Diversity and Population Dynamics of Methanogenic Bacteria in a Granular Consortium

Upflow anaerobic sludge blanket bioreactor granules were used as an experimental model microbial consortium to study the dynamics and distribution of methanogens. Immunologic methods revealed a considerable diversity of methanogens that was greater in mesophilic granules than in the same granules 4 months after a temperature shift from 38 to 55°C. During this period, the sizes of the methanogenic subpopulations changed with distinctive profiles after the initial reduction caused by the shift. Methanogens antigenically related to Methanobrevibacter smithii PS and ALI, Methanobacterium hungatei JF1, and Methanosarcina thermophila TM1 increased rapidly, reached a short plateau, and then fell to lower concentrations that persisted for the duration of the experiment. A methanogen related to Methanogenium cariaci JR1 followed a similar profile at the beginning, but it soon diminished below detection levels. Methanothrix rods weakly related to the strain Opfikon increased rapidly, reaching a high-level, long-lasting plateau. Two methanogens related to Methanobrevibacter arboriphilus AZ and Methanobacterium thermoautotrophicum ΔH emerged from very low levels before the temperature shift and multiplied to attain their highest numbers 4 months after the shift. Histochemistry and immunohistochemistry revealed thick layers, globular clusters, and lawns of variable density which were distinctive of the methanogens related to M. thermoautotrophicum ΔH, M. thermophila TM1, and M. arboriphilus AZ and M. soehngenii Opfikon, respectively, in thin sections of granules grown at 55°C for 4 months. Mesophilic granules showed a different pattern of methanogenic subpopulations.     

Direct Characterization of Methanogens in Two High-Rate Anaerobic Biological Reactors

The methanogenic flora from two types of turbulent, high-rate reactors was studied by immunologic methods as well as by phase-contrast, fluorescence, and scanning electron microscopy. The reactors were a fluidized sand-bed biofilm ANITRON reactor and an ultrafiltration membrane-associated suspended growth MARS reactor (both trademarks of Air Products and Chemicals, Inc., Allentown, Pa.). Conventional microscopic methods revealed complex mixtures of microbes of a range of sizes and shapes, among which morphotypes resembling Methanothrix spp. and Methanosarcina spp. were noticed. Precise identification of these and other methanogens was accomplished by antigenic fingerprinting with a comprehensive panel of calibrated antibody probes of predefined specificity spectra. The methanogens identified showed morphotypes and antigenic fingerprints indicating their close similarity with the following reference organisms: Methanobacterium formicicum MF and Methanosarcina barkeri W in the ANITRON reactor only; Methanosarcina barkeri R1M3, M. mazei S6, Methanogenium cariaci JR1, and Methanobrevibacter arboriphilus AZ in the MARS reactor only; and Methanobrevibacter smithii ALI and Methanothrix soehngenii Opfikon in both reactors. Species diversity and distribution appeared to be, at least in part, dependent on the degree of turbulence inside the reactor.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Population dynamics of biofilm development during start-up of a butyrate-degrading fluidized-bed reactor

Summary Population dynamics during start-up of a fluidized-bed reactor with butyrate or butyrate plus acetate as sole substrates as well as biofilm development on the sand substratum were studied microbiologically, immunologically and by scanning electron microscopy. An adapted syntrophic consortium consisting of Syntrophospora sp., Methanothrix soehngenii, Methanosarcina mazei and Methanobrevibacter arboriphilus or Methanogenium sp. achieved high-rate butyrate degradation to methane and carbon dioxide. Desulfovibrio sp., Methanocorpusculum sp., and Methanobacterium sp. were also present in lower numbers. Immunological analysis demonstrated methanogens antigenically related to Methanobrevibacter ruminantium M1, Methanosarcina mazei S6, M. thermophila TM1, Methanobrevibacter arboriphilus AZ and Methanothrix soehngenii Opfikon in the biofilm. Immunological analysis also showed that the organisms isolated from the butyrate-degrading culture used as a source of inoculum were related to M. soehngenii Opfikon, Methanobacterium formicicum MF and Methanospirillum hungatei JF1. Offprint requests to: G. Zellner     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Isolation and Characterization of Methanogenic Bacteria from Landfills

Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of Methanobacterium formicicum, Methanosarcina barkeri, several different immunotypes of Methanobacterium bryantii, and a coccoid methanogen distinct from the reference immunotypes were identified.     

