Stable carbon isotope fractionation as tracer of carbon cycling in anoxic soil ecosystems

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ScienceDirect Stable carbon isotope fractionation as tracer of carbon cycling in anoxic soil ecosystems Martin Blaser and Ralf Conrad While the structure of microbial communities can nowadays be determined by applying molecular analytical tools to soil samples, microbial function can usually only be determined by physiological experiments requiring incubation of samples. However, analysis of stable isotope fractionation might be able to analyse microbial function without incubation in soil samples. We describe the limitations of diagnosing and quantifying carbon flux pathways in soil by using the determination of stable carbon isotope composition in soil compounds and emphasize the importance of determining stable isotope fractionation factors for defined biochemical pathways. Fractionation factors are sufficiently different for some central biochemical pathways in anaerobic degradation of organic carbon. Thus, it is possible to quantify the relative contribution of CH4 production by hydrogenotrophic or aceticlastic methanogenic pathways, and of acetate formation by chemolithotrophic (acetyl-CoA synthase) or heterotrophic (fermentation) pathways. In addition, stable isotope analysis may allow the differentiation between different organic substrates used for degradation, for example, the relative contribution of root exudation versus soil organic matter degradation, provided the different substrates are sufficiently distinct in their isotopic compositions (e.g., mixture of C3 and C4 plants) and the carbon conversion pathways display only small fractionation factors or are identical for the different substrates. Address Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany Corresponding author: Blaser, Martin ([email protected])

Current Opinion in Biotechnology 2016, 41:122–129 This review comes from a themed issue on Analytical biotechnology Edited by Hans-Hermann Richnow and Tillmann Lueders

http://dx.doi.org/10.1016/j.copbio.2016.07.001 0958-1669/# 2016 Elsevier Ltd. All rights reserved.

Introduction Carbon cycling in soil ecosystems is mainly the result of input of carbon substrates by plants and degradation by soil microorganisms. The input by plants is mostly in the form of litter, dead plant material or root exudates. The Current Opinion in Biotechnology 2016, 41:122–129

degradation, however, is achieved by a complex soil microbial community comprising many different biochemical pathways, which depend on the chemical nature of the organic substrate and the environmental conditions (Figure 1). In general, the anaerobic degradation of soil organic matter can be separated in the hydrolysis and fermentation of polymeric substrates to short-chain fatty acids, alcohols, CO2 and H2. Further fermentation results in degradation to acetate, CO2 and H2, which are the dominant precursors of methanogenesis. In anaerobic systems, CH4 and CO2 are stable end products. Oxidation of CH4 usually requires the presence of O2 and methane oxidizing bacteria. Understanding the carbon cycling in soil ecosystems therefore requires knowledge of both the structure and the function of the microbial communities. There has recently been much progress in elucidating the structure of microbial communities by applying molecular analytical tools, in particular sequencing of microbial genes and gene products [1]. These analyses just require sampling of soil and — the sometimes technically demanding — extraction of nucleic acids or proteins. While some information on microbial functioning can be obtained by combining genomic and metaproteomic approaches [2–4] or using stable isotope probing techniques [5,6], the in situ functions of the microbial communities usually can only be analyzed by incubation and measurement of the temporal change of analytically accessible variables. However, analysis of stable isotope signatures in soil samples might overcome this problem, since the isotopic signatures partially reflect the microbial functioning [7]. Our review intends to explore the limits of using stable carbon isotopic signatures for elucidating the microbial functional pathways of organic carbon degradation in soil, anaerobic degradation of small organic molecules originating from hydrolysis and fermentation, and methane production in particular.

