16S rRNA Transcripts Research Articles

Ecological processes shaping highly connected bacterial communities along strong environmental gradients.

Along the river-sea continuum, microorganisms are directionally dispersed by water flow while being exposed to strong environmental gradients. To compare the two assembly mechanisms that may strongly and differently influence metacommunity dynamics, namely homogenizing dispersal and heterogeneous selection, we characterized the total (16S rRNA gene) and putatively active (16S rRNA transcript) bacterial communities in the Pearl River-South China Sea Continuum, during the wet (summer) and dry (winter) seasons using high-throughput sequencing. Moreover, well-defined sampling was conducted by including freshwater, oligohaline, mesohaline, polyhaline, and marine habitats. We found that heterogeneous selection exceeded homogenizing dispersal in both the total and active fractions of bacterial communities in two seasons. However, homogeneous selection was prevalent (the dominant except in active bacterial communities during summer), which was primarily due to the bacterial communities' tremendous diversity (associated with high rarity) and our specific sampling design. In either summer or winter seasons, homogeneous and heterogeneous selection showed higher relative importance in total and active communities, respectively, implying that the active bacteria were more responsive to environmental gradients than were the total bacteria. In summary, our findings provide insight into the assembly of bacterial communities in natural ecosystems with high spatial connectivity and environmental heterogeneity.

Microbial rrn copy number is associated with soil C: N ratio and pH under long-term fertilization

Soil microbial life-history strategies, as indicated by rRNA operon (rrn) copy numbers, strongly influence agro-ecosystem functioning. Long-term N fertilization causes strong and lasting changes in soil properties, yet its impact on microbial strategies remains largely unexplored. Using long-term field experiments across three agro-ecosystems, we consistently found that N fertilization strongly decreased soil C: N ratio and pH, further increasing the community-level rrn copy number, including both average rrn copy number and total 16S rRNA copy number. Soil C: N stoichiometry balanced by N supplement favored the growth of N-dependent copiotrophic species containing high rrn copy numbers (an average of 2.5) and increased their network connections, predominantly contributing to community-level rrn copy number increase. Decreased soil pH caused by N fertilization also favored the growth of some species whose abundances negatively correlated with pH, partially contributing to the community-level rrn copy number increase. By examining the genomes of two dominant species, we found that microorganisms with a higher rrn copy number (6), e.g., Streptomyces scabiei, possessed more genes related to C and N transport and metabolism. In contrast, the Mycobacterium simiae with a lower rrn copy number (1) has more genes associated with secondary metabolite biosynthesis and lipid transport and metabolism. Our finding challenges the concept of microbial life-strategy regulation solely by nutrient availability, highlighting the important contributions of soil stoichiometric balance and pH to microbial strategies in agro-ecosystems under long-term N inputs.

Chemolithoautotrophic bacteria flourish at dark water-ice interfaces of an emerged Arctic cold seep.

Below their ice shells, icy moons may offer a source of chemical energy that could support microbial life in the absence of light. In the Arctic, past and present glacial retreat leads to isostatic uplift of sediments through which cold and methane-saturated groundwater travels. This fluid reaches the surface and freezes as hill-shaped icings during winter, producing dark ice-water interfaces above water ponds containing chemical energy sources. In one such system characterized by elevated methane concentrations - the Lagoon Pingo in Adventdalen, Svalbard (~10mg/L CH4, <0.3mg/L O2, -0.25°C, pH7.9), we studied amplicons of the bacterial and archaeal (microbial) 16S rRNA gene and transcripts in the water pond and overlaying ice. We show that active chemolithoautotrophic sulfur-oxidizing microorganisms (Sulfurimonas, Thiomicrorhabdus) dominated a niche at the bottom of the ice in contact with the anoxic water reservoir. There, the growing ice offers surfaces interfacing with water, and hosts favorable physico-chemical conditions for sulfide oxidation. Detection of anaerobic methanotrophs further suggests that the ice led to a steady-state dark and cold methane sink under the ice throughout winter, in two steps: first methane is oxidized to carbon dioxide and sulfates concomitantly reduced to sulfides by the activity of ANME-1a and SEEP-SRB1 consortia, in a second time energy from sulfides is used by sulfur- oxidizing microorganisms to fix carbon dioxide into organic carbon. Our results underline ice- covered and dark ecosystems as a hitherto overlooked oasis of microbial life and emphasize the need to study microbial communities in icy habitats.

