Monitoring of microbial proteome dynamics using Raman stable isotope probing

Abnormal protein kinetics could be a cause of several diseases associated with essential life processes. An accurate understanding of protein dynamics and turnover is essential for developing diagnostic or therapeutic tools to monitor these changes. Raman spectroscopy in combination with stable isotope probes (SIP) such as carbon-13, and deuterium has been a breakthrough in the qualitative and quantitative study of various metabolites. In this work, we are reporting the utility of Raman-SIP for monitoring dynamic changes in the proteome at the community level. We have used ¹³C-labeled glucose as the only carbon source in the medium and verified its incorporation in the microbial biomass in a time-dependent manner. A visible redshift in the Raman spectral vibrations of major biomolecules such as nucleic acids, phenylalanine, tyrosine, amide I, and amide III were observed. Temporal changes in the intensity of these bands demonstrating the feasibility of protein turnover monitoring were also verified. Kanamycin, a protein synthesis inhibitor was used to assess the feasibility of identifying effects on protein turnover in the cells. Successful application of this work can provide an alternate/adjunct tool for monitoring proteome-level changes in an objective and nondestructive manner.     

稳定同位素探针技术在有机污染物生物降解中的应用

稳定同位素探针技术(Stable isotope probing,SIP)是稳定同位素标记技术和各种分子生物学手段相结合的一系列技术总称。将其应用于探查污染物降解的功能微生物,实现了不经过分离培养直接把微生物的代谢功能、微生物间相互作用与微生物种群结合起来,从而克服了传统分离培养的缺陷,扩大了微生物资源的利用空间,具有广阔的发展前景。本文介绍了稳定同位素探针技术的基本原理和技术路线,对常规PLFA-SIP、DNA-SIP、RNA-SIP的特点进行了阐述和对比;综述了SIP在有机污染物——苯系物、多环芳烃、多氯联苯生物降解方面的研究进展,提出SIP应用于根际研究是今后该技术在生物降解研究中的一个发展方向。     

Identification of cellulolytic bacteria in soil by stable isotope probing

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. ¹³C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the ¹³C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.     

DNA buoyant density shifts during 15N-DNA stable isotope probing

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from ¹⁵N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of ¹⁵N into DNA increased BD by 0.015±0.002 g mL⁻¹ for E. coli and 0.013±0.002 g mL⁻¹ for M. luteus. The DNA BD shift was greatly increased (0.045 g mL⁻¹) when dual isotope (¹³C plus ¹⁵N) labeling was employed. Despite the limited DNA BD shift following ¹⁵N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing

Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.     

Metagenomic stable isotope probing reveals bacteriophage participation in soil carbon cycling

Soil viruses are important components of the carbon (C) cycle, yet we still know little about viral ecology in soils. We added diverse ¹³C-labelled carbon sources to soil and we used metagenomic-SIP to detect ¹³C assimilation by viruses and their putative bacterial hosts. These data allowed us to link a ¹³C-labelled bacteriophage to its ¹³C-labelled Streptomyces putative host, and we used qPCR to track the dynamics of the putative host and phage in response to C inputs. Following C addition, putative host numbers increased rapidly for 3 days, and then more gradually, reaching maximal abundance on Day 6. Viral abundance and virus:host ratio increased dramatically over 6 days, and remained high thereafter (8.42 ± 2.94). From Days 6 to 30, virus:host ratio remained high, while putative host numbers declined more than 50%. Putative host populations were ¹³C-labelled on Days 3–30, while ¹³C-labelling of phage was detected on Days 14 and 30. This dynamic suggests rapid growth and ¹³C-labelling of the host fueled by new C inputs, followed by extensive host mortality driven by phage lysis. These findings indicate that the viral shunt promotes microbial turnover in soil following new C inputs, thereby altering microbial community dynamics, and facilitating soil organic matter production.     

