APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1986, p. 623-6300099-2240/86/100623-08$02.00/0Copyright C 1986, American Society for Microbiology

Acetate Synthesis from H2 plus CO2 by Termite Gut MicrobestJOHN A. BREZNAK* AND JODI M. SWITZER

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824-1101

Received 8 May 1986/Accepted 14 June 1986

Gut microbiota from Reticuliternes flavipes termites catalyzed an H2-dependent total synthesis of acetatefrom CO2. Rates of H2-CO2 acetogenesis in vitro were 1.11 ± 0.37 ,umol of acetate g (fresh weight)-' h-'(equivalent to 4.44 ± 1.47 nmol termite-1 h-1) and could account for approximately 1/3 of all the acetateproduced during the hindgut fermentation. Formate was also produced from H2 + C02, as were small amountsof propionate, butyrate, and lactate-succinate. However, H2-CO2 formicogenesis seemed largely unrelated toacetogenesis and was believed not to be a significant reaction in situ. Little or no CH4 was formed from H2 +CO2 or from acetate. H2-CO2 acetogenesis was inhibited by 02, KCN, CHCl3, and iodopropane and could beabolished by prefeeding R. flavipes with antibacterial drugs. By contrast, prefeeding R. flavipes with starchresulted in almost complete defaunation but had little effect on H2-CO2 acetogenesis, suggesting that bacteriawere the acetogenic agents in the gut. H2-CO2 acetogenesis was also observed with gut microbiota fromProrhinotermes simplex, Zootermopsis angusticollis, Nasutitermes costalis, and N. nigriceps; from the wood-eating cockroach Cryptocercus punctulatus; and from the American cockroach Periplaneta americana. Purecultures of H2-CO2-acetogenic bacteria were isolated from N. nigriceps, and a preliminary account of theirmorphological and physiological properties is presented. Results indicate that in termites, CO2 reduction toacetate, rather than to CH4, represents the main electron sink reaction of the hindgut fermentation and canprovide the insects with a significant fraction (ca. 1/3) of their principal oxidizable energy source, acetate.

Acetate is a metabolite central to the symbiosis betweenwood-eating termites and their intestinal microbiota. It is amajor end product of microbial fermentation in the gut (12)and is an important oxidizable energy source andbiosynthetic precursor for the termites (1, 2, 9, 10, 12, 18). InReticulitermes flavipes termites, acetate production by thehindgut microbiota supports 77 to 100% of the respiratoryrequirement of the insect (12).

Following a study of volatile fatty acid production andutilization in R. flavipes, Odelson and Breznak (12) proposedthat the hindgut fermentation could be viewed essentially asa homoacetic fermentation of cellulose (Table 1). The fer-mentation begins with hydrolysis of cellulose and fermenta-tion of the glucosyl units to 2 acetate + 2 CO2 + 4 H2 byanaerobic, cellulolytic protozoa (reaction A). The 4 H2 + 2CO2 are then presumably consumed by anaerobic acetogenicbacteria to form a third acetate molecule (reaction B). Thethree net acetates are finally taken up from the hindgut andoxidized aerobically by termite tissues to 6 CO2 + 6 H20(reaction C). Although CH4 and H2 are also end products ofthe fermentation and can be easily detected as emissionsfrom live R.flavipes specimens, the amount of these gases isnegligible on a per mole of glucose fermented basis (12).There is experimental support for reactions A (12-14,26,

27) and C (10,12) of Table 1. However, a critical butunconfirmed element of this scheme was reaction B. Accord-ingly, the present study was undertaken. In this paper, wepresent evidence that reaction B occurs in the hindgut ofboth lower and higher termites, that it is carried out bybacteria, and that it outcompetes methanogenesis as themajor electron sink reaction of the hindgut fermentation.(A preliminary report of these findings has been presented

[J. M. Switzer and John A. Breznak, Abstr. Annu. Meet.Am. Soc. Microbiol. 1985, 1108, p. 164]).

* Corresponding author.t Journal article no. 11974 from the Michigan Agricultural Exper-

iment Station.

MATERIALS AND METHODS

Insects and rumen fluid. R. flavipes (Kollar) (Rhino-termitidae) specimens were collected from Dansville andSpring Arbor, Mich., and Janesville, Wis. Termites wereused within 2 h of collection or were maintained in labora-tory culture (12) and used as needed. Prorhinotermessimplex (Hagen) (Rhinotermitidae) specimens were obtainedfrom G. D. Prestwich, State University of New York, StonyBrook. Zootermopsis angusticollis (Hagen) (Hodotermi-tidae) specimens were obtained from A. Stuart, Universityof Massachusetts, Amherst. Nasutitermes costalis(Holmgren) (Termitidae) specimens were obtained from J. F.Traniello, Boston University, Boston, Mass., and N.nigriceps (Holdeman) (Termitidae) specimens were obtainedfrom B. Thorne, Harvard University, Cambridge, Mass. Thelast four species were from laboratory cultures and wereused within 72 h of receipt. Worker termites (i.e., externallyundifferentiated larvae beyond the third instar) were used forall experiments.Specimens of the wood-eating cockroach Cryptocercus

punctulatus Scudder (Cryptocercidae) were collected byD. G. Cochran, Virginia Polytechnic Institute and StateUniversity, Blacksburg, and used within 48 h of receipt.Specimens of the American cockroach Periplaneta ameri-cana L. (Blattidae) were obtained from the Pesticide Re-search Center, Michigan State University, where they hadbeen fed Purina dog chow and water ad libitum.Rumen fluid was obtained from a fistulated dairy cow at

