'rhe Acetylene Ethylene Assay for N2 Fixation: Laboratory ...HARDY ET AL.-ACETYLENE - ETHYLENE ASSAY...

Plant Physiol. (1968) 43, 1185-1207

'rhe Acetylene - Ethylene Assay for N2 Fixation:Laboratory and Field Evaluation'

R. W. F. Hardy, R. D. Holsten, E. K. Jackson, and R. C. BurnsCentral Research Department, Experimental Station, E. I. du Pont de Nemours and Company,

Wilmington, Delaware 19898

Received April 22, 1968.

A bstract. The methodology, characteristics and application of the sensitive C,H.,-C2H4assay for N2 fixation by nitrogenase preparations and bacterial cultures in the laboratory andby legumes and free-living bacteria in situ is presented in this comprehensive report. Thisassay is based on the N9ase-catalyzed reduction of C2Ho to C2H4, gas chromatographic isolationof C2H2 and C9H4, and quantitative measurement with a H,-fiame analyzer. As little as1 ,u,lmole C..H4 can be detected, providing a sensitivity 103-fold greater than is possible with'-5N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described

for the analysis of C,H2-reducing activity in the field. Applications to field samples includedan evaluation of N, fixation by commercially grown sovbeans based on over 2000 analysesmade during the course of the growing season. Assay values reflected the degree of nodulationof soybean plants and indicated a calculated seasonal N2 fixation rate of 30 to 33 kg No fixedper acre, in good agreement with literature estimates based on Kjeldahl analyses. The assaywas successfully applied to measurements of N.> fixation by other symbionts and by free livingsoil microorganisms, and was also used to assess the effects of light and temperature on theNo fixing activity of soybeans. The validity of measuring N., fixation in terms of C,H,reduction was established through extensive comparisons of these activities using definedsystems, including purified N9ase preparations and pure cultures of N9-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveysof N9 fixation, to make detailed comparisons among different N,.fixing symbionts, and torapidly evaluate the effects of cultural practices and environmental factors on N, fixation.The knowledge obtained through extensive application of this assay should provide the basisfor efforts leading to the maximum agricultural exploitation of the N, fixation reaction.

To meet the imminent crisis in the world foodsupply (38) it is imperative that the resources ofthis planet be mobilized as rapidly and effectivelyas possible. Basic to such mobilization is a knowl-edge of the magnitude of the dynamic processes inthe biosphere which affect the availability of nitrogen,the one element most often limiting in the productionof foodstuffs (37). Of paramount importance inthis context is the process of biological nitrogenfixation. Just as photosynthesis utilizes the freelyavailable CO2 of the atmosphere, nitrogen fixationdraws on the unlimited supply of atmosphericnitrogen, and its potential role in increasing nitrogenavailability has long been recognized. In spite ofthe importance of N2 fixation very little accurateinformation is available to define the quantitativeextent to which it occurs in the biosphere (35),

1 Contribution No. 1451.2 The following abbreviations are used: N2ase for

nitrogenase; DTT for dithiothreitol; TES for N-tris(hydroxymethyl) -methyl-2-amino-ethanesulfonic acid; andMES for 2-(N-morpholino) ethanesulfonic acid; DEAEfor diethylatninoethyl.

and virtually nothing is known concerning the effectsof various field practices on No fixation. Thesegaps in our knowledge are attributable to the absenceof effective methods for quantitative measurementof N2 fixation in situ.

In the laboratory, N2 fixation by living organ-isms has been measured by Kjeldahl analvsis (7),l5N-enrichment assayed by mass spectrometry (7),and 13N-incorporation assayed by radioactive count-ing (8, 30) ; N. fixation by nitrogenase (N2ase)2in cell-free extracts has been measured by l5N-enrichment (9), 13N-incorporation (8, 30), micro-Conway diffusion technique coupled with titrimetric(26) or colorimetric analysis of NH3 (14), andN9-H, uptake (27) or H2 evolution (5) assayedmanometrically. These methods are relativelv in-sensitive except for the 13N method, and its appli-cation is extremely limited because of its shorthalf-life (10 minutes). Of these procedures onlyKjeldahl analysis has been used to an appreciableextent for estimating N2 fixation in field samples(35), btut the method is insensitive and time-con-suming. Although isotopic analysis of samples ex-posed to 15N9 in the field has been used to demon-

1185

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

strate N2 fixation in situ, the complexity and expenseof this method have limited widespread applicationto field studies.

The opportunity for a novel approach to N.)fixation analysis arose from demonstrations thatN,ase is a most versatile reducing catalyst (15, 20).The following reductions have been reported:N-O N2 + H,0 (17,21a)N3- N2 + NH3 (19,31)C2H2 C,H4 (13,19,23,32)RCCH RCHCH2 (16a)HCN CH, + NHo and tentati-ely CH.NH.

(16a, 19, 22a)RCN RCH3 + NH3 (16a)RNC CH4 CH4 C2Hr, 4- C318 etc.

(16a, 22a)Sehollhorn and Burris (31) and Dilworth (13)independently observed inhibition of N., fixation byC2H2 with extracts of C. pasteuriamniu: Schollhornand Burris (32) established the competitive natureof this inhibition and Dilworth found C.H. to bereduced to C2H4 in a reaction analogous to thereduction of N, to NH3. The application of thisreaction to a sensitive assay procedure for N.,-fixingactivity was proposed by Hardy and Knight (19):"Utilization of the reduction of HCN to CH4. ofH'4CN to 14tCH3NHQ, or of C2H2 to C.,H,, anddetection of CH4 and C.H, by hydrogen flameionization after gas chromatography or detection of'4CH3NH2 may provide a sensitive new assay fordetection of the N2-fixing system. The gas chro-matographic determination makes possible a rangeof about 10,000 times between minimumi and maxi-mum, in contrast to a 20-fold range with the NH3assay. C,2H, is the preferred assay substrate, sincemore product is formed because of its requirementfor 2 electrons versus 6 electrons for HCN". Sub-sequently, Koch, Evans, and Russell (24, 2.5), Silver(33), Sloger and Silver (34) and SteNart. Fitz-gerald, and Burris (36) have successfully emplovedC.2H2 reduction coupled wiith C2H, detection by H.,flame ionization as an assay for N.ase activity.

Since the original proposal by Hardy and Knight(19), the C2H2-C2H4 assay of N2.-fixing activityhas undergone extensive development in this labora-tory. This paper reports: 1) methodology of C2H..and C2H4 analyses; 2) methodology of the C2H2-C.H assay of N-fixing activity in situt, and 3)characteristics of CJ-1J. reduction by N.\ase in vitroby cultures of N-fixing bacteria and by samples ofthe biosphere in situ. The results indicate that theC,H2-C,H4 assay of N-fixing activity is sensitive.universal, specific, rapid, simple, economical, andquantitative. Since this assay has the potential topromote revolutionary fundamental and practicaladvances, we believe that the C.,H2-C.,H assay ofN2 fixation represents one of the most importantdevelopnients in N2 fixation research. It is empha-sized that the adoption of a consistent procedure bythe various disciplines, e.g., soil science, agronomy,marine biology, plant biology, microbiology, and

biochemistry, whichl viii utilize this method isessential if valid comparisons are to be made amionlgresults obtainied fronm various sources.

Methods

Growth of Cells anid Preparationi of Extracts.Azotobacter vinelaudii, ATCC 12518, was grown onnitrogen-free or urea media. Optical densities ofbacterial cultures were determined in \Vill color-imeter tubes with a 650 mp, filter in a Lumetroncolorimeter. Cells were broken and N,ase waspurified as previously described for N2, N20, N3-,or HCN reductioin experiments (5, 17, 18, 19).N,ase was fractionated into the Mo-Fe proteinfraction (also called Enzyme I) and Fe proteinfraction (also called Enzyme II) (15) by publishedprocedures (4, 22) modified to produce a discretefractionation of the 2 components. Fractions weredesignated as follows: 1) crude extract-super-natant after 35,000 X g for 30 minutes; 2) heatedextract-crucde extract heated for 10 minutes at 60°under 0.5 atm of H., and centrifuged at 35,000 X gfor 1 hour: 3) pre-protamine sulfate precipitate-phosphocellulose resolubilized precipitate between0.0 to 0.1 milg protamine sulfate per mg protein ofheated extract: 4) protamine sulfate precipitate-phosphocellulose resoltubilized precipitate betweeni0.100 to 0.125 mg protamine sulfate per mg proteinof heated extract; 5) protamine sulfate superna-tant-supernatant after 0.125 mg protamine sulfateper mg protein of heated extract; 6) Mo-Fe proteinfractiori-fraction of protamine sulfate precipitateeluted from a DEAE-cellulose column in an anaerobicchamber bv 0.20 M\NaCl + 0.02 M MgCl2 + 5 X10-4 M DTT in 0.02 m' tris H,Cl at pH 7.0 followingpre-elution with 0.15 M NaCl + 0.02 M MgCl2 +5 X i0-4 M DTT in 0.02 M tris H;Cl at pH 7.0;and 7) Fe protein fraction-fraction eluted fromabove colummn by 0.35 Ml NaCl + 0.02 M MgCl2 +5 X 10-4 M DTT in 0.02 M tris-HCl at pH 7.0;All fractions were stored anaerobically with frac-tions 4 and 7 stored at room temperature. Fraction6 contained Fe and 'Mo and fraction 7 contained Feas reported by others (4). Neither fraction washomogeneous by gel electroplhoresis.

Clostridium pasteutriantnum, ATCC 6013, wasgrown on nitrogen-free or NH,ICl media (9). Driedcells were broken by auitolysis as previously describedfor N2, N,,O, N.-, or HCN reduction experiments(9,17. 19). An extremely sharp fractionation ofN2ase into its 2 components was produced by modi-fication of an old procedure (29). Fractions xx-eredesignated as follow-s: 1) crude extract-super-natant after 35,000 X g for 30 minutes of autolysate;2) negative phosphate gel preparation-supernatantfrom crude extract treated with protamine sulfate toremove nucleic acids and with calcium phosphategel to remove inactive protein (29) ; 3) Mo-Fe pro-tein fraction-fraction eluted from DEAE-celluilose

1186


HARDY ET AL.-ACETYLENE - ETHYLENE ASSAY FOR No FIXATION1

between 0.1 to 0.3 m NaCl in 0.01 M KPO4, pH 7.0.after 15 minutes anaerobic batch treatment of aphosphate gel preparation with 8 mg damp DEAE-cellulose per mg protein. This fraction containsMo and Fe anid is stable for months when storedanaerobically at 40. In some cases it was fuirtherpurified by anaerobic chromatography on DEAE-cellulose with elution by 0.29 M NaCl in 0 01 altris HCl, pH 7.0. This fraction is not homogeneouisby gel electrophoresis. 4) Fe-protein fraction-crude extract heated for 10 minutes at 600 under0.5 atm H., and centrifuged at 35,000 X g for 30minutes: this fraction was stored under aniaerobicconditions at room temperature. Fraction 3 or 4could onl- be obtained from N..-grown cells.

