DNA-based Stable Isotope Probing Research Articles

Deciphering the active bacteria involving glucose-triggered priming effect in soils with gradient N inputs

The soil priming effect, which refers to the alteration of soil organic matter (SOM) decomposition due to labile carbon (C) inputs, is widely acknowledged for its impact on C storage in terrestrial ecosystems. However, the impact of chronic nitrogen (N) fertilizer on soil priming effect, particularly in agroecological systems, remains unclear. Here, we utilized soils subjected to varying levels of N fertilization (0, 140, 280, 470, and 660 kg N ha−1 y−1), which were collected from a long-term experimental site. Enzyme activity related to C, N, and phosphorus (P) acquisition was measured using the fluorometric method. Additionally, DNA-based stable isotope probing with 13C-labeled glucose was conducted to explore the role of active bacterial communities (16S rRNA gene analysis) on the priming effect in soils with different N fertilization histories. Glucose addition enhanced the decomposition of native SOM and induced positive priming effects in all soils, which were amplified by the historical N application level. Activity of C-related enzymes essential for soil C decomposition increased following glucose addition, which was positively correlated with the soil priming effect. Active bacterial taxa, primarily Firmicutes, Actinobacteria, and Proteobacteria, were capable of assimilating exogenous glucose-C or native SOM-C. Notably, bacteria assimilating glucose exhibited higher abundance-weighted average ribosomal RNA gene operon copy number than those assimilating SOM, indicating the role of r-strategists in accelerating SOC turnover and increasing C loss. These findings highlight the role of active microbial community attributes on the soil priming effects. This study provides new insights into the intricate processes of C transformation in soils subjected to long-term N management in agroecosystems.

Methane-Driven Perchlorate Reduction by a Microbial Consortium.

The phenomenon of methane oxidation linked to perchlorate reduction has been reported in multiple studies; yet, the underlying microbial mechanisms remain unclear. Here, we enriched suspended cultures by performing methane-driven perchlorate reduction under oxygen-limiting conditions in a membrane bioreactor (MBR). Batch test results proved that perchlorate reduction was coupled to methane oxidation, in which acetate was predicted as the potential intermediate and oxygen played an essential role in activating methane. By combining DNA-based stable isotope probing incubation and high-throughput sequencing analyses of 16S rRNA gene and functional genes (pmoA, pcrA, and narG), we found that synergistic interactions between aerobic methanotrophs (Methylococcus and Methylocystis) and perchlorate-reducing bacteria (PRB; Denitratisoma and Dechloromonas) played active roles in mediating methane-driven perchlorate reduction. This partnership was further demonstrated by coculture experiments in which the aerobic methanotroph could produce acetate to support PRB to complete perchlorate reduction. Our findings advance the understanding of the methane-driven perchlorate reduction process and have implications for similar microbial consortia linking methane and chlorine biogeochemical cycles in natural environments.

The effect of the 13C abundance of soil microbial DNA on identifying labelled fractions after ultracentrifugation

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP.Key points• Appropriate 13C-DNA amount was needed for DNA-SIP.• Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions.• Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled.

The significant contribution of comammox bacteria to nitrification in a constructed wetland revealed by DNA-based stable isotope probing

The discovery of Comammox bacteria (CMX) has changed our traditional concept towards nitrification, yet its role in constructed wetlands (CWs) remains unclear. This study investigated the contributions of CMX and two canonical ammonia-oxidizing microorganisms, ammonia-oxidizing bacteria (AOB) and archaea to nitrification in four regions (sediment, shoreside, adjacent soil, and water) of a typical CW using DNA-based stable isotope probing. The results revealed that CMX not only widely occurred in sediment and shoreside zones with high abundance (5.08 × 104 and 6.57 × 104 copies g−1 soil, respectively), but also actively participated in ammonia oxidation, achieving ammonia oxidation rates of 1.43 and 2.00 times that of AOB in sediment and shoreside, respectively. Phylogenetic analysis indicated that N. nitrosa was the dominant and active CMX species. These findings uncovered the crucial role of CMX in nitrification of sediment and shoreside, providing a new insight into nitrogen cycle of constructed wetlands.

