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Determination of Methanogenic Pathways through Carbon Isotope (δ13C) Analysis for the Two-Stage Anaerobic Digestion of High-Solids Substrates Tito Gehring,*,† Johanna Klang,‡ Andrea Niedermayr,§ Stephan Berzio,† Adrian Immenhauser,§ Michael Klocke,‡ Marc Wichern,† and Manfred Lübken† †

Institute of Urban Water Management and Environmental Engineering, Ruhr-Universität Bochum, Universitätsstrasse 150, Bochum 44801, Germany ‡ Leibniz Institute for Agricultural Engineering Potsdam-Bornim (ATB), Max-Eyth-Allee 100, Potsdam 14469, Germany § Institute of Geology, Mineralogy and Geophysics, Ruhr-Universität Bochum, Universitätsstrasse 150, Bochum 44801, Germany S Supporting Information *

ABSTRACT: This study used carbon isotope (δ13C)-based calculations to quantify the specific methanogenic pathways in a two-stage experimental biogas plant composed of three thermophilic leach bed reactors (51−56 °C) followed by a mesophilic (36.5 °C) anaerobic filter. Despite the continuous dominance of the acetoclastic Methanosaeta in the anaerobic filter, the methane (CH4) fraction derived from carbon dioxide reduction (CO2), f mc, varied significantly over the investigation period of 200 days. At organic loading rates (OLRs) below 6.0 gCOD L−1d−1, the average f mc value was 33%, whereas at higher OLRs, with a maximum level of 17.0 gCOD L−1d−1, the f mc values reached 47%. The experiments allowed for a clear differentiation of the isotope fractionation related to the formation and consumption of acetate in both stages of the plant. Our data indicate constant carbon isotope fractionation for acetate formation at different OLRs within the thermophilic leach bed reactors as well as a negligible contribution of homoacetogenesis. These results present the first quantification of methanogenic pathway (f mc values) dynamics for a continually operated mesophilic bioreactor and highlight the enormous potential of δ13C analysis for a more comprehensive understanding of the anaerobic degradation processes in CH4producing biogas plants.

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INTRODUCTION Biological methane production is the final degradation step of organic matter in a wide range of anaerobic habitats and accounts for the conversion of approximately 2% of the total carbon fixed through photosynthesis.1 Anaerobic technologies based on similar anaerobic consortia as these natural ecosystems can utilize organic substrates for methane production.2 Municipal, agricultural, and many other types of industrial organic wastes are ideal substrates for biomethanation plants that combine waste treatment and energy recovery.3,4 Methanogenesis, the last step in the conversion of these complex substrates to methane, results mainly from the acetate cleavage (acetoclastic pathway) and carbon dioxide reduction with hydrogen (hydrogenotrophic pathway).5 The pioneering studies of Jeris and McCarty6 and Smith and Mah7 indicated that the acetoclastic pathway accounts for approximately twothirds of the methane production in sewage sludge digesters. Most of the current anaerobic digestion models are in line with these findings.8 However, there is an increasing amount of evidence indicating that depending on the substrate characteristics, hydrogenotrophic rather than acetoclastic methanogenesis is the dominant pathway.9−11 This pathway determi© 2015 American Chemical Society

nation has important implications for the design and operation of biomethanation plants, as acetoclastic and hydrogenotrophic methanogens’ growth kinetics differ significantly.12 Nevertheless, the quantification of the methanogenic pathways in bioreactors remains a challenge. Molecular methods allow for the identification of the dominant methanogenic Archaea population13,14 but are neither sufficient for quantifying the specific metabolic activity from hydrogenotrophic and acetoclastic methanogens in a mixed community, nor capable of tracking variations on a short time scale. Accurate and dynamic quantification is possible through methods based on the analysis of carbon isotopes, including (i) tracer experiments with the addition of labeled substrates, 14C or 13C isotopes and (ii) calculations from the depletion of stable heavy carbon isotopes, 13C, in methane against its precursors, acetate or carbon dioxide. Although the latter method requires a high number of parameters,15 only raw Received: Revised: Accepted: Published: 4705

November 19, 2014 February 20, 2015 March 5, 2015 March 5, 2015 DOI: 10.1021/es505665z Environ. Sci. Technol. 2015, 49, 4705−4714

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Environmental Science & Technology

Figure 1. Schematic of the two-stage experimental plant and the different recirculation flows, a, b, c, and d (see SI Table S1 for more detailed information on the flow rates).

