Lysozyme Treatment Makes 16S rRNA Amplicon Sequencing Results Less Biased

2021 ◽  
Author(s):  
Yuting Jiao ◽  
Zijie Gao ◽  
Shiyu Gui ◽  
Lu Ren ◽  
Yongyue Lu ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Community Analysis ◽  
Amplicon Sequencing ◽  
Microbial Community Analysis ◽  
Effective Agent ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
16S Rrna Amplicon Sequencing ◽  
Structure Result ◽  
Total Bacteria

Abstract Background Amplicon sequencing is widely applied in gut bacteria structure analysis. However, the proportion of Gram-positive bacteria may greatly affect the results of microbial community analysis. Lysozyme is an effective agent to extract DNA of Gram-positive bacteria. In this study, we assessed the influence of lysozyme treatment on results of Bactrocere dorsalis rectal bacteria structure. Result The results indicated that the total bacteria content can be significantly increased in lysozyme treated samples. Moreover, rectal bacteria diversity was significantly higher in lysozyme treated samples. A detail analysis revealed that abundance of Gram-positive bacteria significantly increased in samples treated with lysozyme. Conclusion This study indicates that lysozyme treatment before DNA extraction is an effective way to reduce bias in bacteria structure analysis, especially for samples with high proportion of Gram-positive bacteria.

Microbial community analysis of a full-scale membrane bioreactor treating industrial wastewater

2008 ◽  
Vol 58 (8) ◽  
pp. 1589-1594 ◽  
Author(s):  
D. Naidoo ◽  
N. Ramdhani ◽  
F. Bux
Keyword(s):  
In Situ Hybridization ◽  
Microbial Community ◽  
Manufacturing Industry ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Positive Hybridization ◽  
Gram Negative Organisms

A Kubota™ submerged membrane bio-reactor was applied to treat wastewater from a sugar manufacturing industry. To achieve optimal results, fundamental and extended understanding of the microbiology is important. Fluorescence in situ hybridization was used to evaluate the microbial community present. The majority of cells visualized in the sludge flocs by staining with the DNA fluorochrome DAPI, hybridized strongly with a bacterial probe. Probes specific for the alpha-, beta-, and gamma-subclasses of proteobacteria and high G + C Gram positive bacteria were used to characterize the community structures by in situ hybridization. Sampling was carried out over 12 weeks and samples were fixed with 4% paraformaldehyde for gram positive organisms and ice cold ethanol for gram negative organisms. The activated sludge population usually constitutes about 80 to 90% of proteobacteria. However, in this study it was found that a relatively small amount of proteobacteria was present within the system. No positive hybridization signal was observed with any of the applied eubacterial family- level probes.

The microbial community analysis of a 5-stage BNR process with step feed system

2003 ◽  
Vol 48 (8) ◽  
pp. 135-141 ◽  
Author(s):  
H.W. Lee ◽  
S.Y. Lee ◽  
J.O. Lee ◽  
H.G. Kim ◽  
J.B. Park ◽  
...  
Keyword(s):  
16S Rdna ◽  
Community Analysis ◽  
Cell Number ◽  
Total Cell ◽  
Microbial Community Analysis ◽  
Feed System ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cell Counts

The microbial communities of 5-stage BNR activated sludge samples were analyzed using fluorescence in-situ hybridization (FISH) and 16S rDNA characterization. The total cell numbers of each reactor were from 2.36 × 109 cells/ml to 2.83 × 109 cells/ml. From 56.5% to 62.0% of total DAPI cell counts were hybridized to the most bacterial specific probe EUB 338. Among them, b-proteobacteria were most dominant in each tank. The number of phosphate accumulating organisms (PAOs) was almost 50% of the total cell number in anoxic-1 tank, and these results indicate that this process has a high content of denitrifying phosphorus accumulating organisms (dPAOs). In contrast with FISH, 16S rDNA analysis showed that dominant groups were the Cytophaga-Flavobacterium group and high G+C% gram-positive bacteria, which were determined as PAOs in anoxic-1 tank. The beta subclass Proteobacteria did not accumulate a large amount of polyphosphate. The overall results indicate that high G+C% gram-positive bacteria and the Cytophaga-Flavobacterium group might play a key role as dPAOs in this process.

scholarly journals Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis

2015 ◽  
Vol 15 (1) ◽  
Author(s):  
Liyou Wu ◽  
Chongqing Wen ◽  
Yujia Qin ◽  
Huaqun Yin ◽  
Qichao Tu ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Analysis ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Microbial Community Analysis

scholarly journals Gene-Trait Matching and Prevalence of Nisin Tolerance Systems in Lactococus lactis

2021 ◽  
Vol 9 ◽  
Author(s):  
Lieke A. van Gijtenbeek ◽  
Thomas H. Eckhardt ◽  
Lucía Herrera-Domínguez ◽  
Elke Brockmann ◽  
Kristian Jensen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Specific Effect ◽  
Starter Cultures ◽  
Main Mechanism ◽  
Effective Agent ◽  
Gram Positive ◽  
Gram Positive Bacteria