Identification of the bacterial community that degrades phenanthrene sorbed to polystyrene nanoplastics using DNA-based stable isotope probing

In the Anthropocene, plastic pollution has become a new environmental biotope, the so-called plastisphere. In the oceans, nano- and micro-sized plastics are omnipresent and found in huge quantities throughout the water column and sediment, and their large surface area-to-volume ratio offers an excellent surface to which hydrophobic chemical pollutants (e.g. petrochemicals and POPs) can readily sorb to. Our understanding of the microbial communities that breakdown plastic-sorbed chemical pollutants, however, remains poor. Here, we investigated the formation of 500 nm and 1000 nm polystyrene (PS) agglomerations in natural seawater from a coastal environment, and we applied DNA-based stable isotope probing (DNA-SIP) with the 500 nm PS sorbed with isotopically-labelled phenanthrene to identify the bacterial members in the seawater community capable of degrading the hydrocarbon. Whilst we observed no significant impact of nanoplastic size on the microbial communities associated with agglomerates that formed in these experiments, these communities were, however, significantly different to those in the surrounding seawater. By DNA-SIP, we identified Arcobacteraceae, Brevundimonas, Comamonas, uncultured Comamonadaceae, Delftia, Sphingomonas and Staphylococcus, as well as the first member of the genera Acidiphilum and Pelomonas to degrade phenanthrene, and of the genera Aquabacterium, Paracoccus and Polymorphobacter to degrade a hydrocarbon. This work provides new information that feeds into our growing understanding on the fate of co-pollutants associated with nano- and microplastics in the ocean.

Neural network establishes co-occurrence links between transformation products of the contaminant and the soil microbiome

It remains challenging to establish reliable links between transformation products (TPs) of contaminants and corresponding microbes. This challenge arises due to the sophisticated experimental regime required for TP discovery and the compositional nature of 16S rRNA gene amplicon sequencing and mass spectrometry datasets, which can potentially confound statistical inference. In this study, we present a new strategy by combining the use of 2H-labeled Stable Isotope-Assisted Metabolomics (2H-SIAM) with a neural network-based algorithm (i.e., MMvec) to explore links between TPs of pyrene and the soil microbiome. The links established by this novel strategy were further validated using different approaches. Briefly, a metagenomic study provided indirect evidence for the established links, while the identification of pyrene degraders from soils, and a DNA-based stable isotope probing (DNA-SIP) study offered direct evidence. The comparison among different approaches, including Pearson's and Spearman's correlations, further confirmed the superior performance of our strategy. In conclusion, we summarize the unique features of the combined use of 2H-SIAM and MMvec. This study not only addresses the challenges in linking TPs to microbes but also introduces an innovative and effective approach for such investigations.Environmental Implication: Taxonomically diverse bacteria performing successive metabolic steps of the contaminant were firstly depicted in the environmental matrix.

Ridge with no-tillage facilitates microbial N2 fixation associated with methane oxidation in rice soil

Aerobic methane-oxidizing bacteria (MOB) play a crucial role in mitigating the greenhouse gas methane emission, particularly prevalent in flooded wetlands. The implementation of ridge with no-tillage practices within a rice-rape rotation system proves effective in overcoming the restrictive redox conditions associated with waterlogging. This approach enhances capillary water availability from furrows, especially during periods of low rainfall, thereby supporting plant growth on the ridges. However, the microbe-mediated accumulation of soil organic carbon and nitrogen remains insufficiently understood under this agricultural practice, particularly concerning methane oxidation, which holds ecological and agricultural significance in the rice fields. In this study, the ridge and ditch soils from a 28-year-old ridge with no-tillage rice field experiment were utilized for incubation with 13C-CH4 and 15NN2 to estimate the methane-oxidizing and N2-fixing potentials. Our findings reveal a significantly higher net production of fresh soil organic carbon in the ridge compared to the ditch soil during methane oxidation, with values of 626 and 543 μg 13C g−1 dry weight soil, respectively. Additionally, the fixed 15N exhibited a twofold increase in the ridge soil (14.1 μg 15N g−1 dry weight soil) compared to the ditch soil. Interestingly, the result of DNA-based stable isotope probing indicated no significant differences in active MOB and N2 fixers between ridge and ditch soils. Both Methylocystis-like type II and Methylosarcina/Methylomonas-like type I MOB catalyzed methane into organic biomass carbon pools. Soil N2-fixing activity was associated with the 15N-labeling of methane oxidizers and non-MOB, such as methanol oxidizers (Hyphomicrobium) and conventional N2 fixers (Burkholderia). Methane oxidation also fostered microbial interactions, as evidenced by co-occurrence patterns. These results underscore the dual role of microbial methane oxidation – not only as a recognized sink for the potent greenhouse gas methane but also as a source of soil organic carbon and bioavailable nitrogen. This emphasizes the pivotal role of microbial methane metabolism in contributing to soil carbon and nitrogen accumulation in ridge with no-tillage systems.

