Constraining activity and growth substrate of fungal decomposers via assimilation patterns of inorganic carbon and water into lipid biomarkers

Fungi are among the few organisms on the planet that can metabolize recalcitrant C but are also known to access recently produced plant photosynthate. Therefore, improved quantification of growth and substrate utilization by different fungal ecotypes will help to define the rates and controls of fungal production, the cycling of soil organic matter, and thus the C storage and CO2 buffering capacity soil ecosystems. This study combined a dual stable isotope probing approach together with rapid analysis by tandem pyrolysis gas chromatography isotope ratio mass spectrometry (Pyr-GC-IRMS) to determine the patterns of water-derived hydrogen (H) and inorganic C assimilated into lipid biomarkers of heterotrophic fungi as a function of C substrate. The water H assimilation factor (αw) and the inorganic C assimilation for C18:2 fatty acid isolated from five fungal species growing on glucose was lower (0.62±0.01 and 4.7±1.6%, respectively) than for species grown on glutamic acid (0.90±0.02 and 7.4±3.7%, respectively). Furthermore, the assimilation ratio (RIC/αW) for growth on glucose and glutamic acid can distinguish between these two metabolic modes. This dual SIP assay thus delivers estimates of fungal activity and may help to delineate the predominant substrates that are respired among a matrix of compounds found in natural environments. (200 Words) Importance Fungi decomposers play important roles in food webs and nutrient cycling because they can feed on both labile and stable forms of carbon. This study developed and applied a dual stable isotope assay to improve the investigation of fungal activity in the environment. By determining the incorporation patterns of hydrogen and carbon into fungal lipids, this assay delivers estimates of fungal activity and the different metabolic pathways that they employ in ecological and environmental systems. (75 Words)

. Fungi are known to feed on chemically stable forms of organic C, such as cellulose (10) and lignin (11)(12)(13), and are also known to directly access recently produced plant photosynthates (14,15).
Heterotrophic microorganisms who mainly feed on organic substrates also fix a variable amount of inorganic C.This flux acts to replenish intermediates in the tricarbox ylic acid (TCA) cycle that have been released for biosynthesis (16).It has been suggested that 2%-8% of heterotrophic biomass originates from CO 2 incorporated via carboxyla tion reactions (17)(18)(19), largely depending on the redox state of the organic substrate.Although these processes were described decades ago (16,20), the relevance and the (metabolic) controls on how much inorganic C is fixed by heterotrophic microorganisms are poorly understood due to the lack of reliable estimates for most organisms and habitats (19).Despite their prevalence as decomposers, estimates of inorganic C fixation by heterotrophic fungi are scarce (20).
In recent years, hydrogen (H) isotope incorporation from water has been shown to be a useful tracer of microbial community activity in a diverse range of environments (21)(22)(23)(24), as water is required by all microbes and there exists a strong relationship between the stable H isotopic composition of microbial lipids and water, which is the ultimate source of H in all-natural organic compounds (22,25).Strong correlations between H isotopes of water medium and microbial lipids were shown to correlate with the type of growth metabolism (26), with slightly higher δ 2 H values for aerobic heterotrophs, to lower δ 2 H values for phototrophs, to lowest δ 2 H values for chemoautotrophs (26,27).
To fully exploit the potential of stable isotope probing (SIP) experiments, a new lipid-based dual-SIP approach was developed to track total microbial production by adding heavy water (D 2 O), together with labeled 13 C-dissolved inorganic carbon (DIC), to enable estimates of total and autotrophic metabolisms, respectively (28,29).Because bicarbonate is often present in relatively high concentrations in natural systems, adding 13 C-bicarbonate and D 2 O to incubation experiments does not alter ambient biogeo chemical conditions (28).The ratio of D 2 O incorporation and 13 C-DIC assimilation into lipid biomarkers can then distinguish the microbial metabolism of autotrophs (ratio ~1) and heterotrophs (ratio <0.3) (28).
The purpose of this work was to apply the dual-stable isotope approach to deter mine heterotrophic inorganic C fixation and the water H assimilation factor (α W ) for fungal lipid biomarkers produced during growth of pure-culture fungal isolates on labile substrates (glucose and glutamic acid), with the hypothesis that growth substrate will activate alternative metabolic pathways with characteristic assimilation ratios of 13 C versus 2 H.We further tested the applicability of pyrolysis coupled with gas chromatogra phy-mass spectrometry (GC-MS) and isotope ratio mass spectrometry (IRMS) as a rapid, direct analysis of isotope incorporation into fungal biomarkers.

