Biomethane Potential of Beef Cattle Slaughterhouse Waste and the Impact of Co-Digestion with Cattle Feces and Swine Slurry

Abstract

Slaughterhouse waste (SW) poses significant environmental challenges due to its complex composition, but anaerobic digestion offers a way to recover valuable biogas from SW. This study investigated the anaerobic co-digestion of beef cattle slaughterhouse waste (BCSW) with either cattle feces (CF) or swine slurry (SS). The biomethane potential, maximum methane yield (M_max), lag phase duration, and effective digestion time (T_eff) for the individual substrates and the combinations were analyzed. BCSW alone exhibited M_max of 578.5 Nml CH₄/g VS_added with a lag phase of 11 days, while CF and SS alone exhibited M_max of 397.2 and 289.8 Nml CH₄/g VS_added, respectively. Co-digestion of BCSW and SS resulted in M_max increase of 48–75.5%, with negligible effects on T_eff compared to solitary SS digestion. Similarly, co-digestion of BCSW and CF increased M_max by 6.2–40.4%, with no significant impact on T_eff compared to solitary CF digestion. However, both co-digestions led to a reduction in M_max (12.1–27%) when compared to BCSW digestion alone. Co-digestion with SS shortened the lag phase duration by 2.8–7.8 days and accelerated T_eff by 5.8–8.3 days due to SS’s high concentrations of essential micronutrients like cobalt and nickel which aid digestion. This study concluded that co-digestion of BCSW with SS is an effective strategy for enhancing methane production and digestion efficiency, offering a viable approach for proper disposal of BCSW while improving biogas output.

1. Introduction

Annual meat consumption per capita in the Republic of Korea has undergone a significant increase, rising from 13.9 kg in 1980 to 62.5 kg in 2022, which has led to a corresponding rise in waste generation from slaughterhouses [1,2]. In 2023, approximately 158,213 tons of beef cattle slaughterhouse waste (BCSW) were generated in the Republic of Korea, resulting from the slaughter of approximately 1,060,569 cattle [3]. BCSW primarily consists of blood, digestive tract tissues, and internal organs that are not suitable for human consumption. It is rich in organic matter, fat, and protein, making it a potential source of energy-rich material [4].

Previously, BCSW was disposed of through various methods, including ocean dumping, composting, landfilling, and recycling as animal feed. However, since 2012, ocean dumping has been prohibited due to environmental concerns. As a result, there is increasing interest in finding commercially viable alternatives for managing slaughterhouse waste. One such approach involves converting slaughterhouse waste into methane (CH₄), which can be used for energy and heat production [1,4,5].

According to Renggaman et al. [6], the anaerobic digestion of pig slaughterhouse waste (PSW) alone resulted in a prolonged lag phase (λ) of 11 days but with a high methane generation potential (M_max) of 711 Nml CH₄/g VS_added. In contrast, the anaerobic digestion of animal waste such as swine slurry (SS) resulted in a short λ of one day but a lower methane yield of 453.2 Nml CH₄/g VS_added [6]. This suggests that while PSW has a high CH₄ yield but a long λ, and SS has a short λ but a low CH₄ yield, neither material is cost-effective when digested alone. Interestingly, Ware and Power [7] also pointed out that slaughterhouse waste (SW) contains large fat particles in the substrate that lead to limited surface area for hydrolytic bacteria, reducing long-chain fatty acid accumulation and process inhibition. Fat liquefaction, identified as the rate-limiting step in anaerobic digestion, resulted in an extended hydraulic retention time (HRT) of 50 days. Therefore, research to improve biomethane yield, digestibility, and HRT of SW in the anaerobic process is necessary.

A few studies reported the improvement of SW degradability in anaerobic digestion using various methods like batch and anaerobic sequential batch reactors and two-phase anaerobic systems (separation of hydrolytic–acidogenic and methanogenic phases) which also improved the HRT of slaughterhouse wastewater [8,9]. Another promising approach is the co-digestion of SW with other substrates to improve biomethane production and digestion efficiency [4]. Substrate co-digestion involves the simultaneous digestion of two or more organic wastes with complementary properties to improve CH₄ production or digestion efficiency [10,11,12]. This process can dilute inhibitory compounds, provide essential macro- and micronutrients, increase organic matter, and enhance the buffering capacity of the co-digested mixture, thus mitigating the negative effects of inhibitory compounds [6,13]. Numerous studies have demonstrated that co-digestion often yields better results than single-substrate digestion, leading to increased CH₄ production [4,11]. Not many studies used co-digestion of SW, while recently some researchers have published about co-digestion of SW with wheat straw [14] and livestock manures [2] and shown improved methane yield and digestibility of SW.

When selecting substrates for co-digestion, geographic availability and substrate compatibility are key considerations. The compatibility of wastes depends on how well their characteristics, such as nutrient content, complement each other. On the other hand, substrate availability is determined by the proximity of waste sources to biogas facilities [15].

SS is characterized by a high mineral content and relatively low volatile solid (VS) concentration, which limits CH₄ production during anaerobic digestion [15]. In contrast, BCSW contains lower mineral content but higher VS levels. This indicates that anaerobic co-digestion could benefit from the complementary properties of SS and BCSW. To minimize stress and transportation costs, animal farms are often located near slaughterhouses. In the Republic of Korea, approximately 97% of beef cattle slaughterhouses also process swine, indicating their proximity to swine farms [3]. Thus, co-digestion of BCSW with CF or SS may improve anaerobic digestion parameters such as M_max, λ, and effective digestion time (T_eff). However, the co-digestion of BCSW with CF or SS has not been extensively studied. Therefore, this study aims to investigate the characteristics of CF and BCSW, as well as the effects of co-digestion of CF, SS, or BCSW on anaerobic digestion parameters.

