Heterotrophic Fixation of CO2 in Soil

The occurrence of heterotrophic CO₂ fixation by soil microorganisms was tested in several mineral soils differing in pH and two artificial soils (a mixture of silica sand, alfalfa powder, and nutrient medium inoculated with a soil suspension). Soils were incubated at ambient (∼0.05 vol%) and elevated (∼5 vol%) CO₂ concentrations under aerobic conditions for up to 21 days. CO₂ fixation was detected using either a technique for determining the natural abundance of ¹³C or by measuring the distribution of labeled ¹⁴C-CO₂ in soil and bacteria. The effects of elevated CO₂ on microbial biomass (direct counts, chloroform fumigation extraction method), composition of microbial community (phospholipid fatty acids), microbial activity (respiration, dehydrogenase activity), and turnover rate were also measured. Heterotrophic CO₂ fixation was proven in all soils under study, being higher in neutral soils. The main portion of the fixed CO₂ (98–99%) was found in extracellular metabolites while only ∼1% CO₂ was incorporated into microbial cells. High CO₂ concentration always induced an increase in microbial activity, changes in the composition of the microbial community, and a decrease in microbial turnover. The results suggest that heterotrophic CO₂ fixation could be a widespread process in soils.     

自养微生物同化CO_2的分子生态研究及同化碳在土壤中的转化

大气中CO2浓度持续升高和全球气候变暖是亟待解决的重大环境问题。自养微生物在环境中广泛分布,能直接参与CO2的同化,因此研究自养微生物同化CO2的分子生态学机制具有重大的科学意义。以往对自养微生物的研究多针对基因组DNA,从DNA水平揭示了不同生态系统中碳同化自养微生物的种群结构和多样性,但这些微生物在生态系统中的具体功能有待进一步的研究。近年来,随着转录组学研究技术和稳定同位素探针技术(SIP)的发展,自养微生物同化CO2的生态机理研究不断深入,这些研究明确揭示了碳同化自养微生物是河流、湖泊和海洋生态系统中CO2固定作用的驱动者,并新发现了一些具有CO2同化功能的微生物群落。基于国内外有关研究进展,从DNA和RNA水平上对自养微生物同化CO2的分子机理以及稳定同位素探针技术(SIP)在碳同化微生物研究中的应用进行了分析和总结,初步展望了RNA-SIP技术在陆地生态系统碳同化微生物分子生态学研究中的前景。同时,探讨了陆地生态系统同化碳的转化和稳定性机理,以期为深入了解生态系统碳循环过程和应对气候变化提供理论依据。     

Dark microbial CO2 fixation in temperate forest soils increases with CO2 concentration

Dark, that is, nonphototrophic, microbial CO₂ fixation occurs in a large range of soils. However, it is still not known whether dark microbial CO₂ fixation substantially contributes to the C balance of soils and what factors control this process. Therefore, the objective of this study was to quantitate dark microbial CO₂ fixation in temperate forest soils, to determine the relationship between the soil CO₂ concentration and dark microbial CO₂ fixation, and to estimate the relative contribution of different microbial groups to dark CO₂ fixation. For this purpose, we conducted a ¹³C‐CO₂ labeling experiment. We found that the rates of dark microbial CO₂ fixation were positively correlated with the CO₂ concentration in all soils. Dark microbial CO₂ fixation amounted to up to 320 µg C kg^?1 soil day^?1 in the Ah horizon. The fixation rates were 2.8–8.9 times higher in the Ah horizon than in the Bw1 horizon. Although the rates of dark microbial fixation were small compared to the respiration rate (1.2%–3.9% of the respiration rate), our findings suggest that organic matter formed by microorganisms from CO₂ contributes to the soil organic matter pool, especially given that microbial detritus is more stable in soil than plant detritus. Phospholipid fatty acid analyses indicated that CO₂ was mostly fixed by gram‐positive bacteria, and not by fungi. In conclusion, our study shows that the dark microbial CO₂ fixation rate in temperate forest soils increases in periods of high CO₂ concentrations, that dark microbial CO₂ fixation is mostly accomplished by gram‐positive bacteria, and that dark microbial CO₂ fixation contributes to the formation of soil organic matter.     

Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured ¹³CO₂ respired from [¹³C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that ¹³C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [¹³C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [¹³C]phenol, and (iii) 12 daily doses of [¹³C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted ¹³CO₂ evolution by a factor of 10. Furthermore, imaging of ¹³C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated ¹³C into their biomass. PCR amplification and 16S rRNA gene sequencing of ¹³C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.     

Small phytoplankton contribute greatly to CO2-fixation after the diatom bloom in the Southern Ocean

Phytoplankton is composed of a broad-sized spectrum of phylogenetically diverse microorganisms. Assessing CO₂-fixation intra- and inter-group variability is crucial in understanding how the carbon pump functions, as each group of phytoplankton may be characterized by diverse efficiencies in carbon fixation and export to the deep ocean. We measured the CO₂-fixation of different groups of phytoplankton at the single-cell level around the naturally iron-fertilized Kerguelen plateau (Southern Ocean), known for intense diatoms blooms suspected to enhance CO₂ sequestration. After the bloom, small cells (<20 µm) composed of phylogenetically distant taxa (prymnesiophytes, prasinophytes, and small diatoms) were growing faster (0.37 ± 0.13 and 0.22 ± 0.09 division d⁻¹ on- and off-plateau, respectively) than larger diatoms (0.11 ± 0.14 and 0.09 ± 0.11 division d⁻¹ on- and off-plateau, respectively), which showed heterogeneous growth and a large proportion of inactive cells (19 ± 13%). As a result, small phytoplankton contributed to a large proportion of the CO₂ fixation (41–70%). The analysis of pigment vertical distribution indicated that grazing may be an important pathway of small phytoplankton export. Overall, this study highlights the need to further explore the role of small cells in CO₂-fixation and export in the Southern Ocean.Subject terms: Biogeochemistry, Biogeochemistry, Stable isotope analysis, Microbial ecology     

Resonance Raman spectroscopy of carotenoids in Photosystem II core complexes

Resonance Raman (RR) spectroscopy has been used to examine the configuration of the carotenoids bound to Synechocystis PCC 6803 Photosystem II (PS II) core complexes. The excitation wavelengths used (514.5, 488.0, 476.5 and 457.9 nm) span the absorption bands of all of the ~12–17 neutral carotenoids in the PS II core complex. The RR spectra of the two carotenoids associated with the D1–D2 polypeptides (Car₅₀₇ and Car₄₈₉) of the reaction center are extracted via light versus dark difference experiments measured at 20 K. The RR results are consistent with all-trans configurations for both Car₅₀₇ and Car₄₈₉ and indicate that majority of the other carotenoids in the PS II core complex must also be in the all-trans configuration. The configuration of β-carotene is relevant to its proposed function as a molecular wire in the secondary electron-transfer reactions of PS II.     

Non-phototrophic CO 2 fixation by soil microorganisms

Although soils are generally known to be a net source of CO₂ due to microbial respiration, CO₂ fixation may also be an important process. The non-phototrophic fixation of CO₂ was investigated in a tracer experiment with ¹⁴CO₂ in order to obtain information about the extent and the mechanisms of this process. Soils were incubated for up to 91 days in the dark. In three independent incubation experiments, a significant transfer of radioactivity from ¹⁴CO₂ to soil organic matter was observed. The process was related to microbial activity and could be enhanced by the addition of readily available substrates such as acetate. CO₂ fixation exhibited biphasic kinetics and was linearly related to respiration during the first phase of incubation (about 20–40 days). The fixation amounted to 3–5% of the net respiration. After this phase, the CO₂ fixation decreased to 1–2% of the respiration. The amount of carbon fixed by an agricultural soil corresponded to 0.05% of the organic carbon present in the soil at the beginning of the experiment, and virtually all of the fixed CO₂ was converted to organic compounds. Many autotrophic and heterotrophic biochemical processes result in the fixation of CO₂. However, the enhancement of the fixation by addition of readily available substrates and the linear correlation with respiration suggested that the process is mainly driven by aerobic heterotrophic microorganisms. We conclude that heterotrophic CO₂ fixation represents a significant factor of microbial activity in soils.     