Lactococcus lactis cheese starter cultures typically contain a mix of many strains and may include variants that produce and/or tolerate the antimicrobial bacteriocin nisin. Nisin is well-established as an effective agent against several undesirable Gram-positive bacteria in cheese and various other foods. In the current study, we have examined the effect of nisin on 710 individual L. lactis strains during milk fermentations. Changes in milk acidification profiles with and without nisin exposure, ranging from unaltered acidification to loss of acidification, could be largely explained by the type(s) and variants of nisin immunity and nisin degradation genes present, but surprisingly, also by genotypic lineage (L. lactis ssp. cremoris vs. ssp. lactis). Importantly, we identify that nisin degradation by NSR is frequent among L. lactis and therefore likely the main mechanism by which dairy-associated L. lactis strains tolerate nisin. Insights from this study on the strain-specific effect of nisin tolerance and degradation during milk acidification is expected to aid in the design of nisin-compatible cheese starter cultures.

scholarly journals To rarefy or not to rarefy: Enhancing microbial community analysis through next-generation sequencing

2020 ◽  
Author(s):  
Ellen S. Cameron ◽  
Philip J. Schmidt ◽  
Benjamin J.-M. Tremblay ◽  
Monica B. Emelko ◽  
Kirsten M. Müller
Keyword(s):  
Microbial Community ◽  
Community Analysis ◽  
Amplicon Sequencing ◽  
Microbial Community Analysis ◽  
List Type ◽  
Sequencing Data ◽  
Water And Wastewater Treatment ◽  
Library Size ◽  
Size Selection ◽  
The Impact

AbstractThe application of amplicon sequencing in water research provides a rapid and sensitive technique for microbial community analysis in a variety of environments ranging from freshwater lakes to water and wastewater treatment plants. It has revolutionized our ability to study DNA collected from environmental samples by eliminating the challenges associated with lab cultivation and taxonomic identification. DNA sequencing data consist of discrete counts of sequence reads, the total number of which is the library size. Samples may have different library sizes and thus, a normalization technique is required to meaningfully compare them. The process of randomly subsampling sequences to a selected normalized library size from the sample library—rarefying—is one such normalization technique. However, rarefying has been criticized as a normalization technique because data can be omitted through the exclusion of either excess sequences or entire samples, depending on the rarefied library size selected. Although it has been suggested that rarefying should be avoided altogether, we propose that repeatedly rarefying enables (i) characterization of the variation introduced to diversity analyses by this random subsampling and (ii) selection of smaller library sizes where necessary to incorporate all samples in the analysis. Rarefying may be a statistically valid normalization technique, but researchers should evaluate their data to make appropriate decisions regarding library size selection and subsampling type. The impact of normalized library size selection and rarefying with or without replacement in diversity analyses were evaluated herein.Highlights▪ Amplicon sequencing technology for environmental water samples is reviewed▪ Sequencing data must be normalized to allow comparison in diversity analyses▪ Rarefying normalizes library sizes by subsampling from observed sequences▪ Criticisms of data loss through rarefying can be resolved by rarefying repeatedly▪ Rarefying repeatedly characterizes errors introduced by subsampling sequences

scholarly journals Long-Term Mesophilic Anaerobic Co-Digestion of Swine Manure with Corn Stover and Microbial Community Analysis

2020 ◽  
Vol 8 (2) ◽  
pp. 188 ◽  
Author(s):  
Haipeng Wang ◽  
Teng Teeh Lim ◽  
Cuong Duong ◽  
Wei Zhang ◽  
Congfeng Xu ◽  
...  
Keyword(s):  
Corn Stover ◽  
Loading Rate ◽  
Swine Manure ◽  
Preliminary Test ◽  
Community Analysis ◽  
Amplicon Sequencing ◽  
Microbial Community Analysis ◽  
Sequencing Analysis ◽  
Degrading Bacteria

Long-term anaerobic co-digestion of swine manure (SM) and corn stover (CS) was conducted using semi-continuously loaded digesters under mesophilic conditions. A preliminary test was first conducted to test the effects of loading rates, and results indicated the 3 g-VS L−1 d−1 was the optimal loading rate. Based on the preliminary results, a verification replicated test was conducted with 3 g-VS L−1 d−1 loading rate and different SM/CS ratios (1:1, 2:1 and 1:2). Results showed that a SM/CS ratio of 2/1 was optimal, based on maximum observed methane-VSdes generation and carbon conversion efficiency (72.56 ± 3.40 mL g−1 and 40.59%, respectively). Amplicon sequencing analysis suggested that microbial diversity was increased with CS loading. Amino-acid-degrading bacteria were abundant in the treatment groups. Archaea Methanoculleus could enhance biogas and methane productions.

scholarly journals Dehalobium species implicated in 2,3,7,8-tetrachloro-p-dioxin dechlorination in the contaminated sediments of Sydney Harbour Estuary

