Arch Microbiol (1989) 151 : 399- 406 Archives of

Microbiology �9 Springer-Verlag 1989

Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen*

R. C. Greening and J. A. Z. Leedle

The Upjohn Company, Microbiology and Nutrition Research, Kalamazoo, MI 49001, USA

Abstract. Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H2 : CO2 (80 : 20) or N2 : CO2 (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H2:C02. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria sub- sequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H2: CO2 to form acetate in quantities 2 to 8-fold higher than when grown under N2:C02. All presumptive acetogenic isolates were derived from either the 10-7 or 10-s dilutions of rumen contents�9 All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain re- quired, and all were stimulated by, rumen fluid. No spores were observed with phase-contast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bac- teria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utiliza- tion of hydrogen in the rumen ecosystem�9 Strain 139B ( = ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species.

Key words: Acetitomaculum ruminis - Hydrogen oxida- tion - Acetate production - Rumen

Carbon dioxide reducing, hydrogen oxidizing bacteria have been found in a variety of anaerobic environments. Acetogenic bacteria have been isolated from freshwater and marine sediments (Balch et al. 1977; Braun et al. 1979; Leigh et al. 1981 ; M611er et al. 1984; Sleat et al. 1985; Hermann et al. 1987), cistern mud (Braun et al. 1979) and sewage (Braun and Gottschalk 1982; Braun et al. 1979; Genthner et al. 1981; Ohwaki and Hungate 1977) and acetogenic activity

* Portions of this work were presented previously (Greening RC, Leedle JAZ (1987) Abstr Annu Meet Am Soc Microbiol I 131, pp 194)

Offprint requests to." J. A. Z. Leedle

has been reported in animal gastrointestinal tracts such as the termite hindgut (Breznak and Switzer 1986), rodent ceca (Prins and Lankhorst 1977; Breznak and Switzer 1986), and the colon of a captive killer whale (Wilmarth KR, Boone DR, Mah RA (1985) Abstr Annu Meet Am Soc Microbiol I 109, p 164). However, only one H21C02 utilizing acetogenic bacterium has been isolated from the bovine rumen ecosys- tem where the terminal phase of organic matter decompo- sition is dominated by methane production (Hungate 1976). This organism was Eubacterium limosum, isolated originally from milk fed calves (Bryant et al. 1958) and later from a sheep fed an unusual high molasses diet (Genthner et al. 1981). Other attempts to isolate acetogens from the rumen have been unsuccessful (Braun et al. 1979). In our labora- tory, however, presumptive evidence was obtained to suggest that rumen bacteria able to produce acid from hydrogen and carbon dioxide were present in high numbers in cattle fed high and low forage diets. This report documents the enrichment and isolation of bacteria from the rumen of a mature steer fed a typical high forage diet that produce acetate at the expense of molecular hydrogen and carbon dioxide. The organism [strain 139B ( = ATCC 43876)] is named Acetitomaculum ruminis gen. nov., sp. nov.

Materials and methods

Source o f rumen contents. A mature, rumen-fistulated Hereford crossbred steer was fed a maintenance level diet of 5.5 kg (as fed) alfalfa-grass hay and 1.0 kg of a concentrate supplement consisting of (percent of dry matter): coarsely ground corn (70.47), soybean meal, 48% protein (28.19), trace minerals (0.70), vitamin B (0.14), vitamin D3 (0.14) and vitamin E (0.36). The animal was fed once daily at 07.00 h and had free access to water.

Media. All media were prepared anaerobically as described by Balch and Wolfe (1976) and Hungate (1969). The iso- lation medium, AC-11.1, is a modification of the medium of Breznak and Switzer (1986) and contained (g per liter): KH2PO4, 0.28; K2HPO4, 0.94; NaC1, 0.14; KC1, 0.16; MgSO4 �9 7 H20, 0.02; NH4C1, 0.5; CaClz - 2 HzO, 0.001; 10 ml of trace mineral solution per liter (as described by Balch et al. 1979; but modified by the addition of 0.1 g NiC12 �9 6 HzO and 0.01 g Na2SeO3); 10 ml of vitamin solution (which contained, in milligrams per liter of distilled water: sodium ascorbate, 5; biotin, 2; folic acid, 2; pyridoxine

