., 1968b, 1971a
G. max 0.04-0.12/mg fr. wt Sloger, 1969
G. max O.O3/mg fr. wt Stewart et ai., 1967b
G. max 0.02-0.71/mg fr. wt Sprent, 1969, 1971a
G. max (12-35 days) 0.20-0.38/mg fr. wt Holsten et al., 1969
G. mux O.l6/mg fr. wt Koch and Evans, 1966
Aracbis hypogea 0’28/mg fr. wt Hardy et al., 1968b, 1971a
Medicago satiua O+ll/mg fr. wt Hardy et al., 1968b, 1971a
Lathyrus odoratus O.IS/mg fr. wt Hardy et al., 1971a
Phase&s limensis 0’15/mg fr. wt Hardy et al., 1971a
P. coccineus O.l3/mg fr. wt Hardy et a/., 1971a
P. vulgaris 0.30-0.60/mg fr. wt Hardy et al., 1971
Pisum sativum 0.15-0*22/mg fr. wt Hardy et al., 1968b
P. sativum O~ll-O~16/mg fr. wt Schwinghammer et al., 1970
~rifoIium pratense 0’35jmg fr. wt Schw~nghammer et al., 1970
Non-legume
AlnlU O.OS/mg fr. wt Stewart er al., 1967b
A. glutinosa (field) 0-093-O. 13/mg dry wt Akkermans, 1971
A. gfutinosa (lab) 0.15-l .5/mg- dry wt Akkermans, 1971
A. rubra (lab) 0.08-0.13/mg fr.-wt Russell and Evans, 1970
A. rubra (field) 0~022-0~026jmg fr. wt Russell and Evans. 1970
Casuarina 0~025-0~035,‘mg fr. wt Sloger and Silver, i967
C. equisetifo~ia O,Ol/mg fr. wt Silver, 1969
Comptonia 0~01-0~03ima- fr. wt Stewart et al., 1967b
Hippophag rhamnoides (field) 0.13-0.4/mg dry wt Akkermans, i971
Myrica ccrifera O.O25/mg fr. wt Slower and Silver. 1967
M. cerifera 2.4*/mg dry wt Silver and Mague, 1970
M. cerifera O.O6/mg fr. wt Silver, 1969
TABLE 3.-Continued
Experimental system nmoles CzH2 reduced/min References
Nz-Fixing field and natural samples

Marine
Anoxic lakes-Lake Mary 0~008-0~08/1. Brezonik and Harper, 1969
-Lake Mize 0~0004~50/1. Brezonik and Harper, 1969
Florida estuary water 0~003-0~008/1. Brooks et al., 1969
Lake Erie water 0-3’2/mg protein Howard et al., 1970
Florida estuary sediment 0.5-2.5 x 10m6/mg dry wt Brooks et al., 1969
Lake Erie sediment 2 x 10sJ/mg dry wt Howard et al., 1970
Rhizosphere
Oryza sativa root zone 2.5-11 x 10m6/mg root fr. wt Yoshida and Ancajas, 1971
Phylloplane
Gunnera-Nostoc 15*/mg algal dry wt Silvester and Smith, 1969
Mammalian
Human feces 1.3 x 10e5/mg wet wt Bergersen and Hipsley, 1970
Rumen and rabbit caecum 0.0-2.3 x 10-s/mg wet wt Granhall and Ciszuk, 1971
* Converted to dry wt. with following factors: 0.5 for protein to dry wt, 0.08 for N to dry wt.
biphasic and the activation energy values are about 14 kcal/mole above 20°C and 35 kcal/
mole below 20°C; similar activation energies have been found for CpH, reduction by
Clostridium (Burns et al., 1971; Hardy et al., 1968b).
N,[C,H&ixing activity of soybean nodules increases at incubation temperatures from
10 to 20°C is maximum from 20 to 30°C and declines above 30°C (Hardy et al., 1968b).
Other legumes (Dart and Day, 1971; Gibson, 1971) show generally similar responses to
temperature, but differing temperature limits and optima. AZnus glutinosa and Hippophad
rhamnoides nodules show about tenfold increases in N,ase[C,H,] activity from 5 to 20°C
with maximum activity in the area of 20 to 25°C; followed by a sharp decline from 25 to
40°C (Akkermans, 1971; Wheeler, 1971). These effects of temperature indicate that N,ase
concentration is not the limiting component for N,ase activity in nodules.
Effects of growth temperature on N,[C,H,]-fixing activity have been examined. The
range of 15-19°C gave maximum effects with Trifolium subterraneum (Roughley and Dart,
1969). No activity was found during spring and fall tests with A. glutinosa when soil tem-
peratures were 1 I-17°C. Incubation at a constant temperature is used for assay work where
direct comparison of samples is desired (Akkermans, 1971).
N,ase[C,H,] activity of legume nodules is cold labile (Moustafa, 1969a; Schwinghammer,
et al., 1970). Excised nodules lose >90 per cent of N,ase[C,H,] activity when frozen and
lose activity rapidly when stored near 0°C. Excised nodules are more sensitive to cold than
attached nodules.
3. Light. Effects of light on C2H, reduction have been examined with N,-fixing algae,
photosynthetic bacteria, legumes and non-legumes (Anon., 1971; Bergersen, 1970a; Cox
and Fay, 1969; Duong and Tiedje, 1971; Fay, 1970; Hardy et al., 1968a, 1968b; Postgate,
1971a; Stewart et al., 1967a, 1967b, 1971; Stewart and Lex, 1970; Wheeler, 1969,197l; Wong
et al., 1971; Wong and Evans, 1971; Wyatt and Silvey, 1969). Light is essential for NJ&H, ]-
fixation by photosynthetic bacteria and algae and effects of light intensity have been
measured in cultured organisms (Cox and Fay, 1969; Duong and Tiedje, 1971). The action
spectrum of A. cylindrica for N,ase[C,H,] activity corresponds to the absorption spectrum
of chlorophyll a (Fay, 1970); DCMU, an inhibitor of photosynthesis, inhibits N,ase[C,H,]

