Prediction, enrichment, and isolation identify a responsive, competitive community of cellulolytic microorganisms from a municipal landfill

2021 ◽  
Author(s):  
Rebecca Co ◽  
Laura A Hug
Keyword(s):  
Cellulose Degradation ◽  
Amplicon Sequencing ◽  
Contaminated Sites ◽  
Metagenomic Sequencing ◽  
Cellulolytic Bacteria ◽  
Municipal Landfill ◽  
16S Rrna Amplicon Sequencing ◽  
Cellulolytic Microorganisms ◽  
Specific Composition ◽  
Introduced Substrates

Abstract Landfills are engineered, heterogeneously contaminated sites containing large reservoirs of paper waste. Cellulose degradation is an important process within landfill microbial ecology, and these anoxic, saturated environments are prime locations for discovery of cellulases that may offer improvements on industrial cellulose degradation efforts. We sampled leachate from three locations within a municipal landfill, a leachate collection cistern, and groundwater from an adjacent aquifer to identify cellulolytic populations and their associated cellulases. Metagenomic sequencing identified wide-spread and taxonomically diverse cellulolytic potential, with a notable scarcity of predicted exocellulases. 16S rRNA amplicon sequencing detected nine landfill microorganisms enriched in a customized leachate medium amended with microcrystalline cellulose or common paper stocks. Paper-enrichment cultures showed three competition dynamics in response to the specific composition (lignin: hemi-cellulose: cellulose) of the different paper stocks. From leachate biomass, four novel cellulolytic bacteria were isolated, including two with the capacity for cellulolysis at industrially-relevant temperatures. None of the isolates demonstrated exocellulase activity, consistent with the metagenome-based predictions. However, there was very little overlap between metagenome-derived predicted cellulolytic organisms, organisms enriched on paper sources, or the isolates, suggesting the landfill cellulolytic community is at low abundance but able to rapidly respond to introduced substrates.

Comparative metagenomic discovery of the dynamic cellulose-degrading process from a synergistic cellulolytic microbiota

2020 ◽  
Author(s):  
Ming Yang ◽  
Jingjing Zhao ◽  
Yue Yuan ◽  
Xiaoyi Chen ◽  
Fan Yang ◽  
...  
Keyword(s):  
High Efficiency ◽  
Cellulose Degradation ◽  
Structural Complexity ◽  
Spatial Diversity ◽  
Clear Understanding ◽  
Metagenomic Sequencing ◽  
Cellulolytic Microorganisms ◽  
Large Molecular Weight ◽  
Synergistic Degradation ◽  
New Perspective

Abstract Background As a green and sustainable bioenergy source, cellulose is difficult to degrade due to its large molecular weight and high structural complexity. Many cellulolytic microorganisms can secrete a series of enzymes to synergistically catalyze the cellulose degradation with a high efficiency. However, the inability to cultivate most of them, as well as their spatial diversity and temporal variability, limit the clear understanding of the relative area. Results To reveal the dynamic process of cellulose biodegradation, we cultivated a microbiota (FP) with efficient cellulose-degrading ability and compared the different stages of filter paper degradation. Ion chromatography and comparative metagenomic sequencing revealed that the diversity of FP enhanced as the complexity of hydrolysates increased, and the disturbance of FP was greater in early-intermediate than intermediate-final period. Sporocytophaga myxococcoides and Cohnella sp. CIP 111063 dominated the synergistic degradation of cellulose in early-intermediate and intermediate-late stages, respectively. Totally, 432 genes were annotated to cellulolytic pathways, and 363 and 231 unannotated genes were speculated to be related to the degradation of cellulose to cellodextrin/cello-oligosaccharide and cellobiose, respectively. Finally, according to the temporal changes in hydrolysates, community structure and gene abundances, a dynamic cellulose-degrading pathway was designed, which involved key cellulolytic species and enzymes in FP. Conclusions The cellulose-degrading pathway was established based on the dynamic analysis in microbiota FP, suggesting the collaboration and competition between species during cellulose degradation. Our work should provide a new perspective for the subsequent identification of key cellulolytic strains and enzymes and clarification the mechanism of cellulose biodegradation.

