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.

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.

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.

Soil drying legacy does not affect phenanthrene fate in soil but modifies bacterial community response.

Alteration of the structure of soil microbial communities following the elimination of hydrophobic organic pollutants (e.g., polycyclic aromatic hydrocarbons, PAHs) is generally assessed using DNA-based techniques, and soil is often required to dry prior to pollutant addition, to facilitate a better mix when establishing microcosms. However, the drying practice may have a legacy effect on soil microbial community structure, which would in turn influence the biodegradation process. Here, we used 14C-labeled phenanthrene to examine the potential side effects of precedent short-term drought events. The results indicate that the drying practice had legacy effects on soil microbial community structure, illustrated by irreversible shifts in the communities. The legacy effects had no significant impact on phenanthrene mineralization and non-extractable residue formation. However, they altered the response of bacterial communities to PAH degradation, leading to a decrease in the abundance of potential PAH degradation genes plausibly attributed to moderately abundant taxa. Based on a comparison of the varied effects of different drying intensity levels, an accurate description of microbial responses to phenanthrene degradation strongly relies on the establishment of stable microbial communities before PAH amendment. Concurrent alterations in the communities resulting from environmental perturbation could greatly mask minor alterations from the degradation of recalcitrant hydrophobic PAH. In practice, to minimize the legacy effects, a soil equilibration step with a reduced drying intensity is indispensable.

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.

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.

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.

Effects of polycyclic aromatic hydrocarbon structure on PAH mineralization and toxicity to soil microorganisms after oxidative bioremediation by laccase.

While bioremediation using soil microorganisms is considered an energy-efficient and eco-friendly approach to treat polycyclic aromatic hydrocarbon (PAH)-contaminated soils, a variety of polar PAH metabolites, particularly oxygenated ones, could increase the toxicity of the soil after biodegradation. In this study, a typical bio-oxidative transformation of PAH into quinones was investigated in soil amended with laccase using three PAHs with different structures (anthracene, benzo[a]anthracene, and benzo[a]pyrene) to assess the toxicity after oxidative bioremediation. The results show that during a 2-month incubation period the oxidation process promoted the formation of non-extractable residues (NERs) of PAHs, and different effects on mineralization were observed among the three PAHs. Oxidation enhanced the mineralization of the high-molecular-weight (HMW) PAHs (benzo[a]anthracene and benzo[a]pyrene) but inhibited the mineralization of the low-molecular-weight (LMW) PAH (anthracene). The inhibition of anthracene suggests increased toxicity after oxidative bioremediation, which coincided with a decrease in soil nitrification activity, bacterial diversity and PAH-ring hydroxylating dioxygenase gene copies. The analysis of PAH metabolites in soil extract indicated that oxidation by laccase was competitive with the natural transformation processes of PAHs and revealed that intermediates other than quinone metabolites increased the toxicity of soil during subsequent degradation. The different metabolic profiles of the three PAHs indicated that the toxicity of soil after PAH oxidation by laccase was strongly affected by the PAH structure. Despite the potential increase in toxicity, the results suggest that oxidative bioremediation is still an eco-friendly method for the treatment of HMW PAHs since the intermediates from HMW PAHs are more easily detoxified via NER formation than LMW PAHs.

Isolation of diverse pyrene-degrading bacteria via introducing readily utilized phenanthrene.

Bacteria able to degrade pyrene play a critical role in the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). However, the traditional isolation procedure only obtains strains related to the genus Mycobacterium. The aim of the present study was to develop a modified method to isolate taxonomically distinct pyrene-degrading strains. The results indicated that replacing pyrene with phenanthrene in the isolation step improved the isolation efficiency. Using the modified method, six PAH degraders belonging to the genera Bosea, Arthrobacter, Paenibacillus, Bacillus, and Rhodococcus were obtained. They were capable of effectively utilizing pyrene (∼100%) as their sole carbon source, and could co-metabolize the degradation of benzo [a]pyrene (26.9-71.5%). In contrast, a small amount of pyrene (5.6%) and benzo [a]pyrene (8.6%) were degraded by a phenanthrene-degrading Sphingobium sp. NS7 under the same conditions. The difference in PAHs degradation between agar plate culture and liquid culture may lead to the low isolation efficiency in the traditional procedure. Hereditary stability analysis showed that PAH degradation capability of the Bosea, Paenibacillus, and Rhodococcus strains were easily lost without PAH pressure, which may partly explain why those strains were difficult to obtain using the traditional method.