Impact of Thermal Pretreatment on the Physicochemical Characteristics and Biomethane Yield Potential of Solid Slaughter Waste from High-Throughput Red Meat Abattoirs Valorized as a Potential Feedstock for Biogas Production

The results for pH, TS, VS, and moisture characterization of abattoir feedstock are presented in Table 2. The pH of red meat abattoir slaughter waste feedstock was measured prior to AD at day 0. pH is a very important parameter in AD as it influences the stability of the digestion process [17,40,41,42]. The pH range for stability of anaerobic digestion has been recommended to be within the range of 6.5 to 8.5, with an optimal range of about 6.8 to 7.2 for stabilized anaerobic digestion and microbial activity [43,44,45]. The pH values for pre-treated samples were observed to be >6.5 in this study in all pre-treated samples (Table 3), thus indicating the ability of pasteurization and sterilization pre-treatments to enhance the stability of abattoir waste feedstock prior to AD. The pH values for pasteurized and sterilized pre-treated samples did not differ significantly (p ≤ 0.05) across all analyzed red meat abattoir slaughter samples, while pH values for untreated and pre-treated samples differed significantly (p ≤ 0.05). This may be attributed to the production of hydroxide ions (OH⁻) being higher than the production of hydrogen ions (H⁺) during pre-treatment. According to Budiyono et al. [46], Islam et al. [47], and Calicioglu et al. [46], methanogens thrive in slightly alkaline to neutral environments (6–8.5). The results observed in this study for both pasteurized and sterilized pre-treated samples fell within the optimal range of 6.5 to 8.5 for stable anaerobic digestion and microbial activity. This indicates that sterilization and pasteurization have the ability to create a more conducive environment for methanogen activity by offering sufficient buffering capacity against pH drop [41,47,48,49]. The results for pH reported in this study were lower as compared to those reported by Budiyono et al. [46], who observed a pH of 7.19 ± 0.06. The difference in results for pH observed in this study and those of Budiyono et al. [46] may be attributed to the difference in feedstock composition, as in their study, the abattoir waste included manure from holding pens and wastewater, which were not included in this study. Results observed in this study were similar to those reported by Bayr et al. [49], where a pH value range of 6.0 to 6.8 was reported.

Results for VS and TS (Table 3) in this study were observed to be higher in pasteurized and sterilized samples as compared to untreated samples for cattle, sheep, and pig abattoir slaughter waste feedstock. The VS content of feedstock in AD is a very important parameter, as it determines the amount of organic matter contained in the feedstock that is biodegradable. The increase in TS contents in both sterilized and pasteurized pre-treated as compared to untreated samples may be attributed to the water contents of the red meat abattoir waste evaporating due to the high temperature, thus indicating that sterilization and pasteurization have the ability to enhance the solubilization of the pre-treated samples [50,51]. Results for TS and VS in this study differed significantly across untreated, pasteurized, and sterilized pre-treated samples (p ≤ 0.05). According to the literature, the VS of abattoir solid slaughter waste, comprising rumen content, intestinal organs, and meat trimmings, were reported to be around 80% of TS [50,51].

According to Jha et al. [51], a VS value ≥ 60% depicts the high proportion of feedstock fraction that is biodegradable and could be utilized as feedstock for mono-digestion for biogas production [16,40]. The VS values across all analyzed red meat abattoir samples in this study were observed to be >60%, with sterilized and pasteurized samples of VS being >72%. This indicated the effects of sterilization and pasteurization pre-treatment’s ability to enhance biodegradability in red meat abattoirs [51,52,53]. These increases in VS can be attributed to the capability of the high temperature of the pasteurization and sterilization imposed to release more volatile compounds within the samples, making them more readily available for digestion by anaerobes [51,52]. The results obtained in this study for TS and VS were similar to those observed by Alarcon et al. [52] and were lower compared to those reported by Ware and Power [14]. The difference in observed results for VS and TS in this study compared to Ware and Power [14] may be owing to the difference in feedstock composition, as in this study only solid red meat abattoir waste was used as a single waste stream (mono-digestion), while in their study the abattoir waste was co-digested with other organic waste such as food waste and activated municipal sludge. The TS and VS results obtained in this study for sterilized pig abattoir waste were similar to those reported by Budiyono et al. [46], who observed VS and TS increase due to thermal pre-treatment using similar pasteurization and sterilization treatment conditions. Budiyono et al. [46] reported a VS of 66.6% in untreated slaughter waste, 76.0% in pasteurized samples, and 74.6% in sterilized samples, and a TS value of 22.4%, 24.1%, and 26.6% for untreated, pasteurized, and sterilized slaughter waste, respectively.

