Biotechnology and Bioprocess Engineering 16: 1044-1052 (2011) DOI 10.1007/s12257-011-0117-4 RESEARCH PAPER Anaerobic Co-digestion of Swine Manure with Energy Crop Residues María José Cuetos, Camino Fernández, Xiomar Gómez, and Antonio Morán Received: 22 March 2011 / Revised: 2 June 2011 / Accepted: 10 June 2011 © The Korean Society for Biotechnology and Bioengineering and Springer 2011 Abstract Anaerobic co-digestion involves the treatment of different substrates with the aim of improving the production of biogas and the stability of the process. In this research, co-digestion of swine manure (SM) and energy crop residues (ECRs) was studied. The mixtures evaluated contained SM combined with maize (Mz), rapeseed (Rs) or sunflower (Sf) residues. Batch and semi-continuous experiments were performed to determine methane (CH4) yields and the behavior of reactors while co-digesting agricultural wastes. Three different proportions of ECRs were tested in batch experiments for co-digestion with SM: 25, 50, and 75% volatile solids (VS). On the basis of the results obtained from batch tests, a mixture with a 50% ECR content was selected for the second stage of the study. Mesophilic reactors with a 3 L working volume were used for semicontinuous experiments. The hydraulic retention time (HRT) was set at 30 days and the reactors were kept under these operational conditions over four HRTs. The addition of ECR to the co-digestion system resulted in a major increase in the amount of biogas produced daily. The highest biogas yield was obtained when co-digesting Rs (3.5 L/day), although no improvement was observed in specific gas production from the addition of the co-substrate. Keywords: anaerobic digestion, rapeseed, maize, swine manure, sunflower 1. Introduction The production of renewable energy, utilization of by- María José Cuetos, Camino Fernández*, Xiomar Gómez, Antonio Morán Institute of Natural Resources (IRENA), University of Leon, Leon 24071, Spain Tel: +34-987-291-841; Fax: +34-987-291-839 E-mail: cferrd@unileon.es products as fertilizers, a reduction in agricultural wastes, and the prevention of environmental pollution are several of the characteristics that have promoted the industrial application of anaerobic digestion for the treatment of wastes. Anaerobic digestion is currently a well-established technology in Europe with large-scale systems developed primarily in countries such as Germany and Denmark [13]. Nevertheless, studies need to be initiated to improve current technologies and encourage the use of biogas [4-7]. Anaerobic co-digestion is defined as a treatment that combines different types of wastes with the aim of increasing biogas yields. This improvement is achieved by balancing the nutrient content and reducing the negative effects on the digestion process of toxic compounds. Moreover, it leads to a more efficient use of equipment and cost-sharing by processing multiple wastes in a single facility [8]. The production of swine in the European Union (EU) is a major agricultural industry. In 2009 there were 152 million head of swine in the EU, with 25 million produced in Spain [9]. It is estimated that 46 million m3 of swine manure (SM) are produced annually in that country [10]. SM is a plentiful source of organic matter that may be used as feedstock in anaerobic digesters. It has a high buffering capacity, which may protect the digestion process against possible failures due to any build-up of volatile fatty acids (VFAs) and any consequential drop in the pH of the system. SM also contains a wide variety of nutrients necessary for bacterial growth. However, another of its characteristics is its high nitrogen content, which may represent a digestion inhibition risk as the result of high levels of ammonia within the digester when manure is digested individually [11,12]. Methanogens are the organisms least tolerant and most likely to cease growing due to ammonia inhibition [13]. Free ammonia concentration has been suggested as the principal factor responsible for inhibition problems. The degree of inhibition depends mainly upon total ammonia Anaerobic Co-digestion of Swine Manure with Energy Crop Residues concentration, pH, and temperature. Different threshold values for free ammonia concentrations have been reported by several authors, ranging from 200 mg of ammonia nitrogen (N-NH3)/L [14] for unadapted populations, to higher values (700 ~ 1,100 mg N-NH3/L) for microorganisms that have previously adapted to their presence [12,15]. For this reason, manure should preferably be codigested with wastes that have a high