Overcoming the Difficulties of Thermophilic Co-Digestion of Sewage Sludge and Beverage Industry Wastes in the Presence of Zeolite

Abstract

The thermophilic anaerobic bioconversion of various wastes is still challenging, mainly due to process instability and economic profitability. This group includes orange wastes (OWs) and brewery spent grain (BSG), the main by-products generated by the beverage industry. In this study, a strategy allowing for improving methane production by the multicomponent co-digestion of sewage sludge (SS), OW, and BSG was proposed. To overcome the difficulties in the thermophilic co-digestion of those wastes, the application of natural zeolite (Z), i.e., clinoptilolite, was proposed. The experiment was performed in the batch mode at a temperature of 55 °C. Four experimental series were conducted with differing feedstock compositions, one of which was a control supplied only by SS. As compared with the control, in the series supplied by OW and OW with BSG, methane production decreased by 20% and 13%, respectively. In turn, significant improvements were achieved in the presence of Z. The most beneficial results were observed in the reactor supplied by SS, OW, and Z, characterized by a methane yield of 420.2 mLCH₄/gVS, which is an increase of almost 14% as compared with the control. In this case, significantly improved stability parameters, as well as decreased presence of inhibitors, i.e., limonene and phenols, were achieved. It was also characterized by enhanced energy balance by 69%, as compared with the control.

1. Introduction

Currently, anaerobic co-digestion (AcD) is recognized as a cost-effective method allowing for improving methane production at wastewater treatment plants (WWTPs). It should be noted that the mono-digestion (AD) of sewage sludge (SS) exhibits numerous shortcomings, including low methane production, a long retention time, as well as poor removal of organic compounds [1]. The main benefits of AcD are an improved nutrient balance, dilution of toxic compounds, ensuring buffer capacity, and enhanced organic load. All these features might lead to synergistic effects between microorganisms resulting in an enhanced digestion rate, and boosted methane production, as well as improved quality of the digestate [2]. Another important factor in favor of this technology is using the existing capacity of infrastructure at WWTPs without incurring significant financial outlays. The implementation of AcD at WWTPs might also ensure additional revenue obtained from waste collection fees and energy surplus [3]. However, the main challenge related to the application of this technology is the selection of the appropriate substrate with complementary characteristics to the main component, i.e., SS. The seasonality and availability of suitable co-substrates on the local market should also be considered [4]. Various organic materials might be applied as SS additives. Substrates with a high C/N ratio and rich in micro- and macro-nutrients are the most desirable. This group includes: the organic fraction of municipal solid wastes [5], crude glycerol [6], fat, oil, and grease [7]. However, due to widespread availability, there is a growing interest in the use of agri-food industry wastes as co-substrates to SS; the most common are rice straw, sugar bagasse, manures, and cow dung [8,9]. Beverage industry by-products might also be applied as SS additives [10]. Globally, this industry is dominated by orange juice and beer production. This sector is responsible for environmental pollution and significant energy demand [6]. Both types of wastes have a valuable composition and are susceptible to anaerobic decomposition; however, lignocellulosic structure and the presence of bioactive compounds might cause low methane production [11,12]. In particular, the essential oil present in OW, i.e., d-limonene, might effectively disturb the AD process, even at small doses, leading to diminished methane production [13]. Previous studies indicated that the inhibitory effect on AD might be found at concentrations exceeding 0.2 g/kg of d-limonene in the digester [14]. D-Limonene inhibition might intensify under thermophilic conditions [15] due to reduced microbial diversity; moreover, thermophilic microbes are more sensitive to environmental stressors [16,17]. However, thermophilic conditions present many benefits, including boosting methane production, shorter retention time, and increased degradation of organic matter. Other advantages include shortening the digester start-up time, enhanced kinetics, and lower risk of contamination due to improved destruction of pathogens [16]. This is due to the increased activity of thermophilic microbes that effectively degrade organic matter. In particular, thermophilic methanogens convert volatile fatty acids (VFAs) into methane more efficiently. Additionally, a higher hydrolysis rate results in the faster breakdown of complex organic materials, making them more accessible for microbial digestion [8]. Another aspect contributing to its increasing popularity is related to the improved solubilization of substrates, which is especially important in the case of recalcitrant lignocellulosic biomass [18]. Nevertheless, the main disadvantages are related to stability problems and the high energy requirement for the digester’s heating. For this reason, new cost-effective solutions are constantly being sought to improve process stability and increase methane production under these temperature conditions.

