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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 significant ly 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. [ABSTRACT FROM AUTHOR]

Understanding the metabolic characteristics and controlled geochemical factors of functional microorganisms in petroleum‐contaminated areas at different locations is pivotal for enhancing pollutant removal strategies. To address the existing research gap in this domain, we employed stable‐isotope‐probing (SIP) with multi‐isotope labeling substrates, combined with 16S amplicon sequencing, metagenomic sequencing, and geochemical factor analysis. Utilizing n‐hexadecane and phenanthrene as model compounds, our study revealed location‐specific differences in the composition of functional microorganisms. Despite these variances, key players such as Pseudomonas, Marinobacter, Alcanivorax, Ochrobactrum, and Sphingomonas consistently emerged as active degraders of n‐hexadecane and/or phenanthrene. Several genera, including Pseudomonas, Ochrobactrum, Alcanivorax, Nitriliruptoraceae, and Sphingobacterium, demonstrated versatility by effectively degrading both contaminants. SIP‐metagenomic binning facilitated the acquisition of genomes from key active degraders, such as Pseudomonas sp., Ochrobactrum sp., Sphingomonas sp., and Shinella sp. This enabled a comprehensive analysis of petroleum hydrocarbon degradation pathways and genes, encompassing PAH dioxygenase genes, alkB genes, phthalate, and salicylate‐related pathways. Environmental factor and variation partitioning analysis revealed that oil pollution significantly influences the functional microbial community (12%), followed by available potassium and available nitrogen. Geochemical parameters and geographic location independently explained 14% and 21% of total variations, respectively. Intriguingly, more than half (51%) of the variation in functional microbial community structure remains unexplained, possibly due to unmeasured environmental variables. Our study contributes valuable insights into the in situ bioremediation mechanism for petroleum‐contaminated soil, elucidating factors influencing functional microbial structures across locations. These findings provide a vital theoretical reference for in situ regulation and bioremediation of petroleum hydrocarbon pollution in diverse environmental contexts. Plain Language Summary: Understanding how microorganisms function in petroleum‐contaminated areas at different locations is crucial for improving pollutant removal strategies. In our study, we used stable‐isotope probing (SIP) and various techniques to analyze the metabolic characteristics and controlled geochemical factors influencing these microorganisms. By focusing on n‐hexadecane and phenanthrene, we identified location‐specific variations in microbial composition, with consistent roles played by key degraders like Pseudomonas, Marinobacter, Alcanivorax, Ochrobactrum, and Sphingomonas. Some genera, including Pseudomonas, Ochrobactrum, Alcanivorax, Nitriliruptoraceae, and Sphingobacterium, demonstrated versatility in degrading both contaminants. SIP‐metagenomic binning allowed genome acquisition for a comprehensive analysis of hydrocarbon degradation pathways. Our findings emphasize the crucial role of these microorganisms in in situ petroleum biodegradation and their bioremediation potential. Environmental factor analysis revealed oil pollution as a significant influencer (12%), followed by AK and AN. Geochemical parameters and geographic location independently explained 14% and 21% of total variations. Over half (51%) of the variation remains unexplained, suggesting unknown environmental factors. Our study provides vital insights into in situ bioremediation mechanisms, elucidating factors influencing microbial structures across locations and offering a theoretical foundation for regulating and remediating petroleum pollution in diverse environmental contexts. Key Points: Our study unveils distinct microbial compositions involved in hydrocarbon degradation, varying across specific locations for the first timeUsing stable‐isotope probing‐metagenomics, we obtained key hydrocarbon degraders' genomes, enhancing pathway analysis and genomic insightsOil pollution (12%) impacts microbial communities; geochemical factors and location contribute 14% and 21% to variations [ABSTRACT FROM AUTHOR]

BackgroundLinking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling.ResultsOur HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13C-AMF hyphosphere DNA from a 13CO2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10-33 atom% 13C), even though the soils' overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera.ConclusionsOur semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract.