Identification of anthracene degraders in leachate-contaminated aquifer using stable isotope probing

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in landfill leachate-contaminated aquifer. It is necessary to identify the microorganisms truly responsible for PAH degradation if bioremediation can be applied as an effective technology. DNA-based stable isotope probing (SIP) in combination with terminal restriction fragment length polymorphism (TRFLP) was used to identify the active anthracene degraders in the contaminated aquifer sediment. One kind of degrader was classified as Variovorax species within class ??-proteobacteria, but another belonged to unclassified bacteria. These findings also suggest novel microorganisms involved in PAH-degrading processes.     

Unraveling uncultivable pesticide degraders via stable isotope probing (SIP)

Uncultivable microorganisms account for over 99% of all species on earth, playing essential roles in ecological processes such as carbon/nitrogen cycle and chemical mineralization. Their functions remain unclear in ecosystems and natural habitats, requiring cutting-edge biotechnologies for a deeper understanding. Stable isotope probing (SIP) incorporates isotope-labeled elements, e.g. ^13?C, ^18?O or ^15?N, into the cellular components of active microorganisms, serving as a powerful tool to link phylogenetic identities to their ecological functions in situ. Pesticides raise increasing attention for their persistence in the environment, leading to severe damage and risks to the ecosystem and human health. Cultivation and metagenomics help to identify either cultivable pesticide degraders or potential pesticide metabolisms within microbial communities, from various environmental media including the soil, groundwater, activated sludge, plant rhizosphere, etc. However, the application of SIP in characterizing pesticide degraders is limited, leaving considerable space in understanding the natural pesticide mineralization process. In this review, we try to comprehensively summarize the fundamental principles, successful cases and technical protocols of SIP in unraveling functional-yet-uncultivable pesticide degraders, by raising its shining lights and shadows. Particularly, this study provides deeper insights into various feasible isotope-labeled substrates in SIP studies, including pesticides, pesticide metabolites, and similar compounds. Coupled with other techniques, such as next-generation sequencing, nanoscale secondary ion mass spectrometry (NanoSIMS), single cell genomics, magnetic-nanoparticle-mediated isolation (MMI) and compound-specific isotope analysis (CSIA), SIP will significantly broaden our understanding of pesticide biodegradation process in situ.     

定量稳定性同位素探针技术及其在微生物生态学研究中的应用

定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率.qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高...     

Unlocking the 'microbial black box' using RNA-based stable isotope probing technologies

Microbial ecologists have long sought to associate the transformation of compounds in the environment with the microbial clades responsible. The development of stable isotope probing (SIP) has made this possible in many ecological and biotechnological contexts. RNA-based SIP technologies represent a significant leap forward for culture-independent 'functional phylogeny' analyses, where specific consumption of a given compound carrying a (13)C signature can be associated with the small subunit ribosomal RNA molecules of the microbes that consume it. Recent advances have led to the unequivocal identification of microorganisms responsible for contaminant degradation in engineered systems, and to applications enhancing our understanding of carbon flow in terrestrial ecosystems.     

Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing

Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [(13)C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.     

Identification of triclosan-degrading bacteria in a triclosan enrichment culture using stable isotope probing

Triclosan, a widely used antimicrobial agent, is an emerging contaminant in the environment. Despite its antimicrobial character, biodegradation of triclosan has been observed in pure cultures, soils and activated sludge. However, little is known about the microorganisms responsible for the degradation in mixed cultures. In this study, active triclosan degraders in a triclosan-degrading enrichment culture were identified using stable isotope probing (SIP) with universally ¹³C-labeled triclosan. Eleven clones contributed from active microorganisms capable of uptake the ¹³C in triclosan were identified. None of these clones were similar to known triclosan-degraders/utilizers. These clones distributed among α-, β-, or γ-Proteobacteria: one belonging to Defluvibacter (α-Proteobacteria), seven belonging to Alicycliphilus (β-Proteobacteria), and three belonging to Stenotrophomonas (γ-Proteobacteria). Successive additions of triclosan caused a significant shift in the microbial community structure of the enrichment culture, with dominant ribotypes belonging to the genera Alicycliphilus and Defluvibacter. Application of SIP has successfully identified diverse uncultivable triclosan-degrading microorganisms in an activated sludge enrichment culture. The results of this study not only contributed to our understanding of the microbial ecology of triclosan biodegradation in wastewater, but also suggested that triclosan degraders are more phylogenetically diverse than previously reported.     

