Effect of H(2)-CO(2) on Methanogenesis from Acetate or Methanol in Methanosarcina spp

Growth of Methanosarcina sp. strain 227 and Methanosarcina mazei on H(2)-CO(2) and mixtures of H(2)-CO(2) and acetate or methanol was examined. The growth yield of strain 227 on H(2)-CO(2) in complex medium was 8.4 mg/mmol of methane produced. Growth in defined medium was characteristically slower, and cell yields were proportionately lower. Labeling studies confirmed that CO(2) was rapidly reduced to CH(4) in the presence of H(2), and little acetate was used for methanogenesis until H(2) was exhausted. This resulted in a biphasic pattern of growth similar to that reported for strain 227 grown on methanol-acetate mixtures. Biphasic growth was not observed in cultures on mixtures of H(2)-CO(2) and methanol, and less methanol oxidation occurred in the presence of H(2). In M. mazei the aceticlastic reaction was also inhibited by the added H(2), but since the cultures did not immediately metabolize H(2), the duration of the inhibition was much longer.     

Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.     

Metabolism of reduced methylated sulfur compounds in anaerobic sediments and by a pure culture of an estuarine methanogen

Addition of dimethylsulfide (DMS), dimethyldisulfide (DMDS), or methane thiol (MSH) to a diversity of anoxic aquatic sediments (e.g., fresh water, estuarine, alkaline/hypersaline) stimulated methane production. The yield of methane recovered from DMS was often 52 to 63%, although high concentrations of DMS (as well as MSH and DMDS) inhibited methanogenesis in some types of sediments. Production of methane from these reduced methylated sulfur compounds was blocked by 2-bromoethanesulfonic acid. Sulfate did not influence the metabolism of millimolar levels of DMS, DMDS, or MSH added to sediments. However, when DMS was added at approximately 2-muM levels as [C]DMS, metabolism by sediments resulted in a CH(4)/CO(2) ratio of only 0.06. Addition of molybdate increased the ratio to 1.8, while 2-bromoethanesulfonic acid decreased it to 0, but did not block CO(2) production. These results indicate the methanogens and sulfate reducers compete for DMS when it is present at low concentrations; however, at high concentrations, DMS is a "noncompetitive" substrate for methanogens. Metabolism of DMS by sediments resulted in the appearance of MSH as a transient intermediate. A pure culture of an obligately methylotrophic estuarine methanogen was isolated which was capable of growth on DMS. Metabolism of DMS by the culture also resulted in the transient appearance of MSH, but the organism could grow on neither MSH nor DMDS. The culture metabolized [C]-DMS to yield a CH(4)/CO(2) ratio of approximately 2.8. Reduced methylated sulfur compounds represent a new class of substrates for methanogens and may be potential precursors of methane in a variety of aquatic habitats.     

In vivo role of three fused corrinoid/methyl transfer proteins in Methanosarcina acetivorans

Methanosarcina acetivorans is able to use carbon monoxide (CO) as the sole source of energy for growth. Its carboxidotrophic growth is peculiar as it involves formation of acetate, formate and methylated thiols, besides methane. Under this condition three proteins homologous to both corrinoid proteins and methyltransferases (MA0859, MA4384 and MA4558) are highly abundant. To address their role in M. acetivorans , a set of single and double mutants, and the triple mutant, was constructed by deletion/disruption of the encoding genes. Phenotypic analysis of the mutants rules out an important role of the methyltransferase homologues in the CO₂ reduction pathway of methanogenesis. Instead, the single and double mutants were affected to various degrees in their capacity to generate dimethylsulphide (DMS) from CO and to form methane from DMS. The triple mutant was unable to produce or metabolize DMS, and could not grow with DMS as the sole energy source, which demonstrates that MA0859, MA4384 and MA4558 are involved in, and required for, methylsulphide metabolism of M. acetivorans . Based on these findings we propose to designate MA0859, MA4384 and MA4558 as m ethyl t ransferases specific for methyl s ulphides, MtsD, MtsF and MtsH respectively.     

