‘Candidatus ferrigenium straubiae’ sp. nov., ‘Candidatus ferrigenium bremense’ sp. nov., ‘Candidatus ferrigenium altingense’ sp. nov., are autotrophic Fe(II)-oxidizing bacteria of the family Gallionellaceae

Iron(II) [Fe(II)] oxidation coupled to denitrification is recognized as an environmentally important process in many ecosystems. However, the Fe(II)-oxidizing bacteria (FeOB) dominating autotrophic nitrate-reducing Fe(II)-oxidizing enrichment cultures, affiliated with the family Gallionellaceae, remain poorly taxonomically defined due to lack of representative isolates. We describe the taxonomic classification of three novel FeOB based on metagenome-assembled genomes (MAGs) acquired from the autotrophic nitrate-reducing enrichment cultures KS, BP and AG. Phylogenetic analysis of nearly full-length 16S rRNA gene sequences demonstrated that these three FeOB were most closely affiliated to the genera Ferrigenium, Sideroxydans and Gallionella, with up to 96.5%, 95.4% and 96.2% 16S rRNA gene sequence identities to representative isolates of these genera, respectively. In addition, average amino acid identities (AAI) of the genomes compared to the most closely related genera revealed highest AAI with Ferrigenium kumadai An22 (76.35–76.74%), suggesting that the three FeOB are members of this genus. Phylogenetic analysis of conserved functional genes further supported that these FeOB represent three novel species of the genus Ferrigenium. Moreover, the three novel FeOB likely have characteristic features, performing partial denitrification coupled to Fe(II) oxidation and carbon fixation. Scanning electron microscopy of the enrichment cultures showed slightly curved rod-shaped cells, ranging from 0.2-0.7 μm in width and 0.5–2.3 μm in length. Based on the phylogenetic, genomic and physiological characteristics, we propose that these FeOB represent three novel species, ‘Candidatus Ferrigenium straubiae’ sp. nov., ‘Candidatus Ferrigenium bremense’ sp. nov. and ‘Candidatus Ferrigenium altingense’ sp. nov. that might have unique metabolic features among the genus Ferrigenium.     

Characterization of wheat straw-degrading anaerobic alkali-tolerant mixed cultures from soda lake sediments by molecular and cultivation techniques

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.     

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 10⁷ cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.     

Phylogenetic composition of enrichment cultures of thermophilic prokaryotes reducing poorly crystalline Fe(III) oxide with and without direct contact between the cells and mineral

Thirty enrichment cultures of thermophilic microorganisms were obtained from Kamchatka terrestrial hydrotherms that reduced insoluble poorly crystalline Fe(III) oxide (ferrihydrite) with and without direct contact between the cells and the mineral. Restricted access to the Fe(III) mineral was achieved by incorporation of ferrihydrite into alginate beads. According to phylogenetic analysis of 22 enrichment cultures by denaturing gradient gel electrophoresis of 16S rRNA gene fragments, Firmicutes were predominant among bacteria in all the variants. Microorganisms of the phylogenetic types Aquificae, Bacteroidetes, Nitrospirae, Planctomycetes, Spirochaetes, Synergistetes, and Thermotogae were also revealed. The archaea revealed belonged to the genera Desulfurococcus, Pyrobaculum, and Thermofilum. In the case of free access to ferrihydrite, most of the phylotypes belonged to genera known for Fe(III) reduction. In the absence of direct contact with the mineral, together with known iron reducers, organisms for which ability to reduce Fe(III) was unknown were detected. Members of the genera Carboxydothermus, Thermoanaerobacter, and Thermotoga were detected most often both in the presence and absence of contact with ferrihydrite. These organisms probably possess efficient mechanisms for Fe(III) reduction within the experimental temperature range. Microbial phylogenetic diversity was higher when acetate, rather than lactate, was used as a potential electron donor.     

Enrichment of Members of the Family Geobacteraceae Associated with Stimulation of Dissimilatory Metal Reduction in Uranium-Contaminated Aquifer Sediments

Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.     

Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring

An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.     

Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and “Methylothermus,” which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57°C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Identification of triclosan-degrading bacteria in a triclosan enrichment culture using stable isotope probing

Triclosan, a widely used antimicrobial agent, is an emerging contaminant in the environment. Despite its antimicrobial character, biodegradation of triclosan has been observed in pure cultures, soils and activated sludge. However, little is known about the microorganisms responsible for the degradation in mixed cultures. In this study, active triclosan degraders in a triclosan-degrading enrichment culture were identified using stable isotope probing (SIP) with universally ¹³C-labeled triclosan. Eleven clones contributed from active microorganisms capable of uptake the ¹³C in triclosan were identified. None of these clones were similar to known triclosan-degraders/utilizers. These clones distributed among α-, β-, or γ-Proteobacteria: one belonging to Defluvibacter (α-Proteobacteria), seven belonging to Alicycliphilus (β-Proteobacteria), and three belonging to Stenotrophomonas (γ-Proteobacteria). Successive additions of triclosan caused a significant shift in the microbial community structure of the enrichment culture, with dominant ribotypes belonging to the genera Alicycliphilus and Defluvibacter. Application of SIP has successfully identified diverse uncultivable triclosan-degrading microorganisms in an activated sludge enrichment culture. The results of this study not only contributed to our understanding of the microbial ecology of triclosan biodegradation in wastewater, but also suggested that triclosan degraders are more phylogenetically diverse than previously reported.     

Bacteria and Genes Involved in Arsenic Speciation in Sediment Impacted by Long-Term Gold Mining

The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one of the world’s largest mining regions known as the “Iron Quadrangle”. A total of 123 As-resistant bacteria were recovered from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing (72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA (20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter. Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the molecular diversity of arrA genes is far from being fully explored deserving further attention.     

Diversity of cultivable bacteria isolated from the water column and bottom sediments of the Kara Sea shelf

In this work, the results of microbiological and molecular genetic investigation of the microorganisms inhabiting the Kara Sea and the adjacent Yenisei and Gydanskii Bays are presented. The microorganisms isolated from the samples collected in the studied area belonged to 4 phyla and 11 genera. Bacteria of two phyla, Firmicutes and Actinobacteria, prevailed; representatives of the Gammaproteobacteria and Bacteroidetes were isolated as well. According to their phenotypic properties, the obtained pure cultures were classified with the genera Streptomyces, Rhodococcus, Micrococcus, Bacillus, Pseudomonas, Acinetobacter, Flavobacterium, and Marinococcus. Analysis of the obtained nucleotide sequences of the 16S rRNA genes confirmed that the isolates belonged to the genus Bacillus. One strain was reidentified as Brevibacillus laterosporus, and two strains were identified Aeromonas piscicola and Plantibacter sp. The results of the study of the enzymatic activity of the obtained pure psychrotolerant cultures suggest that the microbial community is actively involved in the destruction processes occurring in the studied area.     

Microbial diversity in formation water and enrichment cultures from the Gangxi bed of the Dagang terrigenous oilfield (PRC)

Microbial diversity and biogeochemical processes of the Gangxi bed with low-mineral water and a temperature gradient from 35 to 54°C were studied. The 16S rRNA gene clone libraries (over 800 clones) were obtained from microbial DNA isolated from formation water and from the primary enrichment cultures for fermenting, sulfate-reducing, methanogenic, and aerobic organotrophic prokaryotes. While both sulfate reduction and methanogenesis were registered in formation water by radioisotope techniques, the genes of sulfate-reducing prokaryotes were not revealed in the 16S rRNA gene clone library from formation water. The 16S rRNA genes of Methanobacterium congolense and Methanococcus vannielii predominated among archaeal sequences retrieved from formation water, while the genes of Methanothermobacter thermoautotrophicus, Methanomethylovorans thermophila, and Methanoculleus sp. predominated in the combined library from enrichment cultures. In the library of Bacteria 16S rRNA genes from formation water, the genes of thermophilic fermentative bacteria of the family Thermoanaerobacteriaceae predominated; the remaining sequences belonged to mesophiles (genera Brevundimonas, Sphingomonas, Oxalicibacterium, and Stenotrophomonas), the phylum Chloroflexi, and unidentified bacteria. The combined library from enrichment cultures, contained, apart from the sequences of the family Thermoanaerobacteriaceae, the genes of fermentative bacteria (genera Anaerobaculum, Coprothermobacter, Thermanaerovibrio, Soehngenia, Bacteroides, and Aminobacterium and the order Thermotogales), of aerobic hydrocarbon-oxidizing bacteria (genera Pannonibacter and Pseudomonas), and of sulfate reducers (genera Desulfomicrobium, Thermodesulfovibrio, and Desulfotomaculum). High coverage was shown for bacterial (97.6%) and archaeal (100%) clone libraries, indicating that a significant portion of the microbial diversity in the studied communities was revealed.     