Feedstock moisture content is one of the most important factors affecting the efficiency of AD, more especially the hydrolysis phase [54,55]. Although results in previous studies suggest that the efficiency of AD increases as feedstock moisture levels increase [46,54,56], there is no information on optimum moisture levels reported. In support of the studies by Zamri et al. [55], Cvetković et al. [54], and Kabeyi and Olanrewaju [57], a study by Khumalo [54] reported that activated sludge with 95% moisture content had a hydrolysis phase of six days, while sludge with 88% moisture content had a hydrolysis phase of 30 days. Similar observations were also observed in this study in sterilized cattle and pig abattoir slaughter samples (Table 3), as the moisture contents were observed to be 63.09 ± 2.07% and 69.95 ± 0.98%, respectively, and the hydrolysis phase was observed to be slower compared to pasteurized and untreated samples during AD. Sterilization pre-treatment was observed to reduce more moisture content in cattle, sheep, and pig abattoir slaughter waste in this study compared to untreated and pasteurized samples, and the results differed significantly (p 51,52,53]. There was no significant difference (p Table 3).

The results for COD concentration are presented in Table 4; in this study, COD varied from 12,530.35 ± 538.22 mg/L to 15,384.9 ± 333.17 mg/L across the studied abattoir waste samples (p ≤ 0.05). Pasteurization and sterilization pre-treatment in this study was observed to reduce COD concentration in abattoir samples; this may be due to the protein denaturing effect of high temperatures [52,58]. According to Otero et al. [53], Ortner et al. [59], and Obileke et al. [60], reduction in COD in feedstock occurs due to the degradation of complex organic compounds, which in turn increases the stability of the AD process by reducing organic acid production during digestion that will cause pH drop, thus enhancing microbial metabolism during AD. The results in this study were higher than those reported by Khumalo [54], Budiyono et al. [46], and Otero et al. [53], and were observed to be lower than those reported by Ortner et al. [59]; this is owing to the difference in feedstock composition.

The organic carbon produced during AD of red meat abattoir waste is mostly produced during the degradation of carbohydrates by microorganisms [57,58]. Carbon as well as nitrogen are essential nutrients for regeneration and growth of methanogens during the AD process [54,59]. The results for carbon concentrations are presented in Table 4. In this study, carbon concentrations differed significantly (p ≤ 0.05) between untreated abattoir samples and treated abattoir samples. Carbon concentrations for pasteurized and sterilized samples were higher as compared to untreated samples; this may be due to the composition of carbohydrates being mostly carbon and water, and during heating the water evaporated, thus leaving C as the most available element within the feedstock [60]. The results obtained in this study were lower compared to those reported by Khumalo [54] and Ware and Power [14], who observed a C concertation of 65.60 ± 0.3% TS and 65.8% TS, respectively, in solid abattoir waste, which consisted of condemned meat, offal, and blood. In their study, Ware and Power [14] examined solid red meat abattoir feedstock that was co-digested with dissolved air flotation sludge and wastewater, while Khumalo [54] examined solid slaughter waste co-digested with winery sludge waste, whereas in this study, only solid red meat slaughter waste was used for mono-digestion.

Nitrogen (N) concentrations in this study were observed to range from 1.89 ± 0.74% to 2.68 ± 0.81% (p Table 4). The C/N ratio is very important in AD of organic waste, as methanogens use carbon from the degradation of carbohydrates and N from the proteins as an energy source to produce biogas [60,61,62]. The optimum C/N ratio for AD was recommended in previous studies to range from 15 to 30 [62,63,64,65,66,67,68,69,70,71]. The results for the C/N ratios observed in this study (Table 4) ranged from 18.12 ± 0.41 to 23.91 ± 0.88 across the analyzed red meat abattoir waste and were within the recommended range, thus suggesting that abattoir waste can serve as a good substrate as a single waste stream for biogas production [66,72].