Clostridituin biutyricunt, Lactobacilluis leicliin an ii,Bacilluis sitbtilis. Serratia marcescens, Escherichiacoli, Streptococcuts lactis, Saccharomvces cerevisiae,Bacillus cercuts var. mycoides, Pseudo tmonlas acrim-ginosa, Pseudonmo nas fluorescens, Aerobacter aero-genes, Staphylococcius epidermidis, Sarcina lutea,Spirilltini itersonii, Proteuts vulgaris, Alcaliginesfaecalis, and Rhodospirillumii ru(brunt were the kindgifts of Dr. R. Bailey of the University of Delaw-areand were grown onl appropriate media containingfixed nitrogen. Rhizobiumn japoniicuit, ATCC 10324.R. ieliloti, ATCC 10312, R. legumninosarunt, ATCC10004. Rhlizobiuiim sp., ATCC 10317, and R. trifolii,ATCC 10328. w-ere grown on a medium containingin g per liter: K,HPO,4 1.0; KH4P,O,, 1.0;MgS04*7H.,0, 0.36: CaS04*2H.20, 0.17; FeC13*6H.2O,0.005; KNO2. 0.7; yeast extract, 1.0; and mannitol,3.0 (10).

Growth of Legumzes. Field-grown soybeans(GlyFcinie inax Mferr. var. Wayne) were sowvn onMay 15, 1967, in 38-inch rows by a commercialgrower in Chester County, Pennsylvania. Standardagricultural practices including seed inoculation withcommercial inoculum and recommended additiolns ofphosp,horus and potassium but no nitrogen wvereused. In addition, soybeans and other legumes(Phaseoluis vulgaris, JMedicago sativa, Arach is hy-pogea, and Pisuntii sativutm) were grown in sterilizedPerlite using a nitrogen-free nutrient solution ( 1)in a greenhouse or in controlled environment growtlchambers. A normal day-night regime of 16 hours.240, and 8 hours. 180 was maintained in Sherer-Gillett -Model CEI9255-6 chambers operated at maxi-mum light intensity during the light period. Othersamples of legumes and soils collected within aradius of 200 miles of Wilmington include samplesfrom the J1ordan Fertility Plots through the courtesy-of Professors A. Richer and E. S. Lindstrom ofPennsylvania State University, and from the George-towvn Experimental Station through the courtesy ofProfessor R. Cole of the University of Delaware.Indicated times are Eastern Daylight Saving Time.The date of bud opening was recorded as the dateof flowerinig, and the first indication of leaf yellow-ing was recorded as the onset of senescence.

Assays. Reductions of N., or C.,H, byv N2.asepreparations or cultures of bacteria were performedin 40 ml incubation vessels sealed wtith serum caps.For N2ase preparations dithionite '%vas dissolved in0..-free water containing a l)re-determined quantityof acid or base to produce a final pH of 7. Theenergy source and reductant were placed in thesidearm, the extract and other components wereplaced in the main compartment, and the incubationflask was immediately evacuated. After repeatedflushing with the indicated gas, the contents of thesidearm were tipped in to initiate the reaction. Forcultures of bacteria, the incubation was initiated bythe aseptic addition of the bacteria to the sealedincubation vessel containing appropriate medium andgas phase. The reaction mixture or culture wasincubated on a rotary shaker at 300 for the indicatedtime, and the incubation was stopped by the additionof 0.5 ml of 6 N HOSO4. Samples of gas phase wereanalyzed with a mass spectrometer utilizing theinitial gas phase as an internal standard or with aH,-flame ionization detector after gas chroma-tographic separation (see below). Nitrogen fixationby N9ase in extracts was measured by titration ofNH, after micro-diffusioni (26). and N., fixation bycultures was measured by Kjeldahl analysis of 5 mlaliquots. Deuterated ethvlenes were anialyzed in aPerkin-Elmer Model 21 infrared spectrophotometerusing a 3.3 cm micro gas cell.

Assay of Acetylenie aiid Etltylence. In early workan activated alumina column at 1500 and a Perkin-Elmer 880 or 800 gas chromatograplh with a dualH.-flame ionization detector were used (19). Sub-sequently, a one-eighth inch X 10 foot columncontaining 20 % ethyl, N',N'-dimethyl oxalamide on100 to 120 mesh acid-washed firebrick at 00 with aHe flow rate of 30 ml/minute has been found to bemost effective for gas chromatographic separation ofacetylene and ethylene as well as other saturatedand unsaturated hydrocarbons containing up to 4carbons. Modified Perkin-Elmer F-11 gas chroma-tographs equipped with H9-flame ionization detectorsare utilized. Representative retention times in min-utes are: methane, 0.8; ethane. 1.0: ethvlene. 1. 1;propane, 1.4; propylene, 1.9; isobutane, 2.1; butane,2.8; acetylene, 3.8; 1-butene., 4.4; isobutylene, 4.5;allene, 4.8; trans-2-butene, 5.2; cis-2-butene. 6.2;methylacetylene, 10.4. A typical chromatogram of astandard mixture of CJI2 (0.1 atmosphere) andCMH4 (2.5 X 10-4 atmosphere) is shown in figurela, and a chromatogram of C,H2 (0.1 atmosphereinitial pressure) and C..H4 produced by a cultureof N2-grown Clostridiumiit pasteturiauittin in figure lb.The symmetry of C,H2 anid C.H4 peaks and theabsence of other components are indicated in figurelb. A standard curve of peak height vs. C2H4 orC2H, content of injected sample (fig 2) demon-strates the linear response and sensitivity of theassay. Less than 10-12 moles of C2H4 can be de-tected per injected sample of 200 Mul. Ethylene con-tent can be calculated from this standard curve, or

1187


PLANT PHYSIOLOGY

(a)C2H2 (0.1 atm)

(b)C2H2 (0.1 atm)

C2H4 (2.5 x 10-4

C2H2-C2H4STANDARD

106

I)

Lii

"IC

uiCLi

E

E

4 3 2 1 0

N2ase-22 C2H4

4* 3 2TIME (min)

1 0

FIC. 1. T-pical chromatograins of a) a known mix-ture of 200 ,ul of C,H, (0.1 atm) C2H4 (2.5 X 10-4atm) and He to 1 atm, and b) 200 ul of the gas phaseof an incubation after N,ase-catalyzed reduction of 0.1atm C.H,. An ester-amide gas chromatographic columnwas used and detection w-as by hydrogen flame ioniza-tion (see under 'Methods).

alternatively the "built-in" internal standard, C.H..,can be used since both C2H2 and C2H4 are deter-mined. Our broad experience with the C2H2-C.H,assay (over 2000 samples assayed) indicates thatC2H2 is a valid and useful internal standard, sincewith the exception of large nodulated plant rootsless than 2 % of the initial C2H2 (0.1 atmosphere)is converted to C2H4 during a 1 hour incubation.

Reagents. ATP, GTP, CTP, UTP, creatinephosphate, creatine kinase (ATP :creatine phospho-transferase, EC 2.7.1.40), and protamine sulfate wereobtained from Sigma Chemical Company; Na2SO4.reagent grade, from Fisher Scientific Company; He,A, N2, CO, and C2H.2 as highest purity available