2. Materials and Methods

2.1. Samples and Inoculum Preparation

BCSW was obtained from a slaughterhouse in Yeongcheon City, Gyeongsang Province, Republic of Korea, which processed approximately 15,220 beef cattle in 2023 [3]. The BCSW comprised offal (internal organs) and viscera (digestive tract tissues). Blood, brain, spinal cord, and bones were excluded from the study. Blood and bones were excluded from this study as they were processed separately by the slaughterhouse, while the brain and spinal cord were omitted due to biosafety concerns. SS was sourced from a swine farm in Hoengseong County, Gangwon Province, Republic of Korea, while CF was collected from a Hanwoo cattle farm near the swine farm. To ensure homogeneity, BCSW was ground and sieved to a particle size of less than 5 mm, while CF and SS samples were screened using a 5 mm mesh. For ultimate and higher heating value (HHV) analysis, the samples were dried at 105 °C, while wet samples were used for proximate analysis and co-digestion tests. The characteristics of the inoculum and SS were obtained from previously published data [6]. The characteristics of CF and BCSW observed in this study were compared to these published values.

2.2. BMP Analysis and Co-Digestion Experiment

BMP analysis was performed on BCSW and CF using 250 mL serum bottles. The substrate-to-inoculum (S/I) ratio was maintained at 1 on a volatile solid basis (w/w VS_basis), with equal amounts (0.5 g VS) of inoculum and BCSW, CF, or SS added to each bottle. This study used an S/I ratio of 1, because in our previous study [6], that ratio provided the best microbial consortium for the anaerobic digestion. Distilled water was added to achieve a total volume of 200 mL. Control bottles containing only the inoculum were used to account for the biogas generated by the inoculum. Approximately 50 mL of each sample was taken from the containers for initial VS analysis. The headspace of each bottle was flushed with a gas mixture of N₂ and CO₂ (20:80% v/v) to establish anaerobic conditions. The serum vials were sealed with aluminum crimp seals and sterile rubber stoppers.

Co-digestion of BCSW with CF and SS was investigated in five proportions of BCSW (1:0, 0:1, 1:1, 2:1, and 1:2 w/w VS_basis) (

Table 1. Experimental design for anaerobic co-digestion of BCSW with CF or SS and its mixture.

Code	Substrate (% VS Basis) 1			Substrate (g VS) 1			Inoculum (g VS) 1
	BCSW	CF	SS	BCSW	CF	SS
C1	100	0	0	0.5	0	0	0.5
C2	0	100	0	0	0.5	0	0.5
C3	0	0	100	0	0	0.5	0.5
C4	67	33	0	0.33	0.17	0	0.5
C5	50	50	0	0.25	0.25	0	0.5
C6	33	67	0	0.17	0.33	0	0.5
C7	67	0	33	0.33	0	0.17	0.5
C8	50	0	50	0.25	0	0.25	0.5
C9	33	0	67	0.17	0	0.33	0.5

¹ All experiment were performed in triplicate.

). The schematic diagram of the experimental setup is shown in Figure 1. The co-digestion experiment followed the same methodology as the BMP analysis, with an S/I ratio of 1 w/w VS_basis. The BMP results for BCSW, CF, and SS as sole substrates were compared with the results of the co-digestion experiment.

2.3. Analytical Methods

2.3.1. Chemical, Proximate, Elemental, and Higher Heating Value (HHV) Analysis

The chemical composition of BCSW, SS, and CF, including fat, protein, neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose content, was analyzed according to procedures outlined in previous studies [6]. Moisture content, total solids (TSs), volatile solids (VSs), and fixed solids (FSs) were analyzed using the APHA Standard Method [16]. Elemental analysis (C, H, N, S, and O) of BCSW, CF, and SS was conducted based on prior research [6,17]. Dried samples of BCSW, CF, and SS were pulverized, sieved through a 5 mm mesh, and pelletized for HHV analysis using an oxygen bomb calorimeter. Benzoic acid pellets were used for calibration to ensure accurate HHV measurements [6,13].

2.3.2. Biogas Production, Composition, and Specific CH₄ Yield

The manometric method described by Renggaman et al. [6] was used to analyze biogas production. Gas composition was determined using an Agilent HP 6890N equipped with a thermal conductivity detector (TCD) and an HP-PLOT Q column. The specific methane yield (SMY) was calculated using Equation (1) [6]:

$$SMY=\left(MP/VS\right)\left(t_o/t_i\right)$$

(1)

where SMY is the specific CH₄ yield (Nml/g VS), MP is the volume of CH₄ produced (ml), VS is the organic content of the initial sample (g), t_o is the temperature at standard conditions (0 °C or 273 K), and t_i is the experimental temperature (35 °C or 308 K).

2.3.3. Theoretical CH₄ Yield (TMY)

Theoretical methane yield (TMY) was calculated based on the chemical composition of organic waste. In addition to CH₄, anaerobic digestion byproducts such as water (H₂O), carbon dioxide (CO₂), ammonia (NH₃), and hydrogen sulfide (H₂S) were considered (Equation (2)). The reaction coefficient for CH₄ (y) was calculated using Equation (3), and TMY was determined using Equation (4). Finally, anaerobic degradability (D_deg) was calculated using both SMY and TMY with Equation (5) [6]:

$${\mathrm{C}}_{\mathrm{a}}{\mathrm{H}}_{\mathrm{b}}{\mathrm{O}}_{\mathrm{c}}{\mathrm{N}}_{\mathrm{d}}{\mathrm{S}}_{\mathrm{e}}+\mathrm{x}{\mathrm{H}}_2\mathrm{O}\to \mathrm{y}{\mathrm{C}\mathrm{H}}_4+\mathrm{z}{\mathrm{C}\mathrm{O}}_2+\mathrm{d}{\mathrm{N}\mathrm{H}}_3+\mathrm{e}{\mathrm{H}}_2\mathrm{S}$$

