INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 267-274 Vol. 41, No. 2 0020-7713/91/020267-08$02.OO/O Copyright 0 1991, International Union of Microbiological Societies

Characterization of barkeri MST and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-76IT GLORIA M. MAESTROJUAN' AND DAVID R. BOONE172* Departments of Environmental Science and Engineering' and Chemical and Biological Science,2 Oregon Graduate Institute, 19600 N.W. von Neumann Drive, Beaverton, Oregon 97006-1999

Members of the genus Methanosarcina are recognized as major aceticlastic , and several which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species ( MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H, plus , growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCI. The catabolic substrates which supported most rapid growth were H, plus methanol; this combination sometimes allowed growth of a strain under extreme environmental conditions which prevented growth on other substrates. The morphology of all strains was affected by growth conditions.

Although Methanosarcina species have been the subjects 2244). We observed this culture growing on methanol in MS of extensive metabolic studies, other aspects of the charac- medium and never saw cysts or multicellular aggregates terization of these organisms do not meet even the minimal larger than two cells. The predominant morphology was standards for description of methanogenic taxa (9). In this individual cocci (unpublished data). Thus, if cells similar to study we examined the physiology of the following three the cells of this strain were present in Barker's enrichment mesophilic, aceticlastic species: Methanosarcina barkeri, cultures, other multicellular methanogenic pseudosarcinae Methanosarcina mazei, and Methanosarcina vacuolata. must also have been present. This strain appears to conform Methanosarcina acetivorans, the only other mesophilic and neither to Barker's description of Methanococcus mazei nor aceticlastic Methanosarcina species, was not included in to Mah's later description of Methanococcus mazei S-6T (T this study because it has been well characterized (35). = type strain) (23) in its morphology. In order to obtain an The original type strain of M. barkeri, isolated by C. G. T. axenic culture of Methanococcus mazei, Mah (23) per- P. Schnellen (32a), has been lost. In 1966, Bryant isolated formed the enrichment protocol of Barker (6) and isolated strain MS (lo), which has been adopted as the type strain (4). the first axenic culture of a (strain S-6T) which Although this strain is one of the most extensively studied exhibited a life cycle and included morphological forms like methanogens, previous studies have not included definitive those described earlier by Barker (6). Strain 5-6 was desig- determinations of its responses to environmental conditions nated the type strain of Methanococcus mazei, and later this such as pH, temperature, and salinity. Strain 227 (25) is species was transferred to the genus Methanosarcina as another extensively studied strain which DNA hybridization Methanosarcina mazei (24). M. barkeri. studies (37) indicate is a member of Zhilina (43) also isolated a gas-vacuolated Methano- Methanogenic cocci were first described by Groenewege sarcina strain, and she and Zavarzin (46) named this organ- (12) in 1920 and were later observed by Kluyver and van Niel ism Methanosarcina vacuolata. These authors suggested (17). These cocci were physiologically similar to M. barkeri, that this species should include vacuolated Methanosarcina but because of morphological differences between the cocci and M. barkeri, Kluyver and van Niel proposed a new genus strains, although the phylogenetic significance of vacuoles (Methanococcus) for the cocci. Later Barker (6) studied a has not been established. purified, nonaxenic culture and named it Methanococcus Members of M. barkeri, M. mazei, and M. vacuolata can mazei. Pure cultures of this species were not available until be distinguished from other bacteria by the following three more than 40 years later. Zhilina and Zavarzin (44) also characteristics: (i) they can produce from trimethyl- studied an aceticlastic enrichment culture which contained amine, from methanol, and from H, plus CO, or ; (ii) morphological forms similar to those described by Barker as they appear as either coccoid cells, pseudosarcinae, or Methanococcus mazei. Although it is not clear from the aggregates (i.e., biotype 3, 2, or 1 in the system of Zhilina (C0.2 report of Zhilina and Zavarzin (44) whether an axenic culture [42]); and (iii) they grow well in nonsaline medium was obtained, an axenic culture (referred to as Methano- osM) at mesophilic temperatures. Although it is not very sarcina biotype 3) was later deposited in the Oregon Collec- difficult to determine whether an organism belongs within this group of three species, it is much more difficult to assign tion of Methanogens as strain OCM 92 (= 2-558 = DSM it to a particular species because of a lack of distinguishing characteristics. Determination of the environmental conditions which sup- * Corresponding author. port most rapid growth is an important part of the charac-

