Biomedical subjects

S H Zinder

Publications and source records attributed to S H Zinder.

31 records · Page 2Linked to original sources

PubMed1989

Kinetics of Acetate Utilization by Two Thermophilic Acetotrophic Methanogens: Methanosarcina sp. Strain CALS-1 and Methanothrix sp. Strain CALS-1.

The kinetics of acetate utilization were examined for washed concentrated cell suspensions of two thermophilic acetotrophic methanogens isolated from a 58 degrees C anaerobic digestor. Progress curves for acetate utilization by cells of Methanosarcina sp. strain CALS-1 showed that the utilization rate was concentration independent (zero order) above concentrations near 3 mM and that acetate utilization ceased when a threshold concentration near 1 mM was reached. Acetate utilization by cells of Methanothrix sp. strain CALS-1 was concentration independent down to 0.1 to 0.2 mM, and threshold values of 12 to 21 muM were observed. Typical utilization rates in the concentration-independent stage were 210 and 130 nmol min mg of protein for the methanosarcina and the methanothrix, respectively. These results are in agreement with a general model in which high acetate concentrations favor Methanosarcina spp., while low concentrations favor Methanothrix spp. However, acetate utilization by these two strains did not follow simple Michaelis-Menton kinetics.

Journal Article↗

PubMed1988

Hydrogen partial pressures in a thermophilic acetate-oxidizing methanogenic coculture.

Hydrogen partial pressures were measured in a thermophilic coculture comprised of a eubacterial rod which oxidized acetate to H(2) and CO(2) and a hydrogenotrophic methanogen, Methanobacterium sp. strain THF. Zinder and Koch (S. H. Zinder and M. Koch, Arch. Microbiol. 138:263-272, 1984) originally predicted, on the basis of calculations of Gibbs free energies of reactions, that the H(2) partial pressure near the midpoint of growth of the coculture should be near 4 Pa (ca. 4 x 10 atm; ca. 0.024 muM dissolved H(2)) for both organisms to be able to conserve energy for growth. H(2) partial pressures in the coculture were measured to be between 20 and 50 Pa (0.12 to 0.30 muM) during acetate utilization, approximately one order of magnitude higher than originally predicted. However, when DeltaG(f) (free energy of formation) values were corrected for 60 degrees C by using the relationship DeltaG(f) = DeltaH(f) - TDeltaS (DeltaH(f) is the enthalpy or heat of formation, DeltaS is the entropy value, and T is the temperature in kelvins), the predicted value was near 15 Pa, in closer agreement with the experimentally determined values. The coculture also oxidized ethanol to acetate, a more thermodynamically favorable reaction than oxidation of acetate to CO(2). During ethanol oxidation, the H(2) partial pressure reached values as high as 200 Pa. Acetate was not used until after the ethanol was consumed and the H(2) partial pressure decreased to 40 to 50 Pa. After acetate utilization, H(2) partial pressures fell to approximately 10 Pa and remained there, indicating a threshold for H(2) utilization by the methanogen. Axenic cultures of the acetate-oxidizing organism were combined with pure cultures of either Methanobacterium sp. strain THF or Methanobacterium thermoautotrophicum DeltaH to form reconstituted acetate-oxidizing cocultures. The H(2) partial pressures measured in both of these reconstituted cocultures were similar to those measured in the original acetate-oxidizing rod coculture. Since M. thermoautotrophicum DeltaH did not use formate as a substrate, formate is not necessarily involved in interspecies electron transfer in this coculture.

Journal Article↗

PubMed1988

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227.

Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N(2)-grown cultures than in NH(4)-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 muM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 muM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h mg of protein), and they "switched off" acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C(2)H(4) formed h mg of protein when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C(2)H(2) reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.

Journal Article↗

PubMed1985

Nutritional Requirements of Methanosarcina sp. Strain TM-1.

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO(2), and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl(2) . 2H(2)O (13.6 muM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 muM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 muM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO(2) for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO(2). When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH(4) concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH(4) limitation.

Journal Article↗

PubMed1985

Growth substrate effects on acetate and methanol catabolism in Methanosarcina sp. strain TM-1.