(2)

$$\mathrm{y}=\left({\displaystyle \frac{\mathrm{a}}{2}}+{\displaystyle \frac{\mathrm{b}}{8}}-{\displaystyle \frac{\mathrm{c}}{4}}-{\displaystyle \frac{3\mathrm{d}}{8}}-{\displaystyle \frac{\mathrm{e}}{4}}\right)$$

(3)

$$\mathrm{T}\mathrm{M}\mathrm{Y}=1000\mathrm{y}/(12\mathrm{a}+\mathrm{b}+16\mathrm{c}+14\mathrm{d}+32\mathrm{e})\times 22.4$$

(4)

$${\mathrm{D}}_{\mathrm{d}\mathrm{e}\mathrm{g}}=\mathrm{S}\mathrm{M}\mathrm{Y}/\mathrm{T}\mathrm{M}\mathrm{Y}\times 100$$

(5)

where TMY is the theoretical CH₄ yield (N_ml CH₄/g VS) and D_deg is the degree of anaerobic degradation (%).

2.3.4. Kinetic Model

The kinetic analysis of anaerobic digestion focused on the lag phase (λ), cumulative CH₄ yield (CMY), and rate of CH₄ production. The modified Gompertz model (Equation (6)) was used to determine λ, M_max, and R_max [18,19]:

$$\mathrm{M}\left(\mathrm{t}\right)={\mathrm{M}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\mathrm{e}\mathrm{x}\mathrm{p}\left\{-\mathrm{e}\mathrm{x}\mathrm{p}\left[{\displaystyle \frac{{\mathrm{R}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\mathrm{e}}{{\mathrm{M}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}}\left(\lambda -1\right)+1\right]\right\}$$

(6)

where M(t) is the CMY at time t (Nml CH₄/g VS_added), M_max is the maximum CH₄ production potential (Nml CH₄/g VS_added), R_max is the maximum CH₄ production rate (Nml CH₄/g VS_added/day), λ is the lag phase (d), t is time (d), and e is Euler’s number (2.7183). M_max, R_max, λ, T₉₀ (time to reach 90% M_max), and regression coefficient (R²) were determined using non-linear least squares regression with Excel Solver [6]. The effective digestion time (T_eff) was calculated using Equation (7):

$${\mathrm{T}}_{\mathrm{e}\mathrm{f}\mathrm{f}}={\mathrm{T}}_{90}-\lambda$$

(7)

Simulated CH₄ production potential for co-digested mixtures was calculated using Equation (8) [6,18]:

$${\mathrm{M}}_{\mathrm{s}\mathrm{i}\mathrm{m}}={\mathrm{M}}_{\mathrm{B}\mathrm{C}\mathrm{S}\mathrm{W}}\times \mathrm{\%}{\mathrm{Y}}_{\mathrm{B}\mathrm{C}\mathrm{S}\mathrm{W}}+{\mathrm{M}}_{\mathrm{C}\mathrm{F}\hspace{0.17em}\mathrm{o}\mathrm{r}\hspace{0.17em}\mathrm{S}\mathrm{S}}\times \mathrm{\%}{\mathrm{Y}}_{\mathrm{C}\mathrm{F}\hspace{0.17em}\mathrm{o}\mathrm{r}\hspace{0.17em}\mathrm{S}\mathrm{S}}$$

(8)

where M_sim represents the simulated maximum methane production potential of the co-digested mixture (Nml CH₄/g VS_added), M_BCSW is the M_max of BCSW as determined by the modified Gompertz formula (Nml CH₄/g VS_added), %Y_BCSW is the proportion of BCSW in the mixture (%), M_CF or M_SS represents the M_max of CF or SS, respectively, as calculated from the modified Gompertz formula (Nml CH₄/g VS_added), and %Y_CF or %Yss refers to the percentage of CF or SS in the mixture (%).

2.3.5. Synergistic Effect

In co-digestion, each substrate can influence the anaerobic digestion process and affect the amount of methane (CH₄) generated. Thus, it is essential to apply Equation (9) to determine the synergistic relationship observed in this study:

$$\alpha ={\mathrm{M}}_{\mathrm{c}\mathrm{o}-\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}/{\mathrm{M}}_{\mathrm{s}\mathrm{i}\mathrm{m}}$$

(9)

where M_{co−digestion} is the experimental M_max obtained from the modified Gompertz formula (Equation (6)) for the co-digested substrate (Nml CH₄/g VS_added).

The results demonstrated that the α value can serve as a quantitative indicator of whether co-digested substrates exhibit an antagonistic, independent, or synergistic effect. Specifically, an antagonistic effect is indicated by α < 1, an independent effect by α = 1, and a synergistic effect by α > 1 [20].

2.3.6. Statistical Analysis

The anaerobic digestion parameters (SMY, M_max, R_max, λ, T₉₀, T_eff) of BCSW, CF, and SS were compared using a one-way ANOVA and Tukey HSD. The impact of BCSW co-digestion with SS or CF on the anaerobic digestion parameters was also ascertained, with a significance level of 0.05.

3. Results

3.1. Characteristics of BCSW, CF, and SS

The characteristics of BCSW, CF, and SS are presented in

Table 2. Characteristics of BCSW, CF, and SS.