Michigan State University. The fluid was chilled to 4°Cduring transport to the laboratory and was filtered throughthree layers of cheesecloth before being used (within 1 h ofcollection).

14CO2 fixation-reduction assay. Termites were introducedinto an anaerobic glove box (Coy Laboratory Products Inc.,Ann Arbor, Mich.) containing an atmosphere of 90%N2-10% H2 and were degutted with forceps. A total of 10 to50 guts were pooled in a small glass tissue homogenizer tube

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624 BREZNAK AND SWITZER

TABLE 1. General scheme for the symbiotic utilization ofcellulose (nC6H1206) by R. flavipes termitesa

Designa- Reaction Organismtion

A nC6H1206 + 2nH20 -* 2nCH3COOH Hindgut protozoa+ 2nCO2 + 4nH2

B 4nH2 + 2nCO2 -* nCH3COOH + Hindgut bacteria2nH20

C 3nCH3COOH + 6nO2 -* 6nCO2 + R. flavipes6nH20

Net nC6H1206 + 6nO2 -. 6nCO2 + Combined system6nH20

a Adapted from Odelson and Breznak (12).

containing 2.0 ml of buffered salts solution (BSS). BSS (pH7.2) contained 10.8 mM K2HPO4, 6.9 mM KH2PO4, 21.5 mMKCl, 24.5 mM NaCl, and 1.0 mM dithiothreitol. The gutsuspension was homogenized for 1 min, and 0.2 ml of thehomogenate was then added to each of a number of 5-mlglass serum vials (no. 223738; Wheaton Scientific, Millville,N.J.) followed by another 0.2 ml of BSS. The vials were thensealed with butyl rubber stoppers (no. 2048-11800; BellcoGlass, Inc., Vineland, N.J.), fitted with aluminum seals (no.224193; Wheaton), and removed from the glove box. Eachvial was then gassed for 1 min with H2 or N2 by using25-gauge needles as gas entry and exit ports. Reactions werebegun by injecting 0.1 ml of 11 to 12 mM NaH14CO3 preparedin 0.1 M potassium phosphate buffer (pH 7.5). This resultedin a final reaction mixture of 0.5 ml at pH 7.3 ± 0.1 and under1 atm (101.29 kPa) pressure. The exact specific activity ofNaH"4CO3 stock solutions was determined before use butwas approximately 103 dpm/nmol. Vials were incubatedupright at 30°C in a water-bath shaker operating at 200 rpm.

Reactions were terminated by addition of 0.1 ml of 0.05 Macetic acid (carrier) and 0.05 ml of 1 N HCl. After mixing,vials were placed on ice and swept with pure CO2 for 10 min.These steps converted any unreacted H14CO3f to 14CO2,which was flushed into a vented hood. Reaction mixtureswere then brought to pH 7.5 by addition of 0.06 ml of 1 NNaOH and 0.05 ml of 1 M potassium phosphate buffer (pH7.5) and made up to a final volume of 1.0 ml with 0.24 ml ofH2O. Mixtures were then clarified by centrifugation at13,000 x g for 5 min. Duplicate 0.2-ml portions of eachsupernatant fluid were added to scintillation vials for deter-mination of radioactivity; the remainder of the clarifiedsolution was frozen for subsequent analyses.

In the reaction mixtures just described, NaH14CO3 willequilibrate principally as a mixture of H14CO3- and 14CO2.However, in this paper, we will simply use the phrase "CO2fixation" without meaning to prejudge the actual reactivespecies of C1 compound. Moreover, the word fixation refersto the conversion of H14CO3-_14CO2 to an acid-stable, solu-ble (i.e., nongaseous) form of 14C, as would be present insupernatant fluids of clarified reaction mixtures (above).We have termed the conversion of H14CO3-_14CO2 to

14CH4 "14CO2 reduction"; it was determined by sampling theheadspaces of reaction vials (just before termination with acid)and analyzing for '4CH4 as described by Zehnder et al. (29).

Reaction mixtures with cockroach gut homogenates wereset up as described above, except that cockroach guts weredissected out of the insects at the laboratory bench (i.e., inair) and quickly transferred to BSS under N2 and then intothe anaerobic glove box for homogenization and subsequentmanipulation.