Impacts of 3 years of elevated atmospheric CO2 on rhizosphere carbon flow and microbial community dynamics

Carbon (C) uptake by terrestrial ecosystems represents an important option for partially mitigating anthropogenic CO₂ emissions. Short‐term atmospheric elevated CO₂ exposure has been shown to create major shifts in C flow routes and diversity of the active soil‐borne microbial community. Long‐term increases in CO₂ have been hypothesized to have subtle effects due to the potential adaptation of soil microorganism to the increased flow of organic C. Here, we studied the effects of prolonged elevated atmospheric CO₂ exposure on microbial C flow and microbial communities in the rhizosphere. Carex arenaria (a nonmycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown at defined atmospheric conditions differing in CO₂ concentration (350 and 700 ppm) for 3 years. During this period, C flow was assessed repeatedly (after 6 months, 1, 2, and 3 years) by ¹³C pulse‐chase experiments, and label was tracked through the rhizosphere bacterial, general fungal, and arbuscular mycorrhizal fungal (AMF) communities. Fatty acid biomarker analyses and RNA‐stable isotope probing (RNA‐SIP), in combination with real‐time PCR and PCR‐DGGE, were used to examine microbial community dynamics and abundance. Throughout the experiment the influence of elevated CO₂ was highly plant dependent, with the mycorrhizal plant exerting a greater influence on both bacterial and fungal communities. Biomarker data confirmed that rhizodeposited C was first processed by AMF and subsequently transferred to bacterial and fungal communities in the rhizosphere soil. Over the course of 3 years, elevated CO₂ caused a continuous increase in the ¹³C enrichment retained in AMF and an increasing delay in the transfer of C to the bacterial community. These results show that, not only do elevated atmospheric CO₂ conditions induce changes in rhizosphere C flow and dynamics but also continue to develop over multiple seasons, thereby affecting terrestrial ecosystems C utilization processes.     

异养微生物固定CO2的合成生物学研究进展

人类活动造成大气二氧化碳（CO₂）浓度不断升高,使当今世界面临着气候变化的重大危机。微生物CO₂固定为实现地球“碳中和”提供了一条有前景的绿色发展路线。与自养微生物相比,异养微生物具有更快的生长速度和更先进的遗传工具,但是其固定CO₂的能力还很有限。近年来,基于合成生物学技术强化异养微生物CO₂固定受到诸多关注,主要包括优化能量供给、改造羧化途径以及基于异养微生物间接固定CO₂。本综述将围绕上述3个方面重点讨论异养微生物CO₂固定的研究进展,为将来更好地利用微生物CO₂固定技术实现“碳达峰、碳中和”提供参考。     