267 268 MAESTROJUAN AND BOONE INT. J. SYST.BACTERIOL. terization of new species of methanogens (9). Such determi- culture, the amount of methane that accumulates in a culture nations are usually made by keeping all environmental vessel is proportional to the increase in biomass, but this conditions except one constant and measuring the specific measure neglects the biomass added as inoculum (29). growth rates at various values of a single parameter, for Therefore, we added the amount of methane formed by the instance pH. Such an experiment determines the pH value inoculum during its growth to the amount of methane present which allows most rapid growth (often called the optimum in the culture vessel at each time point to obtain a value pH) under the conditions of the assay. However, it is proportional to total biomass. The specific growth rate was unlikely that the effects of environmental growth conditions estimated by performing a least-squares analysis of the (such as pH, temperature, and salinity) are independent, so, logarithm of this value during the period of exponential for instance, one range of pH values may support the most growth. Inocula were grown under conditions similar to the rapid growth at one temperature and a different range of pH test conditions. The inoculum volumes were 5% (for cultures values may support the fastest growth at a second tempera- growing as individual cells or very small clumps) or 10% (for ture. The present study was initiated to determine whether cultures growing as aggregates) of the culture volumes; the optimal conditions for growth are mutually dependent smaller inocula gave inconsistent results, perhaps because it and to further characterize M. barkeri MST and 227, M. was impossible to obtain representative inocula when cells mazei S-6T, and M. vacuolutu Z-761T. were present as small numbers of large clumps. (Portions of the results were reported at the 1990 Annual Analytical methods. The methane in gas samples was Meeting of the American Society for Microbiology, Ana- separated by gas chromatography in a column of activated heim, Calif.) charcoal and was quantified by flame ionization detection. Samples at the pressure of the culture vessel were collected MATERIALS AND METHODS in a 10-pl loop (Fig. 1). Thus, the peak areas of methane were proportional to the partial pressure, rather than to the Bacterial strains and culture methods. The following bac- mole fraction (as when a sample is equalized to atmospheric terial strains were obtained from the Oregon Collection of pressure). When the valve was in the load position, each Methanogens (OCM). M. barkeri MST (= OCM 38T = DSM odd-numbered port was connected to the even-numbered 800T) and 227 (= OCM 35 = DSM 1538), M. rnazei S-6T (= port whose number was one higher (port 1 to port 2, port 3 OCM 26T = DSM 2053T), M. vacuoluta Z-761T (= OCM 85T to port 4, port 5 to port 6, and port 7 to port 8). The gas = DSM 1232T), and Methanosarcina sp. strain 2-558 (= sample was collected via a needle through the stopper of the OCM 92 = DSM 2244 = biotype 3). Strains were cultivated culture vessel (connected to port 3 of the valve via 1.6-mm by using the anaerobic techniques of Hungate (15) with [1/16-in.] stainless steel tubing). Thus, the sample flowed syringes and needles. through the valve (from port 3 to port 4), swept through the The basal culture medium was MS medium (7), which 10-pl loop to port 7, and then flowed to port 8 and into a contained yeast extract, peptones, minerals, and coenzyme previously evacuated, sealed 0.5-ml space. Within a few M and sulfide as reducing agents and was