activity (Stewart and Lex, 1970; Stewart and Pearson, 1970). Samples of marine algae
(Rusness and Burris, 1970; Stewart et al., 1967b, 1971; Stewart and Lex, 1970), legumes
(Bergersen, 1970a; Hardy et al., 1968a, 1968b) and non-legumes (Akkermans, 1971;
Wheeler, 1969, 1971) show diurnal variations in N,[C,H,]-fixation activities with maxima
around midday. The maximum in the case of A. glutinosa is related to photosynthate
available to nodules, and activity declines rapidly upon its depletion, e.g. N,ase[C&,]
activity decreased by 40 per cent within an hour of stem ringing. Legumes and non-legumes
placed in the dark lose N,[C,H,]-fixing activity rapidly over a 12-14 h period; this loss is
not correlated with disappearance of the polymer of ,&hydroxybutyrate from nodules but
rather with low sucrose levels (Hardy et al., 1968b; Wheeler, 1971; Wong et al., 1971;
Wong and Evans, 1971). These rapid responses to light demonstrate the need for adequate
control samples spaced throughout the day for assays of algae, photosynthetic bacteria and
nodulated plants; the need for prompt assay of nodulated legumes after removal from their
native environment is also emphasized.
N,[C,H,]-fixation declines following defoliation of legumes. This may be a response to
photosynthate deficiency as well as injury. N,[C,H,]-fixing activity of legumes is markedly
decreased within 24 h after defoliation and recovers to the activity of an undefoliated con-
trol after about 35 days (Gibson, 1971; Hardy et al., 1968b; Moustafa, 1969b). Leaf emer-
gence and senescence of AZnus and Hippophat; are correlated with seasonal effects of N,
fixation (Akkermans, 1971).
4. pOz. Oxygen produces reversible and, at higher concentrations, irreversible effects on
N,[C,H,]-fixing activity. N,ase is irreversibly inactivated by short exposure to air. The
Fe-protein is more sensitive than the MO-Fe protein. Inactivation by 0, has been followed
using the C2H,-C,H4 assay (e.g., Dalton et al., 1971; Haystead et aZ., 1970; Kelly, 1969;
Moustafa, 1970; Smith and Evans, 1971).
Aerobic N,-fixing organisms such as Azotobacter require aerobic conditions for C2H,
reduction, while strict anaerobic organisms such as Clostridium require anaerobic condi-
tions for C,H, reduction. Facultative anaerobic organisms and photosynthetic organisms
require anaerobic conditions for C2H, reduction. Legume nodules require aerobic condi-
tions for C2H, reduction and a p0, of 0.2-0.5 atm is reported to be optimal. Concentra-
tions of O2 >0.4 atm inhibit C2H, reduction at p&Hz of O-005 atm, but not at p&H2
of 0.1 atm (Bergersen, 1970a; Hardy et al., 1968b; Koch and Evans, 1966; Munson and
Burris, 1969; Pratt et aZ., 1971).
Maximum Nzase activity for aerobic bacteria (Biggins and Postgate, 1969; Dalton and
Postgate, 1969; Drozd and Postgate, 1970a, 1970b; Milekhina et al., 1971a; Postgate, 1971a;
Yates, 1970b) and algae (Neilson et al., 1971; Smith and Evans, 1970, 1971; Stewart, 1971;
Stewart and Lex, 1970; Stewart and Pearson, 1970; Wolk, 1970) occurs at p0, < 0*2atm, e.g.
Mycobacterium flavum 301 has maximum activity at 0.05 atm 0, (Biggins and Postgate,
1969) and A. chroococcum at 0.1 atm O2 (Dalton and Postgate, 1969). Azotobacter
strains with a natural high rate of O2 consumption are less inhibited by high p0, than those
with a natural low rate. The inhibition of Azotobacter N,ase by elevated pOZ is rapidly re-
versible and is suggested to involve a conformational change (Drozd and Postgate, 1970b).
Plectonema boryanum (Stewart and Lex, 1970) and GZoeocapsa (Stewart, 1971) are incubated
under reduced p0, to obtain improved N,ase activity; cell-free N,ase was first prepared
from cells of A. cylindrica grown under anaerobic conditions (Smith and Evans, 1970,
1971). Inhibition by 0, of algal N,[C,H,] fixation was greater on the sea bottom than
at sea level (Bunt et al., 1970).
N,-fixing activity may show greater sensitivity to O2 when measured by C!,H2 reduction
than when measured by N, reduction. Saturating concentrations of C,H, almost completely
inhibit in vitro H2 evolution, while those of Nz inhibit only about 75 per cent. It has been
suggested that the in vivo function of H, evolution by N,ase may be to provide reductant
for O2 via adjacent hydrogenase and thereby decrease pOZ in the vicinity of N,ase. If
C,H, and N, affect H, inhibition in vivo as in vitro, then increased sensitivity to p0, would
be expected for C,H, vs. N, reduction.
An effect of the p0, on the conversion factor, &HZ/N,, has been reported. A ratio of
3-O was obtained at 0.7 atm of 0, and 6.0 at 0.2 atm for soybean nodules (Bergersen,
1970a). Variable C,H,/N,‘ratios observed with Azotobacter were pointed out as a need for
careful matching of conditions for Nz and C2H, reduction. Conversion factors for anaerobic
soil systems are extremely high as already discussed; however, the cause for the unusual
ratios may be other than pOZ.
The above examples document the extreme importance of p0, with respect to N,[C,H,]
fixation. Measurements of activity by any method-C,H, or lSN,-must duplicate in the
assay chamber the p0, of the natural system in order to obtain valid results. Conditions of
p0, should not be maximized in assays of field samples, but should duplicate those of the
native environment. Specially designed sampling equipment may be necessary to satisfy
this requirement with soil samples.
5. Dinitrogen. When acetylene and N, are both present, they compete for electrons in the
Nzase reaction. Acetylene inhibits N, fixation by a mechanism originally suggested to be
competitive and more recently reported to be noncompetitive (Dilworth, 1966; Schollhorn
and Burris, 1966, 1967; Hwang and Burris, 1968). Failure to replace air with an inert gas-O,
mixture before &Hz assay reduces N,[CzH,]-fixing activities of soybean nodules by lo-20
per cent (Hardy et al., 1968b). Algal (Stewart et al., 1971) and non-legume nodule (Akker-
mans, 1971) N,[C,H,] fixation rates showed no advantage of air removal, possibly (in the
case of algae) because 0.2 atm pC2H2, an extremely high level, was used for the algal
samples.
6. pC,H,. The rate of C,H2 reduction is proportional to p&H2 at levels less than required
to saturate the enzyme. For assay, p&H, should be selected to saturate Nzase to the same
degree that it is saturated by ambient N,. The K, of N2 is about 0.1-0.2 atm for Nzase
in vitro and about 0.04 atm for N,-fixing organisms, while the K,,, for C,H, is 0.005 atm
for N,ase in vitro and in vivo (see apparent Michaelis constant). Acetylene pressures of
0 - 10 atm and 0 *02 atm in assays should produce saturation comparable to a pN, of 0 *8 atm
for in vivo and in vitro N,ase, respectively. It may be noted that C,H, is about 60 times as
soluble in water at 25°C as is N,.
Inhibition of growth of C. pasteurianum by a p&Hz of greater than 0.025 atm has been
reported (Brouzes and Knowles, 1971). This effect of C,H, on cell growth further emphasizes
the need for short term assays.
7. Water. Water stresses decrease N,[C,H,] reduction by legume nodules. Storage of
nodules in water or immersion in H,O during assay decreases activity (Hardy et al., 1968b;
Schwinghammer et al., 1970), possibly by diminishing the 0, supply, since increasing p0,
to O-8 atm is reported in one case to restore activity (Sprent, 1969a, 1969b, 1971a, 1971b).
Reduction of water content of detached nodules to 80 per cent of normal fresh weight
decreased N,[C2 H,]-fixing activity, while further dehydration produced irreversible changes
accompanied by loss of C,HZ and N2 reduction. In the case of soil samples, N,[C,H,]-
fixation is usually increased with increasing moisture (Akkermans, 1971; Paul et al., 1971).
8. Injury. Nodular injury decreases activity. Excised nodules are less active than nodulated
roots (Hardy et al., 1968b; Oghoghorie and Pate, 1971), and whole plants may be even
more active than nodulated roots. Nodulated roots are recommended for C,H,-C,H,
assay, and activity comparisons with whole plants are needed.
9. UnidentiJed gas product. An unidentified gas product is reported to inhibit N,[C,H,]
fixation by soybean nodules (Sprent, 1969b). Detached nodules show decreased &Hz
reduction after prolonged incubation. Addition of O2 or removal of CO, had no effect on
activity. This inhibition can be avoided with short term assays.
10. Background ethylene. Ethylene is produced in small amounts by plants, fungi and
bacteria (Freedbairn and Buddenhagen, 1964; Ilag and Curtis, 1968; Young et al., 1951).
Low activity soil samples require correction for non-N,ase-formed C2H4 with a pre-
incubation in the absence of C2H2. Ethylene metabolism has not been reported although
ethylene uptake was found in a rice rhizosphere (Yoshida and Ancajas, 1971) and in soil
samples incubated for 24 h (Abeles et al., 1971).
METHODOLOGY
(A) Incubation
Although no apparatus has been specifically developed for the &Hz-CzH4 assay of Nz
fixation, a number of effective improvised methods have been adopted for analysis of
various N,-fixing systems, including N,ase in vitro, bacterial and algal cultures, legumes
and non-legumes, soil and marine samples, and rhizosphere and phylloplane associations.
Methodology of the C,H,-C,H, assay for these various systems will be summarized with
respect to sample preparation, incubation chamber, gas exchange, p&HZ, incubation
conditions, termination of reaction, and expression of results.
Specific variations or applications in the techniques have been described for measurement
of available phosphorus in marine samples via C2H, reduction by phosphate-starved A.
cylindrica (Stewart et al., 1970), estimation of in situ N,-fixing activity in oceans (Bunt et al.,
1970), marine assays (Brezonik and Harper, 1969; Stewart et al., 1971), and model studies of
soil utilizing a variable p0, column (Brouzes et al., 1971a, 1971b).
1. Sample preparation. Special preparations may be useful with marine, soil, nodulated,
and fungal samples. A polyvinyl chloride Van Dorn sampling bottle has been used to
sample subsurface lake water, and algae were concentrated by filtering through plankton
netting (Stewart et al., 1971). Soils are usually sampled with a borer. Sample sizes have
varied from 0 -78 x 1 cm obtained with a cork borer to cores of 6 x 12 *5 cm taken with a
hydraulic coring unit (Hardy et al., 1968b; Paul et al., 1971; Stewart et al., 1967b). In view
of the great variability of soil activity, large samples are essential to obtain representative
data. Nodulated legumes have been collected as soil cores or as nodulated roots, or excised
nodules following excavation (Hardy et al., 1968b; Roughley and Dart, 1969; Schwing-
hammer et al., 1970; Sloger, personal communication; Sprent, 1969b, 1971a; Stewart
et al., 1967b). Small diameter soil cores have produced lower activities than those obtained
with nodulated roots; substantially larger cores may avoid this problem and would be useful
with soil types or legumes where excavation is difficult. Nodulated roots are generally more
active than excised nodules and their use avoids the time-consuming operation of nodule
removal. Because of their heterogeneous distribution, nodules have been excised from non-
legume roots prior to assay (Akkermans, 1971; Silver and Mague, 1970). Lichens have
been sampled as 1 cm discs of thalli taken with a cork borer (Millbank and Kersbaw, 1970).
2. Incubation chambers. Incubation chambers vary; serum bottles, test tubes, erlenmeyer
flasks, Warburg flasks, Pankhurst tubes (Campbell and Evans, 1969), disposable syringes
(Hardy et al., 1968b, 1971a), plastic bags (Hardy and Holsten, unpublished results), Mason
jars (Sloger, personal communication), micro canopies (Paul et al., 1971) and fermentors for
synchronous cultures (LaRue and Kurz, 1970) have all been used. Rubber stopalls or
rubber tubing is employed to facilitate removal and addition of gas. Uptake and subsequent
release of hydrocarbons by rubber stoppers can produce errors; consequently, rubber seals
should not be reused (Kavanagh and Postgate, 1970). A rubber sealant (R.T.V. 112 self
vulcanizing silicone rubber compound, General Electric Co.) is used by some laboratories
to seal needle puncture ports in rubber seals. Modified Pankhurst tubes are proposed as
simple and inexpensive chambers in which to screen large numbers of faculative and
anaerobic bacteria for NJ&H,] fixation (Campbell and Evans, 1969).
Disposable syringes have been used for several thousand C,H,-C,H, assays of nodulated
roots, soil, and marine samples in the field and Rhizobium-tissue culture symbionts in our
laboratory (Hardy, 1971; Hardy et al., 1968b). Advantages of this technique include
effective exchange of gas, uniform mixing of gas, and facile removal of gas at the conclusion
of incubation. Facility is demonstrated by the 800 field analyses performed per day by a
crew of four. Limited size of available disposable syringes is a disadvantage, and larger
containers such as Mason jars (Sloger, personal communication) and plastic bags (Hardy
and Holsten, unpublished results) have been substituted recently. Plastic bags should be
selected to avoid gas adsorption and permeability. Special chambers for root enclosure
have been used to study N,-fixation in the rhizosphere (Pan and Schmidt, 1969). Cylindrical
micro canopies with a brass base and glass top have been used for in situ measurements of
soil (Paul etal., 1971).
Utilization of a low concentration of C,H, in the inlet gas of a fermentor allows continu-
ous monitoring of N,[C,H,]-fixing activity by measurement of C2H, in the effluent gas
(LaRue and Kurz, 1970). The low concentration of C,H, is sufficient to permit measurement
of N,-fixing activity but does not significantly inhibit actual fixation of N,. Similar systems
are being used to continuously monitor nodulated plants (Lie, 1971), and this technique
merits further application, since it produces minimal alteration of the N,-fixing process,
although profound effects on plant physiology may occur.
3. Gas phase. Gas phase varies with the N,-fixing system being tested. A strictly an-
aerobic phase is required for N,ase preparations and for anaerobic and photosynthetic
bacteria, while varying ~0,‘s are employed for algae and aerobic organisms (see PO,).
Argon or He are commonly used in place of air for anaerobic systems and Ar : 0, : COP
(0*8:0*2:0.04) for aerobic; C,H, is then added to the desired concentration. The gas
phase is changed by evacuation and filling or by flushing, the latter being preferred for
any incubation involving Nz-fixing organisms. In recent field experiments the gas phase
exchange step was replaced by simply adding desired amounts of C,H, to the air-covered
samples (Akkermans, 1971; Sprent, 1971a; Stewart et al., 1970; Stewart and Lex, 1970).
The facility of eliminating gas exchange in the field may outweigh the small error intro-
duced. Football bladders have been used to replace gas cylinders to facilitate &Hz trans-
port at the field site (Stewart et al., 1971; Waughman, 1971).
Acetylene should be obtained as the highest purity gas commercially available. Minimal
purification of C,H, requires traps of concentrated H,S04 and H,O; the conventional
sequence for rigorous purification employs traps of soda-asbestos to remove CO,, water
to remove acetone, concentrated sulfuric acid to remove PH3 and H,S (responsible for the
odor of commercial acetylene; pure acetylene is odorless) and water to remove SOZ.
Generation of C,H, from CaC, immediately before use is practiced by one group (Drozd
and Postgate, 1970a, 1970b). Tablets containing the desired amount of CaC, could provide
an effective method for in situ generation of C,H, in the assay chamber.
S.&B.5/1-E
The pC,H, used in the incubation gas phase covers a wide range and limits the quantita-
tive utility of C,H, reduction data. A literature sampling of pC,H, used revealed that values
as low as 0.002 and as high as 0.25 atm have been used. It is desirable that a specific
pC,H, such as 0.05 or 0.1 atm (see p&H,) become the standard concentration, just as
0.8-I *Oatm is the standard concentration used for N,.
4. Incubation conditions. Temperature, time and illumination will be discussed. Incubation
temperatures for in situ measurements should duplicate the field conditions; other incuba-
tions are usually conducted in the range 20-30” with the exception of mammalian samples
which are incubated at 37-40°C. Duration of the incubation is generally 30-60 min. Some
examples of longer incubation times of up to 48 h are described (Brezonik and Harper,
1969; Paul et al., 1971; Rinaudo et al., 1971; Yoshida and Ancajas, 1971). Long-term
incubations with C,H, may be expected to produce anomolous results (Steyn and Delwiche,
1970) and should not be used. Illumination is used with algae and photosynthetic bacteria;
light intensities of 3.5-65 x IO3 lux (Stewart et aE., 1968; Wolk and Wojciuch, 1971) have
been used, and the important consideration is that the intensity should duplicate the in situ
condition.
5. Termination of reaction. The most satisfactory way of terminating the incubation for
assay purposes is removal of a sample of gas. Evacuated blood analysis tubes using needle
holders and receivers for transfer are an effective means for removing and storing a sample
of product gas phase and are routinely used in our laboratory. They show negligible loss
of C,H4 or C,H, in one week of storage. Other widely used methods of stopping the reaction
include addition of trichloroacetic acid (e.g. Brezonik and Harper, 1969; Schwinghammer
et al., 1970; Stewart et al., 1967b; 1968), or H,S04 (e.g., Roughley and Dart, 1967; Silver
and Mague, 1970) and storage of the gas in the incubation chamber. Failure of reagents
to quench the reaction rapidly because of slow penetration into the sample can cause errors.
Chilling followed by displacement of gas with water has been used with soil samples (Steyn
and Delwiche, 1970) and is subject to the same criticism as chemical termination.
6. Expression of results. N,-fixing activities measured with the C,H,-C,H4 assay are
expressed in terms of moles of C,H, reduced or are converted to moles of N2 fixed using a
C,H,/N, conversion factor of three. Analysis of annual legume symbionts has been based
on activities per plant (Hardy et al., 1968b, 1971a) or per 30 cm of row (Sloger, personal
communication) integrated over a complete growth cycle and directly extrapolated to an
area basis. The relationship between Nz fixed and age of annual symbionts may also be
analysed in terms of three additional parameters-age at initiation, age at loss of exponen-
tial phase, and rate of N, fixation increase-which are useful in evaluation of treatment
effects or genetic differences. Measured specific activities of non-legume nodules during a
season have been coupled with estimated nodule mass per acre to calculate N, fixation/ha
(Akkermans, 1971). A similar approach may be used for algal fixation (Jewel1 and Kulasoo-
riya, 1970), in which case the activity per heterocyst would be measured and extrapolated
on the basis of the number of heterocysts per area or volume. N,[C,H,]-fixation by marine
samples has been calculated by integration of data over one season (Stewart et al., 1971).
(B) CZH4 analysis

1. Fractionation. All reports in which C,H, reduction has been used as an assay of N,ase
activity employ gas chromatography to fractionate product C,H, from C,H, and other
gases. Six different packing materials have been used for the gas chromatographic columns.
Characteristics of the different systems are tabulated in Table 4. Alumina was used initially
but was replaced with materials of shorter retention times, lower operating temperatures,
greater reproducibility and increased sensitivity. A variety of Porapaks, N, R, T and Q,

(Waters Associates, Farmingham, Mass., U.S.A.) are now used by most laboratories and
appear to be quite satisfactory. Our laboratory uses 20 per cent ethyl, N’,N’-dimethyloxal-
amide on 100-200 mesh acid-washed firebrick (Richmond, 1969) (Supelco, Inc., Bellefonte,
Pa, U.S.A.). Attributes of this support include: effective separation of C,H, and CzH4
from other C, to C4 hydrocarbons, high sensitivity enabling analysis of lo-l2 moles of
C,H,/sample, linear response to C,H, over 106-fold range of concentration, parallel res-
ponse to C,H, and C,H, permitting use of C,H, as an internal standard, capacity of 20-25
samples per hour utilizing ‘double injection’, operation at 0°C maintained with an ice bath
and reproducibility of a single column for up to 1 year.
2. Detection system. Hydrogen-flame ionization, suggested as the detector in the initial
proposal for use of C,H, reduction as a N,ase assay (Hardy, 1971), has been the single
detection system used. It provides adequate sensitivity, even necessitating correction for
C,H, present as an impurity in commercial C,H,. More sensitive systems would appear
to offer no advantages until C,H,-free C,H, becomes available.
Simplified systems that would replace the need for the gas chromatographic system would
be desirable for field work in both developed and underdeveloped areas. A possible innova-
tion would be the development of a tube system which would respond by quantitative color
change to C,H, concentration. Such tubes now are used for sampling of various gases
including C2H,; however, C,H, interferes with analyses and sensitivities are not yet
adequate.
REPORTED USES AND CONTRIBUTIONS OF THE C,H,-C,H, ASSAY

Uses of the C,H,-C,H, assay are summarized for N,ase and N,-fixing organisms and
field and natural samples. Major contributions of the technique to the biochemistry and
physiology of N, fixation will be indicated. Many important advances were made possible
by the C,H,-C,H, assay either because of the sensitive and non-destructive character of the
method or because the facility, simplicity and relatively low cost made it attractive for
increasingly more laboratories to investigate N, fixation.
(A) Biochemistry of N,ase