Soils of two Antarctic Dry Valleys exhibit unique microbial community structures in response to similar environmental disturbances

BackgroundIsolating the effects of deterministic variables (e.g., physicochemical conditions) on soil microbial communities from those of neutral processes (e.g., dispersal) remains a major challenge in microbial ecology. In this study, we disturbed soil microbial communities of two McMurdo Dry Valleys of Antarctica exhibiting distinct microbial biogeographic patterns, both devoid of aboveground biota and different in macro- and micro-physicochemical conditions. We modified the availability of water, nitrogen, carbon, copper ions, and sodium chloride salts in a laboratory-based experiment and monitored the microbial communities for up to two months. Our aim was to mimic a likely scenario in the near future, in which similar selective pressures will be applied to both valleys. We hypothesized that, given their unique microbial communities, the two valleys would select for different microbial populations when subjected to the same disturbances.ResultsThe two soil microbial communities, subjected to the same disturbances, did not respond similarly as reflected in both 16S rRNA genes and transcripts. Turnover of the two microbial communities showed a contrasting response to the same environmental disturbances and revealed different potentials for adaptation to change. These results suggest that the heterogeneity between these microbial communities, reflected in their strong biogeographic patterns, was maintained even when subjected to the same selective pressure and that the ‘rare biosphere’, at least in these samples, were deeply divergent and did not act as a reservoir for microbiota that enabled convergent responses to change in environmental conditions.ConclusionsOur findings strongly support the occurrence of endemic microbial communities that show a structural resilience to environmental disturbances, spanning a wide range of physicochemical conditions. In the highly arid and nutrient-limited environment of the Dry Valleys, these results provide direct evidence of microbial biogeographic patterns that can shape the communities’ response in the face of future environmental changes.

Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

An investigation of cyanobacteria, cyanotoxins and environmental variables in selected drinking water treatment plants in New Jersey

Harmful Algal Blooms (HAB) have the potential to impact human health primarily through their possible cyanotoxins production. While conventional water treatments can result in the removal of unlysed cyanobacterial cells and low levels of cyanotoxins, during severe HAB events, cyanotoxins can break through and can be present in the treated water due to a lack of adequate toxin treatment. The objectives of this study were to assess the HAB conditions in drinking water sources in New Jersey and investigate relationships between environmental variables and cyanobacterial communities in these drinking water sources. Source water samples were collected monthly from May to October 2019 and analyzed for phytoplankton and cyanobacterial cell densities, microcystins, cylindrospermopsin, Microcystis 16S rRNA gene, microcystin-producing mcyB gene, Raphidiopsis raciborskii-specific rpoC1 gene, and cylindrospermopsin-producing pks gene. Water quality parameters included water temperature, pH, dissolved oxygen, specific conductance, fluorescence of phycocyanin and chlorophyll, chlorophyll-a, total suspended solids, total dissolved solids, dissolved organic carbon, total nitrogen, ammonia, and total phosphorus. In addition to source waters, microcystins and cylindrospermopsin were analyzed for treated waters. The results showed all five selected New Jersey source waters had high total phosphorus concentrations that exceeded the established New Jersey Surface Water Quality Standards for lakes and rivers. Commonly found cyanobacteria were identified, such as Microcystis and Dolichospermum. Site E was the site most susceptible to HABs with significantly greater HAB variables, such as extracted phycocyanin, fluorescence of phycocyanin, cyanobacterial cell density, microcystins, and Microcystis 16S rRNA gene. All treated waters were undetected with microcystins, indicating treatment processes were effective at removing toxins from source waters. Results also showed that phycocyanin values had a significantly positive relationship with microcystin concentration, copies of Microcystis 16S rRNA and microcystin-producing mcyB genes, suggesting these values can be used as a proxy for HAB monitoring. This study suggests that drinking water sources in New Jersey are vulnerable to forthcoming HAB. Monitoring and management of source waters is crucial to help safeguard public health.