Isotopic signatures, isotope effects, and fractionation factors The 13C isotopic signature of a particular carbon compound is given by its ratio R = 13C/12C and is usually denoted relative to a standard (st) as d13C = 103 (R/ Rst  1) [8,9]. The reactions in a biochemical pathway, especially those involving the cleavage of carbon bonds, often display characteristic fractionation factors (a) or enrichment factors (e = 103[1  a]), which define the extent to which the carbon atoms have been fractionated during the conversion of a substrate to a product. For a reaction (or pathway) A ! B, the fractionation factor is defined as aA,B = (d13CA + 103)/(d13CB + 103) [10]. The www.sciencedirect.com

Stable isotope fractionation in soil carbon cycling Blaser and Conrad 123

Figure 1

C4 plant (mais)

C3 plant (rice) δ13CCO2= –8‰

CO2

assimilation

assimilation

δ13CC3= –25‰

δ13CCH4= –47‰

CO2

δ13CC4= –15‰

CH4

Water

Oxic zone Anoxic zone

acetate, H2/CO2

CO2 fermentation

Soil organic matter

respiration

Current Opinion in Biotechnology

Carbon cycling in soil shown for a flooded ecosystem planted with a C3 plant (i.e. rice) for a non-flooded ecosystem planted with a C4 plant (e.g., maize). The CO2 of the atmosphere is fixed by either RUBISCO (C3) or PEP-carboxylase (C4) resulting in different d13C values of the plant material. Dead plant material, roots and root exudates are the major sources of soil organic matter, which can either be respired to CO2 in oxic soils or fermented to CH4 and CO2 in anoxic soils. Details on the carbon isotope fractionation during anaerobic degradation to CH4 can be found in Figure 2. Delta13C are taken from Refs. [15,90,91].

fractionation factors thus quantify how much a given biochemical reaction (or pathway) discriminates against the substrate molecules containing the heavy isotope 13C. The discrimination itself is caused by the isotope effect. This is either a kinetic isotope effect (KIE) caused by reaction rate constants being larger for substrate with 12C than 13C, or an equilibrium isotope effect (EIE) that is involved in the establishment of chemical equilibria, such as dissociations, protonations and may either prefer 13C or 12 C [11–13]. Biochemical reactions involving carbon often display a KIE, which usually discriminates against the heavy 13C isotope, so that the d13C of the product is always lower than that of the substrate. Biochemical pathways usually consist of a sequence of reactions which may influence the overall fractionation factor to various extents. In many cases the initial reaction has great implications for the overall fractionation in the pathway [14–17]. A special case is branched biochemical pathways leading to more than one product (e.g., A ! B + C). Here, the fractionation factor aA,B is often different from aA,C. Moreover, one branch may discriminate against www.sciencedirect.com

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C while the other discriminates against 13C, although the KIE of all the individual reactions are generally positive [18,19]. Hence, branched pathways may (but not obligatorily) result in a product with d13C larger than that of the substrate, if the other branch results in a product with correspondingly lower d13C fulfilling isotopic mass balance. When using the isotopic signature in microbial biomass for elucidating the pathway by which it has been formed, one is always confronted with the problem of a branched pathway with a catabolic and an anabolic branch, respectively. In summary, stable isotopic fractionation is a general feature of (bio)chemical reactions. If two pathways display sufficiently different fractionation factors, reflected in the difference of d13C between substrate and product these pathways can be differentiated using stable carbon isotope measurements.

Determination of fractionation factors for microbial pathways The compound specific stable isotope analysis (CSIA) of d13C usually requires an isotope ratio mass spectrometer (IRMS), which measures the d13C in CO2. If a particular Current Opinion in Biotechnology 2016, 41:122–129