Biochemical studies of N,ase have employed C,H, reduction as an analytical method or
mechanistic probe. Cell-free N,ase extracts are routinelyassayedwith C,H, reduction (Berger-
sen, 1970b; Biggins and Postgate, 1969; Biggins and Kelly, 1970; Bothe, 1970; Evans and
Smith, 1971; Haystead et al., 1970; Kelly and Lang, 1970; Kennedy, 1970a, 1970b; Koch et
al., 1967a, 1967b, 1967~; Munson and Burris, 1969; Oppenheim et al., 1970; Smith andEvans,
1970,197l; Stewart, 1971; Winter and Arnon, 1970; Winter and Ober, 1971; Yates, 1968).
The first active cell-free extracts of N,ase from legume nodules (Koch et al., 1967a, 1967b)
and blue-green algae (Smith and Evans, 1970, 1971) were reported from laboratories not
previously involved in this area of N, fixation research and utilized the C,H,-C2H4 assay.
Isolation and fractionation of N,ase into MO-Fe protein and Fe protein has been facilitated
with the C,H,-C2H4 assay (Bergersen and Turner, 1970; Burns and Hardy, 1970; Burns
et al., 1970; Evans, 1969; Hardy et al., 1968b; Kelly et al., 1967; Klucas et al., 1968;
Moustafa, 1970; Moustafa and Mortenson, 1968; Taylor, 1969; Vandecasteele and Burris,
1970; Winter and Ober, 1971). Cold lability of N,ase and specifically its Fe protein compo-
nent (Haystead et al., 1970; Kelly, 1969; Moustafa and Mortenson, 1968, 1969; Moustafa
et al., 1968), air inactivation of N,ase (Dalton et al., 1971; Haystead et al., 1970; Kelly,
1969; Moustafa, 1970; Smith and Evans, 1971), including effect of iron-sulfur proteins and
TABLE ~.GAs CHROMATOGRAPHICCOLUMNSLJSED FOR C,H,-CIH, ASSAY
Retention
Flow time (min)
Carrier rate Temperature
Packing material Typical column dimensions gas (ml/min) (“C) CZH4 CzH2 Sensitivity Typical ref.
Alumina 1.8m x 6mm He 88 130 - - - Hardy and Knight, 1967;

Koch and Evans, 1966
Ethyl,N’N’-dimethyl- 3mx3mm He 30 0 1.1 3.8 < 10-12 Hardy et al., 1968b,
oxalamide moles Richmond, 1969
Porapak N 2mx3mm Nz or He 20 55 - - - Roughley and Dart, 1969;
Sloger, personal communi-
cation
Porapak R 1.5-3.6m x 2-4mm Nz 25-60 75 - - - Bergersen and Hipsley, 1970b
Dalton and Postgate, 1969,
Schwinghammer er al.,
1970; Stewart et aI., 1967b,
1968
Porapak T 1.5-2.7m x 0.3-0.4mm NZ 25-60 50-90 - - - Brezonik and Harper 1969;
Granhall and Ciszuk,
1971; Millbank, 1970
Porapak Q + T (1: 1) 1.2m x 3.5mm N2 20 60 1.25 2.0 - Akkermans, 1971
cytochrome on 0, sensitivity (Yates, 1970a) and nature of the oxygen inhibition of nitro-
genase (Wong and Burris, 1971), have utilized C,H, reduction. The C,H,-C,H+ assay
permitted quantitative evaluation of homologous and heterologous recombinations of Fe
and MO-Fe proteins from a variety of aerobic, anaerobic and faculatative anaerobic bac-
teria, and soybean bacteroids (Biggins et al., 1971; Dahlen et al., 1969; Hardy et al., 1968b;
Kelly, 1969; Murphy and Koch, 1971). Identification and isolation of possible electron
sources or donors for Azotobacter, algal and bacteroid N,ase utilized the C,H,-C,H, assay
(Benemann et al., 1969, 1971; Biggins and Postgate, 1971a; Shethna et al., 1971;
Wong and Evans, 1971, Yoch et al., 1969, 1970; Klucas and Evans, 1968a; Koch et al.,
1970a, 1970b; Smith et al., 1971; Wong et al., 1971; Yates and Daniel, 1970; Yoch and
Arnon, 1970). The sensitivity of the C,H,-C,H, assay may be a mixed blessing in assays
such as this, since it permits detection of very low C,H, levels which may lead to the isola-
tion of artifacts.
The sensitivity of the C,H,-C$H, assay made it possible to dispense with the ATP-
generating system and use low substrate levels of ATP to evaluate the kinetics of the ATP
reaction with N,ase (Kennedy, 1970a, 1970b; Moustafa, 1970; Moustafa and Mortenson,
1967), which indicated that the rate-limiting interaction of ATP with N,ase was second
order. The high specificity of N,ase for ATP (Hardy et al., 1968b; Moustafa and Mortenson,
1967) was established with the C,H,-C,H, assay. The absence of negative feedback of
fixed N (Hardy et al., 1968b; Kennedy, 1970b) was also shown using C,H, reduction.
Acetylene was shown not to support formation of HD from D, and H,O by N,ase (Jackson
and Hardy, 1967; Jackson et al., 1968; Kelly, 1968b; Turner and Bergersen, 1969). N,ase-
catalyzed reduction of acetylene analogs (Hardy et al., 1968d; 1971b; Hardy and Jackson,
1967) demonstrated that only one hydrogen of acetylene could be replaced without activity
loss, and this indicated steric limitations of the N,ase site for alkynes. Results of the inhibi-
tion of other alternate substrates of N,ase by C,H, (Burris, 1969) were interpreted as evi-
dence for four substrate sites.
The reduction of C2H, to C,H4 has been used as an assay with which to seek model sys-
tems for N,ase. A molybdothiol-reductant system was found to reduce C,H, to C,H4
and lesser amounts of C,H, with high specificity for molybdenum (Schrauzer and Doemeny,
1971; Schrauzer and Schlesinger, 1970; Schrauzer et al., 1971). This system also reduces
small amounts of Nz and is stimulated by ATP.
(B) Physiology of N,-fixing organisms

1. Bacteria. A number of physiological studies of N, fixation by bacteria have employed
the C,H,-C,H, assay. The effect of PO,, combined N and temperature on N, fixation have
already been discussed. Studies of N,ase[C,H,] activity in synchronous cultures are pro-
viding the first data on the relationship of cellular development and N,ase activity and
effect of exogenous factors on N,ase activity (Kurz and LaRue, 1970; LaRue and Kurz,
1970). Effects of uncouplers and inhibitors have been measured (Milekhina et al., 1971a,
1971b). The light requirement and 0, inhibition of N,ase[C,H,] activity of photosynthetic
bacteria (Pratt et al., 1971) has been explored with the CzH,-CzH4 assay. The C,H,-C,H,
assay was used to demonstrate the first transduction of N,ase[C,H,] from a N,-fixing to
non-N,-fixing mutant of Klebsiellapneumoniae (Streicher et al., 1971), to test non-N,-fixing
mutants (Fisher and Brill, 1969; Simon and Brill, 1971), to evaluate possible repression of
N,ase (St. John and Brill, 1972) and to assess N,[C,H,]-fixing bacteria, e.g., Klebsiella, A.
vinelandii and A. paspali, in the rhizosphere (Evans et al., 1971; Kass et al., 1971; Pan and
Schmidt, 1969). N,-fixing activity was not detected in nodulated leaves of Psychotria
bacteriophila and Ardisia sp., although the endophyte ofP. bacteriophila was active (Becking,
1971; Silver, 1969).
2. Blue-green algae. Greatly expanded investigations of N, fixation by algae at all organi-
zational levels-biochemical, biological and ecological-are a product of the C2H,-C,H4
assay. Factors such as light, PO,, and fixed N have been discussed. Formazan localization
and N,ase[C,H,] activity (Stewart et al., 1969b) were correlated. The C2H2-C2H4 assay
has been intensely applied to determine the localization of N,ase in heterocysts vs. vegeta-
tive cells (Kurz and LaRue, 1971; Smith and Evans, 1970, 1971; Stewart, 1971; Stewart
et al., 1967, 196913, 1971, Stewart and Lex, 1970b, Wolk and Wojciuch, 1971) and was used
to first demonstrate N,[C,H,]-fixing activity in a unicellular alga by a group new to N,
fixation research (Wyatt and Silvey, 1969). N,[C,H,] fixation could not be demonstrated
with isolated spores of Anabaena (Fay, 1969). N,-fixing activity of algal associations, includ-
ing lichens (Henriksson, 1971; Henriksson and Simu, 1971; Millbank, 1972; Millbank
and Kershaw, 1970), cycad root nodules (Stewart et al., 1968) and plant gland associations
(Silvester and Smith, 1969) have been measured with the C,H,-C,H4 assay.
3. Legume symbionts. Investigations of legume symbionts have ranged from studies of
physiological parameters through detailed studies of the time course of NJ&Hz] fixation
to demonstration of N,-fixing activity in a tissue culture symbiosis. The facility of the C,H2-
C2H, assay permitted the detailed correlation of morphology of nodule development and
NJC,H,]-fixing activity (Hardy et al., 1971a; Holsten et al., 1969). Leghemoglobin concen-
tration was correlated in general with N,[C,Hz]-fixing activity (Hardy et al., 1971a; Schwing-
hammer et al., 1970; Tjepkima and Yocum, 1970; Weber et al., 1971), but in early develop-
ment leghemoglobin concentration increases more rapidly than Nzase activity. Leghemo-
globin is not essential for N,ase activity of isolated bacteroids (Koch et al., 1967a). Effects
of temperature, p02, and light have been discussed. Shoot N at harvest and N,[C,H,]-fixing
activity were shown to be highly correlated (Bergersen, 1970a; Schwinghammer et al., 1970).
The C&H,-C,H4 assay has enabled intensive study of age-activity profiles for legumes
(Anon., 1971; Copley and Reuss, 1971; Hardy et aE., 1968a, 1968b, 1968c, 1969a, 1969b,
197la; Roughley and Dart, 1969; Weber et al., 1971). The N,[C,H,]-fixing activity and
N,[C,H,] fixed profiles of field-grown soybeans (S) and peanuts (P) were found to be
similar during 5 successive years. Profiles of soybean N2[C2H2] fixation for two seasons-
1968 and 1969-are shown in Fig. 4. Both soybeans and peanuts exhibit a major exponential
phase. The parameters: (i) age at initiation (20-30 days for S and 35-50 days for P) and loss
(85-95 days for 5’ and 95-100 days for P) of exponential phase, (ii) rate of daily increase
(8-9 per cent for S and 7-10 per cent for P), and (iii) total N, fixed (250-300 mg N,[C,H,]
for S or P) are used for evaluating bacterial, host, and environmental effects on Nz fixation
(Table 5).
The effects of fertilizer nitrogen on legume N, fixation is being intensively re-examined
with the C,H,-C,H, assay (Hardy et al., 1968c, 1969b, 197la; Hardy and Holsten, 1971;
Moustafa, 1969b; Oghoghorie and Pate, 1971; Vitosh, 1970). Fertilizer N inhibits N,[C,H,]
fixation by field peas, soybeans, white clover, and red kidney beans. Additions of NOs-,
NH4+ or urea at 34, 67, 135 kg N/ha to field-grown soybeans at either 0 or 40-50 days of
age produce equivalent inhibitions of N,[C,H,] fixation, e.g., 49-57 per cent at 0 days and
50-63 per cent at 40-50 days for 135 kg N/ha. Foliar urea applied from 50 to 70 days of
age is more inhibitory than soil urea, e.g., 71 vs. 56 per cent for 135 kg N/ha, indicating a
negative sink effect of shoot N on development of the N, fixation system. Average yield
increases by any of the tested forms of fertilizer are < 10 per cent. Soybean meal at 135 kg
N/ha did not inhibit N2[C2HZ] fixation and increased yield by 20 per cent. Other novel N
Np FIXED vs AGE
N2 FIXING ACTIVITY vs AGE
LOG Np FIXED = K-AGE

LOG N2 FIXING ACTIVITY * K-AGE 50
lOO( 1
I
2.
P
/--’ *
B
0
f
ii! 1
\ P lO(
1
I
5 *
1
IF
c? 1968
1969
d
B
i
10
:i
I 1 01
0 50 loo 0 50 100
AGE (DAYS) AGE (DAYS)
FIG. 4. Age: N,[C&]-fixing activity profiles for a single variety of field-grown Glycine rnax
measured with C2H2-CZH4 assay during two seasons.
TABLE 5. N2 FIXATION PARAMETERS

OF SOYBEANSAND PEANUTS
Exponential phase
Initiation Termination Increase NJC&I fixed
Symbiont Age (days) Age (days) %/day mg N/plant kg N/ha*
Soybeans
1967 ND ND ND 233 33
1968 21 86 8.0 279 42
1969 31 94 8.6 267 40
Average 26 90 8.3 260 38
Peanuts
1968 48 96 10.0 256
1969 36 97 6.9 186
Average 42 97 8.5 221
* Calculation based on 142,000 plants/ha in 1967 of Wayne variety and 150.000

plants/acre in 1968 and 1969 of Kent variety.
ND-Not determined.
TABLE 6. N FERTILIZERS: N2[C2Hz] FIXATION AP;D YIELD
Nz[GEi21
Application Fixation Yield
Age
Form* (days) Control (%)
NOs- 0 43 98
NH4+ 0 51 104
Urea 48 109
Protein
NC.&- 40-50
0” 112
50
123
106
NH+ 40-50 37 106
Urea 40-50 44 91
Urea SO-71 29 100
Urea 78-99 53 100
* 135 kg N/ha of indicated form.