RESULTS
All five microscopic fungal species Aspergillus dimorphicus (AD), Cordyceps farinosa (CF), Penicillium janczewskii (PJ), Acremonium polychromum (AP), and Aspergillus brasiliensis (AB), and the yeast species Yarrowia lipolytica (YL) were pure cultures and exhibited a capacity to grow in the modified minimum media Czapek-Dox broth (30), with either glucose or glutamic acid as a sole carbon source and having various enrichment levels of 13 C-DIC and D 2 O.These fungal species were extracted for their membrane lipid fatty acids and analyzed by GC-IRMS for their isotopic composition.The results reported and evaluated in this study include stable isotopic compositions of DIC, CO 2 , water, and the fungal biomarkers C 18:2 and ergosterol.

Fungal growth
CO 2 concentrations in the fungal incubations increased linearly, and the biomass was harvested when the CO 2 headspace concentration reached 4%-6% vol/vol (Fig. 1), which required incubations times ranging between 2 and 4 weeks for glucose and between 4 and 8 weeks for glutamic acid.Incubations with CF grown on glucose were performed in 1-L bottles and reached higher CO 2 concentrations of ~10%.YL grown on glucose did not show any increase in CO 2 concentration in the headspace during the incubation period of 6 weeks.δ 13 C of headspace CO 2 decreased from a maximum value of +5,000‰, measured 3 days after inoculation, to a minimum of approximately +40‰ at the end of the incubation, as CO 2 released during the respiration of unlabeled glucose or gluta mic acid slowly diluted the 13 C-label.Biomass harvested from each incubation bottle amounted to 50-to 100-mg dry weight.

Fatty acid distributions
The fatty acid composition of all strains were similar, except for YL, which contained only four different fatty acids (Fig. 2).The fatty acids C 16:0 , C 18:0 , C 18:1 , and C 18:2 accounted for about 80%-100% of the total lipid content when grown on glucose.The fatty acids C 16:0 and C 18:2 accounted for 80%-94% of the total lipid content when grown on glutamic acid.Higher fatty acid diversity was found for fungi grown on glucose versus those grown on glutamic acid.
The inorganic C incorporation into fungal lipids (Fig. 3) was calculated after Equation 1.
The fatty acid composition for all strains grown on glucose (A) and glutamic acid (B), and determined by conventional GC-flame ionization detector.A slightly higher diversity of fatty acids was found when grown on glucose with the dominant fatty acids C 16:0 , C 18:0 , C 18:1 and C 18:2 accounting for more than 80%.
The dominant fatty acids when grown on glutamic acid were C 16:0 and C 18:2 , accounting for more than 80% of total fatty acid content.Equation 1 Inorganic carbon (IC) assimilation was calculated as the difference in the fraction of 13 C [F 13C = ( 13 C) / ( 13 C + 12 C)] of the lipids harvested from the labeling experiment compared to the natural (control), normalized by the difference in F 13C of the DIC measured at the end of the incubation and the F 13C of the substrate.F was calculated as F 13 C = (R 13C/12C ) / (R 13C/12C + 1), where R is calculated from the δ 13 C ratios as measured with the IRMS equipment using the reverse of the δ notations (δ 13 C = ([ 13 C/ 12 C] sample / [ 13 C/ 12 C] ref − 1) × 1,000 [modified after references (28,32)].

DISCUSSION
We have generated complementary data sets of inorganic C and water H incorporation into fungal lipids using two different approaches.The interpretation of these estimates in the sections below are based on data determined via classical GC analysis, following extraction, purification, and transesterification of lipids as described in Lipid Extraction and Chemical Preparation in Materials and Methods and thus comparable with previous studies that have used this approach [e.g., see references (22,26,33)].We further compare these values with those derived from direct analysis of fungal biomass via pyrolysis-GC (cf.Pyrolysis GC in Materials and Methods), as this approach may promote higher throughput for lipid analyses and thereby may help to constrain inorganic C and water H assimilation factors across more species and carbon substrates.

