Biochemical Methane Potential of Swine Slaughter Waste, Swine Slurry, and Its Codigestion Effect

: The codigestion of slaughter waste with animal manure can improve its methane yield, and digestion parameters; however, limited studies are available for the effectiveness of anaerobic codigestion using swine slaughter waste (SSW) and swine slurry (SS). Hence, this study was conducted to determine the characteristics of SSW and the effect of anaerobic codigestion with (SS) and explored the potential of CH 4 production ( M max ), the lag phase period ( λ ), and effective digestion time ( T eff ). SSW contains fat and protein contents of 54% and 30% dry weight within 18.2% of solid matters, whereas SS showed only 6% and 28% within 4.1% of solid matters, respectively. During sole anaerobic digestion, SSW produced a high M max (711 Nml CH 4 /g VS added ) but had a long duration λ (~9 days); whereas SS produced a low M max (516 Nml CH 4 /g VS added ) but had a shorter duration λ (1 day). Codigestion increased the M max from 22–84% with no signiﬁcant T eff compared to sole SS digestion. However, the low M max of SS and high M max of SSW, resulted in a 7–32% decrease in M max at codigestion compared to SSW sole digestion. Codigestion improved the digestion efﬁciency as it reduced λ (3.3–8.5 days shorter) and T eff (6.5–9.1 days faster) compared to SSW sole digestion. The substrate-to-inoculum ratio of 0.5 was better than 1; the volatile solid and micronutrient availability may be attributed to improved digestion. These results can be used for the better management of SSW and SS for bio-energy production on a large scale.

The SSW samples were collected from a slaughterhouse in Yeongcheon City, Gyeongsang Province, South Korea, which slaughtered around 189,466 swine in 2017 [2]. The samples collected consisted of livestock remains except for blood, brain, bones, and spinal cord. Blood was omitted since it was processed separately by the slaughterhouse. Brain, spinal cord, and skin hairs were also omitted due to safety reasons. The sample mostly contains intestines, feeds left-over in the stomach, and flushing contents. SS was collected from the swine farm in Hoengseong County, Gangwon Province, South Korea. The samples were mixed separately (ground into SSW using a fruit mixer), sieved (<5 mm) and dried at 105 • C for 12 h, and used for chemical, ultimate, and higher heating value (HHV) analysis. The wet sample was utilized for the proximate and BMP analyses. The inoculum was collected from a mesophilic anaerobic digester treating SS (active anaerobic digester in Suwon campus) and used for the BMP experiment. The inoculum was maintained in a serum bottle (250 mL) with the addition of SS and SSW once a month. Before the experiment, the inoculum was degassed for two weeks to deplete any remaining organic materials and gas production. Table 1 shows the characteristics of the inoculum utilized in both BMP and codigestion experiments.