which was operated at room temperature. The LBR temperatures were maintained between 51 and 56 °C, and the temperature of both the UAF and CSTR was 36.5 °C. A sieve plate with 1.5 mm-diameter bores followed by a 40 mm-thick filter mat (30 dpi) retained the particulate material of the LBR. The leachate from the STR was pumped into the LBR with a ceramic centrifugal pump and percolated back by gravity flow. The internal recirculation rate was controlled by the OLR to approximately 100 L gVSadded−1 d−1. The LBRs were operated in batch-fed mode with a solid retention time (SRT) of 21 days, with an LBR being fed every seventh day. A portion of digestate mass, equivalent to 20% of the new substrate charge, was retained in each cycle. A three-channel peristaltic pump recirculated 4.4 L d−1 from the STRUAF to each LBR semicontinually (three cycles per hour), and the exceeding liquid from the three LBRs overflowed back into the STRUAF. Figure 1 presents all of the reactors and recirculation flows in detail. In the overflow connection from the thermophilic STRs to the STRUAF, small openings to the atmosphere with diameter of ∼1 mm were foreseen to allow methane to be degassed from the leachate, thus minimizing the transport of dissolved methane from the STRs to the UAF. This thermophilicoriginated methane could potentially interfere with stable carbon signatures measurements from the UAF. A constant recirculation of 50 mL min−1 was maintained between the UAF and STRUAF with a peristaltic pump. The UAF was designed to allow for a simple biofilm carriersampling strategy to regularly characterize the sessile biomass. A total of 50 polyvinyl chloride (PVC) stripes (395 × 18 × 3 mm3) were used as biofilm carriers, resulting in a surface area of 0.83 m2. These stripes were fixed in a radial disposition from the top of the reactor and could be separately removed without disturbing the remaining biomass of the system. Figure S1 in the Supporting Information (SI) presents a picture of the biofilm carrier support system. The CSTR was operated at a constant hydraulic retention time of 15 days. The CSTR was fed 6 days per week by pumping 600 mL of leachate from the LBR-STRs, and the effluent was returned to the UAF-LBR

gas samples are necessary without interference with the investigated system (e.g., addition of specific substrates or batch cultivations). Therefore, this method is potentially applicable to both laboratory reactors and full-scale plants. Although still a novel approach to analyze engineered systems, methanogenic pathway determination through the analysis of stable isotopes has already been reported for a wide range of anaerobic ecosystems, including crop field soils, boreal peatlands and lake sediments.15 In this study, we evaluate this isotope-based method to determine the acetoclastic and hydrogenotrophic methanogenesis dynamics for an upflow anaerobic filter (UAF) over an experimental duration of 200 days. The UAF was continually fed with leachate from three leach bed reactors (LBRs) degrading high-solid organic substrates (total solids content over 32%). The two-stage plant was operated under increasing organic loading rates (OLRs). In order to guarantee a constant δ13C in the leachate, we used the same substrate throughout the entire experiment. Maize silage (Zea mays) was chosen as the model substrate. In addition, parallel to the UAF, a control continuously stirred tank reactor (CSTR) was fed with leachate from the same LBRs at constant low OLRs. The control CSTR acted as a blank reactor for carbon isotope measurements and served as an indicator of possible variations of isotopic composition from the inflow leachate.

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MATERIALS AND METHODS Experimental Two-Stage Plant. The plant consisted of three thermophilic leach bed reactors followed by a mesophilic anaerobic filter reactor and a mesophilic control CSTR (Figure 1). Each of the LBRs was connected directly to a storage tank reactor (STR), comprising three pairs of reactors: LBR01STR01, LBR02-STR02, and LBR03-STR03. A fourth storage tank reactor, STRUAF, was used for UAF internal recirculation and for the interface between the LBRs and UAF systems. Glass reactors with an inner diameter of approximately 200 mm and a total volume of 13 ± 1 L were utilized, all of which were double jacketed to control temperature, with the exception of STRUAF, 4706

DOI: 10.1021/es505665z Environ. Sci. Technol. 2015, 49, 4705−4714

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Table 1. Organic Loading Rates (OLRs) for the Leach Bed Reactors (LBRs) and the Upflow Anaerobic Filter (UAF) at the Stages S1, S2, and S3a parameter

unit

OLR-S1

OLR-S2

duration

weeks

11

11

OLR LBRs OLR UAF

−1 −1

gVS L d gCOD L−1d−1 gCOD L−1d−1

OLR-S3 6

min.

max.

min.

max.

min.

max

0.7 1.0 1.7b

1.4 2.0 3.7

1.6 2.3 2.0b

2.1 3.0 5.9

2.3 3.2 5.7b

2.7 3.9 17.0

a

Values given on basis of the inflow chemical oxygen demand (COD) and volatile solids (VS). bMinimal values after the three LBRs were fed with the maximal OLR of the specific OLR stage.

Figure 2. Weekly dynamics for the LBRs and UAF during OLR-S1 (white bars and circles), OLR-S2 (gray bars and squares), and OLR-S3 (black bars and triangles). Error bars indicate 95% confidence intervals (n ≥ 4). On the first day of the week, the LBRs were fed. (a) daily leachate VFA concentrations (inflow to the UAF); (b) average leachate VFA (ace: acetate, pro: propionate, but: butyrate, val: valerate, and cap: caproate) distribution in COD equivalents; (c) daily methane production rates summed for the three LBRs; and (d) daily methane production rates of the UAF.

