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.

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.

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.

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.

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.

Stable-Isotope Probing-Enabled Cultivation of the Indigenous Bacterium Ralstonia sp. Strain M1, Capable of Degrading Phenanthrene and Biphenyl in Industrial Wastewater.

To identify and obtain the indigenous degraders metabolizing phenanthrene (PHE) and biphenyl (BP) from the complex microbial community within industrial wastewater, DNA-based stable-isotope probing (DNA-SIP) and cultivation-based methods were applied in the present study. DNA-SIP results showed that two bacterial taxa (Vogesella and Alicyclobacillus) were considered the key biodegraders responsible for PHE biodegradation only, whereas Bacillus and Cupriavidus were involved in BP degradation. Vogesella and Alicyclobacillus have not been linked with PHE degradation previously. Additionally, DNA-SIP helped reveal the taxonomic identity of Ralstonia-like degraders involved in both PHE and BP degradation. To target the separation of functional Ralstonia-like degraders from the wastewater, we modified the traditional cultivation medium and culture conditions. Finally, an indigenous PHE- and BP-degrading strain, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by enrichment of the 16S rRNA gene and distinctive dioxygenase genes in the DNA-SIP experiment. Our study has successfully established a program for the application of DNA-SIP in the isolation of the active functional degraders from an environment. It also deepens our insight into the diversity of indigenous PHE- and BP-degrading communities.IMPORTANCE The comprehensive treatment of wastewater in industrial parks suffers from the presence of multiple persistent organic pollutants (POPs), such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which reduce the activity of activated sludge and are difficult to eliminate. Characterizing and applying active bacterial degraders metabolizing multiple POPs therefore helps to reveal the mechanisms of synergistic metabolism and to improve wastewater treatment efficiency in industrial parks. To date, SIP studies have successfully investigated the biodegradation of PAHs or PCBs in real-world habitats. DNA-SIP facilitates the isolation of target microorganisms that pose environmental concerns. Here, an indigenous phenanthrene (PHE)- and biphenyl (BP)-degrading strain in wastewater, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by DNA-SIP. Our study provides a routine protocol for the application of DNA-SIP in the isolation of the active functional degraders from an environment.

Autochthonous Bioaugmentation-Modified Bacterial Diversity of Phenanthrene Degraders in PAH-Contaminated Wastewater as Revealed by DNA-Stable Isotope Probing.

To reveal the mechanisms of autochthonous bioaugmentation (ABA) in wastewater contaminated with polycyclic aromatic hydrocarbons (PAHs), DNA-stable-isotope-probing (SIP) was used in the present study with the addition of an autochthonous microorganism Acinetobacter tandoii LJ-5. We found LJ-5 inoculum produced a significant increase in phenanthrene (PHE) mineralization, but LJ-5 surprisingly did not participate in indigenous PHE degradation from the SIP results. The improvement of PHE biodegradation was not explained by the engagement of LJ-5 but attributed to the remarkably altered diversity of PHE degraders. Of the major PHE degraders present in ambient wastewater ( Rhodoplanes sp., Mycobacterium sp., Xanthomonadaceae sp. and Enterobacteriaceae sp.), only Mycobacterium sp. and Enterobacteriaceae sp. remained functional in the presence of strain LJ-5, but five new taxa Bacillus, Paenibacillus, Ammoniphilus, Sporosarcina, and Hyphomicrobium were favored. Rhodoplanes, Ammoniphilus, Sporosarcina, and Hyphomicrobium were directly linked to, for the first time, indigenous PHE biodegradation. Sequences of functional PAH-RHDα genes from heavy fractions further proved the change in PHE degraders by identifying distinct PAH-ring hydroxylating dioxygenases between ambient degradation and ABA. Our findings indicate a new mechanism of ABA, provide new insights into the diversity of PHE-degrading communities, and suggest ABA as a promising in situ bioremediation strategy for PAH-contaminated wastewater.

MiR-26a Mediates Ultraviolet B-Induced Apoptosis by Targeting Histone Methyltransferase EZH2 Depending on Myc Expression.

