Long-term storage of obligate anaerobic microorganisms in glycerol

We evaluated the possibility of storing the cultures of obligate anaerobic microorganisms (clostridia, acetogenic and sulfate-reducing bacteria, and methanogenic archaea) in 25% glycerol at ?70°C for a long time (up to 3 years). This method of storage is adequate for preserving cell viability in the majority of obligate anaerobes.     

The taxonomy of obligate anaerobic bacteria

Studies on the chemotaxonomy of obligate anaerobic bacteria have been made. The combination of gas chromatography and mass spectrometry with computer-assisted analysis, permitting the multicomponent analysis of all products of bacterial metabolism and bacterial cell components, has been shown to be a research method, quite suitable for such studies. The chromatographic profiles of the end products of metabolism in anaerobic cultures of different age have been found to differ not in the set and number of peaks indicating various metabolites, but only in the concentration of metabolites, increasing in the process of prolonged incubation. The authors believe that the national microbiological "library" of the chromatographic profiles of anaerobic organisms should be created and the album of typing chromatographic profiles should be published; besides, data on new profiles should regularly appear in magazines.     

A novel anaerobic needle inoculator for strict obligate anaerobic bacteria

Summary An inoculating needle for picking and transferring colonies of strict anaerobic bacteria was built. A self-refilling syringe was adapted to carry a stainless steel needle within the cannula, and a tee junction allowed continuous flushing with dioxygen-free gas.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

Isolation and investigation of anaerobic microorganisms involved in methanol transformation in an underground gas storage facility

High methanol and acetate concentrations (up to 12 and 14 g l⁻¹, respectively) were found in water samples collected at different objects of the North Stavropol underground gas storage facility (UGSF), and significant seasonal variations in the content of these compounds were revealed. The dominant anaerobic microorganisms isolated from these samples during the study belonged to acetogens, methanogens, and sulfate reducers. The results of 16S rRNA gene sequencing and analysis of the physiological properties showed that the isolates were close to the species of Eubacterium limosum, Sporomusa sphaeroides, Methanosarcina barkeri, Methanobacterium formicicum, and Desulfovibrio desulfuricans. The isolated organisms, except for Methanobacterium formicicum, were capable of methylotrophic growth. All strains were characterized by resistance to high methanol concentrations (up to 40–50 g l⁻¹). Their other energy substrate was hydrogen. The combination of the growth characteristics of these strains (pH, temperature, and salinity ranges) was shown to correspond to the ecological situation observed in the UGSF. The results of investigation of the isolated strains suggest that organic acids (acetate, butyrate) found in high concentrations in the initial samples are metabolic products of the revealed acetogens. Based on the established biological peculiarities of the isolated strains of methanogens, acetogens, and sulfate-reducing bacteria, these microorganisms may be considered as the main agents of anaerobic transformation of methanol and some other organic and inorganic compounds in UGSFs.     

Determination of mercury and organomercurial resistance in obligate anaerobic bacteria

A methodology for determining the minimum inhibitory concentration of inorganic and organomercurial compounds for obligate anaerobic bacteria is described. A wide variation in the susceptibility of anaerobic clinical and sewage isolates was observed. Isolates of Bacteroides ruminicola and Clostridium perfringens resistant to mercury were examined for their plasmid content and ability to demonstrate inducible resistance. None of the resistant anaerobes contained any plasmids, while resistant facultative isolates from the same source contained several plasmids. In 24 h, resistant strains of clostridia and Bacteroides volatilized 20 and 43% of the 203Hg2+ added to cultures, while Escherichia coli R100 and a sewage isolate of Enterobacter cloacae volatilized 63 and 27%, respectively, of the added 203Hg2+. Attempts to induce mercury resistance in the aerobic isolates were successful, but no induction was seen in the anaerobes. Thus, mercury resistance in these anaerobic isolates was neither inducible nor plasmid mediated.     

Evolution of microorganisms in thermophilic-dry anaerobic digestion

Microbial population dynamics were studied during the start-up and stabilization periods in thermophilic-dry anaerobic digestion at lab-scale. The experimental protocol was defined to quantify Eubacteria and Archaea using Fluorescent in situ hybridization (FISH) in a continuously stirred tank reactor (CSTR), without recycling solids. The reactor was subjected to a programme of steady-state operation over a range of the retention times from 40 to 25 days, with an organic loading rate between 4.42 and 7.50 kg volatile solid/m3/day. Changes in microbial concentrations were linked to traditional performance parameters such as biogas production and VS removal. The relations of Eubacteria:Archaea and H2-utilising methanogens:acetate-utilising methanogens were 88:12 and 11:1, respectively, during start-up stage. Hydrogenotrophic methanogens, although important in the initial phase of the reactor start-up, were displaced by acetoclastic methanogens at steady-state, thus their relation were 7:32, respectively. The methane yield coefficient, the methane content in the biogas and VS removal were stabilized around 0.30 LCH4/gCOD, 50% and 80%, respectively. Methanogenic population correlated well with performance measurements.     

Origin of spores of anaerobic microorganisms in milk

Summary Samples from silage, hay, milk, air, and faeces were taken at 25 farms in mid-Sweden, farms with different hygienic standards of milk-production being chosen.The correlation between fodder and milk-quality was investigated. The investigation was limited to the transfer of spores of anaerobes to the milk during feeding with silage of various qualities.The role of grassland fodders other than silage in spore-infection of milk is discussed.These studies are still in progress. We are indebted to the Swedish Foundation: Fonden för främjande av forsknings- och försöksverksamheten på jordbrukets område for generous financial support. We also gratefully acknowledge the encouraging interest and support of Professor R. Nilsson.     

