Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community.

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10 mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

Identification of bacteria and fungi responsible for litter decomposition in desert steppes via combined DNA stable isotope probing.

Introduction: Soil microorganisms play crucial roles in determining the fate of litter in desert steppes because their activities constitute a major component of the global carbon (C) cycle. Human activities lead to increased ecosystem nitrogen (N) deposition, which has unpredictable impacts on soil microorganism diversity and functions. Nowadays, it is necessary to further study the succession of these microorganisms in the process of litter decomposition in desert steppe, and explore the effect of N deposition on this process. This issue is particularly important to resolve because it contributes to the broader understanding of nutrient cycling processes in desert steppes. Methods: In this study, DNA stable isotope probing (DNA-SIP) was used to study changes in soil bacterial and fungal community composition and function during 8 weeks of culture of 13C-labeled litter in desert steppes. Results: The results were as follows: (1) Actinomycetota, Pseudomonadota, and Ascomycota are the main microorganisms involved in litter decomposition in desert steppes; (2) N deposition (50 kg ha-1 year-1) significantly increased the relative abundance of some microorganisms involved in the decomposition process; and (3) N deposition likely promotes litter decomposition in desert steppes by increasing the abundances of N cycles bacteria (usually carrying GH family functional genes). Discussion: These findings contribute to a deeper understanding of the C assimilation mechanisms associated with litter residue production, emphasizing the importance of extensive C utilization.

New insights into bioaugmented removal of sulfamethoxazole in sediment microcosms: degradation efficiency, ecological risk and microbial mechanisms.

BACKGROUND: Bioaugmentation has the potential to enhance the ability of ecological technology to treat sulfonamide-containing wastewater, but the low viability of the exogenous degraders limits their practical application. Understanding the mechanism is important to enhance and optimize performance of the bioaugmentation, which requires a multifaceted analysis of the microbial communities. Here, DNA-stable isotope probing (DNA-SIP) and metagenomic analysis were conducted to decipher the bioaugmentation mechanisms in stabilization pond sediment microcosms inoculated with sulfamethoxazole (SMX)-degrading bacteria (Pseudomonas sp. M2 or Paenarthrobacter sp. R1). RESULTS: The bioaugmentation with both strains M2 and R1, especially strain R1, significantly improved the biodegradation rate of SMX, and its biodegradation capacity was sustainable within a certain cycle (subjected to three repeated SMX additions). The removal strategy using exogenous degrading bacteria also significantly abated the accumulation and transmission risk of antibiotic resistance genes (ARGs). Strain M2 inoculation significantly lowered bacterial diversity and altered the sediment bacterial community, while strain R1 inoculation had a slight effect on the bacterial community and was closely associated with indigenous microorganisms. Paenarthrobacter was identified as the primary SMX-assimilating bacteria in both bioaugmentation systems based on DNA-SIP analysis. Combining genomic information with pure culture evidence, strain R1 enhanced SMX removal by directly participating in SMX degradation, while strain M2 did it by both participating in SMX degradation and stimulating SMX-degrading activity of indigenous microorganisms (Paenarthrobacter) in the community. CONCLUSIONS: Our findings demonstrate that bioaugmentation using SMX-degrading bacteria was a feasible strategy for SMX clean-up in terms of the degradation efficiency of SMX, the risk of ARG transmission, as well as the impact on the bacterial community, and the advantage of bioaugmentation with Paenarthrobacter sp. R1 was also highlighted. Video Abstract.

Identification of functional microflora underlying the biodegradation of sulfadiazine-contaminated substrates by Hermetia illucens.

The increasing discharge of antibiotic residues into the natural environment, stemming from both human activities and animal farming, has detrimental effects on natural ecosystems and serves as a significant driving force for the spread of antibiotic resistance. Biodegradation is an important method for the elimination of antibiotics from contaminated substrates, but the identifying in situ microbial populations involved in antibiotic degradation is challenging. Here, DNA stable isotope probing (DNA-SIP) was employed to identify active sulfadiazine (SDZ) degrading microbes in the gut of black soldier fly larvae (BSFLs). At an initial SDZ concentration of 100 mg kg-1, the highest degradation efficiency reached 73.99% after 6 days at 28 °C. DNA-SIP revealed the incorporation of 13C6 from labeled SDZ in 9 genera, namely, Clostridum sensu stricto 1, Nesterenkonia, Bacillus, Halomonas, Dysgonomonas, Caldalkalibacillus, Enterococcus, g_unclassified_f_Xanthomonadaceae and g_unclassified_f_Micrococcaceae. Co-occurrence network analysis revealed that a significant positive correlation existed among SDZ degrading microbes in the gut microbiota, e.g., between Clostridium sensu stricto 1 and Nesterenkonia. Significant increases in carbohydrate metabolism, membrane transport and translation were crucial in the biodegradation of SDZ in the BSFL gut. These results elucidate the structure of SDZ-degrading microbial communities in the BSFL gut and in situ degradation mechanisms.