Parameters 1	BCSW 2	CF 2	SS 2,3	p-Values 4
TS (%FM)	16.7 ± 0.3 b	32.1 ± 0.2 c	4.1 ± 0.2 a	1.41 × 10−7
VS (%DM)	93.2 ± 0.6 b	91.2 ± 2.1 b	67.6 ± 0.8 a	4.44 × 10−5
FS (%DM)	6.8 ± 0.6 a	8.8 ± 2.1 a	32.4 ± 0.8 b	4.44 × 10−5
TKN (%DM)	3.2 ± 0.3 a	2.5 ± 0.3 a	4.5 ± 0.1 b	0.0089
Protein (%DM)	19.8 ± 1.9 a	15.4 ± 1.7 a	28.1 ± 0.9 b	0.0089
Fat (%DM)	57.4 ± 0.3 c	3.1 ± 0.1 a	5.6 ± 0.2 b	3.33 × 10−7
NDF (%DM)	18.9 ± 0.6 a	61.3 ± 0.1 c	27.4 ± 0.8 b	1.14 × 10−5
ADF (%DM)	13.1 ± 0.5 b	23.5 ± 0.6 c	10.7 ± 0.0 a	0.00019
Hemicellulose (%DM)	5.8 ± 0.2 a	37.9 ± 0.5 c	16.7 ± 0.8 b	2.65 × 10−5

¹ %FM: % of fresh matter, %DM: % of dry matter; ² Values expressed as mean ± standard deviation; ³ The data were reported in [6]; ⁴ p-values of one-way ANOVA; ^a,b,c Means in the same row with different lowercase letters differ significantly (p < 0.05); BCSW, beef cattle slaughterhouse waste; CF, cattle feces; SS, swine slurry; TS, total solid; VS, volatile solid; FS, fixed solid; TKN, Total Kjeldahl Nitrogen; NDF, neutral detergent fiber; ADF, acid detergent fiber.

. BCSW exhibited a low TS content of 16.67% on a fresh weight basis, which contrasts with previous reports where TS content ranged from 26% to 65.2% on a fresh weight basis [1,7,9]. This discrepancy could be attributed to the hygienic washing and cleaning procedures at the slaughterhouse prior to sample collection. Despite the low TS content, BCSW showed a high VS content of 93.2% of total solids (% DM), with protein (19.83% DM) and fat (57.4% DM) making up the majority of its composition. The fat and protein levels of BCSW, which range from 17.5% to 58.1% DM and 13% to 33% DM, respectively, are consistent with values reported in previous studies [1,7,9]. The high protein and fat content (volatile matter) indicates that BCSW is a substrate rich in energy.

CF exhibited a high VS content of 91% DM, similar to that of BCSW. Its composition primarily consisted of fat (3.1% DM), protein (15.4% DM), and neutral detergent fiber (61.3% DM), derived from undigested feed. This indicates that while CF has a high VS content like BCSW, it is a lower-energy substrate. However, the high VS content in both CF and BCSW suggests a greater availability of organic matter for anaerobic digestion and conversion to CH₄. Nonetheless, the low fixed solid (FS) content in BCSW (5.4% DM) and CF (8.8% DM) may limit their potential for CH₄ production.

In comparison to BCSW and CF, SS had the lowest TS and VS contents. Moreover, SS exhibited the highest FS content (32.4% DM), indicating a substantial mineral content. Therefore, it is noteworthy to examine the effects of anaerobic co-digestion between substrates with high VS content, such as BCSW and CF, and substrates with complementary VS and FS concentrations, such as BCSW and SS.

3.2. Energy Content of BCSW, CF, and SS

BCSW was shown to have an HHV of 29.26 MJ/kg DM, which is higher than that of other renewable resources. Animal waste ranged from 11.92 to 19.44 MJ/kg DM, while energy crops showed HHV ranging from 14.69 to 20.71 MJ/kg DM [21,22]. The high energy content of BCSW suggests its potential for bioenergy production. CF and SS showed HHV values of 17.5 MJ/kg DM and 17.6 MJ/kg DM, respectively, consistent with several types of animal waste in the Republic of Korea [17]. Furthermore, HHV and VS content were found to positively correlate. However, BCSW, CF, and SS only showed HHV values of 4.88, 5.61, and 0.72 MJ/kg FM, respectively, in terms of fresh matter, indicating that direct heat treatment conversion might not be a cost-effective option [23]. Therefore, biological conversion processes like anaerobic digestion are recommended for converting VS in BCSW, CF, or SS into CH₄, a method proven effective across Europe [24].

3.3. Anaerobic Digestion of BCSW, CF, and SS

Table 3. Ultimate analysis, empirical formula, TMY, SMY, and anaerobic degradability (D_deg) of BCSW, CF, and SS.

Parameter 1	BCSW 2	CF 2	SS 2,3	p-Values 4
Carbon (%DM)	58.4 ± 1.9 c	45.1 ± 2.0 b	37.3 ± 0.3 a	0.002
Hydrogen (%DM)	8.8 ± 0.5 b	5.9 ± 0.3 a	5.2 ± 0.0 a	0.004
Oxygen (%DM)	22.1 ± 3.5 a	31.9 ± 0.1 b	23.7 ± 0.7 a	0.032
Nitrogen (%DM	3.7 ± 0.0 a	2.8 ± 0.4 a	4.8 ± 0.1 b	0.007
Sulfur (%DM)	0.6 ± 0.2 b	0.2 ± 0.0 a	1.0 ± 0.1 b	0.009
Empirical formula	C25H45O7N1S0.1	C60H94O32N3S0.1	C10H16O5N1S0.1
TMY (Nml CH4/g VSadded)	738.8 ± 54.7 b	533.5 ± 22.3 a	529.5 ± 9.0 a	0.014
SMY (Nml CH4/g VSadded)	582.2 ± 3.3 c	431.5 ± 15.4 b	310.1 ± 9.0 a	2.04 × 10−7
Ddeg (%)	78.8 ± 0.4 b	80.9 ± 2.9 b	58.6 ± 1.7 a	1.47 × 10−5

¹ %DM: % of dry matter; TMY: theoretical methane yield (Equation (4)); SMY: specific methane yield from experiment. ² Values are expressed as mean ± standard deviation. ³ The data were also reported in [6]. ⁴ p-values of one-way ANOVA. ^a,b,c Means in the same row with different uppercase letters differ significantly (p < 0.05).

