Stable Isotope Probing Research Articles

Constraining activity and growth substrate of fungal decomposers via assimilation patterns of inorganic carbon and water into lipid biomarkers.

Fungi are among the few organisms on the planet that can metabolize recalcitrant carbon (C) but are also known to access recently produced plant photosynthate. Therefore, improved quantification of growth and substrate utilization by different fungal ecotypes will help to define the rates and controls of fungal production, the cycling of soil organic matter, and thus the C storage and CO2 buffering capacity in soil ecosystems. This pure-culture study of fungal isolates combined a dual stable isotope probing (SIP) approach, together with rapid analysis by tandem pyrolysis-gas chromatography-isotope ratio mass spectrometry to determine the patterns of water-derived hydrogen (H) and inorganic C assimilated into lipid biomarkers of heterotrophic fungi as a function of C substrate. The water H assimilation factor (αW) and the inorganic C assimilation into C18:2 fatty acid isolated from five fungal species growing on glucose was lower (0.62% ± 0.01% and 4.7% ± 1.6%, respectively) than for species grown on glutamic acid (0.90% ± 0.02% and 7.4% ± 3.7%, respectively). Furthermore, the assimilation ratio (RIC/αW) for growth on glucose and glutamic acid can distinguish between these two metabolic modes. This dual-SIP assay thus delivers estimates of fungal activity and may help to delineate the predominant substrates that are respired among a matrix of compounds found in natural environments.IMPORTANCEFungal decomposers play important roles in food webs and nutrient cycling because they can feed on both labile and more recalcitrant forms of carbon. This study developed and applied a dual stable isotope assay (13C-dissolved inorganic carbon/2H) to improve the investigation of fungal activity in the environment. By determining the incorporation patterns of hydrogen and carbon into fungal lipids, this assay delivers estimates of fungal activity and the different metabolic pathways that they employ in ecological and environmental systems.

Raman micro-spectroscopy as a tool to study immunometabolism.

In the past two decades, immunometabolism has emerged as a crucial field, unraveling the intricate molecular connections between cellular metabolism and immune function across various cell types, tissues, and diseases. This review explores the insights gained from studies using the emerging technology, Raman micro-spectroscopy, to investigate immunometabolism. Raman micro-spectroscopy provides an exciting opportunity to directly study metabolism at the single cell level where it can be combined with other Raman-based technologies and platforms such as single cell RNA sequencing. The review showcases applications of Raman micro-spectroscopy to study the immune system including cell identification, activation, and autoimmune disease diagnosis, offering a rapid, label-free, and minimally invasive analytical approach. The review spotlights three promising Raman technologies, Raman-activated cell sorting, Raman stable isotope probing, and Raman imaging. The synergy of Raman technologies with machine learning is poised to enhance the understanding of complex Raman phenotypes, enabling biomarker discovery and comprehensive investigations in immunometabolism. The review encourages further exploration of these evolving technologies in the rapidly advancing field of immunometabolism.

A novel bioprospecting strategy via 13C-based high-throughput probing of active methylotrophs inhabiting oil reservoir surface soil

Methane-oxidizing bacteria (MOB) have long been considered as a microbial indicator for oil and gas prospecting. However, due to the phylogenetically narrow breath of ecophysiologically distinct MOB, classic culture-dependent approaches could not discriminate MOB population at fine resolution, and accurately reflect the abundance of active MOB in the soil above oil and gas reservoirs. Here, we presented a novel microbial anomaly detection (MAD) strategy to quantitatively identify specific indicator methylotrophs in the surface soils for bioprospecting oil and gas reservoirs by using a combination of 13C-DNA stable isotope probing (SIP), high-throughput sequencing (HTS), quantitative PCR (qPCR) and geostatistical analysis. The Chunguang oilfield of the Junggar Basin was selected as a model system in western China, and type I methanotrophic Methylobacter was most active in the topsoil above the productive oil wells, while type II methanotrophic Methylosinus predominated in the dry well soils, exhibiting clear differences between non- and oil reservoir soils. Similar results were observed by quantification of Methylobacter pmoA genes as a specific bioindicator for the prediction of unknown reservoirs by grid sampling. A microbial anomaly distribution map based on geostatistical analysis further showed that the anomalous zones were highly consistent with petroleum, geological and seismic data, and validated by subsequent drilling. Over seven years, a total of 24 wells have been designed and drilled into the targeted anomaly, and the success rate via the MAD prospecting strategy was 83 %. Our results suggested that molecular techniques are powerful tools for oil and gas prospecting. This study indicates that the exploration efficiency could be significantly improved by integrating multi-disciplinary information in geophysics and geomicrobiology while reducing the drilling risk to a greater extent.

