Deep subsurface sulfate reduction and methanogenesis in the Iberian Pyrite Belt revealed through geochemistry and molecular biomarkers.

The Iberian Pyrite Belt (IPB, southwest of Spain), the largest known massive sulfide deposit, fuels a rich chemolithotrophic microbial community in the Río Tinto area. However, the geomicrobiology of its deep subsurface is still unexplored. Herein, we report on the geochemistry and prokaryotic diversity in the subsurface (down to a depth of 166 m) of the Iberian Pyritic belt using an array of geochemical and complementary molecular ecology techniques. Using an antibody microarray, we detected polymeric biomarkers (lipoteichoic acids and peptidoglycan) from Gram-positive bacteria throughout the borehole. DNA microarray hybridization confirmed the presence of members of methane oxidizers, sulfate-reducers, metal and sulfur oxidizers, and methanogenic Euryarchaeota. DNA sequences from denitrifying and hydrogenotrophic bacteria were also identified. FISH hybridization revealed live bacterial clusters associated with microniches on mineral surfaces. These results, together with measures of the geochemical parameters in the borehole, allowed us to create a preliminary scheme of the biogeochemical processes that could be operating in the deep subsurface of the Iberian Pyrite Belt, including microbial metabolisms such as sulfate reduction, methanogenesis and anaerobic methane oxidation.

An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors.

An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test. The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make the test well suited for routine analysis of large numbers of samples and thus for process surveillance during operation of biogas digesters.

Methanogenesis in thermophilic biogas reactors.

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.

Dissolution and immunochemical analysis of the sheath of the archaeobacterium Methanospirillum hungatei GP1.

The sheath of Methanospirillum hungatei GP1 was degraded by three dissolution techniques, which produced a range of soluble products. By using 0.05 M L-arginine buffer (pH 12.6) at 90 degrees C for 10 min, 74% (dry weight) of the sheath was dissolved; however, the solubilized polypeptides were extensively degraded. Treatment with 2% beta-mercaptoethanol and 2% sodium dodecyl sulfate at 90 degrees C in 0.05 M 2(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 9.0) solubilized 42% (dry weight) of the sheath as a group of polypeptides of 30 to 40 kDa. At 100 degrees C for 2 h, 5% beta-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 20 mM EDTA released 74% of the sheath's mass as a group of polypeptides of 10 to 40 kDa. All solubilized products were examined by SDS-polyacrylamide gel electrophoresis, and a range of high- and low-molecular-weight polypeptides was identified. None were glycoproteins. Hoops, which comprise the sheath's structure, were seen by electron microscopy after all of the attempted dissolutions. Monoclonal antibodies were produced against the 10- to 40-kDa range of solubilized products and against the approximately 40-kDa polypeptides, and polyclonal antiserum was produced against an 18-kDa polypeptide. These immunological markers were used in Western immunoblotting and protein A-colloidal gold-antibody probing by electron microscopy to identify the structural location of the various polypeptides. Native sheath, which possesses 2.8-nm particles on its outer surface (M. Stewart, T.J. Beveridge, and G.D. Sprott, J. Mol. Biol. 183:509-515, 1985; P.J. Shaw, G.J. Hills, J.A. Henwood, J.E. Harris, and D.B. Archer, J. Bacteriol. 161:750-757, 1985), presented a gentle wave-form surface in platinum-shadowed specimens. In contrast, the inner face of the sheath was highlighted by ridges lying perpendicular to the longitudinal axis of the sheath and likely corresponded to hoop boundaries. Both the polyclonal and monoclonal antibodies were specific for different faces; polyclonal antibodies labeled the inner face, whereas monoclonal antibodies labeled the outer face. Accordingly, the apparent asymmetry of structure between the two faces of the sheath can be correlated by our immunochemical probing with a distinct asymmetry in the distribution of exposed polypeptides between the faces. The possible implications of this asymmetry for growth and maturation of the sheath are explained.

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.

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.

Quantitative immunologic analysis of the methanogenic flora of digestors reveals a considerable diversity.

To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive significance.

Antigenic distinctiveness, heterogeneity, and relationships of Methanothrix spp.

A detailed immunologic analysis of Methanothrix soehngenii Opfikon (the type species of the genus), Methanothrix sp. strain CALS-1, and Methanothrix concilii GP6 was performed. A variety of poly- and monoclonal antibody probes for a comprehensive panel of reference organisms were used to determine immunogenicity, antigenicity, and relationships. The three organisms are antigenically distinct but interrelated, forming an immunologically cohesive group, weakly related to methanosarcinae. A prominent feature of the organisms was heterogeneity characterized by antigenic diversity and compartmentation, the latter particularly evident in M. soehngenii Opfikon, which was examined more thoroughly. The complexity of the antigenic profile of this strain and the heterogeneity of the group suggest a high degree of phenotypic diversification within the genus.