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carbon compound can be isolated (e.g., by chromatography) and converted to CO2 (e.g., by combustion), the average d13C of all its C atoms can be determined usually using a GC–IRMS or HPLC–IRMS system. For some simple compounds (e.g., CO2, CH4), the d13C can also be determined using laser-based absorption spectrometry, for example, cavity ringdown spectrometers, which allow continuous, non-destructive operation in contrast to IRMS, which can only analyze discrete samples. For reviews on recent instrumental developments see [20,21]. As the fractionation factors are of pivotal importance for the detection of different pathways, knowledge of their magnitudes is mandatory. However, determination of the fractionation factor for a particular pathway requires that there is no interference by other pathways, no restriction by small-scale mass transfer processes or by the so-called commitment to catalysis favoring a non-equilibrium mass flow according to the direction of the pathway taking place inside the microbial cell [16,19,22]. This requirement is usually not met when incubating environmental samples in which an a priori unknown number of pathways may contribute to a particular substrate–product conversion. Fractionation factors have to be determined under well defined conditions, which are usually only met by assaying biochemical reactions or defined microbial cultures in which the desired pathway operates. Then, fractionation factors can be determined either in a closed system (e.g., batch culture) during the course of substrate depletion and product accumulation using the Rayleigh equation for the fractional ( f) turnover of the substrate into the product [23,24]; for the substrate: dr = dri + e[ln(1  f)] and the product: dp = dri  e(1  f)[ln(1  f)]/f, where dri is the initial isotope composition of the reactant, dr and dp the isotope composition of the substrate and product at the instant when f was determined. In an open system (e.g., chemostat) under steady state conditions the isotope fractionation can be directly deduced from the difference in d13C of substrate and product [18,24]; for the substrate: dr = dri + e(1  f) and for the product: dp = dri  ef. This can be simplified to: e = dr  dp. Even in a defined microbial culture the catabolic path of substrate conversion is not completely isolated, as anabolic substrate conversion is taking place simultaneously (i.e., branched pathway). Since the energy yield is usually low (<2 ATP per substrate consumed) in anaerobic metabolism, anabolism contributes accordingly little (usually less than 10%) to total anaerobic substrate consumption. Nevertheless, fractionation during anaerobic anabolism is happening: In methanogenic archaea, fractionation factors during biomass production range from 31 to +7 % [25–28]. Catabolic CH4 production, on the other hand, shows fractionation factors between 25 and 69% for methanogenesis from CO2 [29–32], 73 to Current Opinion in Biotechnology 2016, 41:122–129

83% from methanol [27,28], and 7 to 35% for methanogenesis from acetate [25,27,32–34], respectively. The bias caused by interfering anabolism can be overcome by incubating microbial cells under defined nongrowing conditions (e.g., absence of a nitrogen source). There are many conceptual and practical problems when determining fractionation factors in microbial cultures. While the strongest fractionation reported for hydrogenotrophic methanogenic archaea was achieved under energy limitation, weaker fractionation factors were determined for energy rich conditions [31,32]; temperature had no effect on the fractionation [35]. By contrast the fractionation of acetogenic bacteria was not influenced by changing the energy supply (H2:CO2 ratios) but was influenced by media composition [36] and substrate usage [37]. Additional constraints on the fractionation due to substrate transport across membranes [14,38], cell density [39,40], availability of co-substrates and electron acceptors [22,41– 43] or commitment to catalysis [15,20] have been described in the literature. Likewise the fractionation factors of microbial cultures show strain-specific variability resulting in slightly different fractionation factors for each strain [25,27,28,32,34,44]. For the anaerobic degradation of acetate by methanogenic archaea, sulfur and sulfate reducers it could be shown that not only the pathway usage (acetylCoA pathway or reversed TCA) but likewise the enzyme responsible for acetate activation (acetate kinase vs. acetyl-CoA synthase) impact the overall fractionation of acetate to the respective products [45,46]. For example, the acetoclastic methanogens can be differentiated into members of the Methanosaetaceae which use the acetyl-CoA synthase, as an activating enzyme and showed a relative small isotope fractionation of 7 to 9% [32,34]; while the second group of acetoclastic methanogens the Methanosarcinaceae usually use the acetate kinase to activate the acetate and can use several substrates in addition to acetate. They have a stronger fractionation of 12 to 35% [25,27,33]. As a consequence the fractionation factors reported for microbial pure cultures have to be interpreted in the context of their detailed biochemical pathways and also in the context of the experimental conditions. Even identical conditions may give a relatively large variability of the fractionation factors. For example, 21 independent replicates of the acetogen T. kivui covered a range of eTIC = 63 to 47% (average, 54%) [44]. Such uncertainties have several consequences: For application to environmental samples a range of possible fractionation factors rather than a constant value should be used. Likewise the interpretation of fractionation factors deduced from environmental samples is uncertain in accordance to the ranges observed in pure cultures. Currently, stable isotope fractionation can safely be used to differentiate very strongly fractionating pathways. While investigating more microbial pure cultures [28,35,36, 44,47] and further constraining the environmental variables www.sciencedirect.com