'4CO2 reduction to "4CH4 by rumen fluid (control) wasmeasured by using rumen fluid that was undiluted (except bythe addition of NaH14CO3) and that had been incubated at39°C. In addition, a separate large volume of the same rumenfluid sample (33 ml in a 150-ml serum-stoppered bottle) wasincubated without NaH'4CO3 to determine the rate of totalCH4 production by gas chromatography (12).Throughout the above procedures, strict anaerobic condi-

tions were maintained whenever possible (5). Rubber stop-pers, reaction vials, glassware, and plastic ware were nor-mally held overnight in the anaerobic glove box before beingused to manipulate gut homogenates or other reactants.Gases used were passed over hot copper filings before use toremove any traces of 02. Inhibitors (when used) or specialgas mixtures were added to reaction vials before addition ofthe NaH14CO3.

Analysis of 14CO2 fixation products. Portions of clarifiedsupernatant fluids (above), containing known amounts of14C, were amended with nonradioactive (carrier) sodiumformate, sodium acetate, sodium propionate, sodium butyr-ate, and sodium succinate to give a final concentration of 10mM for each acid. They were then filtered through 0.45-,um-pore-size filter units (no. DDNO400300; Fisher ScientificCo., Pittsburgh, Pa.), and samples of the filtrate (25 to 50 ,ul)were injected into a high-performance liquid chromatogra-phy (HPLC) apparatus (Waters Associates, Inc., Milford,Mass.) to separate organic acids. The column used wasAminex HPX-87H (no. 125-1040; Bio-Rad Laboratories,Rockville Center, N.Y.), and elution was isocratic with0.013 N H2SO4 at ambient temperature. The flow rate was0.6 ml/min at 1,500 lb/in2, and detection was by a Watersrefractometer interfaced with an HP 3390A reporting inte-grator (Hewlett-Packard Co., Palo Alto, Calif.). Fractionseluting from the chromatograph were collected dropwiseinto scintillation vials containing 0.05 to 0.2 ml of 0.1 NNaOH and 0.05 to 0.2 ml of 1.0 M potassium phosphatebuffer to neutralize the acids prior to radioactivity determi-nation. This HPLC system yielded excellent separation oforganic acids, with less than 1% cross-contamination and100% recovery of standard 14C-organic acid mixtures.

Characterization of labeled acetate. Samples of 14C-labeledacetate that had been purified by HPLC of an R. flavipesreaction mixture were pooled and amended with additionalsodium acetate carrier to yield a solution 40 to 50 mM inacetate and 6,000 to 12,000 dpm/ml. This was used for etherextraction and for Schmidt degradation.For ether extraction, one 1-ml portion was acidified with

0.1 ml of 18 N H2SO4, whereas another was made alkalinewith 0.1 ml of 5 N NaOH. Each sample was then extractedthree times by vigorous shaking with 1.0-ml portions ofdiethyl ether. Ether phases were pooled over 1.0 ml of 0.1 Mpotassium phosphate buffer (pH 7.5). The ether was thenevaporated by a stream of N2, and the radioactivity presentin the remaining buffer solution was determined. Samples ofstandard sodium [1-14C]acetate (50 mM; 60,334 dpm/ml)were used as controls for the ether extraction procedure.To determine the position of 14C-label in acetate, samples

were subjected to a Schmidt degradation as described byPhares (15), but with the following exceptions. Acetatesamples for degradation were ca. 40 p.mol each, the reactionvessel was an 18-mm serum stoppered tube, and the liber-ated 14CO2 was swept into tubes containing a CO2 trappingsolution of phenethylamine-methanol (1/1, vol/vol). Controlexperiments were performed with sodium [1-14C]acetate andsodium [2-14C]acetate.To produce 13C-labeled products of 13CO2 fixation, reac-

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H,-CO2 ACETOGENESIS IN TERMITES 625

tions were carried out under H2 as described above, exceptthat the reaction system was scaled up 10-fold, guthomogenates of R. flavipes were first centrifuged at 13,000 xg for 10 min and then suspended in fresh BSS (to reduce thebackground level of 12C-acetate species), and the source of13C°2 was a 10 mM solution of Na213CO3 (90.7% atomenriched) prepared in 0.1 M potassium phosphate buffer (pH7.5). After termination of reactions and clarification, themixtures were filtered as described above and lyophilized,and the dried residue was dissolved in a small volume of99.97% D20. Nuclear magnetic resonance (NMR) spectra ofthe sample were then determined by staff at the NMRFacility, Chemistry Department, Michigan State University.

Feeding experiments. Incubation vessels and conditionswere similar to those described previously (17). Antibioticsapplied to cellulose tablets were 200 pug each of penicillin G,tetracycline, and chloramphenicol (pH 7.0). Starch was fedto termites as ca. 200-mg nuggets of Argo gloss laundrystarch (Best Foods, Englewood Cliffs, N.J.). Nuggets werereplaced periodically to limit the extent of microbial growthon them.

