Stable Isotope Probing Research Articles

Anaerobic selenite-reducing bacteria and their metabolic potentials in Se-rich sediment revealed by the combination of DNA-stable isotope probing, metagenomic binning, and metatranscriptomics

Microorganisms play a critical role in the biogeochemical cycling of selenium (Se) in aquatic environments, particularly in reducing the toxicity and bioavailability of selenite (Se(IV)). This study aimed to identify putative Se(IV)-reducing bacteria (SeIVRB) and investigate the genetic mechanisms underlying Se(IV) reduction in anoxic Se-rich sediment. Initial microcosm incubation confirmed that Se(IV) reduction was driven by heterotrophic microorganisms. DNA stable-isotope probing (DNA-SIP) analysis identified Pseudomonas, Geobacter, Comamonas, and Anaeromyxobacter as putative SeIVRB. High-quality metagenome-assembled genomes (MAGs) affiliated with these four putative SeIVRB were retrieved. Annotation of functional gene indicated that these MAGs contained putative Se(IV)-reducing genes such as DMSO reductase family, fumarate and sulfite reductases. Metatranscriptomic analysis of active Se(IV)-reducing cultures revealed significantly higher transcriptional levels of genes associated with DMSO reductase (serA/PHGDH), fumarate reductase (sdhCD/frdCD), and sulfite reductase (cysDIH) compared to those in cultures not amended with Se(IV), suggesting that these genes played important roles in Se(IV) reduction. The current study expands our knowledge of the genetic mechanisms involved in less-understood anaerobic Se(IV) bio-reduction. Additinally, the complementary abilities of DNA-SIP, metagenomics, and metatranscriptomics analyses are demonstrated in elucidating the microbial mechanisms of biogeochemical processes in anoxic sediment.

Advances and perspectives of using stable isotope probing (SIP)-based technologies in contaminant biodegradation

Stable isotope probing (SIP) is a powerful tool to study microbial community structure and function in both nature and engineered environments. Coupling with advanced genomics and other techniques, SIP studies have generated substantial information to allow researchers to draw a clearer picture of what is occurring in complex microbial ecosystems. This review provides an overview of the advances of SIP-based technologies over time, summarizes the status of SIP applications to contaminant biodegradation, provides critical perspectives on ecological interactions within the community, and important factors (controllable and non-controllable) to be considered in SIP experimental designs and data interpretation. Current trend and perspectives of adapting SIP techniques for environmental applications are also discussed.

Increased uptake of deep soil water promotes drought resistance in mixed forests.

The intensity and frequency of droughts are projected to rise in recent years and adversely affect forests. Thus, information on plant water use and acclimation during and after droughts is crucial. This study used the stable isotope and thermal dissipation probes to detect the water-use adaptation of mixed forests to drought using a precipitation gradient control experiment in the field. The results showed that Platycladus orientalis and Quercus variabilis mainly absorbed stable water from deep soil layers during the drought (32.05% and 28.2%, respectively). The synergetic nocturnal sap flow in both species replenished the water loss, but P. orientalis experienced a greater decline in transpiration acclimation to drought. The transpiration of Q. variabilis remained high since it was mainly induced by radiation. After short-term exposure to drought, P. orientalis majorly obtained shallow soil water, confirming its sensitivity to shallow water. Contrarily, Q. variabilis mainly absorbed stable water from deep soil layers regardless of the soil water content. Therefore, these findings suggest that Q. variabilis cannot physiologically adjust to extreme drought events, possibly limiting their future distributions and altering the composition of boreal forests.

Microbial Catabolic Activity: Methods, Pertinence, and Potential Interest for Improving Microbial Inoculant Efficiency.

Microbial catabolic activity (MCA) defined as the degrading activity of microorganisms toward various organic compounds for their growth and energy is commonly used to assess soil microbial function potential. For its measure, several methods are available including multi-substrate-induced respiration (MSIR) measurement which allow to estimate functional diversity using selected carbon substrates targeting specific biochemical pathways. In this review, the techniques used to measure soil MCA are described and compared with respect to their accuracy and practical use. Particularly the efficiency of MSIR-based approaches as soil microbial function indicators was discussed by (i) showing their sensitivity to different agricultural practices including tillage, amendments, and cropping systems and (ii) by investigating their relationship with soil enzyme activities and some soil chemical properties (pH, soil organic carbon, cation exchange capacity). We highlighted the potential of these MSIR-based MCA measurements to improve microbial inoculant composition and to determine their potential effects on soil microbial functions. Finally, we have proposed ideas for improving MCA measurement notably through the use of molecular tools and stable isotope probing which can be combined with classic MSIR methods. Graphical abstract describing the interrelation between the different parts and the concepts developed in the review.

