FEMS Microbiology Letters 25 (1984) 47-52 Published by Elsevier FEM 01904

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Isolation and characterization of a thermophilic, acetate-utilizing methanogenic bacterium

(Methanogenic bacteria; acetate-decarboxylation; H 2 - C O 2 and formate metabolism)

Birgitte K. A h r i n g and Peter W e s t e r m a n n

Department of Microbial Ecology, Institute of Thallophytes, Oster Farimagsgade 2 D, DK- 1353 Copenhagen K, Denmark

Received 16 January 1984 Accepted 2 February 1984

1. SUMMARY A thermophilic acetate-decarboxylating methanogenic b a c t e r i u m was isolated from a laboratory-scale 60 C sludge digestor. Cells form straight filaments with flat to blunted ends normally consisting of 2-3 cells held together by a sheath-like outer cell wall. The organism uses acetate, H 2 - C O 2 and formate for methanogenesis and growth. With acetate as the sole methanogenic substrate, almost all of the radioactivity from methyl-labelled acetate appeared as methane. Acetate was converted to methane in equimolar amounts with a doubling time of 3 days.

ture optimum near 50C and is therefore only slightly thermophilic. Neither H 2 - C O 2 n o r formate can be used for methanogenesis by
Methanosarcina TM1.

2. I N T R O D U C T I O N Numerous studies have implicated acetic acid as being the principal precursor of methane [1-5]. Nevertheless, only few of the isolated methanogens are able to use acetate as sole carbon and energy source. The only pure culture of a thermophilic acetoclastic methanogen previously described is Methanosarcina TM1 [6]. This organism has a tempera-

Recently, Nozhevnikova and Yagodina [7] and Zinder et al. [5,8] described a thermophilic Methanothrix sp. obtained in enrichment cultures from thermophilic digestors. This methanogen was described as a filamentous rod with flat ends composed of short cells where empty swollen sheaths often could be observed in old filaments. During previous experiments with thermophilic anaerobic digestion of sewage sludge at 60 C [9], we were never able to isolate a Methanosarcina from the digested sludge. Instead, acetate enrichments always resulted in the selection of a straight filamentous rod somewhat similar to the rod found in thermophilic enrichments by Nozhevnikova and Yagodina [7] and Zinder et al. [5,8]. This paper deals with the isolation and characterization of the filamentous rod which besides acetate u s e s H 2 - C O 2 and formate as sole energy and carbon source. In the following the organism will be named thermophilic acetate-utilizing methanogen (TAM) organism.

0378-1097/84/$03.00 1984 Federation of European Microbiological Societies

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3. M A T E R I A L S A N D M E T H O D S

3.1. Source of organism The initial enrichment was made from a thermophilic anaerobic bench scale digestor operating at 6 0 C on sewage sludge obtained from the sewage treatment plant in Usserod, Denmark. 3.2. Media and conditions for cultivation Anaerobic techniques described by Hungate [10] as modified by Bryant [11], and Balch and Wolfe [12] were used. The acetate mineral medium for enrichment and isolation of the TAM-organism was prepared essentially as described by Zehnder et al. [13] except that solution B contained 28.4 g N a z H P O 4 2 H 2 0 and NazSeO 3 was omitted. In the experiments the media were supplemented with yeast extract 0.1% and clarified rumen fluid 3% (v/v). Usually acetate concentration in both mineral and complex media was 6.8 g sodium acetate (50 mM). Experiments were performed either in 20-ml vials (total volume 24.6 ml) closed with butyl rubber stoppers and aluminium crimps [12], or in 0.5-1 serum bottles (total volume 593.5 ml) closed with black butyl rubber stoppers (Belco size 4) and serum caps. Unless stated otherwise, the gas phase in vials and bottles w a s 8 0 % N 2 - 2 0 % C O 2 pressurized to 1 arm overpressure. Incubations were performed at 60 C and all experiments were run in duplicate. 3.3. Enrichment and isolation Enrichment of the TAM organism was performed in a 1-1 serum bottle with a working volume of 750 ml, fitted with a two-hole butyl rubber stopper; one hole serving as the inlet and outlet for culture fluid and medium, the other being the outlet for a tubing leading to a gas collection bottle. The initial enrichment was performed by adding 10 ml sludge from the digestor to 740 ml reduced acetate mineral medium and incubating the bottle in a water bath at 60 C. The serum bottle was operated as a semi-continuous system. At approx. 72-h intervals, the bottle was shaken vigorously after which 150 ml culture fluid was removed and replaced by 150 ml reduced acetate mineral medium giving a mean