The functioning, health, and productivity of soil are intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable-isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a 13CO2 atmosphere into soil. We collected paired rhizosphere and nonrhizosphere soil at 6 and 9 weeks of plant growth and extracted DNA that was then separated by density using ultracentrifugation. Thirty-two fractions from each of five samples were grouped by density, sequenced, assembled, and binned to generate 55 unique bacterial genomes that were ≥70% complete. We also identified complete 18S rRNA sequences of several 13C-enriched microeukaryotic bacterivores and fungi. We generated 10 circularized bacteriophage (phage) genomes, some of which were the most labeled entities in the rhizosphere, suggesting that phage may be important agents of turnover of plant-derived C in soil. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genome bins for traits known to be used in interactions involving bacteria, microeukaryotes, and plant roots and found DNA from heavily 13C-labeled bacterial genes thought to be involved in modulating plant signaling hormones, plant pathogenicity, and defense against microeukaryote grazing. Stable-isotope-informed, genome-resolved metagenomics indicated that phage can be important agents of turnover of plant-derived carbon in soil. IMPORTANCE Plants grow in intimate association with soil microbial communities; these microbes can facilitate the availability of essential resources to plants. Thus, plant productivity commonly depends on interactions with rhizosphere bacteria, viruses, and eukaryotes. Our work is significant because we identified the organisms that took up plant-derived organic C in rhizosphere soil and determined that many of the active bacteria are plant pathogens or can impact plant growth via hormone modulation. Further, by showing that bacteriophage accumulate CO2-derived carbon, we demonstrated their vital roles in redistribution of plant-derived C into the soil environment through bacterial cell lysis. The use of stable-isotope probing (SIP) to identify consumption (or lack thereof) of root-derived C by key microbial community members within highly complex microbial communities opens the way for assessing manipulations of bacteria and phage with potentially beneficial and detrimental traits, ultimately providing a path to improved plant health and soil carbon storage.

Soil bacteria and fungi are known to form niche-specific communities that differ between actively growing and decaying roots. Yet almost nothing is known about the cross-kingdom interactions that frame these communities and the environmental filtering that defines these potentially friendly or competing neighbors. We explored the temporal and spatial patterns of soil fungal (mycorrhizal and nonmycorrhizal) and bacterial cooccurrence near roots of wild oat grass, Avena fatua, growing in its naturalized soil in a greenhouse experiment. Amplicon sequences of the fungal internal transcribed spacer (ITS) and bacterial 16S rRNA genes from rhizosphere and bulk soils collected at multiple plant growth stages were used to construct covariation-based networks as a step toward identifying fungal-bacterial associations. Corresponding stable-isotope-enabled metagenome-assembled genomes (MAGs) of bacteria identified in cooccurrence networks were used to inform potential mechanisms underlying the observed links. Bacterial-fungal networks were significantly different in rhizosphere versus bulk soils and between arbuscular mycorrhizal fungi (AMF) and nonmycorrhizal fungi. Over 12 weeks of plant growth, nonmycorrhizal fungi formed increasingly complex networks with bacteria in rhizosphere soils, while AMF more frequently formed networks with bacteria in bulk soils. Analysis of network-associated bacterial MAGs suggests that some of the fungal-bacterial links that we identified are potential indicators of bacterial breakdown and consumption of fungal biomass, while others intimate shared ecological niches.IMPORTANCE Soils near living and decomposing roots form distinct niches that promote microorganisms with distinctive environmental preferences and interactions. Yet few studies have assessed the community-level cooccurrence of bacteria and fungi in these soil niches as plant roots grow and senesce. With plant growth, we observed increasingly complex cooccurrence networks between nonmycorrhizal fungi and bacteria in the rhizosphere, while mycorrhizal fungal (AMF) and bacterial cooccurrence was more pronounced in soil further from roots, in the presence of decaying root litter. This rarely documented phenomenon suggests niche sharing of nonmycorrhizal fungi and bacteria, versus niche partitioning between AMF and bacteria; both patterns are likely driven by C substrate availability and quality. Although the implications of species cooccurrence are fiercely debated, MAGs matching the bacterial nodes in our networks possess the functional potential to interact with the fungi that they are linked to, suggesting an ecological significance of fungal-bacterial cooccurrence patterns.