Autotrophic growth activity of complete ammonia oxidizers in an upflow biological contact filter for drinking water treatment.

Biological filters effectively remove ammonium from drinking water via nitrification. In a pilot-scale upflow biological contact filter (U-BCF), complete ammonia oxidizers (comammox), which are capable of oxidizing ammonia to nitrate in one cell, were more abundant than ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, little information is available on the contribution of comammox to nitrification. In this study, we evaluated the autotrophic growth activity of comammox associated with biological activated carbon (BAC) in a U-BCF by DNA-stable isotope probing (DNA-SIP). BAC samples collected from the U-BCF were continuously fed mineral medium containing 0.14 mg N L-1 ammonium and 12C- or 13C-labeled bicarbonate for 20 days. DNA-SIP analysis revealed that comammox (clades A and B) as well as AOA assimilated bicarbonate after 10 days of incubation, proving that dominant comammox could contribute to nitrification. Contrarily, AOB remained inactive throughout the observation period. Amplicon sequencing of the 13C-labeled DNA fractions of comammox revealed that specific genotypes other than the most dominant genotype in the original sample were more enriched under the incubation condition for the DNA-SIP experiment. Thus, dominant genotypes of comammox in a U-BCF might utilize organic nitrogen to fuel nitrification in ammonia-limited environments.

New mechanisms of biochar-assisted vermicomposting by recognizing different active di-(2-ethylhexyl) phthalate (DEHP) degraders across pedosphere, charosphere and intestinal sphere.

Biochar-assisted vermicomposting can significantly accelerate soil DEHP degradation, but little information is known about the underlying mechanisms as different microspheres exist in soil ecosystem. In this study, we identified the active DEHP degraders in biochar-assisted vermicomposting by DNA stable isotope probing (DNA-SIP) and surprisingly found their different compositions in pedosphere, charosphere and intestinal sphere. Thirteen bacterial lineages (Laceyella, Microvirga, Sphingomonas, Ensifer, Skermanella, Lysobacter, Archangium, Intrasporangiaceae, Pseudarthrobacter, Blastococcus, Streptomyces, Nocardioides and Gemmatimonadetes) were responsible for in situ DEHP degradation in pedosphere, whereas their abundance significantly changed in biochar or earthworm treatments. Instead, some other active DEHP degraders were identified in charosphere (Serratia marcescens and Micromonospora) and intestinal sphere (Clostridiaceae, Oceanobacillus, Acidobacteria, Serratia marcescens and Acinetobacter) with high abundance. In biochar-assisted vermicomposting, the majority of active DEHP degraders were found in charosphere, followed by intestinal sphere and pedosphere. Our findings for the first time unraveled the spatial distribution of active DEHP degraders in different microspheres in soil matrices, explained by DEHP dynamic adsorption on biochar and desorption in earthworm gut. Our work highlighted that charosphere and intestinal sphere exhibited more contribution to the accelerated DEHP biodegradation than pedosphere, providing novel insight into the mechanisms of biochar and earthworm in improving contaminant degradation.

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.

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

BACKGROUND: We 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. RESULTS: An 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. CONCLUSIONS: The 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.

Anammox bacterial abundance and diversity in different temperatures of purple paddy soils by 13C-DNA stable-isotope probing combined with high-throughput sequencing.

Introduction: Anaerobic ammonium oxidation (anammox) plays a vital role in the global nitrogen cycle by oxidizing ammonium to nitrogen under anaerobic environments. However, the existence, abundance, and diversity of anammox bacteria between different temperatures are less studied, particularly in purple paddy soils. Methods: 13C-DNA stable-isotope probe combined with Illumina MiSeq high-throughput sequencing was employed to explore soil abundance and diversity of anammox bacteria. In doing so, 40-60 cm depth soils from typical purple paddy soils in Chongqing, southwest China, were cultured under 12CO2-labeled and 13CO2-labeled at 35°C, 25°C, 15°C, and 5°C for 56 days. Results and Discussion: Anammox bacteria were not labeled at all by 13CO2 at 5°C. The highest abundance of anammox bacteria was found at 25°C (3.52 × 106~3.66 × 106 copies·g-1 dry soil), followed by 35°C and 15°C (2.01 × 106~2.37 × 106 copies·g-1 dry soil) and almost no increase at 5°C. The relative abundance of Candidatus Jettenia sp. was higher at 25°C and 15°C, while Candidatus Brocadia sp. was higher at 35°C and 5°C. Our results revealed differences in anammox bacteria at different temperatures in purple paddy soils, which could provide a better understanding of soil N cycling regulated by anammox bacteria.

