Determination of methanogenic Archaea abundance in a mesophilic biogas plant based on 16S rRNA gene sequence analysis

2010 ◽  
Vol 56 (5) ◽  
pp. 440-444 ◽  
Author(s):  
I. Bergmann ◽  
E. Nettmann ◽  
K. Mundt ◽  
M. Klocke
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Energy Production ◽  
Methanogenic Archaea ◽  
Biogas Plant ◽  
Raw Material ◽  
Gene Copy ◽  
Rrna Gene ◽  
Methane Formation ◽  
Technical Equipment

Energy production of renewable raw material is of increasing importance for sustainable energy production. As an indispensable prerequirement for further upgrading of technical equipment and operation modes of biogas plants, a deeper knowledge of the microbial community responsible for methane formation is crucial. To overcome these limitations a mesophilic biogas plant converting pig manure, maize silage, and grains of crop was sampled and subsequently analysed by construction of a methanogenic Archaea specific 16S rRNA gene clone library combined with PCR–RFLP analysis and group-specific quantitative real-time PCR (qPCR). Seventy percent of all analysed clones belonged to the order Methanomicrobiales, whereas 13% belonged to Methanosarcinales, 6% belonged to the Methanobacteriales group, and 11% of all detected clones were assigned to the CA11 and Arch1 cluster. Comparable percentages were obtained with qPCR: 84% of all detected 16S rRNA gene copy numbers were affiliated with the Methanomicrobiales, while only 14% belonged to the Methanosarcinales and 2% to the Methanobacteriales order. In conclusion, both approaches detected similar archaeal groups and revealed nearly the same abundance, pointing to a predominance of hydrogenotrophic methanogens in the biogas plant.

scholarly journals GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

2022 ◽  
Vol 12 ◽  
Author(s):  
Ilona A. Ruhl ◽  
Andriy Sheremet ◽  
Chantel C. Furgason ◽  
Susanne Krause ◽  
Robert M. Bowers ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Trees ◽  
Hot Spring ◽  
Gc Content ◽  
Gene Copy ◽  
Marker Genes ◽  
Rrna Gene ◽  
Separate Species ◽  
Distinct Subgroup

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

scholarly journals Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Peter Kusstatscher ◽  
Wisnu Adi Wicaksono ◽  
Alessandro Bergna ◽  
Tomislav Cernava ◽  
Nick Bergau ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Dynamics ◽  
Statistical Tests ◽  
Central Element ◽  
Shannon Index ◽  
Gene Copy ◽  
Rrna Gene ◽  
Core Microbiome ◽  
Defense Strategies

Abstract Background The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments. Results Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108–109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific feature; Burkholderiaceae and Actinobacteriaceae showed similar patterns. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed. Conclusion Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies.

scholarly journals Influences of Infaunal Burrows on the Community Structure and Activity of Ammonia-Oxidizing Bacteria in Intertidal Sediments

2006 ◽  
Vol 73 (4) ◽  
pp. 1341-1348 ◽  
Author(s):  
Hisashi Satoh ◽  
Yoshiyuki Nakamura ◽  
Satoshi Okabe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Sediment Surface ◽  
Intertidal Sediment ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Intertidal Sediments ◽  
Burrow Wall ◽  
Microelectrode Measurements

ABSTRACT Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O2 concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O2 transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH4 + consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH4 + and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O2 transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH4 + consumption capacity. Consequently, the infaunal burrow became an important site for NH4 + consumption in the intertidal sediment.

scholarly journals Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato

2020 ◽  
Author(s):  
Peter Kusstatscher ◽  
Wisnu Adi Wicaksono ◽  
Alessandro Bergna ◽  
Tomislav Cernava ◽  
Nick Bergau ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Dynamics ◽  
Statistical Tests ◽  
Central Element ◽  
Shannon Index ◽  
Gene Copy ◽  
Rrna Gene ◽  
Core Microbiome ◽  
Defense Strategies

Abstract Background The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments.Results Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108- 109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific taxa; Burkholderiaceae and Actinobacteria showed similar pattern. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed.Conclusion Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies.

scholarly journals High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Žana Kapustina ◽  
Justina Medžiūnė ◽  
Gediminas Alzbutas ◽  
Irmantas Rokaitis ◽  
Karolis Matjošaitis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Sequencing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Copy Numbers ◽  
Rrna Sequencing ◽  
Metagenome Sequencing ◽  
Gene Copy Numbers

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1–V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

scholarly journals Comparison of Euryarchaea Strains in the Guts and Food-Soil of the Soil-Feeding Termite Cubitermes fungifaber across Different Soil Types

2004 ◽  
Vol 70 (7) ◽  
pp. 3884-3892 ◽  
Author(s):  
S. E. Donovan ◽  
K. J. Purdy ◽  
M. D. Kane ◽  
P. Eggleton
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rflp Analysis ◽  
Methanogenic Archaea ◽  
16S Rrna Gene Sequencing ◽  
Nutrient Content ◽  
Soil Types ◽  
Rrna Gene ◽  
Soil Communities ◽  
Rrna Gene Sequencing

ABSTRACT Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.

scholarly journals Culture-Independent Characterization of Archaeal Biodiversity in Swine Confinement Building Bioaerosols

2009 ◽  
Vol 75 (17) ◽  
pp. 5445-5450 ◽  
Author(s):  
Benjamin Nehm� ◽  
Yan Gilbert ◽  
Val�rie L�tourneau ◽  
Robert J. Forster ◽  
Marc Veillette ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Molecular Ecology ◽  
Pcr Amplification ◽  
Methanogenic Archaea ◽  
Archaeal Community ◽  
Pig Manure ◽  
Rrna Gene ◽  
Swine Confinement

ABSTRACT It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 108 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.

scholarly journals Abundance, Distribution, and Activity of Fe(II)-Oxidizing and Fe(III)-Reducing Microorganisms in Hypersaline Sediments of Lake Kasin, Southern Russia

2012 ◽  
Vol 78 (12) ◽  
pp. 4386-4399 ◽  
Author(s):  
Maren Emmerich ◽  
Ankita Bhansali ◽  
Tina Lösekann-Behrens ◽  
Christian Schröder ◽  
Andreas Kappler ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sediment Layer ◽  
Hypersaline Environments ◽  
Probable Number ◽  
Content Type ◽  
Southern Russia ◽  
Hypersaline Sediments

ABSTRACTThe extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducingBacteriaandArchaeain pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter−1) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries forBacteriaandArchaearevealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 102to 8.32 × 103iron(II)-oxidizingBacteriaand 4.16 × 102to 2.13 × 103iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of totalBacteria, totalArchaea, and species dominating the iron(III)-reducing enrichment cultures (relatives ofHalobaculum gomorrense,Desulfosporosinus lacus, and members of theBacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.

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