Genome-resolved Metagenomics Research Articles

Mammals show distinct functional gut microbiome dynamics to identical series of environmental stressors.

In our manuscript, we report the first interspecific comparative study about the plasticity of the gut microbiota. We conducted a captivity experiment that exposed wild-captured mammals to a series of environmental challenges over 45 days. We characterized their gut microbial communities using genome-resolved metagenomics and modeled how the taxonomic, phylogenetic, and functional microbial dynamics varied across a series of disturbances in both species. Our results indicate that the intrinsic properties (e.g., diversity and functional redundancy) of microbial communities coupled with physiological attributes (e.g., thermal plasticity) of hosts shape the taxonomic, phylogenetic, and functional response of gut microbiomes to environmental stressors, which might influence their contribution to the acclimation and adaptation capacity of animal hosts.

Genes and genome-resolved metagenomics reveal the microbial functional make up of Amazon peatlands under geochemical gradients.

The Pastaza-Marañón Foreland Basin (PMFB) holds the most extensive tropical peatland area in South America. PMFB peatlands store ~7.07 Gt of organic carbon interacting with multiple microbial heterotrophic, methanogenic, and other aerobic/anaerobic respirations. Little is understood about the contribution of distinct microbial community members inhabiting tropical peatlands. Here, we studied the metagenomes of three geochemically distinct peatlands spanning minerotrophic, mixed, and ombrotrophic conditions. Using gene- and genome-centric approaches, we evaluate the functional potential of the underlying microbial communities. Abundance analyses show significant differences in C, N, P, and S acquisition genes. Furthermore, community interactions mediated by toxin-antitoxin and CRISPR-Cas systems were enriched in oligotrophic soils, suggesting that non-metabolic interactions may exert additional controls in low-nutrient environments. Additionally, we reconstructed 519 metagenome-assembled genomes spanning 28 phyla. Our analyses detail key differences across the geochemical gradient in the predicted microbial populations involved in degradation of organic matter, and the cycling of N and S. Notably, we observed differences in the nitric oxide (NO) reduction strategies between sites with high and low N2 O fluxes and found phyla putatively capable of both NO and sulfate reduction. Our findings detail how gene abundances and microbial populations are influenced by geochemical differences in tropical peatlands.

Metabolically diverse microorganisms mediate methylmercury formation under nitrate-reducing conditions in a dynamic hydroelectric reservoir

Brownlee Reservoir is a mercury (Hg)-impaired hydroelectric reservoir that exhibits dynamic hydrological and geochemical conditions and is located within the Hells Canyon Complex in Idaho, USA. Methylmercury (MeHg) contamination in fish is a concern in the reservoir. While MeHg production has historically been attributed to sulfate-reducing bacteria and methanogenic archaea, microorganisms carrying the hgcA gene are taxonomically and metabolically diverse and the major biogeochemical cycles driving mercury (Hg) methylation are not well understood. In this study, Hg speciation and redox-active compounds were measured throughout Brownlee Reservoir across the stratified period in four consecutive years (2016–2019) to identify the location where and redox conditions under which MeHg is produced. Metagenomic sequencing was performed on a subset of samples to characterize the microbial community with hgcA and identify possible links between biogeochemical cycles and MeHg production. Biogeochemical profiles suggested in situ water column Hg methylation was the major source of MeHg. These profiles, combined with genome-resolved metagenomics focused on hgcA-carrying microbes, indicated that MeHg production occurs in this system under nitrate- or manganese-reducing conditions, which were previously thought to preclude Hg-methylation. Using this multidisciplinary approach, we identified the cascading effects of interannual variability in hydrology on the redox status, microbial metabolic strategies, abundance and metabolic diversity of Hg methylators, and ultimately MeHg concentrations throughout the reservoir. This work expands the known conditions conducive to producing MeHg and suggests that the Hg-methylation mitigation efforts by nitrate or manganese amendment may be unsuccessful in some locations.