Methanethiol degradation in anaerobic bioreactors at elevated pH (8): reactor performance and microbial community analysis

The degradation of methanethiol (MT) at 30 degrees C under saline-alkaline (pH 8-10, 0.5M Na(+)) conditions was studied in a lab-scale Upflow Anaerobic Sludge Blanket (UASB) reactor inoculated with estuarine sediment from the Wadden Sea (The Netherlands). At a sodium concentration of 0.5M and a pH between 8 and 9 complete MT degradation to sulfide, methane and carbon dioxide was possible at a maximum loading rate of 22mmolMTL(-1)day(-1) and a hydraulic retention time of 6h. The presence of yeast extract (100mg/L) in the medium was essential for complete MT degradation. 16S rRNA based DGGE and sequence analysis revealed that species related to the genera Methanolobus and Methanosarcina dominated the archaeal community in the reactor sludge. Their relative abundance fluctuated in time, possibly as a result of the changing operational conditions in the reactor. The most dominant MT-degrading archaeon was enriched from the reactor and obtained in pure culture. This strain WR1, which was most closely related to Methanolobus taylorii, degraded MT, dimethyl sulfide (DMS), methanol and trimethylamine. Its optimal growth conditions were 0.2M NaCl, 30 degrees C and pH 8.4. In batch and reactor experiments operated at pH 10, MT was not degraded.     

Effect of osmotic potential, pH, and temperature on the growth of a helical, motile mycoplasma causing corn stunt disease.

Growth characteristics of corn stunt spiroplasma, a helical, motile mycoplasma, were studied over a range of osmolality, pH, and temperature in a simple medium containing 20% (v/v) agamma horse serum, 1.5% (w/v) PPLO broth, and various concentrations of sucrose. The spiroplasma was able to grow in a wide spectrum of osmolalities from 360 to 1120 mosm. Optimal growth was observed in media that contained 0.25-0.35 M sucrose. The organism became longer and thinner in media adjusted to 0.65 M sucrose or more. The spiroplasma lost helicity and motility immediately after transfer to media at pH 5.4 or lower. Optimal pH for growth was 7.2. No growth was observed at pH lower than 5.4 or higher than 8.0. Optimal temperature for growth was 32 degrees C. Very little or no growth was observed at temperatures lower than 15 degrees C or higher than 35 degrees C.     

Seasonal changes in fluxes of methane and volatile sulfur compounds from rice paddies and their concentrations in soil water

Rice paddies emit not only methane but also several volatile sulfur compounds such as dimethyl sulfide (DMS: CH₃SCH₃). However, little is known about DMS emission from rice paddies. Fluxes of methane and DMS, and the concentrations of methane and several volatile sulfur compounds including hydrogen sulfide (H₂S), carbonyl disulfide (CS₂), methyl mercaptan (CH₃SH) and DMS in soil water and flood water were measured in four lysimeter rice paddies (2.5 × 4 m, depth 2.0 m) once per week throughout the entire cultivation period in 1995 in Tsukuba, Japan. The addition of exogenous organic matter (rice straw) was also examined for its influence on methane or DMS emissions. Methane fluxes greatly differed between treatments in which rice straw had been incorporated into the paddy soil (rice straw plot) and plots without rice straw (mineral fertilizer plot). The annual methane emission from the rice straw plots (37.7 g m^-2) was approximately 8 times higher than that from the mineral fertilizer plots (4.8 g m^-2). Application of rice straw had little influence on DMS fluxes. Significant diurnal and seasonal changes in DMS fluxes were observed. Peak DMS fluxes were found around noon. DMS was emitted from the flood water in the early growth stage of rice and began to be emitted from rice plants during the middle stage. DMS fluxes increased with the growth of rice plants and the highest flux, 15.1 µg m^-2 h^-1, was recorded before heading. DMS in the soil water was negligible during the entire cultivation period. These facts indicate that the DMS emitted from rice paddies is produced by metabolic processes in rice plants. The total amount of DMS emitted from rice paddies over the cultivated period was estimated to be approximately 5–6 mg m^-2. CH₃SH was emitted only from flood water during the first month after flooding.     