Identification of bacteria and fungi responsible for litter decomposition in desert steppes via combined DNA stable isotope probing.

Soil microorganisms play crucial roles in determining the fate of litter in desert steppes because their activities constitute a major component of the global carbon (C) cycle. Human activities lead to increased ecosystem nitrogen (N) deposition, which has unpredictable impacts on soil microorganism diversity and functions. Nowadays, it is necessary to further study the succession of these microorganisms in the process of litter decomposition in desert steppe, and explore the effect of N deposition on this process. This issue is particularly important to resolve because it contributes to the broader understanding of nutrient cycling processes in desert steppes. In this study, DNA stable isotope probing (DNA-SIP) was used to study changes in soil bacterial and fungal community composition and function during 8 weeks of culture of 13C-labeled litter in desert steppes. The results were as follows: (1) Actinomycetota, Pseudomonadota, and Ascomycota are the main microorganisms involved in litter decomposition in desert steppes; (2) N deposition (50 kg ha-1 year-1) significantly increased the relative abundance of some microorganisms involved in the decomposition process; and (3) N deposition likely promotes litter decomposition in desert steppes by increasing the abundances of N cycles bacteria (usually carrying GH family functional genes). These findings contribute to a deeper understanding of the C assimilation mechanisms associated with litter residue production, emphasizing the importance of extensive C utilization.

Identification of the bacterial community that degrades phenanthrene sorbed to polystyrene nanoplastics using DNA-based stable isotope probing

In the Anthropocene, plastic pollution has become a new environmental biotope, the so-called plastisphere. In the oceans, nano- and micro-sized plastics are omnipresent and found in huge quantities throughout the water column and sediment, and their large surface area-to-volume ratio offers an excellent surface to which hydrophobic chemical pollutants (e.g. petrochemicals and POPs) can readily sorb to. Our understanding of the microbial communities that breakdown plastic-sorbed chemical pollutants, however, remains poor. Here, we investigated the formation of 500 nm and 1000 nm polystyrene (PS) agglomerations in natural seawater from a coastal environment, and we applied DNA-based stable isotope probing (DNA-SIP) with the 500 nm PS sorbed with isotopically-labelled phenanthrene to identify the bacterial members in the seawater community capable of degrading the hydrocarbon. Whilst we observed no significant impact of nanoplastic size on the microbial communities associated with agglomerates that formed in these experiments, these communities were, however, significantly different to those in the surrounding seawater. By DNA-SIP, we identified Arcobacteraceae, Brevundimonas, Comamonas, uncultured Comamonadaceae, Delftia, Sphingomonas and Staphylococcus, as well as the first member of the genera Acidiphilum and Pelomonas to degrade phenanthrene, and of the genera Aquabacterium, Paracoccus and Polymorphobacter to degrade a hydrocarbon. This work provides new information that feeds into our growing understanding on the fate of co-pollutants associated with nano- and microplastics in the ocean.