Genome-resolved metagenomics provides insights into the ecological roles of the keystone taxa in heavy-metal-contaminated soils.

Microorganisms that exhibit resistance to environmental stressors, particularly heavy metals, have the potential to be used in bioremediation strategies. This study aimed to explore and identify microorganisms that are resistant to heavy metals in soil environments as potential candidates for bioremediation. Metagenomic analysis was conducted using microbiome metagenomes obtained from the rhizosphere of soil contaminated with heavy metals and mineral-affected soil. The analysis resulted in the recovery of a total of 175 metagenome-assembled genomes (MAGs), 73 of which were potentially representing novel taxonomic levels beyond the genus level. The constructed ecological network revealed the presence of keystone taxa, including Rhizobiaceae, Xanthobacteraceae, Burkholderiaceae, and Actinomycetia. Among the recovered MAGs, 50 were associated with these keystone taxa. Notably, these MAGs displayed an abundance of genes conferring resistance to heavy metals and other abiotic stresses, particularly those affiliated with the keystone taxa. These genes were found to combat excessive accumulation of zinc/manganese, arsenate/arsenite, chromate, nickel/cobalt, copper, and tellurite. Furthermore, the keystone taxa were found to utilize both organic and inorganic energy sources, such as sulfur, arsenic, and carbon dioxide. Additionally, these keystone taxa exhibited the ability to promote vegetation development in re-vegetated mining areas through phosphorus solubilization and metabolite secretion. In summary, our study highlights the metabolic adaptability and ecological significance of microbial keystone taxa in mineral-affected soils. The MAGs associated with keystone taxa exhibited a markedly higher number of genes related to abiotic stress resistance and plant growth promotion compared to non-keystone taxa MAGs.

Gastrointestinal microbiome, resistance genes, and risk assessment of heavy metals in wild giant pandas

The gastrointestinal microbiome (GM) of giant panda (GP) plays an important role in food utilization and health and is also an essential reservoir of resistance genes. Currently, little knowledge is available on the GM, acid resistance genes (AcRGs), antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs) in wild GPs. We sampled the gastrointestinal tract of a dead GP and explored the composition and function of GM and resistance genes through cryo-scanning electron microscopy, metagenomic sequencing, and genome-resolved metagenomics. The concentration of metals in the gastrointestinal lumen, feces, bamboo, and soil was measured by inductively coupled plasma mass spectrometry. Results showed that the composition of the microbiota varied in different gastrointestinal regions. Fecal microbiota was highly associated with small intestinal and colonic microbes. The lignocellulosic cross-linked structure of bamboo was destroyed in the stomach initially and destroying degree increased from stomach to anus. Reconstruction of metagenome-assembled-genomes confirmed that core GM, e.g., Streptococcus, Clostridium, Lactococcus, Leuconostoc, and Enterococcus, carried genes encoding the lignocellulose degradation enzyme. There were no significant differences of resistance genes between gastrointestinal and fecal samples, except MGEs. Multidrug and multi-metal resistance genes were predominant in all samples, while the transposase gene tnpA was the major type of MGE. Significant correlations were observed among the abundance of GM, resistance genes, and MGEs. Gastrointestinal and fecal mercury and chromium were the main metals influencing GM and resistance genes. The content of gastrointestinal and fecal metals was significantly associated with the presence of the same metals in bamboo, which could pose a threat to the health of wild GPs. This study characterized the gastrointestinal microbiome of wild GPs, providing new evidence for the role of the gastrointestinal microbiome in degrading lignocellulose from bamboo and highlighting the urgent need to monitor metal levels in soil and bamboo.

Novel diversity of polar Cyanobacteria revealed by genome-resolved metagenomics.