104

103

i2

x/

~~~ ~~x /ZC2H4 ,x/

,/x, C2H2

_ /X~~~~~~~~/X/,F~~~, /~~~/

- X

x1

.-/.. I ,l II ..I 111 - - -

o- 100 101 1o2 103 104

/FiL moles C2H4 or C2H2

,x

io5 106 107

FIC. 2. A standard curve of peakliheights of C.,H.,and C.,H4 determined with the gas chromatographicsystem of figure 1.

from The Matheson Company. Acetone was re-moved from C2H, by a concentrated H2S04-scrubberand correctionis were made for the C2H4 content ofC,H2. The C,H4 impurity in CoH2 from a givencylinder must be determined daily since it variesinversely Nvith the pressure in the cylinder.

Results

CJH.2 Reduictiont by N,ase in vitro. Reductionof C2Ho to C2H4 by N2ase of cell-free extracts ofA4. vinuelandii was examined with respect to a widevariety of characteristics, and the striking similari-ties between N., fixation and CAH2 reduction arereported in this section.

Requiremnents and Products of CoHo Redutction.Reduction of CoHo to C2H4, like reduction of No to2 NH3 (3, 12, 16, 25, 27), requires an enzyme extractcontaining N2ase, an energy source, and a reductant(table I). A similar energy and reductant require-ment has been reported for reduction of acetyleneby extracts of C. pasteurianiumii and soybean bac-teroids (13, 25, 29a, 32). Extracts of urea-growncells do not have N,ase activity and do not haveC2H,-reducing activity. No C,H4 formation isfound in the absence of C,H,. Detectable amountsof C2H6 or CH4 are not formed by the completesystem capable of reducing C,H, (sensitive analysesindicate that C.H6 can be no more than 0.01 % asabundant as C2F14) ; furthermore, ethylene is notreduced to C2H6 or CH4 by Azotobacter N2ase ina complete system. Thus, the N2ase-catalyzed re-action appears to be quite specific for the reductionof C.H. only to C2H4.

Specific Requirement for ATP. The sensitivityof the C.H2>-C2H4 assay of N,ase permits a deter-

1188

..... .... -A -, ... I

ll

105

I -1


HARDY ET AL.-ACETYLENE - ETHYLENE ASSAY FOR N2 FIXATION

Table 1. Requirements and Product of C2H2 Reduction by Azotobacter N2aseComplete system contained per ml in ,.tmoles: tris-HCl, 50; creatine phosphate (CrP), 56; ATP, 5; Na.2504,

20 (all at pH 7.0); and MgCl2, 5; and in mg proteins: heated extract of N2-grown or NH3-grown A. vinelandii, asindicated, 4; anid creatine kinase (CrK), 0.2. Gas volume 36 ml; liquid volume, 4 ml; incubation time, 30 min; tem-perature, 300. Aliquots of gas phase assayed by gas chromatography on alumina column.

Incubationsystem Gas phase C2H4 C2H6l, CH4

Requirements jAmoles/incubationoomplete, N.- 0.05 atm C2H2 24

PLANT PHYSIOLOGY

z 1.0-0

aD 075-1o)z

°05 -

(-)N

Q 0G25E.Zi

(b)

x/

-

000 0.05 01 0 0 500 1000C2H2(Otm)

PC2H2(atm)

FIG. 4. a) Ethylene formation from C,H. as afunction of pC2H2 by Azotobacter N2ase, and b) plot ofreciprocal velocity evrsus reciprocal pC2H, for deter-mination of Km of C.H.1. Complete incubation system.table I; liquid voltie.e 2 ml; gas volume, 38 ml; time,3.0 miii; gas phase, indicated pC.,H, plus He to 1 atm.

and reductant-depenident ATPase activity of N'2ase.This inhibition at 0.5 atmosphere C21H1, may be dueto C9H. or to possible trace impurities in C.H_.A typical plot of reciprocal velocity of CH.,1 reduc-tion vs. reciprocal pC,H., is shown in figure 4b.Estimated Michaelis constants of 0.002 to 0.009atmosphere of C2,14, with an average value of0.004 atmosphere. have been obtained. A tentativeKmii of 0.01 atm lhas been reported for clostridial

Tal-)le III. Inliil)tiozn of A TP-Depcndcnt H., Ez.'llionIby N, and C.,H,

Complete s\ stem, table I; liqjuid volume 2 ml; gasvoltime 38 ml; N2ase preparation, heated extract of A.zinelandii, 7.5 mg; gas phase, as indicated plus H-e to 1atm. Hydrogen determined by mass spectrometric analysisof gas plhase w-ith He as an internal standard.

Addedlsubstrate

NoneN., 0.5 atmC..H.,. 01 atm

H, AH.,

mum.noles per minper mg protein

71 018 5311 61

% Inhibitionof H.,

evoltitioi3

0

7585

N2ase (13). Michael'is constants of 0.05 to 0.17atmosphere of N2 have been reported for N.,ase( 14,19, 25, 27). Based on partial pressures. theestimated Km of C2H, is only about 5 % that of No.Based on the calculated concentrations of C.,H2 andNo in an aqueous solution, the estimated Kmi2 ofCo0H.,. 0.1 to 0.3 mM, is similar to that of N.. 0.03to 0.1 mM.

Inhibition of H, Evolutioni. The ATP-dependentH.-evolving activity of N2ase is decreased b N2,N3 , or N\O reduction, and the decrease in H11evol\ved is equivalent in electrons to those requiredfor reduction of No to 2 NH. N.- to N NNH3,or N,0 to NT. + H10 (4, 17. 19 25). Redtuctionof C.,H., to C,H, also inhibits H. evolution byclostridial (13. 32) and A4zotobacter N.,ase ('21).Inhibition by a saturating level of C.H.. may beg,reater thani by that of No. c.g., 85 % for C.H. and75 % for No (table III).

.Stoichliomnetr'v of C2H, Rediuctioni. An excellentbalance exists between the concomiiitanit decrease inC.,H.,. increase in C,H., and decrease in H. evolu-tion (table IV) during N.ase-catalyzed reductionof C.,H2,. Thus, reduction of C.,H. (lecreasedC.0. by 13.8 ,umoles and increased GM,4 by 14.5,lnmoles, supporting the following relationslhip:

N,asenCH2 - 4 nC.2H4

The (lecrease of 12.3 umoles in H. evolution (equiva-lent to 24.6 umoles of electrons) produced by CGH.reduction corresponds to the formation of 14.5 jimolesof C,,H4 (equivalent to 29 Mmoles of electrons) andin(licates the following electron balanice:

(11H evolved) -(119- H. evolvedC,H4 formed) .+ 2113

\ddition of 0.18 atmosphere CO inhibited CH.,i-eduction and restored H1 evolution. Since the lossof C.,H., can be accounted for as CGH4 and the lossof electrons evolved as H2 can be accounted for asthe electrons required for C2H2, reduction, no sig-nifica

HARDY ET AL.-ACETYLENE - ETHYLENE ASSAY FOR No FIXATION

N2-.'2NH3

z0

4

: 1.6z'- 1.2

z 0.8

;0.4

atm CO

0.000

.000II .1

2 6 10I

p N2(t m )

C2H2 -* C2H4

z0

lmD 0.4zO0.3

0 0.20

E 0.1E4.

atm CO

0.ooi

0.000

1100 250 400

pC2H12(otm)

FIG. 5. Competitive inhibition of C,H.-.C,H4 andN2->2NH3 by CO using Azotobacter N0ase. Completeincubation system, table I; liquid volume. 2 ml; gasvolume, 38 ml; time, 30 min; gas phase, indicated pC,H.,or pNo plus indicated pCO and He or A to 1 atm. N2is replaced by A as control for N->2NH,.

the electrons used for ethylene formation indicatesthat at least the proposed electron-activating site ofN.ase (15, 20) is involved in oH> reduction, ashas been proposed for N.0 and N.j reductions(17, 19, 21a).

Inhibitiont of C2IIH Reduiction by, CO. Carbonmonoxide is a competitive inhiibitor of No fixation('15, 20, 25a). Figure 5 demonstrates that CO isalso a competitive inhibitor of CoH.> reduction byAzotobacter NYase. Furthermore. the similar COinhibition constants of 2.9 X 10-4 and 3.1 X 10-4atmosphere for N2 fixation and C.,H2 reduction.respectively, provide indirect support that the sub-strate-complexin,g site of N2ase (15, 20) is involvedin the reduction of CoH. as %-ell as N,.

8

xz0

6 -

o x NH4Cxz, 4 C

0~~~~~~~~

E _0.o) N

0 40 80 200

NoCl or NH4Cl (mM)FIG. 6. Ini}1bition of C2H2-*COH4 by NH4C1 or

NaCI. Complete incubation system with AzotobacterN,ase, table I; liquid volume, 2 ml; gas volume, 38 mi;time, 30 min; gas phase, 0.1 atm C..H.,, 0.9 atmn He;NH4Cl or KCI as indicated.

Effect of NH44 and Na+ on C2H2 -* CGH4 Re-dutction. Ammonia is the product of N2 fixation;however, N2 fixation appears to be relatively insensi-tive to added NH4+ (9). This insensitivity suggeststhat the product of N2 fixation does not effectivelycompete with N, for the substrate-complexing siteof N2ase and that NH4' does not control activitiesof N,ase associated with electron-activation. Reduc-tion of C,H2 to C2H4 provides an opportunity todetermine if there is a specific effect of NH4' onother N,ase-catalyzed reductions. Figure 6 indi-cates no specific inhibition by NH4', since C2H.reduction is equally sensitive to NH4' or Na+ with50 % inhibition produced by 50 to 70 m-s NH,Clor NaCl.

2.0zw

0cr

> . 1.5

u) E0 .-J c-w -

. 1.0

0cj( O

0 0

X 0.5nGI0E

E o.0.

400 30° 200 1lo

3.2 3.4 3.6

TX 103

FicK. 7. Arrheniius plots of C,H2-*C,H4, N.,->2NH,and ATP-*>ADP + Pi by ALotobacter N.,ase in therange 100 to 400. Complete incubation system, table I;liquid volume, 2 ml; gas volume, 38 ml; time, 30 minfor COH. or NO reduction, 15 min for ATP hydrolvsis;gas phase, 0.1 atm C,H. plus He to 1 atm for CH..H.CH4, 1 atm N., N-ith 1 atm He as control for N.--2NH, and 1 atlmi He for ATP- ADP + P. Poilntsrepresent averages of 3 samples.

Actizvatioi? Eniergy of C,H.. Reduictiont. Theactivation energies for reduction of N., anid otherreactions of N2ase, including ATP-dependent Hoevolution anid reductant-dependent ATPase, havebeen recently- determined (6. 21). A break in theArrhenius plots for all these activities is observednear 200 ith similar but lower activation energiesabove (13-1i kcal/mole) and similar but higheractivation energies below this point (35-50 kcal/mole). Arrhenius plots of C2H,, reduction also showa similar break and similar activation energy (fig /7).

ATP-.-ADP +'Pi

1-

1H

H[_

1191

I


PLANT PHYSIOLOGY

Stereochenitistry of CsHo Reductioni. Ethyleneformed from CGHE by reduction bv AzotobacterN,ase in a 99.8 % D2O system was examined byinfrared spectrophotometry in order to identify thedeuterated species (fig 8). cis-1,2-Dideuteroethylene(843 cm- ) is the major product, as reported forclostridial N,ase (13). A small amount of mono-

z 0.021

0 0Q4

0 8e

5t ,2 3 4 5 6 7 8 9 10 1 12 13 14 15WAVELENGTH (MICRONS)

FIG. 8. Infrared spectrum of deuterated ethylenesproduced by reduction of C,H, by Azotobacter N9ase.Complete incubation system, table I; liquid volume, 10ml; gas volume, 30 ml; time, 30 min; gas phase 01latm C2H2; all reagents in 99.8 % D,0 and protaminesulfate ppt of N9ase resuspended in 99.8 % D,0.