Neural network establishes co-occurrence links between transformation products of the contaminant and the soil microbiome

It remains challenging to establish reliable links between transformation products (TPs) of contaminants and corresponding microbes. This challenge arises due to the sophisticated experimental regime required for TP discovery and the compositional nature of 16S rRNA gene amplicon sequencing and mass spectrometry datasets, which can potentially confound statistical inference. In this study, we present a new strategy by combining the use of 2H-labeled Stable Isotope-Assisted Metabolomics (2H-SIAM) with a neural network-based algorithm (i.e., MMvec) to explore links between TPs of pyrene and the soil microbiome. The links established by this novel strategy were further validated using different approaches. Briefly, a metagenomic study provided indirect evidence for the established links, while the identification of pyrene degraders from soils, and a DNA-based stable isotope probing (DNA-SIP) study offered direct evidence. The comparison among different approaches, including Pearson's and Spearman's correlations, further confirmed the superior performance of our strategy. In conclusion, we summarize the unique features of the combined use of 2H-SIAM and MMvec. This study not only addresses the challenges in linking TPs to microbes but also introduces an innovative and effective approach for such investigations.Environmental Implication: Taxonomically diverse bacteria performing successive metabolic steps of the contaminant were firstly depicted in the environmental matrix.

Ridge with no-tillage facilitates microbial N2 fixation associated with methane oxidation in rice soil

Aerobic methane-oxidizing bacteria (MOB) play a crucial role in mitigating the greenhouse gas methane emission, particularly prevalent in flooded wetlands. The implementation of ridge with no-tillage practices within a rice-rape rotation system proves effective in overcoming the restrictive redox conditions associated with waterlogging. This approach enhances capillary water availability from furrows, especially during periods of low rainfall, thereby supporting plant growth on the ridges. However, the microbe-mediated accumulation of soil organic carbon and nitrogen remains insufficiently understood under this agricultural practice, particularly concerning methane oxidation, which holds ecological and agricultural significance in the rice fields. In this study, the ridge and ditch soils from a 28-year-old ridge with no-tillage rice field experiment were utilized for incubation with 13C-CH4 and 15NN2 to estimate the methane-oxidizing and N2-fixing potentials. Our findings reveal a significantly higher net production of fresh soil organic carbon in the ridge compared to the ditch soil during methane oxidation, with values of 626 and 543 μg 13C g−1 dry weight soil, respectively. Additionally, the fixed 15N exhibited a twofold increase in the ridge soil (14.1 μg 15N g−1 dry weight soil) compared to the ditch soil. Interestingly, the result of DNA-based stable isotope probing indicated no significant differences in active MOB and N2 fixers between ridge and ditch soils. Both Methylocystis-like type II and Methylosarcina/Methylomonas-like type I MOB catalyzed methane into organic biomass carbon pools. Soil N2-fixing activity was associated with the 15N-labeling of methane oxidizers and non-MOB, such as methanol oxidizers (Hyphomicrobium) and conventional N2 fixers (Burkholderia). Methane oxidation also fostered microbial interactions, as evidenced by co-occurrence patterns. These results underscore the dual role of microbial methane oxidation – not only as a recognized sink for the potent greenhouse gas methane but also as a source of soil organic carbon and bioavailable nitrogen. This emphasizes the pivotal role of microbial methane metabolism in contributing to soil carbon and nitrogen accumulation in ridge with no-tillage systems.

Soil warming increases the number of growing bacterial taxa but not their growth rates.

Soil microorganisms control the fate of soil organic carbon. Warming may accelerate their activities putting large carbon stocks at risk of decomposition. Existing knowledge about microbial responses to warming is based on community-level measurements, leaving the underlying mechanisms unexplored and hindering predictions. In a long-term soil warming experiment in a Subarctic grassland, we investigated how active populations of bacteria and archaea responded to elevated soil temperatures (+6°C) and the influence of plant roots, by measuring taxon-specific growth rates using quantitative stable isotope probing and 18O water vapor equilibration. Contrary to prior assumptions, increased community growth was associated with a greater number of active bacterial taxa rather than generally faster-growing populations. We also found that root presence enhanced bacterial growth at ambient temperatures but not at elevated temperatures, indicating a shift in plant-microbe interactions. Our results, thus, reveal a mechanism of how soil bacteria respond to warming that cannot be inferred from community-level measurements.