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hydrochloride, 10; thiamine hydrochloride, 5; riboflavin, 5; nicotinic acid, 5; DL-calcium pantothenate, 5; vitamin Blz, 0.1; p-aminobenzoic acid, 5; lipoic[thioctic]acid, 5; choline chloride, 5; myo-inositol, 5; niacinamide, 5; pyridoxal hydrochloride, 5); NazWO 4 �9 2 HzO, 3 x 10 -4 mM; re- sazurin, 0.001 ; yeast extract (Difco), 0.5; NaHCO3, 6; reduc- ing agent, 10 ml; distilled water, 660 ml; and incubated, clarifed rumen fluid (Leedle and Hespell 1980), 100 ml. Re- ducing agent was prepared under 100% N2 and contained distilled water 200 ml, 5.0 g NazS �9 9 H20 and 5.0 g L- cysteine HC1; pH was adjusted to 10.0 with NaOH. Enrich- ment medium was the isolation medium plus 2- bromoethanesulfonic acid, sodium salt (BES, filter sterilized and added aseptically with a syringe) at a final concentration of 50 mM. Broth media were boiled under an atmosphere of 80% N2 and 20% CO2 and dispensed into 18 x 150 mm culture tubes in 5 ml amounts, stoppered, sealed and auto- claved for t5 min at 121~ The final pH of the medium was 7.15. Agar media were prepared similarly; however, the NaHCO3 and the reducing agent were added as sterile solutions to the sterile, cooled (60 ~ C) molten medium inside an anaerobic glovebox (Coy Laboratory Products, Inc, Ann Arbor, MI, USA; atmosphere of 85% N2, 10% H2 and 5% COz, ambient temperature). After mixing, the medium was poured into 60 x 15 mm plastic Petri plates that had been maintained in the glovebox for at least 48 h. The poured plates were allowed to stand in the glovebox overnight to evaporate excess surface moisture.

Enrichment and isolation procedure. Ruminal fluid was col- lected into a CO2-flushed flask by squeezing composite ruminal contents samples collected at 7 h after the once daily feeding through two layers of cheesecloth. For enrichment, a portion of the mixed fluid was sealed in a tube and placed in the glovebox where serial 10-fold dilutions to 10-8 were made in the enrichment medium. Duplicate tubes were in- oculated with 5% of each dilution. The two sets of tubes were removed from the glovebox and evacuated, then pres- surized to 3 atmospheres (304 kPa), one set with H2:CO2 (80 : 20) and the other with N2 : C02 (80 : 20). Tubes were laid almost horizontally and incubated at 38~ in a gyrotary shaker (New Brunswick, Edison, N J, USA) with 130 revolu- tions per minute. Uninoculated, but pressurized tubes served as controls. The tubes were incubated for 7 days with per- iodic repressurization with the respective gas mixtures. Ali- quots (0.5 ml) from each tube were transferred to fresh me- dia and incubated under the same conditions as above for an additional 7 days. On the third transfer, 0.5 ml of enriched growth from the H 2 : C 0 2 series (10-2 to 10-8) was used to inoculate duplicate tubes, one incubated under H2: CO2 and the other under N2: CO2. The portion of the original dilution series which had been incubated under N2 : CO2 was discard- ed.

For isolation, samples from the 2nd through 7th trans- fers of the H2 : CO2 enrichments were streaked onto plates of AC-11.1 isolation medium. The plates were transferred to incubation vessels (Leedle and Butine 1987) and incubated under 2.5 atmospheres (253 kPa) of H2:CO2 (80:20) at 37 ~ C. Colonies were picked from plates after incubation for 7 days and restreaked for purity. Isolates were inoculated into broth and incubated under either H2 : CO2 or N2 : CO2. Both culture supernatant fluids were analyzed chroma- tographically to determine which isolates were acetogenic. Those which produced at least a 2-fold increment in acetate

in the H 2 : C O 2 tubes over that produced in the N2:CO2 tubes were retained, all others were discarded. Cultures were preserved by lyophilization or in AC-11.1 medium contain- ing glycerol as described by Teather (1982).

Nutritional studies. To assess rumen fluid as a growth factor for the acetogenic bacteria, a source of highly clarified rumen fluid (CRF) was used. Rumen fluid was collected from a steer fed a high forage diet, autoclaved for 15 min, cooled, then centrifuged at 150,000 x g for 2 h at 15~ The super- natant then was passed through a 0.2 txm filter and stored at - 20 ~ C. For the titration experiments, medium AC-11.1 was prepared with 0, 0.1, 0.5, 1, 5 and 10% CRF. Mid-log cultures growing in AC-11.1 with 10% CRF were used as inocula. Three successive transfers in the respective media were done and results reported for the third transfer.

