Explore the vast range of titles available.

Aiming at the isolation of novel enzymes from previously uncultured thermophilic microorganisms, a metagenome library was constructed from DNA isolated from a pilot-plant biogas reactor operating at 55 °C. The library was screened for starch-degrading enzymes, and one active clone was found. An open reading frame of 1,461 bp encoding an α-amylase from an uncultured organism was identified. The amy13A gene was cloned in Escherichia coli , resulting in high-level expression of the recombinant amylase. The novel enzyme Amy13A showed the highest sequence identity (75 %) to α-amylases from Petrotoga mobilis and Halothermothrix orenii . Amy13A is highly thermoactive, exhibiting optimal activity at 80 °C, and it is also highly salt-tolerant, being active in 25 % (w/v) NaCl. Amy13A is one of the few enzymes that tolerate high concentrations of salt and elevated temperatures, making it a potential candidate for starch processing under extreme conditions.

All species from the phylum Thermotogae, class Thermotogae , are currently part of a single family, Thermotogaceae . Using genomic data from 17 Thermotogae species, detailed phylogenetic and comparative genomic analyses were carried out to understand their evolutionary relationships and identify molecular markers that are indicative of species relationships within the phylum. In the 16S rRNA gene tree and phylogenetic trees based upon two different large sets of proteins, members of the phylum Thermotogae formed a number of well-resolved clades. Character compatibility analysis on the protein sequence data also recovered a single largest clique that exhibited similar topology to the protein trees and where all nodes were supported by multiple compatible characters. Comparative genomic analyses have identified 85 molecular markers, in the form of conserved signature indels (CSIs), which are specific for different observed clades of Thermotogae at multiple phylogenetic depths. Eleven of these CSIs were specific for the phylum Thermotogae whereas nine others supported a clade comprising of the genera Thermotoga , Thermosipho and Fervidobacterium . Ten other CSIs provided evidence that the genera Thermosipho and Fervidobacterium shared a common ancestor exclusive of the other Thermotogae and four and eight CSIs in other proteins were specific for the genera Thermosipho and Fervidobacterium , respectively. Two other deep branching clades, one consisting of the genera Kosmotoga and Mesotoga and the other comprising of the genera Petrotoga and Marinitoga , were also supported by multiple CSIs. Based upon the consistent branching of the Thermotogae species using different phylogenetic approaches, and numerous identified CSIs supporting the distinctness of different clades, it is proposed that the class Thermotogae should be divided into three orders ( Thermotogales , Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families ( Thermotogaceae , Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.). Additionally, the results of our phylogenetic/compatibility studies along with the species distribution patterns of 22 identified CSIs, provide compelling evidence that the current genus Thermotoga is comprised of two evolutionary distinct groups and that it should be divided into two genera. It is proposed that the emended genus Thermotoga should retain only the species Thermotoga maritima , Tt. neapolitana , Tt. petrophila , Tt. naphthophila , Thermotoga sp. EMP, Thermotoga sp. A7A and Thermotoga sp. RQ2 while the other Thermotoga species (viz. Tt. lettingae , Tt. thermarum , Tt. elfii , Tt. subterranean and Tt. hypogea ) be transferred to a new genus, Pseudothermotoga gen. nov.

A gram-negative, motile, fermentative, thermophilic bacterium, designated AR80ᵀ, was isolated from a high-temperature oil reservoir in Yabase Oilfield in Akita, Japan. Cells were rod-shaped, motile by means of polar flagella, and formed circular, convex, white colonies. The strain grew at 40–65 °C (optimum 60 °C), 0.5–9 % (w/v) NaCl (optimum 0.5–1 %), pH 6–9 (optimum pH 7.5), and elemental sulfur or thiosulfate serves as terminal electron acceptor. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain AR80ᵀbelonged to the genus Petrotoga and shared approximately 94.5 % sequence similarity with the type species of this genus. The G + C content of genomic DNA was 32.4 mol% while the value of DNA–DNA hybridization between the closest relative species Petrotoga miotherma and AR80ᵀwas 58.1 %. The major cellular fatty acids of strain AR80ᵀconsisted of 18:1 w9c, 16:0, and 16:1 w9c. Based on genetic and phenotypic properties, strain AR80ᵀwas different with other identified Petrotoga species and represents as a novel species, for which the name Petrotoga japonica sp. nov. is proposed. The type strain is AR80ᵀ(=NBRC 108752ᵀ = KCTC 15103ᵀ = HUT 8122ᵀ).