In recent years, the application of zeolites (Z) in various engineering applications has gained increasing attention due to their flexibility and economic profitability [19,20]. They have been applied in membrane separation, photo-catalytic degradation, adsorption, ion exchange, and filtration. These widespread applications have resulted from their properties, including major ion-exchange capacity, significant specific surface area, thermal stability, and lattice strength [21]. In the AD process, Z has been applied to alleviate the inhibition of, e.g., ammonia or phenols owing to their cation exchange capacity [22]. Due to their unique microporous structure, they enrich microbial communities and enable biofilms to develop by providing a suitable surface [23]. They also ensure essential nutrients for microbial metabolism, such as potassium, sulfur, and other trace elements. Additionally, they limit the production of long-chain fatty acids (LCFAs) [24]. The negative impact of LCFA on AD is related to disturbing the nutrient transport into cells due to its presence on microbial surfaces [25].

A strategy allowing for the difficulties of thermophilic co-digestion of SS and beverage industry wastes to be overcome has been developed in this study. For this purpose, the use of natural Z, i.e., clinoliptolite, was proposed. Thus far, the effect of Z application on the AcD of selected substrates has not been investigated, and there have been few studies related to thermophilic AcD in the presence of Z. In this paper, the influence of Z introduction was assessed based on methane production and its kinetics, process stability, as well as removal of organic compounds. The energy balance was determined to examine the profitability of the adopted strategy. The obtained results might constitute a breakthrough in the use of selected organic wastes in thermophilic AD.

2. Materials and Methods

2.1. Substrates and Inoculum

Municipal SS obtained from mechanical–biological WWTP located in Lublin (Poland) was chosen as the main component in this study. This sample was a mixture of primary and excess sludge, both thickened. Under laboratory conditions, both types of sludge were mixed at the volumetric ratio of 60:40 (primary:excess) known to be beneficial due to enhanced biogas production [26]. The inoculum for AD was obtained from the same facility; it was taken from the outlets of mesophilic anaerobic digesters. To adapt to thermophilic conditions, it was kept below 55 °C under an anaerobic environment for two weeks.

OW and BSG were chosen as co-substrates for SS. Ground orange peels were adopted as OW. Oranges were obtained from a local market; before peeling, they were thoroughly washed. Prior to being supplied to the reactor, they were crushed to fraction sizes of 0.5 mm using a laboratory mill. The second waste was BSG, taken from a local craft brewery. After discharging, this sample was ground to the same size fraction as OW and dried at a temperature of 55 °C.

Table 1. Composition of all materials applied in this study (average values and standard deviations are given).

Parameter	Unit	SS	OW	BSG	Inoculum
COD	g/L	46.9 ± 3.2
VFA	mg/L	486 ± 17.4
pH		6.50 ± 0.02			7.94 ± 0.01
Alkalinity	mg/L	663 ± 13.3
TS	g/kg	43.89 ± 2.7	231.7 ± 9.2	961.5 ± 11.4	24.91 ± 0.7
VS	g/kg	35.32 ± 1.7	224.4 ± 17.6	889.5 ± 81.1	17.85 ± 0.55
Phenols	mg/L	2.95 ± 0.7	62.1 ± 3.7	49.8 ± 4.9
Limonene	ppm	nd.	713 ± 37.1	nd.	nd.

nd.—not detected.

lists the composition of those substrates.

The applied Z, i.e., clinoptilolite, was a natural resource-obtained zeolitic tuff from a quarry near Nižný Hrabovec located in Slovakia. After collection, it was dried at a temperature of 105 °C and crushed to the same fraction as other co-substrates. This material was characterized by the following composition: SiO₂ (72.42%), Al₂O₃ (9.48%), loss on ignition (LOI) representing organic matter content (VS) (7.63%), K₂O (4.11%), CaO (3.67%), Fe₂O₃ (1.86%), MgO (0.54%), and TiO₂ (0.26%). This material was characterized by a specific surface area of 18.3 m²/g, volume of micropores of 0.0051 cm³/g, volume of mesopores of 10.65 m²/g, pore radius of 10.0 nm, and cation exchange capacity of 1.38 meq/g [27].

