Cultivation and characterization of functional-yet-uncultivable phenanthrene degraders by stable-isotope-probing and metagenomic-binning directed cultivation (SIP-MDC).

High-throughput identification and cultivation of functional-yet-uncultivable microorganisms is a fundamental goal in environmental microbiology. It remains as a critical challenge due to the lack of routine and effective approaches. Here, we firstly proposed an approach of stable-isotope-probing and metagenomic-binning directed cultivation (SIP-MDC) to isolate and characterize the active phenanthrene degraders from petroleum-contaminated soils. From SIP and metagenome, we assembled 13 high-quality metagenomic bins from 13C-DNA, and successfully obtained the genome of an active PHE degrader Achromobacter (genome-MB) from 13C-DNA metagenomes, which was confirmed by gyrB gene comparison and average nucleotide/amino identity (ANI/AAI), as well as the quantification of PAH dioxygenase and antibiotic resistance genes. Thereinto, we modified the traditional cultivation medium with antibiotics and specific growth factors (e.g., vitamins and metals), and separated an active phenanthrene degrader Achromobacter sp. LJB-25 via directed isolation. Strain LJB-25 could degrade phenanthrene and its identity was confirmed by ANI/AAI values between its genome and genome-MB (>99 %). Our results hinted at the feasibility of SIP-MDC to identify, isolate and cultivate functional-yet-uncultivable microorganisms (active phenanthrene degraders) from their natural habitats. Our findings developed a state-of-the-art SIP-MDC approach, expanded our knowledge on phenanthrene biodegradation mechanisms, and proposed a strategy to mine functional-yet-uncultivable microorganisms.

The uptake and degradation of polychlorinated biphenyls in constructed wetlands planted with Myriophyllum aquaticum.

The unregulated dismantling and improper disposal of electronic waste lead to severe soil contamination by polychlorinated biphenyls (PCBs). Constructed wetlands (CWs) play an important role in PCBs removal as a result of the co-existence of anaerobic and aerobic conditions. However, the effects and mechanisms of different PCBs concentrations in soils on plant uptake and PCBs degradation within CWs are unclear. We evaluated the uptake and degradation of PCBs at different concentrations by Myriophyllum aquaticum (Vell.) Verdc. Planting significantly increased PCBs removal by 8.70% (p < 0.05) in soils with 1500 and 2500 µg/kg PCBs, whereas no significant effect was observed at 500 and 1000 µg/kg. PCBs levels did not significantly affect plant growth and PCBs accumulation. The contribution of plant uptake to PCBs removal was only 0.10-0.12%, indicating that microbial degradation was the dominant pathway for PCBs removal after planting with M. aquaticum. In the treatments with PCBs ≥ 1500 µg/kg, M. aquaticum increased the microbial population, altered the microbial community structure and enriched PCB-degrading bacteria. Functional prediction revealed that microbes in M. aquaticum rhizosphere secreted more peroxidase and glycosyltransferase than non-plant control, which were likely involved in PCBs metabolism.

Unveiling the synergistic mechanism of autochthonous fungal bioaugmentation and ammonium nitrogen biostimulation for enhanced phenanthrene degradation in oil-contaminated soils.

Autochthonous bioaugmentation and nutrient biostimulation are promising bioremediation methods for polycyclic aromatic hydrocarbons (PAHs) in contaminated agricultural soils, but little is known about their combined working mechanism. In this study, a microcosm trial was conducted to explore the combined mechanism of autochthonous fungal bioaugmentation and ammonium nitrogen biostimulation, using DNA stable-isotope-probing (DNA-SIP) and microbial network analysis. Both treatments significantly improved phenanthrene (PHE) removal, with their combined application producing the best results. The microbial community composition was notably altered by all bioremediation treatments, particularly the PHE-degrading bacterial and fungal taxa. Fungal bioaugmentation removed PAHs through extracellular enzyme secretion but reduced soil microbial diversity and ecological stability, while nitrogen biostimulation promoted PAH dissipation by stimulating indigenous soil degrading microbes, including fungi and key bacteria in the soil co-occurrence networks, ensuring the ecological diversity of soil microorganisms. The combination of both approaches proved to be the most effective strategy, maintaining a high degradation efficiency and relatively stable soil biodiversity through the secretion of lignin hydrolytic enzymes by fungi, and stimulating the reproduction of soil native degrading microbes, especially the key degraders in the co-occurrence networks. Our findings provide a fresh perspective of the synergy between fungal bioaugmentation and nitrogen biostimulation, highlighting the potential of this combined bioremediation approach for in situ PAH-contaminated soils.