When Methanosarcina sp. strain TM-1 is grown in medium in which both methanol and acetate are present, growth is biphasic, with methanol used as the primary catabolic substrate during the first phase. To better understand this phenomenon, we grew cells on methanol or on acetate or on both and examined the abilities of anaerobically washed cells to catabolize these substrates. Washed acetate-grown cells incubated with 10 mM acetate, 10 mM methanol, or both substrates together produced methane at initial rates of 325, 3, and 315 nmol min-1 mg of protein-1, respectively. Although the initial rate of methanogenesis from both substrates was nearly identical to the rate for acetate alone, after several hours of incubation the rate was greater for cells provided with both substrates. Studies with 14C-labeled methanol indicated that methanol was catabolized to methane at increasing rates by acetate-grown cells in a manner reminiscent of an induction curve, but only when cells were provided with acetate as a cosubstrate. Acetate was presumably providing energy and carbon for induction of methanol-catabolic enzymes. Methanol-grown cells showed a pattern of substrate utilization significantly different from that of acetate-grown cells, producing methane from 10 mM acetate, 10 mM methanol, or both substrates at initial rates of 10, 280, and 450 nmol min-1 mg of protein-1, respectively. There was significant oxidation of the methyl group of acetate during metabolism of both substrates. Cells grown on methanol-acetate and harvested before methanol depletion (methanol phase) showed catabolic patterns nearly identical to those of methanol-grown cells, including a low rate of methanogenesis from acetate. Cells harvested from methanol-acetate cultures in the acetate phase were capable of significant methanogenesis from either methanol or acetate alone, and the rate from both substrates together was nearly equal to the sum of the rates for the single substrates. When both 10 mM methanol and 10 mM acetate were presented to the acetate-phase cells, there was a preference for the methanol. These results are consistent with a model for regulation in Methanosarcina sp. strain TM-1 in which methanol represses acetate catabolism while methanol catabolism is inducible.

Acetates↗

PubMed1984

Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor: Methanothrix sp. as an Important Aceticlastic Methanogen.

Aceticlastic methanogens and other microbial groups were enumerated in a 58 degrees C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (10 to 10 CFU/ml) and autofluorescent Methanosarcina-like clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 10 to 10/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 mumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent K(m) value obtained for methanogenesis from acetate, 0.3 mumol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. CO(2)-reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 mumol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 mumol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low K(m) value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.

Journal Article↗

PubMed1984

Effects of Temperature on Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor.

The short-term effects of temperature on methanogenesis from acetate or CO(2) in a thermophilic (58 degrees C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with C-labeled methane precursors (CH(3)COO or CO(2)). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60 degrees C and was completely inhibited at 65 degrees C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58 degrees C and did not grow or produce methane at 65 degrees C. An accidental shift of digestor temperature from 58 to 64 degrees C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from CH(3)COO was optimal at 65 degrees C and completely inhibited at 75 degrees C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70 degrees C. Methanogenesis from CO(2) in the sludge was optimal at 65 degrees C and still proceeded at 75 degrees C. A CO(2)-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75 degrees C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65 degrees C produced more methane than sludge incubated at 60 degrees C, and no acetate accumulated at 65 degrees C. Methanogenesis was severely inhibited in sludge incubated at 70 degrees C, but since neither acetate nor H(2) accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.

Journal Article↗

PubMed1984

Selective inhibition by 2-bromoethanesulfonate of methanogenesis from acetate in a thermophilic anaerobic digestor.

The effects of 2-bromoethanesulfonate, an inhibitor of methanogenesis, on metabolism in sludge from a thermophilic (58 degrees C) anaerobic digestor were studied. It was found from short-term experiments that 1 mumol of 2-bromoethanesulfonate per ml completely inhibited methanogenesis from CH(3)COO, whereas 50 mumol/ml was required for complete inhibition of CO(2) reduction. When 1 mumol of 2-bromoethanesulfonate per ml was added to actively metabolizing sludge which was then incubated for 24 h. it caused a 60% reduction in methanogenesis and a corresponding increase in acetate accumulation; at 50 mumol/ml it caused complete inhibition of methanogenesis and accumulation of acetate. H(2), and ethanol.