2.2. Experimental Design and Laboratory Installation

The impact of Z application on the AcD of the chosen substrates was investigated via a batch experiment. For this study, a BioReactor Simulator, supplied by BPC Instruments AB (BPC Instruments AB, Lund, Sweden), was utilized. This apparatus comprised two units, with the first unit containing six anaerobic reactors, each fitted with a mixing system, and submerged in a water bath to maintain a consistent temperature of 55 °C. The second unit was applied to constantly monitor the volume of the generated biogas.

The working volume of each batch reactor was 2000 mL; they contained 1400 mL of inoculum and 400 mL of mixed sludge. The substrate-to-inoculum ratio (S/I) was varied between 0.57 and 0.69 on VS basis, depending on the feedstock composition.

In this study, four experimental series were planned, according to the following assumptions:

• T0—control series, mono-digestion of SS;
• T1—two-component AcD of OW and SS;
• T2—three-component AcD of OW, BSG, and SS;
• T3—three-component AcD of OW and SS with Z application;
• T4—four-component AcD of OW, BSG, and SS with Z application.

In series T1 and T2, the doses of both OW and BSG were 1.5 g. In turn, the amounts of the applied Z were 3.0 and 6.0 g in T4 and T3, respectively. The doses of OW and BSG were adopted according to the protocol presented in the authors’ previous study [28]. In turn, the amount of zeolite was established based on a study conducted by Kotsopouloset [29]. The lowest dose of Z in T4 was related to the introduction of an additional component, i.e., BSG, which is characterized by a high TS content. The use of the same dose as in T3 could contribute to reduced methane production resulting from supplying the digester with excessive TS [30]. The detailed experimental design is presented in Figure 1.

2.3. Analytical Methods

All samples underwent monitoring for several parameters, including pH values, limonene, volatile solids (VSs), total solids (TSs), and chemical oxygen demand (COD). The supernatant, obtained by centrifuging the samples at a rotation speed of 4000 r min⁻¹ for a period of 30 min, was analyzed for ammonia nitrogen (TAN), total alkalinity (TA), phenols, volatile fatty acids (VFAs), and soluble chemical oxygen demand (sCOD). TS and VS were assessed in accordance with The Standard Methods for the Examination of Water and Wastewater [31]. Other measurements i.e., COD, sCOD, TAN, TA, and VFA, were performed using a Hach Lange UV–VIS DR 3900 spectrophotometer (Hach, Loveland, CO, USA) and the analytical methods provided by Hach Lange. The pH of the samples was measured using a CP-511 pH meter (Elmetron, Zabrze, Poland).

The analysis of biogas composition was conducted with a ThermoTrace GC-Ultra gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA), which featured a conductivity detector and divinylbenzene (DVB)-packed columns (RTQ-Bond). Helium was utilized as the carrier gas, flowing at a rate of 1.5 cm³ per minute. The measurement parameters included a detector temperature of 100 °C and injector temperature of 50 °C.

The D-limonene content was also measured using a Trace GC-Ultra gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA). A Supelco capillary column with dimensions of 30 m × 0.25 mm ID × 0.25 μm was utilized. Helium was used as the carrier gas at a flow rate of 1.5 cm³/min. The analysis parameters were set with an inlet temperature starting at 40 °C, followed by a gradual increase of 5 °C/min until reaching 250 °C.

2.4. Kinetics of Methane Production and Energy Balance

Two kinetic models, i.e., modified Gompertz (Equation (1)) and logistic growth (Equation (2)) were applied to evaluate the effect of Z application on the methane production rates, and hence understand the conditions under which methane is generated. The corresponding equations are presented below:

• Modified Gompertz

$$MP\left(t\right)=MP·exp\left(-exp\left({\displaystyle \frac{R_m·e}{MP}}exp\left(\lambda -t\right)+1\right)\right),$$

(1)

• Logistic growth

$$MP\left(t\right)={\displaystyle \frac{MP}{\left(1+\mathrm{e}\mathrm{x}\mathrm{p}(4·R_m·\left(\frac{\lambda -t}{MP}\right)+2\right)}},$$

(2)

where:

• MP(t)—cumulative methane production (mL CH₄/gVS);
• MP—methane production (mL CH₄/gVS);
• R_m—maximum methane production rate (mL CH₄/gVS d);
• e—constant (2.71828);
• λ—lag phase (d);