Single-Cell Analysis Reveals Malignant Cells Reshape the Cellular Landscape and Foster an Immunosuppressive Microenvironment of Extranodal NK/T-Cell Lymphoma.

Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive type of lymphoma associated with Epstein-Barr virus (EBV) and characterized by heterogeneous tumor behaviors. To better understand the origins of the heterogeneity, this study utilizes single-cell RNA sequencing (scRNA-seq) analysis to profile the tumor microenvironment (TME) of NKTCL at the single-cell level. Together with in vitro and in vivo models, the study identifies a subset of LMP1+ malignant NK cells contributing to the tumorigenesis and development of heterogeneous malignant cells in NKTCL. Furthermore, malignant NK cells interact with various immunocytes via chemokines and their receptors, secrete substantial DPP4 that impairs the chemotaxis of immunocytes and regulates their infiltration. They also exhibit an immunosuppressive effect on T cells, which is further boosted by LMP1. Moreover, high transcription of EBV-encoded genes and low infiltration of tumor-associated macrophages (TAMs) are favorable prognostic indicators for NKTCL in multiple patient cohorts. This study for the first time deciphers the heterogeneous composition of NKTCL TME at single-cell resolution, highlighting the crucial role of malignant NK cells with EBV-encoded LMP1 in reshaping the cellular landscape and fostering an immunosuppressive microenvironment. These findings provide insights into understanding the pathogenic mechanisms of NKTCL and developing novel therapeutic strategies against NKTCL.

In Situ Discrimination and Cultivation of Active Degraders in Soils by Genome-Directed Cultivation Assisted by SIP-Raman-Activated Cell Sorting.

The identification and in situ cultivation of functional yet uncultivable microorganisms are important to confirm inferences regarding their ecological functions. Here, we developed a new method that couples Raman-activated cell sorting (RACS), stable-isotope probing (SIP), and genome-directed cultivation (GDC)─namely, RACS-SIP-GDC─to identify, sort, and cultivate the active toluene degraders from a complex microbial community in petroleum-contaminated soil. Using SIP, we successfully identified the active toluene degrader Pigmentiphaga, the single cells of which were subsequently sorted and isolated by RACS. We further successfully assembled the genome of Pigmentiphaga based on the metagenomic sequencing of 13C-DNA and genomic sequencing of sorted cells, which was confirmed by gyrB gene comparison and average nucleotide identity determination. Additionally, the genotypes and phenotypes of this degrader were directly linked at the single-cell level, and its complete toluene metabolic pathways in petroleum-contaminated soil were reconstructed. Based on its unique metabolic properties uncovered by genome sequencing, we modified the traditional cultivation medium with antibiotics, amino acids, carbon sources, and growth factors (e.g., vitamins and metals), achieving the successful cultivation of RACS-sorted active degrader Pigmentiphaga sp. Our results implied that RACS-SIP-GDC is a state-of-the-art approach for the precise identification, targeted isolation, and cultivation of functional microbes from complex communities in natural habitats. RACS-SIP-GDC can be used to explore specific and targeted organic-pollution-degrading microorganisms at the single-cell level and provide new insights into their biodegradation mechanisms.

Unveiling the novel role of ryegrass rhizospheric metabolites in benzo[a]pyrene biodegradation.

Rhizoremediation is a promising remediation technology for the removal of soil persistent organic pollutants (POPs), especially benzo[a]pyrene (BaP). However, our understanding of the associations among rhizospheric soil metabolites, functional microorganisms, and POPs degradation in different plant growth stages is limited. We combined stable-isotope probing (SIP), high-throughput sequencing, and metabolomics to analyze changes in rhizospheric soil metabolites, functional microbes, and BaP biodegradation in the early growth stages (tillering, jointing) and later stage (booting) of ryegrass. Microbial community structures differed significantly among growth stages. Metabolisms such as benzenoids and carboxylic acids tended to be enriched in the early growth stage, while lipids and organic heterocyclic compounds dominated in the later stage. From SIP, eight BaP-degrading microbes were identified, and most of which such as Ilumatobacter and Singulisphaera were first linked with BaP biodegradation. Notably, the relationship between the differential metabolites and BaP degradation efficiency further suggested that BaP-degrading microbes might metabolize BaP directly to produce benzenoid metabolites (3-hydroxybenzo[a]pyrene), or utilize benzenoids (phyllodulcin) to stimulate the co-metabolism of BaP in early growth stage; some lipids and organic acids, e.g. 1-aminocyclopropane-1-carboxylic acid, might provide nutrients for the degraders to promote BaP metabolism in later stage. Accordingly, we determined that certain rhizospheric metabolites might regulate the rhizospheric microbial communities at different growth stages, and shift the composition and diversity of BaP-degrading bacteria, thereby enhancing in situ BaP degradation. Our study sheds light on POPs rhizoremediation mechanisms in petroleum-contaminated soils.