Radioactivity determination. Determination of radioactiv-ity and quench corrections were made as previously de-scribed (21). The scintillation cocktail was ACS countingsolution (Amersham Corp., Arlington Heights, Ill.).

Isolation of bacteria. Serial 10-fold dilutions of termite guthomogenate in BSS were inoculated (2% inoculum) into18-mm serum-stoppered tubes containing 5.0 ml of anaerobicAC-11 medium. Tubes were incubated upright, in a station-ary position, at 30°C under 2 atm of H2-CO2 (80/20, vol/vol)or N2-CO2 (controls). AC-11 medium contained 7.5 mMpotassium phosphate buffer (pH 7.4), 9.4 mM NH4Cl, 2.4mM NaCl, 2.2 mM KCl, 0.08 mM MgSO4, 0.07 mM CaCI2,3 x 10-4 mM Na2WO4, 10 ml of mineral solution per liter(modified from that described in reference 25 by the inclu-sion of 0.01 g each of NiCl2 6H20 and Na2SeO3 per liter), 5ml of vitamin solution (25) per liter, 0.5 g of yeast extract perliter, 10-4 g of resazurin per liter, and 71 mM NaHCO3(autoclaved separately). Before inoculation, an aqueoussuspension of PdCl2 was added to a final concentration of 330mg/liter. The final pH of this medium was 7.4 + 0.1. Fromthe highest-dilution tube showing H2-dependent growth andacetate production, roll tubes (5) were prepared by usingAC-11 medium solidified with 2% agar. Isolated colonieswere picked, transferred to AC-11 broth, and again tested forH2-dependent growth and acetate production. Cultures wereconsidered pure after three successive passages had beenmade in roll tubes, and microscopic examination confirmedthe presence of a single morphological type. When purecultures were obtained, the PdCl2 was replaced with dithio-threitol (final concentration, 1 mM).

Acetate production by broth cultures was determined bygas chromatography as previously described (12), exceptthat SP-1220 (Supelco, Bellefonte, Pa.) was the column-packing material. Culture fluids were first clarified by cen-trifugation at 13,000 x g for 10 min, and the supernatant fluidwas acidified with 8.5% H3PO4 (final concentration) beforeinjection into the gas chromatograph.

Chemicals. Unless noted otherwise, all chemicals usedwere reagent grade and were purchased from commercialsources. 14C-labeled radioactive chemicals were obtainedfrom New England Nuclear Corp., Boston, Mass., exceptfor sodium [1-14C]acetate and sodium [2-14C]acetate, whichwere purchased from Pathfinder Laboratories Inc., St.Louis, Mo., and were gifts from J. M. Tiedje. Na213CO3 waspurchased from B.O.C. Limited, London, U.K.

3.01

2.5[O

c]x

a:2.0

CM0

t1.5E

).i.

Q-

0.5

0 1 2 3TIME (hours)

4 5

FIG. 1. Fixation of 14CO2 to acid-stable soluble products by guthomogenates of R. flavipes under 100% H2 or 100%o N2. Reactionmixtures (0.5 ml) contained 5 gut equivalents, 1.16 ,umol ofNaH14CO3 (specific activity, 1.16 x 106 dpm/,umol), and otherreagents as indicated in Materials and Methods. Each point on thecurves represents a separate reaction mixture.

RESULTS

H2-dependent fixation of CO2 by gut homogenates. Underan atmosphere of H2, gut homogenates of R. flavipes fixed14C02 to an acid-stable, soluble, nongaseous form (Fig. 1).The 1'4CO2 fixation activity was linear for 5 h (the maximumtime tested), was proportional to the amount of gut homog-enate used, and could be abolished by exposinghomogenates to 100°C for 10 min (data not shown). A muchlower level of fixation of 14CO2 was obtained under anatmosphere of N2 (Fig. 1; Table 2).The amount of 14CO2 fixed into acid-stable, soluble prod-

ucts by R. flavipes gut homogenates was always greater thanthe amount of 14CO2 reduced to '4CH4 (Table 2). This wasespecially so for reaction mixtures incubated under H2.Interestingly, exogenously added H2 did little or nothing tostimulate 14CO2 reduction to 14CH4. This suggested that theH2-CO2 methanogenic system of R. flavipes gut homoge-nates was already saturated by endogenous H2 production.There were no obvious differences in rates of 14CO2 fixation-reduction by R. flavipes gut homogenates that could becorrelated with the geographical origin of the insects or thelength of their captivity. Consequently, the data reported inTable 1 are grand means + standard errors of the mean forall determinations.