BMP Analysis and Codigestion Experiment
The BMP of SSW and SS was measured using 250-mL serum bottles. The substrate-toinoculum ratio (S/I ratio, 1 and 0.5) was selected based on the vs. content of the substrate and inocula [5]. The total volume of the digestion was set at 200 mL. The head space was filled with CO 2 and N 2 gas (20:80% volume per volume), and the bottle was sealed with a butyl rubber cap and aluminum crimps and incubated at 35 • C for 50 days. The control experiment was conducted by adding inocula and distilled water only. The codigestion experiment of SSW and SS was performed in five different SSW samples per SS (SSW/SS) ratio of 1:0, 0:1, 2:1, 1:1, and 1:2, on a w/w vs. basis. This was equal to a 100% (946 g VS/L), 0% (676 g VS/L), 67% (875 g VS/L), 50% (811 g VS/L), and 33% (765 g VS/L) vs. basis of SSW content in the codigested mixture ( Table 2). The experiments were also performed at an S/I ratio of 1 and 0.5 w/w vs. basis, and the procedure followed was the same as that for the BMP test. Then, 50 mL of the mixture was sampled for further analysis. Chemical analysis of fat, protein, neutral detergent fiber (NDF), and acid detergent fiber (ADF) was performed in SSW and SS. Fat content was determined by Soxhlet extraction with ether as solvent. Protein content was determined with total Kjeldahl nitrogen (TKN). NDF and ADF contents were determined according to the study by Fernández-Cegrí et al. [19]. Proximate analyses of moisture, TS, VS, and fixed solids (FS) were performed by standard methods [20]. Before ultimate (elemental) analysis, samples were pretreated following the procedure described in the study by Choi et al. [21]. Carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) contents of pretreated samples were analyzed with an elemental analyzer (Flash EA 1112, Thermo Fisher Scientific, Dreieich, Germany). The oxygen (O) content of the samples was analyzed using a Flash 2000 elemental analyzer (Thermo Fisher Scientific, Germany). The mineral contents such as cobalt (Co), iron (Fe), molybdenum (Mo), nickel (Ni), tungsten (W), and zinc (Zn) of SSW and SS were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-7510, Shimadzu Corp., Kyoto, Japan). Before the analysis, the samples were digested with nitric acid-hydrochloric acid (method number 3030F) [20].

HHV Analysis
Dried samples were ground using a mortar and pestle and sieved (5 mm) (DH.Si8021, DAIHAN Scientific, Gangwon-do, Korea), then pelletized using a pellet press (2811, Parr Instrument), and analyzed using an oxygen bomb calorimeter (Model 1341 plain jacket calorimeter, Parr Instrument). Benzoic acid pellets (3415, Parr Instrument) were used to standardize the oxygen bomb calorimeter prior to the analysis.

Biogas Production, Composition, and Specific CH 4 Yield
The biogas production was analyzed using the manometric method, in which constant volume was maintained and headspace pressure increase was measured using a pressure transducer [22]. The excess gas was released regularly and quantified using a glass syringe until the pressure was similar to that at the start of the incubation [23]. The gas composition was analyzed using a gas chromatograph HP 6890N (Agilent Technologies) equipped with an HP-PLOT Q column (Agilent Technologies) and a thermal conductivity detector. The inlet, oven, and detector temperatures were 40 • C, 35 • C, and 200 • C, respectively. The CH 4 content was then utilized to determine CH 4 production and, subsequently, the specific methane yield (SMY) using Equation (1): where SMY is the specific CH 4 yield, in Nml CH 4 /g VS added or NL CH 4 /kg VS added ; MP is the CH 4 production, in mL; vs. is the volatile solid content of initial samples, in g; t 0 is the temperature under a standard condition, 273 K; and t i is the temperature where the experiment was conducted, 308 K.

Theoretical CH 4 Yield (TMY)
Elemental analysis was used to determine the chemical formula of organic waste [5]. TMY can be determined empirically from the chemical formula of organic waste suggested by Symons and Buswell [24], and Boyle [25]. Equation (2) was used to determine theoretical CH 4 from the chemical formula of organic waste [5,26]. This equation considered the production of carbon dioxide (CO 2 ), ammonia (NH 3 ), and hydrogen sulfide (H 2 S) gas, which were the by-products of anaerobic digestion complex substrates: The reaction coefficient for H 2 O (x), CH 4 (y), and CO 2 (z) can be determined using Equation (3): The TMY of organic waste at a standard temperature and pressure of 1 atm and 273 K can be determined using Equation (4) as suggested by Pellera and Gidarakos [27]: where TMY is the theoretical CH 4 yield, in Nml CH 4 /g VS. The degree of anaerobic degradation (D deg ) was determined using Equation (5): where D deg is the degree of anaerobic degradation, in %.