SRSF3/AMOTL1 splicing axis promotes the tumorigenesis of nasopharyngeal carcinoma through regulating the nucleus translocation of YAP1.

Dysregulation of serine/arginine splicing factors (SRSFs) and abnormal alternative splicing (AS) have been widely implicated in various cancers but scarcely investigated in nasopharyngeal carcinoma (NPC). Here we examine the expression of 12 classical SRSFs between 87 NPC and 10 control samples, revealing a significant upregulation of SRSF3 and its association with worse prognosis in NPC. Functional assays demonstrate that SRSF3 exerts an oncogenic function in NPC progression. Transcriptome analysis reveals 1,934 SRSF3-regulated AS events in genes related to cell cycle and mRNA metabolism. Among these events, we verify the generation of a long isoform of AMOTL1 (AMOTL1-L) through a direct bond of the SRSF3 RRM domain with the exon 12 of AMOTL1 to promote exon inclusion. Functional studies also reveal that AMOTL1-L promotes the proliferation and migration of NPC cells, while AMOTL1-S does not. Furthermore, overexpression of AMOTL1-L, but not -S, significantly rescues the inhibitory effects of SRSF3 knockdown. Additionally, compared with AMOTL1-S, AMOTL1-L has a localization preference in the intracellular than the cell membrane, leading to a more robust interaction with YAP1 to promote nucleus translocation. Our findings identify SRSF3/AMOTL1 as a novel alternative splicing axis with pivotal roles in NPC development, which could serve as promising prognostic biomarkers and therapeutic targets for NPC.

Effects of nitrogen stress on uptake and translocation of organophosphate esters by watermifoil (Myriophyllum aquaticum L.) in an aquatic ecosystem.

Although organophosphate esters (OPEs) and nitrogen (N) are normally present in aquatic environments, the effects of the plant uptake, accumulation, and translocation of OPEs in different levels of N remain ambiguous. To better understand these processes, watermifoil (Myriophyllum aquaticum L.) as tested plant was chosen to investigate the effects of different N levels on the uptake and translocation of OPEs by plants in matched water-sediment-plant samples. After two months, we found the root-water concentration factors, root-sediment concentration factors, and translocation factors (TFs) were significantly changed with the levels of N (p < 0.05), implying that the presence of N could alter uptake, accumulation, and translocation of OPEs in M. aquaticum, particularly the process of root absorption. Low concentrations of N could remarkably promote the uptake of OPEs by M. aquaticum. However, when the concentrations of N in water were higher than 200 mg/L, the plants' growth and OPE accumulation by M. aquaticum were obviously inhibited with the elevated N contents. Moreover, the enrichment and environmental transport of OPEs in M. aquaticum seemed to be closely associated with physicochemical parameters; the octanol-water partition coefficient had significant relationships with measured organic carbon-normalized sediment-water partition coefficients and TFs in the present study. Additionally, the substituents and structures of OPEs could also affect the accumulation and translocation of OPEs in M. aquaticum, including the chlorination degree and alkyl chain length. This study could improve our understanding of uptake and translocation of OPEs in aquatic plants under different levels of N.

Spatial redistribution and enantiomeric signatures of hexachlorocyclohexanes in Chinese forest soils: Implications to environmental behavior and influencing factors.