BACKGROUND/AIMS: Ultraviolet B (UVB) damage is the most essential etiological factor in skin carcinogenesis, and apoptosis leads to the efficient elimination of UVB-damaged cells. However, the mechanisms underlying resistance to UVB-induced apoptosis remain unclear. METHODS: HaCaT and A431 cells were used in the present study. Quantitative real-time PCR, single cell PCR, and western blotting were used to examine cancer-related gene expression at the mRNA and protein levels. RESULTS: We report that miR-26a, which is upregulated upon UVB irradiation, promotes UVB-induced apoptosis in HaCaT cells by targeting the histone methyltransferase EZH2. Moreover, the UVB/miR-26a/EZH2 regulatory axis largely depends on the MYC expression level. Interestingly, treatment with EZH2 inhibitors significantly enhanced UVB-induced apoptosis. CONCLUSION: miR-26a/EZH2 might be potential targets for skin cancer prevention and therapy.

Biodegradation of Phenanthrene in Polycyclic Aromatic Hydrocarbon-Contaminated Wastewater Revealed by Coupling Cultivation-Dependent and -Independent Approaches.

The indigenous microorganisms responsible for degrading phenanthrene (PHE) in polycyclic aromatic hydrocarbons (PAHs)-contaminated wastewater were identified by DNA-based stable isotope probing (DNA-SIP). In addition to the well-known PHE degraders Acinetobacter and Sphingobium, Kouleothrix and Sandaracinobacter were found, for the first time, to be directly responsible for indigenous PHE biodegradation. Additionally, a novel PHE degrader, Acinetobacter tandoii sp. LJ-5, was identified by DNA-SIP and direct cultivation. This is the first report and reference to A. tandoii involved in the bioremediation of PAHs-contaminated water. A PAH-RHDα gene involved in PHE metabolism was detected in the heavy fraction of 13C treatment, but the amplification of PAH-RHDα gene failed in A. tandoii LJ-5. Instead, the strain contained catechol 1,2-dioxygenase and the alpha/beta subunits of protocatechuate 3,4-dioxygenase, indicating use of the ß-ketoadipate pathway to degrade PHE and related aromatic compounds. These findings add to our current knowledge on microorganisms degrading PHE by combining cultivation-dependent and cultivation-independent approaches and provide deeper insight into the diversity of indigenous PHE-degrading communities.

Novel bacteria capable of degrading phenanthrene in activated sludge revealed by stable-isotope probing coupled with high-throughput sequencing.

The indigenous microorganisms responsible for degrading phenanthrene (PHE) in activated biosludge were identified using DNA-based stable isotope probing. Besides the well-known PHE degraders Burkholderia, Ralstonia, Sinobacteraceae and Arthrobacter, we for the first time linked the taxa Paraburkholderia and Kaistobacter with in situ PHE biodegradation. Analysis of PAH-RHDα gene detected in the heavy DNA fraction of 13C-PHE treatment suggested the mechanisms of horizontal gene transfer or inter-species hybridisation in PAH-RHD gene spread within the microbial community. Additionally, three cultivable PHE degraders, Microbacterium sp. PHE-1, Rhodanobacter sp. PHE-2 and Rhodococcus sp. PHE-3, were isolated from the same activated biosludge. Among them, Rhodanobacter sp. PHE-2 is the first identified strain in its genus with PHE-degrading ability. However, the involvement of these strains in PHE degradation in situ was questionable, due to their limited enrichment in the heavy DNA fraction of 13C-PHE treatment and lack of PAH-RHDα gene found in these isolates. Collectively, our findings provide a deeper understanding of the diversity and functions of indigenous microbes in PHE degradation.

Ornithinibacillus composti sp. nov., isolated from sludge compost and emended description of the genus Ornithinibacillus.

