Stable Isotope Probing Approach 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.

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.

Warming and altered precipitation independently and interactively suppress alpine soil microbial growth in a decadal-long experiment

Warming and precipitation anomalies affect terrestrial carbon balance partly through altering microbial eco-physiological processes (e.g., growth and death) in soil. However, little is known about how such processes responds to simultaneous regime shifts in temperature and precipitation. We used the 18O-water quantitative stable isotope probing approach to estimate bacterial growth in alpine meadow soils of the Tibetan Plateau after a decade of warming and altered precipitation manipulation. Our results showed that the growth of major taxa was suppressed by the single and combined effects of temperature and precipitation, eliciting 40–90% of growth reduction of whole community. The antagonistic interactions of warming and altered precipitation on population growth were common (~70% taxa), represented by the weak antagonistic interactions of warming and drought, and the neutralizing effects of warming and wet. The members in Solirubrobacter and Pseudonocardia genera had high growth rates under changed climate regimes. These results are important to understand and predict the soil microbial dynamics in alpine meadow ecosystems suffering from multiple climate change factors.

Warming and altered precipitation independently and interactively suppress alpine soil microbial growth in a decadal-long experiment

Warming and precipitation anomalies affect terrestrial carbon balance partly through altering microbial eco-physiological processes (e.g., growth and death) in soil. However, little is known about how such processes responds to simultaneous regime shifts in temperature and precipitation. We used the 18O-water quantitative stable isotope probing approach to estimate bacterial growth in alpine meadow soils of the Tibetan Plateau after a decade of warming and altered precipitation manipulation. Our results showed that the growth of major taxa was suppressed by the single and combined effects of temperature and precipitation, eliciting 40–90% of growth reduction of whole community. The antagonistic interactions of warming and altered precipitation on population growth were common (~70% taxa), represented by the weak antagonistic interactions of warming and drought, and the neutralizing effects of warming and wet. The members in Solirubrobacter and Pseudonocardia genera had high growth rates under changed climate regimes. These results are important to understand and predict the soil microbial dynamics in alpine meadow ecosystems suffering from multiple climate change factors.

Distinct carbon incorporation from 13C-labelled rice straw into microbial amino sugars in soils applied with manure versus mineral fertilizer

Emerging evidences have emphasized the primacy of microorganisms as executor in soil organic C (SOC) formation. Yet, whether and how microbial transformation of crop residue-derived C into necromass is affected by fertilization regimes remains poorly understood. To address this, soils subjected to 3-year applications of chemical fertilizer (NPK) vs manure plus chemical fertilizer (NPKM) were incubated with or without addition of 13C-labelled rice straw for 60 days. The 13C incorporation into amino sugars (microbial necromass biomarker) was identified by using an amino sugar-based stable isotope probing approach. The straw-C were transformed more rapidly into total amino sugars (13C-ASs) in NPK-treated soil, while more pronounced 13C-ASs accumulation occurred in NPKM soil at later stage, indicating temporal microbial C uptake to generate necromass varied depending on fertilization regimes. Moreover, straw-derived fungal C was significantly higher in NPKM over NPK while bacterial counterpart reversed during the incubation. Our results reinforced changes in microbial communities and their C utilization capacities for efficient residue production in different fertilized soils. Additionally, NPKM-treated soil sustained a prolonged but likely more efficient utilization of straw-C with increasing time as evidenced by significantly higher percentage of straw-dervied C enrichment in 13C amino sugar pools by the end of incubation. These findings reveal that fertilization regimes can alter response strategies of microbial transformation of plant C into microbial-derived C. Together with differential contributions of these newly-formed microbial C to SOC pool, our work highlights the key role of microbial anabolisms in building up SOC following crop residue return to soils under different fertilization regimes.

Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

BackgroundStable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput.ResultsHere, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.ConclusionsWe demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

The Formation of Refractory Dissolved Organic Nitrogen From Free Amino Acids in the Ocean

AbstractDissolved organic nitrogen (DON) is actively involved in N cycling and transformation processes in the ocean, yet how this large N pool is formed remains elusive. Here we incubated 15N‐labeled individual amino acids (Ala, Val, and Phe) in seawater and monitored their fates over 21 days. About 25%–45% of Phe‐N and Val‐N were transformed to “uncharacterizable DON,” as compared to only about 6% for Ala‐N, indicating the formation of refractory DON is related to specific amino acids. Through a stable isotope probing approach, 5 Phe‐derived DON molecules from the incubations were found to be present in natural waters, and their possible structures were proposed. These results shed new lights on the formation mechanisms of refractory DON, including the roles of specific amino acids and particular chemical structures that may resist decomposition in millennial time scales.