Autotoxin affects the rhizosphere microbial community structure by influencing the secretory characteristics of grapevine roots.

Autotoxins secreted by roots into the soil can trigger rhizosphere microecological imbalances and affect root secretory properties resulting in conditions such as replanting disease. However, information on the effect of autotoxins on root secretion characteristics and regulation of the composition of rhizosphere microorganisms by altered root exudates is limited. In this study, autotoxin ρ-hydroxybenzoic acid (4-HBA) was added to the soil of potted grapevine seedlings, CO2 pulse-labeling, and DNA stable isotope probing were used to track the rhizosphere microbiome that assimilates root exudates. Bacterial and fungal microbiomes that assimilated plant-derived carbon were identified by high-throughput sequencing. Results showed that 4-HBA treatment altered bacterial and fungal communities in 13C-labeled organisms, with a lower abundance of beneficial bacteria (e.g., Gemmatimonas, Streptomyces, and Bacillus) and a higher abundance of potential pathogen fungi (e.g., Fusarium, Neocosmospora, Gibberella, and Fusicolla) by changing the composition of root exudates. The exogenous addition of upregulated compound mixtures of root exudates reduced the abundance of beneficial bacterial Bacillus and increased the abundance of potential pathogen fungi Gibberella. These results suggest that 4-HBA can alter root secretion properties and altered root exudates may enrich certain potential pathogens and reduce certain beneficial bacteria, thereby unbalancing the structure of the rhizosphere microbial community.

Oceanospirillales containing the DMSP lyase DddD are key utilisers of carbon from DMSP in coastal seawater.

BACKGROUND: Ubiquitous and diverse marine microorganisms utilise the abundant organosulfur molecule dimethylsulfoniopropionate (DMSP), the main precursor of the climate-active gas dimethylsulfide (DMS), as a source of carbon, sulfur and/or signalling molecules. However, it is currently difficult to discern which microbes actively catabolise DMSP in the environment, why they do so and the pathways used. RESULTS: Here, a novel DNA-stable isotope probing (SIP) approach, where only the propionate and not the DMS moiety of DMSP was 13C-labelled, was strategically applied to identify key microorganisms actively using DMSP and also likely DMS as a carbon source, and their catabolic enzymes, in North Sea water. Metagenomic analysis of natural seawater suggested that Rhodobacterales (Roseobacter group) and SAR11 bacteria were the major microorganisms degrading DMSP via demethylation and, to a lesser extent, DddP-driven DMSP lysis pathways. However, neither Rhodobacterales and SAR11 bacteria nor their DMSP catabolic genes were prominently labelled in DNA-SIP experiments, suggesting they use DMSP as a sulfur source and/or in signalling pathways, and not primarily for carbon requirements. Instead, DNA-SIP identified gammaproteobacterial Oceanospirillales, e.g. Amphritea, and their DMSP lyase DddD as the dominant microorganisms/enzymes using DMSP as a carbon source. Supporting this, most gammaproteobacterial (with DddD) but few alphaproteobacterial seawater isolates grew on DMSP as sole carbon source and produced DMS. Furthermore, our DNA-SIP strategy also identified Methylophaga and other Piscirickettsiaceae as key bacteria likely using the DMS, generated from DMSP lysis, as a carbon source. CONCLUSIONS: This is the first study to use DNA-SIP with 13C-labelled DMSP and, in a novel way, it identifies the dominant microbes utilising DMSP and DMS as carbon sources. It highlights that whilst metagenomic analyses of marine environments can predict microorganisms/genes that degrade DMSP and DMS based on their abundance, it cannot disentangle those using these important organosulfur compounds for their carbon requirements. Note, the most abundant DMSP degraders, e.g. Rhodobacterales with DmdA, are not always the key microorganisms using DMSP for carbon and releasing DMS, which in this coastal system were Oceanospirillales containing DddD. Video abstract.