Distinct carbon incorporation from 13C-labelled rice straw into microbial amino sugars in soils applied with manure versus mineral fertilizer

Emerging evidences have emphasized the primacy of microorganisms as executor in soil organic C (SOC) formation. Yet, whether and how microbial transformation of crop residue-derived C into necromass is affected by fertilization regimes remains poorly understood. To address this, soils subjected to 3-year applications of chemical fertilizer (NPK) vs manure plus chemical fertilizer (NPKM) were incubated with or without addition of 13C-labelled rice straw for 60 days. The 13C incorporation into amino sugars (microbial necromass biomarker) was identified by using an amino sugar-based stable isotope probing approach. The straw-C were transformed more rapidly into total amino sugars (13C-ASs) in NPK-treated soil, while more pronounced 13C-ASs accumulation occurred in NPKM soil at later stage, indicating temporal microbial C uptake to generate necromass varied depending on fertilization regimes. Moreover, straw-derived fungal C was significantly higher in NPKM over NPK while bacterial counterpart reversed during the incubation. Our results reinforced changes in microbial communities and their C utilization capacities for efficient residue production in different fertilized soils. Additionally, NPKM-treated soil sustained a prolonged but likely more efficient utilization of straw-C with increasing time as evidenced by significantly higher percentage of straw-dervied C enrichment in 13C amino sugar pools by the end of incubation. These findings reveal that fertilization regimes can alter response strategies of microbial transformation of plant C into microbial-derived C. Together with differential contributions of these newly-formed microbial C to SOC pool, our work highlights the key role of microbial anabolisms in building up SOC following crop residue return to soils under different fertilization regimes.

Iron oxidation coupled with nitrate reduction affects the acetate-assimilating microbial community structure elucidated by stable isotope probing in flooded paddy soil

Acetate is an abundant carbon source that can trigger microbial redox processes in anoxic environments, thereby affecting the microbial community structure, function, and associated element cycling. Here, we investigated the acetate-assimilating microbial community, especially the key microorganisms involved in nitrate-dependent Fe(II) oxidation that are closely associated with soil redox processes in flooded soil. In the present study, DNA-stable isotope probing (DNA-SIP) with labeled acetate (13C) as a carbon source was applied to examine the acetate-assimilating communities associated with nitrate-dependent Fe(II) oxidation. The results showed that NO3− was rapidly reduced in the treatments with NO3− and NO3− + Fe(II). Fe(II) oxidation occurred quickly only in the presence of NO3−. In the treatments with acetate only, the predominant 13C-labeled genera such as Geobacter, Azospira, Azospirillum, Ideonella, and Desulfovibrionia were probably involved in acetate oxidation coupled with the redox processes of NO3−, Fe(III) and SO42− reduction, which are the most important electron acceptors in flooded soils. The addition of NO3− and Fe(II) significantly affected the acetate-assimilating microbial community from the original soil. The enriched genera in 13C heavy fractions were associated with Pseudogulbenkiania, Azospira, Zoogloea, Azoarcus, and Bdellovibrio dominated in the treatments with NO3− and Fe(II). In the treatments with NO3− only, Zoogloea, Azospira, Azoarcus, and Geothrix were the dominant genera. Given the different genera with enrichments in 13C-heavy fractions in different treatments, Zoogloea and Pseudogulbenkiania were identified as key microorganisms associated with NO3− reduction and nitrate-dependent Fe(II) oxidation, respectively. These findings suggest the importance of Zoogloea and Pseudogulbenkiania for C, Fe, and N biogeochemistry and indicate that Fe and N cycling have a great impact on soil biogeochemistry processes.

Dual stable isotopes enhance lipidomic studies in bacterial model organism Enterococcus faecalis.