Benthic microbial mats dominated by Cyanobacteria are important features of polar lakes. Although culture-independent studies have provided important insights into the diversity of polar Cyanobacteria, only a handful of genomes have been sequenced to date. Here, we applied a genome-resolved metagenomics approach to data obtained from Arctic, sub-Antarctic and Antarctic microbial mats. We recovered 37 metagenome-assembled genomes (MAGs) of Cyanobacteria representing 17 distinct species, most of which are only distantly related to genomes that have been sequenced so far. These include (i) lineages that are common in polar microbial mats such as the filamentous taxa Pseudanabaena, Leptolyngbya, Microcoleus/Tychonema and Phormidium; (ii) the less common taxa Crinalium and Chamaesiphon; (iii) an enigmatic Chroococcales lineage only distantly related to Microcystis; and (iv) an early branching lineage in the order Gloeobacterales that is distributed across the cold biosphere, for which we propose the name Candidatus Sivonenia alaskensis. Our results show that genome-resolved metagenomics is a powerful tool for expanding our understanding of the diversity of Cyanobacteria, especially in understudied remote and extreme environments.

Evidence for nontraditional mcr-containing archaea contributing to biological methanogenesis in geothermal springs.

Recent discoveries of methyl-coenzyme M reductase-encoding genes (mcr) in uncultured archaea beyond traditional euryarchaeotal methanogens have reshaped our view of methanogenesis. However, whether any of these nontraditional archaea perform methanogenesis remains elusive. Here, we report field and microcosm experiments based on 13C-tracer labeling and genome-resolved metagenomics and metatranscriptomics, revealing that nontraditional archaea are predominant active methane producers in two geothermal springs. Archaeoglobales performed methanogenesis from methanol and may exhibit adaptability in using methylotrophic and hydrogenotrophic pathways based on temperature/substrate availability. A five-year field survey found Candidatus Nezhaarchaeota to be the predominant mcr-containing archaea inhabiting the springs; genomic inference and mcr expression under methanogenic conditions strongly suggested that this lineage mediated hydrogenotrophic methanogenesis in situ. Methanogenesis was temperature-sensitive , with a preference for methylotrophic over hydrogenotrophic pathways when incubation temperatures increased from 65° to 75°C. This study demonstrates an anoxic ecosystem wherein methanogenesis is primarily driven by archaea beyond known methanogens, highlighting diverse nontraditional mcr-containing archaea as previously unrecognized methane sources.

Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Metagenomic insights into genetic factors driving bacterial niche differentiation between bulk and rhizosphere soils

Cellular motility is crucial for effective colonization of the rhizosphere, but it is not yet clear whether bacterial motility is particularly linked to other genetic traits. Here, we applied genome-resolved metagenomics and phylogenomics to investigate the ecological significance of cellular motility for niche differentiation and the links between the genetic makeup of motile bacteria and rhizosphere colonization within a four-decade maize field experiment. Indeed, highly diverse sets of genes encoding cellular motility, including chemotaxis, flagellar assembly and motility proteins, and utilization of polymeric carbon were the important predictors of bacterial niche differentiation between bulk and rhizosphere soils. This is well exemplified by metagenome-assembled genomes encoding high motility capacity (hmc_MAGs). Their collective abundance was, on average, sixfold higher in rhizosphere soil than in bulk soil. All bulk-soil-derived MAGs showed low motility capacities (lmc). The hmc_MAGs were highly enriched in beneficial traits involved in carbohydrate utilization, assimilatory (nasA) and dissimilatory (nirBD) nitrate reduction, inorganic phosphate solubilization (gcd), and organic phosphate mineralization (phoD). Belonging to the families Sphingomonadaceae, Burkholderiaceae and Steroidobacteraceae, the hmc_MAGs showed a ninefold greater enrichment in these traits than proteobacterial lmc_MAGs and a twofold greater enrichment than 264 genomes publicly available for the above three families, thereby substantiating that a specific rhizosphere effect acted on the microbes represented by the hmc_MAGs. The particular link between the genetic capacities for high cellular motility and increased carbohydrate depolymerization as the key determinant for plant-selected rhizosphere colonization was further substantiated by the analysis of public bulk-rhizosphere soil metagenomes retrieved from wheat and cucumber field sites.