deuteroethylene (1000 cnf') and a possible trace oftrans-1,2-dideuteroethylene (988 cm- ) were founid.These results indicate that neither of the originalhydrogens of acetylene is replaced during reductionand that acetylene may be complexed to the sub-strate-complexing site of N.ase via a "side-on'orientation.

Fractiontation of N9ase and N-Fixing antdC2H9-Reduiciing Activities. Nitrogen-fixing extractsof A. vinelandii were fractionated according to theestablished procedure in this laboratory for N..asepurification. The N2- and C9H.,-reducing activitiesparalleled each other, and the ratio of CGH4 formedto N2 fixed was found to be in the range of 3 to 4.5(table V).

Recomnbiniationi of Mo-Fe Protein and Fe ProteiniFractions of N,ase. Nitrogenase can be separatedinto 2 protein fractions (4, 22). One contains Feand Mo and is called the Mo-Fe protein fractioni:the other contains Fe and is called the Fe proteinfraction. Neither individual protein fractioni lhasbiological activity, but N2ase, the complex formiiedby the protein fractions, is active for No reductioni,

Table V. C,H, and N, Reduction by Azotobacter N,asc PreparationsComplete incubation system, table I; li(quid volume, 2 ml; gas volume, 38 ml: N,ase preparation, as indicated; gas

phase, 0.1 atmi- C,H,, 0.9 atm He for C,H, reduction, 1 atm N, with 1 atm He as control for N, fixation. C.,Hassa!-ed gas chromatographicall; NH3 assa - 2 4assayed gas chromatographically; NH, assayed titrimetrically.

Proteinmig/incubation

N.asepreparationi 1

Heated extractPre-protamineprecipitate

Protamineprecipitate

Protaminesuperniatanit

4.34.0

0.91

3.6

C.,H.-C,H N.,-2NH3,umoles/inicubation

5.00

HARDY ET AL.-ACETYLENE - ETHYLENE ASSAY FOR FIXATION

ATP-dependent H2 evolution, and reductant-depend-ent ATPase (4,22). Nitrogenases of C. pasteuri-anum and A. vinelandii were separated into theirMo-Fe protein and Fe protein fractions as describedunder Methods. Acetylene reduction was determinedwith the individual fractions, the recombination ofthe individual fractions from the same species andthe cross-combination of the proteins from differentspecies (table VI). No nitrogen-fixing activityrenmained in the individual fractions, but it wasrestored by recombination of the fractions. Neitherthe Mo-Fe protein fraction nor the Fe proteinfraction has appreciable C2H2-reducing activity, andthus represent the "lowest activity" fractions thathave been reported, e.g., our best preparations ofMo-Fe protein (Clostridium), Fe protein (Clostri-dium), Mo-Fe protein (Azotobacter), and Fe protein(Azotobacter) have less than 0.04, 0.02, 0.03, and0.7 %, respectively, of their recombined activities.Recombination of the 2 protein fractions of Azoto-bacter produces stimulations up to 123-fold, and ofClostridium up to 1080-fold. A recent report indi-cates an enhancement of activity of 5.5-fold byrecombination of Azotobacter fractions (22). Cross-combination of Mo-Fe protein (Azotobacter) + Feprotein (Clostridium) or Mo-Fe protein (Clostri-ditin) + Fe protein (Azotobacter) produces< 5 % of the C2H2-reducing activity found in therecombination within species experiment. The abovecross-combinations have been reported to produceno N.-fixing activity (11).

These experiments were designed to demonstratethe absence of C2H2-reducing activity in each frac-tion and the presence of this activity in recombinedfractions from the same species. Specific activitieswere not maximized by the addition of an excess ofone fraction to a limiting amount of the fractionwvhose activity is to be maximized.

C2H2 Reduction by Bacterial Cells. Character-istics of the reduction of C2H9 to C.H, by N.,-fixingcultures of A. vinelandii and C. pasteutrianuimitl are

reported in this section. These results indicate thevalidity of the C2H2-C2H4 assay of N2 fixation withliving organisms and complement results reported inthe previous section with N2ase preparations fromAzotobacter and Clostridium.

Requirements and Products of C2H2 Reduction.Acetylene is reduced to C2H4 by N2-'grown cells ofA. vinelandii and C. pasteurianum (table VII).Extracts from these cells contain N2ase and reduceC2H2 to C2H4. Control cultures grown on fixednitrogen sources reduced less than 0.1 % (Azoto-bacter) and less than 5 % (Clostridium) the amountof C2H2 reduced by the N2-grown cultures. Extractsfrom these cells contain little or no N9ase and donot reduce C9H2. Neither ethane nor methane isdetected as a product of acetylene reduction; ethyleneis not reduced to ethane or methane. The decreasein acetylene during reduction equals the increase inethylene. The aerobe Azotobacter requires aerobicconditions for C2H2 reduction, while the anaerobeClostridium reduces acetylene anaerobically. Negli-gible ethylene is formed in the absence of C9H2.Thus, no correction is required for background C,H4.

Time Course. Time courses of acetylene reduc-tion by N2-grown Azotobacter and Clostridium areshown in figures 9, 10 and 13. The rate of C2H2reduction is constant up to 18 to 20 hours for Azoto-bacter and 6 hours for Clostridium. An initial lagis often observed with Azotobacter, presumablybecause of the effect of transfer and dilution. Addi-tion of 40 mM NH4Cl to N2-grown cells decreasesthe rate of C2H2 reduction by 95 % after 4 hours(fig 10). This inhibition is in contrast to theeffect on N,ase in vitro. The C,H2 reduction assavoffers a potent method to further define the rela-tionship of No and fixed nitrogen compounds toinduction and repression of N2ase. The results withAzotobacter in figure 9 permit calculation of acorrelation between C.,H, reduction and No fixation.Ethylene formation stops when 0.2 moles of C..H,harve been formed for each mole of O initially

T.able VII. Distribution of aid Requiruciiwits for CoHo Rcduction b)Y Cultures of Azotobacter and CostridiumiiOne mnl of culture in early log phase was aseptically added to 4 ml of its niitrogen-free growth media for N.,-

grow-n bacteria and its nitrogen supplemented media for NH3- or urea-groN-ii bacteria in a sealed incubation vesselof 40 ml total volume containing the indicated gas phase. Incubation time, 16 hr, temperature, 300. Gas phase assayedchromatographically on ester-amide column.

Cells Gas phase (atm) ,umoles

Organism (X 10-6) /in1cubation A 02 C2H2 He C2H4/hAr

ALzotobacter, Nr,-grows-n 85 0.8 0.2 0.000074Azotolbacter, N2-grow-n 85 0.1 0.9 0.030Azotobacter, N,-grown 85 0 7 0.2 0.1 1.42Clostridiurn, N,-grown 88 1.0

PLANT PHYSIOLOGY

4 8 12 16 20 24 50

TIME (hr)

Table VIII. Cell Dilution and C,H, Redutction bvAzotobacter Culture

Incubation system, table VII, for N2-grow-n Azoto-bacter; inicuibation time, 16 hr; temperature, 300; gasphase, 0.1 atm C2H2, and A:O (0.8:0.2) to 1 atm.Culture serially diluted and cells in original culturecounted in a hemocytometer. Ethylene assaved gas chro-matographically on ester-amide column.

Cells/incubation

9,250,0001.550,000255.00043,0007,000

m,umoles C2H4/hrincubation

183346.00.8750.106

,u,umoles C,H4/lhr cell

0.0200.0220.0240.0210.015

FIG. 9. Time course of C9H,,-*C2H4 reduction bycultures of N,-grown A. vinelandii and C. pasteurianuin.Incubation system, table VII; liquid volume, 5 ml; gasvolume, 35 ml; temperature, 300; gas phase, 0.1 atmC2,H, 0.2 alm 02, 0.7 atm A for Azotobacter and0.1 atm C,H2, 0.9 atm He for Clostridiuni; Azotobacter,250 X 106 cells; Clostridion, 110 X 106 cells. Pointsrepresent averages of 3 samples.

present. Based on aerobic oxidation of glucose,1.25 moles of ethylene are formed per mole ofglucose oxidized. On the basis of one mole of N2fixed per 3 to 4 moles of C2H2 reduced, 0.3 to 0.4mole of N., would be fixed per mole of glucoseoxidized. This calculated ratio of N2 fixed perglucose oxidized, based on C2H1 reduction, is inreasonable agreement with reported experimentalvalues based on direct measurements of No fixa-tion (35).

Cell NVunber. A linear relationship exists be-tween cell number and acetylene reduced (tableVIII). Ethylene formation measured after 16 hours

30 X

z

0NH° - NH

a 20 -z x

In lo NH+~i10 NH+4

E ADDED+ NH

; x I I~~~~~~~~

0 250 500TIME (min)

FIG. 10. Effect of NH4Cl on time course of C,H2-*C,H4 reduction by a culture of N2-grown A. vine-landii. Incubation system, figure 9; 300 X 106; cells;NH4Cl added to 3 flasks at 30 ruin to produce 40) mmNH + Nhile no addition -x a. ma(le to 3 control flasks.

of incubation at 30° is 0.02 u/,umole per lhour perAzotobacter cell over a 10000-fold range of cell con1-centration. The extreme sensitivity of the C.Ho-C9H, assay is indicated; theoreticall-, as few as2 to 3 cells produce sufficient C.H4 for detection bythe HO-flame ionization system.Km of C2H,. Acetylene saturatioln of N'.-grown

clostridial cells occurs between 0.025 and 0.1 atmos-phere (fig lla), and even 0.5 atmosphere is notinhibitory (fig 1lb). The saturation concentrationis similar for NO-grown Azotobacter, buit these cells

z 2(0

m

z

.,

'11 '(rcl

z

0

tDDz_

C,IE'

0

E:1

II0 - --

(~~~~~~b)

x ~ ~ ~ ~

I~~~~~~~~~

I'I;

)0.025 0 05 2 0 5P C2 H2 (at m)

pC2H2(at ml

FIG. 11. a) and b). Ethylene forimiationi frolmi C H.,as a function of pC,H, by culture of N,-grow-\n C. aus-teurianum, and c) plot of reciprocal velocity :zcrsuts re-ciprocal pC,H. for determinatioln of Km of C H.. In-cubation system, table VII; liquid volume. ml;:n dgasphase, in-dicated pC,H2 plus He to 1 atm; points rep-reselnt averages of 3 incuhations.

1194

5C

z

0

!~ 4Cm

C)E

z

"I 30

2C\

E

:C

iI t

- AZOTOBACTER , -

,01

-' CLOSTRIDIUM

/ ~I I1,..M- f .-.



show an as yet unexplained increase in rate of CGH2reduction at 0.2 and 0.5 atmosphere of C.H,. Aplot of reciprocal pC2H, vs. reciprocal rate of CJ112reduction by clostridial cells is shown in figure llc.The range of Michaelis constants with Clostridiumis 0.003 to 0.008 atmosphere C2H2 with an averageof 0.006, while that for A. vinelandii incubated at0.1 atmosphere or less CoHs is 0.003 to 0.006 atmos-phere with an average of 0.005.

