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.     

Detection and Quantitation of Methanogens by Enzyme-Linked Immunosorbent Assay

Antisera to two methanogenic bacteria, Methanosarcina barkeri and a Methanobacterium sp., were raised in rabbits and used to develop an enzyme-linked immunosorbent assay (ELISA) method. ELISA was shown to be a sensitive technique, detecting as little as 4 ng of methanogen protein. The specificities of the antisera toward other methanogens were evaluated, and it was found that the antisera recognized species of the same genus as the immunizing species, but gave very little cross-reaction with methanogens of different genera. ELISA was used to estimate the growth of methanogens in pure culture. In natural environments and in anaerobic digesters methanogens exist as part of a mixed bacterial community, so the possibility of using ELISA to quantitate methanogens in mixed cultures was examined. The two antisera gave very little reaction in ELISA when non-methanogenic bacteria were used as antigens and ELISA was used to quantitate methanogens in an acetate enrichment culture. I conclude that the ELISA is a useful method for quantitating methanogens in defined mixed cultures, but has limited applicability to more complex systems.     

New Method for the Isolation and Identification of Methanogenic Bacteria

A new technique is reported for the rapid growth and detection of methanogenic bacteria by using petri plates. The method employs an anaerobic glove box containing an inner chamber with separate gas-flushing facilities. The numbers of methanogenic bacteria recovered from domestic sewage sludge are comparable to those recovered by other methods. The methanogenic organisms isolated from sludge include Methanosarcina, Methanospirillum, Methanobacterium strain M. o. H., and Methanobacterium formicicum. Identification of colonies containing methanogenic bacteria is facilitated by taking advantage of the unique fluorescence properties of these organisms. Colonies as small as 0.5 mm can be detected by exposing them to long-wave ultraviolet light.     

牛源金黄色葡萄球菌粘附因子Efb与ClfA的免疫生物学特性比较

为评价与比较金黄色葡萄球菌的粘附因子Efb(Extracellular fibrinogen-binding protein)和Clf A(Clumping factor A)的抗原性、粘附特性及其抗血清对金黄色葡萄球菌菌体的识别能力、抗粘附能力及免疫保护力等免疫生物学特性,分别构建Efb和Clf A的重组表达载体,并用纯化的重组蛋白免疫实验动物,分别检测家兔抗血清对菌体的识别能力、抗粘附能力以及小鼠抗血清的抗体效价和免疫保护作用。结果显示,Efb和Clf A均有与纤维蛋白原(Fibrinogen,Fg)结合的能力,且Efb蛋白对纤连蛋白(Fibronectin,Fn)的结合能力优于Clf A(P0.01),Clf A免疫组抗血清对全细菌的识别能力优于Efb免疫组(P0.01)。与对照组相比Efb和Clf A免疫组的抗血清均能显著抑制金黄色葡萄球菌对Fg和Fn的粘附(P0.01),Efb免疫组对金黄色葡萄球菌的粘附抑制优于Clf A,两种粘附因子免疫小鼠后产生的血清抗体效价与对照组比较有显著的升高,抗体滴度可达1∶40 500,攻毒结果显示Efb与Clf A免疫组均对小鼠具有良好的保护效果。该研究结果对Efb在金黄色葡萄球菌的亚单位疫苗的应用奠定了基础。     

Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.     

伤寒沙门菌与甲、乙、丙副伤寒沙门菌质控血清的制备

应用免疫学原理,将伤寒沙门菌O901、H901和甲、乙、丙型副伤寒沙门菌分别制成全菌体抗原,免疫实验兔获取免疫血清。依据伤寒沙门菌和副伤寒沙门菌的抗原成分的异同性,选择适当的吸收菌除去免疫血清中的交叉反应抗体和类属凝集素,而保留其特异性的抗体。通过对诊断菌液的验证试验,证实吸收充分的免疫血清具有质控血清的特性。具备可靠性能的质控血清,适用于伤寒沙门菌与副伤寒沙门菌的菌种检定及其效价检测;亦有利于肥达氏诊断菌液的质量控制。     

The anion-exchange substrate shuttle process: A new approach to two-stage biomethanation of organic and toxic wastes

A novel biomethanation process configuration is described which uses improved biocatalysts to enhance the productivity and stability of waste biomethanation systems. The design facilitates the maintenance of acetogenic and methanogenic bacteria under optimum substrate concentrations far above their K(s) values in a two-stage biomethanation system with closed loop reactors. Volatile fatty acid anions (VFA anions) are provided as substrates for the methanogenic stage by using an anion exchange unit coupled to the acidogenic stage. The slow growing and sensitive acetogenic and methanogenic bacteria are protected from oxygen, cationic pollutants, toxins, and microbial contamination by use of the substrate shuttle process. Due to the closed loop process configuration, washout of the ecoengineered biocatalysts is excluded. The modular system configuration allows industrial mass production of the system components. The new biomethanation process enhances both the BTU content of the gas and the methane production over state-of-the-art anaerobic digestor bioreactor concepts.     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