Inorganic C assimilation into fungal lipids
CO 2 assimilation by heterotrophic organisms is reported to be a measure of both anabolic processes and the catabolic status of the cell, with assimilation, anaplerotic, biosynthesis, and redox balancing reactions playing important roles to ensure the provision of energy and to replenish intermediates in the TCA cycle that have been released for biosynthesis (16,34,35).The by-fixation of inorganic C via anaplerotic pathways typically varies between 1% and 8% of the biomass C, depending on central metabolic pathways, and could further increase to assimilate C substrates that are more reduced than the biomass, as various carboxylase enzymes are employed to match the redox state of the substrate with that of the biomass (16).Whereas inorganic C assimilation by fungi was previously reported to amount to roughly 1%, based primarily on radiocarbon uptake into whole cells (20,36,37), our results suggest that 13 C-labeled inorganic C accounted for 2%-12% of C assimilated into fungal membrane lipid biomarkers, when grown on minimum medium with glucose or glutamic acid as the main C source.Both glucose and glutamic acid are slightly more reduced [degree of reduction (DOR) = 4.0 and 3.6, respectively (38)] than expected for biomass [DOR = 4.25 (19)], suggesting that assimilatory carboxylation should not contribute as strongly as anaplerotic or biosynthesis reactions to inorganic C by-fixation during growth on these substrates.Our findings indicate that inorganic 13 C incorporation into the C 18:2 biomarker was slightly lower during growth on glucose substrate (4.7% ± 1.6%) versus glutamic acid (7.4% ± 3.7%, Fig. 3), which may have involved more carboxylation reactions to replenish central metabolites.For example, during growth on glutamic acid, relatively more carboxylase reactions may be required to replenish acetyl-CoA, thereby increasing the conduit for by-fixation of inorganic C. Consistent with this notion, growth on glutamic acid was slower than glucose across fungal species (Fig. 1).
In contrast to the observations for C 18:2 , inorganic 13 C incorporation into ergosterol was similar for both substrates (6.9% ± 3.3% and 6.8% ± 2.5%, Fig. 3).The different patterns of inorganic C incorporation into C 18:2 versus ergosterol are likely due to their different biosynthetic pathways, where both products start from acetyl-CoA.For fatty acids, acetyl-CoA molecules are merged to malonyl-CoA, and other acetyl-CoA molecules are added repeatedly until the desired chain length for the fatty acid is attained.Ergosterol is synthesized from squalene (C 30 ), which is produced by merging C 5 intermediates of the mevalonate pathway (e.g., isopentenyl-pyrophosphate).Each C 5 unit of squalene stems from the merger of three acetyl-CoA molecules in a decarbox ylation reaction.Squalene further undergoes cyclization to form lanosterol, the main precursor for sterols, through the subsequent removal of three methyl groups (39,40).Before the final ergosterol molecule is produced, another methyl group is added to the backbone (39,40).The removal and/or replacement of acetyl-CoA-derived C during ergosterol biosynthesis may thus make this biomarker less sensitive than fatty acids to the inorganic 13 C incorporation via assimilation or anaplerotic pathways.

Water hydrogen incorporation into fungal lipids
Previous studies have demonstrated that the regression slope between hydrogen isotopes of water medium and microbial lipids (i.e., α W ) varies with the type of metabo lism used during growth, and different organisms may exhibit similar fractionation or α W values when grown on the same substrate (26,41,42).Variability in fatty acid hydrogen isotopic composition is suggested to be a function of the isotope effects of hydrogen transporters and electron acceptors (NADPH and NADH), as they are the main portals for hydrogen incorporation in FA biosynthesis pathways (accounting for around 50%), with the remaining coming directly from environmental water (~25%) and acetyl-CoA (~25%) (24,26,42).In the current study, the water-derived hydrogen contribution to membrane lipids was consistent among fungal species grown on the same substrate and similar to the patterns observed for inorganic C incorporation, with the average α W value of the C 18:2 fatty acid biomarker harvested from glucose incubations being significantly lower than glutamic acid incubations (0.62 ± 0.04 versus 0.90 ± 0.16, P < 0.005).This finding indicates that the central metabolism of glutamic acid resulted in a higher contribution of H from environmental water to C 18:2 .This finding may be explained by the metabolic pathway, where glutamic acid is converted to α-ketoglutarate by transamination, which leads to an exchange of functional groups between the amino acid and the carboxyl acid as well as the addition of water molecules and the involvement of NADP and NADPH before entering the TCA cycle as α-ketoglutarate (43).The contribution of H from the glutamic acid substrate to fatty acids would thereby be lower and replaced by water-derived H, explaining the higher α W value.For ergosterol, α W values for glucose and glutamic acid incubations were not significantly different (0.64 ± 0.13 and 0.68 ± 0.08, respectively), suggesting that ergosterol is less sensitive to H contributions from water in the growth medium.For the synthesis of ergosterol from squalene, at least 10 intermediate steps are needed, which include several steps involving desaturase enzymes (44) which remove hydrogen atoms from the molecule.This might explain the insensitive response of ergosterol to the different metabolic pathways presented in this study.In the context of previously reported α W values for the three domains of life, fungal lipids are more comparable to those of bacteria (0.4-0.9) (24) than those of eukaryotes (0.7-1.2), although only phyto-and zooplankton α W values are reported so far for that domain [see reference (45) and references within].