Similar results were obtained in a limited number of assayswith other lower termites (P. simplex, and Z. angusticollis),higher termites (N. costalis and N. nigriceps), a wood-eatingcockroach (C. punctulatus), and the American cockroach(P. americana) (Table 2). However, only in P. americanawas the rate of 14CO2 reduction to 14CH4 higher than the rateof 14Co2 fixation to nongaseous products.To be sure that our assay system was not seriously

underestimating 14CH4 production, a control experiment was

K /0~~~~~~H2

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3.51 1 1 1 t I

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626 BREZNAK AND SWITZER

TABLE 2. Fixation-reduction of "4CO2 by gut homogenates fromtermites and cockroachesa b

Amt (p.mol) of 14Co2 convertedto following product g (fresh

Insect Incubation weight) of insect-' h-1:Nongaseous 14CH"4C-products

TermitesR. flavipes H2 3.88 ± 1.82c 0.23 ± O.ld

N2 0.45 ± 0.24c 0.22 ± 0.92dP. simplex H2 3.42 0.45

N2 1.65 0.13Z. angusticollis H2 0.99 ± 0.75e 0.18

N2 0.21 ± 0.06e 0.11N. costalis H2 16.21 0.49

N2 2.70 0.54N. nigriceps H2 9.62 0.24

N2 2.32 0.29

CockroachesC. punctulatus H2 0.55

H2-CO2 ACETOGENESIS IN TERMITES 627

TABLE 4. Distribution of 14C label in acetate samples

Sample Distribution of 14C labelSample radioac- in dpm (%)ativity

(dpm) CH3 group COOH group

Standard 14CH3COOH 102,067 98,674 (96.7) 1,656 (1.6)Standard CH314C0OH 50,400 636 (1.3) 50,984 (101.2)R. flavipes 14C-acetateExpt 1 11,352 5,710 (50.3) 6,043 (53.2)Expt 2 11,352 6,022 (53.0) 5,958 (52.5)a Values tabulated for R. flavipes samples were corrected for the small

amount of cross-contamination observed with standard acetate samples,which was assumed to result from the degradation procedure rather than fromradiochemical impurity.

formed when R. flavipes gut homogenates were incubatedwith 14CO2 represented a true net synthesis of acetate. Itarose not merely from an exchange between 14CO2 and thecarboxyl or methyl group of acetate molecules present in thegut homogenate. This was verified by control experiments inwhich sodium [U-14C]acetate was used as substrate in thepresence of nonradioactive CO2. Neither 14CO2 nor 14CH4was formed under an atmosphere of H2 or N2. Therefore, itwas of interest to determine which of the carbon atoms ofacetate was derived from CO2. To do this, R. flavipes guthomogenates were incubated under H2 with 13C02 and theclarified reaction fluids were examined by NMR spectros-copy. Proton NMR spectra revealed high-intensity satellitepeaks that were 66 and 60 Hz equidistant upfield anddownfield from a central 12C-'2C acetate peak, indicating thepresence of a doubly labeled '3C-acetate species. Thesepeaks coincided exactly with those of standard sodium[U-13C]acetate, and it was estimated that about 58% of the13C-acetate molecules formed by the gut homogenates weredoubly labeled. Proton NMR spectra also revealed signalsidentical to those of standard 13C-formate, with a couplingconstant of 196 Hz.

In a different approach, 14C-acetate formed by R. flavipesfrom 14CO2 was purified by HPLC, dried, and subjected to aSchmidt degradation. Results indicated that 14C was distrib-uted equally between the methyl and carboxyl carbon atoms(Table 4). Taken together, these data indicate that R. flavipesgut homogenates can effect a total synthesis of acetate fromCO2 + H2 and that the C from CO' can enter both carbonatoms of acetate with equal facility.

Inhibitors of 14CO2 fixation. H2-dependent 14CO2 fixationby R. flavipes gut homogenates was sensitive to oxygen (Fig.2). A 50% inhibition of activity was seen with approximately10-3.25 atm of 02 (57 Pa); complete inhibition occurred at10-2 atm of 02 (1.01 kPa). 14CO2 fixation was also markedlyinhibited by 5 mM KCN and 50 ,uM CHC13, the lattercompound exerting a preferential inhibition of acetogenesisfrom 14CO2 compared with formicogenesis (Table 5). Bycontrast, another alkylhalide, iodopropane, was only mod-erately inhibitory at 1 mM and affected formicogenesis andacetogenesis equally (ca. 15 to 16% inhibition).