7 s, Soil;F, fohr.
fertilizers compatible with the N, fixation system may be shown to increase yields of soy-
beans (Hardy et al., 1969b, 1971a; Hardy and Holsten, 1971) (Table 6).
Considerable research is occurring in the area of host variety and bacterial strain effects on
N, fixation (Davidson and Reuzer, 1970; Ek-Jander and Fahraeus, 1971; Francis and Alex-
ander, 1971; Ham and Cooper, 1971; Hardy et al., 1968b, 1971a; Sch~nghammer ef al.,
1970; Sloger, 1969; Weber et al., 1971); a major practical use of the C,H,-C,H, assay is the
selection of hosts and bacteria with improved N,-fixing activities. N,[C,H,] fixation by
mid- and late-maturing varieties of soybeans with determinate and indeterminate flowering
characteristics are shown in Fig. 5. {Hardy ef ai., 1971a; Jackson and Hardy, unpublished
results). Mid- and late-maturing soybean varieties fix up to two times as much N,[C,H,]
as early maturing varieties. Soybeans with determinate and indeterminate flowering charac-
teristics and of similar inaturity have similar characteristics, although determinate varieties
may fix up to 25 per cent of total N,[C,H,I fixed before flowering vs. 10 per cent for indeter-
minate varieties. Similar serological patterns of many of t,he varieties demonstrate the con-
trol of the host on all parameters and indicate the importance of varietal as well as bacterial
selection for optimization of symbiotic Nz fixation_
The sensitivity of the C,H,-C,H4 assay made it feasible to attempt the development of
the simplified Rhizobium-tissue culture symbiosis (Holsten et nl., 1970, 1971; Holsten
and Hardy, 1972), which holds potential for elucidation of the factors which control the
in uivn Rhizobium-legume symbiosis. N,fC,Hz]-fixing activity of this system has been in-
creased to about 1 per cent of the natural nodule system. Reduced levels of auxins and
cytokinins increased the degree of bacterial involvement and N,[C,H,]-fixing capacity in
this system.
Non-legume symbiunts. Physiological studies of non-legume symbionts have been accele-
rated through application of the C,H,-C,H4 assay. Temperature and light effects have been
discussed. Effect of noduIe size has been determined with the C,H,-C,H4 assay. Nodules
of 3.5-S -0 mm lobe size had specific NZ[C,Hz]-fixing activities 5 times those of 1 *O-l ‘5 mm
nodules (Akkermans, 1971); in another report (Russell and Evans, 1970) nodule mass was
correlated with N,ase[C,H,] activity. N,[C,H, J-fixing activity only occurred in nodules in
which tetrazolium reduction was demonstrated in the vesicles {Akkermans, i971). No
relationship was found between location of nodules and activity (Silver and Mague, 1970).
N, FIXED vs AGE LOG N, FIXED = K* AGE

I I I 1 /
,hORK
507
j-
IO
1
5-
‘0 25 50 75 100 125 15
wac ,VAT>, AGE [DAYS)
FIG. 5. Age: N,[C&]-fixing activity profiles for several varieties of Glycine mm of various
maturity dates and flowering characteristics (Jackson and Hardy, unpublished results).
Young woody tissue was more active than old. Age-activity profiles (Russell and Evans,
1970) indicated a modest rate of N,[C,H,]fixation during the first thirty days followed by a
rapid increase up to 110 days and decline after 125 days for laboratory grown Alnus rubra.
(C) Refining the list of N,-jixing organisms

Ability to reduce alternate substrates of N,ase has been used to provide a rapid test for
presumptive evidence for N,-fixation (Hill and Postgate, 1969; Millbank, 1969, 1970).
It is emphasized that the C,H,-C,H, assay has great utility in this area because of the
facility and sensitivity of the assay for screening purposes; however, definitive classification
requires confirmation with 15N2.
The C,H,-C,H4 assay has assisted in the elimination of some putative nitrogen fixers
and inclusion of some putative non-nitrogen fixers, Eliminations include : Pseudomonas
(with the exception of Pseudomonas azotogensis strain V and P. methanitrijicans, both of
which may be incorrectly classified), Azotomonas, Nocardia, Bullera, Torulopsis and Pullu-
laria. In the above cases both C,H, reduction and 15Nz fixation tests were negative (Hill and
Postgate, 1969; Millbank, 1969, 1970; Parejko and Wilson, 1968; Whittenbury et al.,
1970). A previous report of N,-fixing activity in extracts of higher plants has not been
verified (Holsten and Hardy, unpublished results) with the C,H,-C,H, assay, and it is
suggested that no eucaryote may have N,-fixing activity (Millbank, 1969). Negative C,H,-
reduction activity is reported for mycorrhizal nodules of Podocarpus (Baylis, 1969; Silver,
1969; Sloger and Silver, 1967). N,[C,H,] fixation by mycorrhiza associated with Pinus
roots (Silver, 1969; Stewart et al., 1967b) is still equivocal.
Various species of Rhizobium grown under normal cultural conditions demonstrated no
N,[C,H,]-fixing activity and this provides the most sensitive report of the absence of N,ase
activity in these organisms. Attempts to cause these organisms to express N,ase activity
70 R. W. F. HARDY, R. C, BURNS AND R. D. HOLSTEN
independent of the host have used the C&H,-C,H4 assay to test for activity. Several other
bacteria previously identified as non-Nz-fixing are also inactive in reducing acetylene
(Hardy et al., 1968b).
A number of Desulfovibrio species, e.g. D. vulgaris, D. gigas and D. desulfuricans have
been shown unequivocally to fix N, on the basis of N-based and C,H, assays (Riederer-
Henderson and Wilson, 1970, Postgate, 1970b) while the C,H, assay has provided tentative
evidence for N, fixation by Thiobacillusferrooxidans (Mackintosh, 1971). A new N,-fixing
alga, Ap~~ail~~orneno~~ $0.~ aquae, previously identified as non-N,-fixing with 15N, assays,
was detected as N,-fixing with the C,H,-C,H4 assay, and its activity confirmed with new
15N, data (Stewart et al., 1967b, 1968). The first example of N,[C,H,] fixation among uni-
cellular algae, e.g., Gloeocapsa, was obtained (Wyatt and Silvey, 1969) with the &HZ-C,H,
assay.
(D) Field and natural sample measurements
The C,H,-C,H4 assay has already been applied to in situ measurements of N&xation
by a variety of organisms in many habitats. Most results are based on minimal analyses.
Because of the variation of Nz fixation during the season, it is strongly recommended that
annual or seasonal estimates be based on integrated results from weekly or biweekly
assays, preferably obtained through several seasons.
1. Legunzes (Table 7). Seasonal profiles (Hardy et al., 19&a, 1968b, 1971a, Weber et al.,
1971) of G. max and Arachis hypogea have been obtained in the Eastern U.S. (Fig. 4).
Calculated values of N,[C,H,] fixed vary from 80 to 105 kg N/ha/season for soybeans.
Less than 25 per cent of the N of the mature plant was provided by N, fixation in these
experiments, which were conducted on soils high in available N. Absolute values of fixation
and per cent N provided by N, will vary with a number of factors, including available N
content of soil; addition of fertilizer N markedly decreases fixation. Seasonal profiles for
other legumes including perennials are unfortunately Iacking and profiles of annual sym-
bionts from a variety of locations are needed.
2. Non-legumes. (Table 7). N,[C,H,] fixation by Alnus gfutinosa and Hippopha2 (Akker-
mans, 1971) in the Netherlands is calculated at 50 and 2-15 kg/ha/year, respectively,
based on seasonal profiles of specific N,[C, HZ]-fixing activity of noduIes and nodular mass/
ha. Older Hippopha~ are more active than younger. Seasonal and temperature effects have
been examined. Annual N,[C,H,]-fixing activity of Myrica cerzjkra in Florida (Silver and
TABLE ~.FIELD MEASUREMENTSOF N,[&H,] HXATION-LEGUMESAND NON-LEGUMES
N,[CzH21 Fixed References
Legumes
Glycirre rnux 250-300 ~/pla~t Hardy etal., 1968b, 1969a, 1971a
or SO-105 kg/hajyr
G. max (135 kg N/ha) 49-63 % of unfertilized Hardy and Holsten, 1971
G. max (early maturity) 90-l 10 mg/plant Hardy et al., 1971a
G. max (late maturity) 170-190 mg/plant Hardy et al., 1971a
Arachis hypogea 180-260 mg/plant Hardy et a/., 1971a
Non-legumes
Alms glutinosa 56 kg/ha/yr Akkermans, 1971
HippophaC (l-2 yr of age) 2 kglhalyr Akkermans, 1971
(IO-15 yr of age) 15 kg/ha/yr Akkermans, 1971
Myrica cerifera 3 *4 kgjhajyr Silver and Mague, 1970
Mague, 1970) was estimated at 3-4 kg/ha on the basis of measurements at only one time of
the year.
3. Soil (Table 8). Monthly N,[C,H,] fixation rates have been obtained for a virgin soil
and cultivated soil in the Canadian Prairies (Paul and Rice, 1970; Paul et al., 1971) and for
fallow, irrigated lawn, native vegetation and wheat fields (Steyn and Delwiche, 1970)
in California. Calculated annual N, [C,H,] fixation by the Canadian soil was about 1 kg/ha/
year while that of the various California soils varied from 2 to 5 kg/ha/year. Other soils have
in general also shown low rates of fixation unless enriched with an energy source or an
energy source in combination with N,-fixing bacteria (Hardy et al., 1971a). Algal crusts on
soils contribute large amounts of fixation where they occur but their occurrence has not
been of major significance in most soils (Akkermans, 1971; Granhall, 1971; Henriksson,
1971a). N,[C,H,]-fixation in a paddy field (Ishizawa et al., 1970) was found to be due to
heterotrophic bacteria as well as blue-green algae and photosynthetic bacteria.
4. Marine (Table 9). A single seasonal profile of N,[C,H,]-fixation has been reported for
Lake Mendota in Wisonsin (Stewart et al., 1971). Activity showed diurnal effects, was quite
variable and correlated with heterocystous content of algae. The calculated annual rate of
N,[C,H,] fixation was 2 *4 kg/ha/year and is similar to the 3 *8 kg/ha/year calculated for an
anoxic lake in Wisconsin (Brezonik and Harper, 1969). Bacteria are important contributors
in the anoxic lakes and in the sediments of lakes or estuaries (Brezonik and Harper, 1969;
Brooks et al., 1969; Howard et al., 1970). Measurements of N,[C,H,]-fixation are
being made in offshore seas as well as inland lakes: Anabaena at 15 m of depth off the coast
of Florida was active (Bunt et al., 1970).
TABLE8. FIELD MEASUREMENT

OF N,[C,H,] FIXATIONIN SOIL
Soil N,[C’&,] Fixed References
Fallow, irrigated lawn, native vegetation and

wheat field soil (Calif., USA) 2-5 kg/ha/yr Steyn and Delwiche, 1970
Virgin and cultivated prairie soil (Canada) 1 kglhalyr Paul and Rice, 1970; Paul
et al., 1971
Coastal plain (SC., USA)-unburned 0.2 g/ha/day Jorgenson and Wells, 1970
-burned 4.0 g/ha/day Jorgenson and Wells, 1970
Agricultural freshly tilled sod loam (Pa., USA) 0.014.30 kg/ha/day Hardy et al., 1971a
+ Azotobacter or Clostridium 0.03-0.28 kg/ha/day Hardy et al., 197la
+ Sucrose up to 1.23 kg/ha/day Hardv et al.. 197la
+ Azotobacter or Clostridium and sucrose up to 6,7kg/ha/day Hard; et al.; 1971a
Agricultural soils (Pa., USA) 0.002X)’ 1 kg/ha/day Hardy et al., 1968b
Jordan fertility plots (Pa., USA) 0.16-1.5 kg/ha/day Hardy et al., 1968b
Virgin prairie (Canada) soil plus vegetation
(May-Sept.) 0.5-9.2 g/ha/day Paul and Rice, 1970; Paul
et al., 1971
Cultivated prairie (Canada) soil 0.0-0.03 g/ha/day Paul and Rice, 1970; Paul
et al., 1971
Forest soil (Japan) O-35 mg/kg dry soil/day Suzuki et al., 1970
Field (Sweden)-algae 15-51 kg/ha/yr Henriksson, 1971
Paddy soil-blue green algae Ishizawa et al., 1970
-photosynthetic bacteria l&200 ng/kg/dry soil/day
-heterotrophic bacteria
Model soil amended with 2 % (w/v) glucose 5-15 mg/kg dry soil/day Brouzes, et al., 1971a,
197lb
Desert grassland with algal crusts (moistened) 72-96 g/ha/day MacGregor and Johnson,
1971
TABLE 9. MEA~UREMENTOFFIELDN~[C~X~]FIXATIONINFRESHWATERANDMARINESAMPLES
N2[C2H2] Fixed References
Water
Lake Mendota (Wise., U.S.A.) 2.4 kg/ha/yr Stewart et al., 1971
Wisconsin lakes 2.4-1800+ ng/l/day Rusness and Burris, 1970;
Stewart et al., 1971
Anoxic lakes
(Lake Mary, Wise., U.S.A.) 3.8 kg/ha/yr Brezonik and Harper, 1969
or 0.1-l. 1 pg/l/day
(Lake Mize, Fla., U.S.A.) O-7 ‘4 pg/l/day Brezonik and Harper, 1969
Estuary (Fla., U.S.A.) 35-l 10 ng/l/day Brooks et aI., 1969
Lake Erie 0.043 pg/mg protein/day Howard et al., 1970
Subtropic sea activity detected Bunt et al., 1970
Sediment
Estuary (Fla., U.S.A.) l-35 rig/g sediment/day Brooks et al., 1969
Lake Erie (U.S.A.) 280 rig/g sediment/day Howard et al., 1970
5. Rhizosphere (Table 10). Several examples demonstrate N,[C,H,]-fixing activity in the