Dual-SIP approach
Dual-SIP experiments with deuterated water (HDO) and 13 C-DIC have been applied to distinguish autotrophic versus heterotrophic modes of C assimilation for pure cultures of bacteria and archaea (26,27) and have also been used to track microbial activity in marine sediments (21,33,46,47).The main objective of this study was to develop the dual-SIP assay to determine inorganic 13 C ( 13 IC) and 2 H incorporation into fungal lipid biomarkers (ratio 13 IC:α W ), which could be further applied to more comprehensively explore the patterns derived from different functional and/or taxonomic groups of fungi.The average 13 IC:α W ratios for C 18:2 were 0.076 ± 0.026 and 0.11 ± 0.057 for glucose and glutamic acid, respectively.This first evidence defines distinct spaces for fungal growth on the different substrates in the ratio plot (Fig. 6) and supports the use of the dual-SIP assay to distinguish the different metabolic modes employed by fungi for growth on sugars versus amino acids.For ergosterol, the 13 IC and α W values determined for both carbon sources could not be distinguished in the ratio plot (Fig. 6), suggesting poor potential for ergosterol to serve as a biomarker of metabolic mode.More data and studies of additional taxa and C substrates are needed to fully exploit the potential of this approach.

Pyr-GC-MS/IRMS
An additional objective of this study was to test the potential of using Pyr-GC-IRMS to determine the isotopic composition of the fungal biomarker C 18:2 and ergosterol for a rapid analysis that eliminates time-and solvent-consuming wet chemistry steps.The application of Pyr-GC-MS for the analysis of whole cells was previously reported for fatty acid quantification (48), which relies on thermal-assisted hydrolysis and methylation via a derivatization agent to enhance detection.In the current study, we used trimethylsulfo nium hydroxide (TMSH) as a derivatization agent because it was reported that it does not cause any isomerization and/or degradation as observed for other TMH solutions (49,50).
For the dual-SIP experiments selected for both analyses, the patterns of 13 IC incorporation and α W values determined by the conventional GC-IRMS protocol were generally redundant with results from the Pyr-GC-IRMS analysis, with the exception of α W values calculated for ergosterol (Fig. 5; Table S2).The average IC incorporation determined for ergosterol was consistent between traditional GC-IRMS and Pyr-GC-IRMS protocols, and while the incorporation trends were consistent, 13 IC incorporation into C 18:2 determined via the traditional GC analyses was mostly higher than their pyrolysis counterparts.This may be due to the improved chromatographic separation between C 18:1 and C 18:2 fatty acid methyl esters (FAMEs) achieved by the longer 120-m column employed for traditional GC-IRMS analyses and/or the greater number of replicates analyzed via this protocol.α W values calculated from the Pyr-GC-IRMS for ergosterol FIG 6 IC and α W value ratio plots for C 18:2 (A) and ergosterol (B).The power of the dual-SIP approach for fungal biomarkers shows distinguished groupings for glucose (yellow) versus glutamic acid (red) for C 18:2 , whereas these groups overlap for ergosterol.IC, inorganic carbon.
are inconsistent with their GC-IRMS counterparts and might be due to the lower signal for ergosterol detected via Pyr-GC-IRMS (Fig. 7) compared to the single, typically large peak observed with traditional GC-IRMS (not shown).Overall, our findings suggest that the Pyr-GC-IRMS protocol for determining stable C and H isotopic composition of C 18:2 represents a robust and fast alternative to the conventional GC-IRMS analysis.
The advantage of the Pyr-GC-IRMS approach was the tandem measurement of fatty acids and ergosterol in the same analytical injection (Fig. 7), which reduced the time of analysis but required high pyrolysis and GC temperatures for optimal detection (51).On the other hand, Pyr-GC analyses required the use of a shorter separation column (30 m) and thus poorer peak resolution because the pyrolysis unit could not withstand the higher backpressure of the 120-m column used for optimal separation of FAMEs.Nevertheless, the IL60 column achieved decent baseline resolution for C 18:2 and allowed for detection of ergosterol in the same injection and negligible interference of other compounds in fungal biomass samples, owing to the high maximum opera tion temperature for this column (300°C), which is a limitation for most polar columns (maximum temperature around 260°C-280°C).