According to Wang et al. [17], Nnaemeka [43], and Kigozi et al. [45], it is important for the C/N ratio not to be higher than 30, as this may result in the quick depletion of nitrogen by bacteria, thus causing carbon to be in excess in the digester, which may result in low biogas yield. The low C/N ratio (43], Kigozi et al. [45], and Xia et al. [38], resulting in excess nitrogen in the digester, leading to the formation of ammonia, thus resulting in an increase in pH (>8.5) and a reduced biogas yield due to microbial inhibition. If the C/N ratio is higher or lower than the recommended range, co-digestion is recommended to increase the digestion process and increase biogas yield [17,38,42,45]. The C/N ratios observed in this study in untreated, pasteurized, and sterilized cattle, sheep, and pig slaughter waste were >10 and 45] and Xia et al. [38] that thermal pre-treatment has the ability to enhance the solubilization of organic matter, thereby enhancing feedstock biodegradability by microorganisms during AD.

The results for NH₄⁺ in this study are presented in Table 4 and ranged from 1.03 ± 0.09 g/L to 3.08 ± 0.31 g/L (p ₄⁺ was higher in sterilized cattle, sheep, and pig samples compared to untreated and pasteurized samples. This may be attributed to the higher temperatures of sterilization compared to pasteurization enhancing Maillard reactions in the feedstock, which may have produced sugar-amino acid compounds with low biodegradability. According to Wang et al. [17], NH₄⁺ concentrations >3000 mg L⁻¹ may cause inhibition of microbial growth during AD. The results obtained in this study were within the recommended limits reported by Wang et al. [17], except for the sterilized cattle abattoir waste, which was observed to have an NH₄⁺ concentration of about 3.08 ± 0.31 g/L.

The studies by Wang et al. [17], Zhang et al. [18], and Renggaman et al. [73] reported that high pH (>8.5) and high temperature (>90 °C) were reported to increase NH₄⁺, thus causing inhibition to microbial activity due to its high permeability to bacterial cell membranes and, as a result, reducing biomethane yield. That was evident in this study, as sterilization of cattle slaughter waste was conducted at 133 °C and resulted in NH₄⁺ > 3000 mg/L. However, Edström et al. [28] indicated that when NH₄⁺ concentration is >3000 mg/L, the AD process, particularly methanogen activity, is inhibited at any pH level, thus resulting in lower biomethane yield. That was also evident in this study, as sterilized cattle slaughter waste samples recorded a pH of 6.96, which was observed to be higher compared to other analyzed red meat slaughter samples, and biomethane yield was lower in sterilized cattle samples compared to untreated and pasteurized cattle samples from day 1 to day 23 HRT. In a similar study, Vavilin et al. [74] reported that an NH₄⁺ concentration of 2500 mg/L inhibited methane production, while an NH₄⁺-N concentration of 3300 mg/L inhibited methanogenesis, completely irrespective of pre-treatment temperature and pH. Similar observations reported by Vavilin et al. [74] were also noted in sterilized pig abattoir waste in this study, where NH₄⁺ was 2.36 ± 0.36 g/L, though lower compared to the reported value of 2500 mg/L; methane production was lowest compared to untreated and pasteurized pig abattoir waste from day 1 to day 8 HRT.

In order to control NH₄⁺ inhibition in AD, previous researchers reported that enhancing methanogens activities by acclimatizing and immobilizing microorganisms in the digester, along with pH (optimal range of 6.8–7.2) and temperature control, may contribute to the reduction of NH₄⁺ concentration [14,17,71,75,76,77]. During this study, to minimize NH₄⁺ toxicity in the digesters, the temperature was kept constant at 37 ± 2 °C (using a water bath) and acclimatization of the microorganism was not performed, which may have resulted in NH₄⁺ toxicity in sterilized cattle and pig abattoir slaughter waste samples. As the AMPTS II is a closed system, it was not possible to monitor the pH value during the AD process, but the pH at day 40 of HRT was observed to range from 7.83 ± 0.06 to 8.11 ± 0.09 across the analyzed red meat abattoir samples in this study, which may indicate that the pH remained slightly alkaline throughout the AD process. The results obtained in this study for NH₄⁺ were lower than those reported by Njoya [16], Tsegaye et al. [78], and Suanon et al. [79], who reported NH₄⁺ concentrations were >4000 mg/L. The results of NH₄⁺ concentrations observed in this study were also lower compared to those previously observed in AD of abattoir waste by Lauterböck et al. [80] and Musa et al. [81], where concentration of NH₄⁺ exceeded 6000 mg/L.