carbon content, so as to improve the carbon/nitrogen (C/N) ratio [16,17]. It has been estimated that 104,731 tons/year of energy crops are produced in Spain [18]. Agricultural wastes may be present in large quantities when no market is available. In such cases, wastes may remain in the fields after harvesting operations. These wastes may be suitable for anaerobic co-digestion with manure, as a result of their considerable carbon content. However, the major drawback when this type of materials is being digested is the lignin content, which may hinder anaerobic degradation. Various pre-treatments have been applied in attempts to improve the biodegradability of agricultural wastes [19]. A reduction in particle size by mechanical pre-treatment leads to an increase in the specific surface available and thus improves gas production in the digester and reduces the technical digestion time [20]. The aim of the research being reported here was to study the performance of reactors co-digesting SM with three types of energy crop residues (ECRs) from: maize (Mz), rapeseed (Rs) and sunflowers (Sf). For this purpose, the influence of the co-substrates on biogas yield under batch conditions was first evaluated. Thereafter, the performance of reactors under semi-continuous operation was studied using a mixture of SM and ECR as substrate. 1045 with a working volume of 250 mL. Solutions were kept in a thermostatic water bath maintained at a mesophilic temperature (35 ± 1°C) and continuously stirred at 250 rpm by magnetic bars. Batch experiments were performed to determine the biochemical methane (CH4) potential from individual substrates and mixtures of crop residues with SM. The duration of experiments was determined by the point at which biogas production stopped completely. All experiments were performed in duplicate and blank reactors were also used, to determine the background gas productivity of the inoculum. In each reactor, substrate and inoculum were introduced and when necessary, tap water was added to attain a total volume of 250 mL. No nutrient solution was added to biochemical CH4 potential tests. The mass of ECRs added to digestion tests was 1.8 g of volatile solids (VS), which was the maximum quantity that could be added to digesters without causing problems with stirring or agglomeration. The inoculum used was diluted to a concentration of 5.0 g/L VS, with 125 mL of prepared inoculum added for digestion tests and 85 mL for codigestion tests. Co-digestion experiments were performed with different ratios of manure to ECR. SM and ECR mixtures were prepared with proportions of 25, 50, and 75% VS. These percentages represent the VS content of the ECR in relation to the total quantity of VS in the mixture. Gas and liquid samples were taken twice a week to measure the composition of the biogas, the pH, and the concentration of VFAs. Gas production was measured daily using a liquid displacement device. The solution used was saturated to 75% with sodium chloride and acidified to pH 2. Adjustments to the pH were made by the addition of an alkaline solution during the first few days of the experiments whenever the pH value fell below 7 units. 2. Materials and Methods 2.1. Substrates and inoculum Three ECRs were used in this study: Mz, Rs and Sf. These residues were collected during the harvest period (September-October) from farms located in Castilla y León (Spain). Specimens were chopped up with a Viking (GB370) agricultural chopper and then further milled using a laboratory grinder to reduce their particle size to less than 3 mm. SM was obtained from a livestock farm located in Alcoba de la Ribera (León, Spain). The manure was stored at 4ºC until required for use. The anaerobic sludge used as inoculum was obtained from the wastewater treatment plant of the city of Leon (Spain). Details of the operating conditions for this digester are available elsewhere [21]. 