rhizosphere, and these indicate the possibly significant contribution of fixed N by the rhizo-
sphere and its population of free-living bacteria. Sand dunes with vegetation were calculated
to fix up to 0.1 kg N,[C,H,]/ha/year while those with no vegetation showed no N,[C,H,]-
fixing activity (Akkermans, 1971). Substantial N,[C,H,] fixation was calculated for paddy
soil with rice plants (Ishizawa et al., 1970; Yoshida and Ancajas, 1971; Rinaudo and Dom-
mergues, 1971) and N,[C,H,]-fixing activity was found in saline soil with vegetation but not
in similar soil without vegetation. Laboratory measurements of the Azotobacter-Paspalum
TABLE 10. MEASUREMENT OF FIELD N2[C2H,]FIXATION-RHIZOSPHERE,PHYLLOPLANE
N2 [C&1 Fixed References
Rhizosphere
Sand dune-no vegetation oo* Akkermans, 1971
+vegetation max. of 0.1 kg/ha/yr Akkermans, 1971
+ vegetation including algae 0.1-0.9 kg/ha/yr Akkermans, 1971
Paddy soil-algae N 10 kg/ha/yr Rinaudo et al., 1971
-rice plants (heterotrophic
bacteria) 9.8-48.8 kg/ha/yr Rinaudo et al., 197 1
Submerged saline soil&corn + Hauke-Pacewiczowa et al., 1969
Saline soil-moist 00 Hauke-Pacewiczowa et al., 1970
-moist + maize 00 Hauke-Pacewiczowa et al., 1970
-waterlogged 00 Hauke-Pacewiczowa et al., 1970
-waterlogged + maize + Hauke-Pacewiczowa et al., 1970
Pine roots-mycorrhiza 00 Silver, 1969
Rice root zone + Yoshida and Ancajas, 1971
Phylloplane
Gunnera-Nostoc up to 72 kg/hr/yr Silvester and Smith, 1969
* 00 Not detected.
association suggest N,[C,H,] fixation rates up to 90 kg N/ha/year (Dabereiner and Dart,

personal communication).
6. ~~~~~Q~~~~e(Table 10). Both extracellular and intracellular associations of the aerial
portions of the plant are included in this section. The Gun~era-~osfoc symbiosis is the
only reported example of an above ground plant association producing a positive C,H2-
C2H4 test, Nodule-bearing leaves of other plants such as Psychotria and Ardisia crispa
(Hofstra and Koch-Bosma, 1970) have been examined. Other N,-fixing phylloplane epi-
phytes which have been reported to be active with other methods (Jones, 1970; Ruinen,
1970) should be tested.
7. mammals (Table I I>. The C,H,-C2H, assay has been used to detect possible N,
fixation by rumen, caecal and intestinal contents of humans, swine, guinea-pigs, sheep,
TABLE il. MEASUREMENT

OF NATURALSAMPLE N2[CZW2] FIXATION-MAMMALS
MammaIs Sample Calcuiated N,[CZHZ] References