Crenarchaeal heterotrophy in salt marsh sediments

Mesophilic Crenarchaeota (also known as Thaumarchaeota) are ubiquitous and abundant in marine habitats. However, very little is known about their metabolic function in situ. In this study, salt marsh sediments from New Jersey were screened via stable isotope probing (SIP) for heterotrophy by amending with a single ¹³C-labeled compound (acetate, glycine or urea) or a complex ¹³C-biopolymer (lipids, proteins or growth medium (ISOGRO)). SIP incubations were done at two substrate concentrations (30–150 μM; 2–10 mg ml⁻¹), and ¹³C-labeled DNA was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes. To test for autotrophy, an amendment with ¹³C-bicarbonate was also performed. Our SIP analyses indicate salt marsh crenarchaea are heterotrophic, double within 2–3 days and often compete with heterotrophic bacteria for the same organic substrates. A clone library of ¹³C-amplicons was screened to find matches to the ¹³C-TRFLP peaks, with seven members of the Miscellaneous Crenarchaeal Group and seven members from the Marine Group 1.a Crenarchaeota being discerned. Some of these crenarchaea displayed a preference for particular carbon sources, whereas others incorporated nearly every ¹³C-substrate provided. The data suggest salt marshes may be an excellent model system for studying crenarchaeal metabolic capabilities and can provide information on the competition between crenarchaea and other microbial groups to improve our understanding of microbial ecology.     

Bacteria and Fungi Respond Differently to Multifactorial Climate Change in a Temperate Heathland,Traced with 13C-Glycine and FACE CO2

It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g⁻¹ soil) of ¹³C-labeled glycine (¹³C₂, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The ¹³C enrichment of respired CO₂ and of phospholipid fatty acids (PLFAs) was determined after 24 h. ¹³C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS).Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated ¹³C in all treatments, whereas fungi had minor or no glycine derived ¹³C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO₂ did suggest increased direct incorporation of glycine in microbial biomass, in particular in G⁺ bacteria, in an ecosystem subjected to elevated CO₂. Warming decreased the concentration of PLFAs in general. The FACE CO₂ was ¹³C-depleted (δ¹³C = 12.2‰) compared to ambient (δ¹³C = ∼−8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern to future changes in substrate availability and climatic factors.     

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO₂) fixation by toxic metals such as cadmium – classified as a priority pollutant – could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO₂ and labelled ¹³C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO₂ fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (μXRF)/micro X-ray absorption near-edge structure (μXANES) at Cd L_III-edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the ¹³C/¹²C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO₂ decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.     

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

Although uncultured microorganisms have important roles in ecosystems, their ecophysiology in situ remains elusive owing to the difficulty of obtaining live cells from their natural habitats. In this study, we employed a novel magnetic nanoparticle-mediated isolation (MMI) method to recover metabolically active cells of a group of previously uncultured phenol degraders, Burkholderiales spp., from coking plant wastewater biosludge; five other culturable phenol degraders—Rhodococcus sp., Chryseobacterium sp. and three different Pseudomonas spp.—were also isolated from the same biosludge using traditional methods. The kinetics of phenol degradation by MMI-recovered cells (MRCs) was similar to that of the original sludge. Stable isotope probing (SIP) and pyrosequencing of the 16S rRNA from the ‘heavy'' DNA (¹³C-DNA) fractions indicated that Burkholderiales spp. were the key phenol degraders in situ in the biosludge, consistent with the results of MRCs. Single-cell Raman micro-spectroscopy was applied to probe individual bacteria in the MRCs obtained from the SIP experiment and showed that 79% of them were fully ¹³C-labelled. Biolog assays on the MRCs revealed the impact of various carbon and nitrogen substrates on the efficiency of phenol degradation in the wastewater treatment plant biosludge. Specifically, hydroxylamine, a metabolite of ammonia oxidisation, but not nitrite, nitrate or ammonia, inhibited phenol degradation in the biosludge. Our results provided a novel insight into the occasional abrupt failure events that occur in the wastewater treatment plant. This study demonstrated that MMI is a powerful tool to recover live and functional cells in situ from a complex microbial community to enable further characterisation of their physiology.     