Stable isotope fractionation in soil carbon cycling Blaser and Conrad 125

influencing the fractionation factors may help to make differentiation of pathways more accurate, another possibility is applying the fractionation of several stable isotopes together, for example, 13C with 2H or 18O [48–50]. These techniques are very valuable in resolving, for example, the anaerobic degradation of organic pollutants where the apparent fractionation factors of the individual isotopes are comparatively small or may be masked by other effects, for example, commitment to catalysis [16,17,22,32,50,51]. Recent application of these combined (clumped) carbon and hydrogen isotope analysis on environmental methane, could show that the microbially derived methane does not follow the thermodynamic equilibrium of abiotically formed methane [52,53,54,55]. However, so far no clumped isotope measurements for microbial pure cultures are available. The data of hydrogen isotope analysis in methanogenic cultures is very limited and restricted to thermophilic hydrogenotrophic methanogens [32,56,57]. The isotopic values of microbial pure cultures can be used to interpret environmental signals. Under certain circumstances, fractionation factors may also be determined directly in soil incubations. However, such determination requires incubation experiments and works only if the operation of microbial pathways can be isolated in situ, for example, by inhibiting alternative pathways. For example, the fractionation factor for hydrogenotrophic methanogenesis can be determined by incubation of soil samples in the presence of CH3F, which specifically inhibits aceticlastic methanogenesis [58,59]. However, this method falls short if CH4 is in addition produced by methylotrophic methanogenesis, which is not inhibited by CH3F [28].

Diagnosis and quantification of carbon flux pathways in soil The measurement of d13C of individual soil carbon compounds may allow the diagnosis of particular biochemical pathways that operated until the time of sampling [60–62]. The usual approach is defining theoretically possible C flux paths that can be differentiated by their fractionation factors and testing them by d13C analysis of their substrates and products. The crucial point is that the pathways to be discriminated must display sufficiently different fractionation factors, reflected in the difference of d13C between substrate and product. Ecological studies sometimes have used tiny differences in the carbon and nitrogen isotopic values for interpretation of underlying food chains using context knowledge (reviewed in [63,64]). In microbial ecology, on the other hand, the underlying biochemical pathways are usually much harder to resolve. We consider two aspects of carbon flux in soil, (i) diagnosis and quantification of the relative contribution of different substrates to the formation of a product; www.sciencedirect.com

and (ii) diagnosis and quantification of the relative contribution of different biochemical pathways to substrate conversion and product formation. (i) Soil organic matter usually originates from biomass that is produced by CO2 fixation. Isotope fractionation is relatively strong, depending on the pathway of CO2 fixation. For example, the pathway of CO2 fixation by C4 plants has a smaller fractionation factor than that of C3 plants resulting in organic carbon that is less depleted in 13C [15,65] (Figures 1 and 2). The isotopic signature of biomass is relatively well preserved in organic matter, since subsequent degradation by respiratory processes displays only small fractionation [66–69]. The same is true for microbial fermentation, which displays only a small fractionation factor [67,70,71,72,73,74]. Therefore, different respiration or fermentation pathways cannot be distinguished using carbon isotope signals alone. However, it is possible to determine the contribution of different substrates to CO2 production provided the d13C of the substrates is sufficiently different. For example, the contribution of the anaerobic degradation of root versus soil organic matter to CO2 production can be quantified, if crops are changed from C3 to C4 plants or vice versa [66,68,69]. Then, soil organic matter has still the typical 13C signature while plant material has a C4 signature. Similarly, this approach can also be used for quantifying the contribution of root exudation versus degradation of soil organic matter (or straw carbon) to production of other compounds, such as microbial biomass, acetate or CH4 [66,75–78,79]. For example, it was shown that most of the CH4 produced in flooded rice field soil is derived from root exudation [77,78]. In case of aerobic degradation to CO2 with negligible isotope fractionation the fraction of C3 ( fC3,CO2) and C4 ( fC4,CO2) organic carbon contributing to total CO2 production is calculated by the following mass balance: d13 CCO2 ¼ d13 C3 f C3 ;CO2 þ ð1f C3 ;CO2 Þd13 CC4 : For the anaerobic degradation to CH4, we have to assume that the overall enrichment factor ðeorg;CH4 Þ for the conversion of organic carbon to CH4 is the same for C3 and C4 organic carbon. This can be written as the following equation: d13 CCH4 ¼ d13 C3 f C3 ;CO2 þ ð1f C3 ;CO2 Þd13 CC4 þ eorg;CH4 : The enrichment factor eorg;CH4 can be calculated from factors in the literature enrichment ði:e:; eCO2 ;CH4 ; eac;CH4 Þ if the path of CH4 production is known: eorg;CH4 ¼ eCO2 ;CH4 f CO2 ; CH4 þ ð1f CO2 ; CH4 Þeac;CH4 : Current Opinion in Biotechnology 2016, 41:122–129