Trophic interactions in the methanogenic microbial community of low-temperature terrestrial ecosystems

The formation of methane in various ecosystems is due to the functioning of an anaerobic community, which combines trophically different groups of microorganisms. The methanogenic microbial community is a complex biological system, which responds to low temperatures by changes in its trophic structure resulting in redistributing matter flows. The enhanced activity of homoacetogenic bacteria at low temperature plays a significant role in this redistribution. Due to their relatively high growth rates and metabolic versatility, homoacetogens can successfully compete with fermenting bacteria and hydrogenotrophic methanogenic archaea for common substrates. The concentration of hydrogen is an important regulatory factor in the psychroactive methanogenic community. At low temperature methanogenic archaea possessing a higher affinity for hydrogen than homoacetogens provide for interspecies H2 transport in syntrophic reactions of fatty acid decomposition. The formation of a balanced community at low temperature is a longtime process. Cold terrestrial ecosystems are dominated by psychroactive (psychrotolerant) microorganisms, which can grow over a wide range of ambient temperatures.     

Aldolases of the lactic acid bacteria

Reciprocal qualitative and quantitative immunological experiments employing an anti-Pediococcus cerevisiae aldolase serum confirmed many of the interspecific relationships demonstrated previously among lactic acid bacteria with antisera prepared against the Streptococcus faecalis fructose diphosphate aldolase. The extent of immunological relatedness observed between the Lactobacillus and Pediococcus aldolases was markedly greater than that noted between Pediococcus and Streptococcus aldolases indicating that the pediococci share closer phylogenetic ties with the rod-shaped lactobacilli than with their spherical counterparts in the streptococci. In addition to confirming the existence of definitive, but distant, relationships between the lactic acid bacteria and certain gram positive nonsporeforming anaerobes, immunological cross-reactivity was also demonstrated between the pediococcal aldolases and those of Aerococcus viridans.This paper is dedicated with deepest appreciation to Prof. Roger Y. Stanier on the occasion of his 60th birthday in token of what his friendship and guidance have meant to me. — J. L.     

High tolerance of methanogens in granular sludge to oxygen

This research assessed the effect of oxygen exposure on the methanogenic activity of anaerobic granular sludges. The toxicity of oxygen to acetoclastic methanogens in five different anaerobic granular sludges was determined in serum flasks with effective gas-to-liquid volumes of 4.65 to 1. The amount of oxygen that caused 50% inhibition of the methanogenic activity after 3 days of exposure ranged from 7% to 41% oxygen in the head space. These results indicate that methanogens located in granular sludge have a high tolerance for oxygen. The most important factor contributing to the tolerance was the oxygen consumption by facultative bacteria metabolizing biodegradable substrates. Uptake of oxygen by these bacteria creates anaerobic microenvironments where the methanogenic bacteria are protected. The results also indicate that methanogens in sludge consortia still have some tolerance to oxygen, even in the absence of facultative substrate for oxygen respiration. (c) 1993 John Wiley & Sons, Inc.     

Anaerobic Phenol Degradation by Microorganisms of Swine Manure

Swine manure contains diverse groups of aerobic and anaerobic bacteria. An anaerobic bacterial consortium containing sulfate-reducing bacteria (SRB) and acetate-utilizing methanogenic bacteria was isolated from swine manure. This consortium used phenol as its sole source of carbon and converted it to methane and CO₂. The sulfate-reducing bacterial members of the consortium are the incomplete oxidizers, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. The methanogenic bacteria of the consortium converted the acetic acid to methane. When a methanogen inhibitor was used in the culture medium, phenol was converted to acetic acid by the SRB, but the acetic acid did not undergo further metabolism. On the other hand, when the growth of SRB in the consortium was suppressed with a specific SRB inhibitor, namely, molybdenum tetroxide, the phenol was not degraded. Thus, the metabolic activities of both the sulfate-reducing bacteria and the methanogenic bacteria were essential for complete degradation of phenol. Received: 31 January 1997 / Accepted: 7 March 1997     

A kinetic model for methanogenesis from whey permeate in a packed bed immobilized cell bioreactor

The fermentation kinetics of methane production from whey permeate in a packed bed immobilized cell bioreactor at mesophilic temperatures and pHs around neutral was studied. Propionate and acetate were the only two major organic intermediates found in the methanogenic fermentation of lactose. Based on this finding, a three-step reaction mechanism was proposed: lactose was first degraded to propionate, acetate, CO(2), and H(2) by fermentative bacteria; propionate was then converted to acetate by propionate-degrading bacteria; and finally, CH(4) and CO(2) were produced from acetate, H(2), and CO(2) by methanogenic bacteria. The second reaction step was found to be the rate-limiting step in the overall methanogenic fermentation of lactose. Monod-type mathematical equations were used to model these three step reactions. The kinetic constants in the models were sequentially determined by fitting the mathematical equations with the experimental data on acetate, propionate, and lactose concentrations. A mixed-culture fermentation model was also developed. This model simulates the methanogenic fermentation of whey permeate very well.     