The VS in the red meat slaughter waste was present in the form of protein, lipid, and carbohydrate contents (Table 4), with a concentration range of 33.45 ± 0.04% TS to 45.8 ± 1.25% TS, 13.21 ± 0.41% TS to 27.3 ± 0.77% TS, and 2.79 ± 0.19% TS to 14.24 ± 0.46% TS, respectively, for proteins, lipids, and carbohydrates across the analyzed samples in this study (p ≤ 0.05). As abattoir waste is characterized by high protein and lipid content, this was also observed in our study, as the concentration of proteins and lipids was higher compared to carbohydrate concentrations. Carbohydrate concentrations in untreated red meat abattoir slaughter samples in this study were observed to be higher than those of pasteurized and sterilized pre-treated samples, and results differed significantly across the analyzed samples (p ≤ 0.05).

The high carbohydrate concentration in untreated red meat slaughter waste was reported previously to be associated with partially digested lignocellulosic material, as well as the crude fiber concentration contained in ruminal content that was included as part of the feedstock in this study [82]. Pasteurization pre-treatments were also observed to increase carbohydrate concentration in cattle, sheep, and pig abattoir slaughter waste samples, attributed to the solubilization of complex molecules to simpler sugars which are easily biodegraded by methanogens during AD [73].

The high protein content in the abattoir samples in this study is consistent with the literature, where it has been reported that abattoir waste is rich in both proteins and lipids [73,74,75]. Pasteurization pre-treatment in this study was observed to increase the protein and lipid concentration as compared to untreated and sterilized samples due to the enhancement of organic matter solubilization. Sterilization had a negative impact on protein concentration, as the values were low compared to control and pasteurization treatments. This may be due to the denaturing effect of the high-temperature sterilization process on proteins [73,75].

Biomethane production for the studied abattoir waste samples in this study were performed with a 40-day HRT and the results are presented in Figure 2, Figure 3 and Figure 4. Biomethane accumulation in this study varied from 1756.33 Nml to 3297.87 Nml (1.77 m³_CH4/kg_VS to 3.30 m³_CH4/kg_VS) for cattle abattoir waste (Figure 2), 1125.4 Nml to 1821.73 Nml (1.23 m³_CH4/kg_VS to 1.82 m³_CH4/kg_VS) for sheep abattoir waste (Figure 3), and 610.67 Nml to 949.57 Nml (0.611 m³_CH4/kg_VS to 0.95 m³_CH4/kg_VS) for piggery waste (Figure 4). Biomethane production was observed from day 1 in all samples, and all pre-treated abattoir waste samples yielded more biomethane as compared to untreated abattoir waste samples at the end of the 40-day HRT. This can be attributed to the pre-treatment breaking down complex molecules into simpler substances, thus making them more bioavailable for degradation by methanogenic bacteria. The degradation of the complex molecules has been observed previously to enhance the hydrolysis stage of the anaerobic digestion process. This observation was also depicted in this study, where sterilized sheep abattoir slaughter waste samples and pasteurized samples yielded more biomethane in the early days of digestion as compared to untreated samples.

The highest methane yields were obtained in sterilized pre-treated cattle waste despite its low biomethane production from day 1 to day 23 (Figure 2) as compared to the control and pasteurized cattle abattoir waste. The delay in methane production may be attributed to the production of long chain fatty acids (LCFAs) from the hydrolysis of fats within the samples, which were reported in previous studies to reduce methane activity [54,74]. Other authors have reported that sterilization causes the accumulation of microbial metabolic waste including the accumulation of metabolic acids which may impact the hydrolysis stage, thus affecting the microbial activity required for AD [14,54,59,60,74]. This also supports the findings reported by Kavuma et al. [15], who observed in their study that sterilization may alter the environmental conditions necessary for microbial growth due to the hypertonic environment caused by the high temperature and pressure during pre-treatment, which in turn reduced gas production during the early days of AD and led to the reduction in food nutrients necessary to support microbial growth.

