Stable Isotope Probing Research Articles

Application of infrared spectroscopy to study carbon-deuterium kinetics and isotopic spectral shifts at the single-cell level

Microbial communities play crucial roles in shaping natural ecosystems, impacting human well-being, and driving advancements in industrial biotechnology. However, associating specific metabolic functions with bacteria proves challenging due to the vast diversity of microorganisms within these communities. In the past decades stable isotope probing (SIP) approaches, coupled with vibrational spectroscopy, have emerged as a novel method for revealing microbial metabolic roles and interactions in complex communities. In this study, we employed various combinations of heavy stable isotopes (D, 13C, 15N, and 18O), to evaluate all possible isotopic spectral shifts in the mid-IR region using Fourier-Transform Infrared (FT-IR) and Optical Photothermal Infrared (O-PTIR) spectroscopy, at both bulk community and single-cell levels. Additionally, we conducted a time-course study to explore the kinetics of CD vibration in Escherichia coli bacteria, allowing time-based sampling and assessment of isotopic labeling kinetics. The FT-IR and O-PTIR, along with the second derivative spectra of E. coli cells cultured in minimal medium supplemented with various combinations of heavy isotopes exhibited notable similarities. Several spectral shifts in primary vibrational peaks were observed due to the incorporation of heavy isotopes into biomolecules. Remarkably, the incorporation of deuterium into amide groups, resulting in the formation of nitrogen-deuterium bonds, caused a shift in amide A and B into the silent region, overlapping with CD signature peaks. The incorporation of 18O into the ester group of lipids and the carbonyl group of proteins resulted in a notable shift to the lower wavenumber region. Additionally, the second derivative of FT-IR spectral data highlighted the integration of 18O into α-helix and β-sheet structures. Furthermore, the spectra, second derivative, and PC-DFA scores plot of FT-IR data collectively illustrated the practicality of monitoring 13C and D incorporation into E. coli bacterial cells within the first 30-min incubation period. Significance: The findings of this study suggest that FT-IR and O-PTIR can serve as efficient tools for monitoring the incorporation of heavy isotopes into bacteria at both the population and single-cell levels. Additionally, the SIP approach allowed us to assign two new deuterium-associated vibrational peaks to their corresponding functional groups, which to the best of our knowledge have not been reported previously.

Warming increases CH4 emissions from rice paddies through shifts in methanogenic and methanotrophic communities

Warming often stimulates methane (CH4) emissions from rice paddies, one of the largest anthropogenic sources of CH4 emissions. However, the responses of methanogenic and methanotrophic communities to warming, particularly within active communities, remain unclear. Therefore, based on a field warming experiment in a rice-wheat system, we investigated the effects of warming on methanogenic and methanotrophic communities, using the DNA stable-isotope probing technology. Our results indicated that warming increased CH4 emissions by 27–49% over two rice growing seasons compared to ambient conditions. Warming significantly increased the abundance of methanogens by 53%, whereas did not affect activities of methanogens and active methanogenic community. Conversely, warming did not influence the abundance of methanotrophs, but reduced activities of methanotrophs by 44%. Notably, warming led to a significant rise in the relative abundance of the active type II methanotrophic community, which exhibits lower CH4 oxidation efficiency. These findings suggest that the observed increase in CH4 emissions under warming conditions is primarily driven by the enhanced abundance of methanogens and the increased presence of less efficient active type II methanotrophs. This study underscores the critical role of active microbial communities in understanding and managing CH4 emissions from rice paddies in a warming world.