Dual stable isotope probes ofdeuterium oxide and 13C fatty acid were demonstrated to probe the lipid biosynthesis cycle of a Gram-positive bacterium Enterococcus faecalis. As external nutrients and carbon sources often interact with metabolic processes, the use of dual-labeled isotope pools allowed for the simultaneous investigation of both exogenous nutrient incorporation or modification and de novo biosynthesis. Deuterium was utilized to trace de novo fatty acid biosynthesis through solvent-mediated proton transfer during elongation of the carbon chain while 13C-fatty acids were utilized to trace exogenous nutrient metabolism and modification through lipid synthesis. Ultra-high-performance liquid chromatography high-resolution mass spectrometry identified 30 lipid species which incorporated deuterium and/or 13C fatty acid into the membrane. Additionally, MS2 fragments of isolated lipids identified acyl tail position confirming enzymatic activity of PlsY in the incorporation of the 13C fatty acid into membrane lipids.

Metagenomic stable isotope probing reveals bacteriophage participation in soil carbon cycling.

Soil viruses are important components of the carbon (C) cycle, yet we still know little about viral ecology in soils. We added diverse 13 C-labelled carbon sources to soil and we used metagenomic-SIP to detect 13 C assimilation by viruses and their putative bacterial hosts. These data allowed us to link a 13 C-labelled bacteriophage to its 13 C-labelled Streptomyces putative host, and we used qPCR to track the dynamics of the putative host and phage in response to C inputs. Following C addition, putative host numbers increased rapidly for 3 days, and then more gradually, reaching maximal abundance on Day 6. Viral abundance and virus:host ratio increased dramatically over 6 days, and remained high thereafter (8.42 ± 2.94). From Days 6 to 30, virus:host ratio remained high, while putative host numbers declined more than 50%. Putative host populations were 13 C-labelled on Days 3-30, while 13 C-labelling of phage was detected on Days 14 and 30. This dynamic suggests rapid growth and 13 C-labelling of the host fueled by new C inputs, followed by extensive host mortality driven by phage lysis. These findings indicate that the viral shunt promotes microbial turnover in soil following new C inputs, thereby altering microbial community dynamics, and facilitating soil organic matter production.

Only a minority of bacteria grow after wetting in both natural and post-mining biocrusts in a hyperarid phosphate mine

Abstract. Biological soil crusts (biocrusts) are key contributors to desert ecosystem functions, therefore, biocrust restoration following mechanical disturbances is imperative. In the Negev Desert hyperarid regions, phosphate mining has been practiced for over 60 years, destroying soil habitats and fragmenting the landscape. In this study, we selected one mining site restored in 2007, and we used DNA stable isotope probing (DNA-SIP) to identify which bacteria grow in post-mining and adjacent natural biocrusts. Since biocrust communities activate only after wetting, we incubated the biocrusts with H218O for 96 h under ambient conditions. We then evaluated the physicochemical soil properties, chlorophyll a concentrations, activation, and functional potential of the biocrusts. The DNA-SIP assay revealed low bacterial activity in both plot types and no significant differences in the proliferated communities' composition when comparing post-mining and natural biocrusts. We further found no significant differences in the microbial functional potential, photosynthetic rates, or soil properties. Our results suggest that growth of hyperarid biocrust bacteria after wetting is minimal. We hypothesize that due to the harsh climatic conditions, during wetting, bacteria devote their meager resources to prepare for the coming drought, by focusing on damage repair and organic compound synthesis and storage rather than on growth. These low growth rates contribute to the sluggish recovery of desert biocrusts following major disturbances such as mining. Therefore, our findings highlight the need for implementing active restoration practices following mining.

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.