scholarly journals Microbial Community Field Surveys Reveal Abundant Pseudomonas Population in Sorghum Rhizosphere Composed of Many Closely Related Phylotypes

2021 ◽  
Vol 12 ◽  
Author(s):  
Dawn Chiniquy ◽  
Elle M. Barnes ◽  
Jinglie Zhou ◽  
Kyle Hartman ◽  
Xiaohui Li ◽  
...  
Keyword(s):  
Single Molecule ◽  
Bacterial Species ◽  
Operational Taxonomic Unit ◽  
Amplicon Sequencing ◽  
Sequence Variant ◽  
Metagenomic Sequencing ◽  
Host Genotype ◽  
16S Rrna Amplicon Sequencing ◽  
The Impact ◽  
Surrounding Soil

While the root-associated microbiome is typically less diverse than the surrounding soil due to both plant selection and microbial competition for plant derived resources, it typically retains considerable complexity, harboring many hundreds of distinct bacterial species. Here, we report a time-dependent deviation from this trend in the rhizospheres of field grown sorghum. In this study, 16S rRNA amplicon sequencing was used to determine the impact of nitrogen fertilization on the development of the root-associated microbiomes of 10 sorghum genotypes grown in eastern Nebraska. We observed that early rhizosphere samples exhibit a significant reduction in overall diversity due to a high abundance of the bacterial genus Pseudomonas that occurred independent of host genotype in both high and low nitrogen fields and was not observed in the surrounding soil or associated root endosphere samples. When clustered at 97% identity, nearly all the Pseudomonas reads in this dataset were assigned to a single operational taxonomic unit (OTU); however, exact sequence variant (ESV)-level resolution demonstrated that this population comprised a large number of distinct Pseudomonas lineages. Furthermore, single-molecule long-read sequencing enabled high-resolution taxonomic profiling revealing further heterogeneity in the Pseudomonas lineages that was further confirmed using shotgun metagenomic sequencing. Finally, field soil enriched with specific carbon compounds recapitulated the increase in Pseudomonas, suggesting a possible connection between the enrichment of these Pseudomonas species and a plant-driven exudate profile.

scholarly journals Bacterial Diversity of Breast Milk in Healthy Spanish Women: Evolution from Birth to Five Years Postpartum

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2414
Author(s):  
Laura Sanjulián ◽  
Alexandre Lamas ◽  
Rocío Barreiro ◽  
Alberto Cepeda ◽  
Cristina A. Fente ◽  
...  
Keyword(s):  
Breast Milk ◽  
16S Rrna ◽  
Human Milk ◽  
Alpha Diversity ◽  
Amplicon Sequencing ◽  
Maternal Body Mass Index ◽  
16S Rrna Amplicon Sequencing ◽  
Spanish Women ◽  
Calcium Magnesium ◽  
Abundant Genus

The objective of this work was to characterize the microbiota of breast milk in healthy Spanish mothers and to investigate the effects of lactation time on its diversity. A total of ninety-nine human milk samples were collected from healthy Spanish women and were assessed by means of next-generation sequencing of 16S rRNA amplicons and by qPCR. Firmicutes was the most abundant phylum, followed by Bacteroidetes, Actinobacteria, and Proteobacteria. Accordingly, Streptococcus was the most abundant genus. Lactation time showed a strong influence in milk microbiota, positively correlating with Actinobacteria and Bacteroidetes, while Firmicutes was relatively constant over lactation. 16S rRNA amplicon sequencing showed that the highest alpha-diversity was found in samples of prolonged lactation, along with wider differences between individuals. As for milk nutrients, calcium, magnesium, and selenium levels were potentially associated with Streptococcus and Staphylococcus abundance. Additionally, Proteobacteria was positively correlated with docosahexaenoic acid (DHA) levels in breast milk, and Staphylococcus with conjugated linoleic acid. Conversely, Streptococcus and trans-palmitoleic acid showed a negative association. Other factors such as maternal body mass index or diet also showed an influence on the structure of these microbial communities. Overall, human milk in Spanish mothers appeared to be a complex niche shaped by host factors and by its own nutrients, increasing in diversity over time.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

