APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p. 955-962Copyright © 1977 American Society for Microbiology

Vol. 33, No. 4Printed in U.S.A.

Citrate, a Specific Substrate for the Isolation of Clostridiumsphenoides

R. WALTHER, H. HIPPE, AND G. GOTTSCHALK*Institut fur Mikrobiologie der Universitat Gottingen* und der Gesellschaft fur Strahlen- und

Umweltforschung mbH, D-3400 Gottingen, West Germany

Received for publication 28 October 1976

With a medium containing citrate as the carbon and energy source, 10clostridial strains were isolated from various mud samples. Characterization ofthese strains revealed that they all belonged to the same species, Clostridiumsphenoides. Strains of this organism obtained from culture collections were alsoable to grow with citrate, whereas 15 other clostridial species tested were not.Citrate was fermented by C. sphenoides to acetate, ethanol, carbon dioxide, andhydrogen. Experiments with stereospecifically 14C-labeled citrate indicated thatcitrate lyase was involved in citrate degradation.

It is well established that a number of or-ganic acids are fermented by clostridial spe-cies. Pyruvate is utilized by Clostridium butyli-cum (19), C. formicoaceticum (1), and C. sac-charolyticum (6). C. butyricum and C. pasteu-rianum grow with pyruvate as the carbon andenergy source (14), whereas C. thermosacchar-olyticum, C. roseum, and C. rubrum utilizethis compound for growth only in the presenceof a hexose such as glucose or fructose (14, 20).Acetate is fermented together with ethanol orL-lactate by C. kluyveri and C. tyrobutyricum,respectively (3, 4). L-Lactate is fermented by C.propionicum (5) and C. formicoaceticum (1). C.tartarivorum grows with D-tartrate and with L-malate and fumarate in the presence of acetate(25). Finally, L-malate and fumarate are uti-lized for growth by C. formicoaceticum (1).

Little is known about the fermentation ofcitrate by anaerobic spore formers. In an articleconcerning clostridia in silage, Gibson (9) men-tioned that "C. sphenoides has shown the abil-ity to ferment both malate and citrate."Using a medium containing citrate as the

energy source, we undertook to isolate anaero-bic spore formers from various mud samples.Several strains capable of growth with citratewere obtained. Interestingly, all of them be-longed to the same species, C. sphenoides. Thispublication describes the isolation and charac-terization of these strains and the course ofcitrate fermentation by C. sphenoides.

MATERIALS AND METHODSBacterial strains. The reference strain of C.

sphenoides (NCIB 10627) was purchased from theNational Collection of Industrial Bacteria, Aber-deen, Scotland. Eighteen strains of 12 clostridial

species were from the German Collection of Microor-ganisms (DSM), Gottingen, Germany, and 20strains of 7 species were from H. J. Kutzner, Darm-stadt, Germany. (The strains are listed in Table 2.)

Media. Citrate medium contained 50 mM triso-dium citrate, 0.03% (NH4)2SO4, 0.06% NaCl, 0.006%CaCl2 *2H2O, 0.2% K2HPO4, 0.34% KH2PO4, 0.4%yeast extract (Difco), 0.2% peptone (Difco), and0.06% sodium thioglycolate. In some experiments,0.03% L-cysteine- HCl * H20 was used instead of so-dium thioglycolate. The final pH was 6.7 to 7.0.Citrate agar medium contained 1.5% agar (Difco).To test for utilization of other substrates, the

medium described above was used except that cit-rate was replaced by the substrate to be tested.Substrate concentrations and final pH were thoserecommended by Holdeman and Moore (12). Whereindicated in the text, the PY and PYG media ofHoldeman and Moore (12) were employed. All mediafor isolation and characterization of the organismswere prepared, as described by Hungate (15), underan atmosphere of oxygen-free nitrogen.

Isolation procedure. Mud samples (1 g, wetweight) were placed in nitrogen-flushed, screw-capculture tubes. After the addition of 9 ml of oxygen-free, sterile 0.03% NaCl, the tubes were closedtightly and then shaken for approximately 1 min.The samples were pasteurized by incubation for 10min at 70'C. A 0.2-ml volume of each was thenwithdrawn and added to a butyl-rubber-stopperedBellco culture tube (16 by 125 mm) containing 3 mlof liquid citrate agar at 50'C. Dilution series weremade in liquid citrate agar. The tubes were thenrolled in an ice bath and incubated at 37'C for 48 to72 h. Single colonies were picked with a Pasteurpipette by the method of (15), inoculated into 10 mlof citrate medium, and incubated at 37'C for 24 h.The transfer of single colonies was repeated at leastthree times for each strain.