Utilization of organic substrates as energy sources were tested in AC-11.1 medium containing 5% CRF using cells from mid-log phase of growth. The compounds were dis- solved in the medium, filter sterilized through 0.2 ~tm syringe filters (Acrodisc, Gelman Sciences, Ann Arbor, MI, USA) or autoclaved depending on their heat stabilities, and added to the medium at the following final concentrations: aro- matic acids, 10 raM; methanol, 6.25 and 62.5 mM; CO, 165 gmol per tube and all others at 0.5%. Hydrogen uptake was measured based on the methods of Robinson et al. (1981). Oxygen sensitivity was determined in stab cultures of 0.4% agar AC-II.1 with 0.5% glucose or formate as growth substrate in which the topmost centimeter was al- lowed to oxidize. Catalase and esculin hydrolysis were deter- mined by the methods of Holdeman et al. (1977).

Analytical methods. Bacterial growth was determined by measuring the optical density at 600 nm with a Perkin-Elmer model LC-55 spectrophotometer. The light path length was 18 ram. The concentrations of C2-C5 volatile fatty acids (VFAs) in the culture medium were measured as described by Cottyn and Boucque (1968) and Supelco (1975) using a modification of an internal standard method (Mayhew and Gorbach 1977). A Varian model 6000 (Walnut Creek, CA, USA) gas chromatograph equipped with a flame ionization detector was used. The glass column was 6 mm O. D. x 2 mm I.D. x2 .4m, silane treated and packed with GP t0% SP-1200/H3PO4 on chromosorb W AW (Supelco Inc., Bellafonte, PA, USA). Chromatograph temperatures were: oven 130 ~ C; inlet 170~ and detector 175 ~ C. Gases were: air 414 kPa (60 psi) and 300 ml/min; hydrogen 276 kPa (40 psi) and 25 ml/min; and nitrogen carrier 531 kPa (77 psi) and 37.5 ml/min. The gas phase of the enrichment cultures was analyzed for methane using a gas chromatograph equipped with a flame ionization detector and a silica gel column.

Mol percent guanine + cytosine. DNA was isolated as de- scribed by Johnson (1981). Cells were lysed by treatment with EDTA, lysozyme and proteinase K. The tool percent guanine plus cytosine was calculated from the inflection point of the melting profile of the isolated DNA using a Gilford model 2600 spectrophotometer with a thermal cuvette, with DNA from an Escherichia coli K12 strain as reference.

Cell wall analysis. Cell wall isolation and purification was done after the methods of Schleifer and Kandler (1972)

and Kandler and Schoberth (1979). Briefly the cells were harvested and washed once with 50 mM potassium phos- phate buffer with 50 mM EDTA (pH 7). Cells were unsuccessfully broken by 3 passages through a French press at 1.38 x 105 kPa (20,000 lbs), but finally broken by sonica- tion (Branson Sonifier) using 5, 1 rain bursts at 80% power. The cell wall suspension was cleaned by 3 treatments of 1% trichloroacetic acid at 70 ~ C, then washed repeatedly with distilled deionized water until the absorbance at 210 nm was zero. The walls were hydrolyzed in vacuo in 6 N HC1 at 110~ for 24 h and the qualitative and quantitative amino acid composition of the total hydrolysates was determined using a Dionex D500 amino acid analyzer (Dionex Corp.). Ornithine and lysine were separated and quantified by derivatization with phenylisothiocyanate using the method of Heinrikson and Meredith (1984). The amount of diaminopimelic acid was quantified as above after sample oxidation using hydrogen peroxide to remove contaminat- ing methionine residues.

Microscopy. Cellular morphology was determined by phase contrast microscopy and electron micrographs were pre- pared from mid-log cells grown under H 2 : C O 2. For scan- ning EM, the bacteria were fixed by a modification of the method of Boyde and Vesley (1972), dehydrated through a graded series to 100% ethanol, and processed for drying by the procedure of Canby et al. (1985) with 2 changes of hexamethyldisilazane. Finally the specimens were air dried. Transmission EM sample preparation was as above through the 100% ethanol step, then the sample was infiltrated with 2 changes of 100% propylene oxide (PO), then a 50:50 mixture of Polybed 812 (Polysciences, Warrington, PA, USA) with PO, then 2 changes of 100% Polybed 812. Samples were allowed to polymerize for 48 to 72 h at 60 ~ C. Ultrathin sections were stained in 3% uranyl acetate aque- ous, washed with distilled water, then re-stained with Rey- nolds (1963) lead citrate. Sections were examined and photographed with a Jeol 1200 EX electron microscope.