Active microbial population dynamics and life strategies drive the enhanced carbon use efficiency in high-organic matter soils.

Microbial carbon use efficiency (CUE) is a critical parameter that controls carbon storage in soil, but many uncertainties remain concerning adaptations of microbial communities to long-term fertilization that impact CUE. Based on H218O quantitative stable isotope probing coupled with metagenomic sequencing, we disentangled the roles of active microbial population dynamics and life strategies for CUE in soils after a long-term (35 years) mineral or organic fertilization. We found that the soils rich in organic matter supported high microbial CUE, indicating a more efficient microbial biomass formation and a greater carbon sequestration potential. Organic fertilizers supported active microbial communities characterized by high diversity and a relative increase in net growth rate, as well as an anabolic-biased carbon cycling, which likely explains the observed enhanced CUE. Overall, these results highlight the role of population dynamics and life strategies in understanding and predicting microbial CUE and sequestration in soil.IMPORTANCEMicrobial CUE is a major determinant of global soil organic carbon storage. Understanding the microbial processes underlying CUE can help to maintain soil sustainable productivity and mitigate climate change. Our findings indicated that active microbial communities, adapted to long-term organic fertilization, exhibited a relative increase in net growth rate and a preference for anabolic carbon cycling when compared to those subjected to chemical fertilization. These shifts in population dynamics and life strategies led the active microbes to allocate more carbon to biomass production rather than cellular respiration. Consequently, the more fertile soils may harbor a greater microbially mediated carbon sequestration potential. This finding is of great importance for manipulating microorganisms to increase soil C sequestration.

The path from root input to mineral-associated soil carbon is dictated by habitat-specific microbial traits and soil moisture

Soil microorganisms help transform plant inputs into mineral-associated soil organic carbon (SOC) – the largest and slowest-cycling pool of organic carbon on land. However, the microbial traits that influence this process are widely debated. While current theory and biogeochemical models have settled on carbon-use efficiency (CUE) and growth rate as positive predictors of mineral-associated SOC, empirical tests are sparse, with contradictory observations. Using 13C-labeling of an annual grass (Avena barbata) under two moisture regimes, we found that microbial traits associated with formation of 13C-mineral-associated SOC varied by soil habitat, as did active microbial taxa and SOC chemical composition. In the rhizosphere, bacterial-dominated communities with fast growth, high biomass, and high extracellular polymeric substance (EPS) production were positively associated with 13C-mineral-associated SOC. In contrast, the detritusphere held communities dominated by fungi and more filamentous bacteria, and with greater exoenzyme activity; there, 13C-mineral-associated SOC was associated with slower microbial growth and lower microbial biomass. CUE was a negative predictor of 13C-mineral-associated SOC in both habitats. Using 13C-quantitative stable isotope probing, we found that the majority of 13C assimilation in the rhizosphere and detritusphere at week 12 of the experiment was performed by very few bacterial and fungal taxa (3–5% of the total taxa that assimilated 13C). Several complementary chemical analyses (13C-NMR, FTICR-MS, and STXM-NEXAFS) suggested that SOC in the rhizosphere had a more oxidized chemical signature, while SOC in the detritusphere had a less oxidized, more lignin-like chemical signature. Our findings challenge current theory by demonstrating that microbial traits linked with mineral-associated SOC are not universal, but vary with soil habitat and moisture conditions, and are shaped by a small number of active taxa. Emerging SOC models that explicitly reflect these interactions may better predict SOC storage, since climate change causes shifts in soil moisture regimes and the ratio of living versus decaying roots.

Arbuscular mycorrhiza convey significant plant carbon to a diverse hyphosphere microbial food web and mineral-associated organic matter.