presents the results of the ultimate analysis, empirical chemical formula, TMY, SMY, and D_deg for BCSW, CF, and SS. For BCSW, the TMY, SMY, and D_deg were 738.8 Nml CH₄/g VS_added, 582.2 Nml CH₄/g VS_added, and 78.8%, respectively. The observed SMY for BCSW aligns with previous studies, where reported SMY values ranged from 232.2 to 609 Nml CH₄/g VS_added. For example, Lee et al. [1] reported an SMY of 520 Nml CH₄/g VS_added for the anaerobic digestion of cattle offal, while untreated and pasteurized cattle offal exhibited SMY values of 515.5 and 650.9 Nml CH₄/g VS_added, respectively [7]. A recent study, however, observed a lower SMY between 232.2 and 250.8 NmL/g VS_added and a D_deg around 53.6 to 57.9% of BCSW [14]. Furthermore, semi-continuous anaerobic digestion of BCSW yielded 410 Nml CH₄/g VS_added [12], which was lower than the SMY achieved through batch anaerobic digestion (609 Nml CH₄/g VS_added) by the same research group [25]. These discrepancies in SMY may be attributed to variations in waste properties. With a D_deg of 78.8% (Table 3), BCSW demonstrates high degradability. However, further analysis of anaerobic digestion parameters is necessary to evaluate BCSW’s feasibility and identify potential strategies for improvement.

For CF, the TMY, SMY, and D_deg were 533.5 Nml CH₄/g VS_added, 431.5 Nml CH₄/g VS_added, and 80.9%, respectively. The corresponding values for SS were 529.5 Nml CH₄/g VS_added, 310.1 Nml CH₄/g VS_added, and 58.6%, respectively. These findings are consistent with previous research, which reported SMY values for CF ranging from 107 to 620 Nml CH₄/g VS_added [2,23,26] and for SS ranging from 204 to 437 Nml CH₄/g VS_added [2,11,24,27]. As shown in Table 3, CF exhibited higher D_deg and SMY values than SS, which may be due to the higher protein content in SS (Table 2). The anaerobic digestion process could be hindered by the presence of ammonia, a byproduct of protein degradation under anaerobic conditions [16]. To better understand the significance of these findings and to explore methods for improving the anaerobic digestion of SS and CF, further research on digestion dynamics is warranted.

The modified Gompertz equation (Equation (6)) was used to calculate M_max, R_max, λ, T₉₀, and T_eff, as shown in

Table 4. Anaerobic digestion parameters of BCSW, CF, and SS estimated by modified Gompertz formula (Equation (6)).

Parameter 1	BCSW 2	CF 2	SS 2,3	p-Values 4
Mmax (Nml CH4/g VSadded)	578.5 ± 14.4 c	397.2 ± 15.3 b	289.8 ± 8.6 a	5.12 × 10−7
Rmax (Nml CH4/g VSadded/d)	30.8 ± 2.6 b	22.0 ± 0.8 a	20.1 ± 0.9 a	0.0005
λ (days)	10.2 ± 1.7 b	8.3 ± 0.4 b	0.2 ± 0.1 a	4.31 × 10−5
R2	0.999	0.998	0.989
T90 (days)	32.7 ± 2.4 b	29.9 ± 0.4 b	17.4 ± 0.3 a	2.71 × 10−5
Teff (days)	22.5 ± 2.3 b	21.6 ± 0.4 b	17.2 ± 0.2 a	0.007

¹ M_max: maximum CH₄ production potential; R_max: maximum CH₄ production rate; λ: lag phase period; R²: correlation coefficient; T₉₀: time required to obtain 90% of M_max; T_eff: effective digestion time (T₉₀ − λ). ² Values are expressed as mean ± standard deviation. ³ The data were also reported in [6]. ⁴ p-values of one-way ANOVA. ^a,b,c Means in same row with different lowercase letters differ significantly (p < 0.05).

. The correlation between predicted CMY and experimental CMY for BCSW, CF, and SS was evaluated to assess the model’s accuracy [18]. The correlation coefficients (R²) in Table 4, which range from 0.989 to 0.999, indicate that Equation (6) provided an excellent fit for the data from BCSW, CF, and SS (Figure 2).

M_max was significantly higher in BCSW than in CF and SS, with a value of 578.5 Nml CH₄/g VS_added (Table 4). In addition to M_max, other critical factors for the anaerobic digestion process include λ and digestion times (T₉₀ and T_eff) [28]. The λ represents the bioavailability of the substrate and the adaptation of methanogens during anaerobic digestion [7]. Table 4 shows the estimated time required for BCSW to adjust to anaerobic conditions. A prolonged λ suggests that bacteria in BCSW may have difficulty accessing the VS. This may be due to the high fat content in BCSW (57.4% DM), as fat must undergo hydrolysis, acidogenesis, and acetogenesis to produce acetic acid, CO₂, and H₂, which are substrates for methanogenesis [29,30]. Similar to BCSW, the anaerobic digestion of PSW [6,15], which also has a high fat content, demonstrated a prolonged λ.

Based on the SMY, D_deg, and M_max values, BCSW shows promise as a substrate for anaerobic digestion. However, the long λ may negatively impact the overall performance of the anaerobic digester, particularly by extending the digestion time. Table 4 provides the T₉₀ and T_eff values for the anaerobic digestion of BCSW. T₉₀, or technical digestion time, refers to the time required to reach 90% of M_max [19]. In this study, the T₉₀ for BCSW was estimated at 32.7 days, and the T_eff was determined to be 22.5 days. BCSW exhibited a low degradation rate, as indicated by significantly higher (p < 0.05) λ, T₉₀, and T_eff values compared to SS. However, no significant difference was observed in λ, T₉₀, or T_eff between BCSW and CF.

