Microbial consortium assembly and functional analysis via isotope labelling and single-cell manipulation of polycyclic aromatic hydrocarbon degraders.

Soil microbial flora constitutes a highly diverse and complex microbiome on Earth, often challenging to cultivation, with unclear metabolic mechanisms in situ. Here, we present a pioneering concept for the in situ construction of functional microbial consortia (FMCs) and introduce an innovative method for creating FMCs by utilizing phenanthrene as a model compound to elucidate their in situ biodegradation mechanisms. Our methodology involves single-cell identification, sorting, and culture of functional microorganisms, resulting in the formation of a precise in situ FMC. Through Raman-activated cell sorting-stable-isotope probing, we identified and isolated phenanthrene-degrading bacterial cells from Achromobacter sp. and Pseudomonas sp., achieving precise and controllable in situ consortia based on genome-guided cultivation. Our in situ FMC outperformed conventionally designed functional flora when tested in real soil, indicating its superior phenanthrene degradation capacity. We revealed that microorganisms with high degradation efficiency isolated through conventional methods may exhibit pollutant tolerance but lack actual degradation ability in natural environments. This finding highlights the potential to construct FMCs based on thorough elucidation of in situ functional degraders, thereby achieving sustained and efficient pollutant degradation. Single-cell sequencing linked degraders with their genes and metabolic pathways, providing insights regarding the construction of in situ FMCs. The consortium in situ comprising microorganisms with diverse phenanthrene metabolic pathways might offer distinct advantages for enhancing phenanthrene degradation efficiency, such as the division of labour and cooperation or communication among microbial species. Our approach underscores the importance of in situ, single-cell precision identification, isolation, and cultivation for comprehensive bacterial functional analysis and resource exploration, which can extend to investigate MFCs in archaea and fungi, clarifying FMC construction methods for element recycling and pollutant transformation in complex real-world ecosystems.

Determination of soil phenanthrene degradation through a fungal-bacterial consortium.

Fungal-bacterial consortia enhance organic pollutant removal, but the underlying mechanisms are unclear. We used stable isotope probing (SIP) to explore the mechanism of bioaugmentation involved in polycyclic aromatic hydrocarbon (PAH) biodegradation in petroleum-contaminated soil by introducing the indigenous fungal strain Aspergillus sp. LJD-29 and the bacterial strain Pseudomonas XH-1. While each strain alone increased phenanthrene (PHE) degradation, the simultaneous addition of both strains showed no significant enhancement compared to treatment with XH-1 alone. Nonetheless, the assimilation effect of microorganisms on PHE was significantly enhanced. SIP revealed a role of XH-1 in PHE degradation, while the absence of LJD-29 in 13C-DNA indicated a supporting role. The correlations between fungal abundance, degradation efficiency, and soil extracellular enzyme activity indicated that LJD-29, while not directly involved in PHE assimilation, played a crucial role in the breakdown of PHE through extracellular enzymes, facilitating the assimilation of metabolites by bacteria. This observation was substantiated by the results of metabolite analysis. Furthermore, the combination of fungus and bacterium significantly influenced the diversity of PHE degraders. Taken together, this study highlighted the synergistic effects of fungi and bacteria in PAH degradation, revealed a new fungal-bacterial bioaugmentation mechanism and diversity of PAH-degrading microorganisms, and provided insights for in situ bioremediation of PAH-contaminated soil.IMPORTANCEThis study was performed to explore the mechanism of bioaugmentation by a fungal-bacterial consortium for phenanthrene (PHE) degradation in petroleum-contaminated soil. Using the indigenous fungal strain Aspergillus sp. LJD-29 and bacterial strain Pseudomonas XH-1, we performed stable isotope probing (SIP) to trace active PHE-degrading microorganisms. While inoculation of either organism alone significantly enhanced PHE degradation, the simultaneous addition of both strains revealed complex interactions. The efficiency plateaued, highlighting the nuanced microbial interactions. SIP identified XH-1 as the primary contributor to in situ PHE degradation, in contrast to the limited role of LJD-29. Correlations between fungal abundance, degradation efficiency, and extracellular enzyme activity underscored the pivotal role of LJD-29 in enzymatically facilitating PHE breakdown and enriching bacterial assimilation. Metabolite analysis validated this synergy, unveiling distinct biodegradation mechanisms. Furthermore, this fungal-bacterial alliance significantly impacted PHE-degrading microorganism diversity. These findings advance our understanding of fungal-bacterial bioaugmentation and microorganism diversity in polycyclic aromatic hydrocarbon (PAH) degradation as well as providing insights for theoretical guidance in the in situ bioremediation of PAH-contaminated soil.

Using rice straw-augmented ecological floating beds to enhance nitrogen removal in carbon-limited wastewater.