Hexachlorocyclohexanes (HCHs) are a group of highly persistent pesticides. The concentrations of HCHs and the enantiomeric fractions of α-HCH in the O- and A-horizons from 30 mountains across China were analyzed in this study. The concentrations of total HCHs ranged from 0.061 to 46.9 ng/g (mean 2.12 ng/g) and 0.046 to 16.1 ng/g (mean 0.792 ng/g) in the O- and A-horizons, respectively. The HCH residues were mainly derived from the historical applications of technical HCH and lindane. Higher concentrations of HCHs were typically found in northern China, and no significant correlations were found between historical technical HCH usage and HCH isomer concentrations in either the O- or A-horizons (p > 0.05). Conversely, the concentrations of HCH isomers were significantly correlated with the environmental parameters (temperature and precipitation), thus indicating a typical secondary distribution pattern. Some HCH isomers tended to be transported northward under the long-term effect of monsoon. Chiral α-HCH was non-racemic in soils and showed preferential degradation of (-) α-HCH in both the O- and A-horizons. The transformation from γ-HCH to α-HCH might alter the enantiomeric signatures of α-HCH in soils. Moreover, the deviation from racemic of α-HCH was positively correlated with the C/N ratio in the A-horizon (p < 0.01), thus suggesting that the C/N ratio could alter the microbial activity and significantly affect the enantioselective degradation extent of α-HCH in soils.

Uptake, accumulation, and translocation of organophosphate esters by watermifoil (Myriophyllum aquaticum) in an aquatic ecosystem: effects of chemical structure and concentrations.

In order to explore the environmental behavior of organophosphate esters (OPEs) in aquatic environment, the accumulation and distribution of OPEs in water, sediment, and plant were investigated. In this study, watermifoil (Myriophyllum aquaticum) were exposed with ten OPEs for concentrations of 200 ng/g, 500 ng/g, 1000 ng/g, and 2000 ng/g, respectively. The concentrations of Σ10OPEs in rhizosphere sediment were higher than those in non-rhizosphere sediment, demonstrating that rhizosphere processes tend to transport OPEs into the rhizosphere sediment. Most of the selected OPEs were not in equilibrium between water and sediment, and trend to retain in sediment. In addition, OPEs with relatively higher hydrophobicity had trend to retained in Myriophyllum aquaticum roots, whereas OPEs with lower hydrophobicity were more likely transported to shoots. In this study, octanol-water partition coefficient (KOW) had significantly positive correlations with organic carbon-normalized soil-water partition coefficients (KOC) and root-water concentration factors (RWCFs), but KOW was negatively correlated with translocation factors (TFs). Moreover, the substituent types and initial levels of OPEs also have impacts on the plant uptake and accumulation. These observations will improve our understanding of the distribution and translocation of OPEs in aquatic environment.

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.

Assessing the bioavailability of antibiotics in soil with the diffusive gradients in thin films (DGT).

The diffusive gradients in thin films (DGT) technique is an excellent method for investigating the dynamic processes of antibiotics in soils. However, whether it is applicable in antibiotic bioavailability assessment is yet to be disclosed. This study employed DGT to determine the antibiotic bioavailability in soil, and compared the results with plant uptake, soil solutions, and solvent extraction methods. DGT exhibited predictive capability for plant taking in antibiotics proved by the significant linear relationship between the DGT based concentration (CDGT) and antibiotic concentration in roots and shoots. Although the performance of soil solution was acceptable based on linear relationship analysis, its stability was weaker than DGT. The results based on plant uptake and DGT indicated the bioavailable antibiotic contents in different soils were inconsistent because of the distinct mobility and resupply of sulphonamides and trimethoprim in different soils, as represented by Kd and Rds, which were affected by soil properties. Plant species played an important role in antibiotic uptake and translocation. Antibiotic uptake by plants depends on antibiotic, plant and soil. These results confirmed the capability of DGT in determining antibiotic bioavailability for the first time. This work provided a simple and powerful tool for environmental risk evaluation of antibiotics in soils.

Stable Isotopic and Metagenomic Analyses Reveal Microbial-Mediated Effects of Microplastics on Sulfur Cycling in Coastal Sediments.

Microplastics are readily accumulated in coastal sediments, where active sulfur (S) cycling takes place. However, the effects of microplastics on S cycling in coastal sediments and their underlying mechanisms remain poorly understood. In this study, the transformation patterns of different S species in mangrove sediments amended with different microplastics and their associated microbial communities were investigated using stable isotopic analysis and metagenomic sequencing. Biodegradable poly(lactic acid) (PLA) microplastics treatment increased sulfate (SO42-) reduction to yield more acid-volatile S and elementary S, which were subsequently transformed to chromium-reducible S (CRS). The S isotope fractionation between SO42- and CRS in PLA treatment increased by 9.1‰ from days 0 to 20, which was greater than 6.8‰ in the control. In contrast, recalcitrant petroleum-based poly(ethylene terephthalate) (PET) and polyvinyl chloride (PVC) microplastics had less impact on the sulfate reduction, resulting in 7.6 and 7.7‰ of S isotope fractionation between SO42- and CRS from days 0 to 20, respectively. The pronounced S isotope fractionation in PLA treatment was associated with increased relative abundance of Desulfovibrio-related sulfate-reducing bacteria, which contributed a large proportion of the microbial genes responsible for dissimilatory sulfate reduction. Overall, these findings provide insights into the potential impacts of microplastics exposure on the biogeochemical S cycle in coastal sediments.