New insight into the mechanism underlying the effect of biochar on phenanthrene degradation in contaminated soil revealed through DNA-SIP.

Biochar has been widely used for the remediation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil, but its mechanism of influencing PAH biodegradation remains unclear. Here, DNA-stable isotope probing coupled with high-throughput sequencing was employed to assess its influence on phenanthrene (PHE) degradation, the active PHE-degrading microbial community and PAH-degradation genes (PAH-RHDα). Our results show that both Low-BC and High-BC (soils amended with 1 % and 4 % w/w biochar, respectively) treatments significantly decreased PHE biodegradation and bioavailable concentrations with a dose-dependent effect compared to Non-BC treatment (soils without biochar). This result could be attributed to the immobilisation of PHE and alteration of the composition and abundance of the PHE-degrading microbial consortium by biochar. Active PHE degraders were identified, and those in the Non-BC, Low-BC and High-BC microcosms differed taxonomically. Sphaerobacter, unclassified Diplorickettsiaceae, Pseudonocardia, and Planctomyces were firstly linked with PHE biodegradation. Most importantly, the abundances of PHE degraders and PAH-RHDα genes in the 13C-enriched DNA fractions of biochar-amended soils were greatly attenuated, and were significantly positively correlated with PHE biodegradation. Our findings provide a novel perspective on PAH biodegradation mechanisms in biochar-treated soils, and expand the understanding of the biodiversity of microbes involved in PAH biodegradation in the natural environment.

Genetic bioaugmentation with triclocarban-catabolic plasmid effectively removes triclocarban from wastewater.

Triclocarban, one of the emerging pollutants, has been accumulating, and it is frequently detected in wastewater. Due to its toxicity and persistence, the efficient removal of triclocarban from wastewater systems is challenging. Genetic bioaugmentation with transferable catabolic plasmids has been considered to be a long-lasting method to clean up pollutants in continuous flow wastewater treatment systems. In this study, bioaugmentation with Pseudomonas putida KT2440, harboring the transferrable triclocarban-catabolic plasmid pDCA-1-gfp-tccA2, rapidly converted 50 µM triclocarban in wastewater into 3,4-dichloroaniline and 4-chloroaniline, which are further mineralized more easily. RT-qPCR results showed that the ratio of the copy number of pDCA-1-gfp-tccA2 to the cell number of strain KT2440 gradually increased during genetic bioaugmentation, suggesting horizontal transfer and proliferation of the plasmid. By using DNA stable isotope probing (SIP) and amplicon sequencing, OTU86 (Escherichia-Shigella), OTU155 (Citrobacter), OTU5 (Brucella), and OTU15 (Enterobacteriaceae) were found to be the potential recipients of the plasmid pDCA-1-gfp-tccA2 in the wastewater bacterial community. Furthermore, three transconjugants in the genera of Escherichia, Citrobacter, and Brucella showing triclocarban-degrading abilities were isolated from the wastewater. This study develops a new method for removing triclocarban from wastewater and provides insights into the environmental behavior of transferrable catabolic plasmids in bacterial community in wastewater systems.

Sulfonamide-metabolizing microorganisms and mechanisms in antibiotic-contaminated wetland sediments revealed by stable isotope probing and metagenomics.

Sulfonamide (SA) antibiotics are ubiquitous pollutants in livestock breeding and aquaculture wastewaters, which increases the propagation of antibiotic resistance genes. Microbes with the ability to degrade SA play important roles in SA dissipation, but their diversity and the degradation mechanism in the field remain unclear. In the present study, we employed DNA-stable isotope probing (SIP) combined with metagenomics to explore the active microorganisms and mechanisms of SA biodegradation in antibiotic-contaminated wetland sediments. DNA-SIP revealed various SA-assimilating bacteria dominated by members of Proteobacteria, such as Bradyrhizobium, Gemmatimonas, and unclassified Burkholderiaceae. Both sulfadiazine and sulfamethoxazole were dissipated mainly through the initial ipso-hydroxylation, and were driven by similar microbes. sadA gene, which encodes an NADH-dependent monooxygenase, was enriched in the 13C heavy DNA, confirming its catalytic capacity for the initial ipso-hydroxylation of SA in sediments. In addition, some genes encoding dioxygenases were also proposed to participate in SA hydroxylation and aromatic ring cleavage based on metagenomics analysis, which might play an important role in SA metabolism in the sediment ecosystem when Proteobacteria was the dominant active bacteria. Our work elucidates the ecological roles of uncultured microorganisms in their natural habitats and gives a deeper understanding of in-situ SA biodegradation mechanisms.