retention time of 15 days. The p H was routinely measured during each feeding and if necessary adjusted to maintain a p H value of about 7.3. Isolation was performed after 8 months of continuous cultivation by serial dilution of the enrichment culture in acetate mineral medium containing 0.1 g / l vancomycin. The highest dilution showing growth was used for preparation of another dilution series and this procedure was repeated twice.

3. 4. Analytic procedures Methane and acetate were detected with a gas chromatograph (Shimadzu 8A) equipped with a flame ionization detector. Methane was quantified on a Porapak T column working isothermically at 100C. Acetate was quantified as described by Ahring and Westermann [9] except that the column temperature was kept constant at 130 C. The detection limit for acetate was 0.5 raM. 3.5. Labeling experiments Experiments with radiotracers were performed as described in the legend of Table 1. 14CH4 and 14CO2 in the headspace was measured by the liquid scintillation method by Zehnder et al. [14]. The method was modified to ensure that no gas escaped during the manipulations, by using 10-ml glass serum vials (total volume 13.2 ml) with black butyl rubber stoppers instead of liquid scintillation vials. Experiments revealed that this had no effect on the counting efficiency of the system, and that there was no interference from 4K contents in the glass (not shown). For determination of 14C02 only 5 ml of toluene-flour mixture was added after absorption of the CO 2. To d e t e r m i n e 14CH4, 10 ml of toluene-flour mixture was used. The amount of methane solubilized in the toluene-flour was determined to 60% by injection of known amounts of methane as described by Zehnder et al. [14]. Label incorporated into the biomass was determined as described by Zinder and Mah [6]. Radioactivity was counted in a Tri-carb 3375 liquid scintillation spectrometer (Packard Instruments Co., Inc.) using the channels-ratio method for quench correction.

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3.5. Electron microscopy Thin sections were prepared essentially as described by Zehnder et al. [13] except that 2% glutaraldehyde in 0.1 M cacodylate buffer was used for the initial fixation and that no agar was used for embedding of the bacteria. Electron microscopy was performed with a JEM 100 CX microscope. 3.6. Chemicals The following radioactive compounds were used: sodium [2-aaC]acetate (specific activity, 51 m C i / m m o l ) , sodium [1-14C]acetate (specific activity, 59 m C i / m m o l ) obtained from New England Nuclear Corp. All other chemicals were reagent grade.

from the last dilution series was used for the present experiments after it was thought plausible that the straight rod was able to utilize H 2 - C O 2 and formate as well as acetate. When growing the organism in roll-tubes on acetate mineral media, small whitish colonies formed paralleling the build-up of methane in the headspace. When individual colonies were picked and transferred to the same medium, no growth could be observed even after prolonged incubation. Occasionally, we were able to obtain growth after picking a colony to acetate complex medium but this culture was never free from contaminants.

4. RESULTS

4.1. Enrichment and &olation After 8 months of continuous enrichment at 6 0 C a very characteristic bacterial population had developed. In microscopic examinations using phase-contrast, a straight filamentous organism of variable length was observed. Only few (102/ml culture fluid) heterotrophic contaminants could be counted on thioglycollate medium (Difco) and AC medium (Difco) incubated at 6 0 C under anaerobic and aerobic conditions. The first dilution-series (MPN) from the enrichment culture showed that the organism had a rather long lag phase ( 3 - 4 weeks) when grown on acetate mineral medium. Experiments showed that this was not due to the presence of 0.1 g/1 of vancomycin, a concentration which was only slightly inhibitory (10%) to the organism (not shown). Methane was found in the 10-10 -7 dilutions after 1.5 months of incubation. No heterotrophic contaminants could be found when the 10 -7 culture was examined and only the straight rod could be observed under the microscope. After the finding that this culture was able to u s e H 2 - C O 2 and formate, the dilution procedure on acetate mineral medium was repeated two times in an attempt to free the organism from possible methanogenic contaminants. The 10 -7 culture