Journal Article↗

PubMed1979

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.

Journal Article↗

PubMed1978

Dimethyl sulfoxide as an electron acceptor for anaerobic growth.

The isolation from lake mud of a bacterium which can use dimethyl sulfoxide (DMSO) as an electron acceptor for growth is described. The isolate, called strain DL-1, was a small, gram negative, non-motile spiral. The sole product of DMSO reduction was dimethyl sulfide (DMS). Other electron acceptors used by the isolate included sulfite, thiosulfate, elemental sulfur, methionine sulfoxide, tetramethylene sulfoxide, nitrate, and oxygen (microaerophilically). Sulfate was not reduced and could not even be assimilated. Lactate or succinate could serve as electron donors, with acetate as the main product. Hydrogen could be used as an electron donor if acetate was present in the medium as a carbon source. The organism has a c-type cytochrome, and most likely uses electron transport phosphorylation during DMSO reduction. Cultures of Desulfovibrio sp., Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris were tested for growth using DMSO as an electron acceptor, and only the Proteus strain grew. Both Proteus and strain DL-1 are versatile at coupling reductions with energy generation. There is a marked resemblance between strain DL-1 and the recently described sulfur-reducing spirillum of Wolfe and Pfennig.

Bacteria↗

PubMed1978

Dimethyl sulphoxide reduction by micro-organisms.

Dimethyl sulphoxide (DMSO) was reduced to dimethyl sulphide by a wide variety of micro-organism, including prokaryotes and eukaryotes, aerobes and anaerobes. Dimethyl sulphone was not reduced by any of the organisms tested. Cell-free extracts of Escherichia coli reduced DMSO using reduced pyridine nucleotides as electron donors. Activity was greater in anaerobically grown cells than in those grown aerobically. Two other sulphoxides, methionine sulphoxide and tetramethylene sulphoxide, substantially inhibited DMSO reduction by extracts. Mutants of E. coli, which were unable to reduce biotin sulphoxide to biotin, were tested for their ability to reduce DMSO in whole cells and extracts. These mutants were in four different gene loci, bisA to bisD. DMSO reductase activity of the mutants was generally less than that of the wild-type strain, and activity depended upon the gene locus involved, the growth medium and the growth conditions. Only the bisA mutant had very low activity under all conditions. All of the bis mutants were able to grow using methionine sulphoxide as a sulphur source, indicating that biotin sulphoxide and methionine sulphoxide are reduced by different enzyme systems. DMSO may be reduced by both of these enzyme systems.

Anaerobiosis↗

PubMed1978

Methane, carbon dioxide, and hydrogen sulfide production from the terminal methiol group of methionine by anaerobic lake sediments.

A significant portion of the sulfide in lake sediments may be derived from sulfur-containing amino acids. Methionine degradation in Lake Mendota (Wisconsin) sediments was studied with gas chromatographic and radiotracer techniques. Temperature optimum and inhibitor studies showed that this process was biological. Methane thiol and dimethyl sulfide were produced in sediments when 1-mumol/ml unlabeled methionine was added. When chloroform (an inhibitor of one-carbon metabolism) was added to the sediments, methane thiol, carbon disulfide, and n-propane thiol were produced, even when no methionine was added. When S-labeled methionine was added to the sediments in tracer quantities (1.75 nmol/ml), labeled hydrogen sulfide was produced, and a roughly equal amount of label was incorporated into insoluble material. Methane and carbon dioxide were produced from [methyl-C]methionine. Evidence is given favoring methane thiol as an intermediate in the formation of methane, carbon dioxide, and hydrogen sulfide from the terminal methiol group of methionine. Methionine may be an important source of sulfide in lake sediments.

Journal Article↗

PubMed1977

Production of volatile sulfur compounds during the decomposition of algal mats.

Blue-green algal mats incubated anaerobically rapidly produce large amounts of volatile sulfur compounds, including hydrogen sulfide, methyl mercaptan, and dimethyl sulfide. The major organic sulfur compound is methyl mercaptan, in contrast to previous results with marine eucaryotic algae. Light inhibited production of volatile sulfur compounds, apparently because the algae then produced O2, rendering the system aerobic.

Anaerobiosis↗