Restoration of deuterium marker for multi-isotope mapping of cellular metabolic activity

Restoration of deuterium marker for multi-isotope mapping of cellular metabolic activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Restoration of deuterium marker for multi-isotope mapping of cellular metabolic activity Nadiia Yamborko, Laura Schwab, Lubos Polerecky, Yalda Davoudpour, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7271395/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Investigation of cellular metabolic activity with stable-isotope probing (SIP) implies the admittance of an isotope tracer into the metabolic pathway. Incubation with several isotope-markers (multi-isotope tracing) is required to trace nutrient metabolization and elucidate inter-cellular interactions in complex hosts and environmental communities. To cope with the lability of cell nutrition, deuterium in heavy 2 H 2 16 O water is employed as a substrate-independent general tracer of metabolic activity. However, the spatially-resolved deuterium tracing is hampered by detection limits due to its relatively low ionization yield and mass-interference issues. In the present work, we comprehensively assess the quantitation of deuterium incorporation into biomass employing the outstanding capabilities of nanoscale Secondary Ion Mass Spectrometry facilitating quantitative analysis of metabolic activity with single-cell or subcellular resolution. The effect of ion-probe-induced material relocation on the acquired pattern in 2 H enrichment has been considered. Analytical expressions are suggested for the restoration of the deuterium fraction from the unresolved C 2 2 H-C 2 1 H 2 mass-interference. Application of the suggested principle of equal relative assimilation and the multi-isotope tracing with the 2 H-marker on a phototrophic symbiotic consortium paves the way to sensing the metabolic interplay among cells, recognition of homeostatic and shifted nutrition, checking for completeness of isotope-labelling and elucidation of nonlabelled substrate contribution. Biological sciences/Biochemistry Biological sciences/Biological techniques heavy water deuterium stable-isotope probing multi-isotope tracing metabolic activity oxygenic photogranule Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Information on microbial-mediated matter conversion scaled up to global biogeochemical cycles [ 1 ] is required for the elaboration of resource-saving and energy-conversion approaches [ 2 ]. The combination of stable-isotope probing and nanoscale secondary ion mass spectrometry (SIP-nanoSIMS) has been efficiently employed to link metabolic activity with the identity of single-cells [ 3 ] for tracing the intercellular nutrient flow and intracellular transformations e.g. [ 4 – 6 ]. In biological and biomedical research, stable-isotope tracers are introduced with isotope-labelled substances (e.g., 15 N-thymidine, 13 C-glucose, 13 C-glutamine, 13 CO 2 gas, 15 N 2 gas, H 13 CO 3 − , 15 NO 3 − , 15 NH 4 + , 2 H 2 O, H 2 18 O) into the nutrition environment or growth medium. Tracing the cellular activity in carbon, nitrogen and hydrogen assimilation at once (multi-isotope tracing) is required for a comprehensive investigation of substrate conversion and metabolic interactions in complex communities. Because of the irreversible ablation of material from a sample area explored upon a SIMS analysis, multi-isotope tracing requires simultaneous detection of secondary ions containing all tracer-isotopes, i.e., 2 H – , 13 C – or 13 C 14 N – and 12 C 15 N – . For a proper decision on an optimal isotope-labelled substance, the lability of cellular nutrition (i.e., dependence of metabolic pathways and rates on nutrients’ chemical and isotopic composition) has to be considered to preserve intracellular homeostasis and to minimise the perturbation of native nutrition scenarios inherent to a studied ecosystem. Isotopic labelling of the growth medium with heavy water [ 7 , 8 ] provides deuterium ( 2 H) or heavy oxygen ( 18 O) as a nutrient-independent tracer of cellular metabolic activity. Deuterated water (heavy 2 H 2 16 O water or the semi-heavy 1 H 2 H 16 O) is a more preferred labelling source than heavy-oxygen water ( 1 H 2 18 O, 1 H 2 17 O or 2 H 2 18 O isotopologues) because the latter is much more expensive due to the more difficult separation of 17 O and 18 O containing isotopologues [ 9 ]. The high relative mass difference between hydrogen isotopes leads to strong hydrogen isotope fractionation [ 10 ] and may impede metabolism, i.e., cause toxicity effects already with 10% of 2 H 2 16 O fraction in the growth medium [ 11 ]. Incorporation of 2 H from heavy water into biomass implies further dilution of the 2 H tracer due to the major atomic fraction of hydrogen in biomass, e.g., 52 atomic percent (at%) according to the Redfield ratio for phytoplankton [ 12 ]. In addition to biotic factors limiting the amount of the 2 H tracer in analysed cells, the relatively low electron affinity of hydrogen atoms results in a moderate yield of secondary 2 H − ions when 16 keV Cs + primary projectiles are employed for nanoSIMS analysis. Nevertheless, nanoSIMS has been successfully applied for 2 H tracing in environmental studies [ 7 , 13 ], in biology [ 14 , 15 ] and in material science e.g. [ 16 ]. Strategies suggested for overcoming construction-related limitations of a serial NanoSIMS 50L instrument imply modification of factory-set hardware settings [ 13 ], hardware upgrade and the derivation of 2 H/ 1 H isotope ratio from polyatomic ions [ 17 ] along with the numerical restoration [ 7 ] (see the Supplementary Information for more details, SI section S1). The primary aim of the present work was to identify optimal isotopologue pairs within the [ 2 H/ 1 H, 12 C 2 H/ 12 C 1 H, 16 O 2 H/ 16 O 1 H, 12 C 2 2 H/ 12 C 2 1 H] series for quantitative high-resolution 2 H mapping that could be done simultaneously with 13 C and 15 N tracing for studying the matter conversion and metabolic interaction in complex environmental systems. The demand for nanoscale lateral resolution is particularly strong when studying metabolic interactions in microbial consortia, where changes in structural composition occur at sub-micrometer spatial scales, even smaller than cell size. To demonstrate the advantages of the multi-isotope SIP-nanoSIMS technique, and particularly the prospects of 2 H as a metabolic tracer in this type of applications, we used oxygenic photogranules (OPGs) as a model system. OPGs comprise a complex microbiome of syntrophically interacting heterotrophic and phototrophic bacteria [ 18 ]. This light-driven ecosystem is exchanging key metabolites when photosynthesis is active. OPGs have proven their effectiveness for wastewater treatment without external aeration [ 19 ] and show potential to compete with the conventional activated sludge process [ 20 ]. The approach of 2 H mapping was optimized on resin-embedded maize-root samples and applied in multi-isotope tracing mode on OPGs. Due to the relatively high yield of C 2 2 H − ions and their robust biomass-featured spatial distribution, the C 2 2 H/C 2 1 H ratio was considered as a rather promising measure of the 2 H fraction, despite the unresolved C 2 2 H − & C 2 1 H 2 − interference. The effect of this interference is reduced due to the decrease in C 2 1 H 2 − ion counts with increasing 2 H fraction [ 14 , 21 ]. Together with the suggested new approach to restoring the 2 H-fraction from polyatomic (C 2 2 H − & C 2 1 H 2 − )/C 2 1 H − ion ratio, the employed principle of equal relative assimilation allowed the elucidation of interplay in nutrition channels and quantitative analysis of cellular metabolic interaction within the OPG consortium. 2. Results and Discussion In the first stage of our study, the efficiency of deuterium mapping with 2 H − , 12 C 2 H − , 16 O 2 H − and 12 C 2 2 H − ion species was evaluated on thick (∼1 mm) resin-embedded maize root samples providing feasibility of long-lasting measurements necessary to reveal features of 2 H spatial distribution in low-abundant 2 H-containing ion counts and to clarify the mass-spectroscopy of corresponding molecular fragments. In the second stage, selected pairs of polyatomic ions were employed for multi-isotope tracing of metabolic activity in photo-hetero-trophic microbial associations (OPGs) enabling quantitative analysis of relative assimilation in carbon, nitrogen and hydrogen simultaneously. The details on multicollector settings, the restoration of 2 H fraction from the unresolved 12 C 2 2 H & 12 C 2 1 H 2 mass-peak, and the effect of an ion-probe-induced material relocation are explained in Supplementary Information (SI, sections S1-S3). 2.1. Mass-spectroscopy of 2 H-containing ion species After carefull alignment of the secondary ion beam, the mass-resolving power (MRP) of about 16.000–18.000 was achieved in the mass-range of polyatomic 2 H-containing ion species. Mass-spectra of 2 H – , 12 C 2 H – , 16 O 2 H – and 12 C 2 2 H – ion-species were acquired from 2 H-labelled and non-labelled maize-root samples (Fig. 1 ). The mass-spectra were acquired with a defocused primary-ion beam in a 64×64 pixels raster over same-size areas of the 2 H-labelled and non-labelled samples to keep counting rates of 2 H-containing ion species for each sample comparable. Obtained counting rates are determined by the electron affinity of the corresponding molecular fragments and by their abundance within the analysed sample fragment. The mass-peak of 2 H – (Fig. 1 , top left) reveals a count rate of 20 counts per second (cps) and is well separated from the mass-peak of 1 H 2 – for the 2 H-labelled sample. The mass-peak of 12 C 2 H – with 50 cps is resolved from 13 C 1 H – (Fig. 1 , top right) and appears as a clear flat-top shoulder at the low-mass side of a partly (≈50%) overlapping 12 C 1 H 2 – peak. The mass-peak of 16 O 2 H – (Fig. 1 , bottom left) delivers 25 cps and overlaps partly (≈50%) with the 17 O 1 H – peak, but it can still be employed for mass-spectrometric quantitation of 2 H content when 16 O 1 H 2 – ion species are not revealed at the high-mass side. Regarding the mass-peak of 12 C 2 2 H – , a relatively high counting rate of 200 cps is delivered from the 2 H-labelled sample (Fig. 1 , bottom right). However, it overlaps to 40% with the mass-peak of 12 C 13 C 1 H – and over 80% with that of 12 C 2 1 H 2 – , where the latter decreases in count rate from 65 cps down to 10 cps when the non-labelled (natural 2 H abundance) and 2 H-labelled samples are compared. This feature of the 12 C 2 1 H 2 – dependence on the 2 H-label content has previously been emphasized [ 21 ] and is comprehensively considered in the present study for the restoration of the 2 H fraction from the unresolved 12 C 2 2 H & 12 C 2 1 H 2 mass-peak. 2.2. Direct measurements and the restoration of natural 2 H abundance The fraction of 2 H in a resin-embedded maize-root sample with a natural isotopic composition was analyzed in the imaging mode of nanoSIMS 50L employing multicollector settings II & III (see Table S1 in SI). The distributions of the measured values among the image pixels are shown with histograms and box-plots in Fig. 2 separately for 2 H fractions derived from the H, CH, OH and C 2 H isotopologue ion count ratios. The median value of the 2 H fraction is close to the natural 2 H abundance (fraction) \(\:{F}_{0}\) = 2 H/( 2 H+ 1 H)×100 = 0.0115 at% when derived from H, CH and OH isotopologue ratios, but appears to be about 2.5 times overestimated ( \(\:{F}_{0}^{{\prime\:}}\) =C 2 2 H/(C 2 2 H+C 2 1 H)×100=0.0296 at%, see Fig. 2 ) due to the unresolved \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}\) & \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{1}{\text{H}}_{2}\) mass peaks (see Fig. 1 and S1). Nevertheless, the high counting rate of \(\:{{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}{\text{H}}_{\:}}^{\--}\) ion species offers an enhanced precision in the quantitation of \(\:{}_{\:}{}^{2}{\text{H}}_{\:}\) fraction. Moreover, the ion-beam induced material-smearing effect, revealed strongly in 2 H maps derived from H, CH and OH isotopic ratios (Fig. S4), is almost not detectable when C 2 H isotopologues are considered for the restoration of the 2 H fraction (Fig. S5 d). Thus, despite the unresolved \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}\) & \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{1}{\text{H}}_{2}\) mass interference, one still has two solid advantages (i.e., enhanced precision and negligible smearing effect) of C 2 H isotopologue ratio consideration for 2 H mapping. With this motivation, a new method for the restoration of 2 H fraction with the unresolved \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}\) & \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{1}{\text{H}}_{2}\) mass peaks has been developed in the present work (SI, section S2). The suggested restoration of 2 H fraction can be implemented with a rather simple expression. In terms of isotope ratio, the restored 2 H/ 1 H value is expressed as $$\:{R}_{r}={R}^{{\prime\:}}-{ϵ}_{{H}_{2}/H}\:$$ 1 , where \(\:{R}^{{\prime\:}}\) is the hydrogen isotope ratio overestimated due to the unresolved \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}\) , and the correcting parameter \(\:{ϵ}_{{H}_{2}/H}\) is defined as the \(\:{{\text{C}}_{2}{\text{H}}_{2}}^{-}/{{\text{C}}_{2}\text{H}}^{-}\) ion-yield ratio (section S2 in SI). The calculation of \(\:{ϵ}_{{H}_{2}/H}\) value employs the difference between CH and C 2 H isotopologue ratios ( \(\:{R}_{0}\) and \(\:{R}_{0}^{{\prime\:}}\) , respectively) acquired on the unlabelled sample with a natural 2 H abundance (see S2 section in SI for more details). \(\:{ϵ}_{{H}_{2}/H}=\left({R}_{0}^{{\prime\:}}-{R}_{0}\right)\times\:\left(1+{R}_{0}\right)=\) (1.6411±0.0012)×10 −4 (2) The median value of the 2 H fraction restored from the unresolved \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}\) & \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{1}{\text{H}}_{2}\) peak employing the Eq. ( 1 ) (shown in blue in Fig. 2 ) is close to the value of the 2 H abundance \(\:{F}_{0}\) =0.0132 ± 0.0045 at% derived from the CH isotopologue ratio \(\:{R}_{0}\) accepted in Eq. (2) for the calculation of \(\:{ϵ}_{{H}_{2}/H}\) value. The distributions derived from the H and OH isotopologue ratios are more stretched and even truncated considerably at 0 at% due to the relatively low counting rates for 2 H – and 16 O 2 H – ions (see Fig. 1 ). In case of such truncated distributions, it may be better to consider a modus value instead of the median. The most stretched distribution, as observed for the OH isotopologues, may be due to the contribution of 16 O 1 H 2 – ion species that are not revealed in the mass-scan at 18 amu for the studied resin-embedded samples (Fig. 1 ) but may show a higher yield from other sample types. 