Rapid screening of indigenous degrading microorganisms for enhancing in-situ bioremediation of organic pollutants-contaminated soil

Organic pollutants (OPs) have caused severe environmental contaminations in the world and aroused wide public concern. Autochthonous bioaugmentation (ABA) is considered a reliable bioremediation approach for OPs contamination. However, the rapid screening of indigenous degrading strains from in-situ environments remains a primary challenge for the practical application of ABA. In this study, 3,5,6-Trichloro-2-pyridinol (TCP, an important intermediate in the synthesis of various pesticides) was selected as the target OPs, and DNA stable isotope probing (DNA-SIP) combined with high-throughput sequencing was employed to explore the rapid screening of indigenous degrading microorganisms. The results of DNA-SIP revealed a significant enrichment of OTU557 (Cupriavidus sp.) in the 13C-TCP-labeled heavy DNA fractions, indicating that it is the key strain involved in TCP metabolism. Subsequently, an indigenous TCP degrader, Cupriavidus sp. JL-1, was rapidly isolated from native soil based on the analysis of the metabolic substrate spectrum of Cupriavidus sp. Furthermore, ABA of strain JL-1 demonstrated higher remediation efficacy and stable survival compared to the exogenous TCP-degrading strain Cupriavidus sp. P2 in in-situ TCP-contaminated soil. This study presents a successful case for the rapid acquisition of indigenous TCP-degrading microorganisms to support ABA as a promising strategy for the in-situ bioremediation of TCP-contaminated soil.

Asymmetric winter warming reduces microbial carbon use efficiency and growth more than symmetric year-round warming in alpine soils

Asymmetric seasonal warming trends are evident across terrestrial ecosystems, with winter temperatures rising more than summer ones. Yet, the impact of such asymmetric seasonal warming on soil microbial carbon metabolism and growth remains poorly understood. Using 18O isotope labeling, we examined the effects of a decade-long experimental seasonal warming on microbial carbon use efficiency (CUE) and growth in alpine grassland ecosystems. Moreover, the quantitative stable isotope probing with 18O-H2O was employed to evaluate taxon-specific bacterial growth in these ecosystems. Results show that symmetric year-round warming decreased microbial growth rate by 31% and CUE by 22%. Asymmetric winter warming resulted in a further decrease in microbial growth rate of 27% and microbial CUE of 59% compared to symmetric year-round warming. Long-term warming increased microbial carbon limitations, especially under asymmetric winter warming. Long-term warming suppressed the growth rates of most bacterial genera, with asymmetric winter warming having a stronger inhibition on the growth rates of specific genera (e.g., Gp10, Actinomarinicola, Bosea, Acidibacter, and Gemmata) compared to symmetric year-round warming. Bacterial growth was phylogenetically conserved, but this conservation diminished under warming conditions, primarily due to shifts in bacterial physiological states rather than the number of bacterial species and community composition. Overall, long-term warming escalated microbial carbon limitations, decreased microbial growth and CUE, with asymmetric winter warming having a more pronounced effect. Understanding these impacts is crucial for predicting soil carbon cycling as global warming progresses.

The ammonium transporter AmtB is dispensable for the uptake of ammonium in the phototrophic diazotroph Rhodopseudomonas palustris

Ammonium (NH4+) transport across cell membranes plays an important role in assimilation or removal of the environmental nitrogen. The membrane protein AmtB has been considered to be the NH4+ transporter that is responsible for the NH4+ uptake. The phototrophic diazotroph Rhodopseudomonas palustris harboring two amtB genes has been widely used in wastewater treatment and bioremediation. However, the role of AmtB in NH4+ uptake remains unclear in R. palustris. Here, we employed an innovative approach combining stable isotope probing (SIP) with Raman spectroscopy to determine the physiological functions of AmtB1 and AmtB2 in R. palustris. This powerful technique allowed us to investigate NH4+ uptake at the single-cell level. The generated R. palustris ΔamtB1 ΔamtB2 mutant lacking AmtB1 and AmtB2 proteins was still capable of utilizing 15NH4+ even the 15NH4+ concentration was as low as 5 μM. These data demonstrate that both of the AmtB proteins are not essential for R. palustris to take up NH4+ regardless of environmental NH4+ levels. However, both AmtB1 and AmtB2 can contribute to NH4+ uptake under nitrogen-limiting conditions. Given that R. palustris primarily expresses AmtB2 in these conditions, AmtB2 plays a more important role in NH4+ uptake compared to AmtB1. In addition, transcriptomic analysis showed that the deletion of the amtB1 and amtB2 genes resulted in the upregulation of many transporter genes, providing potential targets for future investigation of alternative NH4+ uptake systems.