Significance of formate as a CO2 fixation product. Inasmuchas appreciable amounts of formate were produced from"4CO2 by gut homogenates, we wondered whether its accu-mulation was relevant to acetogenesis from 14CO2. To an-swer this question, we first determined the ability ofexogenously added formate to serve as a carbon source foracetogenesis. 14C-formate was readily oxidized to "4CO2 butwas a poor substrate for acetogenesis in either the presenceor absence of CO2 (Table 6, reactions 1 and 2). In fact, littlemore "4C-formate carbon was incorporated into acetate

*> Hs- , | . ,

100

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80

z

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z20

az

CL

C.,.6 .5 .4 .3 -2 -1

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FIG. 2. Inhibition by 02 of H2-dependent "4CO2 fixation. Reac-tion conditions were similar to those described for Fig. 1. Theabscissa represents initial 02 concentration. Each point representsthe mean of three independent reaction mixtures incubated for 3 h.

under these conditions than in homologous control vials withheat-inactivated homogenates (reactions 7 and 8). As ex-pected, "'CO2 was readily incorporated into acetate andformate under H2 (reaction 5 versus reaction 6). However,the addition of unlabeled formate to similar reaction mix-tures did not dilute (i.e., did not compete with) the amount of"'CO2 carbon entering acetate (reactions 3 and 4). Fromthese results, it appeared likely that the bulk of "4C-formateformation from "4CO2 arose from organisms in the homoge-nate that are unable to reduce the "4CO2 to acetate.A different approach to ascertaining the significance of

formate production from "4CO2 was the examination of theeffect of H2 concentration on "4C-product formation. Thisapproach was prompted by our notice that the 14C-acetate/"4C-formate ratio was routinely higher in reactionmixtures incubated under N2 (i.e., controls) than in thoseincubated under H2 (see, e.g., Tables 5 and 6). The results ofsuch an experiment are quite striking (Fig. 3). As can beseen, acetogenesis from "4CO2 was far less sensitive thanwas formicogenesis to a decrease in H2 concentration from100 to 10%. In this range of H2 concentration, formico-

TABLE 5. Inhibitors of H2-C02 acetogenesis and formicogenesisby R. flavipes gut homogenates

Amt (Mmol) of "4CO2Inhibitor added (concn) atmosphere fixed g (fresh weight)-'

Formate Acetate

None H2 9.00 12.81None N2 0.38 1.17KCN (5 mM) H2 0.25 0.11CHCl3 (50 ,M) H2 9.88 3.82lodopropane (1 mM) H2 7.62 10.70

a Fixed-time assay: 8-h incubation.

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628 BREZNAK AND SWITZER

TABLE 6. Evaluation of formate as a substrate for acetogenesisAmt of productb (nmol of

Reaction Gas 14C incorporated gutno. Substrate' phase equivalent I)

CO2 Formate Acetate

1 H14COOH H2 64.13 1.672 H14COOH + CO2 H2 20.29 1.733 HCOOH + 14CO2 H2 23.00 10.994 HCOOH + 14CO2 N2 0.62 3.455 14CO2 H2 16.47 8.756 14CO2 N2 0.46 0.867 H14COOH (boiled)c H2 0.07 0.928 H14COOH + CO2 H2 0.04 0.54

(boiled)a Reaction mixtures (0.5 ml, total) contained 5 gut equivalents and 1.02

,umol of NaCOOH or 1.15 /Mmol of NaHCO3 (i.e., CO2) or a mixture of theseas indicated. Specific activities of Na"4COOH and NaH14CO3 were 2,238 and702 dpm nmol-1, respectively. Mean recovery of 14C label in these experi-ments was 101%.

b Fixed-time assay; 3 h.Gut homogenate was boiled for 10 min at 100°C before use in reaction

mixtures.

genesis decreased by 86%, whereas acetogenesis decreasedby only 23%. At 1% H2, the 14C-acetate/14C-formate ratioreached a maximum of 6.0. Neither the acetogenic nor theformicogenic system ever became saturated, even at 100%H2 (Fig. 3). This probably reflected the technical difficulty ofmaximizing H2 transfer between the gas and liquid phases in

8.0 1

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100 10 0.1 0

INCUBATION ATMOSPHERE (% H2)

FIG. 3. Effect of H2 concentration on acetogenesis and

formicogenesis from 14C02. Reaction conditions were similar to

those described for Fig. 1. Reaction mixtures were incubated for 5.5

h at the initial H2 concentration (percentage in N2) indicated. Closed

circles indicate the distribution of '4C in 35-p.l samples of clarified

reaction mixtures. Symbols: A, acetate; F, formate.

v I.UNTRUL

80

42 60

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0480

40

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LU

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0 2 4 6 8 10FEEDING TIME (DAYS)

FIG. 4. Effect of antibiotic prefeeding on the H2-dependent14C02 fixation activity of R. flavipes gut homogenates. Reactionconditions were similar to those described for Fig. 1. Individualreaction mixtures were incubated for 3 h.

reaction vials. Attempts to use 100% H2 at pressures greaterthan 1 atm were not made in the present study.

Iniplication of bacteria in H2-CO2 acetogenesis. When R.flavipes termites were fed with cellulose tablets impregnatedwith antibacterial drugs, H2-dependent 14CO2 fixation activ-ity of gut homogenates declined rapidly and was virtuallyundetectable at 4 days (Fig. 4). By contrast, little or nochange in 14CO2 fixation activity was observed with guthomogenates prepared from control termites, which werefed only cellulose. After 9 days of feeding, hindgut protozoaremained abundant and actively motile in both the drug-fedand control termites.