Sources of strains isolated. The sources of thestrains were as follows: C2 (DSM 614), sewage plantof Davis, Calif.; L6, L9/2. L9/3, and L9/4, mud from

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956 WALTHER, HIPPE, AND GOTTSCHALK

river Leine, Gottingen, Germany; N2, mud from theriver Nab, Regensburg; R8/1 and R8/2, mud from apool near Regensburg; Z2, waste water pool of thesugar factory, Northeim; Clx, ditch mud, Gottingen.

Physiological characterization. Gelatin liquefac-tion, digestion of meat and milk, hydrolysis of escu-lin, lipase and lecitinase activity, and the formationof indole were tested by the methods of Holdemanand Moore (12). H2S production (lead acetate papermethod) was examined in citrate medium supple-mented with 1% casein. Reduction of nitrate wasdetermined as described by Skerman (30).

Analytical procedures. Enzymatic methods wereemployed for quantitative determinations of ethanol(2), citrate (24), and acetate (13). Quantitative de-terminations of the fermentation products (see Ta-ble 1) were carried out by using gas chromatographywith Chromosorb G AW DMCS (60/80 mesh) plus 5%silicon gum rubber UC1 W-982. A Perkin-Elmermodel 3920 gas chromatograph equipped with aflame ionization detector was used.Growth experiments were carried out in Bellco

tubes with 10 ml of medium or in 5-liter flasks.Turbidity was measured with a Zeiss PM 4 spectro-photometer at 600 nm in cuvettes with 1-cm lightpath.

Fermentation balances. To determine the prod-ucts formed, a fermentation train was used resem-bling the one described by Dawes et al. (7). Cellsfrom the early stationary phase of growth were har-vested, washed with oxygen-free 0.1 M potassiumphosphate buffer (pH 6.9), and suspended in 5 ml of0.1 M potassium phosphate buffer (pH 6.6). Thefermentation train was carefully flushed with nitro-gen; 30 ml of the above buffer and 5 ml of cellsuspension (0.4 g [wet weight]/ml) were added to thefermentation vessel. After incubation of the cell sus-pension for 10 min with stirring, fermentation wasstarted by addition of 1 mmol of citrate in 10 ml ofwater. The suspension was incubated for 4 to 6 h at40°C, and the fermentation was terminated by theaddition of 0.5 ml of phosphoric acid. During thefermentation and for 30 min thereafter, a stream ofnitrogen was allowed to pass slowly through theapparatus. CO2 and H2 were determined as de-scribed by Dawes et al. (7).DNA base composition. Citrate-grown cells were

harvested at the end of the logarithmic growthphase and washed with a solution containing 0.15 MNaCl and 0.1 M ethylenediaminetetraacetate, pH8.0. Isolation and purification of deoxyribonucleicacid (DNA) were carried out by the method of Mar-mur (22). The guanine-plus-cytosine (G+C) contentof the DNA was determined by the thermal denatur-ation method (21). Determination was carried outwith a Pye Unicam SP 1800 spectrometer with anautomatic cuvette changer. The temperature wasraised at a rate of 30°C per h (Lauda U3-S15 thermo-stat coupled to a Lauda P 120 linear temperatureprogrammer). The temperature was measured di-rectly in the DNA sample cuvettes with Pt 100-resistant sensors and a Doric digital thermometer.Absorbance and temperature were read every min-ute and printed by a PA BCD-Moduprint. The melt-ing temperature was determined graphically after

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correction of absorbance values for thermal solventexpansion. Calf thymus and Bacillus laterosporus(DSM 25) DNAs were used as references. The G+Ccontent was calculated from the melting tempera-ture by the method of Marmur and Doty (23).

