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A novel halophilic archaeon, designated NK23 T , was isolated from an inland saline soil sampled from Xinjiang, China. The cells of strain NK23 T were observed to be pleomorphic, to stain Gram-negative and form red-pigmented colonies on agar plates. The strain can grow at 25-50 °C (optimum 37 °C), at 0.9-4.8 M NaCl (optimum 2.1 M), at 0-1.0 M MgCl 2 (optimum 0.05 M) and at pH 6.5-9.5 (optimum pH 7.0). The polar lipids were found to be phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, mannosyl glucosyl diether, sulfated mannosyl glucosyl diether, and three minor unidentified glycolipids, which were chromatographically identical to those detected in Haloprofundus (Hpf.) marisrubri CGMCC 1.14959 T . On the basis of 16S rRNA gene and rpoB' gene sequence similarities and phylogenetic analysis, strain NK23 T was found to be related to Hpf. marisrubri CGMCC 1.14959 T (97.8% and 94.1% similarities, respectively). The average nucleotide identity values and in silico DNA-DNA hybridization values between strain NK23 T and Hpf. marisrubri SB9 T were 85.22% and 29.3%, respectively. The DNA G+C content of the novel strain was determined to be 65.29 mol%. Based on the phenotypic and chemotaxonomic data, together with phylogenetic relationships, strain NK23 T (= CGMCC 1.14944 T  = JCM 30670 T ) is considered to represent a new species of the genus Haloprofundus, for which the name Haloprofundus halophilus sp. nov. is proposed.

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 degrees C. Methane was the only end product in balanced microbial community with H2/CO2 as a substrate, besides acetate was produced as an intermediate at temperatures below 10 degrees C. The activity of different microbial groups of methanogenic community in the temperature range of 6-28 degrees C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 degrees C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H2/CO2-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.

The butyrate-degrading Syntrophospora bryantii degrades butyrate and a propionate-degrading strain (MPOB) degrades propionate in coculture with the hydrogen- and formate-utilizing Methanospirillum hungatii or Methanobacterium formicicum. However, the substrates are not degraded in constructed cocultures with two Methanobrevibacter arboriphilus strains which are only able to consume hydrogen. Pure cultures of the acetogenic bacteria form both hydrogen and formate during butyrate oxidation with pentenoate as electron acceptor and during propionate oxidation with fumarate as electron acceptor. Using the highest hydrogen and formate levels which can be reached by the acetogens and the lowest hydrogen and formate levels which can be maintained by the methanogens it appeared that the calculated formate diffusion rates are about 100 times higher than the calculated hydrogen diffusion rates.

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35-40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetition of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.

The influence of a high energy substrate, i.e. sucrose, on the granular sludge yield and the development of different types of granular sludge was investigated by using Upflow Anaerobic Sludge Bed (UASB) reactors fed with synthetic wastewater. The feed COD was a mixture of volatile fatty acids (VFA) i.e., 20, 40, and 40% of the COD as C2-, C3-, and C4-VFA, respectively. Furthermore, experiments were carried out in which 10 and 30% of the VFA COD was substituted with sucrose. The following distinctly different types of granules were observed in each testrun: in the reactor fed with solely VFA, black (B) and white (W) granules developed; in the reactor fed with a mixture of 90% VFA and 10% sucrose, three types of granules i.e., B, W, and grey (G) granules could be seen; in the reactor fed with 70% VFA and 30% sucrose, only W and G granules were found. The granular sludge yield increased proportional to the amount of sucrose COD. At steady-state performance of the reactors, specific acidogenic (SAA) and methanogenic (SMA) activity tests on these granules revealed that B granules had the highest SMA with low SAA. The W granules had very high SMA with low SAA. G granules gave the highest SAA with a considerable SMA. Measurement of coenzyme F420 revealed that B granules consist mainly of acetoclastic methanogens. The fore-mentioned tests were supplemented with analyses of the wash-out cells present in the reactor effluent and the results suggested that acidogens, if present, prevail at the granule surface. The B granules were particularly rich in Ca, Mn, and Zn minerals. The size distribution analysis showed that the granule diameter increased in the following order: B < W < G granules. The biogas bubbles tended to adhere to the B and W granules but not so strongly to the G granules.