Genome-Resolved Metagenomics of Nitrogen Transformations in the Switchgrass Rhizosphere Microbiome on Marginal Lands

Switchgrass (Panicum virgatum L.) remains the preeminent American perennial (C4) bioenergy crop for cellulosic ethanol, that could help displace over a quarter of the US current petroleum consumption. Intriguingly, there is often little response to nitrogen fertilizer once stands are established. The rhizosphere microbiome plays a critical role in nitrogen cycling and overall plant nutrient uptake. We used high-throughput metagenomic sequencing to characterize the switchgrass rhizosphere microbial community before and after a nitrogen fertilization event for established stands on marginal land. We examined community structure and bulk metabolic potential, and resolved 29 individual bacteria genomes via metagenomic de novo assembly. Community structure and diversity were not significantly different before and after fertilization; however, the bulk metabolic potential of carbohydrate-active enzymes was depleted after fertilization. We resolved 29 metagenomic assembled genomes, including some from the ‘most wanted’ soil taxa such as Verrucomicrobia, Candidate phyla UBA10199, Acidobacteria (rare subgroup 23), Dormibacterota, and the very rare Candidatus Eisenbacteria. The Dormibacterota (formally candidate division AD3) we identified have the potential for autotrophic CO utilization, which may impact carbon partitioning and storage. Our study also suggests that the rhizosphere microbiome may be involved in providing associative nitrogen fixation (ANF) via the novel diazotroph Janthinobacterium to switchgrass.

A survey on computational strategies for genome-resolved gut metagenomics.

Recovering high-quality metagenome-assembled genomes (HQ-MAGs) is critical for exploring microbial compositions and microbe-phenotype associations. However, multiple sequencing platforms and computational tools for this purpose may confuse researchers and thus call for extensive evaluation. Here, we systematically evaluated a total of 40 combinations of popular computational tools and sequencing platforms (i.e. strategies), involving eight assemblers, eight metagenomic binners and four sequencing technologies, including short-, long-read and metaHiC sequencing. We identified the best tools for the individual tasks (e.g. the assembly and binning) and combinations (e.g. generating more HQ-MAGs) depending on the availability of the sequencing data. We found that the combination of the hybrid assemblies and metaHiC-based binning performed best, followed by the hybrid and long-read assemblies. More importantly, both long-read and metaHiC sequencings link more mobile elements and antibiotic resistance genes to bacterial hosts and improve the quality of public human gut reference genomes with 32% (34/105) HQ-MAGs that were either of better quality than those in the Unified Human Gastrointestinal Genome catalog version 2 or novel.

Gut microbiome responds compositionally and functionally to the seasonal diet variations in wild gibbons

Wild animals may encounter multiple challenges especially food shortage and altered diet composition in their suboptimal ranges. Yet, how the gut microbiome responds to dietary changes remains poorly understood. Prior studies on wild animal microbiomes have typically leaned upon relatively coarse dietary records and individually unresolved fecal samples. Here, we conducted a longitudinal study integrating 514 time-series individually recognized fecal samples with parallel fine-grained dietary data from two Skywalker hoolock gibbon (Hoolock tianxing) groups populating high-altitude mountainous forests in western Yunnan Province, China. 16S rRNA gene amplicon sequencing showed a remarkable seasonal fluctuation in the gibbons’ gut microbial community structure both across individuals and between the social groups, especially driven by the relative abundances of Lanchnospiraceae and Oscillospiraceae associated with fluctuating consumption of leaf. Metagenomic functional profiling revealed that diverse metabolisms associated with cellulose degradation and short-chain fatty acids (SCFAs) production were enriched in the high-leaf periods possibly to compensate for energy intake. Genome-resolved metagenomics further enabled the resolving metabolic capacities associated with carbohydrate breakdown among community members which exhibited a high degree of functional redundancy. Our results highlight a taxonomically and functionally sensitive gut microbiome actively responding to the seasonally shifting diet, facilitating the survival and reproduction of the endangered gibbon species in their suboptimal habitats.