Methane Oxidation Coupled to Selenate Reduction in a Membrane Bioreactor under Oxygen-Limiting Conditions.

Microbial methane oxidation coupled to a selenate reduction process has been proposed as a promising solution to treat contaminated water, yet the underlying microbial mechanisms are still unclear. In this study, a novel methane-based membrane bioreactor system integrating hollow fiber membranes for efficient gas delivery and ultrafiltration membranes for biomass retention was established to successfully enrich abundant suspended cultures able to perform methane-dependent selenate reduction under oxygen-limiting conditions. The microbial metabolic mechanisms were then systematically investigated through a combination of short-term batch tests, DNA-based stable isotope probing (SIP) microcosm incubation, and high-throughput sequencing analyses of 16S rRNA gene and functional genes (pmoA and narG). We confirmed that the methane-supported selenate reduction process was accomplished by a microbial consortia consisting of type-II aerobic methanotrophs and several heterotrophic selenate reducers. The mass balance and validation tests on possible intermediates suggested that methane was partially oxidized into acetate under oxygen-limiting conditions, which was consumed as a carbon source for selenate-reducing bacteria. High-throughput 16S rRNA gene sequencing, DNA-SIP incubation with 13CH4, and subsequent functional gene (pmoA and narG) sequencing results collectively proved that Methylocystis actively executed partial methane oxidation and Acidovorax and Denitratisoma were dominant selenate-reducing bacteria, thus forming a syntrophic partnership to drive selenate reduction. The findings not only advance our understanding of methane oxidation coupled to selenate reduction under oxygen-limiting conditions but also offer useful information on developing methane-based biotechnology for bioremediation of selenate-contaminated water.

Exploring novel alkane-degradation pathways in uncultured bacteria from the North Atlantic Ocean.

Microbes play a significant role in the degradation of petroleum hydrocarbons in the oceans, yet little is known about the native bacteria that metabolize hydrocarbons before an oil spill. The Faroe-Shetland Channel (FSC) is a deepwater subarctic region of the North Atlantic with prominent oil production and a diverse microbial community associated with the degradation of petroleum. Here, we combine DNA-based stable-isotope probing (DNA-SIP) with metagenomics to elucidate the metabolic underpinnings of native alkane-degrading bacteria from the FSC. From two 13C n-hexadecane SIP experiments using seawater from 5 and 700 m depths in the FSC, we obtained 42 metagenome-assembled genomes (MAGs) belonging to 19 genera, including two previously overlooked hydrocarbon-degrading bacteria, Lentibacter (Alphaproteobacteria) and Dokdonia (Bacteroidetes). Diversity surveys indicated Lentibacter were dominant members of the FSC, constituting up to 17% of these communities. Many of the SIP-enriched MAGs (20/42) encoded a complete alkane oxidation pathway, including alkane monooxygenase (AlkB), rubredoxin reductase (AlkT), and rubredoxin-2 (AlkG). Fourteen Aphaproteobacteria MAGs lacked AlkG for electron transfer. Instead, they encoded novel disulfide isomerases with iron-binding cysteine motifs conserved across rubredoxins. Dokdonia lacked AlkT and AlkG, however, their central alkane-degradation catabolic pathways were complete. We describe previously unrecognized bacteria capable of hydrocarbon degradation, including the dominant genera Lentibacter, which may continuously purge hydrocarbons released from oil exploration activities in the FSC. This advances the understanding of the diversity and physiologies of alkane degradation in the North Atlantic and provides evidence of new mechanisms used to metabolize alkanes. IMPORTANCE Petroleum pollution in the ocean has increased because of rapid population growth and modernization, requiring urgent remediation. Our understanding of the metabolic response of native microbial communities to oil spills is not well understood. Here, we explored the baseline hydrocarbon-degrading communities of a subarctic Atlantic region to uncover the metabolic potential of the bacteria that inhabit the surface and subsurface water. We conducted enrichments with a 13C-labeled hydrocarbon to capture the fraction of the community actively using the hydrocarbon. We then combined this approach with metagenomics to identify the metabolic potential of this hydrocarbon-degrading community. This revealed previously undescribed uncultured bacteria with unique metabolic mechanisms involved in aerobic hydrocarbon degradation, indicating that temperature may be pivotal in structuring hydrocarbon-degrading baseline communities. Our findings highlight gaps in our understanding of the metabolic complexity of hydrocarbon degradation by native marine 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.