A Gram-stain positive, aerobic, motile, endospore-forming and rod-shaped bacterium, designated GSS05(T), was isolated from a sludge compost sample and was characterized by means of a polyphasic taxonomic approach. Growth was observed to occur with 0-3 % (w/v) NaCl (optimum 1 %), at pH 5.5-10 (optimum pH 7.5) and at 15-50 °C (optimum 37 °C). According to the results of a phylogenetic analysis, strain GSS05(T) was found to belong to the genus Ornithinibacillus and to be related most closely to the type strains of Ornithinibacillus halotolerans and Ornithinibacillus contaminans (96.5 and 95.1 % 16S rRNA gene sequence similarity, respectively). The peptidoglycan amino acid type was determined to be A4ß. The major respiratory quinone was identified as menaquinone-7 (MK-7). The polar lipid profile of strain GSS05(T) was found to contain a predominance of diphosphatidylglycerol, moderate amounts of phosphatidylglycerol and minor amounts of two unknown phospholipids and two unknown lipids. The G+C content of genomic DNA was determined to be 42.1 mol%. The dominant cellular fatty acids were identified as iso-C15:0 and anteiso-C15:0. The phenotypic, chemotaxonomic, phylogenetic and genotypic data indicated that strain GSS05(T) represents a novel species of the genus Ornithinibacillus, for which the name Ornithinibacillus composti sp. nov. is proposed. The type strain is GSS05(T) (=CCTCC AB 2013261(T) = KCTC 33192(T)).

Paenibacillus guangzhouensis sp. nov., an Fe(III)- and humus-reducing bacterium from a forest soil.

A Gram-reaction-variable, rod-shaped, motile, facultatively aerobic and endospore-forming bacterium, designated strain GSS02(T), was isolated from a forest soil. Strain GSS02(T) was capable of reducing humic substances and Fe(III) oxides. Strain GSS02(T) grew optimally at 35 °C, at pH 78 and in the presence of 1% NaCl. The predominant menaquinone was MK-7. The major cellular fatty acids were anteiso-C(15:0) and iso-C(16:0) and the polar lipid profile contained mainly phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol, with moderate amounts of two unknown aminophospholipids and a minor amount of one unknown lipid. The DNA G+C content was 53.4 mol%. Comparative 16S rRNA gene sequence analysis showed that strain GSS02(T) was related most closely to Paenibacillus terrigena JCM 21741(T) (98.1% similarity). Mean DNA-DNA relatedness between strain GSS02(T) and P. terrigena JCM 21741(T) was 58.8 ± 0.5%. The phylogenetic, chemotaxonomic and phenotypic results clearly demonstrated that strain GSS02(T) belongs to the genus Paenibacillus and represents a novel species, for which the name Paenibacillus guangzhouensis sp. nov. is proposed. The type strain is GSS02(T) ( =KCTC 33171(T) =CCTCC AB 2013236(T)).

Bacillus haikouensis sp. nov., a facultatively anaerobic halotolerant bacterium isolated from a paddy soil.

A Gram-stain positive, rod-shaped, endospore-forming and facultatively anaerobic halotolerant bacterium, designated as C-89(T), was isolated from a paddy field soil in Haikou, Hainan Province, People's Republic of China. Optimal growth was observed at 37 °C and pH 7.0 in the presence of 4% NaCl (w/v). The predominant menaquinone was identified as MK-7, the major cellular fatty acids were identified as anteiso-C(15:0) and iso-C(15:0), and the major cellular polar lipids were identified as phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unknown phospholipids. The peptidoglycan type was determined to be based on meso-DAP. Based on 16S rRNA gene sequence similarity, the closest phylogenetic relatives were identified as Bacillus vietnamensis JCM 11124(T) (98.8% sequence similarity), Bacillus aquimaris JCM 11545(T) (98.6%) and Bacillus marisflavi JCM 11544(T) (98.5%). The DNA G+C content of strain C-89(T) was determined to be 45.4 mol%. The DNA-DNA relatedness values of strain C-89(T) with its closest relatives were below 18%. Therefore, on the basis of phylogenetic, chemotaxonomic, and phenotypic results, strain C-89(T) can be considered to represent a novel species within the genus Bacillus, for which the name Bacillus haikouensis sp. nov., is proposed. The type strain is C-89(T) (=KCTC 33545(T) = CCTCC AB 2014076(T)).

Bacillus huizhouensis sp. nov., isolated from a paddy field soil.