Abstract Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13C-AMF hyphosphere DNA from a 13CO2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13C), even though the soils’ overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera. Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract

Productive oxygen minimum zones are regions dominated by heterotrophic denitrification fueled by sinking organic matter. Microbial redox-sensitive transformations therein result in the loss and overall geochemical deficit in inorganic fixed nitrogen in the water column, thereby impacting global climate in terms of nutrient equilibrium and greenhouse gases. Here, geochemical data are combined with metagenomes, metatranscriptomes, and stable-isotope probing incubations from the water column and subseafloor of the Benguela upwelling system. The taxonomic composition of 16S rRNA genes and relative expression of functional marker genes are used to explore metabolic activities by nitrifiers and denitrifiers under decreased stratification and increased lateral ventilation in Namibian coastal waters. Active planktonic nitrifiers were affiliated with Candidatus Nitrosopumilus and Candidatus Nitrosopelagicus among Archaea, and Nitrospina, Nitrosomonas, Nitrosococcus, and Nitrospira among Bacteria. Concurrent evidence from taxonomic and functional marker genes shows that populations of Nitrososphaeria and Nitrospinota were highly active under dysoxic conditions, coupling ammonia and nitrite oxidation with respiratory nitrite reduction, but minor metabolic activity toward mixotrophic use of simple nitrogen compounds. Although active reduction of nitric oxide to nitrous oxide by Nitrospirota, Gammaproteobacteria, and Desulfobacterota was tractable in bottom waters, the produced nitrous oxide was apparently scavenged at the ocean surface by Bacteroidota. Planctomycetota involved in anaerobic ammonia oxidation were identified in dysoxic waters and their underlying sediments, but were not found to be metabolically active due to limited availability of nitrite. Consistent with water column geochemical profiles, metatranscriptomic data demonstrate that nitrifier denitrification is fueled by fixed and organic nitrogen dissolved in dysoxic waters, and prevails over canonical denitrification and anaerobic oxidation of ammonia when the Namibian coastal waters and sediment–water interface on the shelf are ventilated by lateral currents during austral winter. [ABSTRACT FROM AUTHOR]

Abstract Background Biological interaction affects diverse facets of microbial life by modulating the activity, diversity, abundance, and composition of microbial communities. Aerobic methane oxidation is a community function, with emergent community traits arising from the interaction of the methane-oxidizers (methanotrophs) and non-methanotrophs. Yet little is known of the spatial and temporal organization of these interaction networks in naturally-occurring complex communities. We hypothesized that the assembled bacterial community of the interaction network in methane hotspots would converge, driven by high substrate availability that favors specific methanotrophs, and in turn influences the recruitment of non-methanotrophs. These environments would also share more co-occurring than site-specific taxa. Results We applied stable isotope probing (SIP) using 13C-CH4 coupled to a co-occurrence network analysis to probe trophic interactions in widespread methane-emitting environments, and over time. Network analysis revealed predominantly unique co-occurring taxa from different environments, indicating distinctly co-evolved communities more strongly influenced by other parameters than high methane availability. Also, results showed a narrower network topology range over time than between environments. Co-occurrence pattern points to Chthoniobacter as a relevant yet-unrecognized interacting partner particularly of the gammaproteobacterial methanotrophs, deserving future attention. In almost all instances, the networks derived from the 13C-CH4 incubation exhibited a less connected and complex topology than the networks derived from the unlabelledC-CH4 incubations, likely attributable to the exclusion of the inactive microbial population and spurious connections; DNA-based networks (without SIP) may thus overestimate the methane-dependent network complexity. Conclusion We demonstrated that site-specific environmental parameters more strongly shaped the co-occurrence of bacterial taxa than substrate availability. Given that members of the interactome without the capacity to oxidize methane can exert interaction-induced effects on community function, understanding the co-occurrence pattern of the methane-driven interaction network is key to elucidating community function, which goes beyond relating activity to community composition, abundances, and diversity. More generally, we provide a methodological strategy that substantiates the ecological linkages between potentially interacting microorganisms with broad applications to elucidate the role of microbial interaction in community function.