2.3. Application on a complex microbial community With the established multi-isotope tracing approach, the SIP-nanoSIMS methodology was applied on oxygenic photogranules (OPG) to check its applicability and prospects for studying metabolic interactions in the complex microbial consortia. OPGs are microbial aggregates with diameters ranging from several hundreds of micrometers to four to five millimeters. These roughly spherical aggregates float freely in an agitated aqueous system, but settle rapidly when mixing is stopped. OPGs harbor a microbial community dominated by heterotrophic and phototrophic bacteria. These two groups of microorganisms are believed to syntrophically exchange heterotrophically produced CO 2 and photosynthetically produced O 2 [ 18 ]. CO 2 may be produced from an externally provided carbon source, for example, organic compounds contained in wastewater. Alternatively, hetetrotrophs may produce CO 2 from the conversion of mainly phototrophically produced extracellular polymeric substances contained in the photogranules. This is the case in our experiment where only inorganic carbon is added in form of HCO 3 − as it can only be assimilated by autotrophic microorganisms. In studied photogranules, the majority of autotrophs are phototrophic filamentous cyanobacteria of the order Oscillatoriales [ 18 ]. These cyanobacteria are not known to fix N 2 in the presence of another more accessible nitrogen source. The 15 N-labelled ammonium chloride was provided as a nitrogen source for heterotrophs and phototrophs. Deuterated water was providing 2 H as a tracer of biosynthetic OPG activity. With the reliable 2 H reconstruction from the unresolved \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}\) & \(\:{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{1}{\text{H}}_{2}\) mass-peak (described in SI, section S2), the correcting parameter \(\:{ϵ}_{{H}_{2}/H}\) =(1.4750 ± 0.0014)×10 − 4 was derived and the multicollector settings V (Table S1 ) were adopted for simultaneous tracing of H, C, N isotopes in parallel with the mapping of cellular 32 S and 31 P ( 31 P 1 H, see Fig. S6) upon the analysis of OPG samples. The histograms of biomass-related pixel distribution according to the biomass isotopic enrichments in 2 H, 13 C and 15 N (Fig. 3 a) are not directly comparable because of the different natural abundance of these isotopes (shown with horizontal dashed lines) and due to their different fractions in the growth substrate. Isotopic enrichment of each biomass volume-unit (voxel of about 60x60x100 nm³, 3D analog of a 2D-pixel) within cell-confining regions of interest (RoIs, see RoI-definition example in Fig. S7) was derived from corresponding ion-count ratios and converted with the Eq. ( 3 ) into the relative assimilation \(\:{K}_{A}^{f}\) (Fig. 3 b) representing the assimilated amount \(\:{E}_{a}\) of tracer-derived H, C or N expressed relatively to the final cellular amount \(\:{E}_{f}\) of the corresponding element achieved after the incubation [ 22 ]. $$\:{K}_{A}^{f}=\frac{{E}_{a}}{{E}_{f}}=\frac{{R}_{f}-{R}_{i}}{\left(1+{R}_{f}\right)\times\:\left\{{F}_{gs}\times\:\left(1+{R}_{i}\right)-{R}_{i}\right\}}=\frac{{F}_{f}-{F}_{i}}{{F}_{gs}-{F}_{i}}$$ 3 In the Eq. ( 3 ), \(\:R\) is the heavy-to-light isotope ratio, \(\:F\) is the corresponding atom fraction of heavy isotope, \(\:F=R/(R+1)\) , subscripts i and f refer to the values before and after the incubation ( initial and final ), and \(\:{F}_{gs}\) is the fraction of heavy isotope in the isotope-labelled tracer-substance contributing to the growth substrate. The relative assimilation has also been calculated for each single cell as the mean value over biomass volume-units (voxels) within the corresponding cell-confining RoI (shown with yellow contours in Fig. S7 a,b). Fig. S9 shows the distribution of single-cells in their relative assimilation of carbon, nitrogen and hydrogen to be well reproduced with the distribution of biomass-units in the corresponding relative assimilation calculated for each cellular-related voxel. Features of the cell distribution in relative assimilation are better revealed with a higher count of subcellular biomass volume-units (voxels). Therefore, voxel-resolved data were considered for quantitative evaluation of the relative elemental assimilation in the present work. To account for the dilution of 13 C and 2 H isotope-tracers with fixative [ 23 ] and embedding materials, values of relative assimilation \(\:{K}_{A}^{f}\left({}_{\:}{}^{12}\text{C}{}_{\:}{}^{13}\text{C}/{}_{\:}{}^{12}{\text{C}}_{2}\right)\) and \(\:{K}_{A}^{f}\left({}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{2}\text{H}/{}_{\:}{}^{12}{\text{C}}_{2}{}_{\:}{}^{1}\text{H}\right)\) , computed with the corresponding C 2 and C 2 H isotopologue ratios, were corrected with 1.136 multiplication factor and 0.03 at% offset, derived as the slope b and y-intercept a of the linear function fitting \(\:{K}_{A}^{f}\left({}_{\:}{}^{13}\text{C}{}_{\:}{}^{14}\text{N}/{}_{\:}{}^{12}\text{C}{}_{\:}{}^{14}\text{N}\right)=a+b\times\:{K}_{A}^{f}\left({}_{\:}{}^{12}\text{C}{}_{\:}{}^{13}\text{C}/{}_{\:}{}^{12}{\text{C}}_{2}\right)\) relation (see Fig. S10). In Fig. 4 , the spatial distribution of cellular relative assimilation is visualized in a thin section through the outer green part of a photogranule. Distribution of single-cells in relative assimilation activity \(\:{K}_{A}^{f}\) (Fig. 4 a-d) facilitates the differentiation between consortium-representative species via quantitative analysis of their elemental assimilation efficiency. With the map derived for the relative carbon assimilation in the OPG-consortium (Fig. 4 a) its autotrophic members fixing 13 CO 2 can be clearly recognized as elongated cells arranged in filaments (see Fig. S7 a,c) typical for cyanobacteria usually found in OPG [ 18 ]. Complete filaments are not seen in the figures, as the imaged area represents a thin section on which only part of the filaments happens to be positioned in the sectioning plane. All metabolically active cells in OPG show the fraction of hydrogen incorporated from water (Fig. 4 b) to be in a similar range as the fraction of carbon supplied via 13 CO 2 fixation by autotrophs (Fig. 4 a,e, y-axis). One can recognize cyanobacteria cells (yellow RoIs in Fig. S7 a) with almost equal relative assimilation of C and H (Fig. 4 e, black). This feature in the metabolic activity of cyanobacteria would suggest their homeostatic nutrition keeping cellular elemental Redfield ratio almost constant, however the availability of 15 NH 4 + boosts synthesis of N-rich molecules (e.g., proteins) revealed as about 5-fold increase in relative assimilation of N (Fig. 4 f, black). Cyanobacteria are known to store nitrogen in the form of so-called structured granules enriched in cyanophycin granule polypeptide [ 24 , 25 ]. Nevertheless, proportional assimilation of carbon and nitrogen makes it easy to recognize cyanobacteria when overlaying frames a, b and c of Fig. 4 , yielding Fig. 4 d. Cyanobacteria are coloured in the RGB-overlay from bright pink (slightly prevailing C assimilation over H assimilation) to magenta and purple spots with 2 H prevailing assimilation. Bright pink-red areas can be identified also in between cyanobacterial cells (Fig. 4 d, lower part) and may be ascribed to carbon-rich extracellular polymeric substances (EPS) synthesised de novo from photosynthetically assimilated 13 CO 2 . Typical cyanobacteria found in OPG belong to the order Oscillatoriales , which are filamentous gliding cyanobacteria [ 18 ]. Bright EPS spots immediately adjacent to cyanobacterial cells could represent EPS excreted for the gliding motility. All cells appearing bright green in the overlay (Fig. 4 d) may be assigned to heterotrophic bacteria (marked with yellow RoIs in Fig. S7 b) possessing intensive nitrogen assimilation (Fig. 4 c,d in red) from the supplied 15 NH 4 + . This high nitrogen assimilation (Fig. 4 f in red, y-axis; Table 1 , column III) implies the obvious necessity of carbon recycling from the unlabelled carbon sources, e.g., EPS or dissolved organic matter pool (DOM), for maintaining the elemental biomass-stoichiometry upon the limited carbon supply with 13 C-labelled phototrophic exometabolites from cyanobacteria (Fig. 4 e,f in red, x-axis; Table 1 , column I). These heterotrophic bacteria consume 2 H from water for fatty-acid synthesis involving NADPH/NADP + , whereas more ammonium-derived 1 H is supplied together with 15 N for protein synthesis resulting in slightly lower 2 H enrichment of heterotrophs as compared with cyanobacteria (Fig. 4 e, y-axis; Table 1 , column V). Table 1 Element- and source-resolved distribution of the relative assimilation \(\:{K}_{A}^{f}\) represented for OPG members with median values and median absolute deviation as Med ± MAD [at%]. Values derived from the SIP-nanoSIMS data # are shown in bold (with white symbols in Fig. 4 e,f). Assimilation due to the syntrophic interaction between cyanobacteria and heterotrophs is shown in *grey-filled cells. Element Carbon Nitrogen Hydrogen Source 13 CO 2 recycled DOM 15 NH 4 + heavy water (33% of 2 H) 15 NH 4 + & recycled DOM I II III IV V Cyanobacteria (Comments) 0.66 ± 0.09 # *2.43 ± 0.21 3.09 ± 0.19 # 0.65 ± 0.09 # *2.44 ± 0.21 20% of photosynthetic C 80% from the shared reduced organic matter 100%, i.e., single nitrogen supply 20% via photosynthetic incorporation 80% from growth medium inv. shared reduced OM Heterotrophs (Comments) * 0.10 ± 0.03 # 5.98 ± 0.85 6.08 ± 0.85 # 0.38 ± 0.14 # 5.70 ± 0.86 2% from shared cyanobacterial exometabolites 98% from the recycled organic matter 100%, i.e., single nitrogen supply 6% from growth medium inv. 2% of cyanobacterial exometabolites 94% from growth medium inv. reduced OM With 1 mM 15 NH 4 + as a not-limiting nitrogen source in the nutrition medium, its relative assimilation reached 10% for heterotrophic cells (Fig. 4 c,f in red), whereas 2–3 times lower N-assimilation activity of cyanobacterial filaments (Fig. 4 f in black) was revealed due to their rate-limiting carbon supply via CO 2 fixation. It is important to note the distribution pattern of bacterial cells around phototrophs – they are always close neighbours with a mutualistic interaction between phylogenetically unrelated members in the OPG community. Assuming the elemental stoichiometry of each OPG member remains stable [ 26 ] during the time of incubation with an isotope-labelled substrate, one may expect close median values of relative assimilation ( \(\:{K}_{A}^{f}\) ) in all elements (H, C and N) for representatives within each group (autotrophs and heterotrophs). With this assumption and the median values of relative assimilation derived with the isotope-labelled nutrition part (Fig. 4 e,f; median values shown with white symbols), the fractions of carbon and hydrogen from the recycled DOM and 15 NH 4 + -related hydrogen, assimilated by cyanobacteria and heterotrophs Table 1 (columns II and V), were derived as follows. With its non-limiting content in the growth medium, 15 NH 4 + was considered as the easily-accessible prevailed nitrogen source providing all, i.e., 100% of cellular nitrogen gained by OPG consortium during the incubation. The relative nitrogen assimilation of 3.09 ± 0.19 at% ( \(\:{K}_{A}^{f}\) , Med ± MAD) for cyanobacteria and 6.08 ± 0.85 at% for heterotrophs (Table 1 , column III; Fig. 4 f, y-axis, median values shown with white symbols) imply 2-fold difference in their nitrogen-specific growth rate $$\:\gamma\:=\frac{-{{log}}_{2}\left(1-{K}_{A}^{f}\right)}{t}=-\frac{{ln}\left(1-{K}_{A}^{f}\right)}{t\times\:{ln}\left(2\right)}$$ 4 $$\:\varDelta\:\gamma\:=\frac{\partial\:\gamma\:}{\partial\:{K}_{A}^{f}}\times\:\varDelta\:{K}_{A}^{f}=\frac{1}{t\times\:\text{ln}\left(2\right)}\times\:\frac{1}{\left(1-{K}_{A}^{f}\right)}\times\:\varDelta\:{K}_{A}^{f}$$ 5 returning 0.76 ± 0.05 at%/h and 1.51 ± 0.22 at%/h respectively with the cultivation time \(\:t\) =6 hours. Carbon fractions supplied from 13 CO 2 via photosynthesis (0.66 ± 0.09 at%) and exometabolite sharing (0.10 ± 0.03 at%, syntrophic share with heterotrophs) by cyanobacteria were derived from nanoSIMS data as \(\:{K}_{A}^{f}\) median values (Table 1 , column I) shown in Fig. 4 e,f (x-axes, median-value symbols in white). Carbon fraction assimilated from the recycled DOM (Table 1 , column II) by cyanobacteria (2.43 ± 0.21 at%, share from heterotrophic OM reducers) and by heterotrophs (5.98 ± 0.85 at%) was calculated as the difference between the corresponding fraction of assimilated nitrogen (column III) and the fraction of carbon from 13 CO 2 (column I). Such a calculation is valid when i) the 15 NH 4 + can be considered as the prevailed nitrogen source and ii) the assumed above preservation of biomass stoichiometry is fulfilled implying equal relative assimilation of all elements (C, N and H) by each group of OPG consortium. The cellular fraction of hydrogen originating from the supplied 15 NH 4 + and recycled DOM (column V) was calculated for cyanobacteria (2.44 ± 0.21 at%, involving a major share from heterotrophic OM reducers) and for