fixed
Human Feces O-170 ng/g/day Bergersen and Hipsley, 1970

Swine Caecal contents Bergersen and Hipsley, 1970
Guinea-pig Whole animal O~D~~,anima~,~ay Bergersen and Hipsiey, 1970
Sheep Rumen contents O-325 ~g/g~day
or I.1 mg~rumen/day Granhatl and Ciszuk, 1971
Sheep Filtrate of rumen contents 1 .O mg/rumen/day Elleway et al., 1971
Sheep Rumen contents Postgate, 1970b, 1971a
Steer Filtrate of rumen contents 2:: Elleway et al., 1971
Steer Rumen contents 10 mg/rumen/day Hardy et al., 1968b
Camel Filtrate of rumen contents AD Elleway ei al., 1971
Goat Rumen contents 130 ng/gIday Granhatl and Ciszuk, 1971
Reindeer Feces O-175 ng/day Granhall and Ciszuk, 1971
Rabbit Caecal contents 85 &day Granhall and Ciszuk, 1971
AD-Activity detected.
cattle, cameIs, goats, reindeer, and rabbits (Bergersen and Hipsley, 1970; E&way et a/.,
1971; Granhall and Ciszuk, 1971; Hardy et al., 1968b; Postgate, 197laj. Traces of activity
were observed in all cases with greater activity found under anaerobic than aerobic condi-
tions. Isolated organisms included K. aerogenes, Desulfotomaculum, and C. pasteurianum
(Bergersen and Hipsley, 1970; Postgate, 1970b, 1971a). Acetylene inhibits CH, formation
indicating significant effects of C&H, on rumen metabolism and the need for caution in
interpretation of results for N2[C,H,] fixation with mammalian samples. measurement of
C2H, reduction by a guinea pig encfosed with CzHz and an O2 : inert gas mixture for a one-
day period provided results for a natural system while all other analyses used samples of
rumen or intestinal material. Calculated N,[C,H;] fixed was about 1 pg, 1 mg and 10 mg
N/animal/day for guinea pigs, sheep and steers, respectively. Contribution of possible N2
fixation to the animals’ nutrition is insignificant, representing only about O-01 per cent of
the daily requirement.
The number and variety of the contributions, tabulated above, and the analytical advant-
ages of the C2H,-C,H4 assay validate the almost ubiquitous utility of this technique for
studies of N, fixation by N,ase, N,-fixing organisms and natural samples. No other tech-
nique of equivalent utility is available.
REFERENCES
ABELES F. B., CRACKERL. E., FL.ORENCE L. E. and LEATHERG. R. (1971) Fate of air pollutants: removal
of ethylene, sulfur dioxide and nitrogen dioxide by soil. Science 173,914-916.
AKKERMANSA. D. L. (1971) Nitrogen Fixation and Nodulation of Abuts and HippophaZ under Natural
Conditions. Doctoral Thesis, University of Leiden.
ANON. (1971) Rothamsted Experimental Station Report for 1970, Part 1, pp. 84-87.
BAYLISG. T. S. (1969) Mycorrhizal nodules and growth of Podocarpris in nitrogen-poor soil. Nature, Land.
223,1385-1386.
BECKINGJ. H. (1971) The physiological significance of the leaf nodutes of Psychotria. In Eiological Nitrogen
Fixation in NaturalandAgriculturalKabitats (T. A. Lie and E. G. Mulder, Eds) PI. Soil Spec. Vol. 361-374.
BELL F. and NUTMANP. S. (1971) Experiments on nitrogen fixation by nodulated lucerne. In Biologicnl
Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds) PI. Soil,
Spec. Vol., 231-264.
BENEMANNJ. R., YOCH D. C., VALENTINER. C. and ARNON D. I. (1969) The electron transport system in
nitrogen fixation by Atotobacter-I. Azotoflavin as an electron carrier. Proc. natl. Acad. Sri. U.S.
64,1079-1086.
BENEMANNJ. R., YOCH D. C., VALENTINER. C. and ARNON D. I. (1971) The electron transport system in
nitrogen fixation by Azotubacter-III. Requirements for NADPH-slIpported nitrogenase activity,
Biochim. biophys. Acta 226,205~212.
BERGERSEN F. J. (1970a) The quantitative relationship between nitrogen fixation and the acetylene-reduction
assay. Aust. J. biol. Sri. 23,1015-1025.
BERGERSENF. J. (1970b) The biochemistry of symbiotic nitrogen fixation in legumes. Abstract of Tenth
international Congress Microbiology, Mexico City, Mexico, p. 220.
BERGERSEN F. J. and HIPSLEYE. H. (1970) The presence of N&ixing bacteria in the intestines of man and
animals. J. gen. Microbial. 60,61-65.
BERGERSEN F. J. and TURNERG. L. (1970) Gel filtration of nitrogenase from soybean root nodule bacteroids.
Siochim. biophys. Acta 214, 28-36.
BIGGINSD. R. and KELLY M. (1970) interaction of nitrogenase from ~lebsiella pnea~noniae with ATP or
cyanide. Biochi~n. biophys. Acta 205,288-299.
BIGGINS D. R., KELLY M. and POSTGATE J. R. (1971) Resolution of nitrogenase of Mycobacterium flavum
301 into two components and cross reaction with nitrogenase components from other bacteria. Eur.
J. Riochem. 20, 140-143.
BIGGINSD. R. and POSTGATEJ. R. (1969) Nitrogen fixation by cultures and cell-free extracts of Mycobac-
terium jlavum 301. J. gen. Microbial. 56, 181-193.
BIGGINSD. R. and POSTGATEJ. R. (197Ia) Nitrogen fixation by extracts of Mycobacterium javum 301.
Use of natural electron donors and oxygen-sensitivity of cell-free preparations. .&r. J. Biochem. 19,
_- - s .
408-41
BIGGINSD. R. and POSTGATEJ. R. (1971b) Confusion in taxonomy of a nitrogen-axing bacterium currently
classified as ~ycobacfer~um flavum 301. J. gen. Microbiul. 65.1199123.
BOTHEH. (1970). Photosynthetische stickstoffixierung mit einem iellfreien extrakt aus der blaualge Anabaena
cylindrica. Ber. Dtsch. Ges. Bd. 83,421432.
BREZONIKP. L. and HARPERC. L. (1969) Nitrogen fixation in some anoxic lacustrine environments. Science
164,1277-1278.
BROCKWELLJ. (1971) An appraisal of an IBP experiment on nitrogen fixation by nodulated legumes. In
Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds)
PI. Soil Spec. Vol., 265-272.
BRINKS R. D. JR., PUTNAMH. D. and BREZONIKP. L. (1969) Biological nitrogen fixation in an estuarine
environment. Eacteriol. Proc. p. 35.
BROU~ESR., MAYFIELDC. I. and KNOWLESR. (1971a) Effect of oxygen partial pressure on nitrogen fixation
and acetylene reduction in a soil system. Agron. Abs. p. 80.
BROUZESR., MAYFIELDC. I. and KNOWLESR. (1971b) Effect of oxygen partial pressure on nitrogen fixation
and acetylene reduction in a sandy loam soil amended with glucose. In Biological Nitrogen Fixation in
Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds) PI. Soil Spec. Vol., 481494.
BRONZESR. and KNOWLESR. (1971) Inhibition of growth of Clostridiumpasteurianum by acetylene: implica-
tion for nitrogen fixation assay. Can. J. Microbial. 17, 1483-1490.
BULENW. A., LECOMTEJ. R., BURNSR. C. and HINKSONJ. (1965) Nitrogen fixation studies with aerobic and
photosynthetic bacteria. In Non-Heme Iron Proteins: Role in Energy Conversion. (A. San Pietro, Ed.)
pp. 261-274. The Antioch Press, Yellow Springs, Ohio.
BUNT J. S., COOKSEYK. E., HEEB M. A., LEE C. C. and TAYLORB. F. (1970) Assay of algal nitrogen fixation
in the marine subtropics by acetylene reduction. Nature, Land. 227, 1163-I 164.
BURNSR. C. (1969) The nitrogenase system from Azotobucter: activation energy and divalent cation require-
ment. Biochim. biophys. Acta 171,253-259.
BURNS R. C., FUCHSMANW. H. and HARDY R. W. F. (1971) Nitrogenase from vanadium-grown Azoto-
bncter: isolation, characteristics and mechanistic implications. Biochem. biophys. Res. Commun. 42,
353-358.
BURNS R. C. and HARDY R. W. F. (1970) Crystallization and properties of MO-Fe protein of Azotobacter
nitrogenase. Federation Proc. 29,404.
BURNSR. C. and HARDYR. W. F. (1972) Purification of nitrogenase and crystallization of its MO-Fe pro-
tein. In Methods in Enzymology, Photosynthesis and Nitrogen Fixation (A. San Pietro, Ed.), Vol. 23B,
In press. Academic Press, New York.
BURNSR. C., HOLSTENR. D. and HARDYR. W. F. (1970) Isolation by crystallization of the MO-Fe protein
of Azotobacter nitrogenase. Biochem. biophys. Res. Commun. 39,90-99.
BURRISR. H. (1969) Progress in the biochemistry of nitrogen fixation. Proc. Roy. Sot. London B172, 339-354.
BURRIS R. H. and WILSON P. W. (1957) Methods for measurement of nitrogen fixation. In Methods in
Enzymology (S. P. Colowick and N. 0. Kaplan, Eds), Vol. 4, pp. 355-367, Academic Press, New York.
CAMPBELLN. E. R., DULAR R., LEES H. and STANDINGK. G. (1967) The production of r3N2 by 50 MeV
protons for use in biological nitrogen fixation. Can. J. Microbial. 13,387-399.
CAMPBELLN. E. R. and EVANSH. J. (1969) Use of Pankhurst tubes to assay acetylene reduction by faculta-
tive and anaerobic nitrogen-fixing bacteria. Can. J. Microbial. l&1342-1343.
COPLEYP. W. and REUSS J. 0. (1971) In uivo acetylene reduction assay for symbiotic nitrogen fixation.
Agron. Abs. p. 81.
Cox R. M. and FAY P. (1969) Special aspects of nitrogen fixation by blue-green algae. Proc. Roy. Sot.
London B172,357-366.
DAESCHG. and MORTENSON L. E. (1968a) Sucrose catabolism in Clostridiumpasteuriunum and its relation to
N2 fixation. J. Bucteriol. 96, 340-351.
DAESCHG. and MORTENSON L. E. (1968b) Regulation of N,-fixation in Clostridiumpasteuriunum. Bacterial.
Proc. p. 132.
DAHLENJ. V., PAREJKOR. A. and WILSONP. W. (1969) Complementary functioning of two components
from nitrogen-fixing bacteria. J. Bacterial. 98,325-326.
DALTONH., MORRISJ. A., WARD M. A. and MORTENSONL. E. (1971) Purification and some properties of
molybdoferredoxin, a component of nitrogenase from Clostridiumpasteurianum. Biochemistry 10,2066-
2072.
DALTONH. and POSTGATEJ. R. (1969) Growth and physiology of Azotobncter chroococcum in continuous
culture. J. gen. Microbial. 56, 307-319.
DART P. J. and DAY J. (1971) Effects of incubation temperature and oxygen tension on nitrogenase activity
of legume root nodules. In Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie
and E. G. Mulder, Eds) PI. Soil Spec. Vol., 167-l 84.
DAVIDSONF. and REUSZERH. W. (1970) Influence of Rhizobium japonicum strain and soybean (Glycine
max) variety on nitrogen fixation. Bacterial. Proc. p. 2.
DILWORTHM. J. (1966) Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteuriunum.
Biochim. biophys. Acta 127,285-294.
DILWORTHM. J., SUBRAMANIAN D., MUNSONT. 0. and BURRISR. H. (1965) The adenosine triphosphate
requirement for nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Biochim. biophys.
Acta 99,486-503.
DUONG T. P. and TIEDJEJ. M. (1971) Factors affecting growth and acetylene reduction of photosynthetic
bacteria isolated from lake sediments. Agron. Abs. p. 81.
DROZD J. and POSTGATEJ. R. (1970a) Interference by oxygen in the acetylene-reduction test for aerobic
nitrogen-fixing bacteria. J. gen. Microbial. 60,427429.
DROZD J. and POSTGATEJ. R. (1970b) Effects of oxygen on acetylene reduction, cytochrome content and
respiratory activity of Azotobacter chroococcum. J. gen. Microbial. 63,63-73.
EK-JANDCRJ. and FAHRAEUSG. (1971) Adaptation of Rhizobium to subarctic environment in Scandinavia.
In Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds)
PI. SoilSpec. Vol., 129-137.
ELLEWAYR. F., SABINEJ. R. and NICHOLASD. J. D. (1971) Acetylene reduction by rumen microflora.
Arch. Mikrobiol. 76,277-29 1.
EVANS H. J. (1969) Preparation and some properties of the nitrogenase system from legume nodule bac-
teroids. Abstracts of Eleventh InternationalBotanical Congress, Seattle, Washington, p. 57.
EVANS H. J., CAMPBELLN. E. R. and HILLS S. (1971) A symbiotic nitrogen-fixing bacterium from the
surfaces of nodules and roots of legumes. Can. J. Microbial. 18, 13-21.
EVANSM. C. W. and SMITHR. V. (1971) Nitrogen fixation by the green photosynthetic bacterium Chloro-
pseudomonas ethylicum J. gen. Microbial. 65,95-98.
FAY P. (1969) Metabolic activities of isolated spores of Anabaena cylindrica. J. exptl. Bot. 20, 100-109.
FAY P. (1970) Photostimulation of nitrogen fixation in Anabaena cylindrica. Biochim. biophys. Acta 216,
353-356.
FAY P. and FOGG G. E. (1962) Studies on nitrogen fixation by blue-green algae-III. Growth and nitrogen
fixation in Chlorogloeafritschii Mitra. Arch. Mikrobiol. 42,310-321.
FISHER R. J. and BRILL W. J. (1969) Mutants of Azotobacter vinelandii unable to fix nitrogen. Bacterial.
Proc. p. 148.
FRANCISA. J. and ALEXANDERM. (1971) Acetylene reduction by effective and ineffective soybean nodules.
Bacterial. Proc. p. I,
FREEDBAIRNH. T. and BUDDENHAGEN I. W. (1964) Ethylene production by Pseudomonas solanacearum.
Nature, Lond. 202,313-314.
FUCHSMANW. H. and HARDYR. W. F. (1970) Model studies on nitrogenase (N,ase): acrylonitrile reduction.
Bacterial. Proc. p. 148.
FUCHSMANW. H. and HARDYR. W. F. (1972) Nitrogenase-catalyzed acrylonitrile reductions. Bioinorganic
Chem. 1,197-215.
GALLONJ. R., LA RUE T. A. and KURZ W. G. W. (1971) Characteristics of nitrogenase activity in broken
cell preparations of the blue-green alga Gloeocapsa sp. LB 795. Can. J. Microbial. 18, 327-360.
GIBSONA. H. (1971) Factors in the physical and biological environment affecting nodulation and nitrogen
fixation by legumes. In Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie
and E. G. Mulder, Eds) PI. SoilSpec. Vol., 139-152.
GRANHALLU. (1971) Acetylene reduction by blue-green algae isolated from Swedish soils. Oikos 21,330-332.
GRANHALLU. and CISZUKP. (1971) Nitrogen fixation in rumen contents indicated by the acetylene reduction
test. J. gen. Microbial. 65,91-93.
HAM G. E. and COOPERR. L. (1971) Correlation of laboratory estimates of symbiotic nitrogen fixation with
soybean seed yields in the field. Agron. Abs. p. 82.
HARDY R. W. F. (1971) Determination of Nitrogen-Fixing Activity based on Conversion of Acetylene to
Ethylene. U.S. Patent 3,591,458.
HARDYR. W. F., BURNSR. C., FRY K. T. and HOLSTENR. D. (1969a) Biochemical, chemical and biological
aspects of N, fixation. Abstracts of Eleventh International Botanical Congress, Seattle, Washington,
p. 85.
HARDYR. W. F., BURNSR. C., HEBERTR. R., HOLSTENR. D. and JACKSONE. K. (1971a) Biological nitrogen
fixation: a key to world protein. In Biological Nitrogen Fixation in Natural and Agricultural Habitats
(T. A. Lie and E. G. Mulder, Eds) PI. Soil Spec. Vol. 561-590.
HARDYR. W. F., BURNSR. C. and PARSHALLG. W. (1971b) The biochemistry of Nz fixation. Advan. Chem.
100,219-247.
HARDY R. W. F. and HOLSTENR. D. (1971). N fertilization of soybeans: effects on N fixation and yield.
Agron. Abs. p. 82.
HARDY R. W. F., HOLSTENR. D., JACKSONE. K. and BURNSR. C. (1968a) The C,H2-C,H, assay for NZ
fixation: laboratory and field evaluation. PI. Physiol. 43 (Suppl.) 9.
HARDY R. W. F., HOLS-~ENR. D., JACKSONE. K. and BURNSR. C. (1968b) The acetylene-ethylene assay
for Nz fixation: laboratory and field evaluation. PI. Physiol. 43, 1185-1207.
HARDYR. W. F., HOLSTENR. D., JACKSONE. K. and BURNSR. C. (1968~) In situ Nz fixation by the C,H,-
C,H, assay. Agron. Abs. p. 93.
HARDY R. W. F., HOLSTENR. D., JACKSONE. K. and BURNSR. C. (1969b) Symbiotic Nz fixation: age-
activity relationship and differential inhibition by various fertilizers. Agron. Abs. p. 95.
HARDYR. W. F. and JACKSONE. K. (1967) Reduction of model substrates-nitriles and acetylenes- by nitro-
genase (N,ase). Federation Proc. 24,725.
HARDY R. W. F. and KNIGHT E., JR. (1966) Reductant-dependent adenosine triphosphatase of nitrogen-
fixing extracts of Azotobacter vinelandii. Biochim. biophys. Acta 122,520-531.
HARDY R. W. F. and KNIGHT E., JR. (1967) ATP-dependent reduction of azide and HCN by Nz-fixing
enzymes of Azotobacter vinelandii and Clostridium pasteurianum. Biochim. biophys. Acta 139, 69-90.
HARDYR. W. F., KNIGHT E., JR. and JACKSONE. K. (1967) Substrate versatility of nitrogenase. Bacterial.
Proc. p. 112.
HARDYR. W. F., PARSHALLG. W., JACKSONE. K., HOL~TENR. D. and BURNSR. C. (1968d) Nitrogenase:
biochemistry and postulated mechanism of a most versatile enzyme. Abstracts of American Chemical
Society Meeting, San Francisco, April.
HAUKE-PACEWICZOWA T., BALENDREAU J. and DOMMERGUES Y. (1969) Effect of submersion on microbial
molecular nitrogen fixation in corn rhizosphere. C. r. hebd. Acad. Sci., Ser. D 269, 1356-1358.
HAUKE-PACEWICZOWA T., BALENDREAUJ. and DOMMERGUESY. (1970) Fixation microbienne de l’azote
dans un sol salinin Tunisien. Soil Biol. Biochem. 2,47-53.
HAYSTEADA., ROBINSONR. and STEWARTW. D. P. (1970) Nitrogenase activity in extracts of heterocystous
and non-heterocystous blue-green algae. Arch. Mikrobiol. 74,235-243.
HENRIKSSONE. (1971) Algal nitrogen fixation in temperate soils. In Biolopical Nitro,een Fixation in Natural
andAgricul&ral~ab~tats (T. A. Lie and E. G. Milder, Eds) PI. Soil Siec. Vol., 415-419.
HENRIKSSON E. and SIMUB. (1971) Nitrogen fixation by lichens. Oikos 22,119-121.
HILL S. and POSTGATE J. R. (1969) Failure of putative nitrogen-fixing bacteria to fix nitrogen. J. gen. Micro-
biol. S&277-285.
HOFSTRAJ. J. and KOCH-BOSMAT. (1970) Some aspects of the leaf nodule symbiosis in Ardisia crispa.
Acta Bot. Neerl. 19,665-670.
HOLSTENR. D., BURNSR. C., HARDYR. W. F. and HEBERTR. R. (1971) Rhizobium-plant cell interaction:
establishment of an in nitro symbiosis. Nuture,Lond. 232,173-178.
HOL~TENR. D. and HARDYR. W. F. (1972) Nz-fixing plant bacterial symbiosis in tissue culture. In Methods
in Enzymology, Photosynthesis and Nitrogen Fixation (A. San Pietro, Ed.) Vol. 23B, in press.
Academic Press, New York.
HOL~TENR. D., HEBERTR. R., BURNSR. C. and HARDYR. W. F. (1970) Symbiotic N&ixation in plant cell
cultures. Bacterial. Proc. p. 149.
HOLSTENR. D., HEBERTR. R., JACKSONE. K., BURNSR. C. and HARDYR. W. F. (1969) Correlation of N,
fixation and legume morphology. Bucteriol. Proc. p. 149.
HOWARDD. L., FREA J. I., PFISTERR. M. and DUGAN P. R. (1970) Biological nitrogen fixation in Lake
Erie. Science 169,61-62.
HWANGJ. and BURRISR. H. (1968) Binding sites of nitrogenase. Federation Proc. 27, 639.
ILAG T. and CURTISR. W. (1968) Production of ethylene by fungi. Science 159, 1357-1358.
ISHIZAWAS., SUZUKI T. and ARARAGIM. (1970) Trend of free-living nitrogen fixers in paddy soil. Proceed-
ings Second Symposium on Nitrogen Fixation and Nitrogen Cycle (H. Takahashi, Ed) Sendai, pp. 28-40.
JACKSONE. K. and HARDY R. W. F. (1967) Nitrogenase-catalyzed HD exchange in Azotobacter uinehmdii.
PI. Physiol. 42, (Suppl.) 38.
JACKSONE. K., PARSHALLG. W. and HARDYR. W. F. (1968) Hydrogen reactions of nitrogenase. Formation
of the molecule HD by nitrogenase and by an inorganic model. J. biol. Chem. 243,4952-4958.
JENG D. Y., DEVANATHAN T. and MORTENSONL. E. (1969) Components of cell-free extracts of Clostridium
pasteurianum as required for acetylene reduction and Nz fixation. Biochem. biophys. Res. Commun.
35, 6255633.
JEWELLW. J. and KULASOORIYA S. A. (1970) The relation of acetylene reduction to heterocyst frequency in
blue-green algae. J. exptl. Bot. 21, 874-880.
JONESK. (1970) Nitrogen fixation in the phyllosphere of the Douglas Fir, Pseudotsuga douglasii. Ann. Bot.
34,239-244.
JORGENSON J. R. and WELLSC. G. (1970) Non-symbiotic Nz-fixation in soil influenced by prescribed burning.
Agron. Abs. p. 159.
KASS D. L., DROSDOFFM. and ALEXANDER M. (1971) Nitrogen fixation by Azotobacterpaspah in association
with bahiagrass (Paspalum notatum). Proc. Soil Sci. Sot. Am. 35,286-289.
KAVANAGHE. P. and POSTGATEJ. R. (1970) Absorption and release of hydrocarbons by rubber closures: a
source of error in some biological assays. Lab. Practice 19,159-160.
KELLY M. (1968a) The kinetics of the reduction of isocyanides, acetylenes and the cyanide ion by nitro-
genase preparations from Azotobacter chroococcum and the effects of inhibitors. Biochem. J. 107, l-5.
KELLY M. (1968b) Hydrogen-deuterium exchange reactions catalyzed by nitrogenase. Biochem. J. 109,
322-324.
KELLY M. (1968~) Properties of purified nitrogenase of Azotobacter chroococcum. Biochim. biophys. Acta
171,9-22.
KELLY M. (1969) Comparisons and cross reactions of nitrogenase from Klebsiellu pneumoniue, Azotobacter
vinelandii, and Bacilluspolymyxa. Biochim. biophys. Acta 191,527-540.
KELLYM., KLUCASR. V. and BURRISR. H. (1967) Fractionation and storage of nitrogenase from Azotobacter
vinelandii. Biochem. J. 105,3C-5C (1967).
KELLY M. and LANG G. (1970) Evidence from Mossbauer spectroscopy for the role of iron in nitrogen
fixation. Biochim. bipohys. Acta 223,86-104.
KENNEDYI. R. (1970a) Properties of nitrogenase from legumes. Proc. Austr. Biochem. Sot. 3,11.
KENNEDYI. R. (1970b) Kinetics of acetylene and cyanide reduction by the N2-fixing system of Rhizobium
Iupini. Biochim. biophys. Acta 222,135-144.
KLUCAS R. V. (1969) Nitrogen fixation assessment by acetylene reduction. Proceedings Eutrophication-
Biostimtdation Assessment Workshop IV. (E. J. Middlebrooks et aI., Eds) pp. 109-116. Pacific Northwest
Water Laboratory, Corvallis, Oregon.
KLUCAS R. V. and EVANS H. J. (1968) An electron donor system for nitrogenase-dependent acetylene
reduction by extracts of soybean nodules. Pt. Physiol. 43,1458-1460.
KLUCAS R. V., KOCH B., RUSSELLS. A. and EVANS H. J. (1968) Purification and some properties of the
nitrogenase from soybean (Glycine max Merr.) nodules. PI. Physiot. 43,1906-1912.
KOCH B. and EVANSH. J. (1966) Reduction of acetylene to ethylene by soybean root nodules. PI. Physiol.
41,1748-1750.
KOCH B., EVANSH. J. and RUSSELLS. (1967a) Reduction of acetylene and nitrogen gas by breis and cell-free
extracts of soybean root nodules. PI. Physiol. 42,466-468.
KOCH B., EVANSH. J. and RUSSELLS. (1967b) Properties of the nitrogenase system in cell-free extracts of
bacteriods from soybean root modules. Proc. natl. Acad. Sci. U.S. 58, 1343-1350.
KOCH B., EVANSH. J. and RUSSELLS. (1967~) Reduction of nitrogen gas and acetylene by cell-free. extracts
of soybean nodule bacteroids. PI. Physiol. 42 (Suppl.) 38.
KOCH B. L., WONG P. P., RUSSELLS. A., HOWARDR. and EVANSH. J. (1970a) Some properties of non-heme
iron protein of soybean (Glycine max Merr.) nodules. PI. Physiol. 46, (Suppl.) 45.
S.B.B. S/l-F
78 R. W. F. HARDY, R, C. BURNS AND R. D. HOLSTEN
KOCH B. L., WANG P. P., RUSSELLS. A., HOWA~CD R. and EVANSH. J. (1970b) Puri~cation and some pro-
perties of a non-heme iron protein from the bacteroids of soya bean (G&&e max Merr.) nodules.
B&hem. J. 118,773-781.
KURZ W. G. W. and LA RUE T. A. (1970) Nitrogenase in synchronized Azotobacter. Abstracts of Tenth
International Congress Microbiology, Mexico City, Mexico, p. 142.
KURZ W. G. W. and Ln RUE T. A. (1971) Nitrogenase in Anabaena~os-uquae filaments lacking heterocysts.
Nutur~issenchafte~~ 58,147.
LA RUE T. A. and KURZ W. G. W. (1970) Continuous determination of nitrogenase. Abstracts of Temh
International Congress Microbiology, Mexico City, Mexico, p. 142.
LIE T. A. (1971) Symbiotic nitrogen fixation under stress conditions. In B~o~ogi~a~Niirogen Fixation in
NatiIrala~d~r~~~~ltura~Hab~tats(T. A. Lie and E. G. Mulder, Eds) Pl. SoilSpee. Vol., 117-127.
MACGREGORA. N. and JOHNSOND. E. (1971) Capacity of desert algal crusts to fix atmospheric nitrogen.
Proc. Soil Sci. Sot. 35,843-844.
MACKINTOSHM. E. (1971) Nitrogen fixation by Tttiobaciltus ferrooxidans species J. gen. Microbial. 66, i-ii.
MCK~NNA C. E., BENEMANN J. R., RIPLEYL. R. and TRAYLORT. G. (1971) Vanadium nitrogenase and the
role of molybdenum in biological nitrogen fixation. Federation Proc. 30,1291.
MC KENNA C. E., BENEMANN J. R. and TRAYLORT. G. (1970) A vanadium containing nitrogenase prepara-
tion: implications for the role of molybdenum in nitrogen fixation. Biochem. biophys. Res. Commun, 41,
ISOl-1508.
MILEK~I~A E. I., RAKHLEEVAE. E. and IVANOVT. D, (197la) Influence of both the oxygen partial pressure
and the substrate concentration on acetylene r~uction by intact cells of Azofobacter v~ne~a~~~. Izw.
Akad. Nauk SSSR, Ser. Biol. 255-260.
MI~EKHINAE. I., RAKHLEEVAE. E., IVANOVI. D. and MILLER U. M. (1971b) Influence of oxidative phos-
phorylation uncouplers and inhibitors on nitrogen fixation and acetylene reduction in Azotobacter
vinelandii cells. Izv. Akad. Nauk SSSR, Ser. Biol. 415-422,
MILLBANK J. W. (1969) Nitrogen fixation in moulds and yeasts-a reappraisal. Arch. ~ikrob~o~. 68, 32-39,
MILLBANKJ. W. (1970) The effect of conditions of low oxygen tension on the assay of nitrogenase in moulds
and yeasts using the acetylene reduction technique. Arch. hEkrobiol.72,375-377.
MKLI.~ANKJ. W. (1972) Nitrogen metabolism in lichens-IV. The nitrogenase activity of the Nostoc phyco-
Mont in Peltigera canina. New Phytol. 71, l-10.
M~L~~A~~ J. W. and KERSHAWK. A. (1370) Nitrogen metabolism in lichens-III. Nitrogen fixation by
internal Cenha/odia in Lobariu oulmonoria. New Phvtot. 69.595-597.
MORTENSONL.E. (1961) A simple-method for measurntg nitrogen fixation by cell-free enzyme preparations
of Clostridiumpasteurianutn. Anal. Biochern. 2,216”.220.
MOUSTAFAE. (1969a) Cold lability of the nitrogen fixation system in excised legmninous nodules. Phytochem.
8.993-995.
MOU&_FA E. (1969b) Use of acetyIene reduction to study the effect of nitrogen fertilizer and defoliation on
nitrogen fixation by field-grown white ciover. New Zealund J. agric. Res. I&691-696.
MOUSTAFA E. (1970) Purification of the cold-labile component of the Azotobacter nitrogenase. Biochim,
biophys. Acta 206,178-180.
MOUSTAFAE. and MOUTE~SO~L. E. (1967) Acetylene reduction by nitrogen fixing extracts of C~ostridinm
pasteurjana~z: ATP requirement and inhibition by ADP. Nature, Land. 216,241-242.
MOUSTAFA E. and MORTENSONL. E. (1968) Determination of azoferredoxin activity using cold inactivated
crude extracts of Clostridiumpasteurianum. Anal. Biochem. 24,226-231.
MOUSTAPAE. and MORTENSON L. E. (1969) Properties of azoferredoxin purified from nitrogen-fixing extracts
of ~~astridiumpastelirjanum. Biochim. biophys. Acta 172,106-l 15.
MOUSTAFAE., MORETENSON L. E. and BUI P. T. (1968) Azoferredoxin, the cold-labile component of nitro-
genase from Clostr~diu~~pasteuria~zum. Bacteriof. Proc. p. 132.
MUNSON T. 0. and BURRIS R. H. (1969) Nitrogen fixation by Rhodospirillam rubrum grown in nitrogen-
limited continuous culture. f. Bncteriof. 97, 1093-1098.
MIJRP~V P. M. and KOCH B. L. (1971) Compatability of the coillponents of nitrogenase from soybean
bacteroids and free-living nitrogen-fixing bacteria. Biochim. biophys. Acta 253,295-297.
NEILSON A,. RIPPKAR. and KUNISAWAR. (1971) Heterocyst formation and nitrogenase synthesis in Anabaena
species: A kinetic study, Arch. Mikrobiol. 76,139-l%.
NICHOLAS D. J. D., SILV~STER D. J. and FOWLERJ. F. (1961) Use of radioactive nitrogen in studying nitrogen
fixation in bacterial cells and their extracts. Nature, Land. 189,634-636.
OOHOGHOR~E C. G. 0. and PATE5. S. (1971) The nitrate stress syndrome of the field pea (Pisum aruensa I_.).
Jn ~~o~agical Nitrogen Fixation in Nataral and Agricuitura~ Habitats (T. A. Lie and E. G. Mulder, Eds)
Pf. Soit Spec. Vol., 185-202.
OPPENHEIM .I., FISHER R. J., WILSONP. W. and MARCUS L. (1970) Properties of a soluble nitrogenase in
Azotobacter. J. Bacterioi. 101,292-296.
PAN S. and SCHMIDTE. L. (1969) Nitrogen-ping bacteria in controlled soil: plant root system. Bucteriof.
Proc. p. 14.
PAREJKOR. A. and WILSONP. W. (1968) Taxonomy of Azotomonas species. J. Bacterial. 95, 143-146.
PAREJKOR. A. and WILSONP. W. (1970) Regulation of nitrogenase synthesis by Klebsiella pneumoniae.