Kinetic Model
The biogas production curve during anaerobic digestion of the complex organic material corresponds to a slower flat curve [28]. Thus, the lag phase (λ) is also an important factor determining anaerobic digestion efficiency, as well as the cumulative CH 4 yield (CMY) and CH 4 production rate [29]. Using the modified Gompertz formula, λ can be estimated as follows [30]: where M(t) is the CMY at digestion time t, in NmL CH 4 /g VS added ; M max is the maximum CH 4 production potential, in NmL CH 4 /g VS added ; R max is the maximum CH 4 production rate, in NmL CH 4 /g VS added /day; λ is the lag phase period, in days; t is the observation time, in days; and e is the exp(1) = 2.7183. A nonlinear least-squares regression analysis was performed using Excel solver add-in to determine M max , R max , λ, and correlation coefficient (R 2 ) of the produced model. In addition, the Excel solver was also used to estimate T 90 that is the time required to obtain 90% M max . Using T 90 and λ, the effective digestion time (T eff ) can be calculated by using Equation (7): where T eff is the effective digestion time, in days; and T 90 is the time required to obtain 90% M max , in days. The simulated maximum CH 4 production potential of the codigested mixture (M sim ) was calculated by the proportion of SSW and SS in the mixture and the M max was estimated using the modified Gompertz formula for the sole SSW or SS anaerobic digestion, as shown in Equation (8) [29]: where M sim is the simulated maximum CH 4 production potential of the codigested mixture obtained from the modified Gompertz formula, in NmL CH 4 /g VS added ; M SSW is the M max of SSW obtained from the modified Gompertz formula, in NmL CH 4 /g VS added ; %Y SSW is the percentage of SSW in the mixture, in %; M SS is the M max value of SS obtained from the modified Gompertz formula, in NmL CH 4 /g VS added ; and %Y SS is the percentage of SS in the mixture, in %.

Synergistic Effect
The synergistic effect is inner reactions produced by the codigestion of different components. Each codigested substrate can influence the CH 4 production rate [31]. The synergistic effect was calculated using Equation (9): (9) where M codigestion is the experimental M max obtained from the modified Gompertz formula (Equation (8)) of the codigested substrate, in NmL CH 4 /g VS added .
The α value determines the type of synergistic relation among codigested substrates. Specifically, α > 1 indicated that codigested substrates have a synergistic effect, α = 1 indicated that codigested substrates work independently during the digestion process, and α < 1 indicated that codigested substrates have an antagonistic effect [31].

Statistical Analysis
The one-tail t-test was performed to compare the anaerobic digestion parameters (SMY, M max , R max , λ, T 90 , and T eff ) of SSW and SS at different S/I ratios. One-way analysis of variance followed by Tukey's honest significant difference test was performed to determine the effect of SSW codigestion with SS on the anaerobic digestion parameters at the same S/I ratio (1 or 0.5). The statistical significance level was set at p < 0.05 for all the analyses.

Characteristics of SSW and SS
The characteristics of SSW and SS are shown in Table 3. SSW showed less TS (18.2%) than previous studies, which reported 27.9% and 55% TS [18,32,33]. This might be attributed to the amount of water used for flushing and cleaning in the slaughterhouse before sample collection. However, the vs. content was noted as 94.57% of TS (% DW) and mainly consisted of protein (30.44% DW) and fat (53.64% DW), indicating an energy-rich substrate. Interestingly, the fat and protein contents were higher than those previously reported in South Korea by Yoon et al. [5] who found 15.1% fat and 40.1% protein DW in SSW. The difference might be due to the offal that increased the fat content. Some studies found that swine offal had fat and protein contents between 41.8 and 65.76%, and 20.1 and 31.6% DW, respectively [18,33]. The TS content of SS was observed to be 4.1% with 67.6% vs. of DW, indicating a high mineral (FS) content. The protein (28.1% DW) and NDF (27.4% DW) contributed to the vs. content, which originated from the swine manure and wasted feed [7,8]. Moreover, the manure itself contained undigested feed material from the digestive tract. The TS and vs. contents of SS were within the ranges between 0.6 and 12.6% DW and between 56 and 84% DW, as previously reported in South Korea, respectively [9]. SSW contains higher vs. and fat contents than SS, while SS showed higher FS, NDF, ADF, and hemicellulose contents than SSW. Fat is an energy-rich substance, indicating that SSW has a higher energy content than SS. Additionally, a high vs. content indicated that more organic matter was available in SSW to be converted into CH 4 during anaerobic digestion. SSW showed lower mineral contents (6.82% as FS) than SS (32.4%), which might inhibit the CH 4 generation rate when used alone for the digestion. Therefore, the codigestion of SSW with SS might contribute enough minerals for the microbes in the digestion, resulting in an enhanced CH 4 production.