Distribution of Methanogenic and Sulfate-Reducing Bacteria in Near-Shore Marine Sediments

The distribution of methanogenic and sulfate-reducing bacteria was examined in sediments from three sites off the coast of eastern Connecticut and five sites in Long Island Sound. Both bacterial groups were detected at all sites. Three distributional patterns were observed: (i) four sites exhibited methanogenic and sulfate-reducing populations which were restricted to the upper 10 to 20 cm, with a predominance of sulfate reducers; (ii) three sites in western Long Island Sound exhibited a methanogenic population most abundant in sediments deeper than those occupied by sulfate reducers; (iii) at one site that was influenced by fresh groundwater, methanogens and sulfate reducers were numerous within the same depths; however, the number of sulfate reducers varied vertically and temporally with sulfate concentrations. It was concluded that the distributions of abundant methanogenic and sulfate-reducing bacteria were mutually exclusive. Methanogenic enrichments yielded all genera of methanogens except Methanosarcina, with the methanobacteria predominating.     

Evolutionary relationships among bacterial carbamoyltransferases

An immunological approach was used for the study of ornithine carbamoyltransferase (OTCase) evolution in bacteria. Antisera were prepared against the anabolic and catabolic OTCases of Pseudomonas aeruginosa and Aeromonas formicans as well as against OTCase and putrescine carbamoyltransferases from Streptococcus faecalis; these antisera were then tested against the unpurified OTCases, either anabolic or catabolic, of 34 bacterial strains. Extensive cross-reactions were observed between the antisera to catabolic OTCases from P. aeruginosa, A. formicans and S. faecalis and the catabolic enzymes from other species or genera. These antisera cross-reacted also with the anabolic OTCases of strains of the Enterobacteriaceae but not with the anabolic OTCases of the same species or of other species or genera. The cross-reaction measured between the antisera against P. aeruginosa anabolic OTCase and the anabolic OTCases of other Pseudomonas were largely in agreement with the phylogenic subdivision of Pseudomonas proposed by N. J. Palleroni. The correlation was also significantly higher with the anabolic enzyme of an archaeobacterium, Methanobacterium thermoaceticum, than with the catabolic or anabolic OTCases from other genera in the eubacterial line. The antiserum raised against A. formicans anabolic OTCase was quite specific for its antigen and appeared to be raised against the heaviest of the various oligomeric structures of the enzyme.     

A novel biosynthesis of medium chain length alpha-ketodicarboxylic acids in methanogenic archaebacteria

Gas chromatographic-mass spectrometric analysis on the distribution of alpha-ketodicarboxylic acids in various bacteria determined that alpha-ketoglutarate and alpha-ketoadipate are widely distributed in all the bacteria examined, whereas alpha-ketopimelate and alpha-ketosuberate are found only in the methanogenic archaebacteria. Labeling experiments with stable isotopes indicated that each of these acids arises from alpha-ketoglutarate by repeated alpha-ketoacid chain elongation. The final product in this series of reactions, alpha-ketosuberate, serves in the methanogenic bacteria as the biosynthetic precursor to the 7-mercaptoheptanoic acid portion of 7-mercaptoheptanoylthreonine phosphate, a methanogenic coenzyme.     

Metabolic interactions between anaerobic bacteria in methanogenic environments

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 is 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.     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Acetogenesis and acetotrophy in a hexanoate-catabolizing microbial association isolated from anoxic landfill

Metabolism of the key intermediates, acetate and hydrogen, in anaerobic hexanoate catabolism by an interacting microbial association isolated from a landfill was examined in the presence of sulphate. Hydrogen (the β-oxidation product of hexanoate and butyrate), was competitively utilized by the component sulphate-reducing bacteria whereas in the absence of sulphate an interaction between H₂-utilizing acetogenic and methanogenic bacteria facilitated H₂ removal, with acetate catabolism restricted to methanogenic bacteria. A possible mechanism for energy conservation was suggested in which excess electrons were stored as acetate for subsequent usage as an energy source.