Res u l t s

Enrichment and isolation

After the second transfer of the enrichment series the headspace pressure in all tubes incubated for 7 days under

401

H2 :COz decreased (data not shown). After subsequent sub- culture the pressure decreases were apparent after only a few days, indicating that the gas was being consumed by the enriched microbial population. Also, it became evident that more turbidity occurred at the highest dilutions (10 -8 ) in the H2:CO2 enrichments than in the N2:CO2 enrichments (data not shown). Analysis of the VFAs in the enrichments showed progressively higher acetate levels with increasing dilutions and subsequent to the second transfer, the 10 - s through 10 -8 dilution tubes showed increased amounts of acetate from one transfer to the next in the H 2 : C O 2 series while little change occurred in the N 2 : C O 2 series. Within a dilution, the amount of acetate increased by as much as 50 to 80 mM in the H2: CO2 enrichment cultures. Propionate, isobutyrate, and butyrate increased only very slightly in both the H2 : C02 and Nz : CO2 enrichments, with their mean acid levels at the 2nd, 4th and 7th transfers of 0.7, 0.1 and 0.2 raM, respectively (data not shown). No changes occurred in the levels of isovalerate and valerate. No methane was detected in the headspace of any of the enrichment tubes.

Ninety colonies were isolated and purified from the H2 : CO2 enrichments. Of these, 24 met the criterion of pro- ducing at least 2-fold more acetate under Hz:CO2 than under Nz:COz. All were from either the 10 -v or 10 .8 di- lutions of the original rumen contents sample. Based on morphology and growth characteristics, five rumen acetogenic bacterial strains (20A, 40, 40C, 139B and 190A4) were selected for further study.

Characterization

Fermentation acid production. The C2-C5 volatile fatty acid profiles of these bacteria after growth in AC-I 1.1 containing 5% CRF under a headspace of H2:CO2 are presented in Table 1. The amount of acetate produced in the H2:CO2 tubes was about 7 times greater than the acetate level in the N2:CO2 tubes for all 5 isolates. Mean propionate concen- tration in the H2 : C02 cultures was 5.4 mM, twice the level in the N2 : COg cultures. Isobutyrate, butyrate and isovalerate were produced at levels of 0.3, 0.5 and 0.1 mM, respectively.

Colonial and cellular morphology and ultrastructure. Colonies of all strains formed on AC-I 1.1 agar isolation medium were opaque to translucent, circular, 2 - 3 mm in diameter, buff

Table 1. Fermentation acid profiles of rumen acetogenic bacteria after growth in AC 11.1 medium under H2:C02

Strain Gas phase Acid (raM)a No.

Acetic Propionic Isobutyric Butyric Isovaleric Valeric

20A N2 :CO2 13.0 2.6 0.2 1.8 0.3 ND b H2 : CO2 76.9 4.8 0.4 2.2 0.4 0.3

40 N2 :CO2 13.2 2.6 0.2 1.8 0.3 ND H2 :CO2 99.7 5.6 0.5 2.4 0.5 0.4

40C Nz: CO2 16.8 2.7 0.2 1.9 0.3 ND He :CO2 99.8 5.6 0.5 2.3 0.4 0.3

139 B N2 : CO2 14.5 2.6 0.2 1.9 0.3 ND H2:C02 98.2 5.4 0.4 2.3 0.4 0.4

190A4 N2:CO2 13.8 2.6 0.2 1.8 0.3 0.3 H2 : C02 98.8 5.6 0.5 2.4 0.4 0.3

Uninoculated N2 : CO2 4.9 t.2 ND 1.3 0.2 0.2 Medium Hz : CO2 4.9 1.2 ND 1.3 0.2 0.2