Epsilonproteobacteria Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas

Recent studies have indicated that chemoautotrophic Epsilonproteobacteria might play an important role, especially as anaerobic or microaerophilic dark CO₂-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO₂-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of Epsilonproteobacteria to dark CO₂ fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [¹⁴C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10^{5 14}CO₂-fixing prokaryotic cells ml⁻¹ were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10^{4 14}CO₂-fixing cells ml⁻¹ were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance. ¹⁴CO₂-incorporating cells belonged exclusively to the domain Bacteria. Among these, members of the Epsilonproteobacteria were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the Sulfurimonas subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO₂-fixing group. In conclusion, Epsilonproteobacteria were found to be mainly responsible for chemoautotrophic activity in the dark CO₂ fixation maxima of the Black Sea and central Baltic Sea redoxclines. These Epsilonproteobacteria might be relevant in similar habitats of the world's oceans, where high dark CO₂ fixation rates have been measured.     

Efficient CO2 fixation by surface Prochlorococcus in the Atlantic Ocean

Nearly half of the Earth''s surface is covered by the ocean populated by the most abundant photosynthetic organisms on the planet—Prochlorococcus cyanobacteria. However, in the oligotrophic open ocean, the majority of their cells in the top half of the photic layer have levels of photosynthetic pigmentation barely detectable by flow cytometry, suggesting low efficiency of CO₂ fixation compared with other phytoplankton living in the same waters. To test the latter assumption, CO₂ fixation rates of flow cytometrically sorted ¹⁴C-labelled phytoplankton cells were directly compared in surface waters of the open Atlantic Ocean (30°S to 30°N). CO₂ fixation rates of Prochlorococcus are at least 1.5–2.0 times higher than CO₂ fixation rates of the smallest plastidic protists and Synechococcus cyanobacteria when normalised to photosynthetic pigmentation assessed using cellular red autofluorescence. Therefore, our data indicate that in oligotrophic oceanic surface waters, pigment minimisation allows Prochlorococcus cells to harvest plentiful sunlight more effectively than other phytoplankton.     

Significance of archaeal nitrification in hypoxic waters of the Baltic Sea

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread, and their abundance in many terrestrial and aquatic ecosystems suggests a prominent role in nitrification. AOA also occur in high numbers in oxygen-deficient marine environments, such as the pelagic redox gradients of the central Baltic Sea; however, data on archaeal nitrification rates are scarce and little is known about the factors, for example sulfide, that regulate nitrification in this system. In the present work, we assessed the contribution of AOA to ammonia oxidation rates in Baltic deep basins and elucidated the impact of sulfide on this process. Rate measurements with ¹⁵N-labeled ammonium, CO₂ dark fixation measurements and quantification of AOA by catalyzed reporter deposition–fluorescence in situ hybridization revealed that among the three investigated sites the highest potential nitrification rates (122–884 nmol l⁻¹per day) were measured within gradients of decreasing oxygen, where thaumarchaeotal abundance was maximal (2.5–6.9 × 10⁵ cells per ml) and CO₂ fixation elevated. In the presence of the archaeal-specific inhibitor GC₇, nitrification was reduced by 86–100%, confirming the assumed dominance of AOA in this process. In samples spiked with sulfide at concentrations similar to those of in situ conditions, nitrification activity was inhibited but persisted at reduced rates. This result together with the substantial nitrification potential detected in sulfidic waters suggests the tolerance of AOA to periodic mixing of anoxic and sulfidic waters. It begs the question of whether the globally distributed Thaumarchaeota respond similarly in other stratified water columns or whether the observed robustness against sulfide is a specific feature of the thaumarchaeotal subcluster present in the Baltic Deeps.     

Application of <Superscript>13</Superscript>C mineral carbon for assessment of the primary production of organic matter in aquatic environments

The possibility of measuring the rates of light and dark CO₂ assimilation using ¹³C carbonate was demonstrated on Lake Kichier (Marii El). The application of methods utilizing the stable ¹³C and the radioactive ¹⁴C isotopes resulted in comparable values of the rates of light and dark CO₂ fixation. Due to its absolute environmental safety, the method with ¹³C mineral carbon can be recommended as an alternative to radioisotope methods for qualitative measurements of CO₂ fixation rates in aquatic ecosystems.     

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.