scholarly journals Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses

2010 ◽  
Vol 76 (22) ◽  
pp. 7451-7458 ◽  
Author(s):  
Yao Pan ◽  
Levente Bodrossy ◽  
Peter Frenzel ◽  
Anne-Grethe Hestnes ◽  
Sascha Krause ◽  
...  
Keyword(s):  
Microbial Community ◽  
Soil Sample ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Community Composition ◽  
Diversity Indices ◽  
Internal Standards ◽  
Microbial Biogeography ◽  
Yield And Quality ◽  
Gradient Gel Electrophoresis

ABSTRACT With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a “quantifiable” bias.

Spatial variation in microbial community structure, richness, and diversity in an alluvial aquifer

2012 ◽  
Vol 58 (9) ◽  
pp. 1135-1151 ◽  
Author(s):  
P.G. Medihala ◽  
J.R. Lawrence ◽  
G.D.W. Swerhone ◽  
D.R. Korber
Keyword(s):  
Community Structure ◽  
Microbial Community ◽  
Spatial Variability ◽  
Microbial Communities ◽  
Microbial Community Structure ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Community Composition ◽  
Alluvial Aquifer ◽  
Gradient Gel Electrophoresis

Relatively little is known regarding the spatial variability of microbial communities in aquifers where well fouling is an issue. In this study 2 water wells were installed in an alluvial aquifer located adjacent to the North Saskatchewan River and an associated piezometer network developed to facilitate the study of microbial community structure, richness, and diversity. Carbon utilization data analysis revealed reduced microbial activity in waters collected close to the wells. Functional PCR and quantitative PCR analysis indicated spatial variability in the potential for iron-, sulphate-, and nitrate-reducing activity at all locations in the aquifer. Denaturing gradient gel electrophoresis analysis of aquifer water samples using principal components analyses indicated that the microbial community composition was spatially variable, and denaturing gradient gel electrophoresis sequence analysis revealed that bacteria belonging to the genera Acidovorax , Rhodobacter , and Sulfuricurvum were common throughout the aquifer. Shannon’s richness (H′) and Pielou’s evenness (J′) indices revealed a varied microbial diversity (H′ = 1.488–2.274) and an even distribution of microbial communities within the aquifer (J′ = 0.811–0.917). Overall, these analyses revealed that the aquifer’s microbial community varied spatially in terms of composition, richness, and metabolic activity. Such information may facilitate the diagnosis, prevention, and management of fouling.

scholarly journals Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition

2000 ◽  
Vol 66 (12) ◽  
pp. 5488-5491 ◽  
Author(s):  
Robert I. Griffiths ◽  
Andrew S. Whiteley ◽  
Anthony G. O'Donnell ◽  
Mark J. Bailey
Keyword(s):  
Microbial Community ◽  
Ribosomal Dna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Community Composition ◽  
Pcr Amplification ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Natural Environments ◽  
Dna And Rna ◽  
Gradient Gel Electrophoresis

ABSTRACT A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

scholarly journals Inhibiting Methanogenesis in Rumen Batch Cultures Did Not Increase the Recovery of Metabolic Hydrogen in Microbial Amino Acids

2019 ◽  
Vol 7 (5) ◽  
pp. 115 ◽  
Author(s):  
Emilio M. Ungerfeld ◽  
M. Fernanda Aedo ◽  
Emilio D. Martínez ◽  
Marcelo Saldivia
Keyword(s):  
Amino Acids ◽  
Energy Efficiency ◽  
Microbial Community ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Microbial Community Composition ◽  
Fermentation Products ◽  
Batch Cultures ◽  
Gradient Gel Electrophoresis

