rRNA Gene Copy Numbers Research Articles

Quantitation of residual host cell DNA in recombinant adeno-associated virus using droplet digital PCR.

Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with risks of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116-bp and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with the copy numbers of three reference genes (EIF5B, DCK, and HBB). Results showed that 88.6-97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate contaminated residual host cell DNA. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products.

Diversity and distribution of archaeal and bacterial nitrifiers in deep oceans

Thaumarchaeota and Nitrospinae are the main drivers of nitrification in global oceans. However, their vertical and horizontal distributions throughout seawater columns remain unclear. Here, we conducted high-throughput sequencing of 16S rRNA genes and quantified putative nitrifying genes of microbial communities within seawater samples from the full depths of the Central Indian Ocean and the Western Pacific Ocean waters. Thaumarchaeota and Nitrospinae exhibited clear compositional distinctions between the two oceanic regions. Vertical variation in their assemblages was also apparent among all samples and within regions. However, their core operational taxonomic units (OTUs) were shared between the two regions and belonged to diverse phylogenetic clades that coincided with specific vertical niches. Further, their relative abundances were significantly and positively associated with Acidobacteriota, SAR324, Chloroflexi, and SAR406 phyla across and within regions, suggesting potential metabolic interactions. Regardless of oceanic regions, the maximal copy numbers of Thaumarchaeotal 16S rRNA and ammonia monooxygenase subunit A genes were both observed at 150–200 m depth, approximately overlapping with the primary nitrite maximum (PNM, 150 m), just below the deep chlorophyll a maximum (DCM, 125 m and 150 m); the maximal Nitrospina 16S rRNA gene copy numbers were located deeper (500 m), where nitrate concentrations had already increased.

Response of microbial communities to nutrient removal in coastal sediment by using ecological concrete.

Ecological concrete (eco-concrete) is a kind of environment-friendly material with porous characteristics. In this study, the eco-concrete was used to remove the total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC) in marine coastal sediment. The bacterial communities in sediment and on eco-concrete surface were also investigated by using high-throughput sequencing and quantitative PCR of 16S rRNA gene. We found that the mean removal efficiencies of TN, TP, and TOC in treatment group were 8.3%, 8.4%, and 12.3% after 28days. The bacterial community composition in the treatment group was significantly different from that in the control group on day 28. In addition, the bacterial community composition on eco-concrete surface was slightly different from that in sediment, and the copy numbers of 16S rRNA gene were higher on eco-concrete surface than in sediment. The types of eco-concrete aggregates (gravel, pebble, and zeolite) also had effects on the bacterial community composition and 16S rRNA gene copy numbers. Furthermore, we found the abundant genus Sulfurovum increased significantly on eco-concrete surface in the treatment group after 28days. Bacteria belonging to this genus were found having denitrification ability and were commonly detected in bioreactors for nitrate removal. Overall, our study expands the application scopes of eco-concrete and suggests that the bacterial communities in eco-concrete can potentially enhance the removal efficiency of nutrients in coastal sediment.

Microbial Diversity Biased Estimation Caused by Intragenomic Heterogeneity and Interspecific Conservation of 16S rRNA Genes.

The 16S rRNA gene has been extensively used as a molecular marker to explore evolutionary relationships and profile microbial composition throughout various environments. Despite its convenience and prevalence, limitations are inevitable. Variable copy numbers, intragenomic heterogeneity, and low taxonomic resolution have caused biases in estimating microbial diversity. Here, analysis of 24,248 complete prokaryotic genomes indicated that the 16S rRNA gene copy number ranged from 1 to 37 in bacteria and 1 to 5 in archaea, and intragenomic heterogeneity was observed in 60% of prokaryotic genomes, most of which were below 1%. The overestimation of microbial diversity caused by intragenomic variation and the underestimation introduced by interspecific conservation were calculated when using full-length or partial 16S rRNA genes. Results showed that, at the 100% threshold, microbial diversity could be overestimated by as much as 156.5% when using the full-length gene. The V4 to V5 region-based analyses introduced the lowest overestimation rate (4.4%) but exhibited slightly lower species resolution than other variable regions under the 97% threshold. For different variable regions, appropriate thresholds rather than the canonical value 97% were proposed for minimizing the risk of splitting a single genome into multiple clusters and lumping together different species into the same cluster. This study has not only updated the 16S rRNA gene copy number and intragenomic variation information for the currently available prokaryotic genomes, but also elucidated the biases in estimating prokaryotic diversity with quantitative data, providing references for choosing amplified regions and clustering thresholds in microbial community surveys. IMPORTANCE Microbial diversity is typically analyzed using marker gene-based methods, of which 16S rRNA gene sequencing is the most widely used approach. However, obtaining an accurate estimation of microbial diversity remains a challenge, due to the intragenomic variation and low taxonomic resolution of 16S rRNA genes. Comprehensive examination of the bias in estimating such prokaryotic diversity using 16S rRNA genes within ever-increasing prokaryotic genomes highlights the importance of the choice of sequencing regions and clustering thresholds based on the specific research objectives.