Four New Species of Aspergillus Subgenus Nidulantes from China.

Aspergillus subgenus Nidulantes includes species with emericella-like ascomata and asexual species. Subgenus Nidulantes is the second largest subgenus of Aspergillus and consists of nine sections. In this study, agricultural soils were sampled from 12 provinces and autonomous regions in China. Based on primary BLAST analyses, seven of 445 Aspergillus isolates showed low similarity with existing species. A polyphasic investigation, including phylogenetic analysis of partial ITS, ß-tubulin, calmodulin, and RNA polymerase II second largest subunit genes, provided evidence that these isolates were distributed among four new species (Aspergillus guangdongensis, A. guangxiensis, A. sichuanensis and A. tibetensis) in sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes. Illustrated morphological descriptions are provided for each new taxon.

The synergistic toxicity effect of di(2-ethylhexyl)phthalate and plant growth disturbs the structure and function of soil microbes in the rhizosphere.

Di-(2-ethylhexyl) phthalate (DEHP) is a dominant phthalic acid ester in the environment and commonly occurs at high concentration in agricultural soils. Its influence on the soil microbial community has been widely reported, while research related to its effects on microbial structure, function, and interactions in the rhizosphere, a microbial hotspot region in the terrestrial ecosystem, is still limited. This study investigated the response of microbes in the rhizosphere to DEHP contamination. DEHP reduced microbial quantity, shifted the microbial community structure, and enriched the soil bacteria with potential DEHP degraders. Although the rhizosphere can alleviate DEHP toxicity, DEHP still played an important role in microbial community construction in the rhizosphere. Interestingly, some microbes were influenced by the synergistic toxicity effect of DEHP addition and plant growth, and there were significant differences in their relative abundance and alpha diversity in soil treated with both DEHP and planting compared to soils with just DEHP spiking or planting. The genes related to cell motility, metabolism of terpenoids and polyketides, protein families, genetic information processing, and replication and repair pathways changed only in soil treated with both DEHP and planting further proved the existence of synergistic toxicity. Anyway, the impact of DEHP on microbial function in the rhizosphere was important with 52.42‰ of the genes being changed. The change in cell motility, biofilm formation, and genes related to the quorum sensing pathway might affect the relationship between microbes, which play a crucial role in ecosystem function. This was further proven by changes in the microbial co-occurrence pattern. Our results can benefit risk evaluation of DEHP to microbial community in the rhizosphere, which is important for the effective function of terrestrial ecosystems and soil health.

Change of the structure and assembly of bacterial and photosynthetic communities by the ecological engineering practices in Dianchi Lake.

Cyanobacterial bloom challenges the aquatic ecosystem and ecological restoration is an effective approach for cyanobacterial bloom control, but the change of aquatic community after ecological restoration is still unclear. Dianchi Lake is an eutrophic lake with frequent cyanobacterial blooms in China, and recent ecological restoration projects in Caohai (north part) have a satisfactory performance. In this study, we collected 249 water samples at 23 sites from Dianchi Lake to explore the relationships between water physicochemical variables and aquatic microbial communities. Water physicochemical variables in Waihai (south part) intensively changed along time, whereas those in Caohai did not. Photoautotrophic communities were significantly divergent between Caohai and Waihai. Waihai had a lower diversity of photoautotrophic community, containing higher abundance of Cyanophyceae (89.9%) than Caohai (42.7%). Nutrient level and Cyanophyceae only exhibited strong correlations in Wahai (p < 0.05). Redundancy analysis and microbial ecological network suggested that microbial communities in Caohai had a higher stability. Deterministic process dominated the microbial assembly (50-80% for bacteria and >90% for photoautotrophs), and particularly in Caohai. Our results unraveled that the structure and assembly of bacterial and photoautotrophic communities significantly changed after ecological restoration, offering valuable suggestions that photosynthetic diversity should be focused for other ecological restoration projects.

Elsholtzia splendens promotes phenanthrene and polychlorinated biphenyl degradation under Cu stress through enrichment of microbial degraders.