Microbial Populations Involved in Cycling of Dimethyl Sulfide and Methanethiol in Freshwater Sediments

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.     

Isolation and Characterization of a Thermophilic Strain of Methanosarcina Unable to Use H(2)-CO(2) for Methanogenesis

A thermophilic strain of Methanosarcina, designated Methanosarcina strain TM-1, was isolated from a laboratory-scale 55 degrees C anaerobic sludge digestor by the Hungate roll-tube technique. Penicillin and d-cycloserine, inhibitors of peptidoglycan synthesis, were used as selective agents to eliminate contaminating non-methanogens. Methanosarcina strain TM-1 had a temperature optimum for methanogenesis near 50 degrees C and grew at 55 degrees C but not at 60 degrees C. Substrates used for methanogenesis and growth by Methanosarcina strain TM-1 were acetate (12-h doubling time), methanol (7- to 10-h doubling time), methanol-acetate mixtures (5-h doubling time), methylamine, and trimethylamine. When radioactively labeled acetate was the sole methanogenic substrate added to the growth medium, it was predominantly split to methane and carbon dioxide. When methanol was also present in the medium, the metabolism of acetate shifted to its oxidation and incorporation into cell material. Electrons derived from acetate oxidation apparently were used to reduce methanol. H(2)-CO(2) was not used for growth and methanogenesis by Methanosarcina strain TM-1. When presented with both H(2)-CO(2) and methanol, Methanosarcina strain TM-1 was capable of limited hydrogen metabolism during growth on methanol, but hydrogen metabolism ceased once the methanol was depleted. Methanosarcina strain TM-1 required a growth factor (or growth factors) present in the supernatant of anaerobic digestor sludge. Growth factor requirements and the inability to use H(2)-CO(2) are characteristics not found in other described Methanosarcina strains. The high numbers of Methanosarcina-like clumps in sludges from thermophilic digestors and the fast generation times reported here for Methanosarcina TM-1 indicate that Methanosarcina may play an important role in thermophilic methanogenesis.     

Role of Methanogens and Other Bacteria in Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments

The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 μmol of DMS was stoichiometrically converted into 112 μmol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.     

Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

Anoxic salt marsh sediments were amended with dl-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant (1 to 10 muM) concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 muM) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO(2) and only small amounts of CH(4). At higher (>100 muM) concentrations of methionine, the proportion of the methyl-sulfur group converted to CH(4) increased. The results of this study demonstrate that methionine and DMSP are potential precursors of methylated sulfur compounds in anoxic sediments and that the microbial community is capable of metabolizing volatile methylated sulfur compounds.     

Anaerobic methanethiol degradation and methanogenic community analysis in an alkaline (pH 10) biological process for liquefied petroleum gas desulfurization

Anaerobic methanethiol (MT) degradation by mesophilic (30 degrees C) alkaliphilic (pH 10) communities was studied in a lab-scale Upflow Anaerobic Sludge Bed (UASB) reactor inoculated with a mixture of sediments from the Wadden Sea (The Netherlands), Soap Lake (Central Washington), and Russian soda lakes. MT degradation started after 32 days of incubation. During the first 252 days, complete degradation was achieved till a volumetric loading rate of 7.5 mmol MT/L/day, and sulfide, methane, and carbon dioxide were the main reaction products. Temporary inhibition of MT degradation occurred after MT peak loads and in the presence of dimethyl disulfide (DMDS), which is the autooxidation product of MT. From day 252 onwards, methanol was dosed to the reactor as co-substrate at a loading rate of 3-6 mmol/L/day to stimulate growth of methylotrophic methanogens. Methanol was completely degraded and also a complete MT degradation was achieved till a volumetric loading rate of 13 mmol MT/L/day (0.77 mmol MT/gVSS/day). However, from day 354 till the end of the experimental run (day 365), acetate was formed and MT was not completely degraded anymore, indicating that methanol-degrading homoacetogenic bacteria had partially outcompeted the methanogenic MT-degrading archea. The archeal community in the reactor sludge was analyzed by DGGE and sequencing of 16S rRNA genes. The methanogenic archea responsible for the degradation of MT in the reactor were related to Methanolobus oregonensis. A pure culture, named strain SODA, was obtained by serial dilutions in medium containing both trimethyl amine and dimethyl sulfide (DMS). Strain SODA degraded MT, DMS, trimethyl amine, and methanol. Flow sheet simulations revealed that for sufficient MT removal from liquefied petroleum gas, the extraction and biological degradation process should be operated above pH 9.     