Activation Energy of CGH, Reductiont. Theeffect of incubation temperature on CGH2 reductionby clostridial cells was determined over the range10' to 350. A close analogy with the in vitro resultson N2ase is observed which suggests that the limitingfactor in growth may be N9ase activity, and further-more that this is related specifically to a propertv ofthe N2ase enzyme per se, rather than to reactionswhich furnish energy or reductant to the enzyme.An Arrhenius plot of the cellular activities is shownin figure 12. Results from 200 to 350 form a linearplot with a calculated activation energy of 13 to 15kcal/mole; results from 10° to 200 are not co-linearwith those from 200 to 350, and a much higheractivation energy, approximately 50 kcal/mole, isestimated for the lower temperatures.

C.,H. Reduction anid No Fixation. Acetvlenereduction by N,-grown Azotobacter cultures wascompared with N2 fixation and increase in OD bvidentical cultures incubated with air under the sameconditions. In all cases the 3 parameters measturedshowed parallel increases with time of incubation.The late-log phase culture (fig 13) did not reduice

+06350 30° 250 200 15° 100

+0.6 _

+04z

0

+02mD

z 0x0 _ x

02A -042

0E -064

° -0.6

-0.

-l.o x32 3 3 3.4 35 36

FIG. 12. Arrhenius plot of C2H,--C2H4 by cultureof N,-grown C. pasteurianuni in the range of 100 to350. Incubation system, table VII; liquid volume, 5 ml;gas volume, 35 ml; temperature as indicated; time, 1 hr;gas phase, 0.1 atm C2H,, 0.9 atm He.

0

a

2 4 0 2

0

20 X

z

6

0

12 2

c)D s ":n

E

TIME (hr)

FIG. 13. Acetylene reduction, AOD650 and N., fixa-tion by culture of N2-grown A. vinelandii in a) latelog phase, and b) early log phase of growth. Incubationsystem, table VII; liquid volume, 40 ml; gas volume,290 ml; temperature, 300; gas phase, 0.1 atm CH92,0.2 atm O,, 0.7 atm A. Initial OD650 of late log phaseculture after dilution with fresh media was 0.087, whilethat of early log phase culture was 0.112. Samples ofgas phase and culture were analyzed for C,H, andC2H4 by gas chromatography and for fixed nitrogenby Kjeldahl analysis at indicated times.

CJi-.,. fix N. nor increase in optical density duringthe initial hour of incubation; in contrast the earlylog phase culture (fig 13) showed uniformly positiveresponses during the initial hour. The ratio ofmoles of N.. fixed to moles of C,H4 formed is 3to 4.5.

Distribution of C,H,-Reducing Activity. Theabsence of significant C2H.-reducing activity in avariety of organisms grown under non-N2-fixingconiditions further establishes the validity of therelationslhip between C2H,-reducing and N,-fixingability. Organisms tested included Clostridimn bu-tvricunt under anaerobic conditions on complete

Table IX. C2H2 Reduction by Selected N2-Fixing andnon-N2-Fixing Bacterial Cultures

One ml of indicated culture in log phase of growthadded to 4 ml of its respective media. Total volume ofincubation vessel, 40 ml; gas phase, 0.1 atm C2H2, 0.9atm He for anaerobes, 0.1 atm C2H2, 0.2 atm °2. 0.7atm A for aerobes; incubation time, 60 min; temperature,300. Ethylene assayed gas chromatographically on ester-amide column. Similar results obtained with 0.01 atmC2H2.

i&molesOD650mt C2H4/ N2-f ixing

Organisms of culture hr-incubation abilityAzotobacter 0.30 1.58 +

vinelandiiClostridium 0.19 1.62 +

pasteurianuntRhizobiuni 0.3 0.0002 -

japonicuinR. melliloti 0.3

PLANT PHYSIOLOGY

mediumii and Lactobacillus leichmianii, Bacilluis sub-tilis, Bacillus cer-eus var. niycoides, Serratia mzar-cescents, Escherichia coli, Streptococcuis lactis,Sacchlaro inyces cerevisiae, Pseudomonas aeruginosa,Pseuidoinontas fluorescens, Aerobacter aerogenes,StaPhylococcus epiderinidis, Sarcina lutea, Spirilluntitersonili, Proteus vulgaris, Alcaligenes faecalis, andRhodospirillurn rubrum under aerobic conditions oncomplete medium. All had


DECAPITATEPLANTS AT SOIL LINE

THE C2H2 -- C2H4 ASSAY FOR N2- FIXING ACTIVITY

STEP I -BIOSPHERE SAMPLE PREPARATON

NODULATED PLANTS SOIL

SOIL BORE

HYD"OPHERE

,F.z

REMOVAL OF ROOTSYSTEM WITH OR

ATTACHED NODULES

\ STEPAS

TNSERT NODULATEDROOT SYSTEM

i L i_ & f

SOIL BORE OVERROOT

STEP 3 -REPLAEMENT OF AIR

2-TRANSFER TO;SAY CHAMBER

FLUSH SX

A O2 AEROBIC FIXERS1-4-A 02 CO2 PHOTOSTNTHIETIC FIXERS

A ANEROBIC FIXERS

STEP 4-SEALINGASSAY CHAMBER

STOPALL

STEP -ADDITIONOF C2H2 MIXTURE

INO SMOKING__f _o

ADD 20mis C2H2 MIXTURE TOASSAY CHAMBER AND FLUSHADO ANOTHER 20mIs C2H2 MIXTUREAND INCUBATE

STEP T - INCUBATION

LNO SMOKING

INCUBATE FORONE HOUR UNDERIN SITU CONDITIONS

40mis A 02. 02 CO or AH2 PLUS lOm' CZH21-C2H2

BEADS

A 02 AEROBIC FIXERS

A 02 CO2 PHOTOSYNTHETIC FIXERS

>4 0 A ANEROBIC FIXERSBER

MIX GAS BY SHAKING

STEPS -REMOVAL ANDMIXING OF GAS PHASE

[NO SMOKING

GAS IS FORCED FROM ASSAYCHAMBER INTO GAS RECEIVER

STEP 9 - C2H2 C2H4 ASSAY BY FLAME IONIZATION AFTER GAS CHROMATOGRAPHY

I NO SMOKING 1

200). FROM THE GAS RECEIVER

STEPIO-CONVERSION OF C2H2 --O C2H4 ACTIVITY TO N2 2 NH5 ACTIVITY

FIG. 14. Steps in C2H2-*C2H4 assay for N2-fixing aotivity of the biosphere, including samples of nodulatedplants, soi-l, or hydrosphere.

1197


1198 PLANT PHYSIOLOGY

Table X. Distribution of C,H,-Reducing Activity in SoYbean FieldSamiples collected betwxeen 8 to 9 A\m and immediately assaved as outUi;ned in figure 14. Gas plhase, 0.1 to 0.2 atIn

C.,H., plus indicated gases A:0., (0.8:0.,) or He to 1 atm; root soil bore xolumle 45 ml; total gas phase volume, 40ml il'lubationi time, 1 hr.

SamiipleRoot -oil boreRoot soil boreRoot soil boreRoot soil boreNodtllated roo)tSoil arouniid

niodu!lated rootSoil bore betxweerows 0- 4" deep

4. S" deep8-12" deep

Gasphase

A:0.He:C.,H.,Ak:0..:C.H.,A:0.,:C,H.,A:0,:C.,H.,

A :0.,:C.,H,AD2:C2H2A:0.,:C.,H..

for C. -L. redutctioni (table X, ref. 23). Failure toreplace air (N.,) with A:02 results in a 10 to 20%decrease in C.H., reduction (table XI).

Timiie Course. The rate of C.,H12 reduction bynodulated roots or root soil bores of soybeans isconstaint up to 60 mlinutes (fig 15) wvith the standardsystemii described in figure 14. However, it isrecommiienided that heavily nodulated roots be assayedfor a shorter time (30 min) since the rate for such

z°40

0 30-

z 20

j 10

E co) 30 60 90 120 150 180 210 240

TIME (min)

Fic.. 15. Time course of C9H,-->C2H4 reduction bynodulated soy,bean roots. Incubation system, figure 14,except that anial-ses w-ere made from 0 to 24,0 uill.Glass bead,- ere added to assay chambers to facilitatemnixio of gas phase.

,umoles C,H4/dav.sample

0.0011.870.026

171195

m,utmoles C.H4/day*IlIg fr xvt nodule

0.0013 74

168172

0.035

0.0330.0300 028

rug fr Nvtnodule

10305031030

01114

0

00)0

samples decreases shortly after 60 miniutes. Thisdecrease is presumably due to O. depletion, sincesamples wvhich were reflushed with A :O., and re-gassed with the C2H., mixture showed activitiesduring a 1 to 2 hour incubation that were comparableto those observed during a 0 to 1 hour incuibation(tables XI, XIII).

Interval Between Samitpling and Assay. Nodu-lated roots and root soil bores of soybeans wereassayed at 0, 2, 6.5, and 13 hours after sampling(fig 16). Values are expressed on the basis ofnodular efficiency, mptmoles C..H. per mig fr wtnodule per day, in order to compensate for thevariable nodulation of samples. The results incdicatethe importance of a minimum interval ( 0-2 hr)between sanmpling and assay in order to obtain valtueswhich are representative of the in situ activity.Consequently, all the results reported in this paperunless otherwise indicated were obtained from assaysinitiated within 30 minutes after collection.

Samnple Variability atnd Reproducibility. Thesample variability with respect to C.2H,-reducingactivitv of field-growin soybeans is shownii in table

Tahle XI. Effcti of Air on C,H,-Reducing Activity of SolcwansSampeles N\ eie collected betwxeen 8 to 9 \M aud assaved from 0 to 1 and acain from 1 to 2 lhr as ouitlined in

fi'igure 14. Air (-N.,) replacel A :2 wN-lere inidicated.

knioles C,H4/ mjumoles C,H.,/day mug fr- -wtSample Gas phase day.sample *mlig fr\wtnodule noduile

Nodulated root0-1 hlr A :0., :C2,H, 659 197 33331-2 hir A :0. :C2H., 750 225 33330-1 lhr A :0.. :C.,H. 410 91 45111-2 lihr Air :C.fH. 328 73 4511

Root soil bore0-1 hr A:0.,:C2H, 185 79 23401-2 hr A:0. :CH.2 190 81 23400-1 hr A:O,:C,H. 289 142 20471-2 hr Air:C.,C.. 266 130 2047

I_

I


HARDY ET AL.-ACETYLENE - ETHYLENE ASSAY FOR N., FIXATION

0

z

E

I

IT

400w

300

200

100o~

° 2 4 6 8 10 12 14

INTERVAL BETWEEN SAMPLING AND ASSAY (hr)

FIG. 16. Reduction of C,H2-*C2H4 by nodulatedsoybeain root or soybean root soil bore assayed at varioustimes after collection between 8 to 9 ANI. Incubationsystem, figure 14. Each point represents the averageof 5 individual samples.

XII. The standard deviation of C,H9-reducing ac-tivity of root soil bores or nodulated roots collectedat 15, 25, 29, and 44 days post-flowering is 25 to35 % while that of nodular efficiency is 17 to 22 %for root soil bores and 5 to 10 % for nodulated roots.Thus, expression of activity on the basis of nodularefficiency substantially decreases the variability dueto differences in nodule weights.

The excellent reproducibility of C,H2-reducingactivity of field-grown soybeans is shown in tableXIII. Samples collected at various times afterflowering show C,H2-reducing activities during a1 to 2 hour assay that are 89 to 107 % of the re-spective activities determined during a 0 to 1 hourassay.

Km of C9H.,. Nodulated roots of soybeans aresaturated by 0.025 to 0.2 atmosphere CoHo (fig

Table XII. Sample Variability of Field-Grown Soybeans for C,H,, RedufctionAll samples collected from different areas of the same field between 8 to 9 A'M on indicated day and assayed

immediatelv as described in figure 14. Each value represents average of 5 samples.

m,umoles C9H4/ mg fr wtSampling time ,umoles C,H4/ mg fr w-t nodule/(days post-flowering) davysample nodule.dav sample

Root soil bores15 33 94 355

47 72 65968 84 81243 62 701

Avg 48 79 63225 105 101 1035

190 153 123890 180 501116 149 776

Avz 125 146 888

Nodulated root29 513 176 2908

514 144 3577785 166 4731

Axg 604 162 3739

44 751 226 3319412 242 1697582 234 2508