The elevated λ, T₉₀, and T_eff values observed in BCSW may be attributed to its high fat and low mineral content. As mentioned, fats must first be converted into volatile fatty acids before the methanogenesis process can begin. Additionally, the low FS content (Table 2) suggests a deficiency in minerals, which are essential for efficient anaerobic digestion. To optimize BCSW digestion, it is crucial to avoid prolonged λ, T₉₀, and T_eff, as these extended timeframes can increase operational costs and reduce the economic viability of the treatment process. Despite this challenge, the high M_max indicates that BCSW holds significant potential as a substrate for anaerobic digestion. One possible strategy to shorten λ, T₉₀, and T_eff during BCSW digestion is co-digestion with other substrates [4].

SS had the lowest M_max compared to BCSW and CF (Table 4). However, SS exhibited the lowest values for λ, T₉₀, and T_eff, indicating that the microorganisms in SS had early access to VS during anaerobic digestion. The high moisture content of SS (95.9%) likely facilitated the solubility of nutrients and VS, making them more readily available for microbial consumption. The lower VS concentration in SS resulted in lower M_max and SMY (p < 0.05) compared to BCSW and CF. However, the low VS content also indicated that SS was rich in minerals, which are beneficial for the growth and metabolism of methanogens. The lower λ, T₉₀, and T_eff values observed for SS suggest that co-digestion with SS could help improve digestion efficiency in BCSW by reducing these parameters. From an economic perspective, the low VS content in SS led to lower SMY and M_max, suggesting that anaerobic digestion of SS alone is not cost-effective. Therefore, increasing the VS content in SS through co-digestion with BCSW may enhance CH₄ production and improve the overall process efficiency.

CF, in comparison to SS, had a higher M_max (397 Nml CH₄/g VS_added) (Table 4). However, it exhibited λ of 8.3 days, similar to that of BCSW, suggesting that bacteria in CF also had limited early access to VS. Table 2 indicates that CF had higher TS and NDF concentrations compared to SS, implying that extended hydrolysis and acidogenesis phases were necessary to generate CO₂, H₂, and volatile fatty acids for methanogenesis. Additionally, the lower FS content in CF compared to SS suggests a potential shortage of easily accessible minerals for methanogens. As a result, CF may not be an ideal co-digestion substrate for BCSW when aiming for optimal biogas production. However, further investigation into the anaerobic co-digestion of CF and BCSW could yield interesting insights.

3.4. Co-Digestion of BCSW with CF or SS

Table 5. Anaerobic digestion parameters from co-digestion of BCSW with CF or SS.

Parameter 1	Sole Digestion 2			Co-digestion between BCSW and CF 2			Co-digestion between BCSW and SS 2			p-Values 3
	C1	C2	C3	C4	C5	C6	C7	C8	C9
Mmax (Nml CH4/g VSadded)	578.5 ± 14.4 d	397.2 ± 15.3 b	289.8 ± 8.6 a	557.9 ± 16.7 d	496.8 ± 12.2 c	422.0 ± 13.8 b	508.5 ± 35.8 c	484.0 ± 8.9 c	430.7 ± 16.9 b	2.58 × 10−12
Rmax (Nml CH4/g VSadded/d)	30.8 ± 2.6 c	22.0 ± 0.8 a	20.1 ± 0.9 a	30.8 ± 1.4 c	29.6 ± 1.8 c	24.7 ± 0.3 b	42.5 ± 1.9 d	34.5 ± 0.9 c	35.0 ± 0.7 c	5.49 × 10−12
λ (days)	10.2 ± 1.7 c	8.3 ± 0.4 c	0.2 ± 0.1 a	8.2 ± 1.0 c	7.5 ± 1.7 b,c	7.1 ± 1.6 b,c	7.4 ± 0.1 b,c	4.9 ± 0.6 b	2.4 ± 0.1 a,b	1.37 × 10−8
R2	0.999	0.998	0.989	0.998	0.999	0.999	0.998	0.998	0.997
T90 (days)	32.7 ± 2.4 d	29.9 ± 0.4 c,d	17.4 ± 0.3 a	29.9 ± 1.7 c,d	27.5 ± 1.5 c	27.5 ± 1.7 c	21.7 ± 0.5 b	21.7 ± 0.3 b	17.1 ± 0.4 a	2.05 × 10−11
Teff (days)	22.5 ± 2.3 c	21.6 ± 0.4 c	17.2 ± 0.2 b	21.7 ± 0.8 c	20.1 ± 0.9 c	20.4 ± 0.8 c	14.3 ± 0.5 a	16.8 ± 0.2 a,b	14.7 ± 0.3 a,b	5.83 × 10−9

¹ M_max: maximum CH₄ production potential; R_max: maximum CH₄ production rate; λ: lag phase period; R²: correlation coefficient; T₉₀: time required to obtain 90% of M_max; T_eff: effective digestion time (T₉₀ − λ). ² Values are expressed as mean ± standard deviation. ³ p-values of one-way ANOVA. ^a,b,c,d Means in same row with different lowercase letters differ significantly (p < 0.05). The BCSW content in co-digested mixtures was 100% (C1), 0% (C2 and C3), 67% (C4 and C7), 50% (C5 and C8), and 33% (C6 and C9) on a volatile solid (VS) basis.

summarizes the effects of co-digesting BCSW with CF or SS on anaerobic digestion parameters, calculated using Equation (6). To evaluate the model’s accuracy, predicted CMY was compared with experimental CMY for co-digestion between BCSW and CF and between BCSW and SS [18]. Equation (6) provided an excellent fit for the data, with correlation values (R²) ranging from 0.989 to 0.999 (Figure 3), confirming the model’s reliability.