Co-contamination of heavy metals and organic pollutants is widespread in the environment. Metal-tolerant/hyperaccumulating plants have the advantage of enhancing co-operation between plants and rhizospheric microbes under heavy metal stress, but the underlying mechanism remains unclear. In the present study, the effects of Elsholtzia splendens and Lolium perenne on the rhizospheric microbial community and degraders of phenanthrene (PHE) and polychlorinated biphenyls (PCBs) were investigated. The results showed E. splendens could tolerate high Cu concentrations, while L. perenne was sensitive to Cu toxicity. Although Cu played the most important role in microbial community construction, both E. splendens and L. perenne caused shifts in the rhizospheric microbial community. For PHE and PCB degradation, L. perenne was more efficient under low Cu concentrations, whereas E. splendens performed better under high Cu concentrations. This difference can be attributed to shifts in the degrader community and key degradation genes identified by stable isotope probing. Moreover, higher abundances of various genes for organic pollutant degradation were observed in the rhizosphere of E. splendens than L. perenne based on gene prediction under high Cu stress. Our study reveals underlying mechanism of the advantages of heavy metal-tolerant plants for organic pollutant removal in soils co-contaminated with heavy metals.

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.

The influence of anaerobic dechlorination on the aerobic degradation of PCBs in e-waste-contaminated soils in an anaerobic-aerobic two-stage treatment.

The combination of microbial reductive dechlorination and aerobic oxidation (RD-AO) process was proposed to be a promising strategy for extensive bioremediation of highly chlorinated polychlorinated biphenyls (PCBs). Nonetheless, experimental evidence on the impact of the RD on subsequent AO in anaerobic-aerobic two-stage treatment remains scarce. The present study applied stable-isotope probing (SIP) to explore the RD-AO mediated degradation of PCBs in an e-waste-contaminated soil. The RD-AO treatment resulted in 37.1 % and 48.2 % degradation of PCB180 and PCB9, respectively, while the PCB9 degradation efficiency decreased compared to the sole AO (81.2 %). The inhibition of PCB aerobic degradation might be caused by the alteration of aerobic bacterial community, which was proved by a higher abundance of anaerobic bacteria and a lower abundance of aerobic bacteria being observed in the aerobic stage of RD-AO. Further evidence was obtained using DNA-SIP that the anaerobic stage altered the PCB degraders' community structures and changed three of the five degraders. There were four lineages (Arenimonas, Steroidobacter, Sulfurifustis, and Thermoanaerobacterales) identified as PCB degraders for the first time. Interestingly, three of them were found in RD-AO microcosm, suggesting that anaerobic-aerobic two-stage treatment can recruit novel bacteria involved in PCBs aerobic degradation. The present study provided novel insight into the synergistic integration of anaerobic and aerobic processes for extensive degradation of highly chlorinated PCBs.

Identifying the Active Phenanthrene Degraders and Characterizing Their Metabolic Activities at the Single-Cell Level by the Combination of Magnetic-Nanoparticle-Mediated Isolation, Stable-Isotope Probing, and Raman-Activated Cell Sorting (MMI-SIP-RACS).

Magnetic-nanoparticle-mediated isolation coupled with stable-isotope probing (MMI-SIP) is a cultivation-independent higher-resolution approach for isolating active degraders in their natural habitats. However, it addresses the community level and cannot directly link the microbial identities, phenotypes, and in situ functions of the active degraders at the single-cell level within complex microbial communities. Here, we used 13C-labeled phenanthrene as the target and developed a new method coupling MMI-SIP and Raman-activated cell sorting (RACS), namely, MMI-SIP-RACS, to identify the active phenanthrene-degrading bacterial cells from polycyclic aromatic hydrocarbon (PAH)-contaminated wastewater. MMI-SIP-RACS significantly enriched the active phenanthrene degraders and successfully isolated the representative single cells. Amplicon sequencing analysis by SIP, 13C shift of the single cell in Raman spectra, and the 16S rRNA gene from single cell sequencing via RACS confirmed that Novosphingobium was the active phenanthrene degrader. Additionally, MMI-SIP-RACS reconstructed the phenanthrene metabolic pathway and genes of Novosphingobium, including two novel genes encoding phenanthrene dioxygenase and naphthalene dioxygenase. Our findings suggested that MMI-SIP-RACS is a powerful method to efficiently and precisely isolate active PAH degraders from complex microbial communities and directly link their identities to functions at the single-cell level.