Non-selective microbiota reduction after the elicitation of a seaweed's immune response.

Pattern-triggered immunity (PTI) is an integral part of the innate immune system of many eukaryotic hosts, assisting in the defence against pathogen invasions. In plants and animals, PTI exerts a selective pressure on the microbiota that can alter community composition. However, the effect of PTI on the microbiota for non-model hosts, including seaweeds, remains unknown. Using quantitative polymerase chain reaction complemented with 16S rRNA gene and transcript amplicon sequencing, this study profiled the impact that PTI of the red seaweed Gracilaria gracilis has on its microbiota. PTI elicitation with agar oligosaccharides resulted in a significant reduction in the number of bacteria (by >75% within 72 h after treatment). However, the PTI elicitation did not cause any significant difference in the community diversity or structure. These findings demonstrated that PTI can be non-selective, and this might help to maintain a stable microbiota by uniformly reducing bacterial loads.

Water aging and the quality of organic carbon sources drive niche partitioning of the active bathypelagic prokaryotic microbiome

AbstractDue to the scarcity of organic matter (OM) sources in the bathypelagic (1000–4000 m depth), prokaryotic metabolism is believed to be concentrated on particles originating from the surface. However, the structure of active bathypelagic prokaryotic communities and how it changes across environmental gradients remains unexplored. Using a combination of 16S rRNA gene and transcripts sequencing, metagenomics, and substrate uptake potential measurements, here we aimed to explore how water masses aging and the quality of OM influence the structure of the active microbiome, and the potential implications for community function. We found that the relative contribution of taxa with a free‐living lifestyle to the active microbiome increased in older water masses that were enriched in recalcitrant OM, suggesting that these prokaryotes may also play a substantial role in the bathypelagic metabolism of vast areas of the ocean. In comparison to particle‐associated prokaryotes, free‐living prokaryotes exhibited lower potential metabolic rates, and harbored a limited number of two‐component sensory systems, suggesting they have less ability to sense and respond to environmental cues. In contrast, particle‐associated prokaryotes carried genes for particle colonization and carbohydrate utilization that were absent in prokaryotes with a free‐living lifestyle. Consistently, we observed that prokaryotic communities inhabiting older waters displayed reduced abilities to colonize particles, and higher capabilities to use complex carbon sources, compared to communities in waters with a higher proportion of labile OM. Our results provide evidence of regionalization of the bathypelagic active prokaryotic microbiome, unveiling a niche partitioning based on the quality of OM.

Environmental processes and health implications potentially mediated by dust-borne bacteria.

Understanding microbial migration and survival mechanisms in dust events (DEs) can elucidate genetic and metabolic exchange between environments and help predict the atmospheric pathways of ecological and health-related microbial stressors. Dust-borne microbial communities have been previously characterized, but the impact and interactions between potentially active bacteria within transported communities remain limited. Here, we analysed samples collected during DEs in Israel, using amplicon sequencing of the 16S rRNA genes and transcripts. Different air trajectories and wind speeds were associated not only with the genomic microbial community composition variations but also with specific 16S rRNA bacterial transcripts. Potentially active dust-borne bacteria exhibited positive interactions, including carbon and nitrogen cycling, biotransformation of heavy metals, degradation of organic compounds, biofilm formation, and the presence of pathogenic taxa. This study provides insights into the potential interactive relationships and survival strategies of microorganisms within the extreme dust environment.