In order to estimate the possible energy gains obtained as a result of the implementation of this strategy, an energy balance was carried out. The evaluation was performed for anaerobic digesters with a capacity of 2500 m³ and hydraulic retention time of 15 d for the winter period (the most unfavorable temperature conditions). The energy balance evaluation was conducted according to the protocol presented in the authors’ previous study [32]. The applied equations with descriptions are presented below:

$$D_{MP}=MP·LVS,$$

(3)

$$Q_{TM}=D_{MP}·H_V,$$

(4)

$$Q_{HF}=q_F·\left(T-T_F\right)·C_{SS},$$

(5)

$$Q_{HL}=24·\left(T-T_{air}\right)·C_{HL}·A,$$

(6)

$$A=3.14·D^2,$$

(7)

$$Q_{TD}=\mathsf{\alpha }·\left(Q_{HF}+Q_{HL}\right),$$

(8)

$$P=\left({\displaystyle \frac{Q_{TM}-Q_{TD}}{Q_{TD}}}\right)100\%,$$

(9)

where:

• D_MP—daily methane production (m³ CH₄/d);
• LVS—volatile solids load in the feedstock (kg VS/d);
• Q_TM—evaluated thermal energy obtained from the combustion of methane (MJ/d);
• Q_HF—thermal energy required for heating the feedstock (MJ/d);
• Q_HL—thermal energy required to cover the heat loss through the walls of the digester (MJ/d);
• C_SS—specific heat of SS (4200 kJ/m³ K);
• D—diameter of the cylindrical part of the digester (m);
• A—surface of the digester walls (m²);
• Q_TD—total energy demand (MJ/d);
• H_V—heating value of methane, (35.8 MJ/m³),
• C_V—calorific value of methane, (10 kWh/m³);
• T—adopted temperature for AD (K);
• T_air—air temperature (K);
• T_F—feedstock temperature (K);
• C_HL—heat loss coefficient by permeation through the walls of the digester, (4.0 kJ m²/h K);
• q_F—feedstock flow rate (m³/d);
• P—profit of thermal energy (%);
• α—margin factor, (1.1).

2.5. Adsorption Capacity of the Applied Z

To evaluate the effect of the applied Z on AD performance, the adsorption capacity test was conducted. The experiment was conducted in a batch mode under thermophilic conditions using an Erlenmeyer flask. The following equations were applied to determine the zeolite adsorption capacity (q) with regard to TAN, COD, d-limonene, and phenols:

$$q={\displaystyle \frac{\left(C_i-C_e\right)V}{m}},$$

(10)

where:

• C_i—content of the selected parameter in an initial sample (mg/L);
• C_e—equilibrium content of the selected parameter (mg/L);
• V—volume of the solution (L);
• m—dose of applied the adsorbent (g).

2.6. Statistical Analysis

Utilizing Statsoft Statistica software (version 13), an analysis of variance (ANOVA) was conducted with a significance level of p < 0.05. The evaluation of kinetic coefficients was carried out using a non-linear regression method. The relationships between the results were quantified using the Pearson correlation coefficient (R) and the determination coefficient (R²).

3. Results and Discussion

3.1. Removal of Organic Compounds and Process Stability

Figure 2 shows that the improvement of VS removal was achieved in all co-digestion series. Major growths of approx. 6% were found in T2 and T4; importantly, both series were supplied by BSG (Figure 2a). The VS removal was established at levels of 63 and 62.7% in T2 and T4, respectively. Enhanced removal of TS was observed as well. Significant growths were found in T3 and T4 in the presence of Z. Improvements of 25 and 16% were noticed in T3 and T4, respectively, compared with the control. This effect occurred despite discharging a significant amount of TS with Z to feedstock, indicating the improvement in process performance (Figure 2b). Similarly, enhanced removal of COD was achieved in the presence of all co-substrates (the highest for T4); however, there were no statistical differences between any of the co-digestion series (Figure 2c). The previous studies demonstrated that zeolites might reduce the overall organic content in the digester due to the large surface area, as well as microporous structure and surface [19,33]. However, the clinoptilolite applied in this study indicated a relatively low adsorption capacity of 3.47 mg/g. A similar finding was observed in the study conducted by Halim et al. [34], in which Z indicated the lowest adsorption capacity of COD compared with that of other conductive materials. Therefore, in this study, the achieved effect of enhanced organic compounds removal is rather related to providing a surface for microorganism immobilization and the reduction of stress caused by the presence of inhibitors [35,36,37]. However, further detailed research should be conducted.