Oral microbiome changes associated with the menstrual cycle in healthy young adult females.

The relationship between the menstrual cycle and the oral microbiome has not been clarified. The purpose of this study was to assess potential changes in the oral microbiome of healthy young adults using 16S rRNA-based sequencing. Eleven females (aged 23-36 years) with stable menstrual cycles and without any oral problems were recruited. Saliva samples were collected before brushing every morning during the menstrual period. Based on basal body temperatures, menstrual cycles were divided into four phases, namely the menstrual, follicular, early luteal, and late luteal phases. Our results showed that the follicular phase had a significantly higher abundance ratio of the Streptococcus genus than the early and late luteal phases, whereas the abundance ratios of the Prevotella 7 and Prevotella 6 genera were significantly lower in the follicular phase than those in the early and late luteal phases and that in the early luteal phase, respectively. Alpha diversity by the Simpson index was significantly lower in the follicular phase than that in the early luteal phase, and beta diversity showed significant differences among the four phases. Using the relative abundance data and copy numbers of the 16S rRNA genes in the samples, the bacterial amounts in the four phases were compared, and we observed that the follicular phase had significantly lower amounts of the Prevotella 7 and Prevotella 6 genera than the menstrual and early luteal phase, respectively. These results indicate reciprocal changes with the Streptococcus genus and Prevotella genera, particularly in the follicular phase. In the present study, we showed that the oral microbiome profiles are affected by the menstrual cycles of healthy young adult females.

Nested PCR Approach for petB Gene Metabarcoding of Marine Synechococcus Populations.

The molecular diversity of marine picocyanobacterial populations, an important component of phytoplankton communities, is better characterized using high-resolution marker genes than the 16S rRNA gene as they have greater sequence divergence to differentiate between closely related picocyanobacteria groups. Although specific ribosomal primers have been developed, another general disadvantage of bacterial ribosome-based diversity analyses is the variable number of rRNA gene copies. To overcome these issues, the single-copy petB gene, encoding the cytochrome b6 subunit of the cytochrome b6f complex, has been used as a high-resolution marker gene to characterize Synechococcus diversity. We have designed new primers targeting the petB gene and proposed a nested PCR method (termed Ong_2022) for metabarcoding of marine Synechococcus populations obtained by flow cytometry cell sorting. We evaluated the specificity and sensitivity of Ong_2022 against the standard amplification protocol (termed Mazard_2012) using filtered seawater samples. The Ong_2022 approach was also tested on flow cytometry-sorted Synechococcus populations. Samples (filtered and sorted) were obtained in the Southwest Pacific Ocean, from subtropical (ST) and subantarctic (SA) water masses. The two PCR approaches using filtered samples recovered the same dominant subclades, Ia, Ib, IVa, and IVb, with small differences in relative abundance across the distinct samples. For example, subclade IVa was dominant in ST samples with the Mazard_2012 approach, while the same samples processed with Ong_2022 showed similar contributions of subclades IVa and Ib to the total community. The Ong_2022 approach generally captured a higher genetic diversity of Synechococcus subcluster 5.1 than the Mazard_2012 approach while having a lower proportion of incorrectly assigned amplicon sequence variants (ASVs). All flow cytometry-sorted Synechococcus samples could be amplified only by our nested approach. The taxonomic diversity obtained with our primers on both sample types was in agreement with the clade distribution observed by previous studies that applied other marker genes or PCR-free metagenomic approaches under similar environmental conditions. IMPORTANCE The petB gene has been proposed as a high-resolution marker gene to access the diversity of marine Synechococcus populations. A systematic metabarcoding approach based on the petB gene would improve the characterization/assessment of the Synechococcus community structure in marine planktonic ecosystems. We have designed and tested specific primers to be applied in a nested PCR protocol (Ong_2022) for metabarcoding the petB gene. The Ong_2022 protocol can be applied to samples with low DNA content, such as those obtained by flow cytometry cell sorting, allowing the simultaneous assessment of the genetic diversity of Synechococcus populations and cellular properties and activities (e.g., nutrient cell ratios or carbon uptake rates). Our approach will allow future studies using flow cytometry to investigate the link between ecological traits and taxonomic diversity of marine Synechococcus.