Functional analysis of an anaerobic m-xylene-degrading enrichment culture using protein-based stable isotope probing

A sulfate-reducing consortium maintained for several years in the laboratory with m-xylene as sole source of carbon and energy was characterized by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of PCR-amplified 16S rRNA genes and stable isotope probing of proteins (Protein-SIP). During growth upon m-xylene or methyl-labeled m-xylene (1,3-dimethyl-(13)C(2)-benzene), a phylotype affiliated to the family Desulfobacteriaceae became most abundant. A second dominant phylotype was affiliated to the phylum Epsilonproteobacteria. In cultures grown with methyl-labeled m-xylene, 331 proteins were identified by LC-MS/MS analysis. These proteins were either not (13)C-labeled (23%) or showed a (13)C-incorporation of 19-22 atom% (13)C (77%), the latter demonstrating that methyl groups of m-xylene were assimilated. (13)C-labeled proteins were involved in anaerobic m-xylene biodegradation, in sulfate reduction, in the Wood-Ljungdahl-pathway, and in general housekeeping functions. Thirty-eight percent of the labeled proteins were affiliated to Deltaproteobacteria. Probably due to a lack of sequence data from Epsilonproteobacteria, only 14 proteins were assigned to this phylum. Our data suggest that m-xylene is assimilated by the Desulfobacteriaceae phylotype, whereas the role of the Epsilonproteobacterium in the consortium remained unclear.     

Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (¹³C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (¹³C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived ¹³C in each of the agricultural soils (“candidate” phylum TM7 and an unclassified Sphingomonadaceae).     

Detection of active butyrate-degrading microorganisms in methanogenic sludges by RNA-based stable isotope probing

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the (13)C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.     

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.Subject terms: Microbiology, Biological techniques, Metabolism, Biogeochemistry, Microbial ecology