From the SS perspective, anaerobic co-digestion with BCSW increased the M_max from 289.8 Nml CH₄/g VS_added (SS single digestion) to 431–508 Nml CH₄/g VS_added (BCSW and SS co-digestion). This represents a 48–75.5% increase in M_max compared to SS digestion alone. A similar result was also reported by Jo et al. [2], where anaerobic co-digestion of SS and SW (1:1 VS basis) resulted in SMY increased (419.9 Nml CH₄/g VS_added) compared to single SS digestion (264.1 Nml CH₄/g VS_added). Furthermore, T_eff was not adversely affected by co-digestion, showing no significant difference compared to single SS digestion (Table 5). However, T₉₀ was impacted by co-digestion with BCSW, particularly when BCSW constituted 67% (C7) and 50% (C8) on a VS basis in the co-digested mixture (Table 5). This suggests that a higher proportion of BCSW in the mixture increases the digestion time. Interestingly, single BCSW digestion exhibited the highest T₉₀, likely due to the higher fat content in BCSW, which degrades more slowly despite its high CH₄ potential [31]. Notably, the co-digested mixture containing 33% BCSW (C9) had a T₉₀ similar to that of SS digestion alone.

From the CF perspective, co-digestion with BCSW increased the M_max from 397.2 Nml CH₄/g VS_added (CF single digestion) to 422–557.9 Nml CH₄/g VS_added (BCSW and CF co-digestion), representing a 6–40% increase in M_max compared to CF digestion alone. A similar result was also reported by Jo et al. [2], where anaerobic co-digestion of CF and SW (1:1 VS basis) resulted in SMY increased (356.9 Nml CH₄/g VS_added) compared to single CF digestion (107.0 Nml CH₄/g VS_added). However, co-digestion did not significantly affect the λ or T_eff compared to BCSW digestion alone (p > 0.05). Despite the increase in M_max, co-digestion of CF and BCSW may not be the most effective option, as M_max decreased by 3–27% compared to single BCSW digestion.

The M_max of BCSW and SS in all co-digested mixtures was lower than the M_max of solitary BCSW digestion (p < 0.05). Specifically, the M_max values obtained from co-digestion of BCSW and SS for mixtures containing 67%, 50%, and 33% BCSW were 508.5, 484, and 430.7 Nml CH₄/g VS_added, respectively. Compared to individual BCSW digestion, the M_max decreased by 3.7% to 25.6%. Previous studies have also documented that anaerobic co-digestion of BCSW with other substrates results in a lower M_max than individual BCSW digestion. For example, Pagés-Díaz et al. [25] reported an M_max of 570 Nml CH₄/g VS_added from anaerobic co-digestion of BCSW with livestock manure (2:1 VS basis), compared to 668 Nml CH₄/g VS_added from individual BCSW digestion. A recent study showed that anaerobic co-digestion of CF and PSW (1:1 VS basis) resulted in SMY of 356.9 Nml CH₄/g VS_added, while co-digestion between SS and PSW (1:1 VS basis) also resulted in SMY of 419.9 Nml CH₄/g VS_added. These were lower compared to single PSW digestion that exhibited SMY of 500.2 Nml CH₄/g VS_added [2].

The lower M_max observed from co-digestion of BCSW with CF or SS can be attributed to the lower M_max and fat content in CF and SS. Fat has a CH₄ potential of 990 Nml CH₄/g VS_added, which is higher than that of carbohydrates and proteins, with CH₄ potentials of 415 and 633 Nml CH₄/g VS_added, respectively [31]. However, the fat content in CF and SS was only 3.1% and 5.6% DM, respectively (Table 2). Additionally, CF had the highest NDF content (61.3% DM), which has a lower CH₄ potential compared to protein. This resulted in a lower M_max for CF, despite its VS content being comparable to that of BCSW.

Co-digestion of BCSW and SS had a significant impact on λ, T_eff, and T₉₀ (Table 5). Anaerobic digestion of BCSW alone resulted in significantly higher values for λ, T_eff, and T₉₀ compared to the co-digested mixtures of BCSW and SS (p < 0.05). Co-digestion reduced the λ from 10.2 days (BCSW alone) to 2.4–7.4 days (BCSW and SS combined). It also decreased the T_eff from 22.6 days (BCSW alone) to 14.3–16.8 days (BCSW and SS combined). Similarly, T₉₀ was reduced from 32.7 days (BCSW alone) to 17.1–21.7 days (BCSW and SS co-digestion). These findings indicate that co-digestion of BCSW and SS enhanced anaerobic digestion efficiency, as demonstrated by the shorter λ, T₉₀, and T_eff values compared to solitary BCSW digestion.

The higher digestion rate observed in the co-digested mixture of BCSW and SS, compared to BCSW digestion alone, can be attributed to the greater availability of VS and minerals for bacteria.

Table 6. Comparison of mineral content between BCSW, CF, and SS.

Mineral (mg/Kg)	BCSW 1	CF 1	SS 1,2	p-Values 3
Cobalt (Co)	0.7 ± 0.3 a	2.2 ± 0.1 b	10.4 ± 0.1 c	3.00 × 10−5
Iron (Fe)	1111.5 ± 14.7 a	975 ± 346.1 a	10,872 ± 135.8 b	3.52 × 10−5
Molybdenum (Mo)	8.9 ± 5.8 a	0.9 ± 0.1 a	13.0 ± 0.1 a	0.076
Nickel (Ni)	3.4 ± 0.7 a	4.9 ± 0.4 a	25.0 ± 0.1 b	3.52 × 10−5
Tungsten (W)	0.0 ± 0.0 a	5.5 ± 0.5 b	22.1 ± 0.0 c	1.06 × 10−5
Zinc (Zn)	626.8 ± 195.3 a	576.3 ± 43.2 a	2239 ± 19.9 b	0.001

¹ Values are expressed as mean ± standard deviation. ² The data were also reported in [6]. ³ p-values of one-way ANOVA. ^a,b,c Means in the same row with different lowercase letters differ significantly (p < 0.05).

shows that SS contains higher concentrations of essential trace elements such as zinc (Zn), cobalt (Co), iron (Fe), molybdenum (Mo), nickel (Ni), and tungsten (W) compared to BCSW, indicating a higher nutritional value in the co-digested mixture. Additionally, the high moisture content of SS (95.9% FW) suggests that most of the VS and minerals in the co-digested mixture may be in a soluble form, which could explain the improved digestive efficiency (shorter λ, T₉₀, and T_eff) observed for the co-digested mixture compared to BCSW alone.

