Shotgun metagenomic analysis reveals new insights into bacterial community profiles in tempeh

Abstract Objective Amplicon sequencing targeting 16S ribosomal RNA (rRNA) has been widely used to profile the microbial community from fermented food samples. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in estimating microbial community composition. As potential paraprobiotics sources, a comprehensive profiling study of tempeh microbial ecology could contribute to tempeh product development. This study employed a shotgun metagenomic approach, where metagenome fragments from tempeh samples were sequenced directly for taxonomic and functional profiling analysis. Results Taxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from the shotgun metagenomic analysis in all tempeh samples. In terms of composition, this shotgun metagenomic study revealed that Proteobacteria was the most abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed gene based on this metagenomic analysis. The metagenome-assembled genomes (MAGs) results from the binning pipeline could reveal almost complete whole genome sequence of Lactobacillus fermentum, Enterococcus cecorum, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

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Shotgun Metagenomic Analysis Reveals New Insights into Bacterial Community Profiles in Tempeh

10.21203/rs.3.rs-42618/v3 ◽

2020 ◽

Author(s):

Adi Yulandi ◽

Antonius Suwanto ◽

Diana Elizabeth Waturangi ◽

Aris Tri Wahyudi

Keyword(s):

Microbial Community ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Food Samples ◽

Iron Complex ◽

Fermented Food ◽

Metagenomic Analysis ◽

Whole Genome Sequence ◽

Sequencing Analysis ◽

Functional Profiling

Abstract Objective: Amplicon sequencing targeting 16S ribosomal RNA (rRNA) has been widely used to profile the microbial community from fermented food samples. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in estimating microbial community composition. As potential paraprobiotics sources, a comprehensive profiling study of tempeh microbial ecology could contribute to tempeh product development. This study employed a shotgun metagenomic approach, where metagenome fragments from tempeh samples were sequenced directly for taxonomic and functional profiling analysis.Results: Taxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from the shotgun metagenomic analysis in all tempeh samples. In terms of composition, this shotgun metagenomic study revealed that Proteobacteria was the most abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed gene based on this metagenomic analysis. The metagenome-assembled genomes (MAGs) results from the binning pipeline could reveal almost complete whole genome sequence of Lactobacillus fermentum, Enterococcus cecorum, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

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Shotgun Metagenomic Analysis Reveals New Insights into Bacterial Community Profiles in Tempeh

10.21203/rs.3.rs-42618/v2 ◽

2020 ◽

Author(s):

Adi Yulandi ◽

Antonius Suwanto ◽

Diana Elizabeth Waturangi ◽

Aris Tri Wahyudi

Keyword(s):

Microbial Community ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Food Samples ◽

Iron Complex ◽

Fermented Food ◽

Metagenomic Analysis ◽

Whole Genome Sequence ◽

Sequencing Analysis ◽

Functional Profiling

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Shotgun Metagenomic Analysis Reveals New Insights into Bacterial Community Profiles in Tempeh

10.21203/rs.3.rs-42618/v1 ◽

2020 ◽

Author(s):

Adi Yulandi ◽

Antonius Suwanto ◽

Diana Elizabeth Waturangi ◽

Aris Tri Wahyudi

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Iron Complex ◽

Metagenomic Analysis ◽

Whole Genome Sequence ◽

Sequencing Analysis ◽

16S Rrna Analysis ◽

Functional Profiling

Abstract Objective: Amplicon sequencing targeting 16S ribosomal RNA (rRNA) has been widely used for the profile analysis of the microbial community from fermented food samples. Previous results of 16S rRNA analysis metagenome showed that Firmicutes was the dominant phylum in tempeh. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in the estimation of microbial community composition. An alternative approach known as shotgun metagenomic might be able to avoid this limitation. In this study, we employed total metagenomic DNA fragments that were sequenced directly for taxonomic dan functional profiling analysis.Results: Taxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from the direct shotgun metagenomic analysis in all tempeh samples. In terms of composition, this shotgun metagenomic study revealed that Proteobacteria was the most abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed genes based in this metagenomic analysis. The binning pipeline could reveal almost complete whole genome sequence of Lactobacillus fermentum, Enterococcus cecorum, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