Metabolic independence drives gut microbial colonization and resilience in health and disease

BackgroundChanges in microbial community composition as a function of human health and disease states have sparked remarkable interest in the human gut microbiome. However, establishing reproducible insights into the determinants of microbial succession in disease has been a formidable challenge.ResultsHere we use fecal microbiota transplantation (FMT) as an in natura experimental model to investigate the association between metabolic independence and resilience in stressed gut environments. Our genome-resolved metagenomics survey suggests that FMT serves as an environmental filter that favors populations with higher metabolic independence, the genomes of which encode complete metabolic modules to synthesize critical metabolites, including amino acids, nucleotides, and vitamins. Interestingly, we observe higher completion of the same biosynthetic pathways in microbes enriched in IBD patients.ConclusionsThese observations suggest a general mechanism that underlies changes in diversity in perturbed gut environments and reveal taxon-independent markers of “dysbiosis” that may explain why widespread yet typically low-abundance members of healthy gut microbiomes can dominate under inflammatory conditions without any causal association with disease.

Genome-resolved metagenomics revealed metal-resistance, geochemical cycles in a Himalayan hot spring.

The hot spring microbiome is a complex assemblage of micro- and macro-organisms; however, the understanding and projection of enzymatic repertoire that access earth's integral ecosystem processes remains ambivalent. Here, the Khirganga hot spring characterized with white microbial mat and ions rich in sulfate, chlorine, sodium, and magnesium ions is investigated and displayed the examination of 41 high and medium qualified metagenome-assembled genomes (MAGs) belonged to at least 12 bacterial and 2 archaeal phyla which aids to drive sulfur, oxygen, iron, and nitrogen cycles with metabolic mechanisms involved in heavy metal tolerance. These MAGs possess over 1749 genes putatively involved in crucial metabolism of elements viz. nitrogen, phosphorus, and sulfur and 598 genes encoding enzymes for czc efflux system, chromium, arsenic, and copper heavy metals resistance. The MAGs also constitute 229 biosynthetic gene clusters classified abundantly as bacteriocins and terpenes. The metabolic roles possibly involved in altering linkages in nitrogen biogeochemical cycles and explored a discerned rate of carbon fixation exclusively in archaeal member Methanospirillum hungatei inhabited in microbial mat. Higher Pfam entropy scores of biogeochemical cycling in Proteobacteria members assuring their major contribution in assimilation of ammonia and sequestration of nitrate and sulfate components as electron acceptors. This study will readily improve the understanding of the composite relationship between bacterial species owning metal resistance genes (MRGs) and underline the exploration of adaptive mechanism of these MAGs in multi-metal contaminated environment. KEY POINTS: • Identification of 41 novel bacterial and archaeal species in habitats of hot spring • Genome-resolved metagenomics revealed MRGs (n = 598) against Cr, Co, Zn, Cd, As, and Cu • Highest entropies of N (0.48) and Fe (0.44) cycles were detected within the MAGs.

O2 partitioning of sulfur oxidizing bacteria drives acidity and thiosulfate distributions in mining waters

The acidification of water in mining areas is a global environmental issue primarily catalyzed by sulfur-oxidizing bacteria (SOB). Little is known about microbial sulfur cycling in circumneutral pH mine tailing impoundment waters. Here we investigate biological sulfur oxidation over four years in a mine tailings impoundment water cap, integrating aqueous sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. The microbial community is consistently dominated by neutrophilic, chemolithoautotrophic SOB (relative abundances of ~76% in 2015, ~55% in 2016/2017 and ~60% in 2018). Results reveal two SOB strategies alternately dominate across the four years, influencing acid generation and sulfur speciation. Under oxic conditions, novel Halothiobacillus drive lower pH conditions (as low as 4.3) and lower [S2O32−] via the complete Sox pathway coupled to O2. Under anoxic conditions, Thiobacillus spp. dominate in activity, via the incomplete Sox and rDSR pathways coupled to NO3−, resulting in higher [S2O32−] and no net significant acidity generation. This study provides genomic evidence explaining acidity generation and thiosulfate accumulation patterns in a circumneutral mine tailing impoundment and has significant environmental applications in preventing the discharge of sulfur compounds that can impact downstream environments. These insights illuminate opportunities for in situ biotreatment of reduced sulfur compounds and prediction of acidification events using gene-based monitoring and in situ RNA detection.