The hypertonic environment due to sterilization has been reported in previous studies to further result in the osmotic potential of the abattoir feedstock being higher than that of the microbial cell cytoplasm, thus affecting the growth of microorganisms in the digester, thereby reducing gas production [14,52,53,60,61]. This effect of reduced biogas production in the early days of AD was also evident in this study in sterilized cattle abattoir slaughter waste (Figure 2) and pig abattoir slaughter waste (Figure 4). Sterilization and pasteurization pre-treatment of red meat abattoir slaughter waste had a significant impact on the biomethane production yield (p Table 5).

The earlier start of biomethane production in all samples in this study may be attributed to the fact that the inoculum that was used already contained active microorganisms that were adapted to the reaction conditions and the type of feedstock in the digester, thereby reducing the lag phase [83,84]. The biomethane production of untreated and pre-treated samples in this study was observed to reach stable methane production by 28–30 days HRT and AD was terminated when daily biomethane yield was less than 1% of the total biomethane volume produced throughout the AD process for four consecutive days, in accordance with Ware and Power [14]. Termination of AD was performed at day 40 of HRT.

Biomethane yield in pasteurized samples in this study was observed to be higher than the untreated and sterilized samples (Figure 2, Figure 3 and Figure 4); this may suggest that pasteurization produced significant thermal particle disintegration and hydrolysis, realizing more readily biodegradable compounds [20,51,52,53,54,59,60]. Biomethane accumulation of sterilized cattle and pig abattoir slaughter waste started slow, and only picked up at day 20 and day 9, respectively. This may be due to the particle disintegration and the hydrolysis process being affected by high sterilization temperature, which may have denatured proteins in the feedstock, thereby affecting the chemical structure and composition of the nutrients needed for microbial growth [22,29,54]. The impact of sterilization pre-treatment on protein denaturation is also evident in this study, where protein concentrations for sterilized samples were lower as compared to control and pasteurized samples (Table 4). Thus, sterilization leads to lower bioavailability of biodegradable compounds.

Even though biomethane accumulation in sterilized samples started slow as compared to untreated and pasteurized samples, both sterilized and pasteurized samples were observed to have produced higher biomethane when the experiment was terminated at day 40 HRT. This increase due to thermal pre-treatment, with positive effects on anaerobic digestion performance, has been reported for many different types of organic waste, i.e., for sewage sludge by Oludare et al. [75] and pig manure by Edström et al. [28].

The results obtained in this study for biomethane accumulation in pasteurized and sterilized cattle abattoir waste were higher compared to those reported by Li et al. [20] (0.46 m³CH₄/kg_VS to 0.58 m³CH₄/kg_VS) [26] (0.23 m³CH₄/kg_VS to 0.62 m³CH₄/kg_VS) and Palatsi et al. [84] (0.63 m³CH₄/kg_VS to 0.78 m³CH₄/kg_VS). The difference in biomethane yield in this study as compared to that reported by Rodriguez-Abalde et al. [25], Palatsi et al. [84], and Hejnfelt and Angelidaki [27] may be due to the difference in HRT, operational temperature, inoculum used and the physicochemical properties of the feedstock. The results of biomethane accumulation for cattle abattoir waste in this study were also higher than those reported by Yenigün and Demirel [77], who observed a methane yield of 0.681 m³CH₄/kg_VS during AD of slaughter waste (cattle rumen contents mixed with blood). This may be due to the composition of the feedstock used as well as the AD operational temperature, as in their study rumen content, fat, and blood were used as feedstock with an AD operation temperature of 55 °C. The results for cattle abattoir waste observed in this study were also higher compared to those reported by Ware and Power [14], where the authors reported a biomethane yield of about 0.64 m³CH₄/kg_VS. The AD of cattle abattoir slaughter waste feedstock in a study by Ware and Power [14] was operated under a mesophilic temperature of 39 ± 2 °C, which was slightly higher than the 37 ± 2 °C that was used in this study, which could be the reason for the difference in biomethane yield observed. It was also noted that the C/N ratio is a critical parameter for anaerobes in AD; the C/N ratio in this study was higher compared to those reported by Ware and Power [14], which may be the other contributor to higher biomethane yield observed in this study. The results for piggery biomethane accumulation in this study were observed to be in a range similar to those reported by Díaz et al. [78], where biomethane accumulation for pasteurized and sterilized pre-treated piggery abattoir waste ranged from 0.58 m³CH₄/kg_VS to 0.96 m³CH₄/kg_VS. The biomethane accumulation observed in this study was also higher than that observed by Bayr et al. [49], Rodriguez-Abalde et al. [25], and Hejnfelt and Angelidaki [27], who reported a biomethane accumulation of 0.43 m³CH₄/kg_VS, 0.58 m³CH₄/kg_VS, and 0.23–0.62 m³CH₄/kg_VS, respectively, for pasteurized pig abattoir waste.