Arbuscular mycorrhizal fungi (AMF) transport substantial plant carbon (C) that serves as a substrate for soil organisms, a precursor of soil organic matter (SOM), and a driver of soil microbial dynamics. Using two-chamber microcosms where an air gap isolated AMF from roots, we 13CO2-labeled Avena barbata for 6 wk and measured the C Rhizophagus intraradices transferred to SOM and hyphosphere microorganisms. NanoSIMS imaging revealed hyphae and roots had similar 13C enrichment. SOM density fractionation, 13C NMR, and IRMS showed AMF transferred 0.77 mg C g-1 of soil (increasing total C by 2% relative to non-mycorrhizal controls); 33% was found in occluded or mineral-associated pools. In the AMF hyphosphere, there was no overall change in community diversity but 36 bacterial ASVs significantly changed in relative abundance. With stable isotope probing (SIP)-enabled shotgun sequencing, we found taxa from the Solibacterales, Sphingobacteriales, Myxococcales, and Nitrososphaerales (ammonium oxidizing archaea) were highly enriched in AMF-imported 13C (> 20 atom%). Mapping sequences from 13C-SIP metagenomes to total ASVs showed at least 92 bacteria and archaea were significantly 13C-enriched. Our results illustrate the quantitative and ecological impact of hyphal C transport on the formation of potentially protective SOM pools and microbial roles in the AMF hyphosphere soil food web.

Identifying Active Rather than Total Methanotrophs Inhabiting Surface Soil Is Essential for the Microbial Prospection of Gas Reservoirs.

Methane-oxidizing bacteria (MOB) have long been recognized as an important bioindicator for oil and gas exploration. However, due to their physiological and ecological diversity, the distribution of MOB in different habitats varies widely, making it challenging to authentically reflect the abundance of active MOB in the soil above oil and gas reservoirs using conventional methods. Here, we selected the Puguang gas field of the Sichuan Basin in Southwest China as a model system to study the ecological characteristics of methanotrophs using culture-independent molecular techniques. Initially, by comparing the abundance of the pmoA genes determined by quantitative PCR (qPCR), no significant difference was found between gas well and non-gas well soils, indicating that the abundance of total MOB may not necessarily reflect the distribution of the underlying gas reservoirs. 13C-DNA stable isotope probing (DNA-SIP) in combination with high-throughput sequencing (HTS) furthermore revealed that type II methanotrophic Methylocystis was the absolutely predominant active MOB in the non-gas-field soils, whereas the niche vacated by Methylocystis was gradually filled with type I RPC-2 (rice paddy cluster-2) and Methylosarcina in the surface soils of gas reservoirs after geoscale acclimation to trace- and continuous-methane supply. The sum of the relative abundance of RPC-2 and Methylosarcina was then used as specific biotic index (BI) in the Puguang gas field. A microbial anomaly distribution map based on the BI values showed that the anomalous zones were highly consistent with geological and geophysical data, and known drilling results. Therefore, the active but not total methanotrophs successfully reflected the microseepage intensity of the underlying active hydrocarbon system, and can be used as an essential quantitative index to determine the existence and distribution of reservoirs. Our results suggest that molecular microbial techniques are powerful tools for oil and gas prospecting.

Soil Humic Acid Stimulates Potentially Active Dissimilatory Arsenate-Reducing Bacteria in Flooded Paddy Soil as Revealed by Metagenomic Stable Isotope Probing.