heterotrophs (5.70 ± 0.86 at%) as the difference between the fraction of assimilated nitrogen (column III) and the hydrogen fraction incorporated from heavy water (Table 1 , column IV) shown in Fig. 4 e (y-axis, median-value symbols in white). For a microbial group with the elemental stoichiometry of its biomass preserved, one may estimate the contribution of non-labelled nutrition sources with either i) an exclusive source of at least one element (C, N or H) isotope-labelled, or with ii) a known value of the growth- or division-rate for a target cell-type of a studies consortium. Assuming the DOM to be depleted in nitrogen, we considered 15 NH 4 + as an exclusive nitrogen source for OPGs in this study. This assumption allowed for the quantitative evaluation of non-labelled nutrient supply (Table 1 ) and for the calculation of cell-division rates (equations 4 , 5 ) for cyanobacteria and heterotrophs. In another way, relative assimilation \(\:{K}_{A}^{f}\) may also be calculated with a known growth-rate value µ for the biomass of a target cell-type or corresponding cell-division rate γ. $$\:{K}_{A}^{f}=1-{e}^{-\mu\:\times\:t}=1-{2}^{-\gamma\:\times\:t}$$ 6 With the value of relative assimilation acquired in this way, one can further analyse the completeness in isotope-labelling of nutrient supply by comparing the Eq. ( 6 ) output with \(\:{K}_{A}^{f}\) values derived from the changes in isotopic composition of a corresponding cell-type upon their incubation with an isotope-labelled substrate. The photogranules that were incubated in the experiments here had a net heterotrophic activity. The required carbon for this activity was derived from the supplied 13 CO 2 and via EPS conversion. EPS is typically present in sample quantities in photogranules as it is produced for example for cyanobacterial gliding motility. However, contrasting environmental conditions, e.g., illumination, as well as photogranule properties, e.g., size, may partition autotrophic and heterotrophic activities differently. Especially for biotechnological applications of photogranule biomass, controlling the specific activities of the photogranule community by varying the environmental conditions are of interest. The procedure of metabolic flux analysis suggested here allows us to generate these data in further experiments. Our study of metabolomic exchange between cyanobacteria and other bacteria in photogranules shows the expected metabolic shifts in carbon fixation (Calvin cycle pathway) depending on light availability [ 27 ]. It is worth noting that the fraction of hydrogen incorporated from heavy water within 0.001-1 at% range (Fig. 3 b) was analysed with NanoSIMS 50L delivering good ion-counting statistics from each biomass unit confined in single voxels (3D-pixels). The dynamic range of hydrogen isotopic analysis starting from its natural abundance (Fig. 2 , 3 a) facilitates the efficient differentiation between single-cells and cellular compartments according to their metabolic activity. The incorporation of hydrogen from heavy water (Fig. 4 e, y-axis) is revealed for all types of cells regardless of functional activity and nutrition mode (whether autotrophic or heterotrophic). In this way, 2 H from water is proved as a universal tracer in all biosynthetic processes being primarily supplied to the sites of the most intensive biosynthesis like DNA/RNA-synthesis sites and synthesis of service enzymes around bacterial chromosome (see the overlay of P and H-incorporation maps in Fig. S8). This mode of stable-isotope labelling ( 2 H from deuterated water) allows the tracing of dividing cells in a population [ 14 ]. Hydrogen is incorporated from water into cell metabolism by either hydrolysis, osmotic interactions, or most importantly by hydrogen incorporation into NADPH synthesis [ 28 ]. NADPH is a co-factor for metabolic reactions in the Calvin cycle, lipid and nucleic acid syntheses, where NADPH is required as a reducing agent (hydrogen source). NADPH and its oxidized form NADP + are utilised by all forms of cellular life [ 29 ]. 3. Conclusions and outlook The multi-isotope tracing of metabolic activity in a complex OPG microbiome allowed us to differentiate between two populations of phototrophic and heterotrophic bacteria and to reveal the syntrophic interaction between these inhabitants of the phototrophic symbiotic consortium via analysis of the interplay in their nutrition channels. With the example of nutrition analysis on the OPG consortium, we demonstrated the prospects in application of the suggested approach for e.g., optimization of wastewater treatment processes. The employed stoichiometry-preservation principle constitutes a framework for metabolic flux analysis in complex systems, implying each representative group of the studied consortium shows equal relative assimilation of different elements (e.g., C, N and H). Homeostatic 13 CO 2 fixation was revealed for autotrophic inhabitants of OPG consortium with relative assimilation of carbon following the fraction of hydrogen incorporated from heavy water (Fig. 4 e, S9 a, black). In this case, the complete isotope-labelling of phototrophic nutrition channel was achieved with heavy 2 H 2 O water and 13 CO 2 involved in the photosynthetic assimilation process. Major fraction (80–98%) of assimilated carbon and hydrogen was found to be supplied via recycling of OM (Table 1 , columns II, V) converted by heterotrophic reducers to DOM, i.e., beyond the isotope-traced pathway. Such an incomplete isotope-labelling of nutrient supply is revealed as a considerable discrepancy in the relative assimilation of different elements within a defined geno- or phenotypic group of a microbial consortium [ 30 ]. The study results emphasised the integrating role of 2 H labelling through deuterated water in complex systems with various life strategies and metabolic types. All metabolically active cells in the studied OPG consortium revealed the incorporation of deuterium from heavy water. Unlike 13 C and 15 N tracers, a major fraction of 2 H-marker from heavy water stays incorporated into the biomass upon cellular metabolic activity (i.e., detectable with nanoSIMS). For example, the respiration process implies the synthesis of NADH, NADPH etc. with incorporation of 2 H from heavy water, whereas 13 CO 2 is released. The achieved reliable restoration of deuterium fraction already from its natural 0.0115 at% abundance proves SIP-nanoSIMS capability to sense and localize extremely low biosynthetic activity with relative assimilation \(\:{K}_{A}^{f}\) starting in the ∼0.001 at% range (Fig. 3 b) corresponding to metabolic rate of ∼10 − 5 h − 1 upon cellular maintenance [ 31 ]. Employing this high sensitivity for the quantitation of metabolic heterogeneity among single cells of a microbial population [ 32 ] makes it feasible to trace microevolutionary processes and reveal obvious and non-obvious anthropogenically elicited changes in the environment. 4. Methods 4.1. Multi-isotope labelling and preparation of oxygenic photogranules OPGs were grown in a sequential batch reactor as previously described [ 33 ]. The photogranules were transported in darkness at room temperature to the nanoSIMS facility at UFZ in Leipzig. Isotope labelling incubations were performed under light in a medium containing heavy water (33 vol.% of 2 H 2 O), H 13 CO 3 − ( 13 CO 2 – 87 at% 13 C) and 15 NH 4 + (93 at% 15 N). The final concentrations of the labelled nutrients were 1 mM 15 NH 4 + and 2 mM H 13 CO 3 − . After 6 hours of incubation, OPGs were fixed with 2% paraformaldehyde in cacodylate buffer (pH 7.4, 0.1 M) at 4˚C overnight and washed two times with the buffer. Dehydration was performed with ethanol series (30%, 50%, 70%, 80%, 90%, and 3 times 100%) for 15 min each. Low-viscosity LR White resin infiltration was done using 1:3, 1:2, 1:1, 2:1, and 3:1 of resin:ethanol mixture, each for 45 min, followed by pure LR White resin for one hour and overnight. Finally, the resin-infiltrated OPG sample was cured in an oven at 60°C for two days. The polymerized sample block was trimmed using a Leica EM TRIM2 trimmer and sectioned with Leica EM UC7 ultramicrotome employing a freshly-prepared glass knife. Sections of 300 nm thickness were placed on a 10 mm diameter As:Si-wafer and coated with a 10 nm of gold/palladium (80/20 weight ratio; Plano, Germany) conductive layer using Leica EM SCD 500 sputter-coater (Leica Microsystems, Germany) at 35 mA of Ar + current against Au/Pd target kept at -0.5 kV potential. 4.2. Maize-root 2 H labelling and sample preparation A long-lasting sample of resin-embedded plant root was prepared to facilitate the optimization of different 2 H mapping approaches in this study. This was necessary because the alignment of a NanoSIMS 50L instrument for multi-isotope tracing, involving the comparison of 2 H fraction in [ 2 H/ 1 H, 12 C 2 H/ 12 C 1 H, 16 O 2 H/ 16 O 1 H, 12 C 2 2 H/ 12 C 2 1 H] series of isotopologue ratios, takes several hours. Additionally, due to the low natural abundance of deuterium (0.0115 at%) and a relatively low yield of 2 H containing secondary ions, the alignment and corresponding measurements of 2 H fraction have to be performed with the current of primary Cs + ions increased up to 4–6 pA implying an enhanced consumption of the sample material. Wild-type maize seeds provided by the Institute of Crop Science and Resource Conservation, University of Bonn, were first surface sterilized [ 34 ]. Plant growth involving 2 H-labelling with 40% heavy water ( 2 H 2 O, 99.8 at% 2 H, Sigma Aldrich, Germany) was performed according to [ 35 ]. The primary 1 cm root tip was harvested after 96 hours. The root fixation, LR White resin infiltration, curring and trimming have been performed in the same way as described for the OPG samples (section 2.1). LR white block with a root crossection in its trimmed face was mounted in 10 mm diameter ring (fitting the nanoSIMS “Biology” sample holder) and coated with 10 nm of gold/palladium as OPG thin-section samples (section 2.1). 4.3. NanoSIMS analysis and data processing The analysis of multi-elemental isotope ratios was implemented with a serial NanoSIMS 50L #134 instrument (AMETEK, CAMECA, France) at the UFZ in Leipzig. A set of 15 secondary ion species ( 1 H − , 2 H – , 12 C 1 H – , 12 C 2 H – , 16 O 1 H – , 16 O 2 H – , 12 C 2 − , 12 C 13 C – , 12 C 2 1 H − , 12 C 14 N − , 12 C 2 2 H – , 12 C 15 N − , 13 C 14 N − , 31 P − , 32 S − and 31 P 1 H − ) were detected with seven detectors upon different mass-assignment configurations involving deflector-plate switching (see Results and Discussion for more details). Measurements were conducted with 15×120 µm (width x height) nominal size of the entrance slit, 40×1800 µm exit slits, 150×150 µm aperture and an energy slit cutting off 30% of secondary ions in their energy-distribution tail. Caesium (Cs) pre-implantation was performed with 16 keV Cs + in 200 pA beam rastering within 100×100 µm 2 area for 30 min. Within these pre-implanted areas, 15×15 µm 2 or 25×25 µm 2 fields of view (FoV) were scanned in a 512×512 pixel raster using a 4 pA primary Cs + beam with a dwelling time of 2 ms/pixel. To ensure sufficient counting statistics, data from the same FoV were acquired over 160 scans with the deflector-plate voltages switching between two values every second scan. Processing of the acquired data was done with a modified version of the open-source Look@NanoSIMS software [ 36 ] (details in SI section S4). The data acquired with each scan were corrected for the lateral drift in the secondary electron intensity map (Esi) and all detected planes were accumulated for each ion species. The accumulated 12 C 14 N − map, which is a proxy of the intrinsic cellular biomarkers (Fig. S6), was then used to draw regions of interest (RoIs) corresponding to plant tissues (maize sample) or microbial cells (OPG sample). Finally, the isotope ratios in the RoI pixels were exported in a text format suitable for further statistical analysis. Declarations Competing interests: authors declare no competing interests in relation to the work described. Funding Declaration The reported study was partly supported by the Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group advised grant (MetaSCOPE) of the Paul. G Allen Family Foundation. N.Y. acknowledge the funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – “MicroBridges” Satellite-Project in SPP2089 “MicroZym” Project 403664479 (BL 1560/2–2; AOBJ683257). The contribution of L.S. and C.V. was supported by the German Federal Ministry of Education and Research (BMBF) and the initiative “Twenty20 – Partnership for Innovation” for funding the H 2 -UGS project of HYPOS network (Grant 03ZZ0721). This work benefited from the activity of K.M. and J.H. in the frame of the Environmental Biotechnology and Biorefinery Facility (Bio2E) of INRAE-LBE ( doi.org/10.15454/1.557234103446854E12 ) and funded partly by the National French Funding Agency ANR project PSST (grant number ANR-16-CE04-0001). N.M. was funded by the Novo Nordisk Foundation through an NNF Young Investigator Award, Grant NNF22OC0071609 ReFuel. Y.D. and H.B. acknowledge the funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project 403641683 (RI-903/7 − 1). Research work of Y.D. was supported by Deutsche Forschungsgemeinschaft Integration of Refugee Scientists and Academics. Author Contribution H.-H.R. and H.S. conceived and planned the study; Y.D., K.M., J.H., H.B. and N.M. performed the growth experiments and isotope-labeling; Y.D., H.B. and H.S. contributed to sample preparation; L.S. and H.S. carried out the nanoSIMS experiment; L.S. and H.S. developed the F(2H)-restoration method refined further together with L.P. and H.B.; L.S., N.Y., L.P. and H.S. performed the data evaluation; L.P. enhanced the Look@NanoSIMS software to allow multi-isotope analyses; Y.D., K.M., J.H., H.