Agricultural biomass used as solid carbon substrates in ecological floating beds (EFBs) has been proven to be applicable in nitrogen removal for carbon-limited wastewater treatment. However, the subtle interactions among plants, rhizosphere microorganisms, and supplementary carbon sources have not been thoroughly studied. This study combined rice straw mats with different aquatic macrophytes in EFBs to investigate denitrification efficiency in carbon-limited eutrophic waters. Results showed that rice straw significantly enhanced the nitrogen removal efficiency of EFBs, while enriching nitrogen-fixing and denitrifying bacteria (such as Rhizobium, Rubrivivax, and Rhodobacter, etc.). Additionally, during the denitrification process in EFBs, rice straw can release humic acid-like fraction as electron donors to support the metabolic activities of microorganisms, while aquatic macrophytes provide a more diverse range of dissolved organic matters, facilitating a sustainable denitrification process. These findings help to understand the synergistic effect of denitrification processes within wetland ecosystems using agricultural biomass.

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.

Differences among active toluene-degrading microbial communities in farmland soils with different levels of heavy metal pollution.

Heavy metals can severely influence the mineralisation of organic pollutants in a compound-polluted environment. However, to date, no study has focused on the effects of heavy metals on the active organic pollutant-degrading microbial communities to understand the bioremediation mechanism. In this study, toluene was used as the model organic pollutant to explore the effects of soils with different levels of heavy metal pollution on organic contaminant degradation in the same area via stable isotope probing (SIP) and 16 S rRNA high-throughput sequencing. Heavy metals can seriously affect toluene biodegradation and regulate the abundance and diversity of microbial communities. SIP revealed a drastic difference in the community structure of active toluene degraders between the unpolluted and heavy metal-polluted soils. All SIP-identified degraders were assigned to nine bacterial classes, among which Alphaproteobacteria, Gammaproteobacteria, and Bacilli were shared by both treatments. Among all active degraders, Nitrospira, Nocardioides, Conexibacteraceae, and Singulisphaera were linked to toluene biodegradation for the first time. Notably, the type of active degrader and microbial diversity were strongly related to biodegradation efficiency, indicating their key role in toluene biodegradation. Overall, heavy metals can affect the microbial diversity and alter the functional microbial communities in soil, thereby influencing the removal efficiency of organic contaminants. Our findings provide novel insights into the biodegradation mechanism of organic pollutants in heavy metal-polluted soils and highlight the biodiversity of microbes involved in toluene biodegradation in compound-polluted environments.

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.

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.

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.

Complete pentachlorophenol biodegradation in a dual-working electrode bioelectrochemical system: Performance and functional microorganism identification.

Bioelectrochemical system (BES) can effectively promote the reductive dechlorination of chlorophenols (CPs). However, the complete degradation of CPs with sequential dechlorination and mineralization processes has rarely achieved from the BES. Here, a dual-working electrode BES was constructed and applied for the complete degradation of pentachlorophenol (PCP). Combined with DNA-stable isotope probing (DNA-SIP), the biofilms attached on the anodic and cathodic electrode in the BES were analyzed to explore the dechlorinating and mineralizing microorganisms. Results showed that PCP removal efficiency in the dual-working BES (84% for 21 days) was 4.1 and 4.7 times higher than those of conventional BESs with a single anodic or cathodic working electrode, respectively. Based on DNA-SIP and high-throughput sequencing analysis, the cathodic working electrode harbored the potential dechlorinators (Comamonas, Pseudomonas, Methylobacillus, and Dechlorosoma), and the anodic working enriched the potential intermediate mineralizing bacteria (Comamonas, Stenotrophomonas, and Geobacter), indicating that PCP could be completely degraded under the synergetic effect of these functional microorganisms. Besides, the potential autotrophic functional bacteria that might be involved in the PCP dechlorination were also identified by SIP labeled with 13C-NaHCO3. Our results proved that the dual-working BES could accelerate the complete degradation of PCP and enrich separately the functional microbial consortium for the PCP dechlorination and mineralization, which has broad potential for bioelectrochemical techniques in the treatment of wastewater contaminated with CPs or other halogenated organic compounds.

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.

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.

Glycyrrhiza Polysaccharide Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice.