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO₂ and H₂O--a conversion that is carried out by a consortium of bacteria and archaea. Especially in the municipal plants dedicated towards waste treatment, the reactor feed may vary considerably, exposing the resident microbiota to a changing variety of substrates. To evaluate how and if such changes influence the microbiology, an established biogas plant (6,600 m³, up to 600 m³ biogas per h) was followed over the course of more than 2 years via polymerase chain reaction-denaturing gradient gel electrophoresis of 16S rRNA genes and subsequent sequencing. Both the bacterial and the archaeal community remained stable over the investigation. Of the bacterial consortium, about half of the sequences were in decreasing order of occurrence: Thermoacetogenium sp., Anaerobaculum mobile, Clostridium ultunense, Petrotoga sp., Lactobacillus hammesii, Butyrivibrio sp., Syntrophococcus sucromutans, Olsenella sp., Tepidanaerobacter sp., Sporanaerobacter acetigenes, Pseudoramibacter alactolyticus, Lactobacillus fuchuensis or Lactobacillus sakei, Lactobacillus parabrevis or Lactobacillus spicheri and Enterococcus faecalis. The other half matched closely to ones from similar habitats (thermophilic anaerobic methanogenic digestion). The archaea consisted of Methanobrevibacter sp., Methanoculleus bourgensis, Methanosphaera stadtmanae, Methanimicrococcus blatticola and uncultured Methanomicrobiales. The role of these species in methane production is discussed.

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.

Thermotogae species are currently identified mainly on the basis of their unique toga and distinct branching in the rRNA and other phylogenetic trees. No biochemical or molecular markers are known that clearly distinguish the species from this phylum from all other bacteria. The taxonomic/evolutionary relationships within this phylum, which consists of a single family, are also unclear. We report detailed phylogenetic analyses on Thermotogae species based on concatenated sequences for many ribosomal as well as other conserved proteins that identify a number of distinct clades within this phylum. Additionally, comprehensive analyses of protein sequences from Thermotogae genomes have identified >60 Conserved Signature Indels (CSI) that are specific for the Thermotogae phylum or its different subgroups. Eighteen CSIs in important proteins such as PolI, RecA, TrpRS and ribosomal proteins L4, L7/L12, S8, S9, etc. are uniquely present in various Thermotogae species and provide molecular markers for the phylum. Many CSIs were specific for a number of Thermotogae subgroups. Twelve of these CSIs were specific for a clade consisting of various Thermotoga species except Tt. lettingae , which was separated from other Thermotoga species by a long branch in phylogenetic trees; Fourteen CSIs were specific for a clade consisting of the Fervidobacterium and Thermosipho genera and eight additional CSIs were specific for the genus Thermosipho . In addition, the existence of a clade consisting of the deep branching species Petrotoga mobilis, Kosmotoga olearia and Thermotogales bacterium mesG1 was supported by seven CSIs. The deep branching of this clade was also supported by a number of CSIs that were present in various Thermotogae species, but absent in this clade and all other bacteria. Most of these clades were strongly supported by phylogenetic analyses based on two datasets of protein sequences and they identify potential higher taxonomic grouping (viz. families) within this phylum. We also report 16 CSIs that are shared by either some or all Thermotogae species and some species from other taxa such as Archaea, Aquificae, Firmicutes, Proteobacteria, Deinococcus, Fusobacteria, Dictyoglomus, Chloroflexi and eukaryotes. The shared presence of some of these CSIs could be due to lateral gene transfers between these groups. However, no clear preference for any particular group was observed in this regard. The molecular probes based on different genes/proteins, which contain these Thermotogae-specific CSIs, provide novel and highly specific means for identification of both known as well as previously unknown Thermotogae species in different environments. Additionally, these CSIs also provide valuable tools for genetic and biochemical studies that could lead to discovery of novel properties that are unique to these bacteria.

Anaerobic incubations using crude oil and brine from a North Sea reservoir were conducted to gain increased understanding of indigenous microbial community development, metabolite production, and the effects on the oil–brine system after addition of a complex carbon source, molasses, with or without nitrate to boost microbial growth. Growth of the indigenous microbes was stimulated by addition of molasses. Pyrosequencing showed that specifically Anaerobaculum, Petrotoga, and Methanothermococcus were enriched. Addition of nitrate favored the growth of Petrotoga over Anaerobaculum. The microbial growth caused changes in the crude oil–brine system: formation of oil emulsions, and reduction of interfacial tension (IFT). Reduction in IFT was associated with microbes being present at the oil–brine interphase. These findings suggest that stimulation of indigenous microbial growth by addition of molasses has potential as microbial enhanced oil recovery (MEOR) strategy in North Sea oil reservoirs.

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.

H sub(2) production by Petrotoga miotherma, Thermosipho africanus, Thermotoga elfii, Fervidobacterium pennavorans, and Thermotoga neapolitana was compared under microaerobic conditions. Contrary to these previously reported strains being strict anaerobes, all tested strains grew and produced H sub(2) in the presence of micromolar levels of O sub(2). T. neapolitana showed the highest H sub(2) production under these conditions. Microscopic counting techniques were used to determine growth curves and doubling times, which were subsequently correlated with optical density measurements. The Biolog anaerobic microtiter plate system was used to analyze the carbon source utilization spectrum of T. neapolitana and to select non-metabolized or poorly metabolized carbohydrates as physiological buffers. Itaconic acid was successfully used as a buffer to overcome pH-induced limitations of cell growth and to facilitate enhanced production of CO-free H sub(2).