2.2. Batch experiments Batch digestion tests were carried out in Erlenmeyer flasks 2.3. Semi-continuously operated reactors Four completely stirred tank reactors were used to evaluate the digestion of SM and mixtures with ECRs. The reactors were made of methacrylate, being cylindrical in shape and having a heating jacket through which water was circulated. They were stirred continuously with two triple-blade propellers to prevent particulate material from floating. The reactors were labelled in accordance with the substrates fed into them. Thus, R_SM referred to the reactor treating SM, and similarly for the various mixtures with ECRs. A mixture with a 50% content of ECR was selected for the second stage of the study on the basis of the results obtained from batch tests in the previous experimental stage. Reactors were initially started up with an adaptation period intended to acclimatize anaerobic microorganisms to conditions with inhibitory concentrations of ammonia [22]. The reactors were operated with a hydraulic retention 1046 Biotechnology and Bioprocess Engineering 16: 1044-1052 (2011) time (HRT) of 50 days over a 30-day period. These conditions led to the application of an organic loading rate (OLR) of 0.7 kg VS/m3/day for R_SM, while the codigesting systems had an OLR of 1.4 kg VS/m3/day. Thereafter, the HRT was decreased to 30 day, equivalent to an OLR of 1.2 kg VS/m3/day for R_SM, and 2.3 kg VS/m3/ day for co-digesting reactors. These operating conditions were evaluated for four consecutive HRTs. The daily production of biogas was measured using a reversible liquid displacement device provided with a wet tip counter. Gas and liquid samples were taken twice a week to monitor biogas composition, pH, chemical oxigen demand (COD), total solids (TS), VS, ammonia, and VFA concentrations. Table 1. Chemical characteristics of inoculum and substrates 2.4. Analytical techniques Kjeldahl nitrogen, TS, VS, COD, alkalinity, ammonium, and pH were determined in accordance with American Public Health Association (APHA) standard methods [23]. Cellulose, hemi-cellulose, and lignin content were estimated by an analysis in duplicate of neutral detergent fiber, acid detergent fiber, and crude fiber [24] in ground samples using an ANKOM 200 Fiber Analyzer. Total organic carbon was determined by following the Walkey-Black method [25], involving oxidation with 1 N potassium dichromate (K2Cr2O7) and 96% sulphuric acid (H2SO4) solutions for 30 min. The organic carbon content was calculated by back-titration with a solution of 0.5 N Fe(NH4-SO4)2·6H2O and organic carbon was determined on the basis of an organic matter content to organic carbon ratio of 1.7241. The organic carbon was subsequently divided by the total nitrogen to obtain the C/N ratio. Biogas composition was analysed using a gas chromatograph (Varian CP 3800 GC) equipped with a thermal conductivity detector. A column 4 m long, packed with HayeSepQ80/100, followed by a molecular sieve column 1m long, was used to separate CH4, CO2, N2, H2, and O2. The carrier gas was helium and the columns were operated at 331 kPa at a temperature of 50ºC. VFAs were analysed using a gas chromatograph (Varian CP 3800 GC) equipped with a Nukol capillary column (30 m × 0.25 mm × 0.25 µm) from Supelco (Bellefonte, PA, USA) and a flame ionization detector. The carrier gas was helium. The temperature of the injector was 250ºC and the temperature of the oven was initially set at 150ºC for 3 min and thereafter increased to 180ºC. 3. Results and Discussion 3.1. Batch digestion experiments The characteristics of the ECRs and SM used are given in Characteristics C/N TS VS TKN(g/kg TS) NH4+ (mg/L) Cellulose (%) Hemicellulose (%) Lignin (%) I SM Mz Rs Sf 6.2 20.1a 13.6a 46.7 650 − − − 1,010.2 1,055a 1,035a 1,032.2 1,282 − − − 64.7 672b 644b 8.2 − 23.6 29.7 7.4 60.1 397b 363b 8.6 − 40.5 21.6 16.6 52.6 888b 771b 9.7 − 23.7 15.2 12.0 a Values expressed in g/L. Values expressed in g/kg. b Table 1. The ECRs showed a high C/N ratio, which indicates that these substrates may be suitable for co-digestion with SM. However, the lignin content of these substrates was also high. The presence of lignin may hinder the break-down of organic matter, thus resulting in a fraction of the substrate possibly remaining unavailable for microbial break-down. Fig. 1A shows the results from batch digestion tests. SM and Mz gave the highest CH4 yields from the batch digestion of individual substrates. The lower values obtained for Rs and Sf may be associated with the higher lignin content of these substrates. The anaerobic microflora probably could not access the organic material encapsulated within lignin structures. With regard to the shapes of curves obtained from digestion tests of individual substrates, an extended lag phase was observed when digesting SM. This result may indicate unfavourable conditions for anaerobic microflora. However, once the organisms acclimatized, the digestion proceeded at a high rate. A similar trend was reported by Lobato et al. [26] when evaluating the digestion of SM under batch conditions at a mesophilic temperature. These authors obtained values for the two manures that they tested of 276 ± 10 and 512 ± 19 mL CH4/g VS added. Under similar conditions Chae et al. [27] reported