Productive oxygen minimum zones are regions dominated by heterotrophic denitrification fueled by sinking organic matter. Microbial redox-sensitive transformations therein result in the loss and overall geochemical deficit in inorganic fixed nitrogen in the water column, thereby impacting global climate in terms of nutrient equilibrium and greenhouse gases. Here, geochemical data are combined with metagenomes, metatranscriptomes, and stable-isotope probing incubations from the water column and subseafloor of the Benguela upwelling system. The taxonomic composition of 16S rRNA genes and relative expression of functional marker genes are used to explore metabolic activities by nitrifiers and denitrifiers under decreased stratification and increased lateral ventilation in Namibian coastal waters. Active planktonic nitrifiers were affiliated with Candidatus Nitrosopumilus and Candidatus Nitrosopelagicus among Archaea, and Nitrospina, Nitrosomonas, Nitrosococcus, and Nitrospira among Bacteria. Concurrent evidence from taxonomic and functional marker genes shows that populations of Nitrososphaeria and Nitrospinota were highly active under dysoxic conditions, coupling ammonia and nitrite oxidation with respiratory nitrite reduction, but minor metabolic activity toward mixotrophic use of simple nitrogen compounds. Although active reduction of nitric oxide to nitrous oxide by Nitrospirota, Gammaproteobacteria, and Desulfobacterota was tractable in bottom waters, the produced nitrous oxide was apparently scavenged at the ocean surface by Bacteroidota. Planctomycetota involved in anaerobic ammonia oxidation were identified in dysoxic waters and their underlying sediments, but were not found to be metabolically active due to limited availability of nitrite. Consistent with water column geochemical profiles, metatranscriptomic data demonstrate that nitrifier denitrification is fueled by fixed and organic nitrogen dissolved in dysoxic waters, and prevails over canonical denitrification and anaerobic oxidation of ammonia when the Namibian coastal waters and sediment–water interface on the shelf are ventilated by lateral currents during austral winter.

The rise and extensive spread of antimicrobial resistance (AMR) has become a growing concern, and a threat to the environment and human health globally. The majority of current AMR identification methods used in clinical setting are based on traditional microbiology culture-dependent techniques which are time-consuming or expensive to be implemented, thus appropriate antibiotic stewardship is provided retrospectively which means the first line of treatment is to hope that a broad-spectrum antibiotic works. Hence, culture-independent and single-cell technologies are needed to allow for rapid detection and identification of antimicrobial-resistant bacteria and to support a more targeted and effective antibiotic therapy preventing further development and spread of AMR. In this study, for the first time, a non-destructive phenotyping method of optical photothermal infrared (O-PTIR) spectroscopy, coupled with deuterium isotope probing (DIP) and multivariate statistical analysis was employed as a metabolic fingerprinting approach to detect AMR in Uropathogenic Escherichia coli (UPEC) at both single-cell and population levels. Principal component-discriminant function analysis (PC-DFA) of FT-IR and O-PTIR spectral data showed clear clustering patterns as a result of distinctive spectral shifts (C–D signature peaks) originating from deuterium incorporation into bacterial cells, allowing for rapid detection and classification of sensitive and resistant isolates at the single-cell level. Furthermore, the single-frequency images obtained using the C–D signature peak at 2,163 cm−1 clearly displayed the reduced ability of the trimethoprim-sensitive strain for incorporating deuterium when exposed to this antibiotic, compared to the untreated condition. Hence, the results of this study indicated that O-PTIR can be employed as an efficient tool for the rapid detection of AMR at the single-cell level.

Members of genus Nitrospira represent the most diverse nitrite-oxidizing bacteria (NOB) and are ubiquitous in terrestrial ecosystems, which complete the nitrification process by converting nitrite to nitrate. NOB has shown physiological adaptation to a wide range of environmental conditions, including extreme pH and salinity. However, little is known about their ecological performances and adaptation features in salt-affected soils. Here, we revealed the genomic adaptations of active NOB in a saline-sodic soil (solonetz) with high Na+ stress (20.9 cmolc exchangeable Na+ kg−1 soil) and pH (9.64), using approaches of 13C-DNA stable-isotope tracing and amplicon-based and metagenomic sequencing. Two novel Nitrospira lineage IV–affiliated genomes ("Nitrospira sp. Sol IV1" and "Nitrospira sp. Sol IV2") were identified by 13C-labeled metagenomic assemble and represented the primary contributors in the solonetz. The genomic analysis of Sol IV1 and IV2 revealed various adaptive strategies for salt tolerance, including direct Na+ extrusion/H+ import, compatible-solute accumulations, and highly efficient energy supply. In contrast, the Nitrospira lineages I and V with much less salt-adaptation features co-dominated the nitrite oxidation in an adjacent nonsaline farmland soil converted from the solonetz, indicating salt tolerance–based niche differentiation of distinct soil NOB phylotypes. This study provides the first direct evidence for autotrophic nitrite oxidation of Nitrospira lineage IV in soil and their critical ecological role in regulating nitrogen (N) cycling in hypersaline terrestrial ecosystems, expanding our understanding of the habitat occupation range and metabolic versatility of nitrifying communities. [ABSTRACT FROM AUTHOR]