used with a seconds pressures equalized so that the portion of the bicarbonate buffer system in equilibrium with a CO,. gas sample remaining in the loop was at the pressure of the phase (pH 6.5). After the medium was dispensed into indi- culture vessel. During this time, carrier gas flowed through vidual vessels, it was sterilized by autoclaving. The pH of the valve (from port 6 to port 5) and to the column of the gas the medium was adjusted by replacing the headspace gas in chromatograph. To analyze the sample in the loop, the valve the vessels with N, or an N,-CO, mixture (a 7:3 N,-CO, was turned to the inject position, which connected each mixture resulted in a pH of 7.2, and 100% N, resulted in a pH odd-numbered port to the even-numbered port whose num- of 8.2) and by adding NaOH or HCl solutions to obtain pH ber was one lower (port 1 to port 8, port 3 to port 2, port 5 values higher than 8.2 or lower than 6.5. The pH usually to port 4, and port 7 to port 6). Thus, the carrier gas now varied less than 0.1 to 0.2 pH unit during growth except in flowed through the valve (from port 6 to port 7), through the some poorly buffered media (those with pH values less than sample loop, again through the valve (from port 4 to port 5), 6). The growth rates in poorly buffered media were deter- and then to the column. The entire sample contained in the mined from the early portions of the growth curves, when loop was thus placed in the flow path to the chromatograph the pH values varied less than 0.2 pH unit. and analyzed. Meanwhile (while the valve was in the inject Soluble catabolic substrates were added from sterile, position), a vacuum pump connected to port 1 drew a anoxic stock solutions to final concentrations of 50 mM, and vacuum on the 0.5-ml space, which was necessary to draw H, was added by pressurizing the preparation with pure H, the subsequent sample through the loop. While the valve (final partial pressure, 101 kPa). All cultures containing was in the inject position, the sample collection line was added H, were incubated on a shaker. The culture vessels connected to port 2, which was sealed to block the flow. We were 121-ml serum vials which contained 20 ml of culture found that this method for analysis of methane gave peak medium. areas that were proportional to methane partial pressures at Specific growth rates were determined during growth from partial pressures between 10 Pa and 200 kPa. the amount of methane formed, which served as an estimate of the amount of biomass present. Because Methanosarcina RESULTS AND DISCUSSION strains often grow as aggregates, other methods for estimat- ing biomass are unsatisfactory; the presence of various sizes pH optima and effects of catabolic substrates. The Metha- of clumps prevents the use of turbidity as a measure of nosarcina strains generally grew faster at pH values below 7 biomass, and other methods which quantify biomass in than at pH values above 7 (Fig. 2). Depending on the liquid samples (such as protein analysis or measurement of catabolic substrate, these strains grew rapidly at pH values dry weight) are inaccurate because it is impossible to obtain below 5. We found that the choice of catabolic substrate small, representative samples of cell suspensions when small usually had only a minor effect on the optimal pH for growth, numbers of large clumps are present (16, 22, 41). Assuming except that growth on methanol plus H, was most rapid at that the growth yield is constant during the growth of each decidedly low pH values. More rapid growth on H, plus VOL. 41, 1991 CHARACTERIZATION OF METHANOSARCINA STRAINS 269