-H.R., and C.V. contributed to the interpretation of the results; N.Y. and H.S. took the lead in writing the manuscript; all authors contributed to manuscript revision, read and approved the submitted version. Acknowledgement We acknowledge the access to the analytical facilities of the Centre for Chemical Microscopy (ProVIS) at the Helmholtz Centre for Environmental Research in Leipzig, which is supported by the European Regional Development Funds (EFRE - Europe funds Saxony) and the Helmholtz Association. Support of ProVIS development by Helmholtz Munich (AG Sheikh "Vascular Epigenetics" Helmholtz Institute for Metabolic, Obesity and Vascular Research) in the frame of the Meta-SCOPE project is greatly acknowledged. The comprehensive support of cultivation and sample-preparation actions by Katja Nerlich and Jasmin Voigt is greatly appreciated. Data Availability The datasets generated and analysed during the current study are available in the Google repository, https://drive.google.com/drive/folders/1iPocpciynqBhEu5QOU-qrUmrcPzcnpZK?usp=sharing References Foster, R. A., Sztejrenszus, S. & Kuypers, M. M. 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Microbiol. 14 (4), 1009–1023 (2012). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationrevised.docx Cite Share Download PDF Status: Published Journal Publication published 07 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 Dec, 2025 Editor assigned by journal 20 Dec, 2025 Reviews received at journal 17 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers invited by journal 09 Dec, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 14 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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09:21:05","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137086,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/7138421b75f00911795d68c7.html"},{"id":97419489,"identity":"c782669c-a4e6-4b7f-a2b8-aef5bd7e990c","added_by":"auto","created_at":"2025-12-04 08:13:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":411991,"visible":true,"origin":"","legend":"\u003cp\u003eMass-spectra acquired in the range of \u003csup\u003e2\u003c/sup\u003eH, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH and \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH mass-peaks on \u003csup\u003e2\u003c/sup\u003eH-labelled root sample (filled circles) and on a root of natural isotopic composition (open circles). Horizontal mass-range bars show neighbouring mass-peak positioning with 40\u0026nbsp;µm width of exit slits. Mass-peak centering is shown with the “0”-point in lateral upper x-axes. Spectra were averaged over 40 scans for 15×15\u0026nbsp;µm\u003csup\u003e2\u003c/sup\u003e sample areas with 2.16\u0026nbsp;s accumulation per point in 0.15\u0026nbsp;V steps.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/2585fe9508403c18caf7b70d.png"},{"id":97419494,"identity":"da3d8e67-d537-4cd6-929a-917ae9592f87","added_by":"auto","created_at":"2025-12-04 08:13:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110349,"visible":true,"origin":"","legend":"\u003cp\u003eSingle-pixel distribution in \u003csup\u003e2\u003c/sup\u003eH fraction derived from the hydrogen isotopic ratio in H, CH, OH and C\u003csub\u003e2\u003c/sub\u003eH isotopologue pairs acquired from a resin-embedded maize-root sample with a natural isotopic composition. The distribution, restored from the unresolved \u0026nbsp;\u0026amp;\u0026nbsp;\u0026nbsp;peak according to equation\u0026nbsp;(1) is shown in blue.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/e95c8b72c474421dd7a1e4f0.png"},{"id":97666800,"identity":"d5261a09-9db4-46dd-aba1-4a1056a1dc72","added_by":"auto","created_at":"2025-12-08 09:22:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153141,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution histograms of biomass-related pixels in the \u003csup\u003e2\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN fractions (a) and in the relative H, C and N assimilation (b) for 5 analyzed areas of OPG thin section. Horizontal dashed lines in the frame (a) show the natural abundance of the corresponding isotopes. The boxplots next to the histograms summarize the distributions with their median and the Q\u003csub\u003e16\u003c/sub\u003e and Q\u003csub\u003e84\u003c/sub\u003e quantiles. Interquantile Q\u003csub\u003e1-99\u003c/sub\u003e range is shown with horizontal whiskers.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/c1d51d0e5cbcf7e033aa01c1.png"},{"id":97419491,"identity":"1a61de05-ae07-4506-af8b-068f33a701b3","added_by":"auto","created_at":"2025-12-04 08:13:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1580870,"visible":true,"origin":"","legend":"\u003cp\u003eRelative metabolic activity \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e\u003csup\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sup\u003e \u0026nbsp;of complex microbial consortium in oxygenic photogranules (OPG) revealed with multi-isotope (\u003csup\u003e2\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN) SIP-nanoSIMS. Separate frames show the relative assimilation in fraction (at%) of assimilated elements: carbon\u0026nbsp;– frame\u0026nbsp;a; hydrogen\u0026nbsp;– frame\u0026nbsp;b; nitrogen\u0026nbsp;–frame\u0026nbsp;c. Frame\u0026nbsp;d shows the RGB-overlay of frames\u0026nbsp;a, b and c. Frames\u0026nbsp;e, f: scatterplots of pixels assigned to the biomass of cyanobacteria (in black) and to heterotrophs (in red) according to the fraction of assimilated carbon and hydrogen – frame\u0026nbsp;e; and according to carbon and nitrogen relative assimilation – frame\u0026nbsp;f; median values and median absolute deviations (Med±MAD) are shown with white symbols.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/d66aab9a11440b713d75640f.png"},{"id":100070490,"identity":"1b521370-a1ac-43d0-bd84-153b476ae35d","added_by":"auto","created_at":"2026-01-12 16:17:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4066893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/fec260bd-f87e-44ce-a716-1f5b21532ad0.pdf"},{"id":97666637,"identity":"1a5991c2-d655-46fe-9f00-536496b70232","added_by":"auto","created_at":"2025-12-08 09:21:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5973994,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationrevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-7271395/v1/0ca2344f923965a8130f2071.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Restoration of deuterium marker for multi-isotope mapping of cellular metabolic activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInformation on microbial-mediated matter conversion scaled up to global biogeochemical cycles [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] is required for the elaboration of resource-saving and energy-conversion approaches [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The combination of stable-isotope probing and nanoscale secondary ion mass spectrometry (SIP-nanoSIMS) has been efficiently employed to link metabolic activity with the identity of single-cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] for tracing the intercellular nutrient flow and intracellular transformations e.g. [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In biological and biomedical research, stable-isotope tracers are introduced with isotope-labelled substances (e.g., \u003csup\u003e15\u003c/sup\u003eN-thymidine, \u003csup\u003e13\u003c/sup\u003eC-glucose, \u003csup\u003e13\u003c/sup\u003eC-glutamine, \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e gas, \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e gas, H\u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003eO, H\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO) into the nutrition environment or growth medium.\u003c/p\u003e\u003cp\u003eTracing the cellular activity in carbon, nitrogen and hydrogen assimilation at once (multi-isotope tracing) is required for a comprehensive investigation of substrate conversion and metabolic interactions in complex communities. Because of the irreversible ablation of material from a sample area explored upon a SIMS analysis, multi-isotope tracing requires simultaneous detection of secondary ions containing all tracer-isotopes, i.e., \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e\u0026ndash;\u003c/sup\u003e or \u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e14\u003c/sup\u003eN\u003csup\u003e\u0026ndash;\u003c/sup\u003e and \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e15\u003c/sup\u003eN\u003csup\u003e\u0026ndash;\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor a proper decision on an optimal isotope-labelled substance, the lability of cellular nutrition (i.e., dependence of metabolic pathways and rates on nutrients\u0026rsquo; chemical and isotopic composition) has to be considered to preserve intracellular homeostasis and to minimise the perturbation of native nutrition scenarios inherent to a studied ecosystem. Isotopic labelling of the growth medium with heavy water [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] provides deuterium (\u003csup\u003e2\u003c/sup\u003eH) or heavy oxygen (\u003csup\u003e18\u003c/sup\u003eO) as a nutrient-independent tracer of cellular metabolic activity. Deuterated water (heavy \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e16\u003c/sup\u003eO water or the semi-heavy \u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e16\u003c/sup\u003eO) is a more preferred labelling source than heavy-oxygen water (\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO, \u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e17\u003c/sup\u003eO or \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e18\u003c/sup\u003eO isotopologues) because the latter is much more expensive due to the more difficult separation of \u003csup\u003e17\u003c/sup\u003eO and \u003csup\u003e18\u003c/sup\u003eO containing isotopologues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe high relative mass difference between hydrogen isotopes leads to strong hydrogen isotope fractionation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and may impede metabolism, i.e., cause toxicity effects already with 10% of \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e16\u003c/sup\u003eO fraction in the growth medium [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Incorporation of \u003csup\u003e2\u003c/sup\u003eH from heavy water into biomass implies further dilution of the \u003csup\u003e2\u003c/sup\u003eH tracer due to the major atomic fraction of hydrogen in biomass, e.g., 52 atomic percent (at%) according to the Redfield ratio for phytoplankton [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to biotic factors limiting the amount of the \u003csup\u003e2\u003c/sup\u003eH tracer in analysed cells, the relatively low electron affinity of hydrogen atoms results in a moderate yield of secondary \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions when 16 keV Cs\u003csup\u003e+\u003c/sup\u003e primary projectiles are employed for nanoSIMS analysis. Nevertheless, nanoSIMS has been successfully applied for \u003csup\u003e2\u003c/sup\u003eH tracing in environmental studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], in biology [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and in material science e.g. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Strategies suggested for overcoming construction-related limitations of a serial NanoSIMS 50L instrument imply modification of factory-set hardware settings [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], hardware upgrade and the derivation of \u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e1\u003c/sup\u003eH isotope ratio from polyatomic ions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] along with the numerical restoration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] (see the Supplementary Information for more details, SI section S1).\u003c/p\u003e\u003cp\u003eThe primary aim of the present work was to identify optimal isotopologue pairs within the [\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH] series for quantitative high-resolution \u003csup\u003e2\u003c/sup\u003eH mapping that could be done simultaneously with \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN tracing for studying the matter conversion and metabolic interaction in complex environmental systems. The demand for nanoscale lateral resolution is particularly strong when studying metabolic interactions in microbial consortia, where changes in structural composition occur at sub-micrometer spatial scales, even smaller than cell size. To demonstrate the advantages of the multi-isotope SIP-nanoSIMS technique, and particularly the prospects of \u003csup\u003e2\u003c/sup\u003eH as a metabolic tracer in this type of applications, we used oxygenic photogranules (OPGs) as a model system. OPGs comprise a complex microbiome of syntrophically interacting heterotrophic and phototrophic bacteria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This light-driven ecosystem is exchanging key metabolites when photosynthesis is active. OPGs have proven their effectiveness for wastewater treatment without external aeration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and show potential to compete with the conventional activated sludge process [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe approach of \u003csup\u003e2\u003c/sup\u003eH mapping was optimized on resin-embedded maize-root samples and applied in multi-isotope tracing mode on OPGs. Due to the relatively high yield of C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions and their robust biomass-featured spatial distribution, the C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH/C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH ratio was considered as a rather promising measure of the \u003csup\u003e2\u003c/sup\u003eH fraction, despite the unresolved C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026amp; C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e interference. The effect of this interference is reduced due to the decrease in C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e ion counts with increasing \u003csup\u003e2\u003c/sup\u003eH fraction [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Together with the suggested new approach to restoring the \u003csup\u003e2\u003c/sup\u003eH-fraction from polyatomic (C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026amp; C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e)/C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e ion ratio, the employed principle of equal relative assimilation allowed the elucidation of interplay in nutrition channels and quantitative analysis of cellular metabolic interaction within the OPG consortium.