The layered costs and benefits of translational redundancy.

The rate and accuracy of translation hinges upon multiple components - including transfer RNA (tRNA) pools, tRNA modifying enzymes, and rRNA molecules - many of which are redundant in terms of gene copy number or function. It has been hypothesized that the redundancy evolves under selection, driven by its impacts on growth rate. However, we lack empirical measurements of the fitness costs and benefits of redundancy, and we have poor a understanding of how this redundancy is organized across components. We manipulated redundancy in multiple translation components of Escherichia coli by deleting 28 tRNA genes, 3 tRNA modifying systems, and 4 rRNA operons in various combinations. We find that redundancy in tRNA pools is beneficial when nutrients are plentiful and costly under nutrient limitation. This nutrient-dependent cost of redundant tRNA genes stems from upper limits to translation capacity and growth rate, and therefore varies as a function of the maximum growth rate attainable in a given nutrient niche. The loss of redundancy in rRNA genes and tRNA modifying enzymes had similar nutrient-dependent fitness consequences. Importantly, these effects are also contingent upon interactions across translation components, indicating a layered hierarchy from copy number of tRNA and rRNA genes to their expression and downstream processing. Overall, our results indicate both positive and negative selection on redundancy in translation components, depending on a species' evolutionary history with feasts and famines.

Impact of different nitrogen additions on microbes and exopolysaccharides excretion in cyanobacterial biocrusts

Recently, it has been found that nitrogen (N) deposition strongly affects the coverage of biocrusts. However, we know little about the response of exopolysaccharides (EPSs), the key cementing material in the formation and stability of biocrusts, to N deposition. Three N-sources including nitrate, ammonia and urea were added to biocrusts at three rates (2 mg/g, 4 mg/g, 8 mg/g) to evaluate the effect of N additions on the growth of biocrusts and the abundance of EPS. Our results showed 2 mg/g of nitrate–N had no obvious effect on the cyanobacterial biomass, while 4 and 8 mg/g of nitrate–N inhibited the growth of Microcoleus vaginatus, the dominant cyanobacterium in biocrusts, but promoted other cyanobacteria growth. Ammonia-N and urea-N strongly decreased the cyanobacterial biomass, indicated by chlorophyll-a and 16 s rRNA gene copy-numbers. On the whole, N additions had a positive impact on the α-biodiversity of biocrusts. However, Ammonia-N and urea-N shifted the bacterial communities from more Cyanobacteria to more Proteobacteria and Actinobacteria. Notably, lesser-N (2 mg/g) promoted the excretion of EPSs, while greater-N (8 mg/g) had the opposite effect, and the total proportion of rhamnose and fucose in EPSs decreased in all treatment groups. N additions (except 2 mg/g of nitrate–N) reduced cyanobacterial biomass and affected the bacterial communities in biocrusts, which would obstruct the development and succession of biocrusts. Meanwhile, the simultaneous reductions of the EPSs contents and proportion of rhamnose and fucose in EPSs may further reduce stability and persistence of cyanobacterial biocrusts, after N additions.

Ammonia-Oxidizing Bacteria Maintain Abundance but Lower amoA-Gene Expression during Cold Temperature Nitrification Failure in a Full-Scale Municipal Wastewater Treatment Plant.