Reassembly of active ammonia oxidizing bacteria following invasion of exogenous microbiome in an acidic soil

Ammonia oxidation is the rate-limiting step of the global nitrogen cycle, and it is mainly catalyzed by ammonia-oxidizing bacteria (AOB) and archaea (AOA). AOA can carry out nitrification even in acidic soils, whereas AOB activity was not consistently observed in acidic soils. In this study, we manipulated two environmental variables, i.e., the microbial composition and ammonium source (urea and ammonium sulphate), in an acidic forest soil and tested their impacts on AOB abundance, diversity and activity. Soil microcosms were established either using the native forest soil (FS) or after amending the forest soil with 5% (w/w) agricultural soil (AFS) to inoculate new, exogenous microbes. Under the same microcosm incubation condition for 28 days, AOB did not grow in FS but they grew in AFS, suggesting that exogenous microbial invasion could stimulate AOB activity in the acidic soil. Urea amendment led to higher growth of AOB than ammonium sulphate amended in AFS microcosms. DNA-based stable isotope probing revealed that the active AOB lineages were dominated by Nitrosospira cluster 3a.2 and Nitrosospira cluster 10 in the ammonium sulphate supplied soil, but shifted to co-dominance of Nitrosospira cluster 3a.2 and Nitrosospira cluster 1 after urea amendment, demonstrating differential preferences of ammonia sources of phylogenetically distinct AOB lineages. Additionally, AOB abundance showed lower stability than AOA in soils subjected to these two environmental changes, suggesting AOB are more important in determining the fate of soil nitrification following short-term environmental disturbances.

Partial S(0)-driven autotrophic denitrification process facilitated the quick natural enrichment of anammox bacteria at room temperature

Anaerobic ammonium oxidation (anammox) is well-known to be an environmental and promising biotechnology. However, the natural enrichment of anammox bacteria is still a challenging topic. In this study, partial S(0)-driven autotrophic denitrification (PSAD) was developed to stably supply nitrite, and natural enrichment of anammox bacteria was rapidly realized in a single sequencing moving bed biofilm reactor at room temperature. With the initiation of PSAD, anammox bacteria spontaneously emerged within 12 days, and PSAD-anammox coupling system was realized successfully. And then, the influent concentration of ammonium continuously increased to the same concentration as the nitrate, and the mean total nitrogen removal efficiency reached 92.77 %, which was mainly contributed by anammox. Moreover, the coupling of PSAD and anammox reduced the risk of sulfate emissions. cDNA high throughput sequencing revealed that the relative abundance of Candidatus Brocadia and Thiobacillus reached 39.03 % and 13.48 % at the 88th day. Oligotyping analysis illustrated that GATTTAAT and GTCCCA were the dominant Ca. Brocadia and Thiobacillus oligotypes in PSAD-anammox coupling system, respectively. DNA-based stable isotope probing further deciphered that Thiobacillus was the actual performer of PSAD and supported the nitrite for anammox bacteria in PSAD-anammox coupling system. Overall, this work provided a new strategy to naturally enrich anammox bacteria.

Methanotrophy Alleviates Nitrogen Constraint of Carbon Turnover by Rice Root-Associated Microbiomes.

The bioavailability of nitrogen constrains primary productivity, and ecosystem stoichiometry implies stimulation of N2 fixation in association with carbon sequestration in hotspots such as paddy soils. In this study, we show that N2 fixation was triggered by methane oxidation and the methanotrophs serve as microbial engines driving the turnover of carbon and nitrogen in rice roots. 15N2-stable isotope probing showed that N2-fixing activity was stimulated 160-fold by CH4 oxidation from 0.27 to 43.3 μmol N g–1 dry weight root biomass, and approximately 42.5% of the fixed N existed in the form of 15N-NH4+ through microbial mineralization. Nitrate amendment almost completely abolished N2 fixation. Ecophysiology flux measurement indicated that methane oxidation-induced N2 fixation contributed only 1.9% of total nitrogen, whereas methanotrophy-primed mineralization accounted for 21.7% of total nitrogen to facilitate root carbon turnover. DNA-based stable isotope probing further indicated that gammaproteobacterial Methylomonas-like methanotrophs dominated N2 fixation in CH4-consuming roots, whereas nitrate addition resulted in the shift of the active population to alphaproteobacterial Methylocystis-like methanotrophs. Co-occurring pattern analysis of active microbial community further suggested that a number of keystone taxa could have played a major role in nitrogen acquisition through root decomposition and N2 fixation to facilitate nutrient cycling while maintaining soil productivity. This study thus highlights the importance of root-associated methanotrophs as both biofilters of greenhouse gas methane and microbial engines of bioavailable nitrogen for rice growth.