some difference in our culture medium. M. barkeri MST and A 227 also grew well on H, plus CO, in complex medium, but M. vacuolata Z-761T grew poorly, as suggested by Zhilina and Zavarzin (46). The inability of M. vacuolata to grow under such conditions may be an important distinguishing characteristic and supports the view of Zhilina and Zavarzin (46) that this bacterium is a distinct species. However, the To sample ability to catabolize H, plus CO, may not be a stable collection characteristic for Methanosarcina strains or may depend on the culture medium or other conditions. For instance, M. rnazei S-6T was originally reported to be unable to use H, plus CO, rapidly, but we found that it could. We had expected growth rates on methanol to be similar to growth rates on trimethylamine because the catabolic path- To column ways should differ only in the initial step of methyl transfer (14). However, growth rates on trimethylamine generally LOAD were slower. In most cases the ranges of pH values which wSample loop w supported growth were similar regardless of whether meth- anol or trimethylamine was the substrate; the exception was M. rnazei S-6T, which in media having pH values of more than 7 grew much faster on methanol than on trimethyl- amine. It is possible that growth on trimethylamine was B inhibited by ammonia formed during its catabolism, but such an inhibition might then have been evident in batch cultures, in which the amount of ammonia increases during growth. We observed no decreases in the growth rates of batch cultures on trimethylamine until the catabolic substrate was exhausted. These data generally confirm findings from other To sample studies of M. barkeri 227 (5, 11, 25) and M. rnazei S-tjT, collection MC3, and LYC (23, 38, 40). Unlike Hutten et al. (16), we found no acidification of culture medium during growth on methanol (or significant alkalinization during growth on acetate), perhaps because our medium was more strongly buffered. Our data were generally consistent with those of previ- ously published studies, but there were some differences between our data and those of Hutten et al. (16); most INJECT notably, we observed good growth of M. barkeri MST on H, 77'Sample loop plus CO, (as did Ferguson and Mah [ll]), whereas Hutten et al. found (16) that growth on this substrate was poor (only FIG. 1. Valve connections for analysis of gas samples. This apparatus collected a 10-pl sample of gas maintained at the pressure twice as fast as growth on acetate). of the sample bottle, thus allowing direct analysis of partial pres- The growth rates of cultures grown on acetate were sures of gases. (A) Valve in the load position. The dark lines generally about 10-fold lower than the growth rates of between numbered ports indicate which ports were connected. (B) cultures grown on methanol or on methanol plus H, and Connections made by the valve in the inject position. about 4- to 5-fold lower than the growth rates of cultures grown on trimethylamine. The maximum specific growth rate which we measured for M. rnazei S-6T grown on acetate was about 0.013 h-' (doubling time, 51 h), in contrast to a methanol than on methanol alone has been observed previ- previous report (23) of a doubling time of 16.6 h. Also, ously (ll), but we found that at higher pH values M. barkeri Peinemann et al. (28) reported more rapid growth of M. MST and M. rnazei S-6T grew faster on methanol in the barkeri Fusaro than we found for M. barkeri MST or 227 absence of H,. The finding that Methanosarcina strains are growing on acetate, and Yang and Okos (41) found growth slightly acidophilic confirms data obtained by Hutten et al. rates that were about twofold higher than our growth rates. (16) but contrasts with the results of several other reports These differences may be attributed to differences in culture (lo, 45, 46). It also is consistent with our unpublished media. For instance, our medium did not contain sludge findings that Methanosarcina morphotypes predominate in supernatant, whose presence could have allowed the more acetate-containing enrichment cultures maintained below rapid growth found by Mah (23). Furthermore, if inoculum- pH 7 and Methanothrix morphotypes predominate in cul- produced methane was not included in the calculations, the tures maintained above pH 7. The low pH optima of Meth- specific growth rates may have been overestimated, espe- anosarcina strains may also have caused difficulties in cially when they were based on time points early in the enriching Methanosarcina cultures from digestors (25). growth curve. The higher rates of Yang and Okos (41) may M. rnazei S-tjT grew well on H, plus CO, in this study and also be attributed to differences in methods of calculation. in a study performed by Harris (13), in contrast to the results The growth rates which we measured are consistent with the of previous studies in which workers used strain S-6T (23,24) growth rates of other aceticlastic methanosarcinae and with and M. mazei MC3 (38). This discrepancy may have been the inability of acetate-degrading methanosarcinae signifi- due to changes which may have occurred in the culture or to cantly to colonize the rumen, which has a solids retention 270 MAESTROJUAN AND BOONE INT. J. SYST.BACTERIOL.