Conclusion and future perspective
The purpose of this work was to apply the dual-SIP approach for determining heterotro phic CO 2 fixation and water H assimilation into fungal lipid biomarkers when grown on labile substrates (glucose and glutamic acid) available in soil systems.Our findings suggest that fungi solicit distinguishable isotope effects during the incorporation of 13 C-DIC and HDO into their membrane lipids when growing on glucose versus glutamic acid as the C substrate.We could show that the heterotrophic inorganic C incorporation into fungal biomarkers was within the expected range of 2%-8% ( 35), and we report for the first time the water H assimilation factor values for fungal biomarkers, which range between 0.58 and 1.15 and are broadly consistent with α W values reported for Separation of FAMEs for the fungal biomarker C 18:2 was successfully achieved with both methods.Advantage of the Pyr-GC method includes the measurement of both FAMEs and ergosterol in the dry biomass, whereas for the conventional method, sterol and FAME analyses are pre-extracted and analyzed separately.TLE, total lipid extract.heterotrophic bacteria.Ergosterol was less informative as a fungal biomarker in these studies in comparison to the more distinguishable signals exhibited by C 18:2 ; however, it may serve as a potential marker for a distinction between labile and more stable (recalcitrant) C sources.Future application of this dual-SIP assay to explore fungal activity in environmental settings has the advantages that both isotope labels can be added to environmental incubations with minimal alteration of the natural habitat; fungal lipid production can be estimated by HDO incorporation alone; and the 13 IC/α W ratio can inform which C substrates support their growth.Environmental case studies applying the dual-SIP assay described here should also choose an appropriate label dose for each isotope: 2 / 1 H-water label strength should be commensurate with the expected production of the biomarker over the fossil pool and may range up to H 2 O values of +8,000‰; similarly, the dose of 13 / 12 C-DIC must consider background DIC levels and heterotrophic production during the incubation.For this study, estimates of 13 C incorporation into fungal lipids were calculated based on 13 F-DIC values at the end of the incubation.Furthermore, maintaining 13 C-enrichment of the DIC pool also requires a closed headspace and especially requires that oxygen levels are monitored during aerobic, dual-SIP incubations.
Additional growth experiments that more comprehensively define 13 IC:α W ratios imparted during growth on more stable forms of soil organic carbon (e.g., lignin) across a larger diversity of fungal taxa are needed to better constrain the interpretive ability of isotope fractionation signals and evaluate the potential of this approach for environmen tal studies.This effort will be supported by the applicability of pyrolysis coupled with GC-MS and IRMS for direct analysis of fungal biomass demonstrated in this study.

Cultivation and harvesting
Pure cultures of microscopic fungi (Table S1), AD strain BCCO 20_2442, CF strain BCCO 20_1579, PJ strain BCCO 20_0265, AP strain BCCO 20_1588, AB strain CCM 8222, and one yeast species YL strain MUCL 054012 were incubated at 25°C in the dark in precombusted 2-L Schott bottles containing 50 mL of a autoclaved modified minimum media Czapek-Dox broth (30).The fungal inocula were prepared using sterile ultrapure water for sampling the spores, which were washed and centrifuged with sterile MilliQ water three times before being inoculated (1 mL) with approximately 10 6 spores/mL under a biological safety cabinet using sterile utensils and solutions.The growth medium contained per liter: 4-g organic C source (C 6 H 12 O 6 , glucose and C 5 H 9 NO 4 , glutamic acid, tested separately); 3-g NaNO 3 ; 1-g K 2 HPO 4 ; 0.5-g MgSO 4 ; 0.5-g KCl; 0.01-g FeSO 4 ; and 0.09-g NaHCO 3 .The pH was adjusted to 7.3.Dual-SIP experiments were performed using 13 C-bicarbonate ( 13 C-DIC and NaH 13 CO 3 ) and D 2 O according to Table 1, which were added to the medium after 0.2-µm sterile filtration.Each fungal species was grown in triplicate with non-labeled substrates (treatment 1), with δ 2 H of the medium water adjusted to (i) 100‰ δ 2 H and 10% of 13 C-DIC (treatment 2), (ii) 200‰ δ 2 H and 10% 13 C -DIC (treatment 3), and (iii) 400‰ δ 2 H and 10% 13 C-DIC (treatment 4).The Schott bottles were kept closed to prevent the labeled 13 C-DIC from outgassing.Headspace samples for CO 2 analysis (2 mL) were collected every 2-3 days from each bottle into helium-flushed (10 min at 100 mL/min) 12-mL exetainer vials (Exetainer; Labco Limited, UK) to monitor fungal growth and ensure O 2 availability.For the experiments with the species AD and CF growing on