Identifying bioaugmentation candidates for bioremediation of polycyclic aromatic hydrocarbons in contaminated estuarine sediment of the Elizabeth River, VA, USA.

Estuarine sediments near former creosoting facilities along the Elizabeth River (Virginia, USA) are contaminated by polycyclic aromatic hydrocarbons (PAHs). In this study, we interrogated the bacterial community of the Elizabeth River with both culture-based and culture-independent methods to identify potential candidates for bioremediation of these contaminants. DNA-based stable isotope probing (SIP) experiments with phenanthrene and fluoranthene using sediment from the former Republic Creosoting site identified relevant PAH-degrading bacteria within the Azoarcus, Hydrogenophaga, and Croceicoccus genera. Targeted cultivation of PAH-degrading bacteria from the same site recovered 6 PAH-degrading strains, including one strain highly similar to Hydrogenophaga sequences detected in SIP experiments. Other isolates were most similar to organisms within the Novosphingobium, Sphingobium, Stenotrophomonas, and Alcaligenes genera. Lastly, we performed 16S rRNA gene amplicon microbiome analyses of sediment samples from four sites, including Republic Creosoting, with varying concentrations of PAHs. Analysis of these data showed a striking divergence of the microbial community at the highly contaminated Republic Creosoting site from less contaminated sites with the enrichment of several bacterial clades including those affiliated with the Pseudomonas genus. Sequences within the microbiome libraries similar to SIP-derived sequences were generally found at high relative abundance, while the Croceicoccus sequence was present at low to moderate relative abundance. These results suggest that Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation, while Croceicoccus spp. might be good targets for bioaugmentation in these sediments. Furthermore, this study demonstrates the value of culture-based and culture-independent methods in identifying promising bacterial candidates for use in a precision bioremediation scheme. KEY POINTS: • This study highlights the importance of using multiple strategies to identify promising bacterial candidates for use in a precision bioremediation scheme. • We used both selective cultivation techniques and DNA-based stable isotope probing to identify bacterial degraders of prominent PAHs at a historically contaminated site in the Elizabeth River, VA, USA. • Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation in Elizabeth River sediments, while Croceicoccus spp. might be good targets for bioaugmentation.

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.

Robustness of the partial nitrification-anammox system exposing to triclosan wastewater: Stress relieved by extracellular polymeric substances and resistance genes

The partial nitrification-anammox (PN/A) process is a promising method for the treatment of municipal wastewater. It is necessary to clarify the responses of PN/A system to antimicrobial agent triclosan (TCS) widely existed in the influent of wastewater treatment plants. In this study, it was found that PN/A system was robust to cope with 0.5 mg/L TCS. Specifically, the control reactor reached 80% total nitrogen removal efficiency (TNRE) on day 107, while the reactor feeding with 0.5 mg/L TCS reached the same TNRE on day 84. The results of the activity test, high-throughput sequencing and DNA-based stable isotope probing showed that 0.5 mg/L TCS did not impede the performance of ammonia oxidizing archaea, ammonia oxidizing bacteria (Nitrosomonas) and anammox bacteria (Candidatus Brocadia and Ca. Kuenenia), but significant inhibited the nitrite oxidizing bacteria (Nitrospira and Ca. Nitrotoga) and denitrifying bacteria. The influent TCS led to the increase of EPS content and enrichment of four resistance genes (RGs) (intI1, sul1, mexB, and tnpA), which might be two principal mechanisms by which PN/A can resist TCS. In addition, functional bacteria carrying multiple RGs also contributed to the maintenance of PN/A system function. These findings improved the understandings of antimicrobial effects on the PN/A system.