Comparison of soil microbial abundances and co-occurrence networks in the volcanic soil of the cone and crater

Volcanism changes the earth's surface building environments where new ecosystems can be constructed. However, the effects of volcanic environmental conditions (e.g. soil moisture, soil organic matter, and available K) on soil microbial communities remains unclear. In the present study, soil samples were collected from two natural habitats (the cone and crater) in Nushan Volcano, China, to investigate the abundances (absolute and relative), compositions, and functions of soil bacterial and fungal communities. The results showed higher 16S rRNA copies, and ITS rRNA copies in the crater compared to the cone. The bacterial and fungal diversities in the crater were higher than in the cone. The soil of the cone and crater displayed distinct bacterial and fungal community compositions based on the nonmetric multidimensional scaling (NMDS). Co-occurrence network analysis revealed the interspecific relationship of soil bacterial and fungal communities was closer in the cone with more links. The number of keystone taxa in the cone was higher than that in the crater. Meanwhile, Nordella and Arthrobacter in bacteria and Stilbella, Tausonia, and Marasmius in fungi were found as the keystone taxa. Moreover, structural equation models (SEM) revealed that soil physicochemical properties had the greatest impact on the composition of bacteria and fungi. Overall, the results advance further research on the effects of microbes on the functions and services of associated ecosystems in extreme environments.

2453. Wastewater-Based Surveillance of Vancomycin-Resistant Enterococci in Hospitals

Abstract Background Vancomycin-resistant Enterococcus (VRE) is an important nosocomial infection that may increase patient morbidity, mortality, and healthcare costs. We have adapted wastewater-based surveillance (WBS) as a novel tool to comprehensively and inclusively monitor the burden of VRE in tertiary acute care hospitals. Herein, we demonstrate our ability to detect, quantify and track VRE dynamically over time across a range of scales. Methods Wastewater (WW) was collected from three hospitals in Calgary, AB: Rockyview General Hospital (RGH; 615 beds), Peter Lougheed Centre (PLC; 517 beds), and Foothills Medical Centre (FMC via three independent sites – A, B, C; 1100 beds). Three WW treatment plants (WWTPs; BBW, PCW, and FCW) serving the entire City of Calgary were sampled as community controls. DNA was extracted from WW pellets obtained following centrifugation. A multiplexed qPCR assay was adapted and used to quantify the abundances of the vanA and vanB resistance gene copies. Copy numbers were assessed as raw (copies per mL of WW processed) or normalized against three fecal biomarker genes: total bacterial 16S rRNA, human 18S rRNA, and Bacteroides HF183 16S rRNA. Differences between hospitals and controls were determined with Mann-Whitney tests (GraphPad Prism version 9.0). Results Samples from the hospitals and WWTPs in Calgary, AB collected over 12 weeks demonstrated that all hospitals had 100-1000X higher mean aggregate abundances of both vanA and vanB relative to community-based WWTPs when assessed as raw or normalized by each fecal biomarker (Figure 1; only total bacterial 16S rRNA is shown, p&amp;lt; 0.001, Mann-Whitney). Within one individual hospital (RGH), each of vanA and vanB abundances follows similar trends over a 12-week period, regardless of whether the values were reported as raw or normalized with the three different fecal biomarker genes (Figure 2; only vanA is shown).Figure 1.Mean aggregate copy numbers of vanA and vanB in wastewater measured by qPCR. VRE vanA and vanB gene abundances normalized as a ratio against total bacterial 16S rRNA copies demonstrate significantly higher abundances of both vanA and vanB in hospitals than the WWTP community controls.Figure 2.Comparison of vanA gene copies assessed as raw versus normalized as measured by qPCR from Rockyview General Hospital (RGH). VRE vanA gene abundances in Rockyview General Hospital (RGH) WW assessed as raw and normalized as a ratio against three different fecal biomarkers (total bacterial 16S rRNA, human 18S rRNA and Bacteroides HF183 16S rRNA) demonstrated similar trends in vanA gene abundances over time. Conclusion WBS is a unique real-time tool that can be adapted to monitor the abundance of VRE across a range of scales. This tool has the potential to augment antimicrobial stewardship and infection prevention and control programs to better understand the contributing factors to selection and colonization. Disclosures All Authors: No reported disclosures

Intact polar lipidome and membrane adaptations of microbial communities inhabiting serpentinite-hosted fluids.

