Iron-corroding methanogen isolated from a crude-oil storage tank.

Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe(0)) granules, which was then incubated anaerobically at 37 degrees C under an N(2)-CO(2) atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ(T), which produced methane at a rate expected from the abiotic H(2) production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ(T) culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO(3).

Colonization of Black Smokers by Hyperthermophilic Microorganisms.

Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but they house hyperthermophiles in substantial amounts in later stages. No hard data exist on the mechanisms by which hyperthermophiles colonize newly erupted black smokers. Here I propose a scenario - based on various experimental data - for how hyperthermophiles colonize black smokers. Hyperthermophiles which are present in cold sea water in minute amounts are transferred by chance to the outside of black smokers and react within seconds to the high temperature by very fast movements. After reaching an optimal temperature region they scan the surface via a zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms.

Methanococcus igneus sp. nov., a novel hyperthermophilic methanogen from a shallow submarine hydrothermal system.

A novel hyperthermophilic strictly chemolithoautotrophic member of the genus Methanococcus was isolated from a shallow (depth: 106 m) submarine vent system at the Kolbeinsey ridge, Iceland. The isolate grew between 45 and 91 degrees C with an optimum around 88 degrees C (doubling time: 25 min). It differs from Methanococcus jannaschii in its 16S rRNA sequence, its non-hybridizing DNA, and its selenium-independent growth. Therefore, the isolate represents a new species which we name Methanococcus igneus. Type strain is isolate "Kol 5" (DSM 5666).

[The composition and distribution of some kinds of anaerobic microorganisms in Yinqion basin].

Under the strictly anaerobic conditions, the population of Sulfate-reducing bacteria, fermentative bacteria and methanogenic bacteria of serial samples got from erect sections of different Sedimentary of Yingqiong basin (a typical marine sedimentary environment) were measured by MPN method. The morphology of different kinds of bacteria and the metabolic types of methanogen and methanogenic activity were observed. The relation between population of bacteria and some indexes were compared. The results show that SRB present in all of the samples. The distribution of SRB and fermentative bacteria have no interrelation with the depth of samples but SRB has interrelation with the SO(4)2- concentration, and fermentative bacteria has negative interrelation with the contents of organic matter. Two kinds of methanogen present in all of the samples. They belong to Methanobacterium and Methanococcus, respectively. Their types of nutriment are H2/CO2.

Treatment of low-strength soluble wastewater using an anaerobic baffled reactor (ABR).

Treatment of low-strength soluble wastewater (COD approximately 500 mg/L) was studied using an eight chambered anaerobic baffled reactor (ABR). At pseudo steady-state (PSS), the average total and soluble COD values (COD(T) and COD(S)) at 8h hydraulic retention time (HRT) were found to be around 50 and 40 mg/L, respectively, while at 10h HRT average COD(T) and COD(S) values were of the order of 47 and 37 mg/L, respectively. COD and BOD (3 day, 27 degrees C) removal averaged more than 90%. Effluent conformed to Indian standards laid down for BOD (less than 30 mg/L). Reactor effluent characteristics exhibited very low values of standard deviation indicating excellent reactor stability at PSS in terms of effluent characteristics. Based on mass balance calculations, more than 60% of raw wastewater COD was estimated to be recovered as CH(4) in the gas phase. Compartment-wise profiles indicated that most of the BOD and COD got reduced in the initial compartments only. Sudden drop in pH (7.8-6.7) and formation of volatile fatty acids (VFA) (53-85 mg/L) were observed in the first compartment due to acidogenesis and acetogenesis. The pH increased and VFA concentration decreased longitudinally down the reactor. Residence time distribution (RTD) studies revealed that the flow pattern in the ABR was neither completely plug-flow nor perfectly mixed. Observations from scanning electron micrographs (SEM) suggest that distinct phase separation takes place in an ABR.

Methanococcus aeolicus sp. nov., a mesophilic, methanogenic archaeon from shallow and deep marine sediments.

Three strains of CO(2)-reducing methanogens were isolated from marine sediments. Strain PL-15/H(P) was isolated from marine sediments of the Lipari Islands, near Sicily and the other two strains, Nankai-2 and Nankai-3(T), were isolated from deep marine sediments of the Nankai Trough, about 50 km from the coast of Japan. Analysis of the cellular proteins and 16S rRNA gene sequences indicated that these three strains represented a single novel species that formed a deep branch of the mesophilic methanococci. Phylogenetic analysis indicated that the three strains were most closely related to Methanothermococcus okinawensis (95 % 16S rRNA gene sequence similarity). However, strains PL-15/H(P), Nankai-2 and Nankai-3(T) grew at temperatures that were more similar to those of recognized species within the genus Methanococcus. Strain Nankai-3(T) grew fastest at 46 degrees C. Results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strains PL-15/H(P), Nankai-2 and Nankai-3(T) from closely related species. The name Methanococcus aeolicus sp. nov. is proposed, with strain Nankai-3(T) (=OCM 812(T)=DSM 17508(T)) as the type strain.

Methanococcus infernus sp. nov., a novel hyperthermophilic lithotrophic methanogen isolated from a deep-sea hydrothermal vent.