Energy Content of SSW and SS
SSW showed a high energy content with an HHV of 28.43 MJ/kg DW, which was higher than that of any of the renewable resources. Figure 1 shows that energy crops had an HHV between 14.69 and 20.71 MJ/kg DW [34,35], whereas the livestock manure collected in South Korea had an HHV between 11.92 and 19.44 MJ/kg DW [21]. Palm kernels had the highest HHV (21 MJ/kg DW) among the energy crops, while SS had the highest HHV (17.6 MJ/kg DW) among the livestock waste. The high HHV of SSW indicated that it had the potential to be used as a substrate for bio-energy production. SS had an HHV of 17.6 MJ/kg DW, and was within the range (11.9-19.44 MJ/kg DW) of HHVs from livestock waste in South Korea [21]. Moreover, it was suggested that the HHV and vs. had a positive correlation [21,34,35]. In the case of fresh weight, the SSW and SS (Table 3) exhibited an HHV of 5.17 and 0.72 MJ/kg FW, respectively. This showed that the physical energy valorization from SSW and SS was not sustainable due to the small amount of energy that could be recovered from the thermal treatment of fresh SSW and SS [26]. Thus, alternative technology to recover energy from SSW and SS is necessary. Any VS-containing substrates can be used in anaerobic digestion process for making biogas (VS converted to biogas) and successfully applied in large-scale digester systems across Europe [36]. Thus, anaerobic digestion could be an alternative technology to recover energy from SSW and SS.
an HHV between 14.69 and 20.71 MJ/kg DW [34,35], whereas the livestock manure collected in South Korea had an HHV between 11.92 and 19.44 MJ/kg DW [21]. Palm kernels had the highest HHV (21 MJ/kg DW) among the energy crops, while SS had the highest HHV (17.6 MJ/kg DW) among the livestock waste. The high HHV of SSW indicated that it had the potential to be used as a substrate for bio-energy production. SS had an HHV of 17.6 MJ/kg DW, and was within the range (11.9-19.44 MJ/kg DW) of HHVs from livestock waste in South Korea [21]. Moreover, it was suggested that the HHV and vs. had a positive correlation [21,34,35]. In the case of fresh weight, the SSW and SS (Table 3) exhibited an HHV of 5.17 and 0.72 MJ/kg FW, respectively. This showed that the physical energy valorization from SSW and SS was not sustainable due to the small amount of energy that could be recovered from the thermal treatment of fresh SSW and SS [26]. Thus, alternative technology to recover energy from SSW and SS is necessary. Any VS-containing substrates can be used in anaerobic digestion process for making biogas (VS converted to biogas) and successfully applied in large-scale digester systems across Europe [36]. Thus, anaerobic digestion could be an alternative technology to recover energy from SSW and SS.  Table 4 shows the ultimate analysis, empirical chemical formula, TMY, SMY, and Ddeg of SSW and SS. The S/I ratio had a significant effect on the SMY and Ddeg of SSW and SS (p < 0.05). The SMY and Ddeg of SSW were 611.5 and 711.2 Nml CH4/g VSadded, and 84.3 and 98% at an S/I ratio of 1, and 0.5, respectively. TMY was observed at 725.5 Nml CH4/g vs. of SSW. Following this finding, Yoon et al. [5] reported that the anaerobic digestion of SSW showed an SMY of 357-589 Nml CH4/g VSadded. The anaerobic digestion of swine offal showed a high SMY of 866 Nml CH4/g VSadded [18], mixed SSW (blood, meat, fat, and flour). A study in Denmark showed the highest SMY of 620 Nml CH4/g VSadded [32], and SSW (meat tissue, fat, bristles, and intestinal wastes) from Poland showed an SMY of 839.2 Nml CH4/g VSadded [14]. A wide range of SMY from SSW occurred because of variations in the SSW and inoculum characteristics.  Table 4 shows the ultimate analysis, empirical chemical formula, TMY, SMY, and D deg of SSW and SS. The S/I ratio had a significant effect on the SMY and D deg of SSW and SS (p < 0.05). The SMY and D deg of SSW were 611.5 and 711.2 Nml CH 4 /g VS added , and 84.3 and 98% at an S/I ratio of 1, and 0.5, respectively. TMY was observed at 725.5 Nml CH 4 /g vs. of SSW. Following this finding, Yoon et al. [5] reported that the anaerobic digestion of SSW showed an SMY of 357-589 Nml CH 4 /g VS added . The anaerobic digestion of swine offal showed a high SMY of 866 Nml CH 4 /g VS added [18], mixed SSW (blood, meat, fat, and flour). A study in Denmark showed the highest SMY of 620 Nml CH 4 /g VS added [32], and SSW (meat tissue, fat, bristles, and intestinal wastes) from Poland showed an SMY of 839.2 Nml CH 4 /g VS added [14]. A wide range of SMY from SSW occurred because of variations in the SSW and inoculum characteristics.