Detection and quantification of microorganisms in anaerobic bioreactors

The presence of sulfate in anaerobic reactors can trigger competitive and syntrophic interactions between various groups of microorganisms, such as sulfate reducers, methanogens and acetogens. In order to steer the reactor process in the direction of sulfidogenesis or methanogenesis, it is essential to get insight into the population dynamics of these groups of microorganisms upon changes in the reactor operating conditions. Several methods exist to characterize and quantify the microbial sludge composition. Combining classical microbiological and modern molecular-based sludge characterization methods has proven to be a powerful approach to study the microbial composition of the anaerobic sludge.     

Proteiniclasticum sediminis sp. nov., an obligate anaerobic bacterium isolated from anaerobic sludge

Antonie van Leeuwenhoek - An obligate anaerobic bacterial BAD-10 T was isolated from anaerobic acetochlor-degrading sludge. The strain was Gram-stain negative, curved rod-shaped,...     

厌氧铁氧化菌研究进展

厌氧铁氧化菌(AFOM)是微生物学、地质学和环境学领域的重大发现.研究AFOM对于认识铁地质层形成,促进铁、氮、碳等元素的地球生物化学循环,丰富微生物学内容,开发厌氧铁氧化工艺,以及探索原始地球环境和外星生命现象,均有重要意义.本文综述了AFOM的研究进展,介绍了AFOM的存在生境,探讨了AFOM的物种多样性及其营养特性和代谢特性,阐述了AFOM在地质学、微生物学和环境学领域的潜在作用,并展望了AFOM在新物种发掘、代谢机理揭示以及开发应用等方面的研究方向.     

Novel proteins for homocysteine biosynthesis in anaerobic microorganisms

The metabolic network for sulfide assimilation and trafficking in methanogens is largely unknown. To discover novel proteins required for these processes, we used bioinformatics to identify genes co‐occurring with the protein biosynthesis enzyme SepCysS, which converts phosphoseryl‐tRNA^Cys to cysteinyl‐tRNA^Cys in nearly all methanogens. Exhaustive analysis revealed three conserved protein families, each containing molecular signatures predicting function in sulfur metabolism. One of these families, classified within clusters of orthologous groups (COG) 1900, possesses two conserved cysteine residues and is often found in genomic contexts together with known sulfur metabolic genes. A second protein family is predicted to bind two 4Fe‐4S clusters. All three genes were also identified in more than 50 strictly anaerobic bacterial genera from nine distinct phyla. Gene‐deletion and growth experiments in Methanosarcina acetivorans, using sulfide as the sole sulfur source, demonstrate that two of the proteins (MA1821 and MA1822) are essential to homocysteine biosynthesis in a background lacking an additional gene for sulfur insertion into homocysteine. Mutational analysis confirms the importance of several structural elements, including a conserved cysteine residue and the predicted 4Fe‐4S cluster‐binding domain.     

Enrichment of anaerobic benzene-degrading microorganisms by in situ microcosms

Microcosms filled with different solids (sand, lava, Amberlite XAD-7) were exposed for 67 days in the sulfidic part of a groundwater monitoring well downstream of the source zone of a benzene-contaminated aquifer and subsequently incubated in the laboratory. Benzene was repeatedly degraded in several microcosms accompanied by production of sulfide, leading to stable benzene-degrading enrichment cultures. In control microcosms without filling material, benzene was initially degraded, but the benzene-degrading capacity could not be sustained. The results indicate that long-term physiologically active benzene-degrading microorganisms were attached to surfaces of the solids. The biodiversity and attachment behavior of microorganisms in the in situ microcosms was assessed by confocal laser scanning microscopy and single-strand conformation polymorphism (SSCP) analysis, followed by sequencing of dominant SSCP bands. The microbial community was composed of several different Bacteria, representing members of Clostridia, Bacteroidales, all subgroups of the Proteobacteria, Verrucomicrobia, Nitrospira, Chloroflexi and Chlorobi. Only a few archaeal sequences could be retrieved from the communities. The majority of phylotypes were affiliated to bacterial groups with a possible functional relationship to the bacterial sulfur cycle, thus indicating that the microbial community in the investigated aquifer zone depends mainly on inorganic sulfur compounds as electron donors or acceptors, a finding that corresponds to the geochemical data.     

Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.     

Potential application of glycerol in the production of plant beneficial microorganisms

Journal of Industrial Microbiology & Biotechnology - This review highlights the importance of research for development of biofertilizer and biocontrol products based on the use of glycerol for...     

厌氧铁氧化菌研究进展

厌氧铁氧化菌(AFOM)是微生物学、地质学和环境学领域的重大发现.研究AFOM对于认识铁地质层形成,促进铁、氮、碳等元素的地球生物化学循环,丰富微生物学内容,开发厌氧铁氧化工艺,以及探索原始地球环境和外星生命现象,均有重要意义.本文综述了AFOM的研究进展,介绍了AFOM的存在生境,探讨了AFOM的物种多样性及其营养特性和代谢特性,阐述了AFOM在地质学、微生物学和环境学领域的潜在作用,并展望了AFOM在新物种发掘、代谢机理揭示以及开发应用等方面的研究方向.