The generation of hydrogen and reduced carbon compounds during serpentinization provides sustained energy for microorganisms on Earth, and possibly on other extraterrestrial bodies (e.g., Mars, icy satellites). However, the geochemical conditions that arise from water-rock reaction also challenge the known limits of microbial physiology, such as hyperalkaline pH, limited electron acceptors and inorganic carbon. Because cell membranes act as a primary barrier between a cell and its environment, lipids are a vital component in microbial acclimation to challenging physicochemical conditions. To probe the diversity of cell membrane lipids produced in serpentinizing settings and identify membrane adaptations to this environment, we conducted the first comprehensive intact polar lipid (IPL) biomarker survey of microbial communities inhabiting the subsurface at a terrestrial site of serpentinization. We used an expansive, custom environmental lipid database that expands the application of targeted and untargeted lipodomics in the study of microbial and biogeochemical processes. IPLs extracted from serpentinite-hosted fluid communities were comprised of >90% isoprenoidal and non-isoprenoidal diether glycolipids likely produced by archaeal methanogens and sulfate-reducing bacteria. Phospholipids only constituted ~1% of the intact polar lipidome. In addition to abundant diether glycolipids, betaine and trimethylated-ornithine aminolipids and glycosphingolipids were also detected, indicating pervasive membrane modifications in response to phosphate limitation. The carbon oxidation state of IPL backbones was positively correlated with the reduction potential of fluids, which may signify an energy conservation strategy for lipid synthesis. Together, these data suggest microorganisms inhabiting serpentinites possess a unique combination of membrane adaptations that allow for their survival in polyextreme environments. The persistence of IPLs in fluids beyond the presence of their source organisms, as indicated by 16S rRNA genes and transcripts, is promising for the detection of extinct life in serpentinizing settings through lipid biomarker signatures. These data contribute new insights into the complexity of lipid structures generated in actively serpentinizing environments and provide valuable context to aid in the reconstruction of past microbial activity from fossil lipid records of terrestrial serpentinites and the search for biosignatures elsewhere in our solar system.

The effects of indigenous microorganisms and water treatment with ion exchange resin on Cu-Ni flotation performance

Mineral processing utilizes large amounts of water and aims to reduce water consumption by recirculation and closing the water loops. This results in accumulation of chemical and biological contaminants in process water that may have adverse outcomes on the process performance. To optimize water quality suitable for each process step and plant, knowledge of both chemical and biological effects are needed as well as techniques to best remove the contaminants. This study focused on the consequences of microorganisms, enriched from the actual process earlier, on the flotation performance in the multi-metal Kevitsa mine in Northern Finland and the applicability of ion exchange for the removal of dissolved sulfur species and microorganisms from water. The increase of microbial load from the original 106 to added 107 16S rRNA copies mL−1 affected positively the flotation selectivity, especially in the case of nickel. Two tested water types, process water (PW) and final tailings water (FT), behaved slightly differently. In the Cu flotation phase added microorganisms did not affect the Cu recovery of FT but decreased significantly the recovery of Cu in PW. With equal Cu grade, the recovery was as high as approximately 25 percentage points lower. However, added microorganisms in both water types decreased notably the recovery of Ni in Cu concentrate (18 to 37 %-points). At the same time the amount of Ni recovered in the Ni concentrate increased by 18 to 33 %-points with added microorganisms. Visually the froth layer was higher and more stable in the Ni flotation in experiments with added microorganisms compared to experiments without added microorganisms. The concentrations of dissolved sulfate and thiosulfate ions were low in the studied waters compared to operations treating massive sulfide ores and did not significantly affect the flotation performance. For this reason, the IX water treatment was not required for these ions. However, the IX treatment proved to be effective in removing both sulfur species and microorganisms. The use of dissolved air flotation (DAF) was a successful pretreatment for ion exchange in removal of microorganisms. However, microorganisms are not usually taken into consideration when process performance or water cleaning techniques are designed and optimization could result generally in even better outcome.