\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cp\u003eIn the first stage of our study, the efficiency of deuterium mapping with \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e and \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e ion species was evaluated on thick (\u0026sim;1 mm) resin-embedded maize root samples providing feasibility of long-lasting measurements necessary to reveal features of \u003csup\u003e2\u003c/sup\u003eH spatial distribution in low-abundant \u003csup\u003e2\u003c/sup\u003eH-containing ion counts and to clarify the mass-spectroscopy of corresponding molecular fragments. In the second stage, selected pairs of polyatomic ions were employed for multi-isotope tracing of metabolic activity in photo-hetero-trophic microbial associations (OPGs) enabling quantitative analysis of relative assimilation in carbon, nitrogen and hydrogen simultaneously. The details on multicollector settings, the restoration of \u003csup\u003e2\u003c/sup\u003eH fraction from the unresolved \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH \u0026amp; \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e mass-peak, and the effect of an ion-probe-induced material relocation are explained in Supplementary Information (SI, sections S1-S3).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Mass-spectroscopy of \u003csup\u003e2\u003c/sup\u003eH-containing ion species\u003c/h2\u003e\u003cp\u003eAfter carefull alignment of the secondary ion beam, the mass-resolving power (MRP) of about 16.000\u0026ndash;18.000 was achieved in the mass-range of polyatomic \u003csup\u003e2\u003c/sup\u003eH-containing ion species. Mass-spectra of \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e and \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e ion-species were acquired from \u003csup\u003e2\u003c/sup\u003eH-labelled and non-labelled maize-root samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe mass-spectra were acquired with a defocused primary-ion beam in a 64\u0026times;64 pixels raster over same-size areas of the \u003csup\u003e2\u003c/sup\u003eH-labelled and non-labelled samples to keep counting rates of \u003csup\u003e2\u003c/sup\u003eH-containing ion species for each sample comparable. Obtained counting rates are determined by the electron affinity of the corresponding molecular fragments and by their abundance within the analysed sample fragment. The mass-peak of \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, top left) reveals a count rate of 20 counts per second (cps) and is well separated from the mass-peak of \u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e for the \u003csup\u003e2\u003c/sup\u003eH-labelled sample. The mass-peak of \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e with 50 cps is resolved from \u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, top right) and appears as a clear flat-top shoulder at the low-mass side of a partly (\u0026asymp;50%) overlapping \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e peak. The mass-peak of \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, bottom left) delivers 25 cps and overlaps partly (\u0026asymp;50%) with the \u003csup\u003e17\u003c/sup\u003eO\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e peak, but it can still be employed for mass-spectrometric quantitation of \u003csup\u003e2\u003c/sup\u003eH content when \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e ion species are not revealed at the high-mass side. Regarding the mass-peak of \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, a relatively high counting rate of 200 cps is delivered from the \u003csup\u003e2\u003c/sup\u003eH-labelled sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, bottom right). However, it overlaps to 40% with the mass-peak of \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003eand over 80% with that of \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e, where the latter decreases in count rate from 65 cps down to 10 cps when the non-labelled (natural \u003csup\u003e2\u003c/sup\u003eH abundance) and \u003csup\u003e2\u003c/sup\u003eH-labelled samples are compared. This feature of the \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e dependence on the \u003csup\u003e2\u003c/sup\u003eH-label content has previously been emphasized [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and is comprehensively considered in the present study for the restoration of the \u003csup\u003e2\u003c/sup\u003eH fraction from the unresolved \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH \u0026amp; \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e mass-peak.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Direct measurements and the restoration of natural \u003csup\u003e2\u003c/sup\u003eH abundance\u003c/h2\u003e\u003cp\u003eThe fraction of \u003csup\u003e2\u003c/sup\u003eH in a resin-embedded maize-root sample with a natural isotopic composition was analyzed in the imaging mode of nanoSIMS 50L employing multicollector settings II \u0026amp; III (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in SI). The distributions of the measured values among the image pixels are shown with histograms and box-plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e separately for \u003csup\u003e2\u003c/sup\u003eH fractions derived from the H, CH, OH and C\u003csub\u003e2\u003c/sub\u003eH isotopologue ion count ratios.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe median value of the \u003csup\u003e2\u003c/sup\u003eH fraction is close to the natural \u003csup\u003e2\u003c/sup\u003eH abundance (fraction) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{F}_{0}\\)\u003c/span\u003e\u003c/span\u003e=\u003csup\u003e2\u003c/sup\u003eH/(\u003csup\u003e2\u003c/sup\u003eH+\u003csup\u003e1\u003c/sup\u003eH)\u0026times;100\u0026thinsp;=\u0026thinsp;0.0115 at% when derived from H, CH and OH isotopologue ratios, but appears to be about 2.5 times overestimated (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{F}_{0}^{{\\prime\\:}}\\)\u003c/span\u003e\u003c/span\u003e=C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH/(C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH+C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH)\u0026times;100=0.0296 at%, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) due to the unresolved \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}\\)\u003c/span\u003e\u003c/span\u003e\u0026amp; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{1}{\\text{H}}_{2}\\)\u003c/span\u003e\u003c/span\u003e mass peaks (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1). Nevertheless, the high counting rate of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}{\\text{H}}_{\\:}}^{\\--}\\)\u003c/span\u003e\u003c/span\u003e ion species offers an enhanced precision in the quantitation of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{2}{\\text{H}}_{\\:}\\)\u003c/span\u003e\u003c/span\u003e fraction. Moreover, the ion-beam induced material-smearing effect, revealed strongly in \u003csup\u003e2\u003c/sup\u003eH maps derived from H, CH and OH isotopic ratios (Fig. S4), is almost not detectable when C\u003csub\u003e2\u003c/sub\u003eH isotopologues are considered for the restoration of the \u003csup\u003e2\u003c/sup\u003eH fraction (Fig. S5 d). Thus, despite the unresolved \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}\\)\u003c/span\u003e\u003c/span\u003e \u0026amp; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{1}{\\text{H}}_{2}\\)\u003c/span\u003e\u003c/span\u003e mass interference, one still has two solid advantages (i.e., enhanced precision and negligible smearing effect) of C\u003csub\u003e2\u003c/sub\u003eH isotopologue ratio consideration for \u003csup\u003e2\u003c/sup\u003eH mapping. With this motivation, a new method for the restoration of \u003csup\u003e2\u003c/sup\u003eH fraction with the unresolved \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}\\)\u003c/span\u003e\u003c/span\u003e \u0026amp; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{1}{\\text{H}}_{2}\\)\u003c/span\u003e\u003c/span\u003e mass peaks has been developed in the present work (SI, section S2).\u003c/p\u003e\u003cp\u003eThe suggested restoration of \u003csup\u003e2\u003c/sup\u003eH fraction can be implemented with a rather simple expression. In terms of isotope ratio, the restored \u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e1\u003c/sup\u003eH value is expressed as\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{R}_{r}={R}^{{\\prime\\:}}-{ϵ}_{{H}_{2}/H}\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}^{{\\prime\\:}}\\)\u003c/span\u003e\u003c/span\u003e is the hydrogen isotope ratio overestimated due to the unresolved \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}\\)\u003c/span\u003e\u003c/span\u003e, and the correcting parameter \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{ϵ}_{{H}_{2}/H}\\)\u003c/span\u003e\u003c/span\u003e is defined as the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\text{C}}_{2}{\\text{H}}_{2}}^{-}/{{\\text{C}}_{2}\\text{H}}^{-}\\)\u003c/span\u003e\u003c/span\u003e ion-yield ratio (section S2 in SI). The calculation of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{ϵ}_{{H}_{2}/H}\\)\u003c/span\u003e\u003c/span\u003e value employs the difference between CH and C\u003csub\u003e2\u003c/sub\u003eH isotopologue ratios (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{0}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{0}^{{\\prime\\:}}\\)\u003c/span\u003e\u003c/span\u003e, respectively) acquired on the unlabelled sample with a natural \u003csup\u003e2\u003c/sup\u003eH abundance (see S2 section in SI for more details).\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{ϵ}_{{H}_{2}/H}=\\left({R}_{0}^{{\\prime\\:}}-{R}_{0}\\right)\\times\\:\\left(1+{R}_{0}\\right)=\\)\u003c/span\u003e\u003c/span\u003e (1.6411\u0026plusmn;0.0012)\u0026times;10\u003csup\u003e\u0026minus;4\u003c/sup\u003e (2)\u003c/p\u003e\u003cp\u003eThe median value of the \u003csup\u003e2\u003c/sup\u003eH fraction restored from the unresolved \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}\\)\u003c/span\u003e\u003c/span\u003e\u0026amp; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{1}{\\text{H}}_{2}\\)\u003c/span\u003e\u003c/span\u003e peak employing the Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (shown in blue in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) is close to the value of the \u003csup\u003e2\u003c/sup\u003eH abundance \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{F}_{0}\\)\u003c/span\u003e\u003c/span\u003e=0.0132\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0045 at% derived from the CH isotopologue ratio \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{R}_{0}\\)\u003c/span\u003e\u003c/span\u003e accepted in Eq.\u0026nbsp;(2) for the calculation of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{ϵ}_{{H}_{2}/H}\\)\u003c/span\u003e\u003c/span\u003e value.\u003c/p\u003e\u003cp\u003eThe distributions derived from the H and OH isotopologue ratios are more stretched and even truncated considerably at 0 at% due to the relatively low counting rates for \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e and \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e ions (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In case of such truncated distributions, it may be better to consider a modus value instead of the median. The most stretched distribution, as observed for the OH isotopologues, may be due to the contribution of \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e ion species that are not revealed in the mass-scan at 18 amu for the studied resin-embedded samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) but may show a higher yield from other sample types.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Application on a complex microbial community\u003c/h2\u003e\u003cp\u003eWith the established multi-isotope tracing approach, the SIP-nanoSIMS methodology was applied on oxygenic photogranules (OPG) to check its applicability and prospects for studying metabolic interactions in the complex microbial consortia. OPGs are microbial aggregates with diameters ranging from several hundreds of micrometers to four to five millimeters. These roughly spherical aggregates float freely in an agitated aqueous system, but settle rapidly when mixing is stopped. OPGs harbor a microbial community dominated by heterotrophic and phototrophic bacteria. These two groups of microorganisms are believed to syntrophically exchange heterotrophically produced CO\u003csub\u003e2\u003c/sub\u003e and photosynthetically produced O\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. CO\u003csub\u003e2\u003c/sub\u003e may be produced from an externally provided carbon source, for example, organic compounds contained in wastewater. Alternatively, hetetrotrophs may produce CO\u003csub\u003e2\u003c/sub\u003e from the conversion of mainly phototrophically produced extracellular polymeric substances contained in the photogranules. This is the case in our experiment where only inorganic carbon is added in form of HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e as it can only be assimilated by autotrophic microorganisms. In studied photogranules, the majority of autotrophs are phototrophic filamentous cyanobacteria of the order Oscillatoriales [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These cyanobacteria are not known to fix N\u003csub\u003e2\u003c/sub\u003e in the presence of another more accessible nitrogen source. The \u003csup\u003e15\u003c/sup\u003eN-labelled ammonium chloride was provided as a nitrogen source for heterotrophs and phototrophs. Deuterated water was providing \u003csup\u003e2\u003c/sup\u003eH as a tracer of biosynthetic OPG activity.