Synthesis and fermentation of [14C]citrate sam-ples. [4-14C]citrate and [5-14C]citrate were synthe-sized from [2-'4C]- and [1-14C]acetyl phosphate, re-spectively. The reaction mixture contained in 1 ml:0.1 M potassium phosphate buffer (pH 8.0); lithium[14C]acetyl phosphate (ca. 106 cpm), 1 mM sodiumoxaloacetate, 0.4 mM coenzyme A, 50 U of phospho-transacetylase, and 4.4 U of citrate synthase. [1-'4C]citrate and [6-14C]citrate were synthesized from[4-'4C]- and [1-_4C]aspartate, respectively. The reac-tion mixture contained in a volume of 2 ml: 22 mMtris(hydroxymethyl)aminomethane-hydrochloridebuffer (pH 8.0), 6 mM lithium acetyl phosphate, 1mM sodium a-ketoglutarate, 50 U of phosphotrans-acetylase, 4 U of glutamate oxaloacetate trans-aminase, 4.4 U of citrate synthase, 0.5 mM coen-zyme A, and ['4C]aspartate (ca. 2.2 x 106 cpm). Thereaction mixtures were incubated for 30 min at roomtemperature. The reaction was stopped by heatingthe solutions in a boiling-water bath for 3 min.Radioactive citrate was isolated by chromatographyon Dowex 1-formate by the method of Gottschalkand Barker (10) and purified by paper chromatog-raphy in the butanol-(2)-formic acid system ofHirsch (11).

Fermentation of the radioactive citrate samplesby resting cells of C. sphenoides C2 was determinedby using the fermentation apparatus describedabove. The amounts of cells used and of citrateadded were the same as in the determination offermentation balances. After the fermentation wasterminated, the cells were removed by centrifuga-tion, and a portion of the supernatant was applied toa Dowex 1-formate column (75 by 8 mm). Ethanolwas eluted with 3 column volumes of water, andacetate was eluted with the same volume of 0.2 Mformic acid. The remaining ['4C]citrate was elutedwith 4 M formic acid. The eluate was collected in 1-ml fractions, which were counted in Unisolve 1 in aliquid scintillation spectrometer (Packard Tri-Carb,model 3375). To determine the radioactivity presentin CO2, a portion of the NaOH from the C02-collect-ing unit was transferred to a Warburg vessel. CO2was liberated by the addition of phosphoric acid andadsorbed by a 1 M solution of hyamine hydroxydepresent in the central well. This solution was trans-ferred to vials and counted in Unisolve 1.Enzymes and chemicals. .Citrate synthase (110 U/

mg), citrate lyase (8 U/mg), malate dehydrogenase(1,200 U/mg), glutamate oxaloacetate transaminase(200 U/mg), phosphotransacetylase (1,000 U/mg),acetate kinase (200 U/mg), ethanol dehydrogenase(300 U/mg), lithium acetyl phosphate, and oxaloace-tate were purchased from Boehringer, Mannheim,Germany. DL-[4-'4C]aspartate, DL- [ 1-_4C]aspartate,and [1-_4C]acetic anhydride were obtained from theRadiochemical Centre, Amersham, England, and[2-14C]acetic anhydride was from New England Nu-clear Corp., Boston, Mass. Lithium ['4C]acetyl phos-phate was synthesized from ['4C]acetic anhydride by

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the method of Kornberg et al. (18).

RESULTSA number of citrate-fermenting sporeformers

were isolated from various mud samples by theHungate technique. When these strains werecharacterized by the methods of Holdeman andMoore (12), it became evident that they closelyresembled one another (Table 1). Except forstrains R8/1 and R8/2, they fermented all of thecarbohydrates tested as substrates. None of thestrains was able to liquify gelatin, degrademeat, or reduce nitrate; all strains formed H2S.Glucose was fermented to acetate and ethanolas the principle nongaseous products. Formatewas formed in small amounts. Trace amountsof butyrate were detectable in the fermentationbroth of only one strain. These results indicatedthat all of the strains isolated were closely re-

lated to C. sphenoides. In Table 1 the physio-logical properties of the reference strain NCIB10627 and the species description from Bergey'sManual are given for comparison.The G+C content of the DNA from four

strains was determined (Table 1). The resultswere in agreement with the value for C. sphe-noides, 41 mol% G+C (17). Because of similar-ity in phenotypic characteristics and G+C con-tent, these four strains, and probably the othersisolated, can be regarded as belonging to the

FERMENTATION OF CITRATE 957

above-mentioned species. Interestingly, itseems that citrate was a rather specific sub-strate for the isolation of C. sphenoides. This isalso suggested from the experiments summa-rized in Table 2. Thirty-nine strains of 16 clos-tridial species were tested for their ability togrow with citrate; they included five additionalstrains of C. sphenoides. To exclude possibleinhibitory effects of high citrate concentrations(16), the growth experiments were carried outat three different substrate concentrations. Allfive C. sphenoides strains grew with citrate(Table 2), although these strains were origi-nally isolated in media not containing citrate.Of the other strains tested, only one C. sporo-genes strain utilized citrate.