a yield of 403 mL CH4/g VS added. The results obtained in the work being reported here were in accordance with those findings, with a value of 357 ± 34 mL CH4/g VS added. The CH4 potential of manure comes from the digestion of the organic components in the feces and in the straw used as bedding material, which are mainly carbohydrates, proteins, and lipids. In theory, the CH4 yield from carbohydrates (415 mL/g VS) is lower than that from proteins (496 mL/g VS) or lipids (1,014 mL/g VS) [28]. In view of this, although no analysis of the protein and lipid content was performed during the experiments, it can be assumed that this substrate was composed mainly of carbohydrates and proteins. In contrast, when particulate substrates are being di- Anaerobic Co-digestion of Swine Manure with Energy Crop Residues 1047 Fig. 1. (A) Methane (CH4) yield of individual substrates. Symbols: (-◇-), maize (Mz); (- △-), rapeseed (Rs); (- ■ -), sunflower (Sf); (- ◆-), swine manure (SM). CH4 yield of co-digestion mixtures of SM with (B) Mz, (C) Sf, and (D) Rs. (-◆-), mixtures were prepared with a content of volatile solids (VS) of 25% of energy crop residue (ECR); (- ■ -), 50% VS of ECR; and (-△-), 75% VS of ECR. gested, the process is generally limited by the hydrolysis phase [29,30] and it may then be described by first order kinetics [31]. This is the case for the ECR curves obtained from these digestion tests, with the exception of Sf, as may be seen in Fig. 1A. The shape of Rs and Mz curves match those for the category of slowly degradable substrates, as defined by Labatut et al. [32]. However, in the case of Sf the shape of the curve indicates a high break-down rate for the material readily accessible to micro-organisms, but the lignin content of this substrate is likely to have hindered the breaking down of a considerable amount of the organic matter, thus leading to low CH4 yields. CH4 yield curves for mixtures studied are also shown in Figs. 1B, 1C, and 1D. A reduction in the lag phase was observed when co-substrate was added. The shape of the curves can in all cases be seen as sigmoidal. This characteristic shape has also been reported in batch digestion tests by several authors [33,34]. The lowest CH4 yield was obtained from co-digestion with Sf in a proportion of 75:25. As previously commented, this substrate was characterized by high lignin content and thus a low CH4 yield was to be expected. The addition of SM to Sf may have resulted in a synergistic effect, since the outcomes of the digestion tests with mixtures having 25:75 and 50:50 proportions were similar. This behavior was also observed with the co-digestion of the other two ECRs, the mixtures thus having similar values for CH4 yield with different proportions of ECR. Although co-digestion with Rs and Mz led to similar values for CH4 yield, differences were observed in the pattern of break-down. The addition of the co-substrates produced preferential breaking-down, which could be observed through changes in the rate of CH4 production. The addition of agricultural residues as co-substrates has been reported to increase the production of biogas significantly [35]. Differences between our results and a previous report may be explained by the diminutive particle size used by Wu et al. [35]. The crop residues (corn stalks, oat straw and wheat straw) were first cut into small sections and then ground into fine particles, under 40 mesh size (0.422 mm) before being added to digesters, thus improving the degradability of the co-substrate particles. However, the present results are in accordance with those reported in the literature when considering batch conditions. Fujita et al. [36] studied the co-digestion of corn and manure in mesophilic conditions (reporting a value of 267 L CH4/Kg VS added) and thermophilic conditions (reporting a value of 305 L CH4/Kg VS added). Varying values for the codigestion of manure with wheat straw can be found in the literature, with reports of 322 L CH4/Kg VS added [37] and 148 L CH4/Kg VS added [28]. Fig. 2 shows the details of VFA concentrations for 1048 Biotechnology and Bioprocess Engineering 16: 1044-1052 (2011) Fig. 2. Volatile fatty acid (VFA) profiles obtained from the digestion of Sf (A), Mz (B), and SM (C). Symbols: (- ◆-), acetic acid; (- □-), propionic acid; and ( × ), butyric acid. digestion and Fig. 3 shows co-digestion tests. In the case of the digestion of Rs, no detectable amount was found; i.e., the values were below the detection limit. This substrate was also characterized by its high lignin content, as was Sf. From Fig. 2 