With regard to sCOD, the increase in its content that occurred within AD was related to the destruction of complex organic matter AD and the release of soluble organic matter. The lowest value of release efficiency was achieved in T4, indicating its effective use by microbes in the AcD process.

Another crucial indicator that should be evaluated in AcD is the effect of introducing co-substrates on process stability. Thus, the pH, TA, VFA, and TA-to-VFA ratio were controlled in this study. It should be noted that thermophilic conditions are susceptible to disturbances in process stability; therefore, their content should be investigated.

Table 2. Process stability in experimental series with inhibitor content (average values with standard deviations are shown).

Series	pH		TA		VFA		TAN		Phenols		D-Limonene
	-		mg/L		mg/L		mg/L		mg/L		ppb
	F	D	F	D	F	D	F	D	F	D	F	D
T0	6.50 ± 0.04	7.8 ± 0.1	663 ± 14.3	6463 ± 21.4	486 ± 3.4	1028 ± 12.4	59.4 ± 4.3	178 ± 11.2	2.95 ± 0.5	27.90 ± 2.3	18.27 ± 1.4	21.47 ± 2.1
T1	5.59 ± 0.02	7.72 ± 0.06	650 ± 7.8	5950 ± 24.1	661 ± 4.7	1245 ± 11.4	65.4 ± 3.7	210 ± 7.8	5.03 ± 0.7	23.40 ± 3.1	2359.5 ± 14.3	839.67 ± 24.1
T2	5.96 ± 0.07	7.68 ± 0.07	647 ± 9.8	5672 ± 17.6	645 ± 5.7	1487 ± 9.7	64.2 ± 4.9	194 ± 14.3	7.45 ± 0.7	33.80 ± 3.4	2332.1 ± 15.7	524.3 ± 14.2
T3	6.43 ± 0.03	8.34 ± 0.1	690 ± 4.8	6399 ± 15.1	604 ± 4.8	788 ± 7.8	60.6 ± 5.1	179 ± 14.8	7.11 ± 0.6	15.33 ± 2.1	21,201.7 ± 31.3	451.4 ± 12.3
T4	6.38 ± 0.06	8.23 ± 0.1	672 ± 7.4	6154 ± 11.1	638 ± 6.7	749 ± 8.9	62.3 ± 5.7	169 ± 11.7	8.23 ± 1.1	14.92 ± 2.0	21,660.4 ± 21.4	432.1 ± 11.7

shows that the introduction of OW led to a deterioration in feedstock composition in both T1 and T2, as well as decreases in the pH values and TA contents, with a simultaneous increase in the VFA concentration. The introduction of Z allowed for overcoming the negative effect resulting from the presence of OW. Regarding the feedstock composition in the presence of Z, improvements in both pH and TA occurred, accompanied by a slight reduction in the VFA content. In the presence of Z, the beneficial effect of its application was observed with regard to the TA/VFA ratio. In both co-digestion series with OW application, the highest values were found at 0.21 and 0.26 in T1 and T2, respectively. Those values are close to the limiting value of 0.3 considered for providing stable process performance [38]. In turn, in the corresponding series with Z application, the TA/VFA ratio was significantly reduced at 0.12 in both T3 and T4. This fact was attributed to a significant improvement in the TA content and reduction in VFA in digestate. In T3 and T4, VFA was significantly diminished as compared with the corresponding series without Z application, with related reductions of 40 and 50%. Previous studies indicated that Z might adsorb VFAs, thus preventing acidification and maintaining a more stable pH within AD [39]. Another important effect that might be found in the presence of Z is improving VFA degradation resulting from the enhanced growth of the bacteria responsible for converting VFAs into methane. The improved buffering capacity in the presence of Z is particularly beneficial because it for allows maintaining the optimal conditions for microbial activity, including methanogens [23].

Moreover, in this study, the effect of Z application on inhibitor contents was examined. TAN is considered as the main inhibitor of the AD process. Its negative impact is multifaceted and depends on various conditions, such as pH or temperature. Its inhibition was revealed through the decrease in methane production and