Treatment
Replicates δ 2 H of water (label strength) δ 13 C-DIC (label strength) glucose, 1-L bottles were used, and neither CO 2 concentration nor isotopic composition were measured for AD.At the end of the experiment, the mycelia were filtered from the growth medium using an autoclaved sterile miracloth placed on a sterile funnel on a sterile 50-mL falcon tube.The mycelia on the miracloth were washed three times with sterile MilliQ water, then the mycelia were dried by first placing the miracloth on dry paper, then transferring to pre-weighed 50-mL falcon tubes.The biomass of the fresh (wet) weight was recorded, and samples were stored at −20°C until lyophilization, after which the dry weight of each sample was determined and stored at −20°C until further analysis.

Growth monitoring
Headspace CO 2 of fungal incubations (1 mL) were measured for CO 2 concentration on a Hewlett Packard HP 5890 Series II GC (Agilent Technologies, Inc, Santa Clara, USA) coupled with a thermal conductivity detector (TCD) equipped with a 30-m HP-PLOT/Q (polystyrene-divinylbenzene, 0.53-mm ID, 0.40-µm df; Agilent Technologies, Inc).The detector temperature was set to 130°C; the oven was set to 70°C; and the system was operated with helium at a flow rate of 1 mL/min.

Bligh and Dyer
Lyophilized fungal biomass was extracted using a modified Bligh and Dyer protocol (52).Samples were sonicated for 10 min in four steps with a mixture of dichlorome thane (DCM)-methanol (MeOH)-water (1.0:2.0:0.8,vol/vol/vol) by using 30-mL solvent per extraction step.A phosphate buffer (8.7-g/L KH 2 PO 4 , pH 7.4) was used for the first two steps, and a trichloroacetic acid buffer (50-g/L CCl 3 COOH, pH 2) was used for the final two steps.After each extraction step, the samples were centrifuged at 1,250 rpm for 5 min, and the supernatants were collected in a pre-combusted separation funnel.Phase separation was induced by addition of DCM and MilliQ, and the organic phase was drawn off and collected in a pre-combusted Schott bottle.The aqueous phase was washed three more times with DCM, and then the pooled organic phase was washed three more times with de-ionized MilliQ water.Finally, the organic phase was collected as the total lipid extract (TLE) and was evaporated under a stream of argon and stored at −20°C.All vials used for sample handling and storage were pre-combusted at 450°C for 6 h, and syringes for sample transfer were pre-rinsed with DCM:MeOH (5:1 vol/vol).

Saponification
Thirty percent of the TLE was dried and saponified for fatty acid analysis, using 1 mL 6% KOH in MeOH for 3 h at 80°C (53).After cooling, 1 mL of 0.05 KCl was added.Neutral lipids were extracted four times using 1-mL n-hexane.The rest of the solution was treated with 10% HCl to reach a pH value of 1, and then free fatty acids were extracted four times using 1-mL n-hexane.Following that, all extracts were dried, mixed with 1-mL 20% BF 3 in MeOH and heated at 70°C for 1 h to convert free fatty acids into FAMEs.The solutions were extracted four times with 1-mL n-hexane.The combined extracts were evaporated under argon and stored at −20°C until further analysis.

HDO isotope analysis
Liquid samples were transferred into 1.5-mL glass vials (32 × 11.6 mm, Fisher Scientific) and then measured using Triple Liquid Water Isotope Analyzer (Los Gatos Research), which is based on the principle of high-resolution laser absorption spectroscopy.Samples were dispensed into the instrument using an autosampler (PAL3 LSI, ABB company) and a 1.2-µL syringe (Hamilton).Samples were measured and evaluated against prepared laboratory standards of known isotopic composition.
The isotopic ratios of these laboratory standards were verified by measuring against international standards (VSMOW2 and SLAP2).To check the quality control, the measurements of the samples were also interspersed with periodic measurements of the prepared verification samples with known isotopic composition.The final isotopic composition (δ 2 H) was determined using LIMS software.Analytical error in case of δ 2 H was <1.5‰.