Cells of the isolated strains of C. sphenoideswere peritrichous flagellated. Figure 1 shows anegatively stained cell of strain C2. Cells of C.sphenoides grown either on citrate or on glu-cose frequently showed asymmetric cell divi-sion (Fig. 2). With glucose as the substrate, thecells were normally larger than with citrate.When the latter substrate was employed, ahigh percentage of sporulated cells could beseen; many of them were wedge-shaped, whichis typical for C. sphenoides (Fig. 2D).

In the medium employed, growth of C.sphenoides was dependent upon the presence ofcitrate; only slight growth occurred in its ab-

TABLE 1. Physiological characteristics of C. sphenoides a strains

Determina-NCIB ~~~~~~~~~~~~~~~~~~~SpeciesDetermina- NCIB C2 L6 L9/2 L9/3 L9/4 N2 R8/1 R8/2 Z2 Clx descrip-tion (29)

pyb _

Sucrose a a a a a a a w w a a +Glucose a a a a a a a w w a a +Maltose a a a a a a a w - a a +Fructose a a a a a a a w - a w +Lactose w a a a a a w - - w w vMannitol w a w w a a a a w a a +Mannose a a a a a a a a a a a +Xylose a a a a a a a - - a a +Gelatin°bIndole + + + + + + + + + + + +MeatbH2S + + + + + + + + + + + +Esculin hy- + + + + + + + + + + +

drolysisbStarch pH - - - - - - - - - - wN03 reduc- - - - - - - - - - - - v

tionLipaseb NT - - NT NT NT NT - NT - - -Lecithinaseb NT - - NT NT NT NT - NT - - -Milkb c, g, a c, g, a g, a g, w g, w g, w NT g, w g, w g, w c, g, a c or -G+C content NT 41.55 41.22 NT NT NT 41.15 41.0 NT NT NT 41.0Products from A2f A2F A2f A2f A2f A2f A2f A2f A2f A2F A2F A23PYGb (3) (ic) (ic) (b, ic)a Abbreviations: A, acetic acid (concentration, >10 mM); a, acid (pH below 5.5); b, butyric acid; c, curd of milk; F, formic

acid (concentration, >10 mM); f, formic acid (concentration, <10 mM); g, gas; ic, isocaproic acid; v, variable; w, weak acid(pH 5.6 to 6.0); -, no acid or negative reaction; 2, ethanol; 3, propanol; NT, not tested; parentheses, trace amounts.

bMedium as described by Holdeman and Moore (12).

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958 WALTHER, HIPPE, AND GOTTSCHALK APPL. ENVIRON. MICROBIOL.

TABLE 2. Utilization of citrate by clostridia a

Citrate utilization at a concn

Species Strains (mM) of:47.5 17 3.4

C. bifermentans DSM 630, DSM 631 NT - -C. butyricum W100, W3a, Lille, S10, SB55 NT - -

C. cochlearium DSM 666, DSM 667 NT - -

C. histolyticum DSM 627 NT - -

C. lentoputrescens 6391 NT - -

C. paraputrificum DSM 661 NTC. pasteurianum DSM 525C. perfringens DSM 628, DSM 629 NTC. putrificum Lille, 13/7 NTC. roseum DSM 51C. rubrum DSM 53C. sphenoides NCIB 10627, S265, H18b, W2, SM4, Zil5 + NT NTC. sporogenes DSM 633, DSM 634, C39, Zi2O, Zi2l - NT

C31 + NT NTC. tertium DSM 663, Z22, Jl NTC. tetanomorphum DSM 665 NT

DSM 528 - NT NTC. tyrobutyricum DSM 663, DSM 664, W4,B NT

a The strains without a culture collection number were obtained from H. J. Kutzner, Darmstadt,Germany. DSM, German Collection of Microorganisms, Gottingen; NCIB, National Collection of IndustrialBacteria, Aberdeen. NT, Not tested.

t~~~~~~~~~~~~~~

1pm

;t b ¢ e *f

-l.I+3'". ,W4m ,~WeaFIG. 1. Negatively stained cell of C. sphenoides C2. Staining was done with 4% uranyl acetate, and the

micrograph was taken in a Philipps EM 301 electron microscope (Philipps, Eindhoven).