In a reciprocal experiment, we attempted to preferentiallyeliminate protozoa from hindguts of R. flavipes termites byfeeding the insects with starch-a technique known to selec-tively defaunate the lower termite Mastotermes darwiniensis(23). This technique also proved effective with R. flavipes,and after 5 weeks the starch-fed group had lost all of theirlarge, cellulolytic protozoa (Trichonympha, Pyrsonympha,and Dinenympha species). Only bacteria and some of thesmaller protozoa (Monocercomonas and Trichomonas spe-cies) remained. Nevertheless, this treatment resulted in onlya moderate decrease in H2-dependent 14CO2 fixation activitycompared with that in a control group fed with cellulose,which had a hindgut microflora that was normal in appear-ance (Table 7).These results strongly suggested that gut bacteria medi-

ated H2-CO2 acetogenesis in termites. Nevertheless, variousattempts to isolate such organisms from R. flavipes (ourprincipal termite for study) resulted in more than a year offailure and frustration, particularly since we could readilyenrich for and isolate H2-CO2 methanogens from this ter-mite, as well as H2-CO2 acetogens from other anaerobic

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TABLE 7. Effect of starch diet on H2-dependent '4CO2 fixation by R. flavipes gut homogenates'

Dietb Amt (/imol) of "CO, fixed g/ Distribution of 14C (%)CDie ~~~~~(fresh weight)- t h- le SFormate Acetate Propionate Butyrate L-SdStarch 1.32 48.5 42.0 -0.8 0.7 2.4Cellulose 3.14 22.9 68.7 0.4 0.8 1.1

a Individual samples for analyses contained 742 to 6,995 dpm/ml.b Termites were maintained on diets for 5 weeks prior to dissection and assay.' Tabulated values were corrected for "CO2 fixed under an atmosphere of N,.d Lactate or succinate or both.

habitats such as swine waste lagoon sediment and rat cecalcontents (unpublished). However, we have recently isolatedseveral strains of H2-CO2 acetogenic bacteria from guthomogenates of N. nigriceps, a termite species that containsonly bacteria, and no protozoa, in its gut. The acetogenicbacteria are gram-negative, strictly anaerobic motile rodsapproximately 1 by 3 to 8 ,um in size. When growing withH2-CO2 (80/20, vol/vol) as substrates, they produce up to 80mM acetate as product. These organisms are currently understudy in our laboratory.

DISCUSSION

Results presented in this paper show that termite gutbacteria can effect a total synthesis of acetate from CO2 andH2. The H2-CO2 acetogenic activity of termite gutmicrobiota was greater than that of cockroach species ex-amined, and among the termites N. costalis and N. nigricepsexhibited the highest rates (Tables 2 and 3). The reason forthis is unknown but may be related to the fact thatNasutitermes is a genus of termite that contains only bacte-ria, and no protozoa, in its gut. Of the two cockroachesexamined, C. punctulatus, which has both bacteria andcellulolytic protozoa in its hindgut and which is a speciesclosely related to termites phylogenetically (8), had greaterH2-CO2 acetogenic activity than P. americana (Tables 2 and3). Although the relatively small volume of termite guts (ca.0.7 ,ul for R. flavipes; 20), and hence the limited amount ofmicrobial material available, hampered an unequivocal dem-onstration of substrate-product stoichiometry, the reactionmost likely proceeds as follows: 4H2 + 2CO2 ->CH3COOH+ 2H20 (AG"' = -104.6 kJ per reaction; 22). H2-CO2acetogenesis by termite gut homogenates was sensitive to 02as well as to other recognized inhibitors of H2-CO2acetogenesis such as KCN (3), CHCl3 (19), and iodopropane(24) (Fig. 2; Table 5).

Results obtained with R. flavipes gut homogenates supportthe model for symbiotic cellulose utilization in this species oftermite as proposed by Odelson and Breznak (12) and asoutlined in Table 1. For example, it has been shown thattotal hindgut acetogenesis in R. flavipes occurs at a rate of5.8 to 12.4 ,umol of acetate formed g (fresh weight)-' h-1(12). According to the model of Odelson and Breznak,H2-CO2 acetogenesis should contribute 1/3 of that acetate,i.e., it should occur at a rate of about 2 to 4 ,umol g-1 h-1.The specific rate of H2-CO2 acetogenesis for R. flavipesdetermined in the present study was 1.11 + 0.37 ,umol g-h-1, with the highest rate observed being 1.76 ,umol g-1 h-1.We believe that these in vitro rates are sufficiently close tothe predicted rate to validate the hypothesis that, in R.flavipes, C02 reduction to acetate is the main electron sinkreaction in the hindgut fermentation. Additional support forthis conclusion derives from the relatively low rates of CH4and H2 emission from R. flavipes (12); from the relativelylow rates of 14CH4 production from 14C02 by gut