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Shotgun Metagenomic Analysis Reveals New Insights on Bacterial Community Profiles in Tempeh

10.1101/2020.03.12.988444 ◽

2020 ◽

Author(s):

Adi Yulandi ◽

Diana Elizabeth Waturangi ◽

Aris Tri Wahyudi ◽

Antonius Suwanto

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Iron Complex ◽

Fermented Food ◽

Metagenomic Analysis ◽

Sequencing Analysis ◽

16S Rrna Analysis ◽

Functional Profiling

AbstractObjectiveAmplicon sequencing targeted 16S ribosomal RNA (rRNA) has been widely used for the analysis profile of the microbial community from fermented food samples. Previous results of 16S rRNA analysis metagenome showed that Firmicutes was the dominant phylum in tempeh. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in the estimation of microbial community composition. An alternative approach known as shotgun metagenomic might be able to avoid this limitation. In this study, we employed total metagenomic DNA fragments sequenced directly for taxonomic dan functional profiling analysis.ResultTaxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from direct shotgun metagenomic analysis in all tempeh samples. In terms of composition, the shotgun metagenomic study revealed that Proteobacteria was the most relatively abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed genes based on metagenome from tempeh samples.

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Reduced metagenome sequencing for strain-resolution taxonomic profiles

Microbiome ◽

10.1186/s40168-021-01019-8 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Lars Snipen ◽

Inga-Leena Angell ◽

Torbjørn Rognes ◽

Knut Rudi

Keyword(s):

Microbial Community ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Good Alternative ◽

Shotgun Sequencing ◽

Least Square ◽

Full Potential ◽

Shotgun Metagenomics ◽

Metagenome Sequencing ◽

Reference Genomes

Abstract Background Studies of shifts in microbial community composition has many applications. For studies at species or subspecies levels, the 16S amplicon sequencing lacks resolution and is often replaced by full shotgun sequencing. Due to higher costs, this restricts the number of samples sequenced. As an alternative to a full shotgun sequencing we have investigated the use of Reduced Metagenome Sequencing (RMS) to estimate the composition of a microbial community. This involves the use of double-digested restriction-associated DNA sequencing, which means only a smaller fraction of the genomes are sequenced. The read sets obtained by this approach have properties different from both amplicon and shotgun data, and analysis pipelines for both can either not be used at all or not explore the full potential of RMS data. Results We suggest a procedure for analyzing such data, based on fragment clustering and the use of a constrained ordinary least square de-convolution for estimating the relative abundance of all community members. Mock community datasets show the potential to clearly separate strains even when the 16S is 100% identical, and genome-wide differences is < 0.02, indicating RMS has a very high resolution. From a simulation study, we compare RMS to shotgun sequencing and show that we get improved abundance estimates when the community has many very closely related genomes. From a real dataset of infant guts, we show that RMS is capable of detecting a strain diversity gradient for Escherichia coli across time. Conclusion We find that RMS is a good alternative to either metabarcoding or shotgun sequencing when it comes to resolving microbial communities at the strain level. Like shotgun metagenomics, it requires a good database of reference genomes and is well suited for studies of the human gut or other communities where many reference genomes exist. A data analysis pipeline is offered, as an R package at https://github.com/larssnip/microRMS.

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

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11475-9 ◽

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

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Evaluation of the microbial community structure of potable water samples from occupied and unoccupied buildings using16S rRNA amplicon sequencing

10.1101/2020.07.17.209346 ◽

2020 ◽

Author(s):

Kimothy L Smith ◽

Howard A Shuman ◽

Douglas Findeisen

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Water Samples ◽

Ad Hoc ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

The Other ◽

Water Usage ◽

16S Rrna Amplicon Sequencing ◽

Oxford Nanopore

AbstractWe conducted two studies of water samples from buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location with matched water sample types. One of the buildings had been continuously occupied, and the other substantially vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings. Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in Study 1 and 8.3% of samples in Study 2.SynopsisComparison of microbial community composition in the plumbing of occupied and unoccupied buildings during the COVID-19 pandemic shutdown.