Concatenated 16S rRNA sequence analysis improves bacterial taxonomy.

Background: Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Advances in DNA sequencing and bioinformatics workflows have facilitated sophisticated genome-based methods for microbial taxonomy although sequencing of the 16S rRNA gene is widely employed to identify and classify bacterial communities as a cost-effective and single-gene approach. However, the 16S rRNA sequence-based species identification accuracy is limited because of the occurrence of multiple copies of the 16S rRNA gene and higher sequence identity between closely related species. The availability of the genomes of several bacterial species provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. Methods: Sequences of the 16S rRNA genes were retrieved from the whole genomes available in the Genome databases. With defined criteria, four 16S rRNA gene copy variants were concatenated to develop a species-specific reference library. The sequence similarity search was performed with a web-based BLAST program, and MEGA software was used to construct the phylogenetic tree. Results: Using this approach, species-specific 16S rRNA gene libraries were developed for four closely related Streptococcusspecies ( S. gordonii, S. mitis, S. oralis, and S. pneumoniae). Sequence similarity and phylogenetic analysis using concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. Conclusions: The approach is very effective in classifying genetically closely related bacterial species and may reduce misclassification of bacterial species and genome assemblies.

Gut bacterial population and community dynamics following adult emergence in pest tephritid fruit flies

Gut microbiota are important contributors to insect success. Host-microbe interactions are dynamic and can change as hosts age and/or encounter different environments. A turning point in these relationships the transition from immature to adult life stages, particularly for holometabolous insects where there is radical restructuring of the gut. Improved knowledge of population and community dynamics of gut microbiomes upon adult emergence inform drivers of community assembly and physiological aspects of host-microbe interactions. Here, we evaluated the bacterial communities of the pest tephritid species melon fly (Zeugodacus cucurbitae) and Medditeranean fruit fly (medfly, Ceratitis capitata) associated with the pupae life stage and timepoints immediately following adult eclosion. We used a combination of culturing to determine cultivatable bacterial titers, qPCR to determine 16S-rRNA SSU copy numbers, and 16S V4 sequencing to determine changes in communities. Both culturing and qPCR revealed that fly bacterial populations declined upon adult emergence by 10 to 100-fold followed by recovery within 24 h following eclosion. Titers reached ~ 107 CFUs (~ 108 16S rRNA copies) within a week post-emergence. We also observed concurrent changes in amplicon sequence variance (ASVs), where the ASV composition differed overtime for both melon fly and medfly adults at different timepoints. Medfly, in particular, had different microbiome compositions at each timepoint, indicating greater levels of variation before stabilization. These results demonstrate that tephritid microbiomes experience a period of flux following adult emergence, where both biomass and the makeup of the community undergoes dramatic shifts. The host-microbe dynamics we document suggest plasticity in the community and that there may be specific periods where the tephritid gut microbiome may be pliable to introduce and establish new microbial strains in the host.

Inhibitory Effects of Ammonia on Archaeal 16S rRNA Transcripts in Thermophilic Anaerobic Digester Sludge

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH4+-N/L), but did not change much at lower ammonia concentrations (3 g NH4+-N/L) compared with the 0 g NH4+-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity.

Accounting for 16S rRNA copy number prediction uncertainty and its implications in bacterial diversity analyses

16S rRNA gene copy number (16S GCN) varies among bacterial species and this variation introduces potential biases to microbial diversity analyses using 16S rRNA read counts. To correct the biases, methods have been developed to predict 16S GCN. A recent study suggests that the prediction uncertainty can be so great that copy number correction is not justified in practice. Here we develop RasperGade16S, a novel method and software to better model and capture the inherent uncertainty in 16S GCN prediction. RasperGade16S implements a maximum likelihood framework of pulsed evolution model and explicitly accounts for intraspecific GCN variation and heterogeneous GCN evolution rates among species. Using cross-validation, we show that our method provides robust confidence estimates for the GCN predictions and outperforms other methods in both precision and recall. We have predicted GCN for 592605 OTUs in the SILVA database and tested 113842 bacterial communities that represent an exhaustive and diverse list of engineered and natural environments. We found that the prediction uncertainty is small enough for 99% of the communities that 16S GCN correction should improve their compositional and functional profiles estimated using 16S rRNA reads. On the other hand, we found that GCN variation has limited impacts on beta-diversity analyses such as PCoA, NMDS, PERMANOVA and random-forest test.