Table XIII. Reproducibility of C H,-Reducing Activity of Field-Grown SoybeansSo-bean root soil bores or soybean nodulated roots collected between 8 to 9 AMi and assayed as in figure 14, from

0 to 1 hr and agairn from 1 to 2 hr after collection. Each value is average of 5 individual samples.

m,amoles CAH4/mg mg fr wtSampling time ,umoles C2H4/sample.day fr wt nodule.day nodule/(days post-flowering) 0-1 hr 1-2 hr 0-1 hr 1-2 hr sampleRoot soil bore

0 12 12 74 79 1580 8 9 71 83 1132 12 11 65 58 191

44 177 166 56 53 3140Nodulated root

44 752 742 226 223 3319

_ONODULATED ROOT

x

N

- ROOT SOIL%L\BORE ' ,

1199

- --I


PLANT PHYSIOLOGY

*E

p CCH2a t m )20(a) (b) )

figure15

20 0

C\C C\d

17a)o

BL 0 0.05 0.1 Zi- 0 500 1000Pt2H2e atm) PC2H2(atm)

FIG. 17. a) Ethylene formation from CHec as afun-tction of pCtH2 by nodulated roots of soybeans, andb) plot of reciprocal velocity versus reciprocal pC2H2for determination of Km of C.He.Incubation system,figure 14.

17a) soybean root soil bores showed identical ac-tivities when incubated with a CrHi gas mixture of0.1 to 0.4 atmosphere CuHr. Activity was decreasedby0.5p atmosphere CaH2. This decrease might bedue to direct inhibition by CG2H2 or to indirectinhibition by the concomitant depletion of 0t. Anaverage Km of 0.007 atmosphere CmHe is obtainedfrom a plot of reciprocal pCdHavs. reciprocal rateof CnH4 formation. This value is somewhat lowerthan that reported for exchised sobean nodules (23).

Temperatuire. The effect of temperature of in-cubation on C,HAreduction by nodulated roots orroot soil bores of soybeans was less pronounced thanobserved with eitherNaase preparations or bacterialcultures. Activity was lower at 10CC to 150 andpossibly at 350 than at 200 to 300 (table XIVT). Incontrast, preliminary results suiggest that the tem-

perature of growth has a m-ore marked effect on

CSI-H,reducing activity. Nodulated rootsfcromplants aintained in growth cabinets for periods ofi to 14 days at 3e00 had only 10 to 20 % of theC.,H2-redticing activity of those at 200.

CadHt-Reducing Activityanh d Calct lated N., Fixa-tionl Dutring One Growing Seasont. Acetylene-re-

ducing activity of field-growuen soybeans was deter-mined during a complete growth and maturation

Table XIV. Effect of Temper-ature- on C,H,, Reduictioniby Soybeans

Soy,bean root soil bores or nodulated roots collected

between 8 to 9 AM, flushed with A :02 as described infigure 14, equiliibrated for 19 min in a water bath atindicated temperature, then incubated for 1 hr after

addition of C2H2 mixture. Values are the averagesof 3 nodulated roots or root soil bores.

Incubation Nodulate root Root soil boretemp

deg m,umoles C2H4 mig fr wt nodule-day10 2915 86 ...20 214 15625 176 20630 250 18835 151 151

cycle. Both the root soil bore (fig 18) and nodu-lated root (fig 19) techniques were used. Sampleswere collected on 41 different days for the root soilbore assays and on 27 different days for the nodu-lated root analyses. The C,H9-reducing activitywas determined as described in figure 14 utilizing1 hour incubations. Results are expressed on a 24hour basis (fig 18a and 19a). The fresh weightof nodules per sample was tabulated (fig 18b and19b), and the nodular efficiency of C,H., reduction,expressed as mumnoles C,H4 formed per mg freshweight nodule per day, was calculated (fig 18c and19c). The averages of all analyses on an individualday and during each week are shown.

Nitrogen fixation, as measured by the C,H.2-C2H4assay, was found to parallel the nitrogen demand of

SOYBEAN ROOT SOIL BORESAMPLES / WEEK2 0 30 38 66 70 30 70 55 40 15 15 10 10

IC)3oo x-

0~~~~~~~~C*~ ICCexE°-,< xx

E b100- -

,,4 00-w x

300- x

z 2000t, u

X0

z .oo- r -

ZL~~~~~x

N8 30 CC XM

a1000-E X xts \v

04000-00z

0 U U

°>30- z A C'~z 200030- 0 2

B x-100- xC- -x

< 0- r1d--Ci-.-.z~

2 0 0 0 0 1 2 8 182

JUNE JULY AUGUST SEPTEMBERFIG. 18. Summars of a) C2H2>-C.H4 reducing

activity-, b) mg fr xs-t nodule, and c) m,umoles C.,H2-C9H4 per mg fr wt nodule per day by soyrbean root soilbores at various stages from initiation of activityr throughflowering and maturation to loss of activity. Incubationsystem, figure 14; root soil bore volume, 45 ml; gasphase volume, 40 ml. Flowering indicated by initialbud opening and senescence indicated by initial y-ellowingof leaves. The average of all samples assayed eachl day(x), during each week - and the average of m,umolesC92H*C H4 per mg fr wvt nodule per dax for theperiod from flowering to senescence (- ) arerecorded. The number of soil bores of soybean rootsassayed each wueek is recorded. Samples wTere collectedbetween 8 to 9 AM except for July 27 to 28 and August9 to 10 wNhen samlples were collected at various timesas specified in figure 20. Samples were assayed imme-diately under in situ conditions of temperature and mois-ture. Assay time, 1 hr; results expressed on 24 hrbasis.

1200



NODULATED SOYBEAN ROOT

SAMPLES/WEEK 13 38 3 45 15 40 10 20 15 10

220 x-I K

i. -Kx

/ x, %

Do-~~~~~~~~~~~-.

UI~~~~~~~~~~~I

z Li 5.

a.~~~~~~~~.0 L~~~~~~~~~~~~~i

L 8.0 i C-))O X ZLA-~~~~~~~~~~~~~~~~~~~~~~~~-Z60 Z -

0.75 u4KE z C/

00i W WLoH0.50 J O0020-:

o/

K

)0 20 02 KC,H4 reducing ac-tivity, b) mg fr wt nodule and c) m,umoles C,H,-+C2,H4 per mg fr wt nodule per day by nodulated rootsof field-grown soybean plants at stages of developmentsimilar to those in figure 18. Incubation system, figure14; gas phase volume, 40 ml. The average of all samp'lesassayed each day (x), each week ( ), and theaverage m,umoles CoH,-C,H4 per mg fr wt nodule perday for the period from flowering to senescence( - -- ) are shown. The number of nodulated soy-bean roots assayed each week is recorded. Sample col-lection and assay conditions as descri-bed in figure 14.Mg No fixed per plant per day and kg No fixed peracre per day are calculated on the theoretical basis ofone-third N., reduced per C,H, reduced.

the plant. Low C2H2-reducing activity occurre(duntil macroscopic flowering was observed, althoughlactivity could be detected as early as 32 days beforethis time; the utilization of residual nitrogen fer-tilizer during this period may have suppressed N2-fixing activity to some extent. Following floweringthe C2H2-reducing activity increased continuously,reflecting the increasing nitrogen requirement forpod formation and filling. Average weekly activitvincreased from 30 to 299 jumoles C2H4 formed perroot soil bore per day or 84 to 650 /jmoles C,H4formed per plant root per day. The average weeklynodule weight increased from 244 to 2453 mg perroot soil bore or 457 to 3478 mg per nodulated planitroot. After pod filling was complete the C2H.,-re-ducing activity rapidly declined. The decline innodule weight following senescence lagged behindthat of CGH-reducing activity.

The nodule efficiency was relatively constantduring the period from flowering to senescence(fig 18c and 19c). The average weekly efficienciesvaried from 88 to 196 m,umoles C2H4 formed per mgfresh weight nodule per day for root soil bores andfrom 156 to 378 for nodulated roots. The averagenodule efficiency from flowering to senescence was142 m,umoles CGH4 formed per mg fresh weightnodule per day for root soil bores and 220 for nodu-lated roots.

Summation of the weekly averages of C2H.,-re-ducing activity indicates that 22.5 mmoles of C,H,could be formed per plant per season (fig 19a). Thecalculated N2 fixation per acre per season is 30 to33 kg of nitrogen based on: 1) the C2H2-C2H,assay, 2) 142,000 plants per acre, and 3) a theoreticalconversion factor of one-third No fixed for eachC..H formed.

22.5 to 25x

3

28x

10a}

142,000 = 30 to 33 kg N. fixedper acre per season

This calculated value was determined during de-velopment of the C2H,-C,H4 assay and with samples(except for 2 occasions) collected between 8 to9 AM, a period which may represent less than maxi-mal activity (fig 20). However, this value is inexcellent agreement with the average value of 38 kg

-JC]0

0z

E

C-)0

E

E

8 12 4 8 12 4 8 12 4 8

AM PM AM PM

FIG. 20. Diurnal variation of C,H2,--C,H4 reductionby nodulated soybean roots and soybean root soil borescollected in the field at indicated times and assayed im-mediately. a) Samples 3 to 4 days post-flowering, andb) 16 to 17 days post-flowering. Incubation system,figure 14. Each point represents the average of 5 sam-ples. Heavy rainfall occurred during the night andsecond day of experiment in b).

1201

LiJJ 3c

E °3 Z 2CE c

E"IC

z-i 30Ca.Li- 20(00Z 10(

E

o 8C;z< 6(i0.

I 4(

d 2(E


PLANT PHYSIOLOGY

of nitrogen fixed per acre reported for Kjeldahl and15N analyses of N, fixation by soybeans (35). Thiscorrelation provides support for the quantitative re-liability of the C9H9-C9H4 assay performed as out-lined in figure 14.

Diutrnial Variation. The diurnal variation ofC..H.,-reducing activity of field-grown soybeans wasdetermined at 3 to 4 (fig 20a) and 16 to 17 (fig20b) days post-flowering. Activities expressed asm,umoles C.,H4 formed per mg fr wt nodule perhour appear to be maximal for samples collectedfrom noon to 8 PM and minimal for those collectedfrom miidnight to 8 AL. Thus, a close relationshipbetween light and N,-fixing activity is suggested.The effect of light on C.112-reducing activity wasfurther demonstrated with nodulated roots of soy-bean plants in growth cabinets (fig 21). Controlplants maintained on a 16 hour light and 8 hourdark cycle did not show a marked diurnal variation,wvhile experimental plants showed a rapid decline to30 % of control activity after 17 hours of totaldarkness, but still had 15 % of control activity after64 hours of darkness. The initial decline mayreflect the depletion of photosynthate, while theresidual activity may represent utilization of storageproducts.

Heavy rainfall eliminated the normal diurnalvariation (fig 20b). This effect might be due toincreased soil moisture and/or decreased light in-tensity. Saturation of soybean root soil bores withwater decreased their C.H2-reducing activities from170 to 50 m-,nmoles C,H4 per mg fresh weight noduleper day.