Antigenic mosaic of Methanogenium spp.: analysis with poly- and monoclonal antibody probes.

Eight well-characterized Methanogenium strains, including the six described type strains, were analyzed with poly- and monoclonal antibody probes to examine the antigenic mosaic of the genus. The pattern of cross-reactions showed that the mosaic is complex and varies with the strains; thus, these organisms have developed a considerable antigenic diversity, which is expressed in their envelopes. Every strain shared at least one determinant with at least one other strain, demonstrating the antigenic cohesiveness of the group. This finding, together with the fact that most strains displayed a distinctive antigenic fingerprint (notwithstanding the limited number of probes available), emphasizes the potential of antibodies for rapid identification of new isolates and for direct elucidation of Methanogenium strains in microbial mixtures.

Immunologic distinctiveness of archaebacteria that grow in high salt.

The antigenic fingerprints of eight halophilic archaebacteria representing the groups recently outlined by molecular and chemical analyses were determined with calibrated antibody probes. Comparison with the antigenic fingerprints of methanogens encompassing all described families and most genera demonstrated that these two archaebacterial groups are themselves antigenically coherent but immunologically distinct.

Isolation of an antigenically unique methanogen from human feces.

A methanogenic bacterium with the morphological and physiological properties of the genus Methanobrevibacter was isolated from the feces of a Japanese man who excreted methane in his breath. Indirect immunofluorescence staining revealed that the isolate had an antigenicity unrelated to that of any known members of the genus Methanobrevibacter.

Antigenic mosaic of Methanosarcinaceae: partial characterization of Methanosarcina barkeri 227 surface antigens by monoclonal antibodies.

Hybridomas were constructed with spleen cells from mice immunized against Methanosarcina barkeri 227. The reaction with the resulting monoclonal antibodies identified two antigenic determinants. Determinant 8A is present in M. barkeri 227, where it is accessible to antibody on whole bacterial cells. 8A is undetectable in (or absent from) M. barkeri R1M3, an immunologically closely related strain. Determinant 8C is present in both strains, but with M. barkeri 227 it is found only in extracts and cannot be demonstrated in whole cells. It therefore appears to be hidden. A soluble form of antigen 8A (antigen 227) was obtained treating whole M. barkeri 227 cells with absolute methanol. This antigen was further purified by affinity chromatography with antibody 8A. Chemical and immunochemical analyses of these preparations showed that antigen 227 is a high-molecular-weight (4 X 10(5)) structure composed mainly of one carbohydrate, glucose, and small amounts of amino acids. Its solubility properties suggest that this molecule is associated with a lipid moiety.

Six antigenic determinants in the surface layer of the archaebacterium Methanococcus vannielii revealed by monoclonal antibodies.

The immunogenicity and antigenic characteristics of the unique surface layer (S layer) of Methanococcus vanielii was studied with a panel of six monoclonal antibodies. Six surface determinants were identified for the first time, each recognized by one antibody exclusively. The determinants are proteins, located in the S layer, and accessible to antibody in whole, unfixed, as well as formalinized bacteria. Hence the six antigens and antibodies reported here should be useful for rapid identification of new isolates and for taxonomy of methanogens, notably Methanococcaceae. In this connection two novel applications of the slide immunoenzymatic assay were developed for analyses of monoclonal antibodies and their complementary sites in the bacterial envelope.

Antibacterial monoclonal antibodies and the dawn of a new era in the control of infection.

Literature reports concerned with monoclonal antibodies against bacteria, or their toxins, which are pathogens for man and animals were surveyed. These antibodies have important potential uses in human and veterinary pathology and medicine. They are likely to become key elements in a fast progression toward a more complete understanding and control of infectious diseases and of toxin poisoning. A new area of bacteriology relevant to sanitary engineering is also being advanced with the help of antibacterial monoclonal antibodies. This area involves bacteria that produce the biofuel methane, along with other molecules of nutritional value, through a process which brings about the recycling of organic wastes and thereby limits or controls microbial contamination of soil and water.

Dissecting the antigenic mosaic of the Archaebacterium Methanobacterium thermoautotrophicum by monoclonal antibodies of defined molecular specificity.