\u003c/p\u003e\u003cp\u003eWith the reliable \u003csup\u003e2\u003c/sup\u003eH reconstruction from the unresolved \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}\\)\u003c/span\u003e\u003c/span\u003e\u0026amp; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{1}{\\text{H}}_{2}\\)\u003c/span\u003e\u003c/span\u003e mass-peak (described in SI, section S2), the correcting parameter \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{ϵ}_{{H}_{2}/H}\\)\u003c/span\u003e\u003c/span\u003e=(1.4750\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014)\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e was derived and the multicollector settings V (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were adopted for simultaneous tracing of H, C, N isotopes in parallel with the mapping of cellular \u003csup\u003e32\u003c/sup\u003eS and \u003csup\u003e31\u003c/sup\u003eP (\u003csup\u003e31\u003c/sup\u003eP\u003csup\u003e1\u003c/sup\u003eH, see Fig. S6) upon the analysis of OPG samples.\u003c/p\u003e\u003cp\u003eThe histograms of biomass-related pixel distribution according to the biomass isotopic enrichments in \u003csup\u003e2\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) are not directly comparable because of the different natural abundance of these isotopes (shown with horizontal dashed lines) and due to their different fractions in the growth substrate. Isotopic enrichment of each biomass volume-unit (voxel of about 60x60x100 nm\u0026sup3;, 3D analog of a 2D-pixel) within cell-confining regions of interest (RoIs, see RoI-definition example in Fig. S7) was derived from corresponding ion-count ratios and converted with the Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e3\u003c/span\u003e) into the relative assimilation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) representing the assimilated amount \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{a}\\)\u003c/span\u003e\u003c/span\u003e of tracer-derived H, C or N expressed relatively to the final cellular amount \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{f}\\)\u003c/span\u003e\u003c/span\u003e of the corresponding element achieved after the incubation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:{K}_{A}^{f}=\\frac{{E}_{a}}{{E}_{f}}=\\frac{{R}_{f}-{R}_{i}}{\\left(1+{R}_{f}\\right)\\times\\:\\left\\{{F}_{gs}\\times\\:\\left(1+{R}_{i}\\right)-{R}_{i}\\right\\}}=\\frac{{F}_{f}-{F}_{i}}{{F}_{gs}-{F}_{i}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:R\\)\u003c/span\u003e\u003c/span\u003e is the heavy-to-light isotope ratio, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:F\\)\u003c/span\u003e\u003c/span\u003e is the corresponding atom fraction of heavy isotope, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:F=R/(R+1)\\)\u003c/span\u003e\u003c/span\u003e, subscripts \u003cem\u003ei\u003c/em\u003e and \u003cem\u003ef\u003c/em\u003e refer to the values before and after the incubation (\u003cem\u003einitial\u003c/em\u003e and \u003cem\u003efinal\u003c/em\u003e), and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{F}_{gs}\\)\u003c/span\u003e\u003c/span\u003e is the fraction of heavy isotope in the isotope-labelled tracer-substance contributing to the growth substrate.\u003c/p\u003e\u003cp\u003eThe relative assimilation has also been calculated for each single cell as the mean value over biomass volume-units (voxels) within the corresponding cell-confining RoI (shown with yellow contours in Fig. S7 a,b). Fig. S9 shows the distribution of single-cells in their relative assimilation of carbon, nitrogen and hydrogen to be well reproduced with the distribution of biomass-units in the corresponding relative assimilation calculated for each cellular-related voxel. Features of the cell distribution in relative assimilation are better revealed with a higher count of subcellular biomass volume-units (voxels). Therefore, voxel-resolved data were considered for quantitative evaluation of the relative elemental assimilation in the present work.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo account for the dilution of \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e2\u003c/sup\u003eH isotope-tracers with fixative [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and embedding materials, values of relative assimilation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\left({}_{\\:}{}^{12}\\text{C}{}_{\\:}{}^{13}\\text{C}/{}_{\\:}{}^{12}{\\text{C}}_{2}\\right)\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\left({}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{2}\\text{H}/{}_{\\:}{}^{12}{\\text{C}}_{2}{}_{\\:}{}^{1}\\text{H}\\right)\\)\u003c/span\u003e\u003c/span\u003e, computed with the corresponding C\u003csub\u003e2\u003c/sub\u003e and C\u003csub\u003e2\u003c/sub\u003eH isotopologue ratios, were corrected with 1.136 multiplication factor and 0.03 at% offset, derived as the slope \u003cem\u003eb\u003c/em\u003e and y-intercept \u003cem\u003ea\u003c/em\u003e of the linear function fitting \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\left({}_{\\:}{}^{13}\\text{C}{}_{\\:}{}^{14}\\text{N}/{}_{\\:}{}^{12}\\text{C}{}_{\\:}{}^{14}\\text{N}\\right)=a+b\\times\\:{K}_{A}^{f}\\left({}_{\\:}{}^{12}\\text{C}{}_{\\:}{}^{13}\\text{C}/{}_{\\:}{}^{12}{\\text{C}}_{2}\\right)\\)\u003c/span\u003e\u003c/span\u003e relation (see Fig. S10).\u003c/p\u003e\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the spatial distribution of cellular relative assimilation is visualized in a thin section through the outer green part of a photogranule. Distribution of single-cells in relative assimilation activity \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-d) facilitates the differentiation between consortium-representative species via quantitative analysis of their elemental assimilation efficiency. With the map derived for the relative carbon assimilation in the OPG-consortium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) its autotrophic members fixing \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e can be clearly recognized as elongated cells arranged in filaments (see Fig. S7 a,c) typical for cyanobacteria usually found in OPG [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Complete filaments are not seen in the figures, as the imaged area represents a thin section on which only part of the filaments happens to be positioned in the sectioning plane. All metabolically active cells in OPG show the fraction of hydrogen incorporated from water (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) to be in a similar range as the fraction of carbon supplied via \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e fixation by autotrophs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,e, y-axis).\u003c/p\u003e\u003cp\u003eOne can recognize cyanobacteria cells (yellow RoIs in Fig. S7 a) with almost equal relative assimilation of C and H (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, black). This feature in the metabolic activity of cyanobacteria would suggest their homeostatic nutrition keeping cellular elemental Redfield ratio almost constant, however the availability of \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e boosts synthesis of N-rich molecules (e.g., proteins) revealed as about 5-fold increase in relative assimilation of N (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, black). Cyanobacteria are known to store nitrogen in the form of so-called structured granules enriched in cyanophycin granule polypeptide [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Nevertheless, proportional assimilation of carbon and nitrogen makes it easy to recognize cyanobacteria when overlaying frames a, b and c of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, yielding Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed.\u003c/p\u003e\u003cp\u003eCyanobacteria are coloured in the RGB-overlay from bright pink (slightly prevailing C assimilation over H assimilation) to magenta and purple spots with \u003csup\u003e2\u003c/sup\u003eH prevailing assimilation. Bright pink-red areas can be identified also in between cyanobacterial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, lower part) and may be ascribed to carbon-rich extracellular polymeric substances (EPS) synthesised \u003cem\u003ede novo\u003c/em\u003e from photosynthetically assimilated \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e. Typical cyanobacteria found in OPG belong to the order \u003cem\u003eOscillatoriales\u003c/em\u003e, which are filamentous gliding cyanobacteria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Bright EPS spots immediately adjacent to cyanobacterial cells could represent EPS excreted for the gliding motility.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAll cells appearing bright green in the overlay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) may be assigned to heterotrophic bacteria (marked with yellow RoIs in Fig. S7 b) possessing intensive nitrogen assimilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d in red) from the supplied \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. This high nitrogen assimilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef in red, y-axis; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column III) implies the obvious necessity of carbon recycling from the unlabelled carbon sources, e.g., EPS or dissolved organic matter pool (DOM), for maintaining the elemental biomass-stoichiometry upon the limited carbon supply with \u003csup\u003e13\u003c/sup\u003eC-labelled phototrophic exometabolites from cyanobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f in red, x-axis; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column I). These heterotrophic bacteria consume \u003csup\u003e2\u003c/sup\u003eH from water for fatty-acid synthesis involving NADPH/NADP\u003csup\u003e+\u003c/sup\u003e, whereas more ammonium-derived \u003csup\u003e1\u003c/sup\u003eH is supplied together with \u003csup\u003e15\u003c/sup\u003eN for protein synthesis resulting in slightly lower \u003csup\u003e2\u003c/sup\u003eH enrichment of heterotrophs as compared with cyanobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, y-axis; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column V).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eElement- and source-resolved distribution of the relative assimilation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e represented for OPG members with median values and median absolute deviation as Med\u0026thinsp;\u0026plusmn;\u0026thinsp;MAD [at%]. Values derived from the SIP-nanoSIMS data\u003csup\u003e#\u003c/sup\u003e are shown in bold (with white symbols in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f). Assimilation due to the syntrophic interaction between cyanobacteria and heterotrophs is shown in *grey-filled cells.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElement\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eCarbon\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNitrogen\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eHydrogen\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erecycled DOM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eheavy water (33% of \u003csup\u003e2\u003c/sup\u003eH)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e \u0026amp; recycled DOM\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eII\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eIII\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eIV\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCyanobacteria\u003c/p\u003e\u003cp\u003e\u003cem\u003e(Comments)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/b\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e*2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/b\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/b\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e*2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003e20% of photosynthetic C\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003e80% from the shared reduced organic matter\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003e100%, i.e., single nitrogen supply\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003e20% via photosynthetic incorporation\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003e80% from growth medium inv. shared reduced OM\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHeterotrophs\u003c/p\u003e\u003cp\u003e\u003cem\u003e(Comments)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e*\u003cb\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/b\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/b\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003e2% from shared cyanobacterial exometabolites\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003e98% from the recycled organic matter\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003e100%, i.e., single nitrogen supply\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003e6% from growth medium\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003einv. 2% of cyanobacterial exometabolites\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003e94% from growth medium inv. reduced OM\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWith 1 mM \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e as a not-limiting nitrogen source in the nutrition medium, its relative assimilation reached 10% for heterotrophic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,f in red), whereas 2\u0026ndash;3 times lower N-assimilation activity of cyanobacterial filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef in black) was revealed due to their rate-limiting carbon supply via CO\u003csub\u003e2\u003c/sub\u003e fixation. It is important to note the distribution pattern of bacterial cells around phototrophs \u0026ndash; they are always close neighbours with a mutualistic interaction between phylogenetically unrelated members in the OPG community.