Anaerobic Digestion of SSW and SS
In the case of SS, the TMY was 529.5 Nml CH 4 /g VS, whereas the SMY and D deg were 310.1 and 516.3 Nml CH 4 /g VS added and 58.6 and 97.5% at an S/I ratio of 1 and 0.5, respectively. The S/I ratio of 1 indicated a lower degradability in SS than in SSW (84%). This might be due to the low vs. content observed in the SS. Previous studies demonstrated similar methane yields from SS. Zhang et al. [29], and Rodríguez-Abalde et al. [18] reported that the SMY from the anaerobic digestion of swine manure were 358.7 Nml CH 4 /g VS added , and 204 Nml CH 4 /g VS added , respectively. Chae et al. [37] observed 228-437 Nml CH 4 /g vs. at SS feed loads between 5% and 40% (v/v reactor). SSW was a better substrate than the SS in the anaerobic digester as, in this study, a higher degradability and SMY were found. The improvement of D deg and SMY at a low S/I ratio was also observed in a previous study, where Yoon et al. [5] reported that the D deg of the swine intestine residue and swine digestive tract content improved from 77.0 to 85.8% and from 69.9 to 86.3% when the S/I ratio reduced from 1 to 0.5, respectively. The SMY was also improved from 361 to 446 mL CH 4 /g VS added of SSW at an S/I ratio of 1-0.5. The high inocula during batch anaerobic digestion could prevent VFA accumulation at the initial stage of anaerobic digestion and the rapid conversion of VFA into CH 4 [38]; the same results were observed in this study. Moreover, high inoculums can dilute the toxic content in the substrate, which might explain the improvement of the SMY and D deg of SSW and SS at an S/I ratio of 0.5 than 1.
The modified Gompertz formula (Equation (6)) was used to estimate the maximum M max , R max , λ, M max , T 90 , and T eff , and the estimated parameters are shown in Table 5. The estimated CMY from the modified Gompertz formula was plotted against the experimental CMY of SSW and SS to test the model accuracy ( Figure 2). The correlation coefficient (R2) ranged from 0.989 to 0.999 (Table 5), indicating the best fit to the substrate used in the experiment. Previous studies also predicted the same accuracy, where the CMY curve from the anaerobic codigestion of SS, dewatered sewage sludge2 and apple waste was best fitted with the modified Gompertz formula [29,30]. A low S/I ratio resulted in a higher M max for SSW and SS (p < 0.05), whereas SSW had significantly higher M max than SS at both S/I ratios (p < 0.05). The M max was estimated for SSW at 598.7 and 723.7 Nml CH 4 /kg VS added at an S/I ratio of 1 and 0.5, whereas SS showed only 289.8 and 453.2 Nml CH 4 /kg VS added , respectively.
In addition to M max , λ and digestion time (T 90 and T eff ) were also important anaerobic digestion parameters. An indicator of methanogen adaptation to the environment, λ also represented the substrate bio-availability [33,39]. The λ was estimated at 9 and 9.7 days for SSW at an S/I ratio of 0.5 and 1, respectively (Table 5). Following this, Rodríguez-Abalde et al. [18] observed 7 days of λ during the batch anaerobic digestion of SSW with a fat content of 65.7% DW. The long λ indicated that the vs. in SSW was not readily available for the microbes and the microbial adaptation to a high fat content [40,41]. This could be related to a high fat content in the SSW (53.6% DW), and fat requires more time for the anaerobic digestion [40].