DNA-based stable isotope probing deciphered the active denitrifying bacteria and triclosan-degrading bacteria participating in granule-based partial denitrification process under triclosan pressure.

Granule-based partial denitrification (PD) is a technology that can supply stable nitrite for applying anaerobic ammonia oxidation in wastewater treatment, and triclosan (TCS) is a frequently detected antibacterial agent in wastewater treatment plants, therefore it is possible that TCS could enter into wastewater that is treated using PD technology. However, the active microorganisms responsible for PD and TCS removing in granule-based PD system have not been clearly identified and it is currently not clear how TCS affects the PD process. In this study, the impacts of TCS on PD performance, PD microbial community, antibiotic resistance genes (ARGs), active PD bacteria and TCS-degrading bacteria in a granule-based PD system were investigated. 3 mg/L TCS had adverse influence on PD process, but PD system could recover gradually after inhibiting of 10 days. After a period of domestication, PD granular sludge could achieve 10.66% of TCS degradation efficiency and 43.62% of TCS adsorption efficiency. Microbes might increase their resistance to TCS by increasing the secretion of extracellular polymeric substances, and the secretion of protein might play a more pivotal role than the secretion of polysaccharides in resisting TCS. The short-term shock of TCS might cause the propagation of acrA-03, while the long-term operation of TCS could propagate fabK and intI1. DNA stable isotope probing assay indicated that Thauera was active PD bacteria and TCS-degrading bacteria in the granule-based PD system, and it could contribute to nitrite accumulation and TCS degradation, simultaneously.

Metagenomics Combined with Stable Isotope Probe (SIP) for the Discovery of Novel Dehalogenases Producing Bacteria.

Halogenated compounds are one of the largest groups of environmental-hazardous chemicals. The removal of the halogen atom from the substrate is possible by the catalytic activity of a type of enzyme called dehalogenase. Hydrolytic dehalogenases are suggested to be a good biodegradation catalyst for halogenated compounds with potential bioremediation applications. Therefore, the identification of possible bacterial strains that produce dehalogenase is of great importance. Soil microorganisms that are regularly exposed to halogenated pesticides are a major source of hydrolytic dehalogenase. Their proper identification may be useful in the production of high-quality dehalogenase. DNA stable isotope probing (DNA-SIP) is quite a useful technique for the identification of active microorganisms that assimilate specific carbon substrates and nutrients. Metagenomics combined with a stable isotope probe (SIP) technique could therefore be used to detect bacterial dehalogenases in pesticides exposed agricultural soil.

Mechanism of salicylic acid in promoting the rhizosphere benzo[a]pyrene biodegradation as revealed by DNA-stable isotope probing.

Benzo[a]pyrene (BaP) is a typical high-molecular-weight PAH with carcinogenicity. Rhizoremediation is commonly applied to remove soil BaP, but its mechanism remains unclear. The role of inducers in root exudates in BaP rhizoremediation is rarely studied. Here, to address this problem, we firstly investigated the effect of the inducer salicylic acid on BaP rhizoremediation, rhizosphere BaP degraders, and PAH degradation-related genes by combining DNA-stable-isotope-probing, high-throughput sequencing, and gene function prediction. BaP removal in the rhizosphere was significantly increased by stimulation with salicylic acid, and the rhizosphere BaP-degrading microbial community structure was significantly changed. Fourteen microbes were responsible for the BaP metabolism, and most degraders, e.g. Aeromicrobium and Myceligenerans, were firstly linked with BaP biodegradation. The enrichment of the PAH-ring hydroxylating dioxygenase (PAH-RHD) gene in the heavy fractions of all 13C-treatments further indicated their involvement in the BaP biodegradation, which was also confirmed by the enrichment of dominant PAH degradation-related genes (e.g. PAH dioxygenase and protocatechuate 3,4-dioxygenase genes) based on gene function prediction. Overall, our study demonstrates that salicylic acid can enhance the rhizosphere BaP biodegradation by altering the community structure of rhizosphere BaP-degrading bacteria and the abundance of PAH degradation-related genes, which provides new insights into BaP rhizoremediation mechanisms in petroleum-contaminated sites.

Diversity and Metabolic Potentials of As(III)-Oxidizing Bacteria in Activated Sludge.