4.2. Morphology The T A M organism is a straight filamentous organism with flat to blunted ends. The cell length varies widely. Normally the organism consists of 2 - 3 single cells with a mean dimension of 0.8-1 /~m width and 4 5 /~m length (Fig. 1, a,b), but longer filaments with many cells are occasionally observed. The G r a m stain is positive to Gramvariable. When examined by epifluorescence microscopy the organism shows no obvious fluorescence when grown on acetate in contrast to the growth on H 2 - C O 2 or formate, where the organism

Fig. 1. (a) Phase contrast photomicrograph of the TAM organism growing on acetate mineral medium. Bar indicates 11.5 p,m. (b) Photomicrograph of Gram-stained cells and filaments of the TAM organism growing on H 2 - C O 2. Bar indicates 9.5 ptm.

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showed a clear but rapidly lost fluorescence. Thin longitudinal- and cross sections show that the organism has a thick 3-layered cell wall (Fig. 2, a, b,c). The outer layer consists of a sheath-like structure common to all cells in the filament, while the other layers continue around the individual cells

close to the double membrane enveloping the cytoplasma. The details of this wall and the membranes are most clearly seen in lysed cells (Fig. 2d). Empty sheaths are frequently seen in long filaments (Fig. 2e), and intracytoplasmic membranes occur in some of the cells (Fig. 2f).

.....

Fig. 2. (a-f) Thin sections of the TAM organism. (a) Cross section through the cell. Bar indicates 0.17 btm; (b) a short filament showing the outer cell wall common to both ceils (O), and the inner cell walls forming the septum between the individual cells (S). Bar indicates 0.5/~m; (c) part of a filament. Bar indicates 0.6/Lm; (d) fine structure of the 3-layered cell wall in a lysed and a growing cell showing the double membranes (M). Bar indicates 1.5 ~tm; (e) part of a filament with a lysed cell. Bar indicates 0.36 tzm; (f) intracytoplasmatic membranes seen in cross section. Bar indicates 0.2/Lm.

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4. 3. Growth of the organism on acetate

A significant decrease in the lag phase to 3 - 4 days was o b t a i n e d when the organism was grown o n acetate complex m e d i u m s u p p l e m e n t e d with yeast extract and r u m e n fluid b u t these additions had no a p p a r e n t effect on the growth rate of the organism (not shown). Acetate was converted into m e t h a n e in equimolar a m o u n t s during growth in batch cultures as shown in Fig. 3. Based u p o n the exponential m e t h a n e p r o d u c t i o n a d o u b l i n g time of 3 days could be calculated c o m p a r e d with a d o u b l i n g time of 3.8 days when measuring the a b s o r b a n c e of the same culture. The fate of methyl- a n d carboxyl-labelled [14C]acetate is shown in Table 1. The results show that the methyl group of acetate is the p r i m a r y precursor of m e t h a n e from acetate. Most of the label was f o u n d in C O 2 when carboxyl-labelled [J4C]acetate was added. Approx. 2% of the labelled acetate was i n c o r p o r a t e d into the biomass for either positions of acetate.
14-13 12 11 10

I ABSO~ ~-- -A

0.60

_ --

9 ~8

0"50 I
0.45

6
4

3 2 1
5 10 Time 15 days 2O 25

035 I
3O

0.~

Fig. 3. Relationship between methane production ((3), acetate consumption (O) and the change in absorbance (A) during growth of the TAM organism in batch culture with 250 ml acetate complex medium. what similar to the thermophilic Methanothrix sp. [5,7,8] a n d the mesophilic Methanothrix soehngenii [14,15]. Despite this similarity there are certain differences the most striking of which is the ability of the T A M organism to utilize H 2 - C O 2 a n d formate besides acetate. N o z h e v n i k o v a a n d Y a g o d i n a c o n c l u d e d that their t h e r m o p h i l i c organism was u n a b l e to utilize H 2 - C O 2 based on the observation that Methanobacterium-like forms developed when H 2 - C O 2 was i n t r o d u c e d into the gas phase of their Methanothrix-enrichments [7].

4.4. Metabolism of other substrates

The T A M o r g a n i s m grows well on both H 2 - C O 2 a n d formate. N o growth was f o u n d on either m e t h a n o l or t r i m e t h y l a m i n e (not shown).