samples pipes were positioned approximately 1 m above the reactors, as depicted in SI Figure S2. Physical and Chemical Analysis. Total and volatile solids (TS and VS, respectively), total ammonia nitrogen (NH4N), chemical oxygen demand (COD) and volatile fatty acids (VFA) were regularly measured for the leachate and UAF and CSTR effluent samples (the analytical methods are described in the SI). Stable Carbon Isotope Analysis. Biogas isotopic ratios were determined through gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMC) analysis. The carbon dioxide (CO2) and methane (CH4) from the gas samples (30−50 μL) were separated in a Trace GC Ultra (Thermo Scientific, Germany) fitted with a CP Pora Plot Q column (length: 27.5 m, internal diameter: 0.32 mm) at a constant temperature of 60 °C. CH4 oxidation to CO2 was carried out in a GC Combustion III (Thermo Scientific,

system. The liquid volumes as well as the solids and hydraulic retention times (SRT and HRT) of all reactors are provided in SI Table S1. On average, approximately 1-L samples were taken per week and were replaced with tap water, resulting in an HRT of 380 days for the entire system. Details about the operation of the LBRs when applying three OLR stages, OLR-S1, OLR-S2, and OLR-S3, are provided in Table 1. Gas production and composition were individually determined for each LBR-STR pair, as for the UAF and CSTR. The gas flows were measured through a Milligascounters MGC-1 (Ritter, Germany), and the gas composition was measured using an Awiflex gas analyzer (Awite, Germany). Accumulated gas in the headspace of the STRUAF was collected in a 10-L gas bag. The composition and volume were determined at weekly intervals. In the UAF and CSTR, 500 mL glass sample pipes with a PTFE septum allowed for the transport of the gas samples for further stable carbon isotope analysis. The gas 4707


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Environmental Science & Technology Germany) at 960 °C. An IRMS Delta S (Thermo Finnigan, Germany) was used to determine the 13C/12C ratios of the CO2 gas. All measurements were conducted at least three times. Acetate carbon isotope ratios in the leachate were determined at the Max Planck Institute (MPI) for Terrestrial Microbiology, Marburg, Germany, as described by Conrad et al.16 Therefore, the samples were stored at −20 °C before analysis. Maize isotopic analyses were carried out at the Institute for Geology, Leibniz Universität Hannover, Germany. The maize silage samples were dried at 105 °C and milled prior to isotope analysis. The isotopic ratios were determined with an organic elemental analyzer (Thermo Scientific Flash 2000) connected online to a Thermo Scientific Delta V Advantage mass spectrometer. The isotope ratios are given as δ13C values per mil (‰) against the PeeDee Bemnite (PDB) standard. Microbiological Analysis. The quantification of copy numbers for the bacterial and archaeal 16S rRNA genes was carried out using a quantitative 5′-nuclease PCR assay (qPCR) as previously described by Yu et al.17 and Bergmann et al.18 Terminal restriction fragment length polymorphism (TRFLP) analyses were conducted to characterize the archaeal community structure in the mesophilic reactors. In order to identify the detected TRFs a 16S rRNA gene sequence libraries were constructed. according to Rademacher et al.19 DNAsequencing was performed by GATC Biotech AG (Germany). Representative sequences from all operational taxonomic units have been submitted to EMBL database under accession number LN624342−LN624363. The detailed analytical protocol for the microbiological analysis is provided in the SI. Quantification of Methanogenic Pathways. We determined the fraction of CH4 produced from CO2, f mc, by following the calculations proposed by Conrad.15 The definitions of the isotopic fractionation factors for the acetoclastic and hydrogenotrophic methanogenesis, αma and α mc , are discussed in detail below. In addition to f mc determination, we calculated the maximal hydrogen yields from the combined syntrophic volatile fatty acid (VFA) oxidation. The stoichiometry suggested by Batstone et al.20 for acetogenic processes defined the propionate (pro), butyrate (but), and valerate (val) oxidation products. The β-oxidation route was assumed for caproate (cap).6 Therefore, we could determine the maximal fraction of methane derived from hydrogen, f H2,VFA, according to these VFA oxidation reactions. The f mc and f H2,VFA calculation methods are described in detail in the SI. Model Substrate. During our experiments, two different charges of maize silage from the same crop field were utilized. The substrate was stored at −20 °C. Both charges had an average of 33.8% TS and 95.7% (of TS) VS. Analysis from the second maize silage charge indicated an elemental composition of C3.75H6.20O2.58N0.94S0.01 and δ13C values of −12.7 ± 0.3 ‰ PDB (SD for n = 3). This isotopic distribution is in accordance with typical 13C literature data for Zea mays.21