Background: Biological interaction affects diverse facets of microbial life by modulating the activity, diversity, abundance, and composition of microbial communities. Aerobic methane oxidation is a community function, with emergent community traits arising from the interaction of the methane-oxidizers (methanotrophs) and non-methanotrophs. Yet little is known of the spatial and temporal organization of these interaction networks in naturally-occurring complex communities. We hypothesized that the assembled bacterial community of the interaction network in methane hotspots would converge, driven by high substrate availability that favors specific methanotrophs, and in turn influences the recruitment of non-methanotrophs. These environments would also share more co-occurring than site-specific taxa. Results: We applied stable isotope probing (SIP) using 13C-CH4 coupled to a co-occurrence network analysis to probe trophic interactions in widespread methane-emitting environments, and over time. Network analysis revealed predominantly unique co-occurring taxa from different environments, indicating distinctly co-evolved communities more strongly influenced by other parameters than high methane availability. Also, results showed a narrower network topology range over time than between environments. Co-occurrence pattern points to Chthoniobacter as a relevant yet-unrecognized interacting partner particularly of the gammaproteobacterial methanotrophs, deserving future attention. In almost all instances, the networks derived from the 13C-CH4 incubation exhibited a less connected and complex topology than the networks derived from the unlabelledC-CH4 incubations, likely attributable to the exclusion of the inactive microbial population and spurious connections; DNA-based networks (without SIP) may thus overestimate the methane-dependent network complexity. Conclusion: We demonstrated that site-specific environmental parameters more strongly shaped the co-occurrence of bacterial taxa than substrate availability. Given that members of the interactome without the capacity to oxidize methane can exert interaction-induced effects on community function, understanding the co-occurrence pattern of the methane-driven interaction network is key to elucidating community function, which goes beyond relating activity to community composition, abundances, and diversity. More generally, we provide a methodological strategy that substantiates the ecological linkages between potentially interacting microorganisms with broad applications to elucidate the role of microbial interaction in community function. [ABSTRACT FROM AUTHOR]

Acidimicrobiia are widely distributed in nature and suggested to be autotrophic via the Calvin-Benson-Bassham (CBB) cycle. However, direct evidence of chemolithoautotrophy in Acidimicrobiia is lacking. Here, we report a chemolithoautotrophic enrichment from a saline lake, and the subsequent isolation and characterization of a chemolithoautotroph, Salinilacustristhrix flava EGI L10123T, which belongs to a new Acidimicrobiia family. Although strain EGI L10123T is autotrophic, neither its genome nor Acidimicrobiia metagenome-assembled genomes (MAGs) from the enrichment culture encode genes necessary for the CBB cycle. Instead, genomic, transcriptomic, enzymatic, and stable-isotope probing data hinted at the activity of the reversed oxidative TCA (roTCA) coupled with the oxidation of sulfide as the electron donor. Phylogenetic analysis and ancestral character reconstructions of Acidimicrobiia suggested that the essential CBB gene rbcL was acquired through multiple horizontal gene transfer events from diverse microbial taxa. In contrast, genes responsible for sulfide- or hydrogen-dependent roTCA carbon fixation were already present in the last common ancestor of extant Acidimicrobiia. These findings imply the possibility of roTCA carbon fixation in Acidimicrobiia and the ecological importance of Acidimicrobiia. Further research in the future is necessary to confirm whether these characteristics are truly widespread across the clade., (© The Author(s) [2024]. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

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 A spergillus sp. LJD-29 and the bacterial strain P seudomonas 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 13 C-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., Competing Interests: The authors declare no conflict of interest.