Enzymes that serve as co-factors during anaerobic digestion, such as methyltransferase and methyl coenzyme M reductase (MCR), require adequate concentrations of Co and Ni [32]. These enzymes play a critical role in the final stage of the methanogenesis process. Methyltransferase forms the methyl coenzyme M (CH₃-CoM) complex, while MCR catalyzes the reduction of this complex into CH₄ [29].

In contrast, the co-digestion of BCSW and CF revealed that both substrates contained lower amounts of Co and Ni than SS (Table 6). Specifically, the Co content of BCSW, CF, and SS was 0.7, 2.2, and 10.4 mg/kg, respectively, while the Ni content was 3.4, 4.9, and 25.0 mg/kg, respectively. Despite this, the concentrations of Ni and Co in both BCSW and CF were within acceptable ranges for anaerobic digestion, which span from 0.03 to 35 mg/kg [33]. This is corroborated by the M_max, D_deg, and SMY values for BCSW and CF, which demonstrate substantial CH₄ production and anaerobic degradability (Table 3 and Table 4).

However, the longer λ, T₉₀, and T_eff observed during the anaerobic co-digestion of BCSW and CF suggest that Co and Ni may not be readily available at the onset of digestion. The higher TS and VS concentrations in BCSW and CF compared to SS imply that Co and Ni may form mineral complexes with VS, reducing their solubility relative to SS. Thus, hydrolysis and acidification processes are necessary to make Co and Ni accessible to methanogens, resulting in prolonged λ, T₉₀, and T_eff during BCSW and CF co-digestion, similar to that of individual BCSW digestion. When combined with the lower M_max of CF, the co-digested mixture exhibited a lower M_max than individual BCSW digestion, without a significant impact on digestion time. This suggests that CF may not be an ideal co-digestion substrate for BCSW.

Substrate co-digestion can result in either favorable (synergistic) or unfavorable (antagonistic) effects [2,4]. Antagonistic effects occur when the measured M_max is lower than the expected M_max (α < 1), while synergistic effects occur when the measured M_max exceeds the expected M_max (α > 1) [20]. As shown in

Table 7. Results of the synergistic or antagonistic effect produced by the co-digestion of BCSW with CF or SS. The BCSW content in co-digested mixtures was 100% (C1), 0% (C2 and C3), 67% (C4 and C7), 50% (C5 and C8), and 33% (C6 and C9) on a volatile solid (VS) basis.

Code 1	Experimental Mmax 2	Simulated Mmax 2,3	α 4
C1	578.5 ± 14.4	578.5 ± 14.4
C2	397.2 ± 15.3	397.2 ± 15.3
C3	289.8 ± 8.6	289.8 ± 8.6
C4	557.9 ± 16.7	518.1 ± 14.3	0.93 ± 0.01
C5	496.8 ± 12.2	487.8 ± 14.4	0.98 ± 0.01
C6	422.0 ± 13.8	457.6 ± 14.6	1.08 ± 0.01
C7	508.5 ± 35.8	482.3 ± 10.7	1.05 ± 0.05
C8	484.0 ± 8.9	434.2 ± 9.3	1.12 ± 0.02
C9	430.7 ± 16.9	386.1 ± 8.4	1.12 ± 0.02

¹ C1, C2, and C3 represent BCSW, CF, and SS sole digestion; C4 to C6 represent co-digestion experiment between BCSW and CF, while C7-C9 represent co-digestion experiment between BCSW and SS. ² Values are expressed as mean ± standard deviation; units are in Nml CH₄/g VS_added. ³ Simulated M_max was calculated by Equation (8). ⁴ α = experimental M_max/simulated M_max (Equation (9)).

, the co-digestion of BCSW and CF resulted in α < 1 for C4 and C5, indicating an antagonistic effect. This may be due to the similar characteristics of CF and BCSW, both of which have low FS content and high VS content. However, a synergistic effect (α > 1) was observed by increasing the concentration of CF in the co-digestion substrate (C6). This could be attributed to CF’s higher concentration of Co compared to BCSW (Table 6). Nevertheless, as shown in Table 5, co-digestion of BCSW and CF did not significantly improve anaerobic digestion parameters compared to single BCSW digestion, suggesting that CF may not be an optimal co-digestion substrate with BCSW.

In contrast, the experimental M_max for co-digestion of BCSW and SS exhibited an α value greater than 1, indicating a synergistic effect during anaerobic co-digestion (Table 7). The observed synergy can be attributed to the complementary properties of BCSW, which has high VS and fat content, and SS, which is rich in Co and Ni. The influence of co-digestion on nutrient availability, anaerobic digestion parameters, and digestate properties presents a promising area for further investigation.

4. Conclusions

This study demonstrated that the anaerobic co-digestion of BCSW with SS or CF can significantly enhance the efficiency of anaerobic digestion parameters (BMP, M_max, λ, and T_eff). Co-digestion with SS significantly enhanced the anaerobic digestion efficiency (λ, T_eff, and T₉₀) of BCSW, likely due to SS’s higher concentrations of Co and Ni, which are essential for enzymatic activity during anaerobic digestion. The synergy was most evident when BCSW was mixed with SS rather than CF which has a lower nutrient profile. Future studies should focus on optimizing co-digestion ratios and evaluating the long-term stability and scalability of the process to enhance its practical applications in CH₄ production and safe disposal of BCSW.