A Methamsarcinit barked MS 0.15 -a- Methand, MS medium -t- Methanol + H 9 MS medium -+-Acetate, MS medium *-, MS medium - - -.-- Acetate, mineral medium ------Hydro en, mineral medium 0.01 0 -$- TrimetRyimine, MS mediurr 0.10

Methanosarcha &ked 227 0*05/0 1 Methamsarcha barked 227

0.10

Methanosarcha mazei S-6 0 t

1 Metbanosarcha vacuo/afa 2-761

FIG. 2. Specific growth rates of Methanosarcina cultures at various pH values. time of about 1 day. Methanosarcina strains enriched and Growth in mineral medium and mineral medium containing isolated from the rumen (26) may have been growing there acetate as a catabolic substrate. Growth with H, plus CO, as on methyl compounds, such as methanol released from the substrate was generally slower in mineral medium than in pectins. complex medium (MS medium) (Fig. 2B). Cultures of M. VOL.41, 1991 CHARACTERIZATION OF METHANOSARCZNA STRAINS 27 1 vacuolata grew poorly in such media. When inoculated into mineral medium containing H, plus CO,, these cultures produced methane for a short time and stopped after about -+- Methanol, MS medium 30 to 40% of the expected methane was formed. Then these 1 Trimethylamine, MS medlurr cultures were transferred to fresh mineral medium contain- o.ol - ing H, plus CO,, and they again produced a similar amount of methane and stopped. All four Methanosarcina strains grew in mineral medium to which acetate had been added as the sole catabolic substrate (Fig. 2B). Growth on acetate as a catabolic sub- strate was generally slower in medium containing no other organic compounds, except in the case of M. barkeri 227, which usually grew as rapidly in both media. Smith and Mah (33, 34) also reported rapid growth of strain 227 in mineral medium containing added acetate. However, at pH values of 7 and above, growth was significantly faster in complex medium, suggesting that some anabolic enzymes necessary 0*01 for growth with acetate as a sole carbon and energy source t do not function well at higher pH values. Our finding that M. vacuolata growing on acetate was stimulated in complex media contrasts with the findings of a previous report (43). Likewise, during growth on H, plus CO,, the relative effects of pH on growth rate were similar in complex and mineral medium, except in the case of M. barkeri 227. The growth 0-05~0 range of M. barkeri 227 extended to lower pH values in complex medium than in mineral medium. M. mazei S-6T grew in mineral medium containing added acetate, but when a logarithmically growing culture in MS medium containing acetate was inoculated into mineral medium, a long lag phase (2 to 3 months) occurred. This lag may be compared with previously observed lags of starved cells (8), because in this case the inoculum was growing logarithmically with acetate as a substrate. Subsequent transfers resulted in much shorter lag phases before logarithmic growth resumed. Effect of salinity on growth rate. The inhibitory effect of added NaCl was observed in all of the cultures examined, Methanosacha vacuo/BIcB 2-761 when either trimethylamine or methanol was the substrate (Fig. 3). Exceptions to this general observation were M. barkeri 227 and M. vacuolata Z-761T grown on methanol, 0.01 which showed very small increases in growth rate when the concentration of Na+ was increased from 0.1 to 0.125 M (Fig. 3). All of the strains in this study had been enriched and isolated from anaerobic digestors, but all of them grew on trimethylamine in media with salinities near the salinity of the ocean. M. vacuolata Z-761T was the most sensitive to salinities above 0.5 M Naf. It is unlikely that members of these four Methanosarcina species are the predominant trimethylamine degraders in marine environments. Trimethylamine-using methanogens isolated from sulfate-containing marine sources are obligately FIG. 3. Specific growth rates of Methanosarcina cultures grow- halophilic (9); furthermore, these marine strains differ phylo- ing at different salinities. genetically from the strains used in our study in that the marine strains cannot use acetate. Sulfate-reducing bacteria may outcompete Methanosarcina strains for acetate and H, rapidly at 37 to 42°C. Generally, the relative effects of when sulfate is in excess, so in most marine environments temperature were independent of the catabolic substrate or trimethylamine is the only available methanogenic substrate. other culture conditions. For example, M. barkeri MST and M. acetivorans is the only marine methanogen which can M. mazei S-6T grew fastest at 40 to 42"C, regardless of the catabolize acetate, but this methanogen was isolated from a substrate. For other cultures, growth conditions appeared to kelp bed where sulfate probably was depleted (35). shift the temperature optima slightly, but these small shifts MS medium contains 2 g of yeast extract per liter, and the may not be significant. For instance, M. vacuolata Z-761T and glycine betaine from this quantity of yeast extract may have M. barkeri 227 grew more rapidly at 42 than at 37°C when the been taken up by the methanogens and used as a compatible pH was 6.5, but at a less optimal pH value (pH 7.2) the solute, enhancing the ability of the methanogens to tolerate organisms grew more slowly at 42 than at 37°C. This finding salinity (30). contrasts with other reports (45,46) for M. vacuolata Z-761T. Effect of temperature on growth rate. Figure 4 shows that Effects of growth conditions on morphology. The following all four of the strains tested were mesophiles, growing most four different Methanosarcina forms have been described: 272 MAESTROJUAN AND BOONE INT. J. SYST. BACTERIOL.