Mosquito sex and mycobiota contribute to fructose metabolism in the Asian tiger mosquito Aedes albopictus

BackgroundPlant floral nectars contain natural sugars such as fructose, which are a primary energy resource for adult mosquitoes. Despite the importance of carbohydrates for mosquito metabolism, a limited knowledge is available about the pathways involved in sugar assimilation by mosquitoes and their associated microbiota. To this end, we used 13C-metabolomic and stable isotope probing approaches coupled to high-throughput sequencing to reveal fructose-related mosquito metabolic pathways and the dynamics of the active gut microbiota following fructose ingestion.ResultsOur results revealed significant differences in metabolic pathways between males and females, highlighting different modes of central carbon metabolism regulation. Competitive and synergistic interactions of diverse fungal taxa were identified within the active mycobiota following fructose ingestion. In addition, we identified potential cross-feeding interactions between this. Interestingly, there is a strong correlation between several active fungal taxa and the presence of fructose-derived metabolites.ConclusionsAltogether, our results provide novel insights into mosquito carbohydrate metabolism and demonstrate that dietary fructose as it relates to mosquito sex is an important determinant of mosquito metabolism; our results also further highlight the key role of active mycobiota interactions in regulating the process of fructose assimilation in mosquitoes. This study opens new avenues for future research on mosquito-microbiota trophic interactions related to plant nectar-derived sugars.Graphical 8ZSbNdwh9FSfnhR6eQHKUrVideo abstract

Flow-through stable isotope probing (Flow-SIP) minimizes cross-feeding in complex microbial communities

Stable isotope probing (SIP) is a key tool for identifying the microorganisms catalyzing the turnover of specific substrates in the environment and to quantify their relative contributions to biogeochemical processes. However, SIP-based studies are subject to the uncertainties posed by cross-feeding, where microorganisms release isotopically labeled products, which are then used by other microorganisms, instead of incorporating the added tracer directly. Here, we introduce a SIP approach that has the potential to strongly reduce cross-feeding in complex microbial communities. In this approach, the microbial cells are exposed on a membrane filter to a continuous flow of medium containing isotopically labeled substrate. Thereby, metabolites and degradation products are constantly removed, preventing consumption of these secondary substrates. A nanoSIMS-based proof-of-concept experiment using nitrifiers in activated sludge and 13C-bicarbonate as an activity tracer showed that Flow-SIP significantly reduces cross-feeding and thus allows distinguishing primary consumers from other members of microbial food webs.

Disentangling immobilization of nitrate by fungi and bacteria in soil to plant residue amendment

Plant residue incorporation improves soil microbial nitrate immobilization, which in turn alleviates nitrate accumulation, thereby improving N retention capacity and reducing N losses. However, how and why the respective nitrate immobilization by fungi and bacteria, which are dominant microorganisms in soil, responds to residue addition remains unknown. By adopting a novel amino sugar-based stable isotope probing approach, we show that both fungal and bacterial nitrate immobilization increased after Paspalum notatum residue addition. The increased proportion of the former was higher than that of the latter, and their difference became much larger with the increased residue addition rate. Furthermore, 70% and 32% of the variations in fungal and bacterial nitrate immobilization activities were explained by their respective biomass. The relative importance of fungal and bacterial nitrate immobilization shifted in a consistent manner with the relative abundance of fungi and bacteria, and the magnitude of these shifts was related to the residue addition rate. Our work demonstrates strong links between increased residue inputs, alterations in microbial community composition, and enhanced ecosystem functioning of nitrate immobilization and retention in soil.