Can. J. Microbial. 16,681-685.
PAUL E. A. and RICE W. A. (1970) Asymbiotic nitrogen fixation in soil. Abstracts of Tenth International
Congress Microbiology, Mexico City, Mexico p. 141.
PAUL E. A., MYERS R. J. and RICE W. A. (1971) Nitrogen fixation in grassland and associated cultivated
ecosystems. In Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G.
Mulder, Eds) PI. Soil Spec. Vol., 495-507.
POSTGATE J. R. (1970a) Stikstofbinding. Chem. Teck. 25,625-627; or Tropical Sci. 12,223~229.
POSTGATEJ. R. (1970b) N,-fixation by sporulating sulfate-reducing bacteria including rumen strains. J. gen.
Microbial. 63,137-139.
POSTGATEJ. R. (1971a) Biochemical and physiological studies with free-living, nitrogen-fixing bacteria. In
Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds)
PI. SoilSpec. Vol., 551-559.
POSTGATEJ. R. (1971b) Relevant aspects of the physiological chemistry of nitrogen fixation. Symp. Sot.
Gen. Microbial. 21,287-307.
PRATT D. C., BERGADP. L. and HAM G. E. (1971) Nitrogenase activity in a strain of Rhodopseudomonas
sp. containing bacteriochlorophyll b. Bacterial. Proc. p. 139.
RICE W. A. and PAUL E. A. (1971) The acetylene reduction assay for measuring nitrogen fixation in water-
logged soil Can. J. Microbial. 17,1049-1056.
RICHMONDA. B. (1969) Liquid phase for gas chromatographic separation of lower hydrocarbons. J. chro-
matog. Sci. 7,321-322.
RIEDERER-HENDERSON
M. A. and WILSONP. W. (1970) Nitrogen fixation by sulfate-reducing bacteria. J. gen.
Microbial. 61,27-31.
RINAUDOG., BALANDREAU J. and DOMMERGUES Y. (1971) Algal and bacterial non-symbiotic nitrogen fixa-
tion in paddy soils. In Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie
and E. G. Mulder, Eds) PI. SoilSpec. Vol., 471479.
RINAUDOG. and DOMMERGUES Y. (1971) Validity of acetylene reduction method for determination of nitro-
gen fixation in rice rhizosphere. Ann. Inst. Pasteur 121,93.
RIPPKA R., NEIL~ONA., COHEN-BAZIREG. and KUNI~AWAR. (1971) Nitrogen fixation by unicellular blue-
green algae. Arch. Mikrobiol. 76,341-348.
ROUGHLEYR. J. and DART P. J. (1969) Reduction of acetylene by nodules of Trifolium subterraneum as
affected by root temperature, Rhizobium strain and host cultivar. Arch. Mikrobiol. 69, 171-179.
RUINEN J. (1970) The phyllosphere V. The grass sheath, a habitat for nitrogen-fixing microorganisms. PI.
Soil33,661-671.
RUSSELL,S. A. and EVANSH. J. (1970) Assessment of Nitrogen Fixation by Alnus rubra by use of the Acetylene
Reduction Technique. Report to the Pacific Northwest Forest and Range Experiment Station.
RUSNESSD. and BURRISR. H. (1970) Acetylene reduction (nitrogen fixation) in Wisconsin Lakes. Limnol.
Oceanogr. 15,808-813.
ST. JOHN R. T. and BRILL W. J. (1972) Inhibitory effect of methylalanine on glucose-grown Azotobacter
oinelandii. Biochim. biophys. Acta 261, 63-69.
SCHOLLHORN
R. and BURRISR. H. (1966) Study of intermediates in nitrogen fixation. Federation Proc. 24,710..
SCHOLLHORN
R. and BURRIS R. H. (1967) Acetylene as a competitive inhibitor of NP fixation. Proc. Nat1
Acad. Sci. U.S. 58,213-216.
SCHRAUZERG. N. and DOEMENYP. A. (1971) Chemical evolution of a nitrogenase mode!-II. Molybdate-
cysteine and related catalysts in the reduction of acetylene to olefins and alkanes. J. Am. them. Sot.
93,1608-1618.
SCHRAUZERG. N. and SCHLESINGER G. (1970) Chemical evolution of a nitrogenase model. Reduction of
acetylene and other substrates by a molybdenum-thiol catalyst system. J. Am. them. Sot. 92, 1808-1809.
SCHRAUZERG. N., SCHLESINGER G. and DOEMENYP. A. (1971) Chemical evolution of a nitrogenase model-
III. The reduction of nitrogen to ammonia. J. Am. them. Sot. 93,1803-1804.
SCHWINGHAMMER E. A., EVANS H. J. and DAWSONM. D. (1970) Evaluation of effectiveness in mutant
strains of Rhizobium by acetylene reduction relative to other criteria of Nz fixation. PI. Soil 33,192-212.
SHETHNAY. I., STOMBAUGH N. A. and BURRISR. H. (1971) Ferredoxin from Bacillus polymyxa. Biochem.
biophys. Res. Commun. 42,1108-1116.
SILVERW. S. (1967) Biological nitrogen fixation. Science 157,100-102.
SILVERW. S. (1969) Biology and ecology of nitrogen fixation by symbiotic associations of non-leguminous
plants. Proc. Roy. Sot. Lond. B 172,389-399.
SILVERW. S. and MAGUE T. (1970) Assessment of nitrogen fixation in terrestial environments in field con-
ditions. Nature, Land. 227,378-379.
SILVESTER W. B. and SMITHD. R. (1969) Nitrogen fixation by Gunnera-Nostoc symbiosis. Nature, Land. 224,
1231.
SIMON M. A. and BRILL W. J. (1971) Mutant of Clostridium pasteurianum that does not fix nitrogen. J.
Bacterial. 105,65-69.
SISLERF. D. and ZOBELLC. E. (1951) Nitrogen fixation by sulfate-reducing bacteria indicated by nitrogen:
argon ratios. Science 113, 511-512.
SL~GERC. (1969) Symbiotic effectiveness and NZ fixation in nodulated soybean. PI. Physiol. 44, 1666-1668.
SL~GER C. and SILVERW. S. (1967) Biological reductions catalyzed by symbiotic nitrogen-fixing tissues.
Bacterial. Proc. p, 112.
SMITHR. V. and EVANSM. C. W. (1970) Soluble nitrogenase from vegetative cells of the blue-green alga
Anabaena cylindrica. Nature, Land. 225,1253-1254.
SMITH R. V. and EVANS M. C. W. (1971) Nitrogenase activity in cell-free extracts of the blue-green alga
Anabaena cylindrica. J. Bacterial. 105,913-917.
SMITHR. V. , NOY R. J. and EVANSM. C. W. (1971) Physiological electron donor systems to the nitrogenase
of the blue-green alga Anabaena cylindrica. Biochim. biophys. Acta 253,104-109.
SPRENT.J. I. (1969a) Effects of carbon monoxide and oxygen on acetylene reduction by detached soybean
nodules. Abstracts of Eleventh International Botanical Congress, Seattle, Washington, p. 207.
SPRENTJ. I. (1969b) Prolonged reduction of acetylene by detached nodules. Planta 88,372-375.
SPRENTJ. I. (1971a) The effects of water stress on nitrogen-fixing root nodules-I. Effects on the physiology
of detached soybean nodules. New Phytol. 70,9-17.
SPRENT J. I. (1971b) Effects of water stress on nitrogen fixation in root nodules. In Biological Nitrogen
Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds) PI. Soil Spec. Vol., 225-228.
STEWARTW. D. P. (1971) Physiological studies on nitrogen-fixing blue-green algae. In Biological Nitrogen
Fixation in Naturaland AgriculturalHabitats (T. A. Lie and E. G. Mulder, Eds) P1. Soil Spec. Vol., 377-391.
STEWARTW. D. P. (1969) Biological and ecological aspects of nitrogen fixation by free-living micro-organ-
isms. Proc. Roy. Sci. Land. B 172,367-388.
STEWARTW. D. P., FITZGERALDG. P. and BURRISR. H. (1967a) In situ studies on Nz fixation with the acety-
lene reduction technique. Science 158,536.
STEWARTW. D. P., FITZGERALDG. P. and BURRISR. H. (1967b) In situ studies on N, fixation using the
acetylene reduction technique. Proc. natl. Acad. Sci. U.S. 58,2071-2078.
STEWART.W. D. P., FITZGERALDG. P. and BURRIS R. H. (1968) Acetylene reduction by nitrogen-fixing
blue-green algae. Arch. Mikrobiol. 62,336-348.
STEWARTW. D. P., FITZGERALDG. P. and BURRISR. H. (1969a) Determinations of nitrogen fixation activity
and phosphorus availability in Wisconsin lakes utilizing acetylene-reduction assay. Proc. nail. Acad. Sci.
U.S. 64,1433.
STEWARTW. D. P., FITZGERALDG. P. and BURRISR. H. (1970) Acetylene reduction assay for determination
of phosphorus availability in Wisconsin lakes. Proc. natl. Acad. Sci. U.S. 66, 1104-l 111.
STEWARTW. D. P., HAYSTEADA. and PEARSONH. W. (1969b) Nitrogenase activity in heterocysts of blue-
green algae. Nature Land. 224,226-228.
STEWARTW. D. P. and LEX M. (1970) Nitrogenase activity in the blue-green alga Plectonema boryanum
strain 594. Arch. Mikrobiol. 73,250-260.
STEWARTW. D. P., MAGU~T., FITZGERALDG. P. and BURRISR. H. (1971) Nitrogenase activity in Wisconsin
lakes of differing degrees of eutrophication. New Phytol. 70,497-509.
STEWARTW. D. P. and PEARSONH. W. (1970) Effects of aerobic and anaerobic conditions on growth and
metabolism of blue-green algae. Proc. Roy. Sot. Lond. B 175,293-311.
STEYN P. L. and DELWICHEC. C. (1970) Nitrogen fixation by non-symbiotic microorganisms in some southern
California soils. Environ. Sci. Technol. 4,1122-l 128.
STREICHERS., GURNEYE. and VALENTINER. C. (1971) Transduction of the nitrogen fixing genes in Klebsiella
pneumoniae. Proc. natl. Acad. Sci. U.S. 68,1174-l 177.
SIJNDARARAOW. V. B. (1971) Field experiments on nitrogen fixation by nodulated legumes. In BiologicalNitro-
gen Fixation in Natural and Agricultural Habitats (T. A. Lie and E. G. Mulder, Eds) PI. Soil Spec. Vol.,
287-291.
SUZUKIT., TANABEI., ARARAGIM. and KAYOK. (1970) Abundance of free-living nitrogen fixers in Japanese
forest soil-I. Sejiri Japan cedar forest soil and Minamata evergreen forest soil. Proceedings of Second
Symposium on Nz Fixation and Nitrogen Cycle (H. Takahashi, Ed.) Sandai, pp. 4142.
TAYLORK. B. (1969) The enzymology of nitrogen fixation in cell-free extracts of Clostridium qasteurianum.
J. biol. Chem. 2&,171-179. -_ -
TJEPKIMAJ. D. and YOCUM C. S. (1970) Leghemoglobin facilitated oxygen diffusion in soybean nodule
slices. PI. Physiol. 46, (Suppl.) 44.
TURNERG. L. and BERGERSEN F. J. (1969) The relationship between nitrogen fixation and the production of
HD from D, by cell-free extracts of soya-bean nodule bacteroids. Biochem. J. 115, 529-53.5.
VANDECASTEELE J.-P. and BURRISR. H. (1970) Purification and properties of the constituents of the nitro-
genase complex from Clostridiumpasteurianum. J. Bacterial. 101,794-801.
VITOSH M. I. (1970) Substitution of acetylene for nitrogen in studies of biological nitrogen fixation. Chem.
Eng. News. 24 August, p. 24.
WAUGHMANG. J. (1971) Field use of acetylene reduction assay for nitrogen fixation. Oikos 22, 111-113.
WEBERD. F., CALDWELLB. E., SL~GER C. and VEST H. G. (1971) Some USDA studies on the soybean-
Rhizobium symbiosis. In Biological Nitrogen Fixation in Natural and Agricultural Habitats (T. A. Lie
and E. G. Mulder, Eds) PI. Soil Spec. Vol., 293-304.
WHEELERC. T. (1969) The diurnal fluctuation in nitrosen fixation in the nodules of Abuts eiutinosa and
Myricagale.‘New’Phytol. 68,675-682.
WHEELERC. T. (1971) The causation of the diurnal changes in nitrogen fixation in the nodules of Abuts
glutinosa. New Phytol. 70,487495.
WHITTENBURY R., PHILLIPSK. C. and WILKINSONJ. T. (1970) Enrichment, isolation and some properties of
methane-utilizing bacteria. J. gen. Microbial. 61,205-218.
WILSONP. W. and FRED E. B. (1935) The growth curve of a scientific literature. Sci. Monrhly 61,240-250.
WINTERH. C. and ARNOND. I. (1970) The nitrogen fixation system of photosynthetic bacteria. I. Prepara-
tion and properties of a cell-free extract from Chromatium. Biochim. biophys. Acta 197, 170-179.
WINTERH. C. and OBERJ. A. (1971) Preparation of a stable nitrogenase particle from Chromatium strain D.
Federation Proc. 30, 1292.
WOLK C. ?. (1970) Aspects of the development of blue-green algae. Ann. N. Y. Acad. Sci. 175, 641-647.
WOLK C. P. and WOJCIUCH E. (1971) Photoreduction of acetylene by heterocysts. Pfanta 97,126-134.
WONG P. and BURRISR. H. (1971) Nature of oxygen inhibition of nitrogenase. Proc. natl. Acad. Sci. U.S.
68,2899a-2900a.
WONG P., EVANSH. J., KLUCASR. and RUSSELLS. (1971) Investigations into the pathway of electron transport
to the nitrogenase from nodule bacteroids. In Biological Nitrogen Fixation in Natural and Agricultural
Habitats (‘I. A. Lie and E. G. Mulder, Eds) PI. Soil, Spec. Vol., 525-543.
WONG P. P. and EVANS H. J. (1971) Poly+-hydroxybutyrate utilization by soybean (Giycine mux Merr.)
nodules and assessment of its role in maintenance of nitrogenase activity. PI. Physiol. 47, 750-755.
WYATTJ. T. and SILVEYJ. K. G. (1969) Nitrogen fixation by Gloeocapsa. Science 165,908-909.
YATESM. G. (1968) Biochemistry of nitrogen fixation in free-living organisms. Biochem. J. 109,2p.
YATESM. G. (1970a) Effect of non-heme iron proteins and cytochrome c from Azotobacter upon the activity
and oxygen sensitivity of Azotobacter nitrogenase. F.E.B.S. Lett. 8,281-285.
YATES M. G. (1970b) Control of respiration and nitrogen fixation by oxygen and adenine nucleotides in N,-
grown Azotobacter chroococcum. J. gen. Microbial. 60,393-401.
YATESM. G. and DANIELR. M. (1970) Acetylene reduction with physiological electron donors by extracts
and particulate fractions from nitrogen-fixing Azotobacter chroococcum. Biochim. biophys. Acta 197,
161-169.
YOCHD. C. and ARNOND. I. (1970) The nitrogen fixation system of photosynthetic bacteria-II. Chromatium
nitrogenase activity linked to photochemically generated assimilatory power. Biochim. biophys. Acfa
197,180-184.
YOCH D. C., BENEMANNJ. R., ARNON D. I. and VALENTINER. C. (1970) An endogenous electron carrier
for the nitrogenase system of Rhizobium bacteroids. Biochem. biophys. Res. Commun. 38,838-842.
YOCH D. C., BENEMANNJ. R., VALENTINER. C. and ARNOND. I. (1969) The electron transport system in
nitrogen fixation by Azofobacter-II. Isolation and function of a new type of ferredoxin. Proc. natl.
Acad. Sci. U.S. 64,14041410.
YOSHIDAT. and ANCAJASR. R. (1971) Nitrogen fixation by bacteria in the root zone of rice. Proc. Soil Sci.
Sot. 35,156-158.
YOUNG R. E., PRATT H. K. and BIALEJ. B. (1951) Identification of ethylene as a volatile product of the
fungus Penicillium digitatum. PI. Physiol. 26,304310.

