Molecular profiling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae)

The use of nuclear power has been a significant part of the United Kingdom’s energy portfolio with the Sellafield site being used for power production and more recently reprocessing and decommissioning of spent nuclear fuel activities. Before being reprocessed, spent nuclear fuel is stored in water ponds with significant levels of background radioactivity and in high alkalinity (to minimize fuel corrosion). Despite these challenging conditions, the presence of microbial communities has been detected. To gain further insight into the microbial communities present in extreme environments, an indoor, hyper-alkaline, oligotrophic, and radioactive spent fuel storage pond (INP) located on the Sellafield site was analyzed. Water samples were collected from sample points within the INP complex, and also the purge water feeding tank (FT) that supplies water to the pond, and were screened for the presence of the 16S and 18S rRNA genes to inform sequencing requirements over a period of 30 months. Only 16S rRNA genes were successfully amplified for sequencing, suggesting that the microbial communities in the INP were dominated by prokaryotes. Quantitative Polymerase Chain Reaction (qPCR) analysis targeting 16S rRNA genes suggested that bacterial cells in the order of 104–106 mL–1 were present in the samples, with loadings rising with time. Next generation Illumina MiSeq sequencing was performed to identify the dominant microorganisms at eight sampling times. The 16S rRNA gene sequence analysis suggested that 70% and 91% from of the OTUs samples, from the FT and INP respectively, belonged to the phylum Proteobacteria, mainly from the alpha and beta subclasses. The remaining OTUs were assigned primarily to the phyla Acidobacteria, Bacteroidetes, and, Cyanobacteria. Overall the most abundant genera identified were Hydrogenophaga, Curvibacter, Porphyrobacter, Rhodoferax, Polaromonas, Sediminibacterium, Roseococcus, and Sphingomonas. The presence of organisms most closely related to Hydrogenophaga species in the INP areas, suggests the metabolism of hydrogen as an energy source, most likely linked to hydrolysis of water caused by the stored fuel. Isolation of axenic cultures using a range of minimal and rich media was also attempted, but only relatively minor components (from the phylum Bacteroidetes) of the pond water communities were obtained, emphasizing the importance of DNA-based, not culture-dependent techniques, for assessing the microbiome of nuclear facilities.

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Identification of prevalent microbial communities in a municipal solid waste landfill

Water Science & Technology ◽

10.2166/wst.2006.244 ◽

2006 ◽

Vol 53 (8) ◽

pp. 139-147 ◽

Cited By ~ 9

Author(s):

B. Calli ◽

S. Durmaz ◽

B. Mertoglu

Keyword(s):

16S Rrna ◽

Municipal Solid Waste ◽

Microbial Communities ◽

Solid Waste ◽

Molecular Techniques ◽

16S Rrna Genes ◽

Rrna Genes ◽

Fish Analysis ◽

Municipal Solid Waste Landfill ◽

Waste Landfill

To identify the microbial communities in Istanbul, Odayeri Municipal Solid Waste Landfill, leachate samples were collected from different sections at different stabilization phases. In identification of microbial communities in leachate samples, molecular techniques such as FISH, DGGE and cloning based on 16S rRNA and mcrA genes were used. As the chemical and microbiological compositions of the samples were compared, obvious correlations were found between the stability of the landfill section and abundance of active methanogens. On the other hand, there were considerable differences between acidogenic and mature leachate samples in DGGE profiles of archaeal and bacterial 16S rRNA genes. Moreover, in acidogenic leachate samples having BOD5/COD ratio of about 0.5 acetate utilizing Methanosarcina and Methanosaeta species were intensively detected in FISH. Although only very few H2-utilizing methanogens were identified with FISH analysis, most of the clones isolated from mature leachate samples clustered within H2-utilizing Methanobacteriales and Methanomicrobiales according to phylogenetic analysis of 16S rRNA and mcrA clones, respectively.