homogenates in vitro (Table 2); and from the fact that evenunder an in vitro atmosphere of 100% H2, the H2-CO2acetogenic system in gut homogenates was not H2 saturated,i.e., was potentially capable of higher rates (Fig. 3). Presum-ably, H2-CO2 acetogenesis in situ occurs in microenviron-ments that maximize this conversion.Although formate was a significant product of CO2 fixation

by gut homogenates, particularly under an atmosphere of100% H2 (Fig. 3), the bulk of its formation seemed not to becarried out by the same organisms catalyzing H2-CO2acetogenesis (Table 6). Moreover, since formate was notdetected in R. flavipes hindgut fluid (12) and since it is not animportant intermediate in most other anaerobic ecosystems(e.g., the bovine rumen [6]), we do not believe that H2-CO2formicogenesis is a significant electron sink for R. flavipeshindgut fermentation in situ. In this regard, it is also impor-tant to recall that synthesis of acetate from CO2 consumesfour times as many reducing equivalents as does the synthe-sis of an equimolar amount of formate from CO2.

In most anaerobic ecosystems that have been studied,H2-CO2 methanogenesis strongly outcompetes H2-CO2acetogenesis as a terminal electron sink. An extreme exam-ple of this is seen in the rumen fermentation, for whichH2-CO2 methanogenesis occurs to the virtual exclusion ofH2-CO2 acetogenesis (19). Exceptions to this generalityexist, however. For example, Phelps and Zeikus (16) foundthat rates of CO2 reduction to acetate and to CH4 weresimilar in Knaack Lake sediments. Moreover, the popula-tion level of H2-CO2 acetogens in sediments was 100-foldhigher than that of H2-CO2 methanogens. The mildly acidicnature of Knaack Lake (pH 6.2) was thought to be a majorfactor in the enhanced competitive ability of acetogens inthis habitat. In Belham Tarn sediments, H2-CO2 aceto-genesis proceeded at about 50% the rate of H2-CO2 methano-genesis (7). This occurred in late summer, when organiccarbon input into profundal sediments was apparently limit-ing. Our knowledge of H2-CO2 acetogenesis in gastrointes-tinal ecosystems is almost nonexistent. However, Prins andLankhorst (19) showed that H2-CO2 acetogenesis totallyoutcompetes methanogenesis in various rodent ceca. Withrat cecal contents, H2-CO2 acetogenesis produced about 8,umol of acetate g (wet weight)-' h-'. Since the rats used byPrins and Lankhorst contained a total of about 2 g of cecalcontents (19), these data, taken together with those of Yanget al. (28) on the contribution of cecal VFA to rat nutrition,allow one to calculate that H2-CO2 acetogenesis in the cecumcould in theory support 0.25% of the maintenance energyrequirement of the rat. By contrast, and as reported in thispaper, H2-CO2 acetogenesis in R. flavipes termites cansupport 33% of the respiratory requirement of the insect.Therefore, in terms of nutritional significance to the animal,H2-CO2 acetogenesis appears to be far more important totermites than to rodents.We do not yet know why H2-CO2 acetogenesis outcom-

petes methanogenesis in termites. In fact, the latter reaction

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630 BREZNAK AND SWITZER

is rnore favorable thermodynamically (AG"' = -135.6 kJ perreaction for methanogenesis versus -104.6 kJ per reactionfor acetogenesis [22]). As in the Knaack Lake ecosystem,low pH could be a contributing factor. The pH in microbe-packed regions of the guts of Reticulitermes andNasutitermes species can be as low as 5.5 or 6.0, but it canalso extend above neutrality (see Table 1 of reference 11).Other possibilities are that the termite gut acetogens mighthave an intrinsically better H2 uptake system (i.e., lower Kmand higher Vmax) than that of the resident methanogens, orthe acetogens may reside closer to H2-emanating micrositeswithin the gut. In lower termites this could mean living on, orin, cellulolytic protozoa which are the main H2 producers inthe hindgut (12). The termite itself might produce a sub-stance(s) which inhibits methanogenesis or stimulatesacetogenesis, or both. It is hoped that studies with theH2-CO2 acetogenic strains isolated from N. nigriceps willhelp clarify this puzzling issue.

ACKNOWLEDGMENTS

We are indebted to K. Hallenga and L. B. Lee, Michigan StateUniversity NMR Facility, for performing the NMR analyses. We arealso grateful to D. G. Cochran, G. Prestwich, A. Stuart, B. Thorne,and J. Traniello for providing insects and to J. M. Tiedje and M. J.Wolin for helpful discussions.

This research was supported in part by the Agricultural Experi-ment Station of Michigan State University and by National ScienceFoundation grant DCB 83-09367.

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