stage. Table 1 lists the minimum and maximum OLRs applied to the LBRs and the resulting OLR ranges of the UAF from leachate recirculation (see SI Figure S3 for a more detailed description of the OLR regime of the plant). An average VS degradation rate of 66 ± 9% (SD for n = 29) was determined for the three LBRs. The two-stage plant had a CH4 yield of 271 ± 25 L gVSadded−1 (SD for n = 29) over the three OLR stages, which represents 85% of the theoretical maximal CH4 yield based on stoichiometric calculations.4 The average weekly CH4 production for the UAF and for the sum of the three LBRs are shown in Figure 2, parts c and d, respectively. An LBR was fed at the first day of the weekly operational cycle (day 1 in Figure 2). The UAF CH4 production rates also include the gas collected from the STRUAF (∼10% of the total UAF CH4 production). In the LBRs, a small increase of CH4 production from 57 to 64 L week−1 occurred from OLR-S1 to OLR-S2. At OLR-S3, the CH4 production decreased substantially to 42 L week−1 in response to the lower pH values from the leachate (data not shown). In contrast, the CH4 production increase in the UAF at each OLR stage was high, from 13 L week−1 at OLR-S1 to 73 L week−1 at OLR-S3. The bell-shaped curve for CH4 production at OLR-S1 and OLR-S2 is not observed at OLR-S3 (Figure 2d), indicating that the UAF continually operated at its maximal CH4 production rates. In fact, after 3 weeks of operation at OLR-S3, a light acidification of the UAF reactor occurred, and the average pH value dropped to 6.9 for 21 days. Otherwise, the UAF pH values were continually between 7.2 and 7.4. The daily VFA leachate concentrations had a higher variation range than the CH4 production rates; hence, no confidence interval was determined. This large variation can be mainly explained through the discrete (daily) VFA sampling, in contrast to the online biogas measurements, which yield a more precise determination of LBR dynamic outflows. Nevertheless, the average CH4 production for OLR-S1 and OLR-S2 are in accordance with the average VFA loads. This correlation cannot be established adequately for OLR-S3 due to the UAF acidification, as the CH4 production is lower than expected considering the VFA loads. The increased acidifying conditions also led to higher variations for LBR CH4 production during OLR-S3 (Figure 2c). However, the COD distribution among the VFAs (acetate, propionate, butyrate, valerate, and caproate) have only small variations (Figure 2b). Acetate and butyrate together comprise more than 60% of the COD from the leachate VFA during the three OLRs, characterizing a clostridial butyric-type fermentation that is typical for silages.23 The NH4N concentrations and biogas composition of the UAF did not exhibit significant variations during the increasing OLRs. The NH4N concentrations increased from averages of 350 mg L−1 at OLR-S1 to 500 mg L−1 at OLR-S3 (SI Figure S3). The average CH4 contents of the UAF were high at 77 ± 3% (SD for 196 days), similar to the values reported for highrate anaerobic filters.22 Hydrogen partial pressures increased marginally in response to the increasingly high VFA concentrations. The average partial pressures were 22, 34, and 44 ppm for OLR-S1, OLR-S2, and OLR-S3, respectively (data not shown). During all OLR stages, the control CSTR was operated at low OLRs of below 0.7 gCOD L−1d−1, with maximal specific CH4 production rates of 0.3 L Lreactor−1d−1 (see SI Figure S4 for detailed data).

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RESULTS AND DISCUSSION VFA and Methane Production. The operation of batchfed LBRs combined with mesophilic reactors results in variable daily gas production rates and leachate characteristics that depend on the feeding intervals.22 In OLR-S1 and OLR-S2, the maximal leachate VFA concentrations were observed for the 2 days after a LBR feeding (Figure 2a). Thereafter, the VFA levels decreased until a new maize silage feeding, resulting in a reproducible weekly CH4 production pattern for each OLR 4708


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Figure 3. Methanogenic microbial community structure at the start of the experiment (S0) and at the end of the three OLR stages (S1, S2, and S3) for biofilm and sludge samples derived from the UAF and for samples derived from the control CSTR (at days 78 and 190), as determined by qPCR and TRFLP analysis. (a) Abundance of bacterial (gray bars) and archaeal (red bars) 16S rRNA gene copies per nanogram of microbial DNA. The mean value and standard deviation are given (n = 6; two biological replicates, i.e., DNA preparations, each with three technical replicates, i.e., qPCR analyses); (b) relative abundance of terminal restriction fragments (TRFs) for methanogenic Archaea, i.e., TRF-106bp, representative of Methanosaeta spp. (1), TRF-627bp, representative of Methanosarcina spp. (2), TRF-342bp, representative of Methanobacteriales (3), TRF-428bp, representative of Methanomicrobiales (4), and TRF-86bp, TRF-98bp, TRF-470bp, representative of unknown Archaea (5−7). The average value for three measurements is given (three technical replicates, i.e., TRFLP analyses). A value of 100% refers to the sum of fluorescence intensity of all TRFs with relative fluorescence intensity above 3% of the total fluorescence. TRFs with minor fluorescence were excluded from the analysis.

Figure 4. Measured δ13C for the biogas and calculated methanogenic pathway contributions for the control CSTR (n = 10) and UAF during OLR-S1 (n = 8), OLR-S2 (n = 8), and OLR-S3 (n = 14). The boxes indicate the first and third quartiles; the horizontal line indicates the median; the circles indicate the mean; and the whiskers indicate the minimal and maximal values. (a) δ13CCO2; (b) δ13CCH4; (c) Δ13C CO2‑CH4; (d) calculated f mc (black box plots); and f H2,VFA (gray box plots) values.

Methanogenic Archaea Community Structure. The bacterial and archaeal 16S rRNA gene copy numbers were determined by quantitative real-time PCR for samples from the both mesophilic reactors, UAF and CSTR (Figure 3a). Between 104 and 105 copies per nanogram of microbial DNA were found in the biofilm and sludge samples from the UAF. Referred to the total 16S rRNA gene copy number, the relative abundance of archaeal 16S rRNA gene copies was between 14% and 28%. These findings indicate a relative enrichment of methanogens in the UAF compared to the control CSTR, where Archaea accounted for only 1−2% of the total 16S rRNA gene copy

number, which corresponds to the same range reported in previous studies, e.g., for biofilms on PVC carriers.24 The relative abundance of methanogens remained rather stable in the UAF independent of the OLR. The archaeal community structure was characterized in detail with the fingerprinting method TRFLP in combination with a cloning/sequencing approach for TRF identification (Figure 3b). Seven representative TRFs for methanogenic Archaea from the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected. The most dominant TRF in all UAF sessile and suspended microbial biomass samples, i.e., 4709


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Figure 5. Weekly dynamics for the UAF during OLR-S2 (gray; first values in the x-axis) and OLR-S3 (black; second values in the x-axis). Error bars indicate standard errors (n ≥ 3). (a) δ13CCH4 measured data; (b) δ13CCO2 measured data; (c) δ13Cac‑methyl calculated from δ13Cac measured data or from the average δ13Cac (white fill); (d) calculated f mc (circles) and f H2,VFA (diamonds) values; (e) calculated specific acetoclastic methane production; and (f) calculated specific hydrogenotrophic methane production.