Bacterial benz(a)anthracene catabolic networks in contaminated soils and their modulation by other co-occurring HMW-PAHs

Polycyclic aromatic hydrocarbons (PAHs) are major environmental pollutants in a number of point source contaminated sites, where they are found embedded in complex mixtures containing different polyaromatic compounds. The application of bioremediation technologies is often constrained by unpredictable end-point concentrations enriched in recalcitrant high molecular weight (HMW)-PAHs. The aim of this study was to elucidate the microbial populations and potential interactions involved in the biodegradation of benz(a)anthracene (BaA) in PAH-contaminated soils. The combination of DNA stable isotope probing (DNA-SIP) and shotgun metagenomics of 13C-labeled DNA identified a member of the recently described genus Immundisolibacter as the key BaA-degrading population. Analysis of the corresponding metagenome assembled genome (MAG) revealed a highly conserved and unique genetic organization in this genus, including novel aromatic ring-hydroxylating dioxygenases (RHD). The influence of other HMW-PAHs on BaA degradation was ascertained in soil microcosms spiked with BaA and fluoranthene (FT), pyrene (PY) or chrysene (CHY) in binary mixtures. The co-occurrence of PAHs resulted in a significant delay in the removal of PAHs that were more resistant to biodegradation, and this delay was associated with relevant microbial interactions. Members of Immundisolibacter, associated with the biodegradation of BaA and CHY, were outcompeted by Sphingobium and Mycobacterium, triggered by the presence of FT and PY, respectively. Our findings highlight that interacting microbial populations modulate the fate of PAHs during the biodegradation of contaminant mixtures in soils.

Hydrogen stable isotope probing of lipids demonstrates slow rates of microbial growth in soil

The rate at which microorganisms grow and reproduce is fundamental to our understanding of microbial physiology and ecology. While soil microbiologists routinely quantify soil microbial biomass levels and the growth rates of individual taxa in culture, there is a limited understanding of how quickly microbes actually grow in soil. For this work, we posed the simple question: what are the growth rates of soil microorganisms? In this study, we measure these rates in three distinct soil environments using hydrogen-stable isotope probing of lipids with 2H-enriched water. This technique provides a taxa-agnostic quantification of in situ microbial growth from the degree of 2H enrichment of intact polar lipid compounds ascribed to bacteria and fungi. We find that growth rates in soil are quite slow and correspond to average generation times of 14 to 45 d but are also highly variable at the compound-specific level (4 to 402 d), suggesting differential growth rates among community subsets. We observe that low-biomass microbial communities exhibit more rapid growth rates than high-biomass communities, highlighting that biomass quantity alone does not predict microbial productivity in soil. Furthermore, within a given soil, the rates at which specific lipids are being synthesized do not relate to their quantity, suggesting a general decoupling of microbial abundance and growth in soil microbiomes. More generally, we demonstrate the utility of lipid-stable isotope probing for measuring microbial growth rates in soil and highlight the importance of measuring growth rates to complement more standard analyses of soil microbial communities.

Ubiquitous occurrence and functional dominance of comammox Nitrospira in full-scale wastewater treatment plants

The recent discovery of complete ammonia oxidation (comammox) bacteria has fundamentally upended the traditional two-step nitrification conception, but their functional importance in wastewater treatment plants (WWTPs) is still poorly understood. This study investigated distributions of comammox Nitrospira, ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in activated sludge samples collected from 25 full-scale WWTPs. Using quantitative PCR (qPCR) and 16S rRNA gene amplicon sequencing, our results revealed that comammox Nitrospira ubiquitously occurred in all of 25 WWTPs and even outnumbered AOB and AOA with an average abundance of 1∼183 orders of magnitude higher in 19 WWTPs. Moreover, DNA-based stable isotope probing (DNA-SIP) assays validated that comammox Nitrospira actively participated in ammonia oxidation in the three microcosms seeding with activated sludge from three typical WWTPs, in which the ratios of comammox amoA to AOB amoA were at the range of 1∼10, 10∼100 and >100, respectively. Phylogenetic analysis in heavy fractions further indicated that Nitrospira nitrosa (N. nitrosa) was the dominant and active species. We quantified the contribution of ammonia oxidizers based on the currently available kinetic parameters of the representative species and found that comammox made major contributions to ammonia oxidation than other nitrifiers (5 ∼ 106 times that of AOB). The findings not only demonstrate the ubiquitous occurrence of comammox, but also highlight their functional dominance in ammonia oxidation in WWTPs.