Biological arsenite [As(III)] oxidation is an important process in the removal of toxic arsenic (As) from contaminated water. However, the diversity and metabolic potentials of As(III)-oxidizing bacteria (AOB) responsible for As(III) oxidation in wastewater treatment facilities are not well documented. In this study, two groups of bioreactors inoculated with activated sludge were operated under anoxic or oxic conditions to treat As-containing synthetic wastewater. Batch tests of inoculated sludges from the bioreactors further indicated that microorganisms could use nitrate or oxygen as electron acceptors to stimulate biological As(III) oxidation, suggesting the potentials of this process in wastewater treatment facilities. In addition, DNA-based stable isotope probing (DNA-SIP) was performed to identify the putative AOB in the activated sludge. Bacteria associated with Thiobacillus were identified as nitrate-dependent AOB, while bacteria associated with Hydrogenophaga were identified as aerobic AOB in activated sludge. Metagenomic binning reconstructed a number of high-quality metagenome-assembled genomes (MAGs) associated with the putative AOB. Functional genes encoding As resistance, As(III) oxidation, denitrification, and carbon fixation were identified in these MAGs, suggesting their potentials for chemoautotrophic As(III) oxidation. In addition, the presence of genes encoding secondary metabolite biosynthesis and extracellular polymeric substance metabolism in these MAGs may facilitate the proliferation of these AOB in activated sludge and enhance their capacity for As(III) oxidation. IMPORTANCE AOB play an important role in the removal of toxic arsenic from wastewater. Most of the AOB have been isolated from natural environments. However, knowledge regarding the structure and functional roles of As(III)-oxidizing communities in wastewater treatment facilities is not well documented. The combination of DNA-SIP and metagenomic binning provides an opportunity to elucidate the diversity of in situ AOB community inhabiting the activated sludges. In this study, the putative AOB responsible for As(III) oxidation in wastewater treatment facilities were identified, and their metabolic potentials, including As(III) oxidation, denitrification, carbon fixation, secondary metabolite biosynthesis, and extracellular polymeric substance metabolism, were investigated. This observation provides an understanding of anoxic and/or oxic AOB during the As(III) oxidation process in wastewater treatment facilities, which may contribute to the removal of As from contaminated water.

A Synergistic Consortium Involved in rac-Dichlorprop Degradation as Revealed by DNA Stable Isotope Probing and Metagenomic Analysis.

rac-Dichlorprop, a commonly used phenoxyalkanoic acid herbicide, is frequently detected in environments and poses threats to environmental safety and human health. Microbial consortia are thought to play key roles in rac-dichlorprop degradation. However, the compositions of the microbial consortia involved in rac-dichlorprop degradation remain largely unknown. In this study, DNA stable isotope probing (SIP) and metagenomic analysis were integrated to reveal the key microbial consortium responsible for rac-dichlorprop degradation in a rac-dichlorprop-degrading enrichment. OTU340 (Sphingobium sp.) and OTU348 (Sphingopyxis sp.) were significantly enriched in the rac-[13C]dichlorprop-labeled heavy DNA fractions. A rac-dichlorprop degrader, Sphingobium sp. strain L3, was isolated from the enrichment by a traditional enrichment method but with additional supplementation of the antibiotic ciprofloxacin, which was instructed by metagenomic analysis of the associations between rac-dichlorprop degraders and antibiotic resistance genes. As revealed by functional profiling of the metagenomes of the heavy DNA, the genes rdpA and sdpA, involved in the initial degradation of the (R)- and (S)-enantiomers of dichlorprop, respectively, were mostly taxonomically assigned to Sphingobium species, indicating that Sphingopyxis species might harbor novel dichlorprop-degrading genes. In addition, taxonomically diverse bacterial genera such as Dyella, Sphingomonas, Pseudomonas, and Achromobacter were presumed to synergistically cooperate with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. IMPORTANCE Understanding of the key microbial consortium involved in the degradation of the phenoxyalkanoic acid herbicide rac-dichlorprop is pivotal for design of synergistic consortia used for enhanced bioremediation of herbicide-contaminated sites. However, the composition of the microbial consortium and the interactions between community members during the biodegradation of rac-dichlorprop are unclear. In this study, DNA-SIP and metagenomic analysis were integrated to reveal that the metabolite 2,4-dichlorophenol degraders Dyella, Sphingomonas, Pseudomonas, and Achromobacter synergistically cooperated with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. Our study provides new insights into the synergistic degradation of rac-dichlorprop at the community level and implies the existence of novel degrading genes for rac-dichlorprop in nature.