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Geothermal Gases Shape the Microbial Community of the Volcanic Soil of Pantelleria, Italy

mSystems ◽

10.1128/msystems.00517-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Nunzia Picone ◽

Carmen Hogendoorn ◽

Geert Cremers ◽

Lianna Poghosyan ◽

Arjan Pol ◽

...

Keyword(s):

Microbial Community ◽

Greenhouse Gas ◽

Relative Abundance ◽

Hot Spots ◽

Microbial Community Composition ◽

Metagenomic Analysis ◽

Volcanic Soil ◽

Metabolic Potential ◽

Gas Emissions ◽

Geothermal Activity

ABSTRACT Volcanic and geothermal environments are characterized by low pH, high temperatures, and gas emissions consisting of mainly CO2 and varied CH4, H2S, and H2 contents which allow the formation of chemolithoautotrophic microbial communities. To determine the link between the emitted gases and the microbial community composition, geochemical and metagenomic analysis were performed. Soil samples of the geothermic region Favara Grande (Pantelleria, Italy) were taken at various depths (1 to 50 cm). Analysis of the gas composition revealed that CH4 and H2 have the potential to serve as the driving forces for the microbial community. Our metagenomic analysis revealed a high relative abundance of Bacteria in the top layer (1 to 10 cm), but the relative abundance of Archaea increased with depth from 32% to 70%. In particular, a putative hydrogenotrophic methanogenic archaeon, related to Methanocella conradii, appeared to have a high relative abundance (63%) in deeper layers. A variety of [NiFe]-hydrogenase genes were detected, showing that H2 was an important electron donor for microaerobic microorganisms in the upper layers. Furthermore, the bacterial population included verrucomicrobial and proteobacterial methanotrophs, the former showing an up to 7.8 times higher relative abundance. Analysis of the metabolic potential of this microbial community showed a clear capacity to oxidize CH4 aerobically, as several genes for distinct particulate methane monooxygenases and lanthanide-dependent methanol dehydrogenases (XoxF-type) were retrieved. Analysis of the CO2 fixation pathways showed the presence of the Calvin-Benson-Bassham cycle, the Wood-Ljungdahl pathway, and the (reverse) tricarboxylic acid (TCA) cycle, the latter being the most represented carbon fixation pathway. This study indicates that the methane emissions in the Favara Grande might be a combination of geothermal activity and biological processes and further provides insights into the diversity of the microbial population thriving on CH4 and H2. IMPORTANCE The Favara Grande nature reserve on the volcanic island of Pantelleria (Italy) is known for its geothermal gas emissions and high soil temperatures. These volcanic soil ecosystems represent “hot spots” of greenhouse gas emissions. The unique community might be shaped by the hostile conditions in the ecosystem, and it is involved in the cycling of elements such as carbon, hydrogen, sulfur, and nitrogen. Our metagenome study revealed that most of the microorganisms in this extreme environment are only distantly related to cultivated bacteria. The results obtained profoundly increased the understanding of these natural hot spots of greenhouse gas production/degradation and will help to enrich and isolate the microbial key players. After isolation, it will become possible to unravel the molecular mechanisms by which they adapt to extreme (thermo/acidophilic) conditions, and this may lead to new green enzymatic catalysts and technologies for industry.

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pH and Phosphate Induced Shifts in Carbon Flow and Microbial Community during Thermophilic Anaerobic Digestion

Microorganisms ◽

10.3390/microorganisms8020286 ◽

2020 ◽

Vol 8 (2) ◽

pp. 286

Author(s):

Nina Lackner ◽

Andreas O. Wagner ◽

Rudolf Markt ◽

Paul Illmer

Keyword(s):

Microbial Community ◽

Microbial Community Composition ◽

Amplicon Sequencing ◽

Carbon Flow ◽

Rrna Gene ◽

Organic Substrates ◽

Ch4 Production ◽

Ph Conditions ◽

Ph Range ◽

In The Beginning

pH is a central environmental factor influencing CH4 production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0–8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH4 production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values ≤5.5, with the maximum CH4 production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions.

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