δ13C biogas data for the UAF at the three OLRs and for the control CSTR. During the complete carbon isotope measurement phase, a constant increase in δ13C from carbon dioxide (δ13CCO2) occurred at an approximate rate of 1.5‰ every 50 days (SI Figure S5). This increase was similar for the UAF and control CSTR reactor, indicating that the rise of δ13CCO2 values was due to variations in the leachate derived from the LBRs. The constant fractionation through methanogenic processes, in both LBRs and methanogenic reactors, is attributed to this continual rise in the δ13CCO2 values. Additionally, through the long leachate HRT (380 days for the entire plant) high amounts of dissolved inorganic carbon are retained within the system, enhancing the 13C accumulation, as bicarbonate is more enriched in 13C than the outgassing CO2.26 Due to this enrichment of δ13CCO2 isotopes, the apparent fractionation from CO2 to CH4 (Δ13CCO2‑CH4 = δ13CCO2 − δ13CCH4) is a more adequate parameter to compare the variations in isotope signatures. Furthermore, δ13CCH4 exhibited high sensitivity to changes in the maize silage charge during the eighth week at OLR-S1. The minimal δ13CCH4 values for the first substrate charge were approximately 2.0‰ lower than for the second

biofilm and sludge, was TRF-106bp, which was assigned to the acetoclastic genus Methanosaeta (order Methanosarcinales). These findings were supported by confocal laser scanning microscopy, as shown in the SI (Figure S1), where the presence of rod-shaped Methanosaeta is clearly observed. During the experiment, the relative abundance of Methanosaeta in the biofilm remained rather stable at values between 67 and 77%, whereas increasing values from 55 to 84% were recorded in the sludge. Minor amounts of hydrogenotrophic methanogens were detected in the UAF; TRF-342bp indicated the presence of members of order Methanobacteriales, whereas TRF-428bp was assigned to the order Methanomicrobiales. The total abundance of both hydrogenotrophic Archaea orders for the UAF samples varied from 7 to 11% in the biofilm and from 8 to 21% in the sludge samples. In addition, progressive biofilm growth was visually observed at the end of each OLR, reaching a biofilm thickness of approximately 1.2 mm at OLR-S3 (SI Figure S1). The low acetate concentrations in the UAF effluent of less than 150 mg L−1 (excluding the acidification period) and the low NH4N concentrations of less than 500 mg L−1 correspond well with the observed Methanosaeta dominance.25 Carbon Isotope Analysis and Methanogenic Pathway Calculation. Figure 4 provides an overview of the measured 4710


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Environmental Science & Technology charge, causing the higher variation range observed for δ13CCH4 during OLR-S1 (Figure 4b). Despite these variations in δ13CCO2 and δ13CCH4, the Δ13CCO2‑CH4 distributions were relatively similar for OLR-S1 and OLR-S2 (Figure 4c). At OLR-S3, the average Δ13CCO2‑CH4 increased above 6.0‰, indicating a potential change in the fractionation processes and thus in the methanogenic pathways. In the control reactor, the δ13CCH4 values increased nearly parallel to δ13CCO2, resulting in invariant Δ13C CO2‑CH4 values, with an average of 42.3‰ and a difference of only 0.9‰ between the first and third quartiles. Thus, aside from the increase in δ13CCO2, we can assume that the isotopic composition of the organic matter in the leachate was nearly constant. Additionally, δ13C values for acetate, δ13Cac, were determined for leachate samples. The calculation of f mc values proceeded with each set of measured δ13CCO2, δ13CCH4 and δ13Cac data. The δ13C for methyl-acetate was determined assuming the following intramolecular acetate isotopic distribution: δ13Cac‑methyl = δ13C ac − 10.0‰.27 This value corresponds well with the reported δ13Cac and δ13Cac‑methyl differences of −9.2‰ for ethanol-derived acetate28 and of −11.4‰ for glucose-derived acetate.29 We assigned the average δ13Cac‑methyl value of −6.9‰ to calculate f mc in cases in which δ13Cac was not measured. In the UAF, according to TRF identification, no distinction between suspended and sessile biomass was necessary. The acetoclastic activity was assumed to be exclusive to the Methanosaeta genera, as Methanosarcina abundance was always extremely low (Figure 3a). This distinction is important, as the acetoclastic stable carbon isotope fractionation, αma, through Methanosaeta is considerably lower than that for Methanosarcina.30 In our calculations, we adopted an αma of 1.010 according to reported values for Methanosaeta.31 Reported values for the hydrogenotrophic methanogenesis fractionation factor, αmc, varies between 1.031 to 1.077 (i.e., Δmc = 31−77‰), presenting a wider range than for acetoclastic methanogenesis fractionation.15 Indeed, αmc depends on the Gibbs free energies (ΔG) available in the methanogenic environment.32 High free energy availability (low ΔG values) facilitates CO2 activation and results in a reduced fractionation (low αmc); hence, αmc increases under energetically limited environments. Unfortunately, the possibility of an exact calculation of ΔG for hydrogenotrophic methanogenesis based on H2 partial pressures (pH2) measured in the UAF headspace is restricted. First, due to mass transfer limitations, pH2 cannot be used to calculate the dissolved hydrogen concentrations.33 Second, in the biofilm, ideal conditions for direct interspecies hydrogen transfer are available,34 and hydrogen may be consumed before reaching the bulk liquid. In fact, the constant and low hydrogen partial pressures in the UAF are characteristics of an obligate syntrophic growth of methanogens and VFA degraders.34 Thus, a high carbon isotopic fractionation is implied, as this syntrophic growth leaves minimal amounts of energy for the methanogen partner.32,35 Hence, a high αmc value of 1.070 was assumed. The distributions of the calculated f mc values for each OLR in the UAF and for the control CSTR are shown in Figure 4d, together with the corresponding fH2,VFA values. The results from both methods differ significantly, with higher hydrogenotrophic methanogenesis contributions determined through the isotope-