Stable isotope probing (SIP) is widely applied to link specific microbial populations to metabolic processes in the environment and has greatly advanced our understanding of the role of microorganisms in biogeochemical cycling. SIP relies on tracing the incorporation of specific isotopically labeled substrates (e.g., ¹³C, ¹⁵N, ¹⁸O, ²H) into cellular biomarkers or bulk cellular biomass [e.g., 1–4]. SIP is considered a robust technique to identify microbial populations that assimilate a labeled substrate of interest in complex environmental communities. However, cross-feeding can occur when isotopically labeled metabolites are released from a primary consumer and then used by other microorganisms, which subsequently also become isotopically labeled. Likewise, when ¹³C-bicarbonate and unlabeled substrate are supplied to assess the activity of specific chemolithoautotrophs [e.g., 5–7], undesired ¹³C-incorporation can occur due to cross-feeding between chemolithoautotrophs whose activity depends on each other, for example in nitrifiers, where ammonia oxidizers provide nitrite oxidizers with their substrate, nitrite. The uncertainties associated with cross-feeding in SIP studies increase as the incubation time of microbial communities increases. While this phenomenon can be used to study microbial interactions and trophic networks [8–11], cross-feeding can lead to erroneous identification of organisms that are not directly responsible for the process of interest, but are rather connected to primary consumers via a microbial food web [2, 10, 12, 13].We developed an approach that significantly reduces the effect of cross-feeding in SIP studies. For this purpose, a thin layer of microbial cells is placed on a membrane filter, and isotopically labeled substrate is supplied at a fixed concentration by continuous flow, which constantly removes released metabolites and degradation products of primary substrate consumers. While previous SIP studies have employed a continuous flow of medium or substrate [e.g., 6, 14–16], in these studies, cross-feeding still occurred, as large amounts of biomass were placed in a 3D space, which allowed for the exchange of metabolites. Here, we present a proof-of-concept experiment with a nitrifying activated sludge microbial community, which converts ammonia to nitrite by the activity of ammonia-oxidizing bacteria (AOB), and subsequently oxidizes nitrite to nitrate by nitrite-oxidizing bacteria (NOB). In our experiments, the carbon source for both groups of autotrophic nitrifiers (the sludge contained no comammox bacteria [17, 18]) was isotopically labeled inorganic carbon (¹³C–NaHCO₃) and, as the sole electron donor, unlabeled ammonium was provided.In the flow-through approach, AOB, but not NOB, should be ¹³C-labeled because the substrate for NOB (nitrite), produced by AOB is continuously removed and thus the NOB should remain metabolically inactive (Fig. 1). In addition to a regular batch incubation, we included a control incubation, where the flow-through was recirculated to determine the impact of the experimental setup (continuous medium flow and retainment of biomass on a membrane filter) in Flow-SIP on the activity of the bacterial cells (in particular on the NOB as their autotrophic activity is used as a read out for cross-feeding in our experiments) in comparison to the batch experiment. Cross-feeding is expected to occur in both recirculated and batch control incubations, where nitrite is not removed and thus both AOB and NOB conserve energy to fix ¹³C–CO₂. After the experiments, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to identify AOB and NOB and combined with nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify ¹³C-assimilation at the single-cell level for all setups.Open in a separate windowFig. 1Schematic representation of the experimental setup: (left) batch, (center) recirculated, and (right) flow-through incubation.In all incubations, the carbon source for both autotrophic nitrifiers (AOB, yellow and NOB, magenta) was isotopically labeled (i.e., CO₂ as ¹³C–NaHCO₃) and ammonia was provided as the only external energy source (as NH₄Cl). Cross-feeding is expected to occur in the batch and recirculated approaches, where NOB consume nitrite produced via ammonia oxidation by AOB and thus both AOB and NOB incorporate ¹³C–CO₂. In the flow-through approach, only AOB are expected to be ¹³C-labeled, as cross-feeding should be eliminated by the continuous removal of nitrite. Other, non-nitrifier cells are indicated in gray.For these experiments, activated sludge from a Danish municipal wastewater treatment plant was initially treated by sonication to disrupt large flocs. Cells were then either placed on a membrane filter for flow-through incubation and the recirculated control experiment, or incubated in a conventional batch experiment. All experiments were set up using the same amount of biomass, and