In this study, we explore the relationship between community structure and transcriptional activity of ammonia-oxidizing bacteria during cold temperature nitrification failure in three parallel full-scale sequencing batch reactors (SBRs) treating municipal wastewater. In the three reactors, ammonia concentrations increased with declines in wastewater temperature below 15°C. We quantified and sequenced 16S rRNA and ammonia monooxygenase (amoA) gene fragments in DNA and RNA extracts from activated sludge samples collected from the SBRs during the warmer seasons (summer and fall) and when water temperatures were below 15°C (winter and spring). Taxonomic community composition of amoA genes and transcripts did not vary much between the warmer and colder seasons. However, we observed significant differences in amoA transcript copy numbers between fall (highest) and spring (lowest). Ammonia-oxidizing bacteria of the genus Nitrosomonas sp. could maintain their population abundance despite lowering their amoA gene expression during winter and spring. In spite of relatively low population abundance, an amoA amplicon sequence variant (ASV) cluster identified as most similar to the amoA gene of Nitrosospira briensis showed the highest amoA transcript-to-gene ratio throughout all four seasons, indicating that some nitrifiers remain active at wastewater temperatures below 15°C. Our results show that 16S rRNA and amoA gene copy numbers are limited predictors of cell activity. To optimize function and performance of mixed community bioprocesses, we need to collect high-resolution quantitative transcriptomic and potentially proteomic data to resolve the response of individual species to changes in environmental parameters in engineered systems. IMPORTANCE The diverse microbial community of activated sludge used in biological treatment systems exhibits dynamic seasonal shifts in community composition and activity. Many wastewater treatment plants in temperate/continental climates experience seasonal cold temperature nitrification failure. "Seasonal nitrification failure" is the discharge of elevated concentrations of ammonia (greater than 4 mg/liter) with treated wastewater during the winter (influent wastewater temperatures below 13°C). This study aims at expanding our understanding of how ammonia-oxidizing bacteria in activated sludge change in activity and growth across seasons. We quantified the ammonia monooxygenase (amoA) gene and transcript copy numbers using real-time PCR and sequenced the amoA amplicons to reveal community structure and activity changes of nitrifying microbial populations during seasonal nitrification failure in three full-scale sequencing batch reactors (SRBs) treating municipal wastewater. Relevant findings presented in this study contribute to explain seasonal nitrification performance variability in SRBs.

The influence of ZnO nanoparticles on horizontal transfer of resistance genes in lab and soil conditions

Antibiotic resistance is a severe problem that threatens the achievements of modern medicine. Metallic nanoparticles may promote the horizontal transfer of resistance genes due to their toxicity to bacterial cells and metal-induced co-selection mechanisms. In this study, we investigated the toxicity of ZnO nanoparticles to E. coli DH5α laboratory strain and the abundance of soil microbial community. Moreover, the influence of ZnO nanoparticles on resistance gene transfer in laboratory and soil conditions was evaluated. ZnO nanoparticles at concentrations up to 10 mg L−1 reduced the survival of E. coli cells by 14.6% and increased the transformation frequency by almost 1.8 fold. In soil, ZnO nanoparticles at a concentration of 1000 mg kg−1 affected the total abundance of bacteria, causing a decrease in the 16S rRNA gene copy number. We did not detect the presence of 11 target antibiotic resistance genes (sul1, sul2, imp2, imp5, blaCTX-M, ermB, mefA, strB, aadA1, tetA1, tetB), which confer resistance to five classes of antibiotics in soil treated with ZnO nanoparticles. No elevated conjugation frequency was observed in soil microbial communities treated with ZnO nanoparticles. However, the increase in czcA gene copies indicates the spread of genetic elements harbouring metal resistance. The data shows that metallic nanoparticles promote the spread of antibiotic and metal resistance genes. The broad implication of the present research is that the inevitable nanoparticles environmental pollution may lead to the further dissemination of antibiotic resistance and profoundly influence public health.

Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes.

One of the first biological machineries to be created seems to have been the ribosome. Since then, organisms have dedicated great efforts to optimize this apparatus. The ribosomal RNA (rRNA) contained within ribosomes is crucial for protein synthesis and maintenance of cellular function in all known organisms. In eukaryotic cells, rRNA is produced from ribosomal DNA clusters of tandem rRNA genes, whose organization in the nucleolus, maintenance and transcription are strictly regulated to satisfy the substantial demand for rRNA required for ribosome biogenesis. Recent studies have elucidated mechanisms underlying the integrity of ribosomal DNA and regulation of its transcription, including epigenetic mechanisms and a unique recombination and copy-number control system to stably maintain high rRNA gene copy number. In this Review, we disucss how the crucial maintenance of rRNA gene copy number through control of gene amplification and of rRNA production by RNA polymeraseI are orchestrated. We also discuss how liquid-liquid phase separation controls the architecture and function of the nucleolus and the relationship between rRNA production, cell senescence and disease.

Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.

Increased abundances of potential pathogenic bacteria and expressions of inflammatory cytokines in the intestine of oyster Crassostrea gigas after high temperature stress

High temperature stress is a significant threat to the health of oysters, but the effects on their intestinal performances are not well understood. In this study, the effects of high temperature stress on the intestinal histology, immune response and associated microbiota were investigated in Crassostrea gigas after rearing at 20, 25 and 28 °C for 21 days. With the increase of temperature, shortened and shed microvilli as well as increased goblet cells were observed in the intestines of oysters. The transcripts of cytokines CgIL17-5, CgTNF-2 and CgTGF-β and apoptosis-related gene CgCaspase-3 in intestine increased with the increasing temperature. Further, the diversity and composition of the oyster intestinal microbiota changed after high temperature stress. The 16S rRNA gene copy number per ng of DNA in the T25 (5.16 × 105) and T28 (1.63 × 105) groups were higher than that in the control group (8.62 × 104). The Chao 1 index in the T25 (238.00) and T28 (240.17) groups was lower than that in the control group (279.00). The Shannon index decreased progressively with the increasing temperature, with the value in the T28 group (4.44) significantly lower than that in the control group (5.40) (p < 0.05). The abundances of potential pathogenic bacteria such as Acinetobacter, Pseudomonas, Vibrio and Endozoicomonas increased while that of probiotic bacteria Bacillus decreased after high temperature exposure. Functional prediction indicated that the pathways associated with bacterial proliferation were enriched at 25 °C, while those involved in protein synthesis were blocked at 28 °C. Collectively, these results suggested that high temperature stress led to an increased abundances of potential pathogenic bacteria and expressions of inflammatory cytokines in the intestine, which may consequently affect the functional integrity of the intestinal barrier in oysters.

Assessment of the ecotoxicological impact of captan@ZnO35–45nm and captan@SiO2 20–30nm nanopesticide on non-target soil microorganisms – A 100-day case study

Nanopesticide application should enable efficient pest management with smaller doses of an active ingredient. Nevertheless, the environmental risk assessment of nanopesticides is currently in its initial stages due to limited access to nanopesticides. Therefore, we synthesised nanofungicides with captan as an organic active ingredient and ZnO35–45nm or SiO2 20–30nm as nanocarriers (captan@ ZnO35–45nm and captan@ SiO2 20–30nm) and evaluated their environmental risk by testing different microbial parameters as its potential biomarkers. First, physicochemical analysis (SEM-EDS, XPS, and FTIR) confirmed the presence of captan in nanofungicides, and they maintained 43–61 % antifungal efficiency against pathogen fungi compared to captan. Second, a laboratory toxicity assay (spot test) showed that nanofungicides generally revealed 10–100-fold lower growth inhibition of non-target microbial strains compared to captan. Next, the effect of nanofungicides on the abundance, structure and function of non-target soil microorganisms was evaluated during the 100-day microcosm using orchard soil and compared to control, captan, and nanocarriers. The changes in the total number of bacteria, ammonia-oxidising bacteria (AOB) and fungi were enumerated using the copy number of the qPCR approach based on the copy number of 16S rRNA, amoA and ITS genes. The functional potential and microbial structure were estimated based on Community Level Physiological Profiles (CLPPs) and Phospholipid Fatty Acids (PLFAs) profiles. Generally, results indicated that nanofungicides affected soil microorganisms by changing, in different scale, various microbial parameters, but their negative effect was generally lower than pesticide. Although qPCR results revealed the harmful effect of all tested compounds on total bacteria number (16S rRNA) on day 42, and captan@ZnO35–45nm and nanocarrier SiO2 20–30nm still affected amoA gene copy number on day 100, but the total fungal abundance in orchard soil was not affected. Furthermore, the analyses of functional and structural microbial diversity indicated the recovery process that was the fastest for captan@SiO2 20–30nm nanofungicide. On the contrary, ZnO35–45nm increased and prolonged the negative effect of captan in synthesised nanofungicide and generally exerted a more profound and/or longer effect than SiO2 20–30nm nanocarrier. Therefore we conclude that SiO2 20–30nm has better potential to be used as a nanocarrier compared to ZnO35–45nm. More studies are needed but soil microorganisms as sensitive biomarkers should be used for environmental risk assessment of nanopesticides.