Dissimilatory arsenate reduction contributes a large proportion of arsenic flux from flooded paddy soil, which is closely linked to soil organic carbon input and efflux. Humic acid (HA) represents a natural ingredient in soil and is shown to enhance microbial arsenate respiration to promote arsenic mobility. However, the community and function profiles of metabolically active arsenate-respiring bacteria and their interactions with HA in paddy soil remain unclear. To probe this linkage, we performed a genome-centric comparison of potentially active arsenate-respiring bacteria in anaerobic microcosms amended with 13C-lactate and HA by combining stable-isotope probing with genome-resolved metagenomics. Indeed, HA greatly accelerated the microbial reduction of arsenate to arsenite. Enrichment of bacteria that harbor arsenate-respiring reductase genes (arrA) in HA-enriched 13C-DNA was confirmed by metagenomic binning, which are affiliated with Firmicutes (mainly Desulfitobacterium, Bacillus, Brevibacillus, and Clostridia) and Acidobacteria. Characterization of reference extracellular electron transfer (EET)-related genes in these arrA-harboring bacteria supports the presence of EET-like genes, with partial electron-transport chain genes identified. This suggests that Gram-positive Firmicutes- and Acidobacteria-related members may harbor unspecified EET-associated genes involved in metal reduction. Our findings highlight the link between soil HA and potentially active arsenate-respiring bacteria, which can be considered when using HA for arsenic removal.

Identification of Aniline-Degrading Bacteria Using Stable Isotope Probing Technology and Prediction of Functional Genes in Aerobic Microcosms

Aniline, a vital component in various chemical industries, is known to be a hazardous persistent organic pollutant that can cause environmental pollution through its manufacturing, processing, and transportation. In this study, the microcosms were established using sediment with a history of aniline pollution as an inoculum to analyze the aniline biodegradation under aerobic conditions through stable isotope probing (SIP) and isopycnic density gradient centrifugation technology. During the degradation assay, aniline that was 13C-labeled in all six carbons was utilized to determine the phylogenetic identity of the aniline-degrading bacterial taxa that incorporate 13C into their DNA. The results revealed that aniline was completely degraded in the microcosm after 45 and 69 h respectively. The bacteria affiliated with Acinetobacter (up to 34.6 ± 6.0%), Zoogloea (up to 15.8 ± 2.2%), Comamonas (up to 2.6 ± 0.1%), and Hydrogenophaga (up to 5.1 ± 0.6%) genera, which are known to degrade aniline, were enriched in the heavy fractions (the DNA buoyant density was 1.74 mg L−1) of the 13C-aniline treatments. Moreover, some rarely reported aniline-degrading bacteria, such as Prosthecobacter (up to 16.0 ± 1.6%) and Curvibacter (up to 3.0 ± 1.6%), were found in the DNA-SIP experiment. Gene families affiliated with atd, tdn, and dan were speculated to be key genes for aniline degradation based on the abundance in functional genes and diversity in different treatments as estimated using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States version 2 (PICRUSt2) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). This study revealed the functional bacteria and possible degradation genes for aniline degradation in simulated polluted environments through SIP. These findings suggest that important degrading bacteria for the transformation of aniline and potential degradation pathways may be useful in the effective application of bioremediation technologies to remediate aniline-contaminated sites.

Methylocystis dominates methane oxidation in glacier foreland soil at elevated temperature.

Methane-oxidizing bacteria (methanotrophs) play an important role in mitigating methane emissions in various ecological environments, including cold regions. However, the response of methanotrophs in these cold environments to extreme temperatures above the in-situ temperature has not been thoroughly explored. Therefore, this study collected soil samples from Longxiazailongba (LXZ) and Qiangyong (QY) glacier forelands and incubated them with 13CH4 at 35°C under different soil water conditions. The active methanotroph populations were identified using DNA stable isotope probing (DNA-SIP) and high throughput sequencing techniques. The results showed that the methane oxidation potential in LXZ and QY glacier foreland soils was significantly enhanced at an unusually high temperature of 35°C during microcosm incubations, where abundant substrate (methane and oxygen) was provided. Moreover, the influence of soil water conditions on this potential was observed. Interestingly, Methylocystis, a type II and mesophilic methanotroph, was detected in the unincubated in-situ soil samples and became the active and dominant methanotroph in methane oxidation at 35°C. This suggests that Methylocystis can survive at low temperatures for a prolonged period and thrive under suitable growth conditions. Furthermore, the presence of mesophilic methanotrophs in cold habitats could have potential implications for reducing greenhouse gas emissions in warming glacial environments.

Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction.

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga's fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide-a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.

Unexpected carbon utilization activity of sulfate-reducing microorganisms in temperate and permanently cold marine sediments.

Significant amounts of organic carbon in marine sediments are degraded, coupled with sulfate reduction. However, the actual carbon and energy sources used in situ have not been assigned to each group of diverse sulfate-reducing microorganisms (SRM) owing to the microbial and environmental complexity in sediments. Here, we probed microbial activity in temperate and permanently cold marine sediments by using potential SRM substrates, organic fermentation products at very low concentrations (15-30μM), with RNA-based stable isotope probing. Unexpectedly, SRM were involved only to a minor degree in organic fermentation product mineralization, whereas metal-reducing microbes were dominant. Contrastingly, distinct SRM strongly assimilated 13C-DIC (dissolved inorganic carbon) with H2 as the electron donor. Our study suggests that canonical SRM prefer autotrophic lifestyle, with hydrogen as the electron donor, while metal-reducing microorganisms are involved in heterotrophic organic matter turnover, and thus regulate carbon fluxes in an unexpected way in marine sediments.

Microbiome processing of organic nitrogen input supports growth and cyanotoxin production of Microcystis aeruginosa cultures.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.

Ferrihydrite-mediated methanotrophic nitrogen fixation in paddy soil under hypoxia.

Biological nitrogen fixation (BNF) by methanotrophic bacteria has been shown to play an important role in maintaining fertility. However, this process is still limited to aerobic methane oxidation with sufficient oxygen. It has remained unknown whether and how methanotrophic BNF proceeds in hypoxic environments. Herein, we incubated paddy soils with a ferrihydrite-containing mineral salt medium to enrich methanotrophic bacteria in the presence of methane (20%, v/v) under oxygen constraints (0.27%, v/v). The resulting microcosms showed that ferrihydrite-dependent aerobic methane oxidation significantly contributed (81%) to total BNF, increasing the 15N fixation rate by 13-fold from 0.02 to 0.28μmol 15N2 (g dry weight soil) -1 d-1. BNF was reduced by 97% when ferrihydrite was omitted, demonstrating the involvement of ferrihydrite in methanotrophic BNF. DNA stable-isotope probing indicated that Methylocystis, Methylophilaceae, and Methylomicrobium were the dominant methanotrophs/methylotrophs that assimilated labeled isotopes (13C or 15N) into biomass. Metagenomic binning combined with electrochemical analysis suggested that Methylocystis and Methylophilaceae had the potential to perform methane-induced BNF and likely utilized riboflavin and c-type cytochromes as electron carriers for ferrihydrite reduction. It was concluded that ferrihydrite mediated methanotrophic BNF by methanotrophs/methylotrophs solely or in conjunction with iron-reducing bacteria. Overall, this study revealed a previously overlooked yet pronounced coupling of iron-dependent aerobic methane oxidation to BNF and improves our understanding of methanotrophic BNF in hypoxic zones.

In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging.

Sensing and visualization of metabolites and metabolic pathways in situ are significant requirements for tracking their spatiotemporal dynamics in a non-destructive manner. The shikimate pathway is an important cellular mechanism that leads to the de novo synthesis of many compounds containing aromatic rings of high importance such as phenylalanine, tyrosine, and tryptophan. In this work, we present a cost-effective and extraction-free method based on the principles of stable isotope-coupled Raman spectroscopy and hyperspectral Raman imaging to monitor and visualize the activity of the shikimate pathway. We also demonstrated the applicability of this approach for nascent aromatic amino acid localization and tracking turnover dynamics in both prokaryotic and eukaryotic model systems. This method can emerge as a promising tool for both qualitative and semi-quantitative in situ metabolomics, contributing to a better understanding of aromatic ring-containing metabolite dynamics across various organisms.