Microbial insights towards understanding the role of hydrochar in enhancing phenol degradation in anaerobic digestion

Anaerobic digestion (AD) of wastewater is the most promising bioprocess for organic conversion, however, phenol is toxic and resistant to anaerobic degradation. The current study compared the effect of hydrochar and granular activated carbon (GAC) on AD of phenol at four concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L). Results demonstrated that hydrochar significantly improved the methane production rate and reduced the lag phase at all concentrations of phenol. The methane production rate was improved by about 50% at both 100 mg/L and 250 mg/L phenol, while it was raised by >160% at 500 mg/L and 1000 mg/L phenol by hydrochar. The GAC only increased the methane production rate at 500 mg/L and 1000 mg/L due to high adsorption capacity. Further, the adsorption of phenol by hydrochar had no apparent impact on the methane production rate, even though certain amounts of phenol were adsorbed. At 500 mg/L, the amount of methane produced significantly increased, so 16S rRNA transcripts sequencing and metabolomic analysis were conducted. 16S rRNA transcripts sequencing analysis indicated that hydrochar resulted in the enrichment of syntrophic bacteria (e.g., Syntrophorhabdus &Syntrophobacter) and Methanosaeta, which might be related with direct interspecies electron transfer. Further, it was noticed that the growth of Methanobacterium was repressed at 500 mg/L phenol, while hydrochar promoted its growth. Phenol was degraded into L-tyrosine and then followed the benzoate degradation pathway for methane production as revealed by metabolomic analysis. In addition, metabolomic analysis also revealed that hydrochar promoted the degradation of all metabolites and enhanced the phenol degradation into methane.

Effect of Cork Flour Supplementation on the Growth Indices and Rhizosphere Bacterial Communities of Quercus variabilis Seedlings

Substrate and rhizosphere microorganisms are key factors affecting seedling growth; however, the effects of seedling substrates and rhizosphere bacteria on the growth of Quercus variabilis are not completely understood. Here, Q. variabilis seedlings were grown in substrates with and without cork flour, as follows: H substrate (charcoal soil/cork flour/perlite, 1:1:2), S substrate (cork flour/perlite, 1:1), and the control (CK) substrate (charcoal soil/perlite, 3:2). High-throughput sequencing and qPCR were used to investigate the effects of these substrates on seedling growth, physiological indices, and rhizosphere bacterial communities. Root and shoot weights of seedlings grown in H and S substrates were significantly higher than those of seedlings grown in CK. Moreover, H was conducive to chlorophyll synthesis in seedling leaves, and the transpiration rate and intercellular CO2 concentration of the leaves of seedlings grown in H were higher than those of seedlings grown in CK. The number of rhizosphere bacterial 16S rRNA copies was significantly greater in the case of seedlings grown in S than for those grown in H and CK. As well, rhizosphere bacterial richness was higher in seedlings grown in H and S than in those grown in CK. Thus, cork-flour-supplemented substrates are beneficial for seedling growth and development, seedling rhizosphere bacterial abundance and diversity, and the abundance of nitrogen and phosphorus metabolism-promoting microbial taxa.

Identification of diverse antibiotic resistant bacteria in agricultural soil with H218O stable isotope probing combined with high-throughput sequencing

BackgroundWe aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled “heavy” and the unlabelled “light” SIP fractions were sequenced.ResultsAn increase of the 16S rRNA copy numbers in the “heavy” fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) were highly abundant after two days of incubation with antibiotics. Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. In comparison, only two ARGs from the unbinned-assembled light fractions were identified.ConclusionsThe results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.