DNA stable isotope probing revealed no incorporation of 13CO2 into comammox Nitrospira but ammonia-oxidizing archaea in a subtropical acid soil

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Purpose: The recently discovered complete ammonia oxidizers (comammox Nitrospira) challenged our concept of the whole nitrification process, i.e., ammonia oxidation catalyzed by autotrophic ammonia-oxidizing archaea (AOA) and bacteria (AOB), followed by the oxidation of nitrite to nitrate. However, the relative contributions of different ammonia oxidizers to soil nitrification are poorly understood. Materials and methods: Soil samples were collected from five land use types (i.e., cropland, grassland, bushland, transition land, and forest land) from a subtropical soil in Yunnan Province, Southwest China. The quantitative distribution of comammox Nitrospira, AOA and AOB in soils was firstly investigated using the QPCR approach and three of which (cropland, transition land and forest land) with pH below 6 were further examined using DNA stable isotope probing approach with 13CO2. Clone library combined with sequencing was applied to detect the phylogenetic information of active microbial groups. Results and discussion: The results showed that the amoA gene abundance of ammonia oxidizers except AOB was significantly lower in croplands than other land uses. Based on the 13CO2 labeling method, AOA, rather than AOB or comammox Nitrospira, was found to incorporate 13CO2 into their genomes during the incubation in cropland and transition land but not in forest land, suggesting the dominant role of AOA in ammonia oxidation. Phylogenetic analysis of the active AOA group revealed that autotrophic AOA community was mainly affiliated with the cluster of Nitrososphaera in transition land, whereas Nitrososphaera, Nitrosopumilus, and Nitrosotalea in cropland. Conclusions: These findings suggested niche differentiation of AOB, AOA, and comammox Nitrospira in the subtropical acid soil with different land uses, and AOA played a more important role in nitrification process of this acid soil.

Assessment of soil microbial communities involved in cellulose utilization at two contrasting Alpine forest sites

Soil microbial communities involved in 13C-labeled cellulose utilization from two contrasting (in terms of vegetation and climatic conditions) forest sites at different elevations were studied by using a laboratory microcosm experiment and a PLFA (phospholipid fatty acid)-SIP (stable isotope probing) approach considering the effects of incubation temperature (0, 10 and 20 °C) and time (1–4 weeks). The amounts of cellulose-13C assimilated by soil microorganisms and the composition of microbial communities utilizing cellulose were site-dependent. Total, bacterial and actinobacterial biomass incorporated higher amounts of 13C in soil from the deciduous forest site (at the lower elevation), while fungal biomass contained increased amounts of 13C in soil from the coniferous forest site (at the higher elevation). Increasing temperatures resulted in increased amounts of 13C assimilated by the different PLFA-based microbial groups, except fungi, and in significant changes in the community structure of cellulose utilizers. Fungi were better adapted to cold conditions than bacteria. Longer incubation times determined an increase in the amount of 13C incorporated into total and bacterial PLFAs but had no effect on the composition of labeled microbial communities.

Are the microbial communities involved in glucose assimilation in paddy soils treated with different fertilization regimes for three years similar?

The aim of this study was to identify the microorganisms involved in 13C-glucose assimilation and to estimate their variations in response to different fertilization regimes. In a 48 h laboratory incubation, glucose was supplied to paddy soils subjected to application of inorganic fertilizer alone (NPK) or application of combination of inorganic fertilizer and organic manure (NPKM) for 3 years. CO2 production and microbial growth responses to glucose addition were monitored during 52 h flooded incubation. Bacterial and fungal populations involved in 13C-glucose assimilation were identified by using DNA stable isotope probing approach (DNA-SIP) combined with high-throughput sequencing at 48 h after 13C-glucose addition. Bacteria were initially more active at utilizing the exogenous glucose added into the soil than fungi. One hundred and twenty-nine bacterial OTUs related to glucose assimilation were found in the NPK-treated soil, being more abundant than 59 in NPKM-treated soils. Moreover, 10 and 11 glucose assimilation-related OTUs of fungi were found in the NPK and NPKM-treated soils, respectively. DNA-SIP revealed that bacterial genera containing Clostridium and Bacillus and fungal genera including Fusarium, Cylindrocarpon and Paralomus were the dominant assimilators of glucose. Besides these ubiquitous assimilators in both soils, Paenibacillus and Sporomusa in NPK-treated soil and Azotobacter and Nectria in NPKM-treated soil were also found as the dominant assimilators. Under the flooded incubation conditions, the species related to glucose assimilation differed between NPK and NPKM-treated paddy soils for both bacteria and fungi. These results could be helpful for improving the mechanistic understanding of LMWOS cycling processes and enhance our understanding of the major microorganisms involved in glucose assimilation in paddy soils under short-term fertilization regimes.