\u003c/p\u003e\u003cp\u003eAssuming the elemental stoichiometry of each OPG member remains stable [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] during the time of incubation with an isotope-labelled substrate, one may expect close median values of relative assimilation (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e) in all elements (H, C and N) for representatives within each group (autotrophs and heterotrophs). With this assumption and the median values of relative assimilation derived with the isotope-labelled nutrition part (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f; median values shown with white symbols), the fractions of carbon and hydrogen from the recycled DOM and \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-related hydrogen, assimilated by cyanobacteria and heterotrophs Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (columns II and V), were derived as follows.\u003c/p\u003e\u003cp\u003eWith its non-limiting content in the growth medium, \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was considered as the easily-accessible prevailed nitrogen source providing all, i.e., 100% of cellular nitrogen gained by OPG consortium during the incubation. The relative nitrogen assimilation of 3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 at% (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e, Med\u0026thinsp;\u0026plusmn;\u0026thinsp;MAD) for cyanobacteria and 6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85 at% for heterotrophs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column III; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, y-axis, median values shown with white symbols) imply 2-fold difference in their nitrogen-specific growth rate\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\gamma\\:=\\frac{-{{log}}_{2}\\left(1-{K}_{A}^{f}\\right)}{t}=-\\frac{{ln}\\left(1-{K}_{A}^{f}\\right)}{t\\times\\:{ln}\\left(2\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:\\varDelta\\:\\gamma\\:=\\frac{\\partial\\:\\gamma\\:}{\\partial\\:{K}_{A}^{f}}\\times\\:\\varDelta\\:{K}_{A}^{f}=\\frac{1}{t\\times\\:\\text{ln}\\left(2\\right)}\\times\\:\\frac{1}{\\left(1-{K}_{A}^{f}\\right)}\\times\\:\\varDelta\\:{K}_{A}^{f}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ereturning 0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 at%/h and 1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 at%/h respectively with the cultivation time \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:t\\)\u003c/span\u003e\u003c/span\u003e=6 hours.\u003c/p\u003e\u003cp\u003eCarbon fractions supplied from \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e via photosynthesis (0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 at%) and exometabolite sharing (0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 at%, syntrophic share with heterotrophs) by cyanobacteria were derived from nanoSIMS data as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e median values (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column I) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f (x-axes, median-value symbols in white). Carbon fraction assimilated from the recycled DOM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column II) by cyanobacteria (2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 at%, share from heterotrophic OM reducers) and by heterotrophs (5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85 at%) was calculated as the difference between the corresponding fraction of assimilated nitrogen (column III) and the fraction of carbon from \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e (column I). Such a calculation is valid when i) the \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e can be considered as the prevailed nitrogen source and ii) the assumed above preservation of biomass stoichiometry is fulfilled implying equal relative assimilation of all elements (C, N and H) by each group of OPG consortium. The cellular fraction of hydrogen originating from the supplied \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and recycled DOM (column V) was calculated for cyanobacteria (2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 at%, involving a major share from heterotrophic OM reducers) and for heterotrophs (5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 at%) as the difference between the fraction of assimilated nitrogen (column III) and the hydrogen fraction incorporated from heavy water (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, column IV) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee (y-axis, median-value symbols in white).\u003c/p\u003e\u003cp\u003eFor a microbial group with the elemental stoichiometry of its biomass preserved, one may estimate the contribution of non-labelled nutrition sources with either i) an exclusive source of at least one element (C, N or H) isotope-labelled, or with ii) a known value of the growth- or division-rate for a target cell-type of a studies consortium. Assuming the DOM to be depleted in nitrogen, we considered \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e as an exclusive nitrogen source for OPGs in this study. This assumption allowed for the quantitative evaluation of non-labelled nutrient supply (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and for the calculation of cell-division rates (equations \u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e5\u003c/span\u003e) for cyanobacteria and heterotrophs. In another way, relative assimilation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e may also be calculated with a known growth-rate value \u003cem\u003e\u0026micro;\u003c/em\u003e for the biomass of a target cell-type or corresponding cell-division rate γ.\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:{K}_{A}^{f}=1-{e}^{-\\mu\\:\\times\\:t}=1-{2}^{-\\gamma\\:\\times\\:t}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWith the value of relative assimilation acquired in this way, one can further analyse the completeness in isotope-labelling of nutrient supply by comparing the Eq.\u0026nbsp;(\u003cspan refid=\"Equ5\" class=\"InternalRef\"\u003e6\u003c/span\u003e) output with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e values derived from the changes in isotopic composition of a corresponding cell-type upon their incubation with an isotope-labelled substrate.\u003c/p\u003e\u003cp\u003eThe photogranules that were incubated in the experiments here had a net heterotrophic activity. The required carbon for this activity was derived from the supplied \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e and via EPS conversion. EPS is typically present in sample quantities in photogranules as it is produced for example for cyanobacterial gliding motility. However, contrasting environmental conditions, e.g., illumination, as well as photogranule properties, e.g., size, may partition autotrophic and heterotrophic activities differently. Especially for biotechnological applications of photogranule biomass, controlling the specific activities of the photogranule community by varying the environmental conditions are of interest. The procedure of metabolic flux analysis suggested here allows us to generate these data in further experiments. Our study of metabolomic exchange between cyanobacteria and other bacteria in photogranules shows the expected metabolic shifts in carbon fixation (Calvin cycle pathway) depending on light availability [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is worth noting that the fraction of hydrogen incorporated from heavy water within 0.001-1 at% range (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) was analysed with NanoSIMS 50L delivering good ion-counting statistics from each biomass unit confined in single voxels (3D-pixels). The dynamic range of hydrogen isotopic analysis starting from its natural abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) facilitates the efficient differentiation between single-cells and cellular compartments according to their metabolic activity. The incorporation of hydrogen from heavy water (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, y-axis) is revealed for all types of cells regardless of functional activity and nutrition mode (whether autotrophic or heterotrophic). In this way, \u003csup\u003e2\u003c/sup\u003eH from water is proved as a universal tracer in all biosynthetic processes being primarily supplied to the sites of the most intensive biosynthesis like DNA/RNA-synthesis sites and synthesis of service enzymes around bacterial chromosome (see the overlay of P and H-incorporation maps in Fig. S8). This mode of stable-isotope labelling (\u003csup\u003e2\u003c/sup\u003eH from deuterated water) allows the tracing of dividing cells in a population [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hydrogen is incorporated from water into cell metabolism by either hydrolysis, osmotic interactions, or most importantly by hydrogen incorporation into NADPH synthesis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. NADPH is a co-factor for metabolic reactions in the Calvin cycle, lipid and nucleic acid syntheses, where NADPH is required as a reducing agent (hydrogen source). NADPH and its oxidized form NADP\u003csup\u003e+\u003c/sup\u003e are utilised by all forms of cellular life [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Conclusions and outlook","content":"\u003cp\u003eThe multi-isotope tracing of metabolic activity in a complex OPG microbiome allowed us to differentiate between two populations of phototrophic and heterotrophic bacteria and to reveal the syntrophic interaction between these inhabitants of the phototrophic symbiotic consortium via analysis of the interplay in their nutrition channels. With the example of nutrition analysis on the OPG consortium, we demonstrated the prospects in application of the suggested approach for e.g., optimization of wastewater treatment processes. The employed stoichiometry-preservation principle constitutes a framework for metabolic flux analysis in complex systems, implying each representative group of the studied consortium shows equal relative assimilation of different elements (e.g., C, N and H).\u003c/p\u003e\u003cp\u003eHomeostatic \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e fixation was revealed for autotrophic inhabitants of OPG consortium with relative assimilation of carbon following the fraction of hydrogen incorporated from heavy water (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, S9 a, black). In this case, the complete isotope-labelling of phototrophic nutrition channel was achieved with heavy \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003eO water and \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e involved in the photosynthetic assimilation process. Major fraction (80\u0026ndash;98%) of assimilated carbon and hydrogen was found to be supplied via recycling of OM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, columns II, V) converted by heterotrophic reducers to DOM, i.e., beyond the isotope-traced pathway. Such an incomplete isotope-labelling of nutrient supply is revealed as a considerable discrepancy in the relative assimilation of different elements within a defined geno- or phenotypic group of a microbial consortium [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe study results emphasised the integrating role of \u003csup\u003e2\u003c/sup\u003eH labelling through deuterated water in complex systems with various life strategies and metabolic types. All metabolically active cells in the studied OPG consortium revealed the incorporation of deuterium from heavy water. Unlike \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN tracers, a major fraction of \u003csup\u003e2\u003c/sup\u003eH-marker from heavy water stays incorporated into the biomass upon cellular metabolic activity (i.e., detectable with nanoSIMS). For example, the respiration process implies the synthesis of NADH, NADPH etc. with incorporation of \u003csup\u003e2\u003c/sup\u003eH from heavy water, whereas \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e is released.\u003c/p\u003e\u003cp\u003eThe achieved reliable restoration of deuterium fraction already from its natural 0.0115 at% abundance proves SIP-nanoSIMS capability to sense and localize extremely low biosynthetic activity with relative assimilation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{K}_{A}^{f}\\)\u003c/span\u003e\u003c/span\u003e starting in the \u0026sim;0.001 at% range (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) corresponding to metabolic rate of \u0026sim;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e upon cellular maintenance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Employing this high sensitivity for the quantitation of metabolic heterogeneity among single cells of a microbial population [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] makes it feasible to trace microevolutionary processes and reveal obvious and non-obvious anthropogenically elicited changes in the environment.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Multi-isotope labelling and preparation of oxygenic photogranules\u003c/h2\u003e\u003cp\u003eOPGs were grown in a sequential batch reactor as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The photogranules were transported in darkness at room temperature to the nanoSIMS facility at UFZ in Leipzig. Isotope labelling incubations were performed under light in a medium containing heavy water (33 vol.