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Comparison of Microbial Community Compositions of Two Subglacial Environments Reveals a Possible Role for Microbes in Chemical Weathering Processes

Applied and Environmental Microbiology ◽

10.1128/aem.71.11.6986-6997.2005 ◽

2005 ◽

Vol 71 (11) ◽

pp. 6986-6997 ◽

Cited By ~ 186

Author(s):

Mark Skidmore ◽

Suzanne P. Anderson ◽

Martin Sharp ◽

Julia Foght ◽

Brian D. Lanoil

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Microbial Communities ◽

Blot Hybridization ◽

16S Rrna Genes ◽

Rrna Genes ◽

Solute Flux ◽

Carbonate Dissolution ◽

Dot Blot ◽

Dot Blot Hybridization

ABSTRACT Viable microbes have been detected beneath several geographically distant glaciers underlain by different lithologies, but comparisons of their microbial communities have not previously been made. This study compared the microbial community compositions of samples from two glaciers overlying differing bedrock. Bulk meltwater chemistry indicates that sulfide oxidation and carbonate dissolution account for 90% of the solute flux from Bench Glacier, Alaska, whereas gypsum/anhydrite and carbonate dissolution accounts for the majority of the flux from John Evans Glacier, Ellesmere Island, Nunavut, Canada. The microbial communities were examined using two techniques: clone libraries and dot blot hybridization of 16S rRNA genes. Two hundred twenty-seven clones containing amplified 16S rRNA genes were prepared from subglacial samples, and the gene sequences were analyzed phylogenetically. Although some phylogenetic groups, including the Betaproteobacteria, were abundant in clone libraries from both glaciers, other well-represented groups were found at only one glacier. Group-specific oligonucleotide probes were developed for two phylogenetic clusters that were of particular interest because of their abundance or inferred biochemical capabilities. These probes were used in quantitative dot blot hybridization assays with a range of samples from the two glaciers. In addition to shared phyla at both glaciers, each glacier also harbored a subglacial microbial population that correlated with the observed aqueous geochemistry. These results are consistent with the hypothesis that microbial activity is an important contributor to the solute flux from glaciers.

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Different Active Microbial Communities in Two Contrasted Subantarctic Fjords

Frontiers in Microbiology ◽

10.3389/fmicb.2021.620220 ◽

2021 ◽

Vol 12 ◽

Author(s):

Claudia Maturana-Martínez ◽

Camila Fernández ◽

Humberto E. González ◽

Pierre E. Galand

Keyword(s):

Climate Change ◽

Microbial Community ◽

16S Rrna ◽

Microbial Communities ◽

Standing Stock ◽

16S Rrna Genes ◽

Rrna Genes ◽

Biogeochemical Processes ◽

Biogeographic Patterns ◽

Group I

Microorganisms play a crucial role in biogeochemical processes affecting the primary production and biogeochemical cycles of the ocean. In subpolar areas, the increment of the water temperature induced by climate change could lead to changes in the structure and activity of planktonic microbial communities. To understand how the structure of the microbial community in Chilean Patagonian fjords could be affected by climate change, we analyzed the composition of the prokaryotic community (bacteria-archaea) in two fjords (Pia and Yendegaia) with contrasting morphological and hydrological features. We targeted both the standing stock (16S rRNA genes) and the active fraction (16S rRNA transcripts) of the microbial communities during two consecutive austral winters. Our results showed that in both fjords, the active community had higher diversity and stronger biogeographic patterns when compared to the standing stock. Members of the Alpha-, Gamma-, and Deltaproteobacteria followed by archaea from the Marine Group I (Thaumarchaeota) dominated the active communities in both fjords. However, in Pia fjord, which has a marine-terminating glacier, the composition of the microbial community was directly influenced by the freshwater discharges from the adjacent glacier, and indirectly by a possible upwelling phenomenon that could bring deep sea bacteria such as SAR202 to the surface layer. In turn, in the Yendegaia, which has a land-terminating glacier, microbial communities were more similar to the ones described in oceanic waters. Furthermore, in Yendegaia fjord, inter-annual differences in the taxonomic composition and diversity of the microbial community were observed. In conclusion, Yendegaia fjord, without glacier calving, represents a fjord type that will likely be more common under future climate scenarios. Our results showing distinct Yendegaia communities, with for example more potential nitrogen-fixing microorganisms (Planctomycetes), indicate that as a result of climate change, changing planktonic communities could potentially impact biogeochemical processes and nutrient sources in subantarctic fjords.