Improved strategy for presumptive identification of methanogens using 16S riboprinting

The predicted 16S riboprint patterns of 10 restriction endonucleases for 26 diverse methanogens were compared to actual patterns produced on agarose gels. The observed patterns corroborated the expected riboprints. Our analyses confirmed that the endonuclease HaeIII gave the best results generating 15 different riboprint sets. Six of these 15 riboprints represented more than one strain. Of these, three riboprint sets were further differentiated: Methanomicrobium mobile, Methanolacinia paynteri, and Methanoplanus petrolearius were differentiated from each other by the endonuclease AluI; Methanofollis liminatans, Methanospirillum hungatei, and Methanoculleus bourgensis were differentiated from each other by HpaII; and the combination of FokI and MluNI was used to differentiate Methanobrevibacter sp. ZA-10, and Methanobrevibacter arboriphilicus strains DH-1, AZ, and DC from each other. We could not differentiate the following pairs of strains from each other: Methanosarcina mazeii S-6 and C16, Methanobacterium bryantii MoH and MoH-G, Methanobacterium thermoautotrophicum GC-1 and DeltaH, and Methanobrevibacter arborophillicus DC and A2. This riboprint strategy provided a simple and rapid method to presumptively identify 22 of the 26 diverse strains of methanogens belonging to 13 genera from a range of environments.     

Anaerobic degradation of sulphite evaporator condensate in a fixed-bed loop reactor by a defined bacterial consortium

Summary After elucidating the composition of an anaerobic bacterial enrichment culture treating sulphite evaporator condensate (SEC), an effluent in the pulp and paper industry, we built up stepwise a defined mixed culture to convert the organic constituents of SEC (acetate, methanol, furfural) to methane and CO₂. In batch cultures Desulfovibrio furfuralis and Methanobacterium bryantii degraded furfural in the absence of sulphate via inter-species H₂ transfer yielding 0.42 mol methane and 1.87 mol acetate/mol furfural degraded. When Methanosarcina barkeri was added to this diculture, acetate was also transformed to methane yielding 0.93 mol methane/mol acetate converted. This consortium (D. furfuralis, Methanobacterium bryantii and Methanosarcina barkeri) degraded furfural in continuous culture (fixed-bed loop reactor) to 92%, but the conversion of acetate was only 67%. The conversion of acetate could be further improved to 86% by adding 10 mm sulphate to the medium. This resulted in a space time yield of 10.9 g chemical oxygen demand (COD)/1 per day for the overall conversion. With a consortium consisting of M. barkeri, Methanobrevibacter arboriphilus, Methanosaeta concilii and D. furfuralis, a synthetic SEC could be degraded at a space time yield of 13.35 g COD/1 per day. This defined culture degraded all the constituents of SEC at an efficiency of almost 90% compared to an enrichment culture under identical conditions.Offprint requests to: U. Ney     

31P-NMR spectra of methanogens: 2,3-cyclopyrophosphoglycerate is detectable only in methanobacteria strains

The unique compound 2,3-cyclopyrophosphoglycerate occurs at a detectable concentration in the genera Methanobacterium and Methanobrevibacter but not in Methanococcus, Methanospirillum and Methanosarcina, as shown by a 31P-NMR survey of several different methanogens. Metabolic poisons (carbonyl cyanide m-chlorophenylhydrazone and valinomycin) do not decrease the level of the cyclic pyrophosphate in Methanobacterium thermoautotrophicum; therefore, it cannot be a phosphagen, i.e., an energy storage material. 13CO2 is rapidly incorporated into this cyclic compound which represents the major soluble carbon as well as the phosphorus component of this methanobacteria. 13C-NMR analysis demonstrates that the pKa of the 2,3-cyclopyrophosphoglycerate carboxyl group is 2.55. The unusual pseudomurein cell wall structure of methano- and methanobrevibacteria necessitates a high demand on carbohydrate metabolism. For this reason, and the fact that when its concentration is decreased no new phosphorus resonances appear in the high resolution spectra, it is suggested that 2,3-cyclopyrophosphoglycerate has a function in carbohydrate metabolism.     

Nickel requirement and factor F430 content of methanogenic bacteria.

Methanobacterium thermoautotrophicum has been reported to require nickel for growth and to contain high concentrations of a nickel tetrapyrrole designated factor F430. In this communication it is shown that all methanogenic bacteria investigated incorporated nickel during growth and also synthesized factor F430. This was also true for Methanobrevibacter smithii, which is dependent on acetate as a carbon source, and for Methanosarcina barkeri growing on acetate or methanol as energy sources. Other bacteria, including Acetobacterium woodii and Clostridium thermoaceticum, contained no factor F430. It is further shown that two yellow nickel-containing degradation products were formed from factor F430 when heated at pH 7. This finding explains why several forms of factor F430 were found in methanogenic bacteria when a heat step was employed in the purification procedure.     

Methanogens revealed immunologically in granules from five Upflow Anaerobic Sludge Blanket (UASB) bioreactors grown on different substrates

Abstract Methanogens were identified and quantified using antibody probes and the antigenic fingerprinting method in five different kinds of granular sludge taken from five Uplow Anaerobic Sludge Blanket (UASB) bioreactors maintained on different substrates. The methanogenic flora present in each bioreactor was elucidated and expressed in cells per garm dry weight. Autofluorescence, phase-contrast and bright field-microscopy of unstained and Gram-stained preparations were used in parallel with immunotechnology to characterize each methanogenic subpopulation. Ten different methanogens were prevalent in the five bioreactor systems. Methanogens antigenically related to Methanobacterium formicicum MF, Methanobrevibacter arboriphilus AZ and Methanothrix soehngenii Opfikon were found in all five granules, while other methanogens were present in only some. A trend was observed towards a wider diversity of methanogenic subpopulations parallelling an increase in the complexity of the bioreactor's substrate.     