based approach. The lower f H2,VFA at OLR-S3 are likely a result of the previously mentioned UAF acidification, which increased the acetate concentrations in the system. Short-Term Dynamics of the Methanogenic Pathways. Figure 5 depicts the results from daily carbon isotope analysis for two exemplary weeks from OLR-S2 and OLR-S3 and the corresponding calculated methanogenic pathways (other relevant measured data for both weeks are depicted in SI Figure S6). For f mc calculations, in addition to the standard errors of the δ13 C biogas measurements, a δ13C ac‑methyl uncertainty range of ±1.0‰ was considered. The weekly variations in the f mc values were approximately 10% at all OLRs, as depicted for both exemplary weeks (Figure 4d). The determined f H2,VFA exhibited no direct correlation with f mc. The resulting specific CH4 production rates for both methanogenic pathways, acetoclastic (QCH4,ma = QCH4 × (1 − f mc)) and hydrogenotrophic (QCH4,mc = QCH4 × f mc), are depicted in Figure 5, parts e and f. During experimental days 99 and 190, an LBR was fed; the following day, a decrease in δ13CCH4 of approximately 2.0‰ in both curves was observed (Figure 5a), similar to previous observations for C4 plant silage fermentation.36,37 In both experimental weeks, the highest δ13Cac‑methyl values were recorded immediately before a new feeding (first day of the week; Figure 5c). A significant δ13Cac‑methyl decrease accompanying the drop in δ13CCH4 was observed after feeding. The discrimination of light substrates in acetoclastic methanogenesis leads to 13C enrichment of the acetate pool. Thus, δ13Cac‑methyl increases when the acetate consumption is higher than the acetate load, which potentially explains the higher δ13Cac‑methyl at days 99 and 190, where the VFA production rate of the LBRs is the lowest of the week. Moreover, the αma fractionation factor diminishes under low acetate concentrations.30 Hence, the f mc value at day 99 (Figure 5d), where the inflow acetate concentration was below 0.1 gCOD L −1 , may be overestimated. This low acetate concentration effects on α ma , likely influenced the f mc calculations in the control CSTR, which was maintained continually at low OLRs and thus with low VFA concentrations. Moreover, lower VFA concentrations can potentially lead to higher ΔG values (i.e., higher αmc); actually, a higher Δ13CCO2‑CH4 average value was found in the CSTR than in the UAF at OLR-S1 and OLR-S2 (Figure 4c). Therefore, we omit the control reactor in further discussions of methanogenic pathway variations. Carbon Isotope Fractionation for Thermophilic Acetate Formation. According to the δ13C measured data shown here, homoacetogenic acetate formation in the LBRs can be discarded. The average δ13Cac measured in the leachate was +3.1‰, and the homoacetogenic CO2 fractionation to acetate (αCO2,ac values of 1.040 to 1.068)38 would lead to δ13Cac ranging from −52 to −27‰. Moreover, this average enrichment of 15.8‰ in acetate formation (δ13Cmaize = −12.7‰) is considerably higher than the acetate enrichment reported for fermentation processes in pure cultures, in the range of 0 to 3‰.39 The mechanisms of this high enrichment are not investigated here, nor are other published data for acetogenic isotope effects in anaerobic digesters, to the best of our knowledge, available in the open literature at present. However, the invariant Δ13CCO2‑CH4 values determined in the control 4711