Codigestion of SSW with SS
The codigestion effects of SSW with the SS parameters are shown in Table 6. The codigestion increased Mmax from 289.8 and 453.2 Nml CH4/g VSadded to 405.1 and 672.4 Nml CH4/g VSadded at an S/I ratio of 1 and 0.5, respectively. This was equal to a 22%-84% Mmax increase compared to the sole digestion of SS. Moreover, codigestion had no significant effect on T90 and Teff at both S/I ratios. This indicated that SSW codigestion with SS improved digestion efficiency in terms of higher CH4 generation with the help of a greater vs. addition from SSW. The mixing of SSW and SS increased the vs. content due to the high fat content in the SSW, and fat has a higher CH4 production potential than protein and carbohydrates.
Mmax of the codigested substrate at both S/I ratios was significantly lower than that for sole SSW digestion. The Mmax obtained from the codigestion of SSW and SS was 535.1, 482.9, and 405.1 Nml CH4/g VSadded at mixing percentages of 67%, 50%, and 33% of SSW at an S/I ratio of 1, respectively. Meanwhile, at an S/I ratio of 0.5, the Mmax was 672.4, 634.8, and 555.5 Nml CH4/g VSadded for the codigested mixture containing 67%, 50%, and 33% of However, the long λ might affect the overall anaerobic reactor performance, especially the digestion times such as T 90 and T eff (Table 5). T 90 is defined as the time required to obtain 90% of M max [30]. The T 90 calculated in this study was 30.7 and 33 days for SSW at an S/I ratio of 1 and 0.5, respectively. Subtracting T 90 with λ as T eff (effective digestion time) for SSW at an S/I ratio of 1 and 0.5 was 20.9 and 24 days, respectively. There was no significant difference for T 90 and T eff at different S/I ratios; however, these parameters were moderately higher in SSW than SS. T eff indicated that most CH 4 production of SSW requires 20.9-24 days assuming that there is no λ at the beginning of the digestion process. Thus, λ occurrence made the digestion process longer than necessary. A long λ, T 90 , and T eff might be caused by the high fat and low mineral content in the SSW, which takes more degradation time and inadequate nutrients. In practice, a long λ, T 90 , and T eff for waste treatment increase the operational cost, which reduces the economic benefit of the systems; therefore, these parameters must be reduced during anaerobic digestion. The codigestion of SSW with other organic matters might be the solution to reduce those parameters during anaerobic digestion.
Interestingly, SS demonstrated a higher R max with a rapid degradation of vs. at a minimum λ of 1.5 days and 0.2 days and at an S/I ratio of 0.5 and 1, respectively. These indicated that SS might have a more available, soluble, organic compound for a bacterial community in the digester. Compared to SSW, SS produced less M max at both S/I ratios. For instance, the M max of SS was only 48.4% of SSW at an S/I ratio of 1. However, SS had lower λ, T 90 , and T eff which indicated that vs. in SS was easily degradable and soluble. However, SS had low M max and SMY compared to SSW (p < 0.05) due to its low vs. content. SS showed a high FS content (32.4% DW) which indicated that it consisted of rich minerals that might benefit the anaerobic digestion process. On the other hand, a low SMY and M max might indicate the unviable use of SS as a feedstock in anaerobic digestion. Therefore, the codigestion of SS with SSW might help to achieve a better digestion efficiency by reducing the λ, T 90 , and T eff , improving CH 4 production, and thus becoming a viable option.