Methanotrophs are the only biofilters for reducing the flux of global methane (CH4) emissions in water-logged wetlands. However, adaptation of aerobic methanotrophs to low concentrations of oxygen and nitrogen in typical swamps, such as that of the Qinghai–Tibetan Plateau, is poorly understood. In this study, we show that Methylobacter-like methanotrophs dominate methane oxidation and nitrogen fixation under suboxic conditions in alpine swamp soils. Following incubation with 13C-CH4 and 15N-N2 for 90 days under suboxic conditions with repeated flushing using an inert gas (i.e., argon), microbial carbon and nitrogen turnover was measured in swamp soils at different depths: 0–20 cm (top), 40–60 cm (intermediate), and 60–80 cm (deep). Results show detectable methane oxidation and nitrogen fixation in all three soil depths. In particular, labeled carbon was found in CO2 enrichment (13C-CO2), and soil organic carbon (13C-SOC), whereas labeled nitrogen (15N) was detected in soil organic nitrogen (SON). The highest values of labeled isotopes were found at intermediate soil depths. High-throughput amplicon sequencing and Sanger sequencing indicated the dominance of Methylobacter-like methanotrophs in swamp soils, which comprised 21.3–24.0% of the total bacterial sequences, as measured by 13C-DNA at day 90. These results demonstrate that aerobic methanotroph Methylobacter is the key player in suboxic methane oxidation and likely catalyzes nitrogen fixation in swamp wetland soils in the Qinghai–Tibetan Plateau.

Methanotrophs are the only biofilters for reducing the flux of global methane (CH4) emissions in water-logged wetlands. However, adaptation of aerobic methanotrophs to low concentrations of oxygen and nitrogen in typical swamps, such as that of the Qinghai–Tibetan Plateau, is poorly understood. In this study, we show that Methylobacter- like methanotrophs dominate methane oxidation and nitrogen fixation under suboxic conditions in alpine swamp soils. Following incubation with 13C-CH4 and 15N-N2 for 90 days under suboxic conditions with repeated flushing using an inert gas (i.e., argon), microbial carbon and nitrogen turnover was measured in swamp soils at different depths: 0–20 cm (top), 40–60 cm (intermediate), and 60–80 cm (deep). Results show detectable methane oxidation and nitrogen fixation in all three soil depths. In particular, labeled carbon was found in CO2 enrichment (13C-CO2), and soil organic carbon (13C-SOC), whereas labeled nitrogen (15N) was detected in soil organic nitrogen (SON). The highest values of labeled isotopes were found at intermediate soil depths. High-throughput amplicon sequencing and Sanger sequencing indicated the dominance of Methylobacter -like methanotrophs in swamp soils, which comprised 21.3–24.0% of the total bacterial sequences, as measured by 13C-DNA at day 90. These results demonstrate that aerobic methanotroph Methylobacter is the key player in suboxic methane oxidation and likely catalyzes nitrogen fixation in swamp wetland soils in the Qinghai–Tibetan Plateau. [ABSTRACT FROM AUTHOR]

ABSTRACT Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after growing on [13C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [13C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (

Plant-derived carbon (C) is considered fundamental to understand the interaction between rhizosphere microbes and plants in terrestrial ecosystems. Biochar soil amendment may enhance plant performance via changing soil properties or microbial diversity in the rhizosphere. However, our knowledge of how plant-microbiome associations respond to biochar amendment remains rather limited. Herein, 13CO2 steady-state labeling combined with DNA stable-isotope probing was used to characterize soil bacterial communities in the rhizosphere contributing to the utilization of plant-derived C. The diversity of bacteria active in the utilization of root exudates was determined after biochar amendment in a legume-based intercropping system (Vicia faba L., with Zea mays L.). The results showed the biochar application not only changed the bacterial community structure and diversity in the rhizosphere, but also altered bacterial members actively assimilating plant-derived C. There were more labeled species in the biochar-amended soils than the control soils. Compared with the control, the biochar amendment increased the relative abundances of Firmicutes and Bacteroidetes members (i.e., Bacillus, Clostridium, Sporomusa, Desulfosporosinus, and Alicyclobacillus) while decreasing the abundances of Proteobacteria members (e.g., Methylobacterium and Sphingomonas) utilizing plant-derived C. In contrast, slow-growing species of the phyla Acidobacteria, Planctomycetes, and Gemmatimonadetes were barely labeled. The bacteria found stimulated by the biochar amendment are known for their ability to fix nitrogen, solubilize phosphorus, or reduce iron and sulfur, which may potentially contribute to the “biochar effect” in the rhizosphere. This study is the first to provide empirical evidence that biochar amendment can alter the soil bacterial community assimilating plant-derived C; this may have consequences for nutrient cycling and improving plant performance in intercropping systems.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Ammonium concentrations and temperature drive the activities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), but their effects on these microbes in eutrophic freshwater sediments are unclear. In this study, surface sediments collected from areas of Taihu Lake (China) with different degrees of eutrophication were incubated under three levels of nitrogen input and temperature, and the autotrophic growth of ammonia oxidizers was assessed using 13C-labeled DNA-based stable-isotope probing (SIP), while communities were characterized using MiSeq sequencing and phylogenetic analysis of 16S rRNA genes. Nitrification rates in sediment microcosms were positively correlated with nitrogen inputs, but there was no marked association with temperature. Incubation of SIP microcosms indicated that AOA and AOB amoA genes were labeled by 13C at 20°C and 30°C in the slightly eutrophic sediment, and AOB amoA genes were labeled to a much greater extent than AOA amoA genes in the moderately eutrophic sediment after 56 days. Phylogenetic analysis of 13C-labeled 16S rRNA genes revealed that the active AOA were mainly affiliated with the Nitrosopumilus cluster, with the Nitrososphaera cluster dominating in the slightly eutrophic sediment at 30°C with low ammonium input (1 mM). Active AOB communities were more sensitive to nitrogen input and temperature than were AOA communities, and they were exclusively dominated by the Nitrosomonas cluster, which tended to be associated with Nitrosomonadaceae-like lineages. Nitrosomonas sp. strain Is79A3 tended to dominate the moderately eutrophic sediment at 10°C with greater ammonium input (2.86 mM). The relative abundance responses of the major active communities to nitrogen input and temperature gradients varied, indicating niche differentiation and differences in the physiological metabolism of ammonia oxidizers that are yet to be described. [ABSTRACT FROM AUTHOR]

Plant-derived carbon (C) is considered fundamental to understand the interaction between rhizosphere microbes and plants in terrestrial ecosystems. Biochar soil amendment may enhance plant performance via changing soil properties or microbial diversity in the rhizosphere. However, our knowledge of how plant-microbiome associations respond to biochar amendment remains rather limited. Herein, 13CO2 steady-state labeling combined with DNA stable-isotope probing was used to characterize soil bacterial communities in the rhizosphere contributing to the utilization of plant-derived C. The diversity of bacteria active in the utilization of root exudates was determined after biochar amendment in a legume-based intercropping system (Vicia faba L., with Zea mays L.). The results showed the biochar application not only changed the bacterial community structure and diversity in the rhizosphere, but also altered bacterial members actively assimilating plant-derived C. There were more labeled species in the biochar-amended soils than the control soils. Compared with the control, the biochar amendment increased the relative abundances of Firmicutes and Bacteroidetes members (i.e., Bacillus , Clostridium , Sporomusa , Desulfosporosinus , and Alicyclobacillus) while decreasing the abundances of Proteobacteria members (e.g., Methylobacterium and Sphingomonas) utilizing plant-derived C. In contrast, slow-growing species of the phyla Acidobacteria, Planctomycetes, and Gemmatimonadetes were barely labeled. The bacteria found stimulated by the biochar amendment are known for their ability to fix nitrogen, solubilize phosphorus, or reduce iron and sulfur, which may potentially contribute to the "biochar effect" in the rhizosphere. This study is the first to provide empirical evidence that biochar amendment can alter the soil bacterial community assimilating plant-derived C; this may have consequences for nutrient cycling and improving plant performance in intercropping systems. [ABSTRACT FROM AUTHOR]

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 13 C-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.