Nanopore sequencing enables novel detection of deuterium incorporation in DNA

Identifying active microbes is crucial to understand their role in ecosystem functions. Metabolic labeling with heavy, non-radioactive isotopes, i.e., stable isotope probing (SIP), can track active microbes by detecting heavy isotope incorporation in biomolecules such as DNA. However, the detection of heavy isotope-labeled nucleotides directly during sequencing has, to date, not been achieved.In this study, Oxford nanopore sequencing was utilized to detect heavy isotopes incorporation in DNA molecules. Two isotopes widely used in SIP experiments were employed to label a bacterial isolate: deuterium (D, as D2O) and carbon-13 (13C, as glucose). We hypothesize that labeled DNA is distinguishable from unlabeled DNA by changes in the nanopore signal. To verify this distinction, we employed a Bayesian classifier trained on signal distributions of short oligonucleotides (k-mers) from labeled and unlabeled sequencing reads.Our results show a clear distinction between D-labeled and unlabeled reads, based on changes in median and median absolute deviation (MAD) of the nanopore signals for different k-mers. In contrast, 13C-labeled DNA cannot be distinguished from unlabeled DNA. For D, the model employed correctly predicted more than 85% of the reads. Even when metabolic labeling was conducted with only 30% D2O, 80% of the obtained reads were correctly classified with a 5% false discovery rate.Our work demonstrates the feasibility of direct detection of deuterium incorporation in DNA molecules during Oxford nanopore sequencing. This finding represents a first step in establishing the combined use of nanopore sequencing and SIP for tracking active organisms in microbial ecology.

Characteristics of organic amendments induce diverse microbial metabolisms for exogenous C turnover in Mollisols

The response of microbial community to organic amendments is closely related to the amendment-derived carbon (C) turnover, which in turn influences the enhancement of soil organic C, including the quantity and quality. The objective of this study was to quantify the decomposition and retention of exogenous C derived from amendments, and to identify the microbial regulatory mechanisms of exogenous C turnover in response to the characteristics of organic amendments. The 13C-labelled glucose, straw and biochar, characterizing organic amendments with high, medium and low labilities, respectively, were used to conduct a 90-day incubation experiment, in conjunction with stable isotope probing technology and amino sugar molecular biomarkers. It was found that the cumulative mineralization of amendment-derived C increased with increasing the corresponding lability of organic amendment, and the decompositions of the added glucose, straw and biochar, expressed as a percentage of the initially added C, were 53.6 %, 37.0 %, and 0.6 %, respectively. Among all treatments, the addition of straw significantly enhanced the total dissolved organic C (DOC) concentration and its ratio to total organic C by increasing the input of exogenous DOC and reducing the decomposition of native soil DOC. The exogenous C use efficiency by microorganisms was significantly enhanced with decreasing the amendment lability, and showed a positive correlation with the total amendment-derived C. However, the microbial growth efficiency was the highest when straw was added, and was closely related to the concentration of amendment-derived DOC. Different functional groups of soil microbes exhibited distinct capacities to metabolize various organic amendments. Generally, the incorporation of amendment-derived C into microbial biomass was significantly affected by fungi and gram-positive (G+) bacteria, while the relative contributions of microbial communities incorporating amendment-derived C into necromass were decreased in the order of gram-negative (G−) bacteria > fungi > actinomycetes > G+ bacteria. From the perspective of microbial metabolism, the larger incorporations of exogenous C into microbial biomass and necromass were observed in the soil with mediumly labile amendment, which contributed to enhance the quantity and quality of organic C in soil. These results would be informative to optimize the organic amendment strategy for integrated soil fertility management.