Applications of The Acetylene-Ethylene Assay For Measurement of Nitrogen Fixation

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Applications of The Acetylene-Ethylene Assay For Measurement of Nitrogen Fixation

Uploaded by

Copyright:

Available Formats

Soil Bid. Biorhem. Vol. 5, pp. 47-81.

APPLICATIONS OF THE ACETYLENE-ETHYLENE ASSAY

(Accepted 1 March 1972)

Summary-A comprehensive report of the acetylene reduction assay for measurement of Nz

METHODS OF MEASUREMENT OF Nz FIXATION

(A) Growth and morphology

(C) Alternate substrates

N,ase-CATALYZED C,H, REDUCTION

THE Nzase REACTION

EXOGENOUS N, NH, (N2 FIXATION 1

C2H2 C&i., Qli2 REDUCTION 1

(B) Characteristics of N,ase-catalyzed C,H, reduction

TABLE1. APPARENTK, OFC2H2 FORN*ASE, Nz-~~ti% ORGANISMS

5. Specijic activities of N,ase and N,-jixing organismsandsystems. The specific N,[C,H,]-

TABLE2. CZH2/N2 CONVERSION

Experimental system Range Average References

6. Inhibitors. Carbon monoxide is a potent inhibitor of N, fixation and also inhibits

(C) Factors afleeting C,H, reduction

experimental system nmoles C2H2 ~du~d~rnin References

Experimental system nmoles CzH2 reduced/min References

Nz-Fixing field and natural samples

of chlorophyll a (Fay, 1970); DCMU, an inhibitor of photosynthesis, inhibits N,ase[C,H,]

(B) CZH4 analysis

greater reproducibility and increased sensitivity. A variety of Porapaks, N, R, T and Q,

REPORTED USES AND CONTRIBUTIONS OF THE C,H,-C,H, ASSAY

(A) Biochemistry of N,ase

Alumina 1.8m x 6mm He 88 130 - - - Hardy and Knight, 1967;

(B) Physiology of N,-fixing organisms

N2 FIXING ACTIVITY vs AGE

LOG Np FIXED = K-AGE

TABLE 5. N2 FIXATION PARAMETERS

Initiation Termination Increase NJC&I fixed

Symbiont Age (days) Age (days) %/day mg N/plant kg N/ha*

* Calculation based on 142,000 plants/ha in 1967 of Wayne variety and 150.000

TABLE 6. N FERTILIZERS: N2[C2Hz] FIXATION AP;D YIELD

* 135 kg N/ha of indicated form.

N, FIXED vs AGE LOG N, FIXED = K* AGE

(C) Refining the list of N,-jixing organisms

TABLE ~.FIELD MEASUREMENTSOF N,[&H,] HXATION-LEGUMESAND NON-LEGUMES

N,[CzH21 Fixed References

TABLE8. FIELD MEASUREMENT

Soil N,[C’&,] Fixed References

Fallow, irrigated lawn, native vegetation and

N2[C2H2] Fixed References

5. Rhizosphere (Table 10). Several examples demonstrate N,[C,H,]-fixing activity in the

TABLE 10. MEASUREMENT OF FIELD N2[C2H,]FIXATION-RHIZOSPHERE,PHYLLOPLANE

N2 [C&1 Fixed References

association suggest N,[C,H,] fixation rates up to 90 kg N/ha/year (Dabereiner and Dart,

TABLE il. MEASUREMENT

MammaIs Sample Calcuiated N,[CZHZ] References

Human Feces O-170 ng/g/day Bergersen and Hipsley, 1970

PAREJKOR. A. and WILSONP. W. (1970) Regulation of nitrogenase synthesis by Klebsiella pneumoniae.

You might also like