Studies of the degradation of the two isomeric forms of butyrate in different anaerobic environments showed isomerization between n- and i-butyrate. Degradation rates were similar for the different examined systems and degradation rates for n-butyrate degradation were generally higher than for i-butyrate. Degradation rates for n-butyrate ranged from 0.52 to 1.39 day-1, while the rates for i-butyrate were from 0.46 to 1.15 day-1. Production of isomers was not observed when the volatile fatty acid degradation was inhibited by addition of bromoethane sulfonic acid, indicating that isomerization was coupled to the methanogenic degradation of the acid. The degree of isomerization observed during n-butyrate degradation was similar to the degree during i-butyrate degradation. Experiments indicated that the isomerization degree was higher for the thermophilic than for the mesophilic inocula.

The state of the art for thermophilic UASB reactors is discussed focusing on the start-up of UASB reactors, the influence of the waste water composition and temperature on the development and maintenance of thermophilic granules, and the microbial composition and structure of thermophilic granules.

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in "healthy" mesophilic, single-phase sewage sludge digesters accounted for approximately 8-12% of the total community and that Methanosarcinales and Methanomicrobiales constituted the majority of the total methanogen population. Methanobacteriales and Methanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels: Desulfovibrio and Desulfobulbus spp. were the dominant sulfate-reducing populations, Desulfobacter and Desulfobacterium spp. were present at lower levels, and Desulfosarcina, Desulfococcus, and Desulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.

The effects of liquid recirculation on a liquefaction-acidogenic reactor in an anaerobic two-phase digesting system operating with grass-clover silage was studied during 40 days after initiating recirculation of effluent from the methanogenic reactor to the liquefaction-acidogenic reactor. An increase in alkalinity and, thus, an increase in pH from 5.2 to 6.0 occurred in the liquefaction-acidogenic reactor. During the same period, a 10-fold increase (from 0.2 to 1.9 g.l-1.h-1) in the degradation rate of mannitol and an almost 9-fold increase in the activity of hydrogenotrophic methanogens was observed. The estimated number of these bacteria increased by one order of magnitude. The average degradation rate of lactate increased 3-fold, probably as a consequence of the more efficient hydrogen consumption by the hydrogenotrophic methanogens. An observed increase in net mineralization of organic nitrogen compounds was probably the main reason for an enhanced net production of organic acids (from 0.2 to 0.9 g.l-1.d-1). The liquefaction of cellulose and hemicellulose was low from the start of recirculation (3% and 20% reduction, respectively) and did not seem to be affected by the liquid recirculation. This was in accordance with the low number of cellulose degraders (4.0 x 10(2) counts.ml-1) observed. The results from this investigation show that the initiation of liquid recirculation in silage-fed two-phase biogas processes will stimulate the activity of hydrogenotrophic methanogens in the liquefaction-acidogenic reactor. This will lead to more thermodynamically favourable conditions for acidification reactions which are dependent upon interspecies transfer of reducing equivalents.

Gas bubbles entrapped in methanogenic granules subjected to hydrostatic pressure oscillations during recirculation in loop reactors will induce intraparticle liquid flows, and thereby enhance mass transfer in excess of diffusion. This 'breathing particle' concept was clearly demonstrated in a well defined inorganic model system. The experimental results could be described satisfactory with a structured mathematical model, while a 30% improvement is predicted for methanogenic loop reactors as compared to constant pressure systems. It is concluded that acceleration of mass transfer in gas-producing systems offers challenging perspectives for both heterogeneous catalysis and biological fermentations.

A range of granular sludges was taken from industrial anaerobic sludge blanket reactors treating a wide variety of wastewaters and a comparison was made between the polymers which were extractable from the granules and their internal structures. The study of the internal structure, using sequential staining of ultra-thin sections, showed the complexity of granular sludges. Much of the area was occupied by Gram-negative cells and the area which stained positive for protein was found to increase nearer the centre of the granules. This was accompanied by a decrease in the carbohydrate positive areas. Positive areas for lipid were widespread throughout the granules. Changes in the internal structure were observed when the type of wastewater treated by the granules was changed and a comparison between sludges treating the same type of wastewater showed that factors other than the nature of the substrate must be considered as parameters which will affect the structure of the granules. Although an appreciable variation in the granule strengths was noted, it was not possible to relate these differences, on an overall basis, to either the internal structure or the chemical composition of the extracted polymers. However, an examination of data for granules produced during the treatment of nominally similar wastes did suggest that there would be a relationship between polymer composition and granule strength in these cases.