Advantage of 16S rRNA amplicon sequencing in Helicobacter pylori diagnosis

Helicobacter ◽  
2021 ◽  
Author(s):  
Boldbaatar Gantuya ◽  
Hashem B. El Serag ◽  
Batsaikhan Saruuljavkhlan ◽  
Dashdorj Azzaya ◽  
Takashi Matsumoto ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
16S Rrna Amplicon Sequencing

scholarly journals Reversion of Gut Microbiota during the Recovery Phase in Patients with Asymptomatic or Mild COVID-19: Longitudinal Study

2021 ◽  
Vol 9 (6) ◽  
pp. 1237
Author(s):  
Han-Na Kim ◽  
Eun-Jeong Joo ◽  
Chil-Woo Lee ◽  
Kwang-Sung Ahn ◽  
Hyung-Lae Kim ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Gastrointestinal Symptoms ◽  
Amplicon Sequencing ◽  
Recovery Phase ◽  
Fecal Samples ◽  
Intestinal Microbes ◽  
Gut Dysbiosis ◽  
16S Rrna Amplicon Sequencing ◽  
Microbiome Data ◽  
Negative Conversion

Patients with COVID-19 have been reported to experience gastrointestinal symptoms as well as respiratory symptoms, but the effects of COVID-19 on the gut microbiota are poorly understood. We explored gut microbiome profiles associated with the respiratory infection of SARS-CoV-2 during the recovery phase in patients with asymptomatic or mild COVID-19. A longitudinal analysis was performed using the same patients to determine whether the gut microbiota changed after recovery from COVID-19. We applied 16S rRNA amplicon sequencing to analyze two paired fecal samples from 12 patients with asymptomatic or mild COVID-19. Fecal samples were selected at two time points: during SARS-CoV-2 infection (infected state) and after negative conversion of the viral RNA (recovered state). We also compared the microbiome data with those from 36 healthy controls. Microbial evenness of the recovered state was significantly increased compared with the infected state. SARS-CoV-2 infection induced the depletion of Bacteroidetes, while an abundance was observed with a tendency to rapidly reverse in the recovered state. The Firmicutes/Bacteroidetes ratio in the infected state was markedly higher than that in the recovered state. Gut dysbiosis was observed after infection even in patients with asymptomatic or mild COVID-19, while the composition of the gut microbiota was recovered after negative conversion of SARS-CoV-2 RNA. Modifying intestinal microbes in response to COVID-19 might be a useful therapeutic alternative.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

scholarly journals Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing

2021 ◽  
Vol 9 (7) ◽  
pp. 1525
Author(s):  
Can Akpolat ◽  
Ana Beatriz Fernández ◽  
Pinar Caglayan ◽  
Baris Calli ◽  
Meral Birbir ◽  
...  
Keyword(s):  
16S Rrna ◽  
Salt Lake ◽  
Amplicon Sequencing ◽  
Deep Zone ◽  
Biotechnological Potential ◽  
16S Rrna Amplicon Sequencing ◽  
Physico Chemical ◽  
Potential Applications ◽  
A Minor ◽  
Tuz Lake

Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum, Haloarcula, Halorhabdus, Natronomonas, Halosimplex, Halomicrobium, Halorubrum, Halonotius, Halolamina, Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum, Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications.

DNA extraction from concrete v2

2021 ◽  
Author(s):  
Anders Kiledal ◽  
Julia A Maresca
Keyword(s):  
Dna Extraction ◽  
Dental Plaque ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Metagenomic Sequencing ◽  
Sample Analysis ◽  
Larger Sample ◽  
Laboratory Contamination ◽  
Biomass Sample ◽  
Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

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