scholarly journals Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16s rDNA amplicon sequencing

2020 ◽  
Author(s):  
Joeselle M. Serrana ◽  
Kozo Watanabe
Keyword(s):  
Microbial Community ◽  
16S Rdna ◽  
Community Composition ◽  
Membrane Filtration ◽  
Amplicon Sequencing ◽  
Sediment Samples ◽  
Community Profiling ◽  
Significant Difference ◽  
16S Rdna Amplicon Sequencing ◽  
Read Abundance

ABSTRACTSequential membrane filtration as a pre-processing step for the isolation of microorganisms could provide good quality and integrity DNA that can be preserved and kept at ambient temperatures before community profiling through culture-independent molecular techniques, e.g., 16s rDNA amplicon sequencing. Here, we assessed the impact of pre-processing sediment samples by sequential membrane filtration (from 10, 5 to 0.22 μm pore size membrane filters) for 16s rDNA-based community profiling of sediment-associated microorganisms. Specifically, we examined if there would be method-driven differences between non- and pre-processed sediment samples regarding the quality and quantity of extracted DNA, PCR amplicon, resulting high-throughput sequencing reads, microbial diversity, and community composition. We found no significant difference in the quality and quantity of extracted DNA and PCR amplicons between the two methods. Although we found a significant difference in raw and quality-filtered reads, read abundance after bioinformatics processing (i.e., denoising and the chimeric-read filtering steps) were not significantly different. These results suggest that read abundance after these read processing steps were not influenced by sediment processing or lack thereof. Although the non- and pre-processed sediment samples had more unique than shared amplicon sequence variants (ASVs), we report that their shared ASVs accounted for 74% of both methods’ absolute read abundance. More so at the genus level, the final collection filter identified most of the genera (95% of the reads) captured from the non-processed samples, with a total of 51 false-negative (2%) and 59 false-positive genera (3%). Accordingly, the diversity estimates and community composition were not significantly different between the non- and pre-processed samples. We demonstrate that while there were differences in shared and unique taxa, both methods revealed comparable microbial diversity and community composition. We also suggest the inclusion of sequential filters (i.e., pre- and mid-filters) in the community profiling, given the additional taxa not detected from the non-processed and the final collection filter. Our observations highlight the feasibility of pre-processing sediment samples for community analysis and the need to further assess sampling strategies to help conceptualize appropriate study designs for sediment-associated microbial community profiling.

scholarly journals Do initial concentration and activated sludge seasonality affect pharmaceutical biotransformation rate constants?

2021 ◽  
Author(s):  
Tamara J. H. M. van Bergen ◽  
Ana B. Rios-Miguel ◽  
Tom M. Nolte ◽  
Ad M. J. Ragas ◽  
Rosalie van Zelm ◽  
...  
Keyword(s):  
Microbial Community ◽  
Activated Sludge ◽  
Community Composition ◽  
Rate Constants ◽  
Wastewater Treatment Plants ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Initial Concentration ◽  
Different Seasons

Abstract Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (kb) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC–MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and kb values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different kb for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different kb values were measured, depending on initial concentration. In general, biodegradable compounds had a higher kb when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the kb in order to reliably predict the fate of pharmaceuticals in the WWTP. Key points • Biotransformation and sorption of pharmaceuticals were assessed in activated sludge. • Higher initial concentrations resulted in higher biotransformation rate constants for biodegradable pharmaceuticals. • Summer and winter inocula produced slightly different biotransformation rate constants although microbial community composition did not significantly change. Graphical abstract

scholarly journals Effects of Actinomycete Secondary Metabolites on Sediment Microbial Communities

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Nastassia V. Patin ◽  
Michelle Schorn ◽  
Kristen Aguinaldo ◽  
Tommie Lincecum ◽  
Bradley S. Moore ◽  
...  
Keyword(s):  
Microbial Community ◽  
Secondary Metabolites ◽  
Microbial Communities ◽  
Community Composition ◽  
Marine Sediments ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
Next Generation ◽  
Content Type ◽  
Predatory Bacteria