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OLR-S2, the inflow VFA was completely degraded and correlated well with the CH4 production rates. Nevertheless, on average, stoichiometric calculations of hydrogenotrophic methanogenesis, f H2,VFA, were approximately 10−15% lower than the values derived from isotope fractionation (Figure 4d). An inadequacy of the chosen fractionation factors, αma and αmc, could lead to an over- or underestimation of f mc; a detailed discussion of the sensitivity of both parameters can be found elsewhere.31,44 Nevertheless, results of an uncertainty analysis indicate that the f H2,VFA yields are always below the f mc values for a wide range of fractionation parameters (SI Figure S7). Thus, a partial degradation of acetate through syntrophic oxidizing bacteria45 can explain the discrepancies between the two calculation methods. Although Methanosaeta dominance may be indicative of an absence of acetate oxidation,10 anaerobic filters are regarded as a favorable environment for the slow growing syntrophic acetate-oxidizing communities.46 There is evidence that an analysis of fractionation in the acetate-carboxyl group may allow for a more direct differentiation between the acetate oxidation and acetoclastic pathways.47 Implications for Further Studies. The utilization of carbon isotope analysis in biogas plants to determine the dominant methanogenic pathways and to indicate process imbalance has been reported.36,37,48,49 There are also examples of methanogenic pathway quantifications in engineered anaerobic systems;40,41,50,51 nonetheless, to the best of our knowledge, this paper reports the first long-term study confirming the reliability of f mc calculations in a biomethanation plant. The f mc determination allows for a more adequate estimation of the kinetics of acetoclastic and hydrogenotrophic methanogens, which are fundamental for design and operation of anaerobic plants. Additionally, the investigation performed for a two-stage plant allows for a clear differentiation of isotope fractionations related to acetate formation (in the LBRs) and consumption (in the UAF). Another important aspect in the system investigated here is the constant low hydrogen partial pressure in the UAF. In single-stage biogas plants, the hydrogen partial pressures can vary over a wider range,52 which may imply variations in αmc.32 Hence, in further studies considering the methanogenic pathways for biomethanation plants, the determination of αmc values through acetoclastic methanogenesis inhibition15 may be required. An increase in these stable isotope-based methods can be expected with respect to the development of simple analytical methods. The online measurements of CH4 and CO2 stable carbon isotopes in biogas plants through near-infrared laser optical spectrometry has already been demonstrated.53 Hence, the calculation of anaerobic degradation pathways through stable isotope measurements is a striking innovation to complement the current monitoring parameters for anaerobic digestion processes in biomethanation plants.

CSTR present a preliminary line of evidence of constant fractionation in thermophilic reactors at different OLRs. A better understanding of δ13Cac during VFA formation in bioreactors is essential for further anaerobic pathway determination through carbon isotope analysis. Dependence of Methanogenic Pathways on the OLR. The determined f mc values are consistent with the high abundance of the acetoclastic methanogen Methanosaeta. At OLR-S1 and OLR-S2, the contribution of hydrogenotrophic methanogenesis was responsible for 28−37% (the variation range gives the first and third quartile limits in Figure 4d) of the total CH4 production in the UAF. Hence, for OLRs between 1.7 and 5.9 gCOD L−1 d−1, the methanogenic pathway distribution in the UAF was in the same range as reported for sewage sludge digesters.6,7 During OLR-S3, the hydrogenotrophic contribution increased to 39−44% (Figure 4d) in response to the abrupt increase in VFA loads at OLR-S3 (Figures 2a). A higher variation range for f mc was observed in the mesophilic degradation of municipal and cellulosic solid wastes.40,41 In these experiments, Methanosarcina was the dominant methanogenic Archaea and was considered to solely promote this pathway shift because some Methanosarcina species are able to metabolize both hydrogen and acetate.1 Despite of minimal and maximal f mc values of 23% (at OLRS1) and 47% (at OLR-S3), the average variation in the methanogenic pathway contributions determined here was low, i.e., maximal difference of 10% between the three OLR stages (Figure 4d). Hence, it is difficult to obtain a direct correlation with TRF abundances. Furthermore, not only a qualitative variation of the methanogenic pathways occurred, but also the amounts of produced CH4 varied. As shown in Figure 5, parts e and f, the daily maximal CH4 production rates from the acetoclastic methanogenesis is almost identical for both OLRS2 and OLR-S3 weeks (QCH4,ma ≈ 6 L d−1). Conversely, the maximal hydrogenotrophic methanogenesis is two times higher at the OLR-S3 than 91 days earlier at the OLR-S2 (QCH4,mc ≈ 4 and 2 L d−1, respectively). Hence, nearly all increase of the CH4 production at OLR-S3 (Figure 2d) derives from these higher rates of the hydrogenotrophic methanogens. This rise of syntrophic hydrogenotrophic communities and maintenance of the acetoclastic methanogens suggest the formation of a layered sessile biofilm structure.42 Acetoclastic methanogens can be maintained in deeper biofilm layers, as the high acetate concentrations did not result in diffusion limitations, and the syntrophic communities benefit from the high VFA concentrations near the bulk liquid. Alternatively, this observed increase in Δ13CCO2‑CH4 values and high Methanosaeta abundance could also be interpreted as an indicator of direct interspecies electron transfer (DIET)-mediated processes. Recently, the Methanosaeta capability to reduce CO2 into CH4-accepting electrons from Geobacter was reported for microbial aggregates from a UASB reactor treating brewery wastes.43 Methanosaeta activates CO2, forming formyl methanofuran, similar to hydrogenotrophic methanogens.32,43 Hence, both carbon reduction methanogenic processes likely have similar CO2 fractionation factors. Thus, the determination of carbon fractionation has potential to reveal more details of Methanosaeta-dominated systems. Oxidation Pathways of Volatile Fatty Acids. The maximal hydrogenotrophic methanogenesis calculated through VFA oxidation yields differed significantly from the calculations from δ13C measurements (Figures 4d and 5d). At OLR-S1 and

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information for the methanogenic pathway calculations, additional design and operational data of the two-stage experimental plant, pictures of the biofilm carriers from the upflow anaerobic filter, and confocal laser scanning microscopy images of the biofilm and results for the sensitivity analysis of fractionation parameters used for f mc calculation. This material is available free of charge via the Internet at http://pubs.acs.org. 4712