Genome-resolving metagenomics reveals wild western capercaillies (Tetrao urogallus) as avian hosts for antibiotic-resistance bacteria and their interactions with the gut-virome community

The gut microbiome is a critical component of avian health, influencing nutrient uptake and immune functions. While the gut microbiomes of agriculturally important birds have been studied, the microbiomes of wild birds still need to be explored. Filling this knowledge gap could have implications for the microbial rewilding of captive birds and managing avian hosts for antibiotic-resistant bacteria (ARB). Using genome-resolved metagenomics, we recovered 112 metagenome-assembled genomes (MAGs) from the faeces of wild and captive western capercaillies (Tetrao urogallus) (n = 8). Comparisons of bacterial diversity between the wild and captive capercaillies suggest that the reduced diversity in the captive individual could be due to differences in diet. This was further substantiated through the analyses of 517,657 clusters of orthologous groups (COGs), which revealed that gene functions related to amino acids and carbohydrate metabolisms were more abundant in wild capercaillies. Metagenomics mining of resistome identified 751 antibiotic resistance genes (ARGs), of which 40.7 % were specific to wild capercaillies suggesting that capercaillies could be potential reservoirs for hosting ARG-associated bacteria. Additionally, the core resistome shared between wild and captive capercaillies indicates that birds can acquire these ARG-associated bacteria naturally from the environment (43.1 % of ARGs). The association of 26 MAGs with 120 ARGs and 378 virus operational taxonomic units (vOTUs) also suggests a possible interplay between these elements, where putative phages could have roles in modulating the gut microbiota of avian hosts. These findings can have important implications for conservation and human health, such as avian gut microbiota rewilding, identifying the emerging threats or opportunities due to phage-microbe interactions, and monitoring the potential spread of ARG-associated bacteria from wild avian populations.

Co-Occurrence and Cooperation between Comammox and Anammox Bacteria in a Full-Scale Attached Growth Municipal Wastewater Treatment Process.

Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems, including synthetic community constructs; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 ± 0.08 mg-N/g TS-h) in the attached growth phase, with minimal contribution by ammonia-oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen (∼8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2 - 6 mg/L) setpoints indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed the high abundance (relative abundance 6.53 ± 0.34%) of two Brocadia-like anammox populations, while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37 ± 0.03%) and Nitrosomonas-like ammonia oxidizers were even lower (0.12 ± 0.02%). Collectively, our study reports for the first time the co-occurrence and cooperation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system.

Genome-resolved metagenomics inferred novel insights into the microbial community, metabolic pathways, and biomining potential of Malanjkhand acidic copper mine tailings.