ABSTRACT Marine sediments harbor complex microbial communities that remain poorly studied relative to other biomes such as seawater. Moreover, bacteria in these communities produce antibiotics and other bioactive secondary metabolites, yet little is known about how these compounds affect microbial community structure. In this study, we used next-generation amplicon sequencing to assess native microbial community composition in shallow tropical marine sediments. The results revealed complex communities comprised of largely uncultured taxa, with considerable spatial heterogeneity and known antibiotic producers comprising only a small fraction of the total diversity. Organic extracts from cultured strains of the sediment-dwelling actinomycete genus Salinispora were then used in mesocosm studies to address how secondary metabolites shape sediment community composition. We identified predatory bacteria and other taxa that were consistently reduced in the extract-treated mesocosms, suggesting that they may be the targets of allelopathic interactions. We tested related taxa for extract sensitivity and found general agreement with the culture-independent results. Conversely, several taxa were enriched in the extract-treated mesocosms, suggesting that some bacteria benefited from the interactions. The results provide evidence that bacterial secondary metabolites can have complex and significant effects on sediment microbial communities. IMPORTANCE Ocean sediments represent one of Earth's largest and most poorly studied biomes. These habitats are characterized by complex microbial communities where competition for space and nutrients can be intense. This study addressed the hypothesis that secondary metabolites produced by the sediment-inhabiting actinomycete Salinispora arenicola affect community composition and thus mediate interactions among competing microbes. Next-generation amplicon sequencing of mesocosm experiments revealed complex communities that shifted following exposure to S. arenicola extracts. The results reveal that certain predatory bacteria were consistently less abundant following exposure to extracts, suggesting that microbial metabolites mediate competitive interactions. Other taxa increased in relative abundance, suggesting a benefit from the extracts themselves or the resulting changes in the community. This study takes a first step toward assessing the impacts of bacterial metabolites on sediment microbial communities. The results provide insight into how low-abundance organisms may help structure microbial communities in ocean sediments.

scholarly journals A step-by-step sequence-based analysis of virome enrichment protocol for freshwater and sediment samples

2020 ◽  
Author(s):  
Federica Pinto ◽  
Moreno Zolfo ◽  
Francesco Beghini ◽  
Federica Armanini ◽  
Francesco Asnicar ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Microbial Community Composition ◽  
Genetic Material ◽  
Amplicon Sequencing ◽  
Sample Type ◽  
Natural Ecosystems ◽  
Sediment Samples ◽  
Water And Sediment ◽  
Water And Sediment Samples

AbstractCultivation-free metagenomic analysis afforded unprecedented details on the diversity, structure and potential functions of microbial communities in different environments. When employed to study the viral fraction of the community that is recalcitrant to cultivation, metagenomics can shed light into the diversity of viruses and their role in natural ecosystems. However, despite the increasing interest in virome metagenomics, methodological issues still hinder the proper interpretation and comparison of results across studies. Virome enrichment experimental protocols are key multi-step processes needed for separating and concentrating the viral fraction from the whole microbial community prior to sequencing. However, there is little information on their efficiency and their potential biases. To fill this gap, we used metagenomic and amplicon sequencing to examine the microbial community composition through the serial filtration and concentration steps commonly used to produce viral-enriched metagenomes. The analyses were performed on water and sediment samples from an Alpine lake. We found that, although the diversity of the retained microbial communities declined progressively during the serial filtration, the final viral fraction contained a large proportion (from 10% to 40%) of non-viral taxa, and that the efficacy of filtration showed biases based on taxonomy. Our results quantified the amount of bacterial genetic material in viromes and highlighted the influence of sample type on the enrichment efficacy. Moreover, since viral-enriched samples contained a significant portion of microbial taxa, computational sequence analysis should account for such biases in the downstream interpretation pipeline.ImportanceFiltration is a commonly used method to enrich viral particles in environmental samples. However, there is little information on its efficiency and potential biases on the final result. Using a sequence-based analysis on water and sediment samples, we found that filtration efficacy is dependent on sample type and that the final virome contained a large proportion of non-viral taxa. Our finding stressed the importance of downstream analysis to avoid biased interpretation of data.

