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.     

Identification of pseudomurein cell wall binding domains

Methanothermobacter thermautotrophicus is a methanogenic Gram-positive microorganism with a cell wall consisting of pseudomurein. Currently, no information is available on extracellular pseudomurein biology and so far only two prophage pseudomurein autolysins, PeiW and PeiP, have been reported. In this paper we show that PeiW and PeiP contain two different N-terminal pseudomurein cell wall binding domains. This finding was used to identify a novel domain, PB007923, on the M. thermautotrophicus genome present in 10 predicted open reading frames. Three homologues were identified in the Methanosphaera stadtmanae genome. Binding studies of fusion constructs of three separate PB007923 domains to green fluorescent protein revealed that it also constituted a cell wall binding domain. Both prophage domains and the PB007923 domain bound to the cell walls of Methanothermobacter species and fluorescence microscopy showed a preference for the septal region. Domain specificities were revealed by binding studies with other pseudomurein-containing archaea. Localized binding was observed for M. stadtmanae and Methanobrevibacter species, while others stained evenly. The identification of the first pseudomurein cell wall binding domains reveals the dynamics of the pseudomurein cell wall and provides marker proteins to study the extracellular pseudomurein biology of M. thermautotrophicus and of other pseudomurein-containing archaea.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.     

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4±0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

Granular sludge formation in upflow anaerobic sludge blanket (UASB) reactors

The state of the art for upflow anaerobic sludge blanket (UASB) reactors is discussed, focusing on the microbiology of immobilized anaerobic bacteria and the mechanism of granule formation. The development of granular sludge is the key factor for successful operation of the UASB reactors. Criteria for determining if granular sludge has developed in a UASB reactor is given based on the densities and diameters of the granular sludge. The shape and composition of granular sludge can vary significantly. Granules typically have a spherical form with a diameter from 0.14 to 5 mm. The inorganic mineral content varies from 10 to 90% of the dry weight of the granules, depending on the wastewater composition etc. The main components of the ash are calcium, potassium, and iron. The extracellular polymers in the granular sludge are important for the structure and maintenance of granules, while the inorganic composition seems to be of less importance. The extracellular polymer content varies between 0.6 and 20% of the volatile suspended solids and consists mainly of protein and polysaccharides. Both Methanosaeta spp. (formerly Methanothrix) and Methanosarcina spp. have been identified as important aceticlastic methanogens for the initial granulation and development of granular sludge. Immunological methods have been used to identify other methanogens in the granules. The results have showed that, besides the aceticlastic methanogens Methanosaeta spp. and Methanosarcina spp., hydrogen and formate utilizing bacteria are also present, e.g., Methanobacterium formicicum, Methanobacterium thermoautotrophicum, and Methanobrevibacter spp. Microcolonies of syntrophic bacteria are often observed in the granules, and the significant electron transfer in these microcolonies occurs through interspecies hydrogen transfer. The internal organization of the various groups of bacteria in the granules depends on the wastewater composition and the dominating metabolic pathways in the granules. Internal organization is observed in granules where such an arrangement is beneficial for an optimal degradation of the wastewater. A four-step model is given for the initial development of granular sludge. (c) 1996 John Wiley & Sons, Inc.     

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.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Methanogenic bacteria from human dental plaque.

Samples of human dental plaque were examined for the presence of methanogenic bacteria. Of 54 samples from 36 patients, 20 yielded H2/CO2-using methanogenic enrichment cultures. All methanogen-positive samples were from patients with some degree of periodontal disease. The predominant populations in the enrichments had morphologies characteristic of Methanobrevibacter spp. In six enrichments derived from three patients, the common methanogen was antigenically similar to Methanobrevibacter smithii. The same was true for the three methanogenic isolates obtained in axenic culture from a fourth patient. The six enrichments and two of the three isolates were antigenically closer to strain ALI than to PS. Two of the enrichments also had subpopulations with weak antigenic similarity to Methanosphaera stadtmanae. The data indicate that methanogens in the oral cavity of humans are antigenically close to those found in the intestinal tract.     

Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.     

Molecular diversity of methanogens in fecal samples from captive Sumatran orangutans (Pongo abelii)

Methane emissions have been previously detected from orangutans, but characterization of the diversity of methanogens in this species has yet to be completed. This preliminary study identified methanogen producing microorganims, also called methanogens, present in the feces from a colony of captive Sumatran orangutans at the Perth Zoo. All animals were housed in the same enclosure and were fed primarily a frugivorous diet. Methanogens were detected using a 16S rRNA gene clone library. A total of 207 clones were examined, revealing 37 different methanogen 16S rRNA sequences, or phylotypes. Of these, 31 phylotypes represented by 170 clones had 96.4-100% sequence identity to Methanosphaera stadtmanae, four phylotypes (32 clones) had 95.1-100% sequence identity to Methanobrevibacter smithii, while two phylotypes (five clones) had 95.9-97.7% sequence identity to Methanobacterium beijingense. Overall, five possible new species were identified from the clone library. This represents the first report of Msp. stadtmanae, a methanol utilizer, as the most predominant methanogen in the gastrointestinal tract of animals. This is likely due to the increased availability of methanol from the highly frugivorous diet of the orangutans. Further studies are warranted to properly assess the effects of frugivorous diets on the methanogen population.     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

Comparative proteomic analysis of Methanothermobacter themautotrophicus ΔH in pure culture and in co-culture with a butyrate-oxidizing bacterium

To understand the physiological basis of methanogenic archaea living on interspecies H(2) transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H(2) supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F(420)-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed.     

Syntrophic growth on formate: a new microbial niche in anoxic environments

Anaerobic syntrophic associations of fermentative bacteria and methanogenic archaea operate at the thermodynamic limits of life. The interspecies transfer of electrons from formate or hydrogen as a substrate for the methanogens is key. Contrary requirements of syntrophs and methanogens for growth-sustaining product and substrate concentrations keep the formate and hydrogen concentrations low and within a narrow range. Since formate is a direct substrate for methanogens, a niche for microorganisms that grow by the conversion of formate to hydrogen plus bicarbonate--or vice versa--may seem unlikely. Here we report experimental evidence for growth on formate by syntrophic communities of (i) Moorella sp. strain AMP in coculture with a thermophilic hydrogen-consuming Methanothermobacter species and of (ii) Desulfovibrio sp. strain G11 in coculture with a mesophilic hydrogen consumer, Methanobrevibacter arboriphilus AZ. In pure culture, neither Moorella sp. strain AMP, nor Desulfovibrio sp. strain G11, nor the methanogens grow on formate alone. These results imply the existence of a previously unrecognized microbial niche in anoxic environments.     

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     

Use of an alternative Archaea-specific probe for methanogen detection

An alternative 16S rRNA-targeted oligonucleotide probe specific for Archaea was developed and used for detection of methanogens in anaerobic reactors. The designed probe was checked for its specificity by computer-aided comparative sequence analysis. For in situ application, optimal stringency conditions were adjusted by performing whole cell hybridization using target and nontarget organisms. Anaerobic sludge samples were examined by in situ hybridization for methanogenic populations. The relative abundance of methanogens was monitored with epifluorescence microscopy. Individual cells could be visualized with strong fluorescence signals after hybridization with the newly developed probe.