Identification and synergetic mechanism of TCE, H2 and O2 metabolic microorganisms in the joint H2/O2 system

The sole H2 and O2 usually promote chlorinated hydrocarbons (CHCs) biotransformation by several mechanisms, including reductive dechlorination and aerobic oxidation. However, the mechanism of the CHCs transformation in joint H2 and O2 system (H2/O2 system) is still unclear. In this study, the degradation kinetics of trichloroethene (TCE) were investigated and DNA stable isotope probing (DNA-SIP) were used to explore the synergistic mechanism of functional microorganisms on TCE degradation under the condition of H2/O2 coexistence. In the H2/O2 microcosm, TCE was significantly removed by 13.00 μM within 40 days, much higher than N2, H2 and O2 microcosms, and 1,1-DCE was detected as an intermediate. DNA-SIP technology identified three anaerobic TCE metabolizers, five aerobic TCE metabolizers, nine hydrogen-oxidizing bacteria (HOB), some TCE metabolizers utilizing limited O2, and some anaerobic dechlorinating bacteria reductively using H2 to dechlorinate TCE. It is also confirmed for the first time that 3 OUTs belonging to Methyloversatilis and SH-PL14 can simultaneously utilize H2 and O2 as energy sources to grow and metabolize TCE or 1,1-DCE. HOB may provide carbon sources or electron acceptors or donors for TCE biotransformation. These findings confirm the coexistence of anaerobic and aerobic TCE metabolizers and degraders, which synergistically promoted the conversion of TCE in the joint H2/O2 system. Our results provide more information about the functional microbe resources and synergetic mechanisms for TCE degradation.

New insight into the mechanisms of autochthonous fungal bioaugmentation of phenanthrene in petroleum contaminated soil by stable isotope probing

Autochthonous fungal bioaugmentation (AFB) is considered a reliable bioremediation approach for polycyclic aromatic hydrocarbon (PAH) contamination, but little is known about its mechanisms in contaminated soils. Here, a microcosm experiment was performed to explore the AFB mechanisms associated with two highly efficient phenanthrene degrading agents of fungi (with laccase-producing Scedosporium aurantiacum GIG-3 and non-laccase-producing Aspergillus fumigatus LJD-29), using stable-isotope-probing (SIP) and high-throughput sequencing. The results showed that each fungus markedly improved phenanthrene removal, and microcosms with both fungi exhibited the best phenanthrene removal performance among all microcosms. Additionally, AFB markedly shifted the composition of the microbial community, particularly the phenanthrene-degrading bacterial taxa. Interestingly, based on SIP results, strains GIG-3 and LJD-29 did not assimilate phenanthrene directly during AFB, but instead played key roles in the preliminary decomposition of phenanthrene though secretion of different extracellular enzymes to oxidize the benzene ring (GIG-3 bioaugmentation with laccase, and LJD-29 bioaugmentation with manganese and lignin peroxidases). In addition, all functional degraders directly involved in phenanthrene assimilation were indigenous bacteria, while native fungi rarely participated in the direct phenanthrene mineralization. Our findings provide a new mechanism of AFB with multiple fungi, and support AFB as a promising strategy for the in situ bioremediation of PAH-contaminated soil.

Determination of carbon-fixing potential of Bathyarchaeota in marine sediment by DNA stable isotope probing analysis

Determination of carbon-fixing potential of Bathyarchaeota in marine sediment by DNA stable isotope probing analysis

Hyperactive nanobacteria with host-dependent traits pervade Omnitrophota

Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 μm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.

Thrive or survive: prokaryotic life in hypersaline soils

BackgroundSoil services are central to life on the planet, with microorganisms as their main drivers. Thus, the evaluation of soil quality requires an understanding of the principles and factors governing microbial dynamics within it. High salt content is a constraint for life affecting more than 900 million hectares of land, a number predicted to rise at an alarming rate due to changing climate. Nevertheless, little is known about how microbial life unfolds in these habitats. In this study, DNA stable-isotope probing (DNA-SIP) with 18O-water was used to determine for the first time the taxa able to grow in hypersaline soil samples (ECe = 97.02 dS/m). We further evaluated the role of light on prokaryotes growth in this habitat.ResultsWe detected growth of both archaea and bacteria, with taxon-specific growth patterns providing insights into the drivers of success in saline soils. Phylotypes related to extreme halophiles, including haloarchaea and Salinibacter, which share an energetically efficient mechanism for salt adaptation (salt-in strategy), dominated the active community. Bacteria related to moderately halophilic and halotolerant taxa, such as Staphylococcus, Aliifodinibius, Bradymonadales or Chitinophagales also grew during the incubations, but they incorporated less heavy isotope. Light did not stimulate prokaryotic photosynthesis but instead restricted the growth of most bacteria and reduced the diversity of archaea that grew.ConclusionsThe results of this study suggest that life in saline soils is energetically expensive and that soil heterogeneity and traits such as exopolysaccharide production or predation may support growth in hypersaline soils. The contribution of phototrophy to supporting the heterotrophic community in saline soils remains unclear. This study paves the way toward a more comprehensive understanding of the functioning of these environments, which is fundamental to their management. Furthermore, it illustrates the potential of further research in saline soils to deepen our understanding of the effect of salinity on microbial communities.