Effect of Mannan-rich fraction supplementation on commercial broiler intestinum tenue and cecum microbiota

BackgroundThe broiler gastrointestinal microbiome is a potent flock performance modulator yet may also serve as a reservoir for pathogen entry into the food chain. The goal of this project was to characterise the effect of mannan rich fraction (MRF) supplementation on microbiome diversity and composition of the intestinum tenue and cecum of commercial broilers. This study also aimed to address some of the intrinsic biases that exist in microbiome studies which arise due to the extensive disparity in 16S rRNA gene copy numbers between bacterial species and due to large intersample variation.ResultsWe observed a divergent yet rich microbiome structure between different anatomical sites and observed the explicit effect MRF supplementation had on community structure, diversity, and pathogen modulation. Birds supplemented with MRF displayed significantly higher species richness in the cecum and significantly different bacterial community composition in each gastrointestinal (GI) tract section. Supplemented birds had lower levels of the zoonotic pathogens Escherichia coli and Clostridioides difficile across all three intestinum tenue sites highlighting the potential of MRF supplementation in maintaining food chain integrity. Higher levels of probiotic genera (eg. Lactobacillus and Blautia) were also noted in the MRF supplemented birds. Following MRF supplementation, the cecum displayed higher relative abundances of both short chain fatty acid (SFCA) synthesising bacteria and SCFA concentrations.ConclusionsMannan rich fraction addition has been observed to reduce the bioburden of pathogens in broilers and to promote greater intestinal tract microbial biodiversity. This study is the first, to our knowledge, to investigate the effect of mannan-rich fraction supplementation on the microbiome associated with different GI tract anatomical geographies. In addition to this novelty, this study also exploited machine learning and biostatistical techniques to correct the intrinsic biases associated with microbiome community studies to enable a more robust understanding of community structure.

Composition of Prokaryotic Communities in Anaerobic Enrichment Cultures of Crude Oil from Romashkino Oilfield (Russia)

Despite the many papers published on oilfield ecosystems, there is a lack of data about microbial community structure and functioning in many crude oils or oilfields and wells. One of such poorly studied oilfields is supergiant Romashkino situated in Tatarstan (Russia). In the present study, prokaryotic community composition and abundance of functional genes encoding sulfate reduction and rhamnolipid production, was investigated in enrichment cultures of four samples from Romashkino oilfield production wells with different oil/water ratios. The enrichment was conducted under anaerobic conditions, sulfate and nitrate were used as electron acceptors, and acetate – as an electron donor. In enrichment culture A and enrichment culture B, bacteria of the Pseudomonadaceae family predominated with 99% and 93% relative abundance, respectively. In enrichment culture C (obtained on the basis of initial sample contained oil with medium water content) and D (obtained on the basis of initial sample contained oil with very high water content), Pseudomonadaceae were one of the two predominating families, along with Micrococcaceae (enrichment culture C) and Dietziaceae (enrichment culture D). The proportion of dsrA and apsA genes encoding sulfate reduction to 16S rRNA gene copy numbers was quite low and ranged between 0.003% (enrichment cultures A, B) and 0.027% (enrichment culture D). The proportion of rhlB gene encoding rhamnolipid production from the total 16S rRNA gene copy number was 1.10% in sample A and 0.06% in sample B. In enrichment cultures C and D the rhlB gene was not found.

Characteristics of antibiotic resistance gene distribution in rainfall runoff and combined sewer overflow.

Rainfall runoff and combined sewer overflow (CSO) converge with organic waste, nutrients, and microbes from the ground and wastewater. These pollutants promote the spread and transformation of antibiotic resistance genes (ARGs). In this study, four rainfall runoff and one CSO outfall were chosen, and samples were collected to explore the occurrence and distribution of ARGs. The ARGs were extracted from suspended solids and analyzed using metagenomic sequencing. A total of 888 ARG subtypes, belonging to 17 ARG types, were detected in all samples. Eleven ARG types were shared by all the samples. Multidrug resistance genes had the highest relative abundance. Their total relative abundance reached 1.07 ratio (ARG copy number/16S rRNA gene copy number) and comprised 46.6% of all the ARGs. In all samples, the CSO outfall had the highest total relative abundance (8.25 × 10-1 ratio) of ARGs, with a ratio ranging ND (not detected)-3.78 × 10-1 ratio. Furthermore, the relationship between ARG types and environmental factors was determined using redundancy analysis. The results showed that chemical organic demand (COD) and bacterial abundance were positively correlated with most ARG types, including multidrug, bacitracin, aminoglycoside, β-lactam, tetracycline, and sulfonamide. NH3-N, TN, and TP were positively correlated with rifamycin, fosmidomycin, and vancomycin resistance genes. The relationship among the ARG subtypes was investigated using network analyses. The multidrug resistance gene subtypes had the highest frequency of co-occurrence. This study provides insights into the occurrence and distribution of ARGs under non-point source pollution and may contribute to the control of ARGs.