% of \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003eO), H\u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (\u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e \u0026ndash; 87 at% \u003csup\u003e13\u003c/sup\u003eC) and \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (93 at% \u003csup\u003e15\u003c/sup\u003eN). The final concentrations of the labelled nutrients were 1 mM \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and 2 mM H\u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. After 6 hours of incubation, OPGs were fixed with 2% paraformaldehyde in cacodylate buffer (pH 7.4, 0.1 M) at 4˚C overnight and washed two times with the buffer. Dehydration was performed with ethanol series (30%, 50%, 70%, 80%, 90%, and 3 times 100%) for 15 min each. Low-viscosity LR White resin infiltration was done using 1:3, 1:2, 1:1, 2:1, and 3:1 of resin:ethanol mixture, each for 45 min, followed by pure LR White resin for one hour and overnight. Finally, the resin-infiltrated OPG sample was cured in an oven at 60\u0026deg;C for two days. The polymerized sample block was trimmed using a Leica EM TRIM2 trimmer and sectioned with Leica EM UC7 ultramicrotome employing a freshly-prepared glass knife. Sections of 300 nm thickness were placed on a 10 mm diameter As:Si-wafer and coated with a 10 nm of gold/palladium (80/20 weight ratio; Plano, Germany) conductive layer using Leica EM SCD 500 sputter-coater (Leica Microsystems, Germany) at 35 mA of Ar\u003csup\u003e+\u003c/sup\u003e current against Au/Pd target kept at -0.5 kV potential.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Maize-root \u003csup\u003e2\u003c/sup\u003eH labelling and sample preparation\u003c/h2\u003e\u003cp\u003eA long-lasting sample of resin-embedded plant root was prepared to facilitate the optimization of different \u003csup\u003e2\u003c/sup\u003eH mapping approaches in this study. This was necessary because the alignment of a NanoSIMS 50L instrument for multi-isotope tracing, involving the comparison of \u003csup\u003e2\u003c/sup\u003eH fraction in [\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH] series of isotopologue ratios, takes several hours. Additionally, due to the low natural abundance of deuterium (0.0115 at%) and a relatively low yield of \u003csup\u003e2\u003c/sup\u003eH containing secondary ions, the alignment and corresponding measurements of \u003csup\u003e2\u003c/sup\u003eH fraction have to be performed with the current of primary Cs\u003csup\u003e+\u003c/sup\u003e ions increased up to 4\u0026ndash;6 pA implying an enhanced consumption of the sample material. Wild-type maize seeds provided by the Institute of Crop Science and Resource Conservation, University of Bonn, were first surface sterilized [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Plant growth involving \u003csup\u003e2\u003c/sup\u003eH-labelling with 40% heavy water (\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003eO, 99.8 at% \u003csup\u003e2\u003c/sup\u003eH, Sigma Aldrich, Germany) was performed according to [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The primary 1 cm root tip was harvested after 96 hours. The root fixation, LR White resin infiltration, curring and trimming have been performed in the same way as described for the OPG samples (section 2.1). LR white block with a root crossection in its trimmed face was mounted in 10 mm diameter ring (fitting the nanoSIMS \u0026ldquo;Biology\u0026rdquo; sample holder) and coated with 10 nm of gold/palladium as OPG thin-section samples (section 2.1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.3. NanoSIMS analysis and data processing\u003c/h2\u003e\u003cp\u003eThe analysis of multi-elemental isotope ratios was implemented with a serial NanoSIMS 50L #134 instrument (AMETEK, CAMECA, France) at the UFZ in Leipzig. A set of 15 secondary ion species (\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e14\u003c/sup\u003eN\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e15\u003c/sup\u003eN\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e13\u003c/sup\u003eC\u003csup\u003e14\u003c/sup\u003eN\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e31\u003c/sup\u003eP\u003csup\u003e\u0026minus;\u003c/sup\u003e, \u003csup\u003e32\u003c/sup\u003eS\u003csup\u003e\u0026minus;\u003c/sup\u003e and \u003csup\u003e31\u003c/sup\u003eP\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e\u0026minus;\u003c/sup\u003e) were detected with seven detectors upon different mass-assignment configurations involving deflector-plate switching (see Results and Discussion for more details). Measurements were conducted with 15\u0026times;120 \u0026micro;m (width x height) nominal size of the entrance slit, 40\u0026times;1800 \u0026micro;m exit slits, 150\u0026times;150 \u0026micro;m aperture and an energy slit cutting off 30% of secondary ions in their energy-distribution tail. Caesium (Cs) pre-implantation was performed with 16 keV Cs\u003csup\u003e+\u003c/sup\u003e in 200 pA beam rastering within 100\u0026times;100 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e area for 30 min. Within these pre-implanted areas, 15\u0026times;15 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e or 25\u0026times;25 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e fields of view (FoV) were scanned in a 512\u0026times;512 pixel raster using a 4 pA primary Cs\u003csup\u003e+\u003c/sup\u003e beam with a dwelling time of 2 ms/pixel. To ensure sufficient counting statistics, data from the same FoV were acquired over 160 scans with the deflector-plate voltages switching between two values every second scan.\u003c/p\u003e\u003cp\u003eProcessing of the acquired data was done with a modified version of the open-source Look@NanoSIMS software [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (details in SI section S4). The data acquired with each scan were corrected for the lateral drift in the secondary electron intensity map (Esi) and all detected planes were accumulated for each ion species. The accumulated \u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e14\u003c/sup\u003eN\u003csup\u003e\u0026minus;\u003c/sup\u003e map, which is a proxy of the intrinsic cellular biomarkers (Fig. S6), was then used to draw regions of interest (RoIs) corresponding to plant tissues (maize sample) or microbial cells (OPG sample). Finally, the isotope ratios in the RoI pixels were exported in a text format suitable for further statistical analysis.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests:\u003c/h2\u003e\u003cp\u003eauthors declare no competing interests in relation to the work described.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eThe reported study was partly supported by the Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group advised grant (MetaSCOPE) of the Paul. G Allen Family Foundation. N.Y. acknowledge the funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; \u0026ldquo;MicroBridges\u0026rdquo; Satellite-Project in SPP2089 \u0026ldquo;MicroZym\u0026rdquo; Project 403664479 (BL 1560/2\u0026ndash;2; AOBJ683257). The contribution of L.S. and C.V. was supported by the German Federal Ministry of Education and Research (BMBF) and the initiative \u0026ldquo;Twenty20 \u0026ndash; Partnership for Innovation\u0026rdquo; for funding the H\u003csub\u003e2\u003c/sub\u003e-UGS project of HYPOS network (Grant 03ZZ0721). This work benefited from the activity of K.M. and J.H. in the frame of the Environmental Biotechnology and Biorefinery Facility (Bio2E) of INRAE-LBE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.15454/1.557234103446854E12\u003c/span\u003e\u003cspan address=\"10.15454/1.557234103446854E12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and funded partly by the National French Funding Agency ANR project PSST (grant number ANR-16-CE04-0001). N.M. was funded by the Novo Nordisk Foundation through an NNF Young Investigator Award, Grant NNF22OC0071609 ReFuel. Y.D. and H.B. acknowledge the funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; Project 403641683 (RI-903/7\u0026thinsp;\u0026minus;\u0026thinsp;1). Research work of Y.D. was supported by Deutsche Forschungsgemeinschaft Integration of Refugee Scientists and Academics.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.-H.R. and H.S. conceived and planned the study; Y.D., K.M., J.H., H.B. and N.M. performed the growth experiments and isotope-labeling; Y.D., H.B. and H.S. contributed to sample preparation; L.S. and H.S. carried out the nanoSIMS experiment; L.S. and H.S. developed the F(2H)-restoration method refined further together with L.P. and H.B.; L.S., N.Y., L.P. and H.S. performed the data evaluation; L.P. enhanced the Look@NanoSIMS software to allow multi-isotope analyses; Y.D., K.M., J.H., H.-H.R., and C.V. contributed to the interpretation of the results; N.Y. and H.S. took the lead in writing the manuscript; all authors contributed to manuscript revision, read and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the access to the analytical facilities of the Centre for Chemical Microscopy (ProVIS) at the Helmholtz Centre for Environmental Research in Leipzig, which is supported by the European Regional Development Funds (EFRE - Europe funds Saxony) and the Helmholtz Association. Support of ProVIS development by Helmholtz Munich (AG Sheikh \"Vascular Epigenetics\" Helmholtz Institute for Metabolic, Obesity and Vascular Research) in the frame of the Meta-SCOPE project is greatly acknowledged. The comprehensive support of cultivation and sample-preparation actions by Katja Nerlich and Jasmin Voigt is greatly appreciated.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analysed during the current study are available in the Google repository, https://drive.google.com/drive/folders/1iPocpciynqBhEu5QOU-qrUmrcPzcnpZK?usp=sharing\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFoster, R. A., Sztejrenszus, S. \u0026amp; Kuypers, M. 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Look@NanoSIMS\u0026ndash;a tool for the analysis of nanoSIMS data in environmental microbiology. \u003cem\u003eEnviron. Microbiol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (4), 1009\u0026ndash;1023 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"heavy water, deuterium, stable-isotope probing, multi-isotope tracing, metabolic activity, oxygenic photogranule","lastPublishedDoi":"10.21203/rs.3.rs-7271395/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7271395/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInvestigation of cellular metabolic activity with stable-isotope probing (SIP) implies the admittance of an isotope tracer into the metabolic pathway. Incubation with several isotope-markers (multi-isotope tracing) is required to trace nutrient metabolization and elucidate inter-cellular interactions in complex hosts and environmental communities. To cope with the lability of cell nutrition, deuterium in heavy \u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e16\u003c/sup\u003eO water is employed as a substrate-independent general tracer of metabolic activity. However, the spatially-resolved deuterium tracing is hampered by detection limits due to its relatively low ionization yield and mass-interference issues. In the present work, we comprehensively assess the quantitation of deuterium incorporation into biomass employing the outstanding capabilities of nanoscale Secondary Ion Mass Spectrometry facilitating quantitative analysis of metabolic activity with single-cell or subcellular resolution. The effect of ion-probe-induced material relocation on the acquired pattern in \u003csup\u003e2\u003c/sup\u003eH enrichment has been considered. Analytical expressions are suggested for the restoration of the deuterium fraction from the unresolved C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003eH-C\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e1\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e mass-interference. Application of the suggested principle of equal relative assimilation and the multi-isotope tracing with the \u003csup\u003e2\u003c/sup\u003eH-marker on a phototrophic symbiotic consortium paves the way to sensing the metabolic interplay among cells, recognition of homeostatic and shifted nutrition, checking for completeness of isotope-labelling and elucidation of nonlabelled substrate contribution.\u003c/p\u003e","manuscriptTitle":"Restoration of deuterium marker for multi-isotope mapping of cellular metabolic activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 08:13:53","doi":"10.21203/rs.3.rs-7271395/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-20T11:45:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-20T11:44:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T02:33:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15555854058801818617626743166457371494","date":"2025-12-09T14:52:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T14:38:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T11:40:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-14T08:19:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6a513b5-e1dd-47b3-9bbd-7a975078cd6b","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58183150,"name":"Biological sciences/Biochemistry"},{"id":58183151,"name":"Biological sciences/Biological techniques"}],"tags":[],"updatedAt":"2026-01-12T16:13:36+00:00","versionOfRecord":{"articleIdentity":"rs-7271395","link":"https://doi.org/10.1038/s41598-025-33762-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-07 15:58:57","publishedOnDateReadable":"January 7th, 2026"},"versionCreatedAt":"2025-12-04 08:13:53","video":"","vorDoi":"10.1038/s41598-025-33762-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-33762-5","workflowStages":[]},"version":"v1","identity":"rs-7271395","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7271395","identity":"rs-7271395","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}