Role of methanogens and other bacteria in degradation of dimethyl sulfide and methanethiol in anoxic freshwater sediments

The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 micromol of DMS was stoichiometrically converted into 112 micromol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.     

Aerobic and anaerobic degradation of a range of alkyl sulfides by a denitrifying marine bacterium.

A pure culture of a bacterium was obtained from a marine microbial mat by using an anoxic medium containing dimethyl sulfide (DMS) and nitrate. The isolate grew aerobically or anaerobically as a denitrifier on alkyl sulfides, including DMS, dimethyl disulfide, diethyl sulfide (DES), ethyl methyl sulfide, dipropyl sulfide, dibutyl sulfide, and dibutyl disulfide. Cells grown on an alkyl sulfide or disulfide also oxidized the corresponding thiols, namely, methanethiol, ethanethiol, propanethiol, or butanethiol. Alkyl sulfides were metabolized by induced or derepressed cells with oxygen, nitrate, or nitrite as electron acceptor. Cells grown on DMS immediately metabolized DMS, but there was a lag before DES was consumed; with DES-grown cells, DES was immediately used but DMS was used only after a lag. Chloramphenicol prevented the eventual use of DES by DMS-grown cells and DMS use by DES-grown cells, respectively, indicating separate enzymes for the metabolism of methyl and ethyl groups. Growth was rapid on formate, acetate, propionate, and butyrate but slow on methanol. The organism also grew chemolithotrophically on thiosulfate with a decrease in pH; growth required carbonate in the medium. Growth on sulfide was also carbonate dependent but slow. The isolate was identified as a Thiobacillus sp. and designated strain ASN-1. It may have utility for removing alkyl sulfides, and also nitrate, nitrite, and sulfide, from wastewaters.     

Isolation and characterization of an h(2)-oxidizing thermophilic methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55 degrees C). This isolate exhibited a temperature optimum of 55 to 60 degrees C and a maximum near 70 degrees C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H(2)-CO(2) but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 mum in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Dimethyl sulfide metabolism in salt marsh sediments

Abstract Anoxic sediment slurries prepared from Spartina salt marsh soils contained dimethyl sulfide (DMS) at concentrations ranging from 1 to 10 μM. DMS was produced in slurries over the initial 1–24 h incubation. After the initial period of production, DMS decreased to undetectable levels and methane thiol (MSH) was produced. Inhibition of methanogenesis caused a 20% decrease in the rate of DMS consumption, while inhibition of sulfate reduction caused a 80% decrease in DMS consumption. When sulfate reduction and methanogenesis were simultaneously inhibited, DMS did not decrease. DMS contributed about 28% to the methane production rate, while DMS probably contributed only 1% or less to the sulfate reduction rate. Incubation of the sediment slurries under an atmosphere of air resulted in similar DMS consumption compared to anaerobic incubations, but MSH and CH₄ were not evolved.
Sediments from the marsh released significant quantities of DMS when treated with cold alkali, indicating that potentially significant sources of DMS existed in the sediments. Values of base-hydrolyzable DMS as high as 190 μmol per liter of sediment were observed near the sediment surface, and values always decreased with depth in the sediment. Simple flux experiments with small intact sediment cores, showed that DMS was emitted from the marsh surface when cores were injected with glutaraldehyde or molybdate and 2-bromoethanesulfonate (BES), but nit when cores were left uninhibited. These results showed that DMS was readily metabolized by microbes in marsh sediments and that this metabolism may be responsible for reducing the emission of DMS from the marsh surface.     

Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol.

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H(2). Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H(2)-CO(2) by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H(2)-CO(2) may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H(2)-CO(2).     

The effect of temperature and selective agents on the growth of Yersinia enterocolitica serotype O:3 in pure culture

This study examined the individual and combined effects of the selective agents normally present in Yersinia-selective agar (i.e. cefsulodin, irgasan and novobiocin) on the growth kinetics of plasmid-bearing (P+) and plasmid-cured (P-) Yersinia enterocolitica serotype O:3 at 25 and 37 degrees C. Growth studies were carried out in pure culture, and the data obtained were subjected to linear regression analysis to determine lag phase duration(s) and growth rates of the examined strains. In general, the presence of selective agents increased the duration of the lag phase at 37 degrees C, with longer lag phases noted in all cases in which two or more selective agents were present. Growth rates in CIN broth base (CIN NA) and CIN NA plus commercial supplement (SR 109) (CIN) were faster at 37 than 25 degrees C, but in some cultures of incomplete CIN NA broth with less than three supplements added, growth tended to be faster at 25 than 37 degrees C. Generally, plasmid-bearing strains grew slower than plasmid-cured strains in most media at 37 degrees C due to virulence plasmid expression retarding growth. In some instances at 37 degrees C, it was observed that the growth rates of both plasmid-bearing and plasmid-cured strains were comparable, indicating the influence of added selective agent/s negating any effects associated with virulence plasmid expression. The effects of selective agents, incubation temperature and virulence plasmid carriage on the growth kinetics of Y. enterocolitica are discussed.     

Dependence of tetrachloroethylene dechlorination on methanogenic substrate consumption by Methanosarcina sp. strain DCM.

Tetrachloroethylene (perchloroethylene, PCE) is a suspected carcinogen and a common groundwater contaminant. Although PCE is highly resistant to aerobic biodegradation, it is subject to reductive dechlorination reactions in a variety of anaerobic habitats. The data presented here clearly establish that axenic cultures of Methanosarcina sp. strain DCM dechlorinate PCE to trichloroethylene and that this is a biological reaction. Growth on methanol, acetate, methylamine, and trimethylamine resulted in PCE dechlorination. The reductive dechlorination of PCE occurred only during methanogenesis, and no dechlorination was noted when CH4 production ceased. There was a clear dependence of the extent of PCE dechlorination on the amount of methanogenic substrate (methanol) consumed. The amount of trichloroethylene formed per millimole of CH4 formed remained essentially constant for a 20-fold range of methanol concentrations and for growth on acetate, methylamine, and trimethylamine. These results suggest that the reducing equivalents for PCE dechlorination are derived from CH4 biosynthesis and that the extent of chloroethylene dechlorination can be enhanced by stimulating methanogenesis. It is proposed that electrons transferred during methanogenesis are diverted to PCE by a reduced electron carrier involved in methane formation.     

Isolate 761M: a New Type I Methanotroph That Possesses a Complete Tricarboxylic Acid Cycle

A methanotrophic bacterium, isolate 761M, grows slowly with methane as the sole carbon and energy source. Growth was stimulated by peptone, casein hydrolysate, glucose, and acetate plus malate. Sugars other than glucose did not stimulate growth. Growth yields, based on the amount of methane consumed, increased when other carbon sources were present, and less methane carbon was assimilated under these conditions. Methane was obligately required for growth of isolate 761M. This bacterium does not grow on rich media. Isolate 761M was found to possess hexulose phosphate synthase and intracytoplasmic membranes characteristic of other type I methanotrophs. Unlike other type I methanotrophs, this bacterium possessed alpha-ketoglutarate dehydrogenase and oxidized [2-¹⁴C]acetate to carbon dioxide.