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FERMENTATION OF CITRATE 959

FIG. 2. Photomicrographs of C. sphenoides C2 cells grown on glucose for 22 h (A) and 40 h (B) and oncitrate for 22 h (C) and 40 h (D). Arrows indicate asymmetric cell divisions.

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960 WALTHER, HIPPE, AND GOTTSCHALK

sence (Fig. 3). For strain C2, optimum sub-strate concentrations were between 40 and 60mM. Higher citrate concentrations were inhibi-tory.

Table 3 gives the amounts of products formedper millimole of citrate fermented. Acetate andcarbon dioxide were the main products, butethanol and molecular hydrogen were alsoformed.The key enzyme of the anaerobic breakdown

of citrate is citrate lyase. Its stereospecificity issuch that C4 and C5 of citrate yield acetate andthat Cl and C6 become the carboxyl groups ofoxaloacetate (Fig. 4).The further degradation of oxaloacetate to

the C2 compounds ethanol and acetate shouldlead to the formation of CO2 originating fromCl and C6 of citrate. When [14C]citrate samples

1 E60 ~e c

-, ~0

0 10 20 30t;me~~~Q(h)

45C

CO'C30-0 ~~~~~~~0.1M

0

15/ ~~~~~~~0.05

0 10 20 30time (h)

FIG. 3. Growth on C. sphenoides in the presenceand absence of citrate. Symbols: *, increase of opti-cal density in medium with 55 mM citrate; 0, de-crease of citrate concentration; A, increase of opticaldensity in medium without citrate.

TABLE 3. Fermentation of citrate by C. sphenoidesC2a

Substrate and product mmol consumed orformed

Citrate......... 1Acetate .......... 1.67Ethanol ......... 0.36CO2-.............................. 2.03H2 ........... 0.46

C recovery (%) ... ...... 102O/R balance ... ...... 1.03

labeled in positions 1, 4, 5, and 6, respectively,were fermented by cell suspensions of C. sphe-noides C2, the labeling of the fermentationproducts was in accordance with the schemeshown in Fig. 4 (Table 4). Thus, it appearedthat citrate lyase is involved in the degradationof citrate by C. sphenoides and, although it isvery unstable, the enzyme can indeed be de-tected in cell extracts of this organism.

DISCUSSIONIn 1920, C. sphenoides was isolated from

wounds by Douglas et al. (8). Since then, thisorganism has been included in a number ofcomparative studies of clostridial species (9, 12,17, 26, 27, 29, 31, 33). Apart from these taxo-nomic investigations, C. sphenoides has not, toour knowledge, been the subject offurther stud-ies. Similarly, the fermentation of citrate byanaerobic sporeformers has not been studied.The results presented in this publication

demonstrate that the ability to ferment citrateis not common to clostridia, in that a number ofsaccharolytic species tested was unable to do so.When citrate-fermenting sporeformers wereisolated by the direct method as described, e.g.,without enrichment procedures, all strains ob-tained belonged to one species, C. sphenoides.Thus, it appears that of the specimens studiedC. sphenoides was the most common anaerobicsporeformer able to utilize citrate as an energysource. Moreover, all of the C. sphenoidesstrains obtained from the culture collectionswere able to grow with citrate. Since thesestrains were isolated and cultivated in mediathat did not contain citrate, it appears that theability to grow on citrate is a distinct propertyof C. sphenoides. Consequently, the citratemedium employed here can be recommendedfor the selective enrichment and isolation of

4 5CH2-COOH

3 6HOo-C4COOH-

CH2-COOH2 1

4 5CH3-COOH

3 6OC-COOH

CH2-COOH2 1

FIG. 4. Stereospecific breakdown of citrate intoacetate and oxaloacetate.

a The fermentation was carried out as describedin the text. The data are mean values from fourexperiments.