A Gram-stain positive, facultative aerobic bacterium, designated as strain GSS03(T), was isolated from a paddy field soil. The cells were observed to be endospore forming, rod-shaped and motile with flagella. The organism was found to grow optimally at 35 °C at pH 7.0 and in the presence of 1 % NaCl. The strain was classified as a novel taxon within the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The closest phylogenetic relatives were identified as Bacillus psychrosaccharolyticus DSM 6(T) (97.61 %), Bacillus muralis DSM 16288(T) (97.55 %), Bacillus asahii JCM 12112(T) (97.48 %), Bacillus simplex DSM 1321(T) (97.48 %) and "Bacillus frigoritolerans" DSM 8801(T) (97.38 %). The menaquinone was identified as MK-7, the major cellular fatty acid was identified as anteiso-C15:0 and the major cellular polar lipids as phosphatidylethanolamine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and three unknown polar lipids. The DNA G+C content was determined to be 40.2 mol%. The DNA-DNA relatedness with the closest relatives was below 48 %. Therefore, on the basis of all the results, strain GSS03(T) is considered to represent a novel species within the genus Bacillus, for which the name Bacillus huizhouensis sp. nov. is proposed. The type strain is GSS03(T) (=KCTC 33172(T) =CCTCC AB 2013237(T)).

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.

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.

Synergism of endophytic microbiota and plants promotes the removal of polycyclic aromatic hydrocarbons from the Alfalfa rhizosphere.

Endophytic bacteria can promote plant growth and accelerate pollutant degradation. However, it is unclear whether endophytic consortia (Consortium_E) can stabilize colonisation and degradation. We inoculated Consortium_E into the rhizosphere to enhance endophytic bacteria survival and promote pollutant degradation. Rhizosphere-inoculated Consortium_E enhanced polycyclic aromatic hydrocarbon (PAH) degradation rates by 11.5-13.1 % compared with sole bioaugmentation and plant treatments. Stable-isotope-probing (SIP) showed that the rhizosphere-inoculated Consortium_E had the largest number of degraders (8 amplicon sequence variants). Furthermore, only microbes from Consortium_E were identified among the degraders in bioaugmentation treatments, indicating that directly participated in phenanthrene metabolism. Interestingly, Consortium_E reshaped the community structure of degraders without significantly altering the rhizosphere community structure, and strengthened the core position of degraders in the network, facilitating close interactions between degraders and non-degraders in the rhizosphere, which were crucial for ensuring stable functionality. The synergistic effect between plants and Consortium_E significantly enhanced the upregulation of aromatic hydrocarbon degradation and auxiliary degradation pathways in the rhizosphere. These pathways showed a non-significant increasing trend in the uninoculated rhizosphere compared with the control, indicating that Consortium_E primarily promotes rhizosphere effects. Our results explore the Consortium_E bioaugmentation mechanism, providing a theoretical basis for the ecological restoration of contaminated soils.

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.

SIP-metagenomics reveals key drivers of rhizospheric Benzo[a]pyrene bioremediation via bioaugmentation with indigenous soil microbes.

Rhizoremediation and bioaugmentation have proven effective in promoting benzo[a]pyrene (BaP) degradation in contaminated soils. However, the mechanism underlying bioaugmented rhizospheric BaP degradation with native microbes is poorly understood. In this study, an indigenous BaP degrader (Stenotrophomonas BaP-1) isolated from petroleum-contaminated soil was introduced into ryegrass rhizosphere to investigate the relationship between indigenous degraders and rhizospheric BaP degradation. Stable isotope probing and 16S rRNA gene amplicon sequencing subsequently revealed 15 BaP degraders, 8 of which were directly associated with BaP degradation including Bradyrhizobium and Streptomyces. Bioaugmentation with strain BaP-1 significantly enhanced rhizospheric BaP degradation and shaped the microbial community structure. A correlation of BaP degraders, BaP degradation efficiency, and functional genes identified active degraders and genes encoding polycyclic aromatic hydrocarbon-ring hydroxylating dioxygenase (PAH-RHD) genes as the primary drivers of rhizospheric BaP degradation. Furthermore, strain BaP-1 was shown to not only engage in BaP metabolism but also to increase the abundance of other BaP degraders and PAH-RHD genes, resulting in enhanced rhizospheric BaP degradation. Metagenomic and correlation analyses indicated a significant positive relationship between glyoxylate and dicarboxylate metabolism and BaP degradation, suggesting a role for these pathways in rhizospheric BaP biodegradation. By identifying BaP degraders and characterizing their metabolic characteristics within intricate microbial communities, our study offers valuable insights into the mechanisms of bioaugmented rhizoremediation with indigenous bacteria for high-molecular-weight PAHs in petroleum-contaminated soils.