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(13) Klocke, M.; Nettmann, E.; Bergmann, I.; Mundt, K.; Souidi, K.; Mumme, J.; Linke, B. Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass. Syst. Appl. Microbiol. 2008, 31, 190−205 DOI: 10.1016/ j.syapm.2008.02.003. (14) Lübken, M.; Wichern, M.; Letsiou, I.; Kehl, O.; Bischof, F.; Horn, H. Thermophilic anaerobic digestion in compact systems: investigations by modern microbiological techniques and mathematical simulation. Water Sci. Technol. 2007, 56, 19−28 DOI: 10.2166/ wst.2007.729. (15) Conrad, R. Quantification of methanogenic pathways using stable carbon isotopic signatures: a review and a proposal. Org. Geochem. 2005, 36, 739−752 DOI: 10.1016/j.orggeochem.2004.09.006. (16) Conrad, R.; Chan, O.-C.; Claus, P.; Casper, P. Characterization of methanogenic Archaea and stable isotope fractionation during methane production in the profundal sediment of an oligotrophic lake (Lake Stechlin, Germany). Limnol. Oceanogr. 2007, 52, 1393−1406 DOI: 10.4319/lo.2007.52.4.1393. (17) Yu, Y.; Lee, C.; Kim, J.; Hwang, S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol. Bioeng. 2005, 89, 670− 679 DOI: 10.1002/bit.20347. (18) Bergmann, I.; Mundt, K.; Sontag, M.; Baumstark, I.; Nettmann, E.; Klocke, M. Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters. Syst. Appl. Microbiol. 2010, 33, 78−84 DOI: 10.1016/j.syapm.2009.11.004. (19) Rademacher, A.; Nolte, C.; Schönberg, M.; Klocke, M. Temperature increases from 55 to 75 °C in a two-phase biogas reactor result in fundamental alterations within the bacterial and archaeal community structure. Appl. Microbiol. Biotechnol. 2012, 96, 565−576 DOI: 10.1007/s00253-012-4348-x. (20) Batstone, D. J.; Keller, J.; Angelidaki, I.; Kalyuzhnyi, S. V.; Pavlostathis, S. G.; Rozzi, A.; Sanders, W. T. M.; Siegrist, H.; Vavilin, V. A. Anaerobic Digestion Model No.1; IWA Publishing: London, U.K., 2002. (21) Fernandez, I.; Mahieu, N.; Cadisch, G. Carbon isotopic fractionation during decomposition of plant materials of different quality. Global Biogeochem. Cycles 2003, 17, 1−9 DOI: 10.1029/ 2001GB001834. (22) Nizami, A.-S.; Murphy, J. D. Optimizing the operation of a twophase anaerobic digestion system digesting grass silage. Environ. Sci. Technol. 2011, 45, 7561−7569 DOI: 10.1021/es201357r. (23) Sträuber, H.; Schröder, M.; Kleinsteuber, S. Metabolic and microbial community dynamics during the hydrolytic and acidogenic fermentation in a leach-bed process. Energy Sustain. Soc. 2012, 2, 1−10 DOI: 10.1186/2192-0567-2-13. (24) Habouzit, F.; Gévaudan, G.; Hamelin, J.; Steyer, J.-P.; Bernet, N. Influence of support material properties on the potential selection of Archaea during initial adhesion of a methanogenic consortium. Bioresour. Technol. 2011, 102, 4054−4060 DOI: 10.1016/j.biortech.2010.12.023. (25) Fotidis, I. A.; Karakashev, D.; Angelidaki, I. The dominant acetate degradation pathway/methanogenic composition in full-scale anaerobic digesters operating under different ammonia levels. Int. J. Environ. Sci. Technol. 2014, 11, 2087−2094 DOI: 10.1007/s13762013-0407-9. (26) Mook, W. G.; Bommerson, J. C.; Staverman, W. H. Carbon isotope fractionation between dissolved bicarbonate and gaseous carbon dioxide. Earth Planet. Sci. Lett. 1974, 22, 169−176. (27) Conrad, R.; Klose, M.; Lu, Y.; Chidthaisong, A. Methanogenic pathway and archaeal communities in three different anoxic soils amended with rice straw and maize straw. Front. Microbiol. 2012, 3 (4), 1−12 DOI: 10.3389/fmicb.2012.00004. (28) Meinschein, W. G.; Rinaldi, G. G.; Hayes, J. M.; Schoeller, D. a. Intramolecular isotopic order in biologically produced acetic acid. Biomed. Mass Spectrom. 1974, 1, 172−174.

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 234 32 23114; e-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the German Research Foundation DFG (Grants LU 1167/5-1 and KL 2069/3-1), which financed this research. We are grateful to Ralf Conrad and Peter Claus (Max Planck Institute for Terrestrial Microbiology, Marburg, Germany) for the acetate stable carbon isotope analysis, to Ulrich Heimhofer and Christiane Wenske (Institute of Geology, Leibniz Universität Hannover, Germany) for the maize silage stable carbon isotope analysis, and to Edith Nettmann for the confocal laser scanning microscopic biofilm image. We also thank Heinrich Wortmann for supplying the inoculation digestate and the maize silage.

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