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FERMENTATION OF CITRATE 961

TABLE 4. Distribution of radioactivity in fermentation products after fermentation of "'C-citrate samples byresting cells of C. sphenoides C2

Radioactivity in products formed from ['4C]citrateSubstrate and [4-'4C]citrate [5-'4C]citrate [1-'4C]citrate [6-'4C]citrate

product ____________________________________________cpm % cpm % cpm % cpm %

Citrate 255,000 100 208,600 100 209,280 100 369,110 100Acetate 272,765 107 199,409 95.6 6,097 2.9 8,277 2.2Ethanol 22,342 8.7 9,289 4.4 3,675 1.8 6,741 1.8CO2 7,630 2.9 788 0.37 185,115 88.5 337,067 91.3

1 acetate]1 citrate

1 oxaloacetate

1 pyruvate |1 C02

i1 C02

(2 H) 10.4 H2

acetyl-CoA 0.3 ethanol

1p0.7 acetate|

FIG. 5. Pathway used by C. sphenoides for thefermentation of citrate. CoA, Coenzyme A.

this organism.A similar situation exists within the Rhodo-

spirillaceae; as far as known only Rhodopseu-domonas gelatinosa is able to degrade citraterapidly (28, 32), and the isolation of photo-trophic bacteria using citrate as a carbon source

usually yields R. gelatinosa. Anaerobic growthwith citrate is much more common among en-

terobacteria and lactic acid bacteria.Citrate is fermented by C. sphenoides to ace-

tate, carbon dioxide, ethanol, and molecularhydrogen. The labeling experiments performedindicate that citrate lyase is involved in citratedegradation. These results are in agreementwith the fermentation scheme depicted in Fig.5.

ACKNOWLEDGMENTSThis work was initiated when G. G. was a guest in R. E.

Hungate's laboratory in the Department of Bacteriology,University of California, Davis. The advice and the gener-osity of R. E. Hungate are gratefully acknowledged. We are

indebted to Dietmar Vollbrecht (Gottingen) for performing

the gas chromatographic analyzes and to Anna Walther-Mauruschat (G6ttingen) for taking the electron micro-graph.

The financial support of the National Science Founda-tion (Senior Foreign Scientist Fellowship to G. G.), theDeutsche Forschungsgemeinschaft, and Forschungsmitteldes Landes Niedersachsen is gratefully acknowledged.

LITERATURE CITED

1. Andreesen, J. R., G. Gottschalk, and H. G. Schlegel.1970. Clostridium formicoaceticum, nov. spec. Isola-tion, description, and distinction from Clostridiumaceticum and Clostridium thermoaceticum. Arch.Mikrobiol. 72:154-174.

2. Bernt, E., and I. Gutmann. 1970. Athanol. Bestim-mung mit Alkohol-Dehydrogenase und NAD, p.1457-1460. In H. U. Bergmeyer (ed.), Methoden derenzymatischen Analyse, 2nd ed. Verlag ChemieGmbH, Weinheim, Germany.

3. Bhat, J. V., and H. A. Barker. 1947. Clostridium lacto-acetophilum nov. spec. and the role of acetic acid inthe butyric acid fermentation of lactate. J. Bacteriol.54:381-391.

4. Bornstein, B. T., and H. A. Barker. 1948. The nutritionof Clostridium kluyveri. J. Bacteriol. 55:223-230.

5. Cardon, B. P., and H. A. Barker. 1946. Two new amino-acid-fermenting bacteria, Clostridium propionicumand Diplococcus glycinophilus. J. Bacteriol. 52:629-634.

6. Cohen, G. N., and G. Cohen-Bazire. 1948. Fermenta-tion of pyruvate, /3-hydroxy butyrate and of C4-dicar-boxylic acids by some butyric acid-forming orga-nisms. Nature (London) 162:578.

7. Dawes, E. A., D. J. McGill, and M. Midgeley. 1971.Analysis of fermentation products, p. 53-215. In J. R.Norris and D. W. Ribbons (ed.), Methods in microbi-ology, vol. 6A. Academic Press Inc., New York.

8. Douglas, S. R., A. Fleming, and L. Colebrook. 1920.Studies in wound infections. Med. Res. Counc. (G.B.)Spec. Rep. Ser. 57:1-159.

9. Gibson, T. 1965. Clostridia in silage. J. Appl. Bacteriol.28:56-62.

10. Gottschalk, G., and H. A. Barker. 1967. Presence andstereospecificity of citrate synthase in anaerobic bac-teria. Biochemistry 6:1027-1034.

11. Hirsch, P. 1963. CO2-Fixierung durch Knallgasbakter-ien. II. Chromatographischer Nachweis der fruhzeiti-gen Fixierungsprodukte. Arch. Mikrobiol. 46:53-70.

12. Holdeman, L. V., and W. E. C. Moore. 1973. Anaerobelaboratory manual, 2nd ed. Virginia Polytechnic In-stitute and State University, Blacksburg.