Substrate analysis
Substrates (~100 µg) were weighed into tin capsules (8 × 5 mm; Sercon, Crewe, UK) and placed in a helium-flushed carousel autosampler, then introduced to an Elemental Analyzer IsoLink IRMS system (EA IsoLink; Thermo Scientific, Bremen, Germany) equipped with a CHN/NC/N EA combustion/reduction reactor (Sercon) heated to 1,020°C.A pulse of oxygen was introduced to the reactor simultaneously with the sample.The sample gases were quantified via a TCD and then introduced to a MAT 253 Plus IRMS (Thermo Scientific) via the open split of a Conflo IV interface, with helium as the carrier gas.The isotopic composition was determined using Isodat version 3.0 software against the corresponding CO 2 working gas (−4.191‰ for δ 13 C), and the values were corrected for linearity and normalized to the Vienna Pee Dee Belemnite (VPDB) scale using an international IAEA-600 (−27.771‰ for δ 13 C) standard.The analytical error was <0.04‰.

DIC and CO 2 isotopic analyses
Medium water samples (2 mL) or CO 2 headspace (2 mL) gas was injected into a heliumflushed 12-mL exetainer vial (Exetainer, Labco Limited).Water samples were acidified with 0.1-mL 85% H 3 PO 4 and left to equilibrate for at least 24 h before measurement.Headspace CO 2 was purged using a double-holed needle with helium into a 250-µL sample loop then separated via a Carboxen PLOT 1010 (0.53-mm ID; Supelco, Bellefonte, USA) held at 70°C with a flow rate of 0.75 bar and then introduced into the MAT253 Plus IRMS (Thermo Scientific) via a Conflo IV interface.Each sample was injected three times during one analysis.The isotopic composition was determined using Isodat version 3.0 software against the corresponding CO 2 working gas (−4.191‰ for δ 13 C).The values were corrected for linearity and normalized to the VPDB scale using internal house standards and international standards IAEA-603 and IAEA-303A (2.46‰ and 93.3‰ for δ 13 C, respectively).The analytical error was <0.2‰.

FAME analysis
FAMEs were measured using a GC Trace1310 gas chromatograph equipped with an SSL injector, a double gooseneck Topaz splittless liner (Restek, Bellefonte, USA) and a TR-FAME column (70% cyanopropyl polysilphenylene-siloxane; 120 m, 0.25-mm ID, 0.25-µm df; Thermo Scientific).The samples were injected in splitless mode, with the injector temperature at 310°C.The GC oven was held at 60°C for 1 min, then ramped to 150°C at 10°C/min and to 250°C at 4°C/min, then held at 250°C for 15 min.Helium was used as carrier gas with a constant flow of 1.5 mL/min.The column was coupled via a multichannel device splitting the flow to a flame ionization detector (FID) and a mass spectrometer (MS), enabling both peak quantification and identification from one injection.The FID was set to 300°C with flows of air at 350 mL/min, H 2 at 35 mL/min, and N 2 makeup gas at 40 mL/min.The MS (ISQ QD, Thermo Scientific) ion source was set to electron impact ionization (EI) mode at 70 eV and a scan range of m/z 50-500 with a scan time of 0.2 s.The transfer line temperature was set to 280°C, and the ion source was set to 230°C.The scan was started 15 min after the injection to avoid ionization of the solvent peak in the MS.
For FAME identification, the fragmentation patterns and mass spectra were compared to the National Institute of Standards and Technology (NIST) Spectral Library.Quantification was determined from the FID trace, according to the response of the internal standard (2-methyloctadecanoic acid) and following Equation 2: The final concentration is calculated with PeakArea being the integrated area of the unknown compound, PeakSTD being the integrated area of the given standard, and STD as the known concentration of the standard in nanogram, and the percentage of the injected TLE and the mass of the extracted sample in gram (52).
Stable carbon and hydrogen isotope compositions of FAMEs were determined by splitting the flow from the GC column to a GC-Isolink II reactor, coupled with a MAT253 Plus IRMS via a Conflo IV interface; values are expressed in standard delta notation (δ 13 C and δ 2 H).MS information was simultaneously acquired by using the multichannel device described above.For conversion of FAMEs and ergosterol to CO 2 , the combustion reactor (nickel oxide tube with CuO, NiO, and Pt wires) was set to 1,000°C.For conversion of FAMEs and ergosterol to H 2 , the pyrolysis reactor (aluminum tube) was set to 1,420°C.FAMEs were identified by their retention times and fragmentation patterns.The isotopic composition was determined using Isodat version 3.0 software against the correspond ing CO 2 or H 2 working gas (−4.191‰ for δ 13 C and −239.5‰ for δ 2 H).Isotope correc tions for instrument drifts, linearity, and normalization to the VPDB or Vienna standard mean ocean water (VSMOW)/standard light Antarctic precipitation (SLAP) scales were performed according to the response of USGS70 (−30.53‰ for δ 13 C, −183.9‰ for δ 2 H) and USGS72 (−1.54‰ for δ 13 C and 348.3‰ for δ 2 H) reference standards.The analytical error was <0.5‰ and <3.1‰ for δ 13 C and δ 2 H, respectively.