Enhanced carbon use efficiency and warming resistance of soil microorganisms under organic amendment

The frequency and intensity of extreme weather events, including rapid temperature fluctuations, are increasing because of climate change. Long-term fertilization practices have been observed to alter microbial physiology and community structure, thereby affecting soil carbon sequestration. However, the effects of warming on the carbon sequestration potential of soil microbes adapted to long-term fertilization remain poorly understood. In this study, we utilized 18O isotope labeling to assess microbial carbon use efficiency (CUE) and employed stable isotope probing (SIP) with 18O-H2O to identify growing taxa in response to temperature changes (5–35 °C). Organic amendment with manure or straw residue significantly increased microbial CUE by 86–181 % compared to unfertilized soils. The microorganisms inhabiting organic amended soils displayed greater resistance of microbial CUE to high temperatures (25–35 °C) compared to those inhabiting soils fertilized only with minerals. Microbial growth patterns determined by the classification of taxa into incorporators or non-incorporators based on 18O incorporation into DNA exhibited limited phylogenetic conservation in response to temperature changes. Microbial clusters were identified by grouping taxa with similar growth patterns across different temperatures. Organic amendments enriched microbial clusters associated with increased CUE, whereas clusters in unfertilized or mineral-only fertilized soils were linked to decreased CUE. Specifically, shifts in the composition of growing bacteria were correlated with enhanced microbial CUE, whereas modifications in the composition of growing fungi were associated with diminished CUE. Notably, the responses of microbial CUE to temperature fluctuations were primarily driven by changes in the bacterial composition. Overall, our findings demonstrate that organic amendments enhance soil microbial CUE and promote the enrichment of specific microbial clusters that are better equipped to cope with temperature changes. This study establishes a theoretical foundation for manipulating soil microbes to enhance carbon sequestration under global climate scenarios.

Scaling up taxon-specific microbial traits to predict community-level microbial activity in agricultural systems

Soil microorganisms perform many important ecosystem functions including nitrogen (N) cycling which dictates plant productivity in agricultural ecosystems. Despite the importance of these communities, connecting microbial composition with ecosystem function has been a long standing challenge. Taxon-specific substrate assimilation traits, measured with quantitative stable isotope probing (qSIP), may provide a means to scale from microbial community composition to community-level process rates. To test the potential for scaling up taxon-specific N assimilation to predict community-level rates of carbon mineralization, N mineralization, and N immobilization we measured soil properties, microbial activity, and N assimilation using 15N qSIP in soils from six distinct farms. N assimilation, measured as DNA 15N enrichment, varied among taxa and within taxa across farms. Taxon specific N assimilation was aggregated to calculate a community-weighted mean, which when combined with measures of microbial biomass was used to estimate new microbial biomass N production. This estimate of new microbial biomass production reflects the growth of active microbes over the incubation period and related to microbial activity. The new microbial biomass N produced was predictive of soil C and N mineralization rates, explaining 37-47% of the observed variation across the six farming systems. This approach highlights the ability of trait-based methods to relate microbial community structure data to microbially-mediated functional process rates. Such advances may enhance our ability to understand and manage microbially mediated processes, such as N cycling, in both natural and agricultural ecosystems.

Deciphering the active bacteria involving glucose-triggered priming effect in soils with gradient N inputs