Grazing weakens competitive interactions between active methanotrophs and nitrifiers modulating greenhouse-gas emissions in grassland soils

Grassland soils serve as a biological sink and source of the potent greenhouse gases (GHG) methane (CH4) and nitrous oxide (N2O). The underlying mechanisms responsible for those GHG emissions, specifically, the relationships between methane- and ammonia-oxidizing microorganisms in grazed grassland soils are still poorly understood. Here, we characterized the effects of grazing on in situ GHG emissions and elucidated the putative relations between the active microbes involving in methane oxidation and nitrification activity in grassland soils. Grazing significantly decreases CH4 uptake while it increases N2O emissions basing on 14-month in situ measurement. DNA-based stable isotope probing (SIP) incubation experiment shows that grazing decreases both methane oxidation and nitrification processes and decreases the diversity of active methanotrophs and nitrifiers, and subsequently weakens the putative competition between active methanotrophs and nitrifiers in grassland soils. These results constitute a major advance in our understanding of putative relationships between methane- and ammonia-oxidizing microorganisms and subsequent effects on nitrification and methane oxidation, which contribute to a better prediction and modeling of future balance of GHG emissions and active microbial communities in grazed grassland ecosystems.

In-situ active Bisphenol A-degrading microorganisms in mangrove sediments

Bisphenol A (BPA), as both an endocrine disrupting compound and an important industrial material, is broadly distributed in coastal regions and may cause adverse effects on mangrove ecosystems. Although many BPA degraders have been isolated from various environments, the in-situ active BPA-degrading microorganisms in mangrove ecosystem are still unknown. In this study, DNA-based stable isotope probing in combination with high-throughput sequencing was adopted to pinpoint the microbes actually involved in BPA metabolism in mangrove sediments. Five bacterial genera were speculated to be associated with BPA degradation based on linear discriminant analysis (LDA) effect size (LEfSe) analysis, including Truepera, Methylobacterium, Novosphingobium, Rhodococcus and Rhodobacter. The in-situ BPA degraders were different between mudflat and forest sediments. The Shannon index of microbes in heavy fractions was significantly lower than that in light fractions. Besides, phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) demonstrated that the functional genes relevant to cytochrome P450, benzoate degradation, bisphenol degradation and citrate cycle were up-regulated significantly in in-situ BPA-degrading microbes. These findings greatly expanded the knowledge of indigenous BPA metabolic microorganisms in mangrove ecosystems.

Identification of pyrene degraders via DNA-SIP in oilfield soil during natural attenuation, bioaugmentation and biostimulation

Pyrene is a model contaminant of high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs), which are compounds that have potential carcinogenic effects and pose a serious threat to human health. Finding effective pyrene-degrading bacteria is crucial for removing PAHs from soil. In this study, DNA-based stable isotope probing (DNA-SIP) technology was used to investigate pyrene degraders in PAH-contaminated oilfield soil during natural attenuation (NA), bioaugmentation (BA) and biostimulation (BS). The results show that BA played an important role in pyrene degradation with the highest pyrene removal rate (~95%) after 12 days incubation, followed by removal rates of ~90% for NA and ~30% for BS. In addition, 6 novel pyrene degraders were identified, while 12 well-known PAH degraders were demonstrated to participate in the biodegradation of pyrene. Additionally, the external homologous strains introduced during BA promoted the degradation of pyrene, but not by directly participating in the metabolism of the target compound. Rhamnolipid supplementation during BS promoted the involvement of more microorganisms in the degradation of pyrene, which was beneficial to identifying more pyrene degraders via DNA-SIP. These findings provide new insight into the effects of external homologous strains and supplementary rhamnolipids on pyrene degradation.

Characterization of tetracycline-resistant microbiome in soil-plant systems by combination of H218O-based DNA-Stable isotope probing and metagenomics

The emergence and spread of antibiotic resistance have been considered as a global health threat. However, effective methods to identify antibiotic-resistant bacteria (ARB) in complex microbial community are lacking, and the potential transmission pathways of ARB and antibiotic resistance genes (ARGs) in the soil-plant system remain scarce. Here in this study, tetracycline was chosen as the target antibiotic due to its globally wide usage and clinical significance. DNA-based stable isotope probing with H218O was applied to identify the tetracycline-resistant bacteria from soil-plant systems. Eighteen-year organic fertilization significantly changed the composition of the tetracycline-resistant microbiome in the soil-wheat system and resulted in a higher relative abundance of ARGs in the wheat endophyte. Rhizosphere harboring the most diverse ARGs and mobile genetic elements was identified as a hot spot for horizontal gene transfer and an important bridge between bulk soil and wheat endophyte. Micrococcaceae and Sphingomonadaceae carrying ARGs associated with abundant mobile genetic elements, were identified as the core bacterial taxa in long-term manure-amended and untreated soil-wheat systems, respectively. This method contributes to a more precise track of ARB in the environment, and our work depicts the high potential of ARG transfer in the rhizosphere mediated by the core species.