Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

ABSTRACT Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

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ANME-1 archaea drive methane accumulation and removal in estuarine sediments

10.1101/2020.02.24.963215 ◽

2020 ◽

Cited By ~ 2

Author(s):

Richard Kevorkian ◽

Sean Callahan ◽

Rachel Winstead ◽

Karen G. Lloyd

Keyword(s):

16S Rrna ◽

Sulfate Reducing Bacteria ◽

Low Energy ◽

Estuarine Sediments ◽

16S Rrna Genes ◽

Rrna Genes ◽

Sulfate Reducing ◽

Hydrogenotrophic Methanogenesis ◽

Natural Settings ◽

Reducing Bacteria

AbstractUncultured members of the Methanomicrobia called ANME-1 perform the anaerobic oxidation of methane (AOM) through a process that uses much of the methanogenic pathway. It is unknown whether ANME-1 obligately perform AOM, or whether some of them can perform methanogenesis when methanogenesis is exergonic. Most marine sediments lack advective transport of methane, so AOM occurs in the sulfate methane transition zone (SMTZ) where sulfate-reducing bacteria consume hydrogen produced by fermenters, making hydrogenotrophic methanogenesis exergonic in the reverse direction. When sulfate is depleted deeper in the sediments, hydrogen accumulates making hydrogenotrophic methanogenesis exergonic, and methane accumulates in the methane zone (MZ). In White Oak River estuarine sediments, we found that ANME-1 comprised 99.5% of 16S rRNA genes from amplicons and 100% of 16S rRNA genes from metagenomes of the Methanomicrobia in the SMTZ and 99.9% and 98.3%, respectively, in the MZ. Each of the 16 ANME-1 OTUs (97% similarity) had peaks in the SMTZ that coincided with peaks of putative sulfate-reducing bacteria Desulfatiglans sp. and SEEP-SRB1. In the MZ, ANME-1, but no putative sulfate-reducing bacteria or cultured methanogens, increased with depth. Using publicly available data, we found that ANME-1 was the only group expressing methanogenic genes during both net AOM and net methanogenesis in an enrichment. The commonly-held belief that ANME-1 perform AOM is based on the fact that they dominate natural settings and enrichments where net AOM is measured. We found that ANME-1 also dominate natural settings and enrichment where net methanogenesis is measured, so we conclude that ANME-1 perform methane production. Alternating between AOM and methanogenesis, either in a single ANME-1 cell or between different subclades with similar 16S rRNA sequences of ANME-1, may confer a competitive advantage, explaining the predominance of low-energy adapted ANME-1 in methanogenic sediments worldwide.Abstract ImportanceLife may operate differently at very low energy levels. Natural populations of microbes that make methane survive on some of the lowest energy yields of all life. From all available data, we infer that these microbes alternate between methane production and oxidation, depending on which process is energy-yielding in the environment. This means that much of the methane produced naturally in marine sediments occurs through an organism that is also capable of destroying it under different circumstances.

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Decoupled distance-decay patterns between dsrA and 16S rRNA genes among salt marsh sulfate-reducing bacteria

Environmental Microbiology ◽

10.1111/1462-2920.12821 ◽

2015 ◽

Vol 18 (1) ◽

pp. 75-86 ◽

Cited By ~ 17

Author(s):

Angus Angermeyer ◽

Sarah C. Crosby ◽

Julie A. Huber

Keyword(s):

Salt Marsh ◽

16S Rrna ◽

Sulfate Reducing Bacteria ◽

Distance Decay ◽

16S Rrna Genes ◽

Rrna Genes ◽

Sulfate Reducing ◽

Reducing Bacteria

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Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2765-2772.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2765-2772 ◽

Cited By ~ 217

Author(s):

Ashita Dhillon ◽

Andreas Teske ◽

Jesse Dillon ◽

David A. Stahl ◽

Mitchell L. Sogin

Keyword(s):

16S Rrna ◽

Gulf Of California ◽

Sulfate Reducing Bacteria ◽

16S Rrna Genes ◽

Rrna Genes ◽

Terrestrial Organic Matter ◽

Guaymas Basin ◽

Sulfate Reducers ◽

Sulfate Reducing ◽

Reducing Bacteria

ABSTRACT The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.

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Anaerobic Oxidation of o -Xylene, m -Xylene, and Homologous Alkylbenzenes by New Types of Sulfate-Reducing Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.65.3.999-1004.1999 ◽

1999 ◽

Vol 65 (3) ◽

pp. 999-1004 ◽

Cited By ~ 143

Author(s):

Gerda Harms ◽

Karsten Zengler ◽

Ralf Rabus ◽

Frank Aeckersberg ◽

Dror Minz ◽

...

Keyword(s):

Crude Oil ◽

Enrichment Culture ◽

Sulfate Reducing Bacteria ◽

16S Rrna Genes ◽

Rrna Genes ◽

Organic Substrates ◽

Sulfate Reducing ◽

Concomitant Reduction ◽

Reducing Bacteria ◽

Growth Experiments

ABSTRACT Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the δ subclass of the classProteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum andDesulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, ando-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene,m-isoproyltoluene, benzoate, andm-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide.

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Desulfoglaeba alkanexedens gen. nov., sp. nov., an n-alkane-degrading, sulfate-reducing bacterium

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64398-0 ◽

2006 ◽

Vol 56 (12) ◽

pp. 2737-2742 ◽

Cited By ~ 81

Author(s):

Irene A. Davidova ◽

Kathleen E. Duncan ◽

Ok Kyoung Choi ◽

Joseph M. Suflita

Keyword(s):

Type Strain ◽

Novel Species ◽

Sulfate Reducing Bacteria ◽

16S Rrna Genes ◽

Rrna Genes ◽

Oily Wastewater ◽

Sulfate Reducing ◽

Production Water ◽

Large Clusters ◽

Reducing Bacteria

Two novel sulfate-reducing bacteria, strains ALDCT and Lake, which were able to oxidize n-alkanes, were isolated from a naval oily wastewater-storage facility (VA, USA) and from oilfield production water (OK, USA), respectively. The type strain (ALDCT) had a narrow substrate specificity and could grow only with n-alkanes (from C6 to C12), pyruvate, butyrate, hexanoic acid and 4-methyloctanoic acid. Cells of strain ALDCT stained Gram-negative and were slightly curved, short rods with oval ends (2.5–3.0×1.0–1.4 μm), often occurring in pairs. Cells tended to form aggregates or large clusters and were non-motile and did not form endospores. Optimum growth occurred between 31 and 37 °C and at pH 6.5–7.2. NaCl was not required for growth, but salt concentrations up to 55 g l−1 could be tolerated. The DNA G+C content was 53.6 mol%. Phylogenetic analysis of the 16S rRNA genes revealed that strains ALDCT and Lake were closely related, but not identical (99.9 % similarity). The two strains were not closely related to other known alkane-degrading, sulfate-reducing bacteria or to other genera of the Deltaproteobacteria. Therefore, it is proposed that strain ALDCT (=JCM 13588T=ATCC BAA-1302T) represents the type strain of a novel species and genus, with the name Desulfoglaeba alkanexedens gen. nov., sp. nov.

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