Codigestion of SSW with SS
The codigestion effects of SSW with the SS parameters are shown in Table 6. The codigestion increased M max from 289.8 and 453.2 Nml CH 4 /g VS added to 405.1 and 672.4 Nml CH 4 /g VS added at an S/I ratio of 1 and 0.5, respectively. This was equal to a 22-84% M max increase compared to the sole digestion of SS. Moreover, codigestion had no significant effect on T 90 and T eff at both S/I ratios. This indicated that SSW codigestion with SS improved digestion efficiency in terms of higher CH 4 generation with the help of a greater vs. addition from SSW. The mixing of SSW and SS increased the vs. content due to the high fat content in the SSW, and fat has a higher CH 4 production potential than protein and carbohydrates. Table 6. Anaerobic digestion parameters from codigestion of SSW with SS. The SSW content in the codigested mixture was 100% (P1), 0% (P2), 67% (P3), 50% (P4), and 33% (P5) on a vs. basis.  M max of the codigested substrate at both S/I ratios was significantly lower than that for sole SSW digestion. The M max obtained from the codigestion of SSW and SS was 535.1, 482.9, and 405.1 Nml CH 4 /g VS added at mixing percentages of 67%, 50%, and 33% of SSW at an S/I ratio of 1, respectively. Meanwhile, at an S/I ratio of 0.5, the M max was 672.4, 634.8, and 555.5 Nml CH 4 /g VS added for the codigested mixture containing 67%, 50%, and 33% Previous studies also revealed a more reduced M max during the anaerobic codigestion of slaughter waste with other substrates, compared to sole digestion. Borowski and Kubacki [14] observed that SSW alone had an M max value of 839 Nml CH 4 /g VS added , while, during the codigestion with sewage sludge at a mixing ratio of 30% and 50% weight per weight (w/w), the M max ranged from 472.8 to 608.6 Nml CH 4 /g VS added . Rodríguez-Abalde et al. [18] obtained M max of 430 Nml CH 4 /g VS added in the codigestion of 36% SSW with 67% swine manure (SM). This was lower than that for SSW sole digestion (M max of 809 Nml CH 4 /g VS added ). The reduced M max was attributed to the low methane potential in the codigested materials other than SSW.
However, the codigestion of SSW and SS had a significant effect on λ, T 90 , and T eff ( Table 6). The anaerobic digestion of sole SSW had the highest λ, T 90 , and T eff at both S/I ratios. All the codigested mixtures had a significantly lower (p < 0.05) λ, T 90 , and T eff compared to sole SSW digestion ( Table 6). Codigestion shortened the λ from 9 and 9.7 days (sole digestion of SSW) to between 1.2 and 5.7 days (SSW and SS codigestion) at an S/I ratio of 1 and 0.5, respectively. It also shortened the T eff from 20.9 and 24.0 days (SSW sole digestion) to between 13.5 and 15.7 days (SSW and SS codigestion) at an S/I ratio of 1 and 0.5, respectively. T 90 was also shortened from 30.7 and 33 days (SSW sole digestion) to 14.9-20.6 days (SSW and SS codigestion) at a S/I ratio of 1 and 0.5, respectively. The improved digestion properties such as λ, T 90 , and T eff were attributed to the SS characteristics (dissolved OM and micronutrients) than SSW. SS had a high moisture content (95.9% FW), indicating that the vs. and nutrients were mostly present in soluble form. Moreover, Table 7 shows that SS had higher cobalt (Co), iron (Fe), molybdenum (Mo), nickel (Ni), tungsten (W), and zinc (Zn) contents compared to SSW. The last stage of methanogenesis, CH 3 -CoM formation, was facilitated by the enzyme, methyltransferase. Co and Ni are known cofactors for the enzyme reaction [40,42]. This indicated that an adequate concentration of Co and Ni during anaerobic digestion was necessary. Shorter λ, T 90 , and T eff indicated that the codigestion of SSW and SS improved anaerobic digestion efficiency in terms of a shorter digestion time from an SSW perspective.  