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The exact quantification of the relative contribution of different substrates to the degradation process requires that the degradation process has a small fractionation factor or is identical for each substrate. Therefore, application to anaerobic degradation processes is challenging, since large fractionation factors are involved in the conversion of CO2 to acetate or CH4. Furthermore, it can a priori not be excluded that the degradation process is (at least to some extent) different for different substrates (e.g., root exudates versus soil organic matter). (ii) While many respiration or fermentation pathways have only a small discrimination against 13C; there are several anaerobic degradation processes, which display large fractionation factors (up to e = 80% reported for methylotrophic methanogens [27,28]) that can be used for diagnosis and quantification of biochemical pathways (Figure 2). Examples are acetate formation by chemolithotrophic acetogenic bacteria using CO2 as carbon substrate. This biochemical pathway exhibits a very large carbon fractionation (e = 68 to 38%) factor [44] allowing to determine whether acetate has been formed from CO2 or from organic compounds [80–82]. Similarly, CH4 production from CO2 displays a much larger fractionation factor than from acetate (see above), so that the path of CH4 production in anoxic environments can be differentiated [75,83–85]. If soil organic matter is anaerobically degraded to CH4 and CO2, fermentation first produces CO2 and acetate displaying little isotopic fractionation, so that these intermediary products have a similar d13C than the organic matter itself [70,72,74]. Their further conversion, however, results in fractionation with CH4 produced from CO2 being much more depleted in 13C than that from acetate [25,27,32,34]. The residual CO2 and

acetate, on the other hand, become then correspondingly enriched in 13C. These processes cannot only be used to diagnose the occurrence of different pathways, but can even be used to quantify their contributions, provided their fractionation factors are known and the d13C in the substrates (CO2, acetate) and products (CH4) can be measured [75,83–85]. For example, for calculation of the fractions of hydrogenotrophic ðf CO2 ;CH4 Þ and aceticlastic ðf ac;CH4 Þ methanogenesis of total CH4 production it is necessary to determine the d13C in the CH4, CO2 and the methyl group of acetate. With these data the following mass balance equations can be used for calculation:

Figure 2

d13 Cac ¼ d13 CCO2 þ eCO2 ;ac

–8‰ CO2

C3 C4

–25‰ 25‰ Org. g C

15‰ –15‰

<3‰ <–5‰

–55‰

–50‰2

CO2

CH4

–80‰

methanol Current Opinion in Biotechnology

Scheme of carbon flow and stable carbon isotope enrichment factors (e) in methanogenic environments. In grey typical values for C3 and C4 plants as well as atmospheric CO2 are given. The isotope enrichment factors give the rounded average of pure culture experiments; details on the variability are given in the text. 1Fractionation factor is given for pure cultures of Methanosaetacea; the fractionation of Methanosarcinacea is on average 24%. 2The fractionation of hydrogenotrophic methanogenesis is highly flexible and best determined for the respective environment using CH3F as inhibitor for acetoclastic methanogenesis. Current Opinion in Biotechnology 2016, 41:122–129