0.1 5 co-workers suggested (35) that aggregates are a survival Mehmmm&.mqd MS M. mazei. :Methanol M medium stage for If so, then it may be beneficial for the Methanol: ME medium, pH 7.2 organism to form aggregates when culture conditions shift -0- Trimethylamins, MS medium Acetab, MS medium dramatically and to revert to growth as individual cells when 0.1 0 - conditions are agreeable or when the cells have adapted to the new conditions. This hypothesis is supported by the data presented below. In contrast, Robinson (31) has suggested that disaggregation is a mechanism of dispersal and that M. 0.05 mazei responds to unfavorable conditions by disaggregation and release of individual cells. Zhilina and Zavarzin have described thick-walled microcysts which may be a survival structure (44). Krzycki et al. (18) have suggested that aggre- 0 gates may conserve H,, which is given off by Methano- sarcina strains during growth on acetate, with neighboring cells taking up produced H,. However, Boone et al. (8) have 0.10 shown that H, pressures below 100 Pa have little effect on aceticlastic cultures. (i) M. barkeri. M. barkeri MST usually grew as small aggregates of cells which settled to the bottom of the culture 5 0.05 vessels but could be dispersed easily to produce a uniform turbidity. We never found single cells of M. barkeri which 8 were sensitive to lysis by sodium dodecyl sulfate. However, during the slow growth which occurred in mineral medium $0 (H2 plus CO, as the catabolic substrate), the aggregates were 0.15 considerably larger and were often visible to the naked eye, although they were not as large as the aggregates of M. mazei. M. barkeri MST in MS medium containing a high fi concentration (60 mM) of trimethylamine grew as a dense 0.10 R culture of very small aggregates (only two to eight cells per clump). M. barkeri Fusaro growing on methanol also pro- duces aggregates when conditions are poor (32), and M. 0.05 barkeri UBS grows as aggregates in defined medium (32). (ii) M. mazei. Like Harris (13), we found that M. rnazei S-6' could disaggregate to individual coccoid cells during growth in liquid medium and that individual cells (but not 0 aggregates) lysed when they were treated with sodium dodecyl sulfate (9). Previous reports (23, 38) have indicated that cells are resistant to lysis. Although we grew and examined cultures of M. maxi under many different condi- 0.10 tions and found changes in the morphology of cells, we were not able to determine definitively what conditions led to growth as aggregates or individual cells. Methanosarcina 0.05 thermophila TM-lT disaggregates and grows as individual cells in marine media (36), but we found no correlation between the formation of aggregates by M. mazei and the salinity of the culture medium. In general, we observed that 0 slow-growing cultures (i.e., cultures under extreme condi- tions relative to the conditions that supported the maximal growth rate) were more likely to grow as aggregates. How- FIG. 4. Specific growth rates of Methunosavcina cultures grow- ever, often one subculture had a different morphology than ing at different temperatures. another subculture of the same strain under identical condi- tions. We were unable to find a pattern to these inconsisten- cies, but it seemed that cultures which had been subjected to recent shifts in growth conditions were more likely to grow individual cells surrounded by only a protein , as aggregates. This was especially true when the shift was to microcysts (31,44), and small and large aggregates of cells or a less favorable medium, as when cells were transferred pseudosarcinae (13, 21, 23, 31, 32, 42, 44). The morphology from another catabolic substrate to acetate (8, 25, 33). When of the cells could usually be determined by simply looking at the growth conditions of cultures containing individual cells our cultures. Turbid cultures consisted mainly of small were changed profoundly, a long lag phase often ensued, aggregates or individual cells; in contrast, large aggregates with cells sinking to the bottom of the vial. This sediment (when present) could be seen with the unaided eye. Cultures often became viscous, and after several weeks growth containing such aggregates usually were devoid of turbidity. slowly resumed as a few visible aggregates formed. Gas These gross observations were confirmed by microscopic production was very slow at first and increased logarithmi- examination. cally, suggesting that a small fraction of the original inocu- Several hypotheses have been advanced to explain aggre- lum had recovered and begun to grow. A similar phenome- gation and disaggregation as survival strategies. Sowers and non was reported previously (40) for the recovery cultures of VOL. 41, 1991 CHARACTERIZATION OF METHANOSARCINA STRAINS 273