Identification of soil bacteria able to degrade phenanthrene bound to a hydrophobic sorbent in situ

Efficient bioremediation of PAH-contaminated sites is limited by the hydrophobic character and poor bioavailability of pollutants. In this study, stable isotope probing (SIP) was implemented to track bacteria that can degrade PAHs adsorbed on hydrophobic sorbents. Temperate and tropical soils were incubated with 13C-labeled phenanthrene, supplied by spiking or coated onto membranes. Phenanthrene mineralization was faster in microcosms with PAH-coated membranes than in microcosms containing spiked soil. Upon incubation with temperate soil, phenanthrene degraders found in the biofilms that formed on coated membranes were mainly identified as Sphingomonadaceae and Actinobacteria. In the tropical soil, uncultured Rhodocyclaceae dominated degraders bound to membranes. Accordingly, ring-hydroxylating dioxygenase sequences recovered from this soil matched PAH-specific dioxygenase genes recently found in Rhodocyclaceae. Hence, our SIP approach allowed the detection of novel degraders, mostly uncultured, which differ from those detected after soil spiking, but might play a key role in the bioremediation of PAH-polluted soils.

DNA‐, rRNA‐ and mRNA‐based stable isotope probing of aerobic methanotrophs in lake sediment

A stable isotope probing (SIP) approach was used to study aerobic methane-oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with (13) CH(4) and the separation of (13) C-labelled DNA and RNA from unlabelled nucleic acids. The incubation with (13) CH(4) was performed over a 4-day time-course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA-SIP is more sensitive than DNA-SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of (13) C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA-SIP and not by RNA-SIP, suggesting that cross-feeding of the (13) C was primarily detected by DNA-SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross-feeding of methanotroph metabolites by other microorganisms.

Review: Tools for the tract: understanding the functionality of the gastrointestinal tract

The human gastrointestinal tract comprises a series of complex and dynamic organs ranging from the stomach to the distal colon, which harbor immense microbial assemblages that are known to be vital for human health. Until recently, most of the details concerning our gut microbiota remained obscure. Over the past several years, however, a number of crucial technological and conceptual innovations have been introduced to shed more light on the composition and functionality of human gut microbiota. Recently developed high throughput approaches, including next-generation sequencing technologies and phylogenetic microarrays targeting ribosomal RNA gene sequences, allow for comprehensive analysis of the diversity and dynamics of the gut microbiota composition. Nevertheless, most of the microbes especially in the human large intestine still remain uncultured, and the in situ functions of distinct groups of the gut microbiota are therefore largely unknown, but pivotal to the understanding of their role in human physiology. Apart from functional and metagenomics approaches, stable isotope probing is a promising tool to link the metabolic activity and diversity of microbial communities, including yet uncultured microbes, in a complex environment. Advancements in current stable isotope probing approaches integrated with the application of high-throughput diagnostic microarray-based phylogenetic profiling and metabolic flux analysis should facilitate the understanding of human microbial ecology and will enable the development of innovative strategies to treat or prevent intestinal diseases of as yet unknown etiology.

Vegetation composition and soil microbial community structural changes along a wetland hydrological gradient

Abstract. Fluctuations in wetland hydrology create an interplay between aerobic and anaerobic conditions, controlling vegetation composition and microbial community structure and activity in wetland soils. In this study, we investigated the vegetation composition and microbial community structural and functional changes along a wetland hydrological gradient. Two different vegetation communities were distinguished along the hydrological gradient; Caricetum gracilis at the wet depression and Arrhenatheretum elatioris at the drier upper site. Microbial community structural changes were studied by a combined in situ 13CO2 pulse labeling and phospholipid fatty acid (PLFA) based stable isotope probing approach, which identifies the microbial groups actively involved in assimilation of newly photosynthesized, root-derived C in the rhizosphere soils. Gram negative bacterial communities were relatively more abundant in the surface soils of the drier upper site than in the surface soils of the wetter lower site, while the lower site and the deeper soil layers were relatively more inhabited by gram positive bacterial communities. Despite their large abundance, the metabolically active proportion of gram positive bacterial and actinomycetes communities was much smaller at both sites, compared to that of the gram negative bacterial and fungal communities. This suggests much slower assimilation of root-derived C by gram positive and actinomycetes communities than by gram negative bacteria and fungi at both sites. Ground water depth showed a significant effect on the relative abundance of several microbial communities. Relative abundance of gram negative bacteria significantly decreased with increasing ground water depth while the relative abundance of gram positive bacteria and actinomycetes at the surface layer increased with increasing ground water depth.