Mine tailing sites provide profound opportunities to elucidate the microbial mechanisms involved in ecosystem functioning. In the present study, metagenomic analysis of dumping soil and adjacent pond around India's largest copper mine at Malanjkhand has been done. Taxonomic analysis deciphered the abundance of phyla Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi. Genomic signatures of viruses were predicted in the soil metagenome, whereas Archaea and Eukaryotes were noticed in water samples. Mesophilic chemolithotrophs, such as Acidobacteria bacterium, Chloroflexi bacterium, and Verrucomicrobia bacterium, were predominant in soil, whereas, in the water sample, the abundance of Methylobacterium mesophilicum, Pedobacter sp., and Thaumarchaeota archaeon was determined. The functional potential analysis highlighted the abundance of genes related to sulfur, nitrogen, methane, ferrous oxidation, carbon fixation, and carbohydrate metabolisms. The genes for copper, iron, arsenic, mercury, chromium, tellurium, hydrogen peroxide, and selenium resistance were found to be predominant in the metagenomes. Metagenome-assembled genomes (MAGs) were constructed from the sequencing data, indicating novel microbial species genetically related to the phylum predicted through whole genome metagenomics. Phylogenetic analysis, genome annotations, functional potential, and resistome analysis showed the resemblance of assembled novel MAGs with traditional organisms used in bioremediation and biomining applications. Microorganisms harboring adaptive mechanisms, such as detoxification, hydroxyl radical scavenging, and heavy metal resistance, could be the potent benefactions for their utility as bioleaching agents. The genetic information produced in the present investigation provides a foundation for pursuing and understanding the molecular aspects of bioleaching and bioremediation applications.

Anaerobic methane-oxidizing activity in a deep underground borehole dominantly colonized by Ca. Methanoperedenaceae.

The family Ca. Methanoperedenaceae archaea mediates the anaerobic oxidation of methane (AOM) in different terrestrial environments. Using a newly developed high-pressure laboratory incubation system, we investigated 214- and 249-m deep groundwater samples at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Ca. Methanoperedenaceae archaea have been shown by genome-resolved metagenomics, respectively. The groundwater samples amended with 13 C-labelled methane and amorphous Fe(III) were incubated at a pressure of 1.6MPa. After 3-7 days of incubation, the AOM rate was 45.8 ± 19.8nM/day in 214-m groundwater. However, almost no activity was detected from 249-m groundwater. Based on the results from 16S rRNA gene analysis, the abundance of Ca. Methanoperedenaceae archaea was high in the 214-m deep groundwater sample, whereas Ca. Methanoperedenaceae archaea was undetected in the 249-m deep groundwater sample. These results support the in situ AOM activity of Ca. Methanoperedenaceae archaea in the 214-m deep subsurface borehole interval. Although the presence of Fe-bearing phyllosilicates was demonstrated in the 214-m deep groundwater, it needs to be determined whether Ca. Methanoperedenaceae archaea use the Fe-bearing phyllosilicates as in situ electron acceptors by high-pressure incubation amended with the Fe-bearing phyllosilicates.

Selective enrichment, identification, and isolation of diclofenac, ibuprofen, and carbamazepine degrading bacteria from a groundwater biofilm

Diclofenac, ibuprofen, and carbamazepine are three of the most widely detected and most concerning pharmaceutical residues in aquatic ecosystems. The aim of this study was to identify bacteria that may be involved in their degradation from a bacterial biofilm. Selective enrichment cultures in mineral salt solution containing pharmaceutical compounds as sole source of carbon and energy were set up, and population dynamics were monitored using shotgun metagenome sequencing. Bacterial genomes were reconstructed using genome-resolved metagenomics. Thirty bacterial isolates were obtained, identified at species level, and tested regarding pharmaceutical biodegradation at an initial concentration of 1.5 mg l−1. The results indicated that most probably diclofenac biodegrading cultures consisted of members of genera Ferrovibrio, Hydrocarboniphaga, Zavarzinia, and Sphingopyxis, while in ibuprofen biodegradation Nocardioides and Starkeya, and in carbamazepine biodegradation Nocardioides, Pseudonocardia, and Sphingopyxis might be involved. During the enrichments, compared to the initial state the percentage relative abundance of these genera increased up to three orders of magnitude. Except Starkeya, the genomes of these bacteria were reconstructed and annotated. Metabolic analyses of the annotated genomes indicated that these bacteria harbored genes associated with pharmaceutical biodegradation. Stenotrophomonas humi DIC_5 and Rhizobium daejeonense IBU_18 isolates eliminated diclofenac and ibuprofen during the tests in the presence of either glucose (3 g l−1) or in R2A broth. Higher than 90% concentration reduction was observed in the case of both compounds.