There is an interest in controlling rumen methanogenesis as an opportunity to both decrease the emissions of greenhouse gases and improve the energy efficiency of rumen fermentation. However, the effects of inhibiting rumen methanogenesis on fermentation are incompletely understood even in in vitro rumen cultures, as the recovery of metabolic hydrogen ([H]) in the main fermentation products consistently decreases with methanogenesis inhibition, evidencing the existence of unaccounted [H] sinks. We hypothesized that inhibiting methanogenesis in rumen batch cultures would redirect [H] towards microbial amino acids (AA) biosynthesis as an alternative [H] sink to methane (CH4). The objective of this experiment was to evaluate the effects of eight inhibitors of methanogenesis on digestion, fermentation and the production of microbial biomass and AA in rumen batch cultures growing on cellulose. Changes in the microbial community composition were also studied using denaturing gradient gel electrophoresis (DGGE). Inhibiting methanogenesis did not cause consistent changes in fermentation or the profile of AA, although the effects caused by the different inhibitors generally associated with the changes in the microbial community that they induced. Under the conditions of this experiment, inhibiting methanogenesis did not increase the importance of microbial AA synthesis as a [H] sink.

Study of microbial community of brewery-treating granular sludge by denaturing gradient gel electrophoresis of 16S rRNA gene

2001 ◽  
Vol 43 (1) ◽  
pp. 77-82 ◽  
Author(s):  
O.-C. Chan ◽  
W.-T. Liu ◽  
H. H. Fang
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Granular Sludge ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Rrna Gene ◽  
Related Sequence ◽  
Gradient Gel Electrophoresis

The microbial community structure of granular sludge from an upflow anaerobic sludge blanket (UASB) reactor treating brewery effluent was studied by denaturing gradient gel electrophoresis (DGGE). Twelve major bands were observed in the DGGE fingerprint for the Bacteria domain and four bands for the Archaea domain. Of the bacterial bands observed, six were successfully purified and sequenced. Among them, three were related to the gram-positive low G+C group, one to the Delta subclass of the Proteobacteria, one to the Gamma subclass, and one to the Cytophaga group with no close related sequence. The 16S rRNA sequences of the four archaeal bands were closely associated with Methanosaeta concilii and Methanobacterium formicum.

Microbial Community Analysis of Underground Drinking Water Using Denaturing Gradient Gel Electrophoresis and Flow Cytometry Technologies

2014 ◽  
Vol 700 ◽  
pp. 519-524 ◽  
Author(s):  
Jie Liu ◽  
Yan Li Ding ◽  
Mark Bartlam ◽  
Ying Ying Wang
Keyword(s):  
Drinking Water ◽  
Microbial Community ◽  
Rural Areas ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Poyang Lake ◽  
Removal Rate ◽  
Underground Water ◽  
Water Sources ◽  
Gradient Gel Electrophoresis ◽  
Bacteria And Fungi

Underground water is directly used as drinking water in most rural areas of developing countries due to limitations in infrastructure. As an important indicator of drinking water quality, however, microbial quality has been largely ignored for a long time. Microbial quality poses a great threat to the safety of underground drinking water, especially in rural areas. The current study compared microbial abundance and community structure of three different water sources, i.e. underground water, Poyang Lake and Hai River, combined with flow cytometry (FCM) and denaturing gradient gel electrophoresis (DGGE). FCM results showed that the bacterial concentration of underground water is the lowest (1.037×106cell/ml) of the three water sources, but still approximates that of the Poyang Lake. The removal rate of bacteria after filtration through a 0.45μm-pore-size filter is 98.16% in underground water. The removal rate for Poyang Lake and Hai River is much lower (i.e. 66.57% and 74.17% respectively). DGGE profiles demonstrated that the microbial community structure in underground water shares higher similarity to Poyang Lake (51.0% and 53.1% similarity for bacteria and fungi respectively) than Hai River. The microbial diversity index (i.e. Shannon-Weaver index) for bacteria and fungi are 2.906 and 2.847 respectively in underground water, which is lower than in Poyang Lake. The evenness (i.e. Simpson index) of groundwater was lowest among the three water sources tested. The results suggested that groundwater has a complex microbial community and hence it is critical to apply necessary hygienic barriers to remove microbes for the safety of underground drinking water.