The rhizospheric microbiome is clearly affected by plant species and certain of their functional traits. These functional traits allow plants to adapt to their environmental conditions by acquiring or conserving nutrients, thus defining different ecological resource-use plant strategies. In the present study, we investigated whether plants with one of the two nutrient-use strategies (conservative versus exploitative) could influence fungal communities involved in soil organic matter degradation and root exudate assimilation, as well as those colonizing root tissues. We applied a DNA-based, stable-isotope probing (DNA-SIP) approach to four grass species distributed along a gradient of plant nutrient resource strategies, ranging from conservative to exploitative species, and analyzed their associated mycobiota composition using a fungal internal transcribed spacer (ITS) and Glomeromycotina 18S rRNA gene metabarcoding approach. Our results demonstrated that fungal taxa associated with exploitative and conservative plants could be separated into two general categories according to their location: generalists, which are broadly distributed among plants from each strategy and represent the core mycobiota of soil organic matter degraders, root exudate consumers in the root-adhering soil, and root colonizers; and specialists, which are locally abundant in one species and more specifically involved in soil organic matter degradation or root exudate assimilation on the root-adhering soil and the root tissues. Interestingly, for arbuscular mycorrhizal fungi analysis, all plant roots were mainly colonized by Glomus species, whereas an increased diversity of Glomeromycotina genera was observed for the exploitative plant species Dactylis glomerata. [ABSTRACT FROM AUTHOR]

Processes governing the fixation, partitioning, and mineralization of carbon in soils are under increasing scrutiny as we develop a more comprehensive understanding of global carbon cycling. Here we examined fixation by Douglas-fir seedlings and transfer to associated ectomycorrhizal fungi, soil microbes, and full-sibling or nonsibling neighbouring seedlings., Stable isotope probing with 99% 13C- CO2 was applied to trace 13C-labelled photosynthate throughout plants, fungi, and soil microbes in an experiment designed to assess the effect of relatedness on 13C transfer between plant pairs. The fixation and transfer of the 13C label to plant, fungal, and soil microbial tissue was examined in biomass and phospholipid fatty acids., After a 6 d chase period, c. 26.8% of the 13C remaining in the system was translocated below ground. Enrichment was proportionally greatest in ectomycorrhizal biomass. The presence of mesh barriers (0.5 or 35 μm) between seedlings did not restrict 13C transfer., Fungi were the primary recipients of 13C-labelled photosynthate throughout the system, representing 60-70% of total 13C-enriched phospholipids. Full-sibling pairs exhibited significantly greater 13C transfer to recipient roots in two of four Douglas-fir families, representing three- and fourfold increases (+ c. 4 μg excess 13C) compared with nonsibling pairs. The existence of a root/mycorrhizal exudation-hyphal uptake pathway was supported. [ABSTRACT FROM AUTHOR]

Autotrophic denitrification has attracted increasing attention for wastewater with insufficient organic carbon sources. Nevertheless, in situ identification of autotrophic denitrifying communities in reactors remains challenging. Here, a process combining micro-electrolysis and autotrophic denitrification with high nitrate removal efficiency was presented. Two batch reactors were fed organic-free nitrate influent, with H 13 CO 3 − and H 12 CO 3 − as inorganic carbon sources. DNA-based stable-isotope probing (DNA-SIP) was used to obtain molecular evidence for autotrophic denitrifying communities. The results showed that the nirS gene was strongly labeled by H 13 CO 3 − , demonstrating that the inorganic carbon source was assimilated by autotrophic denitrifiers. High-throughput sequencing and clone library analysis identified Thiobacillus -like bacteria as the most dominant autotrophic denitrifiers. However, 88% of nirS genes cloned from the 13 C-labeled “heavy” DNA fraction showed low similarity with all culturable denitrifiers. These findings provided functional and taxonomical identification of autotrophic denitrifying communities, facilitating application of autotrophic denitrification process for wastewater treatment. [ABSTRACT FROM AUTHOR]

Northern peatlands in general have high methane (CH4) emissions, but individual peatlands show considerable variation as CH4 sources. Particularly in nutrient-poor peatlands, CH4 production can be low and exceeded by carbon dioxide (CO2) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO2 to CH4 produced. After [13C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH4 and CO2. The oligotrophic fen had lower CH4 production but produced 3 to 59 times more CO2 than CH4. RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria. The oligotrophic peat with excess CO2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia. Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO2 production in peatlands. [ABSTRACT FROM AUTHOR]