The soil priming effect, which refers to the alteration of soil organic matter (SOM) decomposition due to labile carbon (C) inputs, is widely acknowledged for its impact on C storage in terrestrial ecosystems. However, the impact of chronic nitrogen (N) fertilizer on soil priming effect, particularly in agroecological systems, remains unclear. Here, we utilized soils subjected to varying levels of N fertilization (0, 140, 280, 470, and 660 kg N ha−1 y−1), which were collected from a long-term experimental site. Enzyme activity related to C, N, and phosphorus (P) acquisition was measured using the fluorometric method. Additionally, DNA-based stable isotope probing with 13C-labeled glucose was conducted to explore the role of active bacterial communities (16S rRNA gene analysis) on the priming effect in soils with different N fertilization histories. Glucose addition enhanced the decomposition of native SOM and induced positive priming effects in all soils, which were amplified by the historical N application level. Activity of C-related enzymes essential for soil C decomposition increased following glucose addition, which was positively correlated with the soil priming effect. Active bacterial taxa, primarily Firmicutes, Actinobacteria, and Proteobacteria, were capable of assimilating exogenous glucose-C or native SOM-C. Notably, bacteria assimilating glucose exhibited higher abundance-weighted average ribosomal RNA gene operon copy number than those assimilating SOM, indicating the role of r-strategists in accelerating SOC turnover and increasing C loss. These findings highlight the role of active microbial community attributes on the soil priming effects. This study provides new insights into the intricate processes of C transformation in soils subjected to long-term N management in agroecosystems.

Organic fertilizer significantly mitigates N2O emissions while increase contributed of comammox Nitrospira in paddy soils

Nitrification is the dominant process for nitrous oxide (N2O) production under aerobic conditions, but the relative contribution of the autotrophic nitrifiers (the ammonia-oxidising archaea (AOA), the ammonia-oxidising bacteria (AOB) and the comammox) to this process is still unclear in some soil types. This is particularly the case in paddy soils under different fertilization regimes. We investigated active nitrifiers and their contribution to nitrification and N2O production in a range of unfertilized and fertilized paddy soils, using 13CO2-DNA based stable isotope probing (SIP) technique combined with a series of specific nitrification inhibitors, including acetylene (C2H2), 3, 4-dimethylpyrazole phosphate (DMPP) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). The soils had a long-term history of fertilizer application, including chemical fertilizer only, a mixture of chemical fertilizers (70 %) and chicken manure (30 %) or a mixture of rice straw and chemical fertilizers. 13CO2-DNA-SIP and Illumina MiSeq sequencing demonstrated that comammox clades A.1 and B were active nitrifiers in all fertilized paddy soils. Inhibitor experiment showed that AOB largely contributed to nitrification activity and N2O emission in all paddy soils, while comammox contribution was more significant than AOA. Fertilization considerably altered nitrifiers' relative contribution to nitrification activity and N2O emissions. Applying organic fertilizers significantly decreased the N2O emissions but increased the contribution of comammox to the process. These findings expand the functional ecological niche of comammox, revealing their nitrification role and N2O production in other ecosystems than oligotrophic habitats.

Impact of yeast extract and basal salts medium on 1,4-dioxane biodegradation rates and the microorganisms involved in carbon uptake from 1,4-dioxane

Conventional physical and chemical treatment technologies for 1,4-dioxane can be ineffective and consequently attention has focused on bioremediation. Towards this, the current research investigated the impact of basal salts medium (BSM) and yeast extract on 1,4-dioxane biodegradation rates in microcosms with different soil or sediment (agricultural soil, wetland sediment, sediment from an impacted site). Phylotypes responsible for carbon uptake from 1,4-dioxane were determined using stable isotope probing (SIP), both with and without BSM and yeast extract. Further, putative functional genes were investigated using 1) soluble di-iron monooxygenase (SDIMO) based amplicon sequencing, 2) qPCR targeting propane monooxygenase (large subunit, prmA) and 3) a predictive approach (PICRUSt2). The addition of BSM and yeast extract significantly enhanced 1,4-dioxane removal rates the agricultural soil and impacted site sediment microcosms. The phylotypes associated with carbon uptake varied across treatments and inocula. Gemmatimonas was important in the heavy SIP fractions of the wetland sediment microcosms. Unclassified Solirubacteraceae, Solirubrobacter, Pseudonocardia and RB4 were dominant in the heavy SIP fractions of the agricultural soil microcosms. The heavy SIP fractions of the impacted site microcosms were dominated by only two phylotypes, unclassified Burkholderiaceae and oc3299. SDIMO based amplicon sequencing detected three genes previously associated with 1,4-dioxane. The predicted functional gene analysis suggested the importance of propane monooxygenases associated with Solirubrobacter and Pseudonocardia. Overall, more microorganisms were involved in carbon uptake from 1,4-dioxane in both the wetland and agricultural soil microcosms compared to the impacted site sediment microcosms. Many of these microorganisms have not previously been associated with 1,4-dioxane removal.