Inhibition of methanogenesis by several heavy metals using pure cultures

The effect of different concentrations of nickel, copper and zinc on methanogenesis using pure cultures of Methanobacterium formicicum, Methanobrevibacter arboriphilicus, Methanosarcina thermophila and Methanospirillum hungatei over time (1, 15 and 30 d) was evaluated. methanobacterium formicicum showed the highest resistance to all the metals tested, while Methanospirillum hungatei was the most sensitive strain. All strains were sensitive to copper and zinc (10–250 mg 1^-1, but were much more resistant to nickel (200–1200 mg 1^-1). An adaptation process of the methanogenic pure culture with the toxicants was observed over time, which indicates that the inhibitory effects of heavy metals may be reverted in optimal anaerobic conditions.     

Composition and Role of Extracellular Polymers in Methanogenic Granules

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.     

Sediment Distribution of Methanogenic Bacteria in Lake Erie and Cleveland Harbor

The direct fluorescent-antibody technique was employed to determine the distribution patterns of four species of methanogens in the sediments of Lake Erie and Cleveland Harbor. Methanobacterium ruminantium was the most numerous methanogen found in regions of high-organic-silt sediments. The population of this species ranged from 10⁶ to 10⁹ cells/g of dry sediment. Methanobacterium strain MoH and Methanosarcina barkeri were identified in sand-silt, clay, or sand sediments. These methanogens ranged in density from 10⁶ to 10⁷ cells/g of dry sediment. Methanospirillum hungatii was observed only after an organic enrichment was performed on Cleveland Harbor sediments. The seasonal and selective sediment distribution of these methanogens appears to be related to the type and concentration of carbon as substrate as well as to the activities of heterotrophic and sulfate-reducing bacteria.     

A simple chromatographic procedure for the detection of cyclized archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids

Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.     

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H₂-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.     

Characterization of a Heme-Dependent Catalase from Methanobrevibacter arboriphilus

Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity. This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri.     

Enzyme-Linked Immunosorbent Assays for the Specific and Sensitive Quantification of Methanosarcina mazei and Methanobacterium bryantii

Three microtitration plate enzyme-linked immunosorbent assays (ELISAs) have been developed: a competitive ELISA and a two-site (or indirect sandwich) ELISA for Methanosarcina mazei S6 and a two-site ELISA for Methanobacterium bryantii FR-2. The assays were sensitive, with limits of cell protein detection of 3 ng ml⁻¹, 5 ng ml⁻¹, and 50 ng ml⁻¹, respectively, and showed good precision. The M. mazei assays used monoclonal antibodies and were entirely species specific, showing no cross-reaction with methanogens of other genera or with other species of the same genus. The Methanobacterium bryantii assay, which used two polyclonal antisera, showed only a slight cross-reaction with one other Methanobacterium species but no cross-reaction with methanogens of other genera. The use of the ELISAs for quantitative analysis of mixed cultures and of sewage sludge samples was investigated. Sludge diluted at 1:10³ or more caused no significant interference in any of the three ELISAs. Various cultures of bacteria, methanogens, and nonmethanogens at a protein concentration of 50 μg ml⁻¹ showed no significant interference in the M. mazei competitive assay and the Methanobacterium bryantii two-site assay, although they did cause falsely low results in the M. mazei two-site assay.     

Inhibition of methanogens by n- and iso-volatile fatty acids

n-Butyrate, n-valerate and n-caproate were more inhibitory towards Methanobacterium byrantii, Methanobacterium formicicum and Methanosarcina barkeri than the corresponding iso-acids. Butyrate caused maximum inhibition irrespective of isomer. Methanobacterium bryantii was more sensitive to inhibition than Methanobacterium formicicum.The authors are with the Division of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune 411 004, India.     

Effect of Gramicidin on Methanogenesis by Various Methanogenic Bacteria

Methanogenesis by Methanobacterium thermoautotrophicum strains was extremely sensitive to gramicidin, total inhibition being observed at 0.2 μg/ml. In contrast, methane synthesis by Methanococcus voltae, Methanogenium marisnigri, Methanosarcina mazei, and Methanospirillum hungatei were resistant to the highest concentrations of gramicidin tested (40 μg/ml), although spheroplasts of Methanospirillum hungatei were extremely sensitive. Other species tested showed intermediate sensitivity to gramicidin, methanogenesis inhibition occurring at 4 to 20 μg/ml.