2021 ◽  
Author(s):  
Matthew Lee ◽  
Gan Liang ◽  
Sophie I Holland ◽  
Casey O'Farrell ◽  
Keith Osborne ◽  
...  
Keyword(s):  
Enrichment Culture ◽  
Sediment Cores ◽  
Community Analysis ◽  
Amplicon Sequencing ◽  
Microbial Community Analysis ◽  
Reductive Dehalogenation ◽  
Anthropogenic Activity ◽  
Contamination Source ◽  
16S Amplicon Sequencing ◽  
Sydney Harbour

Polychlorinated dibenzo-p-dioxins and furans (PCDD/F) are some of the most environmentally recalcitrant and toxic compounds. They are naturally occurring and by-products of anthropogenic activity. Sydney Harbour Estuary (Sydney, Australia), is heavily contaminated with PCDD/F. Analysis of sediment cores revealed that the contamination source in Homebush Bay continues to have one of the highest levels of PCDD/F contamination in the world (5207 pg WHO-TEQ g-1) with >50% of the toxicity attributed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) the most toxic and concerning of the PCDD/F congeners. Comparison of congener profiles at the contamination source with surrounding bays and historical data provided evidence for the attenuation of 2,3,7,8-TCDD and other congeners at the source. This finding was supported by the detection of di-, mono- and unchlorinated dibenzo-p-dioxin. Microbial community analysis of sediments by 16S amplicon sequencing revealed an abundance of lineages from the class Dehalococcoidia (up to 15% of the community), including the genus Dehalobium (up to 0.5%). Anaerobic seawater enrichment cultures using perchloroethene as a more amenable growth substrate enriched only the Dehalobium population by more than six-fold. The enrichment culture then proved capable of reductively dechlorinating 2,3,7,8-TCDD to 2,3,7-TCDD and octachlorodibenzo-p-dibenzodioxin to hepta and hexa congeners. This work is the first to show microbial reductive dehalogenation of 2,3,7,8-TCDD with a bacterium from outside the Dehalococcoides genus, and one of only a few that demonstrates PCDD/F degradation in a marine environment.

scholarly journals A critical perspective on interpreting amplicon sequencing data in soil ecological research

2021 ◽  
Author(s):  
Lauren V. Alteio ◽  
Joana Séneca ◽  
Alberto Canarini ◽  
Roey Angel ◽  
Ksenia Guseva ◽  
...  
Keyword(s):  
Statistical Power ◽  
Marker Gene ◽  
Community Analysis ◽  
Amplicon Sequencing ◽  
Data Availability ◽  
Microbial Community Analysis ◽  
Sequencing Data ◽  
Spatio Temporal ◽  
Complementary Techniques ◽  
The Impact

Microbial community analysis via marker gene amplicon sequencing has become a routine method in the field of soil research. In this perspective, we discuss technical challenges and limitations of amplicon sequencing studies in soil and present statistical and experimental approaches that can help addressing the spatio-temporal complexity of soil and the high diversity of organisms therein. We illustrate the impact of compositionality on the interpretation of relative abundance data and discuss effects of sample replication on the statistical power in soil community analysis. Additionally, we argue for the need of increased study reproducibility and data availability, as well as complementary techniques for generating deeper ecological insights into microbial roles and our understanding thereof in soil ecosystems. At this stage, we call upon researchers and specialized soil journals to consider the current state of data analysis, interpretation and availability to improve the rigor of future studies.

Microbial community analysis involved in the aerobic/extended-idle process performing biological phosphorus removal

2013 ◽  
Vol 67 (3) ◽  
pp. 485-493 ◽  
Author(s):  
Tian-jing Zeng ◽  
Guo-jing Yang ◽  
Dong-bo Wang ◽  
Xiao-ming Li ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Phosphorus Removal ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Fish Analysis ◽  
Biological Phosphorus Removal ◽  
Acclimation Period ◽  
Polyphosphate Accumulating Organisms ◽  
Biological Phosphorus ◽  
Total Bacteria

Recently, it has been found that biological phosphorus removal can be achieved in an aerobic/extended-idle (AEI) process using both glucose and acetate as the sole substrate. However, the microbial consortiums involved in glucose-fed and acetate-fed systems have not yet been characterized. Thus the aims of this paper were to investigate the diversities and dynamics of bacterial communities during the acclimation period, and to quantify polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) in the systems. The phylogenetic analysis showed that the microbial communities were mainly composed of phylum Proteobacteria, Bacteroidetes, Chlorobi and another six kinds of unclassified bacteria. Fluorescence in-situ hybridization (FISH) analysis revealed that PAOs and GAOs accounted for 43 ± 7 and 16 ± 3% of all bacteria in the glucose-fed system, and 19 ± 4 and 35 ± 5% of total bacteria in the acetate-fed system, respectively. The results showed that the conventional PAOs could thrive in the AEI process, and a defined anaerobic zone was not necessarily required for putative PAOs growth.

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