Table 7 shows that the SSW only had a Co and Ni content of 0.3 and 2.9 mg/kg, while SS contained 10.4 and 25 mg/kg, respectively. The recommended Co and Ni content for anaerobic digestion was between 0.03 and 35 mg/kg [40]. This indicated that the low Co and Ni content in SSW was still adequate for anaerobic digestion. This was confirmed from the sole digestion of SSW, which showed a high M max , D deg , and SMY (Tables 4 and 5). However, a long λ, T 90 , and T eff observed during the anaerobic digestion of SSW might indicate that both Co and Ni might not be readily available from the start of anaerobic digestion.
Mineral availability is an important factor in anaerobic digestion, and it exists in a soluble (free ions) form, complex form (organic or inorganic), and precipitate form [42][43][44].
A high vs. content of SSW might form a mineral complex with Co and Ni, which makes it unavailable from the start of anaerobic digestion. After the vs. is digested through hydrolysis and acidification processes, the Co and Ni then become available to be utilized by the microorganisms. This might be one cause of the long λ, T 90 , and T eff observed during the anaerobic digestion of SSW.
On the other hand, SS had low VSs with a higher moisture, Co, and Ni content than SSW, which was attributed to the improved digestion (shorter λ, T 90 , and T eff ) in the codigestion mixture compared to sole SSW digestion. Moreover, the effect of SSW and SS codigestion on other minerals and the anaerobic digestion parameters seems to be an interesting topic for further studies. Substrate codigestion can produce either a synergistic or an antagonistic effect. The antagonistic effect occurred when the experimental M max was lower than the simulated M max , whereas the synergistic effect occurred when the experimental M max was higher than the simulated M max [31]. The experimental M max of SSW codigestion, with an SS at an S/I ratio of 1 and 0.5, showed an α value of more than 1 indicating that the synergistic effect occurred during the anaerobic codigestion of SSW and SS ( Table 8). The combination of the high vs. and fat contents of SSW and highly soluble OM, and Co, and Ni contents of SS were the reason for the synergistic effect. Digestate characteristics are further recommended for a detailed analysis of their possible agricultural reuse, especially regarding heavy metals and microbiological parameters.  1 The SSW content in the codigested mixture was 100% (P1), 0% (P2), 67% (P3), 50% (P4), and 33% (P5) on a vs. basis. A indicated that the experiment was conducted at an S/I ratio of 1, while B indicated that the experiment was conducted at an S/I ratio of 0.5. 2 Values are expressed as mean ± standard deviation; unit in Nml CH 4 /g VS added. 3 α = Experimental M max /Simulated M max (Equation (6)).

Conclusions
In this study, we conclude that the SSW and SS have significant biomethane production potential. However, the anaerobic codigestion of SSW and SS improves the λ and T eff , as well as causing a considerable amount of methane production. SSW contributes more organic matter, while SS provides more minerals for the improved digestion. The substrate to inoculums ratio affects methane production, significantly. Experimental and simulated methane yields are correlated. Still, the exact mechanisms of the shorter λ and T eff of SSW codigestion with SS are not clear. Hence, the codigestion effect on mineral availability and anaerobic digestion parameters seems to be an interesting topic for future research.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.