" Means of duplicate tubes after 48 h incubation None detected

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Fig. 1. Phase contrast photomicrograph of strain 139B

color, and had a butyrous consistency and entire margins. Strain 40C had a granular surface, whereas all the other strains were smooth. None of the 5 strains produced dif- fusable pigments. All 5 strains were Gram-positive; however, cultures more than 24 h old stained Gram-negative. Isolate 40C was most commonly Gram-variable. By phase contrast microscopy, all strains were curved, sausage-shaped rods and occurred as single or paired cells or small clumps (Fig. 1). Transmission and scanning electron micrographs typical of strains 40, 40C and 190A4 are shown in Fig. 2a and b, while micrographs typical of the cell shape of strains 20A and 139B are shown in Fig. 3a and b. Spores were not observed in any of the cultures when observed with phase contrast or electron microscopy. Flagella were observed in strains 139B and 190A4 with phase contrast microscopy

Figs. 2, 3 Transmission (a) and scanning (b) micro- graphs of rumen acetogenic bacteria. Mag- nification 2000 x

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Fig. 4 Thin section of strain 139B showing cell wall detail

0.5 '

0 .4 '

go.a

80.2

0 . t

O.O

t2 24 36 48

Time (h)

Fig. 5. Growth of rumen acetogenic bacterial strain 40C under U2 :CO2 (80:20) in medium AC-11.1 with 0% (O), 0.1% (A), 0.5% (Z~), 1% (O), 5% (A) and 10% (B) clarified rumen fluid. Values are means of triplicate tubes on the third transfer

with a flagella stain. A transmission electron micrograph of strain 139B (Fig. 4) revealed a cell wall~ structure of a thick- ness intermediate to Escherichia coli (Gram-negative stan- dard) and Staphylococus aureus (Gram-positive standard). This may explain the tendency toward Gram-variable stain- ing observed in these cultures.

Nutritionalstudies. All 5 strains of rumen acetogenic bacteria were obligately anaerobic, and all were catalase negative. Doubling times for strains 20A, 40, 40C, 139B and 190A4 were 2.8, 1.9, 1.7, 2.1 and 1.6 h, respectively, when grown in isolation medium containing 5% CRF under Hz:CO2 atmosphere (data not shown). The amount of rumen fluid in the medium was titrated to determine its stimulation or requirement for growth by the acetogenic bacterial strains. Rumen fluid was required for growth of only strain 40C but was stimulatory for the others (Fig. 5). The stoichiometry of Ha uptake was related to acetate production with a mean value of 4.1 tool of H2 consumed per mol of acetate formed. The temperature for growth ranged from 37 to 42~ and the optimal pH was near 6.8.

Substrates. All strains grew on formate, CO and glucose, but strain 40C grew only very slowly on glucose (Table 2).

All except strain 40C hydrolyzed and utilized esculin. Strain 139B also utilized cellobiose and fructose. Of the aromatic acids, all but strain 40C used ferulic and syringic acid while only 190A4 used vanillic acid. The fermentation product from all growth substrates was acetate. Isolation medium with 5% CRF and 5 mM Na2SO4 was used to determine sulfate reducing ability. Results showed that these bacteria did not reduce sulfate, as the sulfate concentration in the culture supernatants did not change during an incubation period of 72 h under 3 atmospheres (304 kPa) of 80:20 H2:CO2 (data not shown).

Molpercent guanine + cytosine. The mol% G + C for these strains was 32 to 36% (Table 2).

Cell wall composition. The cell wall amino acid composition was assessed to determine relatedness of our rumen isolates with previously described acetogenic bacteria. The ratio of serine: glutamic acid : alanine: diaminopimelic acid: ornithi- ne: lysine was 1 : 2.6: 2.3 : 0.4:0:1.8. All five strains yielded the same ratio with a standard deviation of 0.1. The presence of both lysine and diaminopimelic acid (cross-linking amino acids) in the cell wall is unusual and may serve to separate rumen acetogenic bacteria from other acetogens (Table 3).

Discussion

Hydrogen oxidizing, carbon dioxide reducing acetogenic bacteria have been isolated from the rumen of sheep fed a high molasses diet (Genthner et al. 1981) and from the rumen of a young calf fed a milk-based diet (Bryant et al. 1958). These isolates, identified as Eubacterium limosum, were able to use a wide variety of organic substrates and produce mixed acids. But these strains were not isolated from typically fed adult ruminants. Braun et al. (1979) were unable to detect H2 plus CO2 utilizing acetogens from the mature bovine rumen. However, we recently reported presumptive evidence that such bacteria were present in the tureen of mature cattle fed normal high or low forage diets (Leedle and Greening 1988). This was the first report of these micro- organisms existing in high population densities in the rumen of a bovine fed a typical high forage diet.