Characterization of methanotrophic community and activity in landfill cover soils under dimethyl sulfide stress

Landfill cover soil is the environmental interface between landfills and the atmosphere and plays an important role in mitigating CH4 emission from landfills. Here, stable isotope probing microcosms with CH4 or CH4 and dimethyl sulfide (DMS) were carried out to characterize activity and community structure of methanotrophs in landfill cover soils under DMS stress. The CH4 oxidation activity in the landfill cover soils was not obviously influenced at the DMS concentration of 0.05%, while it was inhibited at the DMS concentrations of 0.1% and 0.2%. DMS-S was mainly oxidized to sulfate (SO42-) in the landfill cover soils. In the landfill cover soils, DMS could inhibit the expression of bacteria and decrease the abundances of pmoA and mmoX genes, while it could prompt the expression of pmoA and mmoX genes. γ-Proteobacteria methanotrophs including Methylocaldum, Methylobacter, Crenothrix and unclassified Methylococcaceae and α-Proteobacteria methanotrophs Methylocystis dominated in assimilating CH4 in the landfill cover soils. Of them, Methylobacter and Crenothrix had strong tolerance to DMS or DMS could promote the growth and activity of Methylobacter and Crenothrix, while Methylocaldum had weak tolerance to DMS and showed an inhibitory effect. Metagenomic analyses showed that methanotrophs had the genes of methanethiol oxidation and could metabolize CH4 and methanethiol simultaneously in the landfill cover soils. These findings suggested that methanotrophs might metabolize sulfur compounds in the landfill cover soils, which may provide the potential application in engineering for co-removal of CH4 and sulfur compounds.

The contribution shift of ammonia-oxidizing archaea and bacteria to ammoxidation under Ag-NPs/SWCNTs/PS-NPs stressors in constructed wetlands

Ammonia-oxidizing microorganisms (AOMs, archaea (AOA) and bacteria (AOB)) are primarily responsible for the ammoxidation in constructed wetlands (CWs). However, little is known about evaluating the response of AOA and AOB to engineered nanomaterials (ENMs) and quantifying the shift of their contribution to ammoxidation. Herein, we operated a series of CWs exposing to silver nanoparticles (Ag-NPs), single-walled carbon nanotubes (SWCNTs), and polystyrene nano-sized plastics (PS-NPs) with the wastewater-accumulating concentration of ENMs for 180 days. The results showed that the abundance of AOA amoA gene in situ was far lower than that of AOB, while the abundance ratio of AOA to AOB increased by 15 folds after 180-day experiment. Using DNA stable isotope probing (DNA-SIP) experiment, we found that the active AOB microbiota varied substantially but the AOA was more stable across different groups. Furthermore, the co-occurrence analysis proved that ENMs stress increased the negative coexistence pattern of AOA and AOB; predictive functional profiling showed that the ENMs enhanced the functional advantage of AOA by inhibiting AOB (mainly hydroxylamine oxidation process). Finally, the contribution of AOA increased under exposing to SWCNTs (18.35%), PS-NPs (24.92%), and Ag-NPs (32.14%) compared with control group (0.03%) for 180 days. Despite this, AOB was still the primary executant of ammoxidation in CWs. Overall, in our study, the differences in activities and contributions of AOMs were quantified in CWs, and a significantly negative coexistence relationship between AOA and AOB was revealed when exposed to emerging nanomaterials.