scholarly journals Microbial Ecology Dynamics during Rye and Wheat Sourdough Preparation

2013 ◽  
Vol 79 (24) ◽  
pp. 7827-7836 ◽  
Author(s):  
Danilo Ercolini ◽  
Erica Pontonio ◽  
Francesca De Filippis ◽  
Fabio Minervini ◽  
Antonietta La Storia ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Microbial Ecology ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Diversity Indices ◽  
Rrna Gene ◽  
Content Type ◽  
Gradient Gel Electrophoresis ◽  
Plate Counts ◽  
Relative Abundances

ABSTRACTThe bacterial ecology during rye and wheat sourdough preparation was described by 16S rRNA gene pyrosequencing. Viable plate counts of presumptive lactic acid bacteria, the ratio between lactic acid bacteria and yeasts, the rate of acidification, a permutation analysis based on biochemical and microbial features, the number of operational taxonomic units (OTUs), and diversity indices all together demonstrated the maturity of the sourdoughs during 5 to 7 days of propagation. Flours were mainly contaminated by metabolically active genera (Acinetobacter,Pantoea,Pseudomonas,Comamonas,Enterobacter,Erwinia, andSphingomonas) belonging to the phylumProteobacteriaorBacteroidetes(genusChryseobacterium). Their relative abundances varied with the flour. Soon after 1 day of propagation, this population was almost completely inhibited except for theEnterobacteriaceae. Although members of the phylumFirmicuteswere present at very low or intermediate relative abundances in the flours, they became dominant soon after 1 day of propagation. Lactic acid bacteria were almost exclusively representative of theFirmicutesby this time.Weissellaspp. were already dominant in rye flour and stably persisted, though they were later flanked by theLactobacillus sakeigroup. There was a succession of species during 10 days of propagation of wheat sourdoughs. The fluctuation between dominating and subdominating populations ofL. sakeigroup,Leuconostocspp.,Weissellaspp., andLactococcus lactiswas demonstrated. Other subdominant species such asLactobacillus plantarumwere detectable throughout propagation. As shown by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis,Saccharomyces cerevisiaedominated throughout the sourdough propagation. Notwithstanding variations due to environmental and technology determinants, the results of this study represent a clear example of how the microbial ecology evolves during sourdough preparation.

Changes in the structure and diversity of bacterial communities during the process of adaptation to organic wastewater

2010 ◽  
Vol 56 (4) ◽  
pp. 352-355 ◽  
Author(s):  
Junmin Li ◽  
Zexin Jin ◽  
Binbin Yu
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Similarity Index ◽  
Pcr Amplification ◽  
Genotypic Diversity ◽  
Diversity Indices ◽  
Inhibitory Effect ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis

To explore changes in the structure and diversity of activated sludge-derived microbial communities during adaptation to gradual increases in the concentration of wastewater, RAPD–PCR and the combination of PCR amplification of 16S rRNA genes with denaturing gradient gel electrophoresis (DGGE) analysis were used. In bacterial communities exposed to 0%, 5%, 10%, 20%, or 40% wastewater, there were 27, 25, 18, 17 and 16 bands, respectively, based on DGGE data, while there were 69, 83, 97, 86, and 88 bands, respectively, based on RAPD data. The community similarity index among bacterial communities during the process of adaptation to different concentrations of wastewater was different based on DGGE and RAPD data. Based on DGGE and RAPD profiles, the Shannon–Weiner and Simpson’s diversity indices decreased sharply upon exposure to 10% wastewater, indicating that 10% wastewater might be a critical point at which the growth of bacteria could be significantly inhibited and the genotypic diversity could change. This indicated that changes in structure and diversity might have an inhibitory effect on the toxicity of organic matter and that selection and adaptation could play important roles in the changes.

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