Pyrolysis GC
The pyrolysis unit Shimadzu 3030D (Shimadzu, Kyoto, Japan/Frontier Laboratories, Fukushima, Japan) was installed on top of the GC Trace1310 gas chromatograph SSL injector (Thermo Scientific), and the GC was equipped with an SLB-IL60 column [non-bonded; 1,12-di(tripropylphosphonium)dodecane bis(trifluoromethanesulfonyl)imide phase, 30 m, 0.25-mm ID, 0.20-µm df; Supelco].Due to the high restriction and thus backpressure from the 120-m FAME column, a different shorter analytical column (30 m) was used with the pyrolysis injector, which resulted in co-elution of C 18:0 and C 18:1 peaks (Fig. 1).The furnace temperature was set to 650°C, and the interface temperature was set to 370°C.The injector temperature was set to 360°C, and the GC oven was held at 80°C for 1 min then ramped to 175°C at 15°C/min, then ramped to 195°C at 2°C/min and then ramped to 300°C at 10°C/min and held for 7 min at 300°C.Helium was used as carrier gas with a constant flow of 1.5 mL/min and a split flow of 40 mL/min.The column was coupled via a multichannel device to enable simultaneous FID and MS acquisition from one injection.The GC-MS (ISQ QD, Thermo Scientific) ion source was set to EI mode at 70 eV and a scan range of m/z 50-500 with a scan time of 0.2 s was applied.MS scanning started after 8 min to avoid ionization of the solvent peak in the MS.Transfer line temperature was set to 300°C, and the ion source was set to 250°C.The samples (lyophilized biomass, 80 µg-1.5 mg) were weighed into an ultraclean stainless steel Eco-Cup LF (Frontier Laboratories), which were burned with a torch before usage to remove contaminants.FAMEs and ergosterol were detected in the same analytical run.Immediately prior to the measurement, 10-40 µL of TMSH was added on the sample to increase the volatization of the FAME and ergosterol and to improve measurement sensitivity.Identification was performed using fragmentation patterns and the NIST 14 library.Quantification was performed with a calibration regression (0.5-400.0 ng) using an external standard (nonadecanoic acid, C 19 ) for FAMEs and ergosterol (5-20 ng) for ergosterol.The same setup and method were also used to acquire stable carbon and hydrogen isotope composition of FAMEs and ergosterol via a GC-Isolink II device, as explained above.The column flow was split via a multichannel device to acquire MS and isotopic data simultaneously.The analytical error was <0.6‰ and <3.3‰ for δ 13 C and δ 2 H, respectively.

FIG 1
FIG 1 Headspace CO 2 concentrations for the fungal incubation grown on glucose (A) and glutamic acid (B) over time with their respective δ 13 C-CO 2 values in the lower panels.Incubation was stopped when CO 2 concentrations in the headspace reached 4 -6% (v/v), which is indicative that roughly half of the carbon substrate was respired, and based on a mol-to-mol comparison, ensures the incubation was still under aerobic conditions.AB, Aspergillus brasiliensis; AD, Aspergillus dimorphicus; AP, Acremonium polychromum; CF, Cordyceps farinosa; PJ, Penicillium janczewskii; YL, Yarrowia lipolytica.

FIG 4
FIG 4 The water hydrogen assimilation factor (α W values) estimated as the slope of the fractional 2/1 H abundance ( 2 F × 10 3 ) in lipids (y-axis) versus medium water (x-axis).Data are shown for fungal biomarkers C 18:2 (A and B) and ergosterol (C and D) extracted from the different fungal species grown on glucose (A and C) or glutamic acid (B and D) using the conventional GC-IRMS method (see Lipid Extraction and Chemical Preparation in Materials and Methods).AB, Aspergillus brasiliensis; AD, Aspergillus dimorphicus; AP, Acremonium polychromum; CF, Cordyceps farinosa; PJ, Penicillium janczewskii; YL, Yarrowia lipolytica.

FIG 7
FIG 7 Typical flame ionization detector chromatogram of a Cordyceps farinosa FAME/lipid measurement with conventional GC-MS (top) and Pyr-GC-MS (bottom).

TABLE 1
Incubation setup of fungal species for SIP experiments with13C-bicarbonate ( 13 C-DIC) and deuterated water (D 2 O)