it may also be observed that low values were measured during the digestion of this substrate. In the case of Mz, VFA concentrations were slightly higher, but only during the early days of the experiment. These results were influenced by the limitation imposed by the hydrolysis stage. This was not the case for SM. Higher concentrations of these acids were detected at the beginning of the experiment. As the digestion proceeded VFAs were continuously detected over a 25-day period. On the basis of these results, the VFA concentrations reported in co-digestion tests (Fig. 3) may be attributed principally to the acidification of SM. All co-digestion tests presented lower values for VFAs, as the amount of SM in the mixtures was reduced in accordance with the ratios being tested. Acetic acid was the main VFA present in all the batch tests, with the exception of the digestion of Rs, where VFAs were below the detection limit. Values for the VFA concentrations measured in the co-digestion tests at a ratio of 75:25 were much higher than those obtained from the digestion of SM on its own. The extended lag phase observed for CH4 production when SM is being digested may be attributed to nutrient imbalance rather than to a Fig. 3. VFA profiles obtained during batch experiments from co-digestion of mixtures of SM with Rs (A1) 25% VS of ECR, (A2) 50% VS of ECR, (A3) 75% VS of ECR, Sf (B1) 25% VS of ECR, (B2) 50% VS of ECR, (B3) 75% VS of ECR, and Mz (C1) 25% VS of ECR, (C2) 50% VS of ECR, (C3) 75% VS of ECR. Symbols: (-◆-), acetic acid; (- □ -), propionic acid; and ( × ), butyric acid. Anaerobic Co-digestion of Swine Manure with Energy Crop Residues 1049 build-up of VFAs. On the basis of the results obtained from batch tests, mixtures containing 50% of ECR were chosen for performing evaluations under semi-continuous operation. This decision was based on the minor modifications observed in CH4 yield obtained at this ratio as compared to the yield from the digestion of SM alone, and also to avoid possible solid build-up problems which may occur under continuous operation. 3.2. Semi-continuous operation Daily biogas production is shown in Fig. 4A. During the adaptation period (days 1 ~ 30), reactors were characterized by a limited output of biogas, which was in accordance with the low OLR applied. Setting the operational conditions at a HRT of 30 days resulted in a gradual increase in the production of biogas for all reactors evaluated. Codigestion systems presented a higher production of biogas in comparison to that of R_SM. Fig. 4B shows the specific gas production (SGP) for reactors. No improvement in the SGP was observed from the addition of the co-substrates. As such, the synergistic effects which may have been present under batch conditions were not registered under semi-continuous operation. The higher production of biogas obtained from co-digesting systems was explained by the increase in OLR arising from the ECRs. Biogas production was steady in all systems from day 60 onwards, with the exception of R_Rs. Over this period the composition of the gas showed minor variations. However, co-digestion with Rs was characterized by an upward trend in the production of biogas from day 90 onwards. This behavior is likely to be due to an accumulation of VS Fig. 4. (A) Daily biogas production and (B) specific gas production (SGP) for reactor treating swine manure (R_SM) (- ◇-) and co-digesting systems: R_Rs ( ), R_Sf ( ), and R_Mz ( ). within the reactor being transformed at a later stage, thus making a larger amount of material available for microbial break-down. Table 2 shows the average values for parameters recorded during days 70 ~ 150. Co-digestion with Rs presented the highest figure for average daily biogas production. As stated previously, this high value was explained by the accumulation of solids within the reactor, detected during Table 2. Main operational parameters of digesters studied R_Rs, R_Mz, R_Sf, and R_SM under semi-continuous operation (HRT of 30 days) Parameters* Biogas (L/day) SGP biogas (m3/kg/VSfeed) CH4 yield (m3/kg/VSfeed) CH4 (%) CO2 (%) TS (g/L) VS (g/L) VS removal (%) TS (g/L)end VS (g/L)end COD (g/L) pH Alkalinity (g/L) NH4+-N (mg/L) NH3-N (mg/L) R_SM 1.6 ± 0.1 0.46 ± 0.03 0.33 ± 0.03 71.5 ± 1.2 28.5 ± 1.2 39.0 ± 3.9 22.2 ± 2.9 36.6 ± 8.4 35.0 ± 0.4 20.0 ± 0.3 28.6 ± 4.9 8.0 ± 0.1 17.5 ± 0.4 4,580 ± 254.2 418.9 ± 55.5 *Means and standard deviations values obtained from days 70 to150. R_Rs 3.5 ± 0.3 0.51 ± 0.04 0.34 ± 0.03 65.9 ± 3.2 34.1 ± 3.2 60.1 ± 9.1 41.8 ± 9.1 40.3 ± 13.0 136.0 ± 4.1 113.0 ± 4.5 61.2 ± 3.7 7.9 ± 0.1 18.1 ± 1.0 .4438 ± 281.0 332.8 ± 52.4 R_Sf 2.7 ± 0.2 0.39 ± 0.03 0.26 ± 0.02 65.0 ± 2.9 35.0 ± 2.9 60.5 ± 3.6 38.7 ± 2.5 44.7 ± 3.6 87.0 ± 3,2 55.0 ± 2,2 52.7 ± 2.6 7.9 ± 0.1 17.3 ± 0.4 4,368 ± 331.5 319.5 ± 28.7 R_Mz 3.2 ± 0.1 0.46 ± 0.02 0.30 ± 0.01 63.1 ± 2.0 36.9 ± 2.0 54.8 ± 1.4 33.4 ± 1.9 52.3 ± 2.7 91.0 ± 5.0 70.0 ± 4.8 41.2 ± 2.5 7.9 ± 0.1 17.0 ± 0.5 4,440 ± 259.1 346.6 ± 32.4 1050 Biotechnology and Bioprocess Engineering 16: 1044-1052 (2011) the experiment due to the formation of a floating layer of ECR particles. Such an accumulation of particles has previously been reported, with descriptions of the formation of a crust layer in the upper part of the liquid during experiments involving the co-digestion of energy crops and crop residues with cow manure [38]. At the end of the experiments, the reactors were dismantled and their contents were thoroughly homogenized. The amounts of TS and VS measured at the end of the experiment are also presented in Table 2. The homogenized slurry presented a much higher concentration of solids than what was periodically measured in the effluent obtained from sampling ports. In this way, the hypothesis of a late breaking-down phase in system R_Rs was corroborated, since the ECR stayed in the reactor for a period longer than the 30 days established. Although all co-digestion systems presented some accumulation of solids, Mz and Sf residues seemed to be broken down completely during the time they remained in the reactor and the particles that accumulated did not increase biogas production at a later stage. The figures for CH4 yield shown in Table 2 were similar to results reported in the literature with respect to experiments in which manure was co-digested with agricultural wastes. Fujita et al. [36] obtained a value of 0.21 m3/kg VS fed with manure and corn, while Fischer et al. [39] noted values in the range of 0.22 ~ 0.24 m3/kg VS fed for mixtures of manure and wheat straw. Lehtomäki et al. [38] tested a mixture with 30% of straw in the feedstock combined with cow manure and achieved a CH4 yield of 0.21 m3/kg VS fed. Comino et al. [40], during trials with a cow manure and crop silage mixture, recorded CH4 yields between 237 and 249 L CH4/kg VS. The reactors showed a continuous increase in the ammonium concentration as the HRT was reduced from 50 to 30 days. Steady values for ammonium were attained once the reactors completed the second HRT at 30 days. Free ammonia concentrations were maintained within the normal ranges for anaerobic digestion of wastes with a high nitrogen content, as stated by several authors [14,41]. The evolution of VFAs is presented in Fig. 5. VFAs were below the detection limit during the adaptation period. The increase in the OLR resulted in a rapid build-up of VFAs during the first HRT cycle at 30 days. The reactors presented high concentrations of acetic acid. This period was also characterized by an increasing production of biogas from all reactors. Thus, this upward trend may be explain- Fig. 5. VFA concentration of digesters studied A) R_Rs, B) R_Mz, C) R_Sf, and D) R_SM. Symbols: (-◆-), acetic acid; (- □ -), propionic acid; and ( × ), butyric acid. Anaerobic Co-digestion of Swine Manure with Energy Crop Residues ed by the availability of acid intermediaries which were used for further biogas production. Although the steep increase in the OLR produced an imbalance in microbial consortia, microorganisms were capable of adapting to new conditions. As a result, VFAs decreased continuously during the second HRT evaluated, and finally the predominant acid types showed minor variations during the third and fourth HRT periods. 1051 7. 8. 9. 4. Conclusion 10. Co-digestion of SM with three different types of ECR derived from Rs, Mz, and Sf was studied under batch and semi-continuous conditions. SM and Mz gave the highest CH4 yields in batch digestion tests of individual substrates. The lower amounts obtained from the other two substrates may be associated with their higher lignin content. During semi-continuous operation, no improvement in the SGP was observed from the addition of the co-substrates. Synergistic effects which may have been present under batch conditions were not noted during semi-continuous operation. An accumulation of solids within the reactor was observed in all reactors tested. This accumulation led to an upward trend in biogas production for the co-digesting system with Rs residue; however, this was not the case for the Mz and Sf co-digesting reactors. 11. 12. 13. 14. 15. 16. 17. 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