Growth rate as a link between microbial diversity and soil biogeochemistry.

Measuring the growth rate of a microorganism is a simple yet profound way to quantify its effect on the world. The absolute growth rate of a microbial population reflects rates of resource assimilation, biomass production and element transformation-some of the many ways in which organisms affect Earth's ecosystems and climate. Microbial fitness in the environment depends on the ability to reproduce quickly when conditions are favourable and adopt a survival physiology when conditions worsen, which cells coordinate by adjusting their relative growth rate. At the population level, relative growth rate is a sensitive metric of fitness, linking survival and reproduction to the ecology and evolution of populations. Techniques combining omics and stable isotope probing enable sensitive measurements of the growth rates of microbial assemblages and individual taxa in soil. Microbial ecologists can explore how the growth rates of taxa with known traits and evolutionary histories respond to changes in resource availability, environmental conditions and interactions with other organisms. We anticipate that quantitative and scalable data on the growth rates of soil microorganisms, coupled with measurements of biogeochemical fluxes, will allow scientists to test and refine ecological theory and advance process-based models of carbon flux, nutrient uptake and ecosystem productivity. Measurements of in situ microbial growth rates provide insights into the ecology of populations and can be used to quantitatively link microbial diversity to soil biogeochemistry.

The effect of substrate concentration on the methane-driven interaction network

Methane, the primary substrate for aerobic methanotrophs, regulates the rate of methanotrophic activity and shapes the composition of the methane-oxidizing community. Given that methane-derived carbon may fuel the food web in the soil, methane availability can potentially be a key determinant, structuring the network of the interacting methane-oxidizing community. Here, we determined the response of the methane-driven interaction network to different methane concentrations (∼1.5 %v/v, 3 %v/v, and 7 %v/v), indicative of different levels of energy flow through the soil food web, using a stable isotope probing approach with 13C-methane coupled to a co-occurrence network analysis in a microcosm study. The accumulated 13C-atom fraction in the total carbon content increased from 1.08 % (background level) to an average of 7.2 % in the incubation under 7 %v/v methane, indicating that the carbon-flow via the methanotrophs can significantly contribute to the total carbon in the rice paddy soil. The 13C-enriched 16 S rRNA gene sequencing analysis revealed the predominance of gammaproteobacterial methanotrophs and Methylocystis. The composition of the actively growing (13C-labelled) bacterial community was dissimilar in the incubation under ∼3 %v/v than under 1.5 %v/v and 7 %v/v methane. This was also reflected in the co-occurrence network analysis, where the topological properties indicated a more complex and connected network in the incubation under 3 %v/v methane. It thus appears that moderate methane concentrations fostered closer associations among members of the methane-oxidizing community. Overall, our research findings showed that the methanotrophs can contribute to the total soil carbon, and methane concentrations not only shifted the bacterial community, including the methanotrophic composition, but also affected bacterial interactions.

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.

Proteomic stable isotope probing with an upgraded Sipros algorithm for improved identification and quantification of isotopically labeled proteins