d13 Cmc ¼ d13 CCO2 þ eCO2 ;CH4 d13 Cma ¼ d13 Cacmethyl þ eac;CH4 f CO2 ;CH4 þ f ac;CH4 ¼ 1 eCO2 ;CH4 is the isotopic enrichment factor for the hydrogenotrophic formation of CH4 from CO2 (e.g., 50%), and eac;CH4 that for the aceticlastic formation of CH4 from the methyl group of acetate (e.g., 8%). If d13Cac-methyl cannot be determined, it may be approximated from either the d13C of total acetate (d13Cac): d13Cac-methyl  d13Cac  8% or of the d13C of organic C (d13Corg) d13Cac  d13Corg  2% [72]. Analogously, fractions of acetate produced from chemolithotrophic ðf CO2 ;ac Þ and fermentative ( forg,ac) acetogenesis can be calculated by the following mass balance, if d13C of acetate, CO2 and organic C have been measured. d13 Cac ¼ d13 Cac f CO2 ;ac þ ð1f CO2 ;ac Þd13 Cao

d13 Cao ¼ d13 Corg þ eorg;ac

–8‰1

acetate

d13 CCH4 ¼ d13 Cmc f CO2 ;CH4 þ ð1f CO2 ;CH4 Þd13 Cma

f CO2 ;ac þ f org;ac ¼ 1 where eCO2 ;ac is the isotopic enrichment factor for the chemolithotrophic formation of acetate from CO2 via the acetyl-CoA pathway (e.g., 55%), and eac,CH4 that for the fermentative formation of acetate from organic substrate (e.g., 2%).

Conclusions and future perspectives Stable isotope fractionation can be useful for distinguishing different carbon flux pathways provided the ranges covered by the fractionation factors associated with the individual pathways do not overlap. This is nicely the case for anaerobic processes involved in the degradation of organic matter, thus allowing the differentiation and even quantification of methanogenesis by hydrogenotrophic or aceticlastic www.sciencedirect.com

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pathways, and of acetogenesis by chemolithotrophic (acetyl-CoA synthase) or heterotrophic (fermentation) pathways. So far many pathways incorporating C1 compounds have the tendency to strongly discriminate against 13C. Hence other pathways involving C1 compounds are the interest of current (e.g., aerobic or anaerobic methane oxidation [86,87]) and future research projects. In addition, stable isotope analysis allows distinguishing between different substrates used for degradation, for example, quantifying the relative contribution of one versus the other substrate in the degradation process. However, such distinction is only possible if the different substrates are sufficiently distinct in their isotopic compositions and if the carbon conversion pathways display only small fractionation factors or are identical for the different substrates. For both applications the knowledge of the fractionation factor is of crucial importance. More studies on effectors influencing the range of fractionation factors in microbial pure cultures are therefore of great importance. To further resolve the underlying principles and pinpoint the fractionating steps of a pathway and possible influencing factors more biochemical investigations on isolated enzyme systems would also be welcome. In addition new analytical methods, such as cavity ring down spectroscopy, which increase the speed and the ease of the analysis, or clumped isotope techniques, which increase the resolution of isotope effects, will shape the future of carbon isotope studies in soil environments. At the time being, isotopic composition of microbial metabolites and microbial biomass is only measured in relatively bulky samples. Hence, interpretation of the isotope values is integrating over relatively large scales. This is unfortunate, since the microbial populations and the individual microbial cells operate on a microscale. Ideally, one would like to trace carbon utilization on a cellular scale. NanoSims technology allows such measurements, but only after labeling with stable isotopes [88]. The natural abundance can presently not be assessed on a cellular scale with great precision [89]. Less ambitious, but still very desirable, would be the analysis of isotopic values along small gradients, for example, vertical profiles of in CH4, TOC, and acetate in a sediment or along a plant root. Gas extraction for analysis of d13C has to make sure that the sampling procedure itself does not cause fractionation.

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