Isolation and Characterization of Novel Marine-Derived Actinomycete Taxa Rich in Bioactive Metabolites

A unique selective enrichment procedure has resulted in the isolation and identification of two new genera of marine-derived actinobacteria. Approximately 90% of the microorganisms cultured by using the presented method were from the prospective new genera, a result indicative of its high selectivity. In this study, 102 actinomycetes were isolated from subtidal marine sediments collected from the Bismarck Sea and the Solomon Sea off the coast of Papua New Guinea. A combination of physiological parameters, chemotaxonomic characteristics, distinguishing 16S rRNA gene sequences, and phylogenetic analysis based on 16S rRNA genes provided strong evidence for the two new genera (represented by strains of the PNG1 clade and strain UMM518) within the family Micromonosporaceae. Biological activity testing of fermentation products from the new marine-derived actinomycetes revealed that several had activities against multidrug-resistant gram-positive pathogens, malignant cells, and vaccinia virus replication.     

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.     

Molecular analysis of high-affinity methane-oxidizing enrichment cultures isolated from a forest biocenosis and agrocenoses

Methane oxidation by microorganisms inhabiting aerobic soils is a key process involved in the regulation of the concentration of this significant greenhouse gas in the atmosphere; however, the microorganisms responsible for this process remain unknown. Three stable methane-oxidizing cultures were isolated from samples of forest soils (FS) and agricultural soils (AS) of Moscow oblast, as well as from soil samples collected from a Belgian agrocenosis (BS). The obtained enrichment cultures exhibit a high affinity for methane; their k_m values range from 54.2 to 176.8 nM CH₄ and are comparable to those of aerobic soils. Analysis of the fragments of the ribosomal (16S rRNA) and functional (pmoA) genes of methanotrophs by PCR— DGGE and cloning demonstrated the presence of bacteria belonging to the genera Methylocystis in FS, Methylosinus in AS and BS, and Methylocella in BS. It was established that Methylocystis and Methylosinus detected in the enrichment cultures contain the genes encoding the synthesis of the active center of two membrane-bound particulate methane monooxygenases; it is likely that one of these genes (pmoA2) is responsible for the capacity of these microorganisms for oxidation of atmospheric methane.     

High abundance of heterotrophic prokaryotes in hydrothermal springs of the Azores as revealed by a network of 16S rRNA gene-based methods

Two hydrothermal springs (AI: 51 °C, pH 3; AIV: 92 °C, pH 8) were analysed to determine prokaryotic community composition. Using pyrosequencing, 93,576 partial 16S rRNA gene sequences amplified with V2/V3-specific primers for Bacteria and Archaea were investigated and compared to 16S rRNA gene sequences from direct metagenome sequencing without prior amplification. The results were evaluated by fluorescence in situ hybridization (FISH). While in site AIV Bacteria and Archaea were detected in similar relative abundances (Bacteria 40 %, Archaea 35 %), the acidic spring AI was dominated by Bacteria (68 %). In spring AIV the combination of 16S rRNA gene sequence analysis and FISH revealed high abundance (>50 %) of heterotrophic bacterial genera like Caldicellulosiruptor, Dictyoglomus, and Fervidobacterium. In addition, chemolithoautotrophic Aquificales were detected in the bacterial community with Sulfurihydrogenibium being the dominant genus. Regarding Archaea, only Crenarchaeota, were detected, dominated by the family Desulfurococcaceae (>50 %). In addition, Thermoproteaceae made up almost 25 %. In the acidic spring (AI) prokaryotic diversity was lower than in the hot, slightly alkaline spring AIV. The bacterial community of site AI was dominated by organisms related to the chemolithoautotrophic genus Acidithiobacillus (43 %), to the heterotrophic Acidicaldus (38 %) and to Anoxybacillus (7.8 %). This study reveals differences in the relative abundance of heterotrophic versus autotrophic microorganisms as compared to other hydrothermal habitats. Furthermore, it shows how different methods to analyse prokaryotic communities in complex ecosystems can complement each other to obtain an in-depth picture of the taxonomic composition and diversity within these hydrothermal springs.