5. D I S C U S S I O N The general a p p e a r a n c e of the T A M organism is that of a sheathed filamentous organism some-

Table 1 Fate of methyl-labeled and carboxyl-labelled[14 C]acetate during growth of the TAM organism a Label b 14CH3COOCH314COO14CH4 c 1984.7 (90.2%) 64.38 (2.9%) 14CO2 c 156.2 (7.1%) 2060.2 (93.6%) 14C incorporated c 42.18 (1.9%) 46.62 (2.1%) Label metabolized 99.2% 98.6%

Growth was performed in 20-ml serum vials with 15 ml of acetate complex medium (acetate cone. 10 mM), headspace 80% N2-20% CO2. Incubation time was 1.5 month. b Either 2200.10 3 dpm o f 14CH3COO or 2200.10 3 dpm of CH 314COO was added to each of the duplicate vials at the beginning of the experiment. Results expressed as thousands of dpm and as % of total added labelled substrate measured after injection of cone. HC1 into the serum vials to obtain a final pH of 2.0.

52 However, this could be due to a c o m p e t i t i v e favouring of the Methanobacterium-like o r g a n i s m c o m p a r e d to the Methanothrix sp. The finding b y N o z h e v n i k o v a a n d Y a g o d i n a that acetate was only slightly utilized in the presence of H 2 could be explained b y a b i p h a s i c growth p a t t e r n of the o r g a n i s m where H 2 is e x h a u s t e d before acetate is used as has been f o u n d in Methanosarcina spp. [16-18] a n d in our T A M o r g a n i s m ( m a n u s c r i p t in p r e p a r a t i o n ) . Methanothrix soehngenii has not been shown to utilize H 2 - C O 2 or f o r m a t e for growth a n d methanogenesis, although it c o n t a i n s b o t h hyd r o g e n a s e a n d f o r m a t e d e h y d r o g e n a s e [19]. This is expected to be f o u n d in o r g a n i s m s growing on H 2 - C O 2 a n d formate. Z i n d e r a n d K o c h [20] have described a rods h a p e d t h e r m o p h i l i c e n r i c h m e n t culture which was f o u n d to require two organisms c o u p l e d via interspecies h y d r o g e n transfer for m e t h a n o g e n e s i s from acetate. The following facts show that such a relat i o n s h i p is not existing in our T A M cultures: (1) neither of the o r g a n i s m s d e s c r i b e d by Z i n d e r a n d K o c h are s t r a i g h t - s h e a t h e d f i l a m e n t o u s rods that fit the d e s c r i p t i o n of o u r organism; (2) T A M cultures grown on H 2 - C O 2 or f o r m a t e can m e t a b o l i z e acetate without a n y lag p e r i o d ; (3) when the T A M cultures were serial diluted on H 2 - C O 2 further d i l u t i o n series m a d e from the 10 - 7 vials c o u l d s u p p o r t growth to the 10 - 6 dilution on acetate c o m p l e x m e d i u m ; (4) no growth a n d acetate utilization was o b t a i n e d when the T A M cultures were i n c u b a t e d on acetate c o m p l e x medium supplemented with 2-bromoethanesulfonic acid (1 m M ) - an i n h i b i t o r of the m e t h a n e p r o d u c t i o n [21] - a n d sulfate (20 m M ) a n d heavily i n o c u l a t e d with a thermophilic, h y d r o g e n - u s i n g Desulfovibrio sp. isolated from the t h e r m o p h i l i c sludge digestor. T h e effects of the a d d i t i o n s of yeast extract a n d r u m e n fluid on the lag p h a s e of the T A M o r g a n i s m indicates that the acetate m i n e r a l m e d i u m used has to be s u p p l e m e n t e d for o p t i m a l growth. F u r ther c h a r a c t e r i z a t i o n of the factors required is now in progress. ACKNOWLEDGEMENTS W e thank K a r i n Vestberg a n d Jens T h o r u p for technical assistance a n d Lisbeth T h r a n e for electron m i c r o s c o p y help. This w o r k was p a r t i a l l y s u p p o r t e d b y grants (No. 11-3888 a n d No. 11-3982) f r o m The D a n i s h N a t u r a l Science Research Council.

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