13. Holz, G., and H. U. Bergmeyer. 1974. Acetat. Bestim-mung mit Acetatkinase und Hydroxylamin, p. 1574-1578. In H. U. Bergmeyer (ed.), Methoden der enzy-matischen Analyse, 3rd ed. Verlag Chemie GmbH,Weinheim, Germany.

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14. Hugo, H. v., S. Schoberth. V. K. Madan, and G. Gott-schalk. 1972. Coenzyme specificity of dehydrogenasesand fermentation of pyruvate by clostridia. Arch.Mikrobiol. 87:189-202.

15. Hungate, R. E. 1969. A roll tube method for cultivationof strict anaerobes, p. 117-132. In J. R. Norris and D.W. Ribbons (ed.), Methods in microbiology, vol. 3B.Academic Press Inc., New York.

16. Imai, K., I. Banno, and T. lijima. 1970. Inhibition ofbacterial growth by citrate. J. Gen. Appl. Microbiol.16:479-487.

17. Johnson, J. L., and B. S. Francis. 1975. Taxonomy ofthe clostridia: ribosomal ribonucleic acid homologiesamong the species. J. Gen. Microbiol. 88:229-244.

18. Kornberg, A., S. R. Kornberg, and E. S. Simms. 1956.Metaphosphate synthesis by an enzyme from Esche-richia coli. Biochim. Biophys. Acta 20:215-227.

19. Langlykke, A. F., N. H. Peterson, and E. B. Fred.1937. Reductive processes of Clostridium butylicumand the mechanism of formation of isopropyl alcohol.J. Bacteriol. 34:443-453.

20. Lee, C. K., and Z. J. Ordal. 1967. Regulatory effect ofpyruvate on the glucose metabolism of Clostridiumthermosaccharolyticum. J. Bacteriol. 94:530-536.

21. Mandel, M., and J. Marmur. 1968. Use of ultravioletabsorbance-temperature profile for determining theguanine plus cytosine content of deoxyribonucleicacid, p. 195-206. In L. Grossman and K. Moldave(ed.), Methods in enzymology, vol. 12B. AcademicPress Inc., New York.

22. Marmur, J. 1961. A procedure for the isolation of deoxy-ribonucleic acid from micro-organisms. J. Mol. Biol.3:208-218.

23. Marmur, J., and P. Doty. 1962. Determination of the

APPL. ENVIRON. MICROBIOL.

base composition of deoxyribonucleic acid from itsthermal denaturation temperature. J. Mol. Biol.5:109-118.

24. Mollering, H., and W. Gruber. 1966. Determination ofcitrate with citrate lyase. Anal. Biochem. 17:369-378.

25. Mercer, W. A., and R. H. Vaughn. 1951. The character-istics of some thermophilic, tartrate-fermenting an-aerobes. J. Bacteriol. 62:27-37.

26. Reed, R. W. 1942. Nitrate, nitrite and indole reactionsof gas gangrene anaerobes. J. Bacteriol. 44:425-431.

27. Reed, G. B., and J. H. Orr. 1941. Rapid identification ofgas gangrene anaerobes. War Med. 1:493-510.

28. Schaab, Ch., F. Giffhorn, S. Schoberth, N. Pfennig,and G. Gottschalk. 1972. Phototrophic growth ofRho-dopseudomonas gelatinosa on citrate; accumulationand subsequent utilization of cleavage products. Z.Naturforsch. Teil B 27:962-967.

29. Smith, L. DS., and G. Hobbs. 1974. Clostridium, p. 551-572. In R. E. Buchanan and N. E. Gibbon (ed.),Bergey's manual of determinative bacteriology, 8thed. The Williams & Wilkins Co., Baltimore.

30. Skerman, V. B. B. 1967. A guide to the identification ofthe genera of bacteria, 2nd ed. The Williams & Wil-kins Co., Baltimore.

31. Spray, R. S. 1936. Semisolid media for cultivation andidentification of the sporulating anaerobes. J. Bacte-riol. 32:135-155.

32. Weckesser, J., G. Drews, and H. D. Tauschel. 1968. ZurFeinstruktur und Taxonomie von Rhodopseudomo-nas gelatinosa. Arch. Mikrobiol. 65:346-358.

33. Willis, A. T., and G. Hobbs. 1959. Some new media forthe isolation and identification of clostridia. J. Pa-thol. Bacteriol. 77:511-521.

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