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe against Methanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor were Methanosarcina spp. single cells, which is a difficult form of the organism to cultivate in vitro. No reactions were observed with antibody probes raised against Methanothrix soehngenii or Methanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-14C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche of Methanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56 degrees C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55 degrees C could be obtained at 61 degrees C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.

Sulphate-containing wastewaters from the paper and board industry, molasses-based fermentation industries and edible oil refineries present difficulties during anaerobic treatment, leading to problems of toxicity, reduction in methane yield, odour and corrosion. The microbiology and biochemistry of dissimilatory sulphate reduction are reviewed in order to illustrate the potential competition between sulphate reducers and other anaerobes involved in the sequential anaerobic mineralisation process. The theoretical considerations which influence the outcome of competition between sulphate reducers and fermentative, syntrophic, homoacetogenic and methanogenic bacteria are discussed. The actual outcome, under the varying influent organic composition and strength and sulfate concentrations which prevail during digestion of industrial wastewaters, may be quite different to that predicted by thermodynamic or kinetic considerations. The factors governing competitive interactions between SRB and other anaerobes involved in methanogenesis is discussed in the context of literature data on sulphate wastewater treatment and with particular reference to laboratory and full-scale digestion of citric acid production wastewater.

In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH2 coupled to proton or bicarbonate reduction in thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.

Methanogenic archaea convert a few simple compounds such as H2 + CO2, formate, methanol, methylamines, and acetate to methane. Methanogenesis from all these substrates requires a number of unique coenzymes, some of which are exclusively found in methanogens. H2-dependent CO2 reduction proceeds via carrier-bound C1 intermediates which become stepwise reduced to methane. Methane formation from methanol and methylamines involves the disproportionation of the methyl groups. Part of the methyl groups are oxidized to CO2, and the reducing equivalents thereby gained are subsequently used to reduce other methyl groups to methane. This process involves the same C1 intermediates that are formed during methanogenesis from CO2. Conversion of acetate to methane and carbon dioxide is preceded by its activation to acetyl-CoA. Cleavage of the latter compound yields a coenzyme-bound methyl moiety and an enzyme-bound carbonyl group. The reducing equivalents gained by oxidation of the carbonyl group to carbon dioxide are subsequently used to reduce the methyl moiety to methane. All these processes lead to the generation of transmembrane ion gradients which fuel ATP synthesis via one or two types of ATP synthases. The synthesis of cellular building blocks starts with the central anabolic intermediate acetyl-CoA which, in autotrophic methanogens, is synthesized from two molecules of CO2 in a linear pathway.

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation in Methanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05-100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH4+ concentration. The length of the poly-gamma-glutamyl side chain of F420 derivatives turned out to be independent of the concentration of ammonia in the culture medium.

Several elemental metals were examined as potential electron donors for methanogenic bacteria, using both a single tube system where the metal was in direct contact with the cells, and a two-flask system, where metal and cells were not in direct contact, but had contact via the gas phase. With all organisms examined in the direct contact system, Fe degree, Al degree and Zn degree served as electron donors for methanogenesis; some organisms used Ni degree or Sn degree as low-level electron donors. Of the metals tested, methanogenesis from H2 + CO2 was inhibited by direct contact with Zn degree or Cu degree, but not by Fe degree or Al degree. Ni degree and Co degree were inhibitory to some methanogens, with Ni degree being particularly inhibitory to the thermophilic strains tested. With all organisms examined in the two-flask system, Fe degree and Zn degree served as good electron sources for both methanogenesis and growth; Co degree generated a very low level of methane and Cu degree did not work at all. In either system V degree, Ti degree or Cd degree did not serve as electron donors. The results suggest that some elemental metals (notably Fe degree, Al degree and Zn degree) produce gaseous H2 by cathodic depolarization which is then consumed by the methanogen, thus accelerating oxidation of the metal by its metabolic activity. All of these reactions are thermodynamically favorable; however, some other metals that are clearly favorable for such a reaction on thermodynamic grounds (Ti degree and V degree) are very stable and do not serve as electron donors.