During this study, it was observed that pre-treatment had a significant impact on biomethane accumulation (p ≤ 0.05) (Table 5), where a twofold increase was observed in both pasteurized and sterilized cattle, sheep, and piggery abattoir samples at day 40 HRT. Similar observations were also reported by Edström et al. [28], but they observed a fourfold increase in biomethane yield after pasteurization pre-treatment of abattoir waste at 70 °C for 1 h. The high methane yield of pre-treated samples as compared to untreated samples was attributed to the fact that pre-treatment decreased particle sizes, thus leading to an increase in the accessible active sites to which exocellular enzymes can attach and cleave complex macromolecules into simpler and more biodegradable constituents [29].

The high methane yield in pre-treated samples may also be due to the increased accessibility of lipids to the microorganisms resulting from the heat treatments, i.e., pasteurization and sterilization [29,75]. In contrast to the observed results, Hejnfelt and Angelidaki [27] reported that pre-treatment (pasteurization at 70 °C for 1 h and sterilization at 133 °C, 3 bar for 20 min) had no effects on the biomethane accumulation of abattoir waste. Until now, there has been limited or no research conducted on solid sheep abattoir waste and this study may be the first to report on the AD of such feedstock.

The results for biogas composition are presented in Table 6. The biogas composition in this study ranged from 54.69 ± 1.66% to 65.17 ± 3.99% CH₄, 129 ± 10.87 to 177 ± 36.98 ppm NH₃, 24.83 ± 1.17% to 39.4 ± 2.18% CO₂, 0.4 ± 0.00% to 1.6 ± 0.03% O₂, and 113 ± 33.74 ppm to 318 ± 31.56 ppm H₂S. The H₂S has been reported to cause corrosion in digesters and may lead to a further impact on methanogen activity due to sulfide inhibition [76,79]. The results in this study were higher compared to those observed by Kavuma et al. [15], who observed a methane volume range of 40.6% to 50.4%. The methane composition in this study was also higher than that reported by Budiyono et al. [46], where they observed a biogas CH₄ volume of about 48.89%. The methane volumes observed in this study were comparable to those reported by Tsegaye et al. [79], where the CH₄ volume in biogas from AD of abattoir waste ranged from 53.4% to 66%.

The methane volume in this study was lower compared to that reported by Njoya [16], where the CH₄ volume in biogas was observed to be 72.33%. The biogas composition observed in this study was comparable to that reported by Ware and Power [14], where they reported a CH₄ concentration of about 63%.

The volume of CO₂ reported in this study was lower than that reported by Nnaemeka [43], where the reported volume was 47.87%. A study by Njoya [16] reported a CO₂ volume of about 23.33% in biogas produced during AD of abattoir waste, which is lower than the CO₂ volume reported in this study. The CO₂ volume in this study was lower than that reported by Ware and Power [14], where a CO₂ concentration of about 37% was observed during the AD of abattoir waste. The presence of CO₂ and H₂S in biogas in high concentrations results in a low heat value of the produced biogas and reducing CO₂ and H₂S content significantly improves the quality of biogas produced. Pasteurization was observed to result in a lower concentration of CO₂ and H₂S as compared to untreated and sterilization pre-treatment.