Background: Licorice is one of the most ubiquitous herbs in traditional Chinese medicine, with notable anti-inflammatory and antiulcerative effects as well as potent digestive disease therapeutic impacts; yet, its active components and mechanisms remain unclear. There is a lot of evidence that Glycyrrhiza polysaccharide (GPS) has antioxidants, improving intestinal flora, anti-inflammatory effects, etc. Hypothesis/Purpose. Here, we investigated the effects of GPS on dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC) mice and its possible mechanisms. Methods: GPS (100, 200, and 400 mg/kg) or the positive control drug sulfasalazine (SASP) (200 mg/kg) were orally administered to mice for 8 days. Body weight was recorded daily. Symptoms associated with UC, such as disease activity index (DAI), colon length, spleen weight, and mucosal damage were detected. The possible mechanism of GPS ameliorating enteritis symptoms was explored by detecting intestinal permeability and serum levels of inflammatory factors, and changes in intestinal permeability were expressed by serum concentration of FITC-dextran and D-lactic acid. Results: The results demonstrated that GPS administration alleviated UC symptoms in colitis mice, including weight loss, DAI index, shorting colon length, and mucosal damage. Mechanistic evaluation revealed that GPS treatment reduced intestinal permeability and serum levels of inflammatory factors: IL-1, IL-6, and TNF-α, while increasing serum levels of the anti-inflammatory factor IL-10, suggesting that GPS's mechanism in UC is related to reducing intestinal permeability and inhibiting the inflammatory response, with intestinal permeability implicated as the initiating mechanism. Conclusion: This study highlights GPS as a promising therapeutic agent, with high therapeutic efficacy and a good safety profile, for enteritis and beyond.

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.

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.

Unveiling metabolic characteristics of an uncultured Gammaproteobacterium responsible for in situ PAH biodegradation in petroleum polluted soil.

Exploring the metabolic characteristics of indigenous PAH degraders is critical to understanding the PAH bioremediation mechanism in the natural environment. While stable-isotopic probing (SIP) is a viable method to identify functional microorganisms in complex environments, the metabolic characteristics of uncultured degraders are still elusive. Here, we investigated the naphthalene (NAP) biodegradation of petroleum polluted soils by combining SIP, amplicon sequencing and metagenome binning. Based on the SIP and amplicon sequencing results, an uncultured Gammaproteobacterium sp. was identified as the key NAP degrader. Additionally, the assembled genome of this uncultured degrader was successfully obtained from the 13 C-DNA metagenomes by matching its 16S rRNA gene with the SIP identified OTU sequence. Meanwhile, a number of NAP degrading genes encoding naphthalene/PAH dioxygenases were identified in this genome, further confirming the direct involvement of this indigenous degrader in the NAP degradation. The degrader contained genes related to the metabolisms of several carbon sources, energy substances and vitamins, illuminating potential reasons for why microorganisms cannot be cultivated and finally realize their cultivation. Our findings provide novel information on the mechanisms of in situ PAH biodegradation and add to our current knowledge on the cultivation of non-culturable microorganisms by combining both SIP and metagenome binning.

The catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation in oil-contaminated soil.

Rhizoremediation is a potential technique for polycyclic aromatic hydrocarbon (PAH) remediation; however, the catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation remain unclear. To address these issues, stable-isotope-probing coupled with metagenomics and molecular ecological network analyses were first used to investigate the phenanthrene rhizoremediation by three different prairie grasses in this study. All rhizospheres exhibited a significant increase in phenanthrene removal and markedly modified the diversity of phenanthrene degraders by increasing their populations and interactions with other microbes. Of all the active phenanthrene degraders, Marinobacter and Enterobacteriaceae dominated in the bare and switchgrass rhizosphere respectively; Achromobacter was markedly enriched in ryegrass and tall fescue rhizospheres. Metagenomes of 13 C-DNA illustrated several complete pathways of phenanthrene degradation for each rhizosphere, which clearly explained their unique rhizoremediation mechanisms. Additionally, propanoate and inositol phosphate of carbohydrates were identified as the dominant factors that drove PAH rhizoremediation by strengthening the ecological networks of soil microbial communities. This was verified by the results of rhizospheric and non-rhizospheric treatments supplemented with these two substances, further confirming their key roles in PAH removal and in situ PAH rhizoremediation. Our study offers novel insights into the mechanisms of in situ rhizoremediation at PAH-contaminated sites.

Stimulation of phenanthrene and biphenyl degradation by biochar-conducted long distance electron transfer in soil bioelectrochemical systems.

The bioelectrochemical degradation of organic pollutants has attracted considerable attention owing to its remarkable sustainability and low cost. However, the application of bioelectrochemical system (BES) for the degradation of pollutants in soils is hindered by limitations in the effective distance in the soil matrix. In this study, a biochar-amended BES was constructed to evaluate the degradation of organic pollutants. This system was expected to extend the electron transfer distance via conductive biochar in soils. The results showed that biochar pyrolyzed at 900 °C facilitated the degradation of phenanthrene (PHE) and biphenyl (BP) in the soil BES (SBES), reaching 86.4%-95.1% and 88.8%-95.3% in 27 days, respectively. The effective distance of SBESs was estimated to be 154-271 cm away from the electrode, which increased 1.9-3 fold after the addition of biochar. Microbial community and functional gene analysis confirmed that biochar enriched functional degrading bacteria. These findings demonstrate that the promotion of long-distance electron transfer and the formation of soil conductive networks can be achieved by biochar amendment. Thus, this study provides a basis for the effective degradation of for persistent organic pollutants in petroleum-contaminated soils using bioelectrochemical strategy.