Leaf or Pod Remtoval. Removal of leaves de-creased C.H9-reducing activity to 12 % of control

-i

, 48-

'^ 3-EI

Xx NORMAL CYCLE1\ X

j\ 2 TOTAL DARKNESS

E LIGHT DARK DARK5H 7TGA3KGQHT IDAKI iEEHT- XH ARK LIGHT NORMAU 'A S . Ap 10PMA 6AM IOPM 6AM IOPM

2 3 4FIG. 21. Effect of light and darkness on C2H,

C9H4 reduction by nodulated soybean roots from plantsmaiintained in plant growth chamber. Incubation system,figure 14. Each poinlt represents the average of 5 indi-vidual samples. Conitrol cycle, 6 AM to 10 PM, 240,ux-itli maximum light (see under Methods), and 10 PM to6 XalI, 180, total dlarkness. Experimental sample wasplaced in total darkness with normal temperature cycleat 10 PM\ of first day of experiment.

after 1 day, and this activity was still only 14 %of control at 10 dayvs after leaf removal.

Removal of pods at 19 days post-flowering didnot alter C.H..-reducing activity per plant (fig 22a)or mg fresh weight nodule per plant (fig 22b) duringthe following 10 days. However, during the sameperiod control plants increased both C9H2-reducingactivity per plant and mg fresh weight nodules perplant. Thus, the magnitude of No fixation reflectsthe demands of the plant, specifically the pod in thiscase, for nitrogen.

Varieties. Acetylene reduction provides a tech-nique for the determination of differences in theN,-fixing activities of different varieties of legumes.A single exploratory experiment was conducted with

Table XV. C,H., Reduction bha Different Varieities of Field-Grown SoybeansNodulated roots of soybean varieties collected between 2 to 3 Pm on the same day (114 days after planting) were

assayed immliediately for C,H. reduction as indicated in figure 14. Soil temperature, 19 to 20°. C1311-Wabash XC1069-Clark X C1069; and UD-61-1806-EC33243 X D49-249L. Eacb value represents 5 individual roots.

Stage of development Iumoles C.H4/plant.day

m/Lmoles C,H4/mg fr wtnodule day

Earlx -maturing:Verde

AdelplhiaClark

Intermediate-maturinig:C1311C1278KentDelmar

Late-maturing:Dare

YorkUD61 -1806Hill

Yellow-, lossof leaves

" + nodule decayInitial yellowx ing

,,

,,

Green, pods stillfilling

,,

Varietv

1.5

1.11.0

1.1

1.51.8

0.87.0

22.730.2

4.87.91739

35.5

59.2136162

57

98104119

1202



0c-z-ja-N1-.I q

0i

(b)za-N 4000 ,w

0z 2000 x

E

C(c)

\ 300

200EZEOE

It 100__"I

00 5 10

DAYS AFTER POD REMOVALFIG. 22. a to c). Effect of pod removal on C,H.>

C2H reduction by nodulated soybean root or soybeansoil bore. Incubation system, figure 14. Pods wereremoved 19 daYs post-flowering.

varieties of soybeans representing different matura-tion dates. Marked differences found in C9H,-re-ducing activity (table XV) correlated with differ-ences in the stage of maturity at analysis. Varietaldifferences will be further investigated.

C,H2 Reduction by Selected Legumes. Nodu-lated roots of Phaseolus vulgaris, Medicago sativa,Arachis hypogea, and Pisum sativum as well asGlycine max reduce C2H2 to C2H4 (table XVI).The nodular efficiencies, m.mmnoles C2H4 formed permg fresh weight nodule per day, of these legumeswere in the range of 106 to 402, similar to thosereported by Koch and Evans (23) for excised soy-bean nodules, but substantially higher than thosereported by Sloger and Silver (34) and subsequentlyby Stewart, Fitzgerald, and Burris (36) for a varietyof excised legume and non-legume nodules.

Table XVI. C,H., Reduction by, Selected LegumesNodulated roots of the indicated legumes collected

betx,-een 8 to 10 AM and 'nimediately assayed as describedin figure 14.

mJimilo'es C.H4/nig fr w-t Calculated kg N.,

Le-ume nodu'e-dav fixed/acre.dayGl,cine mitaxl 220 0.60Phaseolus zdulgaris2 106Mfedicago satizva2 161 0.47Arachis hypogea2 402 ...Pisuin sativum2 3041 Average value from figure 19 for flowX-ering to sen-

escence period.2 Samples assayed at flowering.

C.,H2 Reduiction by Free-Living Bacteria in Soil.Nitrogen-fixing activities calculated from the C,H2-reducing activities of selected soil samples are tabu-lated in table XVII. The activities varied over500-fold between locations and suggest that N.,-fixa-tion by free-living soil bacteria varies from negligibleto highly significant. Activities under both aerobicand anaerobic conditions were found; the anaerobicactivity was higher in the lower soil levels.

Variations in CGI1-reducing activity of theJordan Fertility Plot samples correlated with nutrientapplications. The 4 plots receiving recommendedadditions of N, P, and K showed similar activities,and the average calculated value of N2 fixation is0.51 kg of No fixed per acre per day. Plots receivingless or no nitrogen showed, in general, greateractivities. The calculated No fixation of the plotreceiving only K is 1.52 kg N2 fixed per acre perday. This is the highest calculated value of N2fixation that we have obtained for soil samplesanalyzed with the C.H,--C.H4 assay. Samples fromall the limed plots had double the activity of thosefrom all the unlimed plots. Initial experiments withsoil to which bacterial cultures were added (Azoto-bacter and Clostridium) showved negligible increasesin C,H2-reducing activity.

Miscellaneous Examitples of C2H2 Reduction.Nitrogen-fixing activity of a variety of miscellaneoussamples of the biosphere, including pond water andrumen contents were assaved by CGH2 reduction.Samples of pond water reduced C2H2 to C2H4 whenthey contained blue-green algae. Negligible C2H4was formed in the absence of C2H2. More extensivestudies of C2H, reduction by blue-green algae havebeen communicated recently (36).

Rumen contents from a fistulated steer reducedC2H., to CH4. Ethylene formation was 10-foldgreater under anaerobic than aerobic conditions.Methane formation was markedly decreased in thepresence of acetylene. The N,-fixing activity of arumen calculated on the basis of the anaerobic resultswas 10 mg No fixed per rumen per day.

i b

1203


Table XVIII. C,H.,-Reducing Activity and Calculated N, Firation by Soil Sa pl1'sSoil bores w ere collected from 12 to 4 Par and immediatelv assayed as indicated in figure 14. Each value repre-

sents the average of 6 samples. Soil bores, 2 X 45 ml for 0 to 6" anid 1 X 45 ml for 0 to 3" and 6 to 9'.

Calculated kg N., fixedSample Special nm,moles C9H4/sample.day /3" or 6" acredaylevel treatmenit Aerobic Anaerobic Aerobic Anaerobic

Chester Co., Pa.0-3"3-6"6-9"

Georgetown, Del.0-3"6-9"

Jordan fertility plots0-6"0-6"'0-6"0-6" No fertilizer0-6"0-6"0-6"0-6"0-6" Recommenided0-6"0-6"0-6"0-6" +3(N), as0-6" Excess burne0-6" +N,P,K0-6" Ground bone0-6" +3(N) as (:0-6" +Lime0-6" -Lime

+Kl+N1+pi

+P,K+N,P+N,K+N,P,K,

d treatmeint2

NaNO, P.1K.d lime

NH4) .,SO4. P,K

1 +K = 100 lb K.O per acre; +N = 24 lb N per acre; +P = 48 lb P,O5 per acre.2 Recommentled treatment = 115 lb N, 130 lb P205,130 lb K.,O per acre.

Discussion

The results reported here support the validity ofthe C2IH2-C.H4 assay as a sensitive and universalanalysis for N-fixing activity. The advantages ofthis assay indicate significant broad applications formeasurement of N. fixation in both laboratory andfield investigations. The essential relationship be-tween C2H2-reducing activity and N2-fixing activityis supported and extended to a most convincingdegree by studies within and among cell-free ex-tracts, bacterial cultures, and symbionts.

This relationship is firmly established by detailedexperiments with Azotobacter preparations whichlead to the conclusion that CdH2 and No evokeidentical responses from N,ase. Convincingly paral-lel responses are observed with respect to: require-ment for ATP and reductant, linear time course,optimum pH, sigmoidal relationship between rateand enzyme concentration, inhibition of H. evolu-tion in an amount equivalent in electrons to thoseuJsed for C.H, or N., reduction, competitive inhibi-tion by CO, relative insensitivity to NH4,, activationenergy of 13 to 15 kcal/mole above 20° and 35 to50 kcal/mole below 20°, activity in extracts from

N,- but not urea-grown cells, distributionl of activitydurinig fractionation, requirement for both the M,o-Feand Fe protein fractions of N.,ase, and relativeinactivity of the clostridial-Azotobacter interspeciesreconmbination of the Mo-Fe and Fe protein fractions.These results provide the most complete correlationbetween N. fixation and C.H., reduction to date.Some of these similarities have been reported forcell-free extracts of bacteroids (24, 25) and C. pas-teurianunt (13, 32). The inhibition of ATP-depend-ent H. evolution by Azotobacter N.,ase in anl amountequivalent to the formation of ethylene establishesthe electron-activating reaction of N.,ase as thesource of electrons for C2,H2 reduction, just as forN., N..O and N2- reductions (15, 20). The similarcompetitive inhibitions of N. fixation anid C.,H.,reduction by CO provide strong indirect support forthe role of the substrate-complexing site of AZoto-bacter N2ase for both C2H2 and N., reduction.

\Vhole cell experiments are completely consistentwith the results obtained in vitro and denmonstratethe parallel C2H2-N2 relationship in in viVo analvses.Thus, cultures of Azotobacter or Clostridium11 reduceC.H., to C.H4 with characteristics similar to fixa-tion of N., which include: anaerobic re(quireinient

312727

6720575

310235

0.00200.001800018

0.0200.015

155940

0.00430.01300.0048

0.0100.106

11,90011,5008,7008 6008,7407,4005,8505,7004,6004,2004,1002,9002,8002,2601,4001,2501,1005,8502,650

1.521.471.121.101.020.950.750.730.590.540.530.370.360.290.270.180.160.750.34

1204 PLANT PHYSIOLOGY



for Clostridium and aerobic requirement for Azoto-bacter. activity in No- but not NH3- or urea-growncells. time course, and ratio of C2H2 reduced to N2fixed of 3 to 4.5. In addition, bacterial specieswithout N..-fixing activity do not possess significantC2H.,-reducing activity.