Four new methane-oxidizing bacteria have been isolated from marine samples taken at the Hyperion sewage outfall, near Los Angeles, CA. These bacteria require NaCl for growth. All exhibit characteristics typical of Type I methanotrophs, except they contain enzyme activities of both the ribulose monophosphate pathway and the serine cycle. All four strains are characterized by rapid growth in liquid culture and on agar plates, and all have temperature optima above 35 degrees C. One strain, chosen for further study, has been shown to maintain broadhost range cloning vectors and is currently being used for genetic studies.

Methanosarcina barkeri grows in defined media with acetate, methanol or carbon dioxide as carbon sources. Methanol is used for methanogenesis at a 5 times higher rate as compared with other substrates. M. barkeri can use the substrates simultaneously, but due to acidification or alkalification of the medium during growth on methanol or acetate, respectively, growth and methano-genesis may stop before the substrates are exhausted. Growth and methano-genesis on methanol or acetate are inhibited by the presence of an excess of H2; the inhibition is abolished by the addition of carbon dioxide, which probably serves as an essential source of cell carbon, in the absence of which methano-genesis ceases.

A number of obligately anaerobic fermentative bacteria are known to degrade a variety of organic substrates such as sugars, amino acids, and others, in the presence of high salt concentrations (up to 3-4 M) to products such as hydrogen, CO2, acetate and higher fatty acids, and ethanol. Our understanding of the fate of these products in hypersaline environments is still extremely limited. The occurrence of bacterial sulfate reduction is well established at salt concentrations of up to 24%; however, the bacteria involved have not yet been isolated in pure culture, and the range of electron donors used is unknown. Halophilic or halotolerant methanogenic bacteria using hydrogen/CO2 or acetate as energy source are notably absent; methanogenesis under hypersaline conditions is probably limited to such substrates as methanol and methylamines, which cannot be expected to be major products of anaerobic degradation of most organic compounds.

The availability of recombinant DNA techniques has enabled the successful genetic analysis and manipulation of a range of C1 utilizing microorganisms. It has resulted in the identification of genes of interest on both plasmids and the chromosome; enabled the linkage of chromosomal genes to be determined; established the function and regulatory patterns of genes essential for utilization of C1 compounds and provided information on the evolution of methanogenic bacteria.

The results of a large number of studies on microorganisms subjected to various degrees of substrate limitation have led to the idea that many species are particularly well adapted to growth at a very low rate at extremely low nutrient concentrations. The possible similarity between this type of bacteria and oligotrophic species is discussed. Some attention is paid to the problem of predicting the competitiveness of microbial species. To this end the apparent specific affinity of an organism for a given substrate is discussed in some detail. It is attempted to bring terminology used in describing this parameter in line with that commonly used in microbial physiology and ecology. Using one particular field study as an example the possible usefulness and limitations of this concept in field studies are discussed.

Methanogenesis involves a novel set of coenzymes as one-carbon and electron carriers. Consequently, metabolic processes of methanogens deviate from those present in non-methanogenic bacteria. Methanogenic bacteria can be classified on the basis of substrate utilization. Group I (24 species) grows at the expense of hydrogen plus CO2 and/or formate and group II (7 species) uses methanol and/or acetate. Hydrogen-consuming methanogens are found as epi- or endosymbionts of anaerobic ciliates.

Methanogenic bacteria gain their energy for growth from the conversion of a number of simple carbon compounds to methane. With a few exceptions all species known to date are able to reduce CO2 at which hydrogen acts as the electron donor. The reduction of CO2 can formally be considered to proceed through the formyl, the formaldehyde and the methyl level of reduction. These C1-units do not occur as free intermediates, but they remain bound to a number of unique coenzymes during the process. In this paper a survey is given of the structures and functions of these compounds; it deals with methanopterin derivatives, carbon dioxide reduction (CDR) factor, factor F430 and coenzyme M derivatives. A model of the process of methanogenesis that integrates previous ones and that allocates a function to the various coenzymes is presented.