In the present study, enrichment of obligately anaerobic acetogenic bacteria from the rumen of a steer fed a typical high forage diet in media containing BES when incubated under a Hi :COg headspace was successful. Since me- thanogenic bacteria are considered the dominant utilizers of

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Table 2. Characteristics of rumen acetogenic bacteria growing in AC 11.1 broth or agar medium

Characteristics Strain

20A 40 40C 139B 190A4

Enrichment dilution level 10 - 8 10 - 8 10 - s 10- a 10 - 7 Gram stain + + + (v) + + Cell size (gin) 0.8 x 0.8 x 1.0 x 0.8 x 1.0 x

2 , 1 - 3 . 0 2 . 0 - 3 . 2 2 . 1 - 4 . 0 2 . 3 - 3 . 2 2 .5 -3 .5 Motility - - - + + Growth in broth (I'-[2 : C 0 2 )

Turbidity granular granular granular homogeneous homogeneous Degree of growth light light light moderate moderate

Catalase . . . . . Mol% G + C 35 32 36 34 36

Substrate utilization" H2: CO2 + b + + + + Cellobiose - - - + - Esculin + + - + + Esculin hydrolysis + + - + + Fructose - - - + - Glucose + + + ~ + + Formate (sodium salt) + + + + + Ferulic acid + + - + + Syringic acid • + - + + Vanillic acid . . . . + CO + + + + +

a Substrates tested but not utilized: adonitol, erythritol, glycerol, maltose, mannitol, pectin, ribose, starch, DL-alanine, betaine, methylamine, lactate, pyruvate, succinate, anisie acid, 3,4-dimethoxybenzoate; gallic acid, p-hydroxybenzoate, 3,4,5-trimethoxybenzoate, methanol, phytic acid b Positive or utilized, _+, weak; - , not used c Slight growth observed in tube used for determination of oxygen sensitivity (in the reduced portion) after incubation for approximately 14 days

Table 3. Mol% G + C and cell wall cross-linking amino acids of selected Gram-positive anaerobic bacteria a. b

Organism Mol% Cross-linking amino acid Reference(s) G + C of D N A

Aeetobacterium woodii 39 D-ornithine

Clostridium barkeri 45 D-lysine (o-ornithine)

Eubacterium limosum 49 D-lysine (D-ornithine)

Clostridium aceticum 33 m-diaminopimelic acid Clostridium lituseburense 27 aspartic acid

Eubacterium tenue 25.9 m-diaminopimelic acid

Peptostreptocoecus asaceharolyticus 31 - 32 L-ornithine D-glutamic acid

Clostridium aminovalericum 33 m-diaminopimelic acid

Acetitomaculum ruminis 34 D-lysine m-diaminopimelic acid

Balch et al. 1977 Kandler and Schoberth 1979 Braun and Gottschalk 1982 Johnson and Francis 1975 Sehleifer and Kandler 1972 Genthner et al. 1981 Tanner et al. 1981 Braun et al. 1981 Tanner et al. 1981 Johnson and Francis 1975 Tanner et al. 1981 Moore and Holdeman 1986 Schleifer and Nimmermann 1973 Huss et al. 1982 Weiss et al. 1981 Johnson and Francis 1975

"Type strain data used b After Tanner et al. 1981

h y d r o g e n in the r u m e n ( H u n g a t e 1976), i n h i b i t i o n o f the r e s iden t m e t h a n o g e n s w i t h BES was necessa ry to p r o v i d e select ive p ressu re to cu l t iva te the h e r e t o f o r e u n d e s c r i b e d r u m e n ace togen ic p o p u l a t i o n . BES, a n a n a l o g o f c o e n z y m e M ( 2 - m e r c a p t o e t h a n e s u l f o n i c acid) w h i c h is u n i q u e to

m e t h a n o g e n s (Ba lch a n d Wol fe 1979), is a p o t e n t i n h i b i t o r o f the r e d u c t i o n o f m e t h y l - c o e n z y m e M in cell ex t rac t s a n d i so la ted who le cells o f severa l m e t h a n o g e n s ( G u n s a l u s et al. 1978; Z e h n d e r e t al. 1980; S m i t h a n d M a h 1981). BES was i nc luded in the e n r i c h m e n t m e d i u m a t 50 m M because th is

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level has been shown to be required to completely inhibit methanogenesis by CO2 reduction in natural populations (Smith and Mah 1981; Bouwer and McCarty 1983; Zinder et al. 1984). With the methanogenic bacteria inhibited, it was possible to successfully enrich for other rumen bacteria that could reduce CO2 at the expense of hydrogen.