Reduction of TS, VS, and COD during anaerobic digestion is referred to as solids/organic load reduction, which occurs when microbes degrade organic substrates [43,72,80]. The reduction efficiency of AD on TS, VS, and COD is represented in Table 7. The results for COD reduction in this study ranged from 73.19 ± 1.23% to 86.87 ± 1.09%, 71.33 ± 2.49% to 81.66 ± 1.99%, and 79.59 ± 0.99% to 87.61 ± 1.19% for cattle, sheep, and pig abattoir waste, respectively. The substrate COD is released in the medium due to microbial activities and their subsequent consumption as carbon sources [80]. Usually, COD removal is one key value reflecting the efficiency of anaerobic digestion of the liquid substrate, and for a typical stable anaerobic digestion system, the value of COD removal is generally above 80% [17]. COD is used as a carbon source by microorganisms during the anaerobic digestion of organic waste [72].

According to Lauterböck et al. [81], COD reduction of >80% shows that AD has excellent and stable performance for COD removal and thus has great potential for the biological treatment of red meat abattoir slaughter waste. The results obtained in this study for COD reduction during AD of red meat slaughter waste were >80% in all pre-treated samples, indicating that pasteurization and sterilization pre-treatment of red meat slaughter has the ability to promote higher COD reduction efficiency. There was a significant difference (p

The results observed in this study for COD removal were lower compared to Musa et al. [82], where the authors observed a >95% COD reduction in AD-treated abattoir waste with HRT ranging from 50 days to 90 days. This can be attributed to the longer HRT of 50 to 90 days as compared to the 40 days HRT in this study, as well as the feeding method, as they used continuous feeding, whereas in this study batch feeding was utilized. The COD removal at day 40 HRT observed by Musa et al. [82] was >83%, which is comparable to the observation of COD removal in this study. The results in this study for COD removal in abattoir waste were also lower as compared to those reported by Hernández et al. [85], where the COD removal efficiency of AD ranged from 82.58% to 91.41% with a 40-day HRT. The results for COD in this study were higher than those observed by Maria et al. [86], where they reported a removal efficacy of about 79% during AD of abattoir waste with 20 days HRT. It should also be noted that comparison for day 20 HRT in this study with that observed by Maria et al. [86] was not possible, as in our study the experiment was a batch AD in a closed system. The results reported in this study for COD were comparable to those reported by Sunada et al. [87], where the researchers observed a COD reduction efficacy of 85.9% with similar HRT and operational AD conditions with pasteurized and sterilized red meat slaughter waste.

The difference in COD removal efficacy observed in this study for pre-treated abattoir samples may be attributed to the impact of the different high temperatures imposed on simple organic compounds, making them more readily available for microorganisms in the digester [87]. High COD removal may also be attributed to longer HRT, which has been observed in previous studies by Musa et al. [82], Hernández et al. [85], Sunada et al. [87] to decrease VFA, thus causing a decrease in the accumulation of these acids in the digester and in turn an increase in COD removal efficacy. With the significant reduction in COD of >70% during the AD of red meat slaughter waste observed in this study, as well as the reduction reported in literature, it is evident the AD can be utilized for both waste management and treatment to minimize the environmental impact COD has in causing the eutrophication of receiving aerosols.

The reduction efficiency at 40 days HRT for VS was observed to range from 78.58 ± 2.01%–80.45 ± 2.55%, 78.53 ± 1.66%–88.65 ± 1.97%, and 81.13 ± 1.36%–84.35 ± 2.79% for cattle, sheep, and pig abattoir waste, respectively (Table 7). The VS reduction efficacy differed significantly between treatments (p 87], where VS reduction efficacy was reported to be 70.44% with 28 days HRT. The results in this study were also higher as compared to those reported by Alfa et al. [88], where the VS reduction was observed to be 47.12%. The high VS reduction during AD indicates that most of the organic matter contained in the cattle, sheep, and pig solid slaughter waste was converted to biogas [89,90].

At 40 days HRT, TS reduction ranged from 68.37 ± 2.01% to 76.98 ± 2.03%, 67.62 ± 1.83% to 78.96 ± 2.44%, 73.42 ± 2.02% to 79.86 ± 2.89% for cattle, sheep, and pig abattoir waste, respectively (Table 7). The TS reduction efficacy differed significantly (p 88], where the TS reduction efficacy in AD of abattoir waste was 53% for anaerobically digested pasteurized cattle abattoir waste. The lower TS reduction observed by Alfa et al. [88] as compared to this study may be due to their HRT of 30 days, whereas in this study HRT was 40 days.