YE,T YE T Betaine YE ash FIG. 5. Effects of culture conditions on the morphology of M. mazei S-6=. Inocula were grown in MS medium containing H, plus CO,; these cells were coccoid. After transfer to mineral media containing various additions (still with H, plus CO, as the catabolic substrate), the cells began to grow as aggregates. Specific growth rates were measured after adaptation of the cultures to the test conditions, and the numbers of transfers required before the morphology reverted to coccoid cells are shown. Abbreviations for additions to mineral medium: YE, 2 g of yeast extract per liter; T, 2 g of Trypticase peptones per liter; Vitamins, 10 mi of a vitamin solution (39) per liter; CoM, 0.5 g of per liter; Ac, 0.5 mM sodium acetate; betaine, 5 mM glycine betaine; YE ash, ash (SSOOC, 1 h) from a preparation containing 2 g of yeast extract per liter. aggregates from single Methanosarcina cells. Such cells plus CO, rapidly (24). However, we found that in our media usually continued to grow as aggregates when they were M. mazei S-6T rapidly catabolizes H, plus CO,, so this is not transferred to fresh medium under identical conditions. a characteristic by which these species can be differentiated. After several transfers, the cells often disaggregated sponta- Also, M. vacuolata, which is known to be closely related to neously (as previously described [40]) and grew as single M. barkeri (4, 46), uses H, plus CO, only poorly. cells. When M. mazei S-6* growing on H, plus CO, as the The major characteristics that distinguish M. mazei from catabolic substrate was shifted from complex (MS) medium the other two species appear to be morphology and pH to less complete medium (such as mineral medium), cells range. M. mazei often forms individual coccoid units which grew as aggregates. Sometimes cells continued to grow as are susceptible to lysis by sodium dodecyl sulfate, whereas aggregates for as many as 10 transfers and then disaggre- M. barkeri and M. vacuolata rarely grow as individual cells, gated into individual cells (Fig. 5). In general, cultures and when they do, these cells remain resistant to lysis. growing under better conditions took less time (fewer trans- Physiologically, M. mazei prefers higher pH values than M. fers) to revert to individual cells than did cultures growing barkeri and M. vacuolata do. under extreme conditions (Fig. 5). (ii) Differentiating M. vacuolata and M. barkeri. M. vacuo- M. mazei LYC disaggregates when the pH is shifted from lata is closely related to M. barkeri, with 61% sequence 6 to 7 (21), and likewise we found that M. mazei S-6T similarity between the type strains (46). M. barkeri cell disaggregated more rapidly at pH values closer to its opti- membranes contain no squalenes, but those of M. vacuolata mum pH (data not shown). do (20,27). Gas vesicles occur in M. vacuolata but not in M. (iii) M. vacuolata. In contrast to previous findings (43, 43, barkeri. However, the taxonomic usefulness of gas vesicles we found that M. vacuolata Z-761T changed during growth in methanogens is questionable because it is not a stable from small to large aggregates. This strain grew as small characteristic (23a). At least two other gas-vacuolated Meth- clumps which appeared as a uniform turbidity during rapid anosarcina strains have been reported (strain W [4] and growth, such as when it was grown on methanol at pH 6. strain FR-1 [l-3]), and if these strains were found to form a Then, when growth was complete or almost complete, larger phylogenetically cohesive group with M. vacuolata Z-761T, aggregates visible to the unaided eye occurred as a sediment it would support the view that M. vacuolata is a species at the bottom of the culture vessel. Also, small aggregates of separate from M. barkeri. M. vacuolata Z-761T grew into larger aggregates when the culture was shifted from complex medium to defined me- ACKNOWLEDGMENTS dium (i.e., mineral medium containing acetate added as the This study was supported by contract 5086-260-1303E from the catabolic substrate). This may be analogous to the shift of M. Gas Research Institute as part of a joint program on methane from mazei S-ST from individual cells to large aggregates under biomass sponsored by the Gas Research Institute and the University similar conditions. of Florida Institute of Food and Agricultural Sciences and by the . (i) Differentiating M. mazei from M. barkeri and Consejo Superior de Investigaciones Cientificas, Spain. M. vacuolata. When M. maxi was transferred to the genus REFERENCES Methanosarcina, its differentiation from M. barkeri was 1. Archer, D. B. 1984. Hydrogen-using bacteria in a methanogenic based mainly on the inability of M. mazei to catabolize H, acetate enrichment culture. J. Appl. Bacteriol. 56:125-129. 274 MAESTROJUAN AND BOONE INT. J. SYST.BACTERIOL.

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