BackgroundProteomic stable isotope probing (SIP) is used in microbial ecology to trace a non-radioactive isotope from a labeled substrate into de novo synthesized proteins in specific populations that are actively assimilating and metabolizing the substrate in a complex microbial community. The Sipros algorithm is used in proteomic SIP to identify variably labeled proteins and quantify their isotopic enrichment levels (atom%) by performing enrichment-resolved database searching.ResultsIn this study, Sipros was upgraded to improve the labeled protein identification, isotopic enrichment quantification, and database searching speed. The new Sipros 4 was compared with the existing Sipros 3, Calisp, and MetaProSIP in terms of the number of identifications and the accuracy and precision of atom% quantification on both the peptide and protein levels using standard E. coli cultures with 1.07 atom%, 2 atom%, 5 atom%, 25 atom%, 50 atom%, and 99 atom% 13C enrichment. Sipros 4 outperformed Calisp and MetaProSIP across all samples, especially in samples with ≥ 5 atom% 13C labeling. The computational speed on Sipros 4 was > 20 times higher than Sipros 3 and was on par with the overall speed of Calisp- and MetaProSIP-based pipelines. Sipros 4 also demonstrated higher sensitivity for the detection of labeled proteins in two 13C-SIP experiments on a real-world soil community. The labeled proteins were used to trace 13C from 13C-methanol and 13C-labeled plant exudates to the consuming soil microorganisms and their newly synthesized proteins.ConclusionOverall, Sipros 4 improved the quality of the proteomic SIP results and reduced the computational cost of SIP database searching, which will make proteomic SIP more useful and accessible to the border community.-J6PT921fQgCkWKyfrRUdgVideo

DNA stable isotope probing reveals the impact of trophic interactions on bioaugmentation of soils with different pollution histories

BackgroundBioaugmentation is considered a sustainable and cost-effective methodology to recover contaminated environments, but its outcome is highly variable. Predation is a key top-down control mechanism affecting inoculum establishment, however, its effects on this process have received little attention. This study focused on the impact of trophic interactions on bioaugmentation success in two soils with different pollution exposure histories. We inoculated a 13C-labelled pollutant-degrading consortium in these soils and tracked the fate of the labelled biomass through stable isotope probing (SIP) of DNA. We identified active bacterial and eukaryotic inoculum-biomass consumers through amplicon sequencing of 16S rRNA and 18S rRNA genes coupled to a novel enrichment factor calculation.ResultsInoculation effectively increased PAH removal in the short-term, but not in the long-term polluted soil. A decrease in the relative abundance of the inoculated genera was observed already on day 15 in the long-term polluted soil, while growth of these genera was observed in the short-term polluted soil, indicating establishment of the inoculum. In both soils, eukaryotic genera dominated as early incorporators of 13C-labelled biomass, while bacteria incorporated the labelled biomass at the end of the incubation period, probably through cross-feeding. We also found different successional patterns between the two soils. In the short-term polluted soil, Cercozoa and Fungi genera predominated as early incorporators, whereas Ciliophora, Ochrophyta and Amoebozoa were the predominant genera in the long-term polluted soil.ConclusionOur results showed differences in the inoculum establishment and predator community responses, affecting bioaugmentation efficiency. This highlights the need to further study predation effects on inoculum survival to increase the applicability of inoculation-based technologies.9HmCi-USiFQRck8tFgyMqrVideo

Single-cell measurement of microbial growth rate with Raman microspectroscopy.

Rates of microbial growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable isotope probing (SIP) is a method for assessing microbial growth and involves measuring the incorporation of an isotopic label into microbial biomass. Here, we assess Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We correlatively measured cells grown in varying concentrations of deuterated water with both Raman spectroscopy and nanoscale secondary ion mass spectrometry (nanoSIMS), generating isotopic calibrations of microbial 2H. Relative to Raman, we find that nanoSIMS measurements of 2H are subject to substantial dilution due to rapid exchange of H during sample washing. We apply our Raman-derived calibration to a numerical model of microbial growth, explicitly parameterizing the factors controlling growth rate quantification and demonstrating that Raman-SIP can sensitively measure the growth of microorganisms with doubling times ranging from hours to years. The measurement of single-cell growth with Raman spectroscopy, a rapid, nondestructive technique, represents an important step toward application of single-cell analysis into complex sample matrices or cellular assemblages.

Reversed oxidative TCA (roTCA) for carbon fixation by an Acidimicrobiia strain from a saline lake.

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.

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.