Although our 5 isolates differ in some of their nutritional characteristics (Table 2) we believe "they are all members of the same species based on the clustering of important metabolic features for hydrogen oxidizing, carbon dioxide reducing bacteria. These features are the ability to grow on H2: CO2, formate, CO and glucose, their non-utilization of lactate or pyruvate, and their similarities of DNA tool% G + C content and cell wall amino acids.

Rumen acetogenic bacterial strains 20A, 40, 40C, 139B and 190A4, differ from other hydrogen oxidizing acetogenic bacteria that have been described. They differ from the genus Clostridium because they do not form endospores. They differ from the genera Acetoanaerobium (Sleat et al. 1985), Sporomusa (M611er et al. 1984; Hermann et al. 1987) and Acetogenium (Leigh et al. 1981) because the isolates stain Gram-positive. Our isolates are different from Eubacterium in tool% G + C, volatile fatty acid profiles, cellular mor- phology and substrate utilization. Acetate is the only prod- uct regardless of whether esculin, formate, cellobiose, fruc- tose or glucose is fermented. The genus which most closely resembles our isolates is Acetobacterium although the DNA base composition for A. woodii is 39 tool% G + C (Balch et al. 1977) and 43 tool% G + C for A. wieringae (Braun and Gottschalk 1982) whereas our isolates' tool% G + C is 32 to 36%. The ratio of amino acids in the cell wall and the presence of both lysine and diaminopimelic acid as cross linking amino acids suggest that our isolates may not belong to a previously described group. Analysis of the 16S rRNA of strain 139B, in comparison to that of a number of low G + C Gram-positive anaerobes, confirmed that these tureen acetogenic bacteria were generally related to the clostridia, but were not specific relatives of any other known acetogenic bacteria (RS Tanner, persc, nal communication.)

We propose that rumen acetogenic bacterial strains 20A, 40, 40C, 139B and 190A4 be placed in a new genus, Acetitomaculum (A.ce.ti.to.ma.cu.lum. L.n. acetum vinegar; L.n. tomaculum sausage; M.L. neut. n. Acetitomaculum vin- egar sausage). The type species for this genus is A. ruminis sp. nov. (ruminis. L. gen. n. ruminis of the rumen), named for its shape and source. A combined generic and specific description follows,

Acetitomaculum gen. nov. and Acetitomaculum ruminis sp. nov.

Morphology. Curved rods measuring 0.8 - 1.0 lam in width and 2 .0-4 .0 gm in length, often occurring in singles, pairs or small clumps. Cells may be flagellated. Gram-positive. Endospores absent.

Colony characteristics. Convex, circular, 2 - 3 mm diameter, entire, translucent, tan in color, with a smooth surface (strain 40C had granular surface).

DNA base composition. 3 2 - 36 tool% G + C.

Temperature relationship. Optimum, 38~ range 34--43 ~ C.

pH Relationship. Optimum, 6.8; range 6.4-7.3.

Physiology. Obligately anaerobic. Ferments formate, glu- cose, cellobiose, fructose and esculin to acetic acid. Oxidizes hydrogen and reduces carbon dioxide according to the fol- lowing equation: 4 H2 + 2 CO2--, CH3COOH + 2 H20. Catalase negative.

Nutrition. Grows in a mineral medium with H2 and C O 2 o r organic substrate. Rumen fluid is stimulatory and may be required.

Source. The rumen of a mature Hereford X Angus crossbred steer fed a high forage diet.

Type strain. Strain 139B, deposited as ATCC 43876 and with the Upjohn Culture Collection as UC strain number 12185.

Acknowledgements. We thank R. S. Tanner for coordinating the 16S rRNA analysis, for assistance in data interpretation, and critical review of this manuscript. We thank T. J. Butine for excellent techni- cal assistance and J.A. Breznak for helpful discussions. We thank C. Cramer who performed the electron microscopy, R. Ulrich for interpretation of the cell wall structure, I. M. Reardon who performed the cell wall analysis, and T.O. MacAdoo for assistance in naming this microorganism.

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Received December 17, 1987/Accepted December 15, 1988

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