An autotrophic, extremely thermophilic methanogen (ME(T)) was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge at a depth of 3000 m. The heavily flagellated cells are motile and coccoid shaped. The new strain growths between 55 and 91 degrees C, with an optimum growth temperature at 85 degree C. The optimum pH for growth is 6.5, and the optimum sea salt concentration for growth is around 25 g l-1. The organism uses H2 and CO2 as the only substrate for growth and methane production. Tungsten, selenium and yeast extract stimulate growth significantly. In the presence of CO2 and H2, the organism reduces elemental sulphur to hydrogen sulphide. The G+C content of the genomic DNA is 33 mol%. As determined by 16S gene sequence analysis, this organism is closely related to Methanococcus jannaschii strain JAL-1T. However, no significant homology was observed between them with DNA-DNA hybridization. It is proposed that this organism should be placed in a new species, Methanococcus infernus. The type strain is ME(T) (= DSM 11812T).

Methanocaldococcus indicus sp. nov., a novel hyperthermophilic methanogen isolated from the Central Indian Ridge.

An autotrophic, hyperthermophilic methanogen, strain SL43(T), was isolated from a deep-sea hydrothermal chimney sample collected on the Central Indian Ridge at a depth of 2420 m. The coccoid, surface-layer-carrying, Gram-negative-staining cells were heavily flagellated and exhibited a slight tumbling motility. The temperature range for growth at pH 6.5 was 50-86 degrees C, with optimum growth at 85 degrees C. The optimum pH for growth was 6.6 and the optimum NaCl concentration for growth was 30 g l(-1). The novel isolate used H(2) and CO(2) as the only substrates for growth and produced methane. Selenium and yeast extract stimulated growth significantly. In the presence of CO(2) and H(2), the organism reduced elemental sulfur to hydrogen sulfide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin or streptomycin. The G+C content of the genomic DNA was 30.7 mol%. On the basis of 16S rRNA gene sequence analysis, this organism was most closely related to Methanocaldococcus infernus ME(T) (3.2 % distance). Its phylogenetic distinctiveness was confirmed by RFLP analysis of the 16S rDNA, a reliable tool for differentiating hyperthermophilic methanococci. On the basis of phylogenetic and physiological characteristics, it is proposed that strain SL43(T) (=DSM 15027(T)=JCM 11886(T)) be designated as the type strain of a novel species, Methanocaldococcus indicus sp. nov.

Characterization of Methanococcus voltaei strain P2F9701a: a new methanogen isolated from estuarine environment.

A new methanogen, designated as strain P2F9701a (= OCM 745), was isolated from a water sample of estuarine environment in Elrin Shi, Taiwan. Cells of strain P2F9701a were motile coccus (0.7 approximately 1.1 micron) with tufts of flagella. Gas vacuoles were observed, and the protein cell wall was composed of S-layer protein subunit with Mr of 74,700. Cells catabolized formate and H2+CO2 to produce methane, but not acetate, methanol, and trimethylamine. Strain P2F9701a grew in the range of 30-42 degrees C, with optimal growth temperature at 37 degrees C, but did not grow below 28 degrees C or above 42 degrees C. This estuarine isolate P2F9701a tolerated well the NaCl concentration between 0.02 and 1.03 m, and the optimal salt for growth was 0.17 m. Although phylogenetic analytic results indicated that P2F9701a belongs to the mesophilic, hydrogenotrophic marine methanogen of Methanococcus voltaei, the occurrence of gas vacuoles, tufts of flagella, eury-halotolerant and steno-thermotolerant characters of strain P2F9701a are different from mesophilic Methanococcus spp. that had been reported.

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.

Methanococcus vulcanius sp. nov., a novel hyperthermophilic methanogen isolated from East Pacific Rise, and identification of Methanococcus sp. DSM 4213T as Methanococcus fervens sp. nov.

An autotrophic, hyperthermophilic methanogen (M7T) was isolated from a deep-sea hydrothermal chimney sample collected on the East Pacific Rise at a depth of 2600 m. The coccoid-shaped cells are flagellated and exhibit a slight tumbling motility. The temperature range for growth at pH 6.5 was 49-89 degrees C, with optimum growth at 80 degrees C. The optimum pH for growth was 6.5, and the optimum NaCl concentration for growth was around 25 g l-1. The new isolate used H2 and CO2 as the only substrates for growth and methane production. Tungsten, selenium and yeast extract stimulated growth significantly. In the presence of CO2 and H2, the organism reduced elemental sulphur to hydrogen sulphide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin and streptomycin. The G + C content of the genomic DNA was 31 mol%. As determined by 16S rDNA gene sequence analysis, this organism was closely related to Methanococcus jannaschii strain JAL-1T. However, despite the high percentage of similarity between their 16S rDNA sequences (97.1%), the DNA-DNA hybridization levels between these strains were less than 5%. On the basis of these observations and physiological traits, it is proposed that this organism should be placed in a new species, Methanococcus vulcanius. The type strain is M7T (= DSM 12094T). During the course of this study, the 16S rDNA sequence analysis placed Methanococcus sp. strain AG86T (= DSM 4213T) as a close relative of M. jannaschii strain JAL-1T. However, the weak level of DNA-DNA hybridization with this strain (< 10%) allowed the proposal that strain AG86T also constitutes a new species, Methanococcus fervens.

Rapid identification of hyperthermophilic methanococci isolated from deep-sea hydrothermal vents.

16S rDNAs amplified by PCR from 22 hyperthermophilic methanococci isolated from deep-sea hydrothermal vents were compared with those of the six type strains of the genus Methanococcus by RFLP analysis. Restriction fragments obtained with Haelli enabied four of the type species to be distinguished. Restrictions with HhaI, BstUI and MspI were necessary to differentiate Methanococcus jannaschii and Methanococcus fervens. The results indicate that the 16S rDNA PCR-RFLP method provides a rapid and reliable tool for the identification of newly isolated hyperthermophilic Methanococcus spp.