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Comparative Analysis of Bacterial and Archaeal Population Structure by Illumina Sequencing of 16S rRNA Genes in Three Municipal Anaerobic Sludge Digesters

10.21203/rs.3.rs-60183/v1 ◽

2020 ◽

Author(s):

Munawwar Ali Khan ◽

Shams Tabrez Khan ◽

Milred Cedric Sequeira ◽

Sultan Mohammad Faheem

Keyword(s):

Wastewater Treatment ◽

16S Rrna ◽

Microbial Communities ◽

Illumina Sequencing ◽

Full Scale ◽

16S Rrna Genes ◽

Rrna Genes ◽

Anaerobic Digesters ◽

Fermentative Bacteria ◽

Better Regulation

Abstract Understanding the microbial communities in anaerobic digesters is important for better regulation, operation, and sustainable management of the sludge produced at various stages of wastewater treatment processes. Microbial communities in the anaerobic digester of the gulf region where the climatic conditions and other factors may impact the incoming feed have not been documented. Archaeal and Bacterial communities of three full-scale anaerobic digesters, namely AD1, AD3 and AD5 were analyzed by Illumina sequencing of 16S rRNA genes. Among bacteria, the most abundant genus was fermentative bacteria Acetobacteroides (Blvii28). Other predominant bacterial genera in the digesters included thermophilic bacteria (Fervidobacterium and Coprothermobacter) and halophilic bacteria like Haloterrigena and Sediminibacter. This can be correlated with the climatic condition in Dubai, where the bacteria in the original feed may be thermophilic or halophilic as much of the water used in the country is desalinated seawater. Propionic acid-producing bacteria like Paludibacter and propionate oxidizing bacteria like W5 were also dominating group and were found in all the digesters. The predominant Archaea include mainly the members of phylum Euryarchaeota and Crenarchaeota belonging to genus Methanocorpusculum, Metallosphaera, Methanocella, and Methanococcus. The highest population of Methanocorpusculum (more than 50% of total Archaea) hydrogenotrophic archaea matches with the high population of Acetobacteroides (Blvii28) and Fervidobacterium bacteria which ferments the organic substrates to acetate and H2. Coprothermobacter, which is known to improve protein degradation by establishing a syntrophy with hydrogenotrophic archaea, was also one of the dominant genera in the digesters. This study, for the first time, contributes to an in-depth understanding of the phylogenetic diversity of a microbial community of three full-scale anaerobic digesters of a municipal wastewater treatment plant in Dubai, UAE.

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High Diversity in Iron Cycling Microbial Communities in Acidic, Iron-Rich Water of the Pyhäsalmi Mine, Finland

Geofluids ◽

10.1155/2019/7401304 ◽

2019 ◽

Vol 2019 ◽

pp. 1-17 ◽

Cited By ~ 10

Author(s):

Malin Bomberg ◽

Jarno Mäkinen ◽

Marja Salo ◽

Päivi Kinnunen

Keyword(s):