The antigenic mosaic of the Archaebacterium Methanobacterium thermoautotrophicum, strain delta H, was analyzed with a panel of six monoclonal antibodies. Five antigenic determinants were identified. One contains N-acetyl-D-glucosamine, another contains N-acetyl-D-galactosamine, and a third contains gamma-glutamylalanine (gamma-Glu-Ala). These residues are not involved, at least as immunodominant epitopes, in the other two determinants, one of which contains L-talosaminuronic acid, a component of pseudomurein found only in Methanobacteriaceae. Each of the five determinants was recognized by one monoclonal antibody exclusively. A sixth antibody recognized a structure containing gamma-Glu-Ala that could be either a sixth determinant or a subdeterminant within the site already recognized as containing gamma-Glu-Ala. We postulate that two of the determinants are strain specific, three are species specific, and one is a common antigen.

Antigenic analysis of Methanomicrobiales and Methanobrevibacter arboriphilus.

Preparation of new antisera has permitted more comprehensive immunological analyses of the two families of Methanomicrobiales. Methanomicrobiaceae and Methanosarcinaceae, and the species Methanobrevibacter arboriphilus. Immunological analysis was carried out with antibody probes against 23 strains, including almost all genera and species of methanogens. The absence of cross-reactions between families of methanogens was confirmed. Methanomicrobium and Methanogenium were found to be immunologically related. Extensive cross-reactions occurred among six strains of Methanosarcinaceae, but none occurred among three strains of M. arboriphilus when tested with the S probe, i.e., the last antiserum dilution of the titration curve's plateau.

Monoclonal antibodies for immunochemical analysis of methanogenic bacteria.

Sixty-nine hybridomas were generated to produce monoclonal antibodies to species of methanogens representing three of the four families accepted at the present time: Methanobacteriaceae, Methanococcaceae, and Methanomicrobiaceae. The antibody of each of 29 hybridomas cross-reacted with a methanogen of the same species or genus as the immunizing (homologous) strain, whereas the antibody of the other 40 cell lines reacted only with the homologous strain. Inhibition-blocking experiments with compounds of known composition and structure were used to define the fine specificity of antibodies of four hybridomas representing the four genera of the methanogens used for immunization. The combining site of the monoclonal antibody against M. thermoautotrophicum delta H examined is specific for a structure in the pseudomurein involving N-acetyl-glucosamine but not the (1-3) linkage or the C-terminus gamma-Glu-Ala of the peptide, both of which are characteristic of pseudomurein, the cell-wall peptidoglycan distinctive of the Methanobacteriaceae. In contrast, the monoclonal antibody to the other methanogen of this family examined, M. arboriphilus DH1, recognizes a determinant involving gamma-Glu-Ala. Thus pseudomurein expresses at least two dissimilar antigenic determinants in different portions of the molecule. The monoclonal antibodies against M. vannielii SB and M. hungatei JF1, whose families do not possess pseudomurein, did not display specificity for analogues of pseudomurein.

Antibody analysis of relationships among methanogenic bacteria.

A bank of antisera to the majority of methanogenic bacteria is now available. Three antibody probes, R, S, and T, were derived from each antiserum in the bank and used for analysis of antigenic relatedness among methanogens by immunofluorescence. The T probe reacted only with the immunizing (or homologous) strain, the S probe gave strong cross-reactions with strains of the same species, and the R probe revealed some interspecies relationships. The results were confirmed and extended by enzyme immunoassays and standard serological methods involving serial dilution analysis, cross-adsorptions, and the use of reference strains. The immunological methods and standardized antibody probes are useful for rapid identification of methanogens and measurements of antigenic relationships which aid in the classification of these bacteria.

Specific antisera and immunological procedures for characterization of methanogenic bacteria.

Specific antisera were raised in rabbits to 19 methanogenic bacteria representing the species available in pure culture at the present time. The antisera were characterized, labeled, and organized in a bank to serve as a source of material for preparation of antibody probes and thus provide standardized reagents for immunological analysis of methanogens. An indirect immunofluorescence procedure was standardized for optimal staining of homologous and heterologous bacterial strains. Two immunoenzymatic assays were developed: (i) a simple slide assay, useful for rapid antibody detection in small samples, antibody titrations, and disclosure of cross-reactions among methanogens, and (ii) a quantitative method. The latter is useful for quantification of antigenic relatedness. Procedural details were developed to obtain optimal bacterial preparations for use as immunogens to raise antibodies in vivo, and as antigens for antibody assay in vitro.

Immunology of archaebacteria that produce methane gas.

The antigenic map of 17 methanogenic bacteria representing the entire range of available species was determined by multiple assay with antibody probes. Four major clusters of antigenically related strains coincide with the females proposed on the basis of 16S ribosomal RNA analysis. Immunological mapping uncovered relationships not yet shown by other methods and allowed identification and classification of two new bacterial isolates.