Ecological stoichiometry drives the evolution of soil nematode life history traits

Ecological stoichiometry is a useful theoretical framework for understanding the sources and controls on nutrient availability that structure the composition and diversity of biotic communities. One such relationship is that organismal development rate is positively linked to cellular Phosphorus (P). We hypothesized that P availability, relative to other nutrients, e.g., nitrogen and carbon, would drive the evolution of traits associated with organismal growth and development. We examined the effects of P availability both in situ and in vitro, on free-living soil nematodes. We found that P-deficient environments produce predictable changes in the ecology and evolution of important life history traits. Our results identify altered rRNA gene copy number and subsequent changes in gene expression and protein synthesis as mechanisms by which P-deficiency influences these traits. These findings have important implications for explaining soil ecological and evolutionary patterns across multiple levels of organization, including the structure and functioning of organisms, populations, communities, and ecosystems.

Impact of innovative nanoadditives on biodigesters microbiome.

Nanoparticles (NPs) supplementation to biodigesters improves the digestibility of biowaste and the generation of biogas. This study investigates the impact of innovative nanoadditives on the microbiome of biodigesters. Fresh cow manure was anaerobically incubated in a water bath under mesophilic conditions for 30 days. Three different NPs (zinc ferrite, zinc ferrite with 10% carbon nanotubes and zinc ferrite with 10% C76 fullerene) were separately supplemented to the biodigesters at the beginning of the incubation period. Methane and hydrogen production were monitored daily. Manure samples were collected from the digesters at different time points and the microbial communities inside the biodigesters were investigated via real-time PCR and 16 S rRNA gene amplicon-sequencing. The results indicate that zinc ferrite NPs enhanced biogas production the most. The microbial community was significantly affected by NPs addition in terms of archaeal and bacterial 16 S rRNAgene copy numbers. The three ZF formulations NPs augmented the abundance of members within the hydrogenotrophic methanogenic phyla Methanobacteriaceae. While Methanomassiliicoccacaea were enriched in ZF/C76 supplemented biodigester due to a significant increase in hydrogen partial pressure, probably caused by the enrichment of Spirochaetaceae (genus Treponema). Overall, NPs supplementation significantly enriched acetate-producing members within Hungateiclostridiaceae in ZF/CNTs, Dysgonomonadaceae in ZF and Spirochaetaceae ZF/C76 biodigesters.

Soil bacterial communities of different crop rotations and yield of succeeding wheat.

Evaluating ecological sustainability and crop productivity of different crop rotation patterns could provide theoretical support for adjusting and optimizing crop planting structure. We set seven treatments with different rotation crops and periods. We used real-time quantitative PCR to determine the abundance of soil bacterial community and 16S rRNA gene amplicon high-throughput sequencing technology to analyze diversity and taxa composition of soil bacterial community. Both soil available nutrients and succeeding wheat yield were measured. The results showed that, compared with the rotation with summer maize, the rotations with summer peanut or soybean in diffe-rent periods reduced soil organic carbon, mineral nitrogen, and available potassium, but significantly increased soil available phosphorus. The 16S rRNA gene copy numbers of soil bacteria in the treatments of rotations with summer peanut or soybean in different periods were significantly decreased, while community richness and diversity were increased. Different rotation crops significantly changed the structure and taxonomic composition of soil bacterial community. Compared with the rotation with summer maize, the rotations with summer soybean in different periods significantly increased the 1000-grain weight and grain yield of succeeding winter wheat. In conclusion, rotations with summer peanut or soybean in different periods could increase soil available phosphorus content and bacterial diversity, and significantly change soil bacterial community structure. In particular, rotation with summer soybean performed best in promoting yield formation of succeeding winter wheat.