16S Rrna ◽

Microbial Diversity ◽

Microbial Communities ◽

Mine Water ◽

Ferrous Iron ◽

Great Part ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Iron Oxidizers

Microbial communities of iron-rich water in the Pyhäsalmi mine, Finland, were investigated with high-throughput amplicon sequencing and qPCR targeting bacteria, archaea, and fungi. In addition, the abundance ofLeptospirillumandAcidithiobacilluswas assessed with genus-specific qPCR assays, and enrichment cultures targeting aerobic ferrous iron oxidizers and ferric iron reducers were established. The acidic (pH 1.4–2.3) mine water collected from 240 m, 500 m, and 600 m depth from within the mine had a high microbial diversity consisting of 63-114 bacterial, 10-13 archaeal, and 104-117 fungal genera. The most abundant microorganisms in the mine water were typical acid mine drainage (AMD) taxa, such as acidophilic, iron-oxidizingLeptospirillum,Acidiphilum,Acidithiobacillus,Ferrovum, andThermoplasma. The fungi belonged mostly to the phylum Ascomycetes, although a great part of the fungal sequences remained unclassified. The number of archaeal 16S rRNA genes in the mine water was between 0.3 and 1.2 × 107copies mL−1in the samples from 500 m and 600 m, but only 3.9 × 103at 240 m and archaea were in general not enriched in cultures. The number of fungal 5.8S rRNA genes was high only in the mine water from 500 m and 600 m, where 0.2–3.4 × 104spore equivalents mL−1were detected. A high number ofLeptospirillum16S rRNA genes, 0.6–1.6 × 1010copies mL−1, were detected at 500 m and 600 m depth and in cultures containing ferrous iron, showing the importance of iron oxidizers in this environment. The abundance of bacteria in general was between 103and 10616S rRNA gene copies mL−1. Our results showed a high microbial diversity in the acid- and iron-impacted waters of the Pyhäsalmi mine, whereLeptospirillumbacteria were especially prominent. These iron oxidizers are also the main nitrogen-fixing microorganisms in this ecosystem.

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Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2577-2587.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2577-2587 ◽

Cited By ~ 96

Author(s):

Manuel Pesaro ◽

Gilles Nicollier ◽

Josef Zeyer ◽

Franco Widmer

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

Stress Responses ◽

Crop Protection ◽

16S Rrna Genes ◽

Cell Counting ◽

Rrna Genes ◽

Soil Dna ◽

Degradation Rates ◽

The Impact

ABSTRACT Prior to registration of crop protection products (CPPs) their persistence in soil has to be determined under defined conditions. For this purpose, soils are collected in the field and stored for up to 3 months prior to the tests. During storage, stresses like drying may induce changes in microbiological soil characteristics (MSCs) and thus may influence CPP degradation rates. We investigated the influence of soil storage-related stress on the resistance and resilience of different MSCs by assessing the impact of a single severe drying-rewetting cycle and by monitoring recovery from this event for 34 days. The degradation and mineralization of the fungicide metalaxyl-M and the insecticide lufenuron were delayed by factors of 1.5 to 5.4 in the dried and rewetted soil compared to the degradation and mineralization in an undisturbed reference. The microbial biomass, as estimated by direct cell counting and from the soil DNA content, decreased on average by 51 and 24%, respectively. The bulk microbial activities, as determined by measuring substrate-induced respiration and fluorescein diacetate hydrolysis, increased after rewetting and recovered completely within 6 days after reequilibration. The effects on Bacteria, Archaea, and Pseudomonas were investigated by performing PCR amplification of 16S rRNA genes and reverse-transcribed 16S rRNA, followed by restriction fragment length polymorphism (RFLP) and terminal RFLP (T-RFLP) fingerprinting. Statistical analyses of RFLP and T-RFLP profiles indicated that specific groups in the microbial community were sensitive to the stress. In addition, evaluation of rRNA genes and rRNA as markers for monitoring the stress responses of microbial communities revealed overall similar sensitivities. We concluded that various structural and functional MSCs were not resistant to drying-rewetting stress and that resilience depended strongly on the parameter investigated.

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Biogas production from hydrothermal liquefaction wastewater (HTLWW): Focusing on the microbial communities as revealed by high-throughput sequencing of full-length 16S rRNA genes

Water Research ◽

10.1016/j.watres.2016.09.052 ◽

2016 ◽

Vol 106 ◽

pp. 98-107 ◽

Cited By ~ 47

Author(s):

Huihui Chen ◽

Jingjing Wan ◽

Kaifei Chen ◽

Gang Luo ◽

Jiajun Fan ◽

...

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

High Throughput ◽

High Throughput Sequencing ◽

Biogas Production ◽

Hydrothermal Liquefaction ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes

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Application of the stochastic labeling methods with random-sequence barcodes for simultaneous quantification and sequencing of environmental 16S rRNA genes

10.1101/072298 ◽

2016 ◽

Cited By ~ 1

Author(s):

Tatsuhiko Hoshino ◽

Fumio Inagaki

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Copy Number ◽

Random Sequence ◽

Digital Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

The 16S Rrna Gene

AbstractNext-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5’ end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103to 5.0 × 104copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

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