Experiments with symbionts establish that theC9H9-N9 correlation is consistently applicable toeven these most complex natural N2-fixing systems.Thus, various legumes also reduce C2H, to C9H4with characteristics similar to fixation of No. Theseinclude: aerobic requirement, activity only in nod-ules possessing leghemoglobin, absence of activityin either the root or infecting bacteria, and similarrate of C[,I4 formation or No fixation per seasonbased on electron requirement.

The characteristics of NYase activity, as exem-plified by C9H9 reduction, are consistent throughthe entire range of organization studied; resultsobtained with the most defined in vitro system applywith equal validity to even the most complex sym-biotic systems. Thus, all systems reduce CGH, toa single significant product, C9H4; none reducesubstrate C..H; all are saturated by 0.02 to 0.2atmosphere of C9H9; the Km values are 0.002 to0.009 atmosphere, and the calculated activationenergies are similar for both N9ase preparationsand bacterial cells with a break in the Arrheniusplots near 200. A common enzyme, N..ase, appearsto be responsible for C.H9 reduction by these diversesystems.

The advantages and disadvantages of the C.2H2-C9H, assay for N.) fixation are summarized in tableXVIII. The advantages emphasize the superiorattributes of this nmethod relative to other assays ofN, fixatioIn.

Sensitivity of C.9H4 detection by flanme ionizationas initially ilidicated by Hardy and Knight (19) isthe critical advantage of the C..H--C9H4 assay. Thesensitivity of this method is related to other methodsof N., fixation by the folloNving comparison: theCJHL-C.9H4 metlhod is to the 15N method as the35N mlletlhodl was to the Kjeldalhl method. Thissensitivity of the C.,H.-C.,H assay makes it possibleto detect lowv levels of N,-fixing activity in bio-sphere samples. bacterial cultures, or N9ase prep-arations. anld to investigate variations in NA-fixingactivity with short-term incubations. It is antici-pated that the list of N..-fixing organisms willuindergo adlditions and possibly some deletions be-cause of the C.H9.-C9H4 assay. The only othermetlhod w-itlh equivalent or greater sensitivity is3N-incorporation: however, the complex productionfacilities anid short half-life restrict the applicationof "3N to limiiited laboratory investigations.

Disadvantatges of the C.2H.2-C,H4 assay includethe inldirect nature of the reaction. Although nodefined samiiple has been found with significantC.H..-reducing activity and without N.,-fixing ac-tivity, thle possibility exists for a non-N.,ase-depend-ent catalysis by biosphere samples of the reduction

of C,.H9 to C2H4. The explosive nature of C2H2 isemphasized. Laboratories accustomed to utilizingthe relatively inert No for 1-N or Kjeldahl analysisare cautioned to employ safe practices with C2H .

As recently as 3 years ago Allison (2) found itnecessary to state "During the past 50 years mucheffort has been devoted to the economic evaluation

Table XVIII. The C2H2-C2H4 Assay for N, Fixation

AdvantagesA) Analytical

Sensitivity. 2NH3'Universality. N2ase preparations, and N,-fixing bac-

teria, blue-green algae and symbionts reduce C2H2-*C2H4,while non-N2-fixing preparations or organisms do not.

Quantitative Relationship of C2H2 reduced to N2fixed of 3 to 4.

Saturation by a low pC9H, -Km (C2H2) of 0.003-0.008 atm c.f. Km(N2) of 0.02 to 0.16 atm.

Specificity of Reaction Product. No significant productother than C&H4.

Low Background. Negligible C,H4 formed in theabsence of C.H2 c.f. the natural background of 15N.

Metabolic Stability of Product. C,H4 is not me-tabolized c.f. the metabolic conversions of inorganic andorganic forms of nitrogen.

DisadvantagesIndirect Nature of Reaction. The possibility exists

for non-N,ase catalysis of this reduction by samples ofthe biosphere.

Explosive Nature of C,H2. C2H2 is a highly ex-plosive gas c.f. N., which is relatively inert.

.1205


PLANT PHYSIOLOGY

of free-living nitrogen-fixing micro-organisms insoils, but we are nearly as much in the dark now----.There is no sound base upon which to make directestimate----", while only a year ago the Subcommitteeof Production Processes of the United States Na-tional Committee for the International BiologicalProgram (37) suggested the following immediateaction with respect to nitrogen fixation: "Providingmeans for the use of available specialized instru-ments (e.g., mass spectrometers) in the accom-plishment of routine analytical determinations."With the advent of the C2H2-C2H4 assay procedure,these statements are no longer relevant. The C.H,-C2H4 assay is as crucial for the optimization ofbiological nitrogen fixation as soil analyses werefundamental to the development of agriculturalfertilizer use.

Acknowledgments

The authlors are indebted to Mrs. A. Richmond fortechnical advice on gas chromiatographic analysis and toMrs. B. Musser, Mrs. S. Lidumils, Miss C. Madden, MissM. Parke, Mr. M. L'ovd, and Mr. C. Orth for skilledtechi-i al assistanice.

Literature Cited

1. AHMED, S. AND H. J. EVANS. 1960. Cobalt: amicronutrient element for the growth of soybeanplants under symbiotic conditions. Soil Sci. 90:205-10.

2. ALLISON, F. E. 1965. Evaluation of incoming andoutgoing processes that affect soil nitrogen. In:Soil Nitrogen. W. V. Bartholomew and F. E.Clark, eds. American Society of Agronomy, In-corporated, Madison, Wisconsin. p 573-606.

3. BULEN, W. A., R. C. BURNS, AND J. R. LECOMTE.1965. Nitrogen fixatioln: hydrosulfite as electrondonor with cell-free preparations of Azotobactervinelandii and Rhodospirillurn rubrumiz. Proc. Natl.Acad. Sci. U.S. 53: 532-39.

4. BULEN, W. A. AND J. R. LECOMTE. 1966. Thenitrogenase system from -4qotobacter: tw-o-enz-merequirement for N., reduction, ATP-dependent H.,evolution, and ATP hydrolysis. Proc. Natl. Acad.Sci. U.S. 56: 979_86.

5. BULEN, W. A., J. R. LECOMTE, R. C. BURNS, ANDJ. HINKSON. 1965. Nitrogen fixation studieswitlh aerobic and photosynthetic bacteria. In: Non-Heme Iron Proteins: Role in Energy Conversion.A. San Pietro, ed. The Antioch Press, YellowSprings, Ohio. p 261-74.

6. BURNS, R. C. 1968. AZlotobactcr Nitrogenase: ac-tivation energy and divalent cation reqtuirement.Biochim. Biophys. Acta: In press.

7. BURRIS, R. H. AND P. W. WILSON. 1957. Methodsfor measurement of nitrogen fixation. In: Meth-ods in Enzymology, Vol. 4. S. P. ColoxN ick andN. 0. Kaplan, eds. Academic Press, New York.p 355-67.

8. CA.NIPBELL, N. E. R., R. DULAR, H. LEES, AND K.G. STANDING. 1967. The production of 13N,by 50 Mev protons for use in biological nitrogenfixation. Can. J. Microbiol. 13: 587-99.

9. CARNAHAN, J. E., L. E. MORTENSON, H. F. MOWER,AND J. E. CASTLE. 1960. Nitrogen f ixation incell-free extracts of Clostridium pasteurianum. Bio-ohim. Biophys. Acta 44: 520-35.

10. DEHERTOGH, A. A., P. A. MAYEUX, AND H. J.EVANS. 1964. The relationship of cobalt require-ment to propionate metabolism in Rhizobiurn. J.Biol. Chem. 238: 246-53.

11. DETROY, R. W., D. F. WITZ, R. A. PAREJKO, ANDP. W. WILSON. 1967. Complementary function-ing of two components required for the reductionof N, from four nitrogen-fixing bacteria. Science158: 526-27.

12. D'EUSTACHIO, A. J. AND R. W. F. HARDY. 1964.Reductants and electron transport in nitrogen fixa-tion. Biochem. Biophys. Res. Commun. 15: 319-23.

13. DILWORTH, M. 1966. Acetylene reduction by nitro-gen-fixing preparations from Clostridium pasteutri-anum. Biochim. Biophy s. Acta 127: 285-94.

14. DILWORTH, M. J., D. SUBRAMANIAN, T. 0. MUN-SON, AND R. H. BURRIS. 1965. The adenosinetriphosphate requirement for nitrogen fixation incell-free extracts of Clostridium pastcurianum.Biochim. Biophys. Acta 99: 486-503.

15. HARDY, R. W. F. AND R. C. BURNS. 1968. Bio-logical Nitrogen Fixation. Ann. Rev. Biochem. 37:331-58.

16. HARDY, R. W. F. AND A. J. D'EUSTACHIO. 1964.The dual role of pyruvate in No fixation. Bio-chem. Biophys. Res. Commun. 15: 314-19.

16a. HARDY, R. W. F. AND E. K. JACKSON. 1967. Re-duction of model substrates-nitriles and acety-lenes-by nitrogenase (N,ase). Federation Proc.24: 725.

17. HARDY, R. W. F. AND E. KNIGHT, JR. 1966. Re-duction of N,O by biological N,-fixing systems.Biochem. Biophys. Res. Commun. 23: 409-14.

18. HARDY, R. W. F. AND E. KNIGHT, JR. 1966. Re-ductant-dependent adenosine triphosphatase of ni-trogen-fixing extracts of Azotobactcr vinelandii.Biochim. Biophys. Acta 132: 520-31.

19. HARDY, R. W. F. AND E. KNIGHT, JR. 1967. ATP-dependent reduction of azide and HiCN by N,-fixing enzymes of Azotobacter zvinelandii andClostridium pasteurianum. Biochim. Biophys.Acta 139: 69-90.

20. HARDY., R. W. F. AND E. KNIGHT, JR. 1968. Thebiochemistry and postulated mechanisms of N2 fix-ation. In: Progress in Phytochemistry. L. Rein-hold, ed. John Wiley and Sons, Ltd. Sussex,England. p 387-469.

21. HARDY, R. W. F., E. KNIGHT, JR., AND E. K. JACK-SON. 1967. Substrate versatility of nitrogenase.Bacteriol. Proc. p 112.

21a. HOCH, G. E., K. C. SCHNEIDER, AND R. H. BURRIS.1960. Hydrogen evolution and exchange, andconversion of N20 to No by soybean root nodules.Biocihim. Biophys. Acta 37: 273-79.

22. KELLY, M., R. V. KLUCAS, AND R. H. BURRIS.1967. Fractionation and storage of nitrogenasefrom .4zotobacter vinclandii. Biochem. J. 105: 3c-5c.

22a. KELLY. M., J. R. POSTGATE, AND R. L. RICHARDS.1967. Reduction of cyanide and isocyanide byiiitrogenase of Azotol)acter chr-oococcumiit. Biochem.J. 102: Ic-2c.

1206



23. KOCH, B. AND H. J. EVANS. 1966. Reduction ofacetylene to ethylene by soybean root nodules.Plant Physiol. 41: 1748-49.

24. KOCH, B., H. J. EVANS, AND S. RUSSELL. 1967.Reduction of acetylene and nitrogen gas by breisand cell-free extracts of soybean root nodules.Plant Physiol. 42: 466-68.

25. KOCH, B., H. J. EVANS, AND S. RuSSELL. 1967.Properties of the nitrogenase system in cell-freeextracts of bacteroids from soybean root nodules.Proc. Natl. Acad. Sci. U.S. 58: 1343-50.

25a. LIND, C. J. AND P. W. WILSON. 1941. Mechani-ism of biological nitrogen fixation. VIII. Car-bon monoxide as an inhibitor for nitrogen fixationby red clover. J. Am. Chem. Soc. 63: 3511-14.

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