the ratio of biomass to medium volume was the same in batch and recirculated control experiments. Incubations were done using mineral medium containing 250 µM NH₄Cl and 2 mM ¹³C–NaHCO₃ for 24 h. Medium flow was maintained at a rate of 26 ml h⁻¹. We did not select a higher flow rate in order to avoid excessive stress by the medium flow on the microbial cells and to minimize the required amounts of media containing isotopically labeled bicarbonate. Furthermore, modeling nitrite advection and diffusion at different flow rates showed that, for example, a tenfold higher flow rate would only marginally reduce nitrite concentrations surrounding the AOB colonies (Fig. S1). In contrast, in a purely diffusive system without continuous flow, our model showed that nitrite would accumulate to significantly higher concentrations around single AOB colonies (Fig. S2). For example, after 24 h, ~23 µM nitrite would accumulate at a distance of 0–100 µm (with no significant decrease over distance) around an AOB colony of 50 cells, which is 230- to 9200-fold higher (depending on the distance to the colony) than modeled nitrite concentrations at the flow rate used in our experiments. Most inorganic metabolites that can directly be taken up into cells behave similar as nitrite in a diffusive system, i.e., they have a similar diffusion coefficient. Larger molecules tend to have an even smaller diffusion coefficient, which would lead to slower diffusion, and thus even more efficient metabolite removal when a medium flow is applied.We monitored nitrification activity via concentration measurements of ammonium, nitrite and nitrate (Fig. S3) and conducted nanoSIMS analyses (Fig. 2) for two successive experiments using sludge collected from the same treatment plant on different days as replication of experimental results (referred to as E1 and E2). Additionally, we confirmed the reproducibility of the method with two further experiments, where nitrification activity was followed (Fig. S4). Details on the experimental setup are given in Fig. 1 and the Supplementary Text.Open in a separate windowFig. 2Single cell isotope probing of nitrifying activated sludge in batch, recirculated, and flow-through incubations.a–c Show representative FISH images of E2 (AOB in yellow; NOB in magenta; other cells counterstained by DAPI in gray) of batch, recirculated, and flow-through incubations, respectively, and d–f show the corresponding nanoSIMS images. Scale bar is 10 µm in all images. g–l Show ¹³C labeling of AOB, NOB, and other cells quantified by nanoSIMS at the single-cell level for E1 (g–i) and E2 (j–l) in batch, recirculated, and flow-through incubations, respectively. We used FISH probe sets targeting AOB (Nitrosomonas oligotropha cluster (Cl6a192), Nitrosomonas eutropha/europea/urea cluster (NEU)) and NOB (Nitrotoga (Ntoga122), Nitrospira Lineage 1 (Ntspa1431), and Nitrospira Lineage 2 (Ntspa1151)), respectively, for differential staining of the two nitrifier groups. In (g–l), dashed lines give the ¹³C natural abundance values of the filter surface. The number of cells analyzed per group is indicated below each boxplot. For each experiment, lower case letters indicate significant difference in ¹³C labeling between groups (AOB, NOB, other cells) within an incubation type and upper case letters indicate significant difference between incubation types for a given group (Kruskal–Wallis test followed by Dunn’s test; Statistics are given in Table S2). Boxplots depict the 25–75% quantile range, with the center line depicting the median (50% quantile) and whiskers encompass data points within 1.5× the interquartile range.In recirculated and batch control incubations, the consumption of ammonium, production of nitrite and nitrate (Fig. S3), and single cell ¹³C-incorporation (Fig. 2) indicated that both AOB and NOB were active. However, nitrification activity (i.e., nitrite and nitrate production) in recirculated incubations were reduced by 57% (E1) and 83% (E2) compared to batch incubations (Fig. S3). The reduced ammonia oxidation activity in the recirculated incubations compared to the batch incubations was also reflected by a 73–82% lower ¹³C-incorporation in AOB cells in the former incubations (Fig. 2, median AOB ¹³C-enrichment in recirculated setup was 3.7 and 3.9 ¹³C-atom%, in batch incubations 20.7 and 14.7 ¹³C-atom% for E1 and E2, respectively). AOB in the flow-through incubations also showed lower ¹³C-enrichment levels (8.2 and 8.5 atom% for E1 and E2, respectively) compared to batch incubations but higher enrichment than in the recirculated incubations. The lower enrichment of AOB in the recirculated compared to the flow-through incubations might be due to an accumulation of compounds leaching from the used tubing (PharMed^® Ismaprene, Table S1), which may negatively affect AOB. Indeed, nitrifiers have previously been reported to be sensitive to various organic compounds [19, 20]. Use of different rubber tubing or replacing rubber tubing by glass might alleviate these effects. AOB ¹³C-enrichment was highest in batch incubations, which could be due to both the lack of stress from the continuous medium flow and the observed reaggregation of the sonicated activated sludge into larger flocs—reminiscent of native activated sludge flocs.As expected, NOB were ¹³C-enriched in both the batch (13.3 and 4.9 atom% for E1 and E2, respectively) and recirculated incubations (7.2 and 4.7 atom% for E1 and E2, respectively). In contrast, as intended, the flow-through setup resulted in a substantial reduction in ¹³C-enrichment of NOB (2.0 atom% for both E1 and E2, respectively; with consistently low ¹³C-enrichment in all NOB cells measured). This demonstrates that Flow-SIP efficiently removed the secondary substrate nitrite released by the AOB primary substrate consumers, thereby strongly limiting cross-feeding between AOB and NOB. The low ¹³C-enrichment of NOB in the flow-through incubations was statistically not significantly different to the ¹³C-enrichment of non-nitrifier cells (Table S2). It is unlikely that this low background ¹³C-enrichment was due to ¹³C-bicarbonate adsorption, as all samples were treated with acid before nanoSIMS analysis. It is, however, possible that at least some of the observed ¹³C-enrichment in NOB and other bacteria is due to anaplerotic reactions leading to C-fixation by background cellular activity rather than substrate-induced autotrophic C-fixation [e.g., 21, 22]. Transfer of ¹³C-labeled metabolites from the autotrophic nitrifiers to non-nitrifier cells was negligible in all incubations, including batch and recirculated incubations (Fig. 2), which was likely due to the short incubation time (24 h). In contrast, other SIP studies using incubation times of several days reported significant C-isotope transfer from nitrifiers to non-nitrifiers [11, 23].Our results demonstrate that Flow-SIP is a promising approach to significantly reduce cross-feeding in complex microbial communities and can be even successfully applied to highly aggregated samples like activated sludge flocs when they are dispersed prior to the experiment. Flow-SIP and conventional SIP are complementary to each other in the analysis of such aggregated or biofilm communities. In such systems, Flow-SIP enables microbial ecologists studying microbial physiologies with drastically reduced cross-feeding, but destroys the spatial arrangement of cells, while conventional SIP retains the 3D architecture, but its results are strongly influenced by cross-feeding. We expect that Flow-SIP is ideally suited for oligotrophic fresh- or seawater samples, which predominantly harbor planktonic cells or small aggregates, and thus do not require any sonication pretreatment before incubation. Furthermore, for such samples, tracer can be directly added to sterile filtered water without the need for using artificial medium, as used for the presented proof-of-principle experiments.Flow-SIP may, after upscaling to label more biomass, also be used in combination with DNA-, RNA- or protein-SIP, which should in comparison to conventional SIP, where cross-feeding is not inhibited, allow microbial ecologists to more precisely identify both previously known and yet unknown primary consumers of a supplied substrate. For example, Flow-SIP with ¹³C-bicarbonate and unlabeled ammonium would allow distinguishing comammox organisms from canonical NOB, as comammox but not the canonical NOB would be active under these conditions together with the canonical ammonia oxidizers. In addition, Flow-SIP has the potential to study direct use of chemically unstable substrates by microorganisms, by distinguishing it from microbial consumption of their chemically formed decomposition products. For example, cyanate, which abiotically decays relatively fast to ammonium and carbon dioxide [24, 25], has previously been shown to serve as energy and nitrogen source for ammonia-oxidizing archaea [25, 26]. Using Flow-SIP, cyanate could be constantly supplied, thereby strongly reducing abiotic decay. At the same time, any abiotically formed ammonium (and ammonium produced by other organisms) would be constantly removed, which should allow identifying ammonia-oxidizing microorganisms that directly use cyanate as a substrate. Furthermore, the presented approach may be coupled to fluorescence-based activity markers, where a substrate of interest and bioorthogonal noncanonical amino acids are supplied and, subsequently, translationally active cells are visualized on an epifluorescence microscope (BONCAT) [27]. In conclusion, Flow-SIP expands the toolbox of microbial ecologists interested in structure–function analyses of microbial communities and will contribute to a more precise understanding of the ecophysiology of bacteria and archaea catalyzing key processes in their natural environments.