scholarly journals Composition and Diversity of the Ocular Surface Microbiota in Patients With Blepharitis in Northwestern China

2021 ◽  
Vol 8 ◽  
Author(s):  
Changhao Wang ◽  
Xiuhong Dou ◽  
Jian Li ◽  
Jie Wu ◽  
Yan Cheng ◽  
...  
Keyword(s):  
16S Rdna ◽  
Relative Abundance ◽  
Ocular Surface ◽  
Amplicon Sequencing ◽  
Northwestern China ◽  
Healthy Control Group ◽  
Control Group ◽  
Eyelid Margin ◽  
16S Rdna Amplicon Sequencing ◽  
Healthy Control

Purpose: To investigate the composition and diversity of the microbiota on the ocular surface of patients with blepharitis in northwestern China via 16S rDNA amplicon sequencing.Methods: Thirty-seven patients with blepharitis divided into groups of anterior, posterior and mixed blepharitis and twenty healthy controls from northwestern China were enrolled in the study. Samples were collected from the eyelid margin and conjunctival sac of each participant. The V3–V4 region of bacterial 16S rDNA in each sample was amplified and sequenced on the Illumina HiSeq 2500 sequencing platform, and the differences in taxonomy and diversity among different groups were compared.Results: The composition of the ocular surface microbiota of patients with blepharitis was similar to that of healthy subjects, but there were differences in the relative abundance of each bacterium. At the phylum level, the abundances of Actinobacteria, Cyanobacteria, Verrucomicrobia, Acidobacteria, Chloroflexi, and Atribacteria were significantly higher in the blepharitis group than in the healthy control group, while the relative abundance of Firmicutes was significantly lower (p < 0.05, Mann-Whitney U). At the genus level, the abundances of Lactobacillus, Ralstonia, Bacteroides, Akkermansia, Bifidobacterium, Escherichia-Shigella, Faecalibacterium, and Brevibacterium were significantly higher in the blepharitis group than in the healthy control group, while the relative abundances of Bacillus, Staphylococcus, Streptococcus, and Acinetobacter were significantly lower in the blepharitis group (p < 0.05, Mann-Whitney U). The microbiota of anterior blepharitis was similar to that of mixed blepharitis but different from that of posterior blepharitis. Lactobacillus and Bifidobacterium are biomarkers of posterior blepharitis, and Ralstonia is a biomarker of mixed blepharitis. There was no significant difference in the ocular surface microbiota between the eyelid margin and conjunctival sac with or without blepharitis.Conclusion: The ocular surface microbiota of patients with blepharitis varied among different study groups, according to 16S rDNA amplicon sequencing analysis. The reason might be due to the participants being from different environments and having different lifestyles. Lactobacillus, Bifidobacterium, Akkermansia, Ralstonia, and Bacteroides may play important roles in the pathogenesis of blepharitis.

scholarly journals Nanoliter-scale next-generation sequencing library-mediated high-throughput 16S rRNA microbial community profiling

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 204-210
Author(s):  
Hui Zhang ◽  
Xiangdan Yu ◽  
Zhe Zhang ◽  
Zhenhua Liu ◽  
Cong Tang ◽  
...  
Keyword(s):  
Microbial Community ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Amplicon Sequencing ◽  
Next Generation ◽  
Library Construction ◽  
Microbial Community Profiling ◽  
Community Profiling ◽  
Sequencing Library ◽  
Generation Sequencing

An ultra-high-throughput workflow for next-generation sequencing library construction at nanoliter scale for amplicon sequencing, termed Smartchip Nanowell Platform for Target Enrichment, was established using a nanodispenser system and a nanoliter-scale PCR chip. To demonstrate its cost and time advantages over conventional methods for library construction, quality control and pooling for large-scale samples, target amplicon sequencing of the 16S ribosomal RNA gene V3-V4 region widely used for microbial community profiling was chosen for comparison. The finding of no significant difference in microbial community profiling between the two methods strongly supports the conclusion that Smartchip Nanowell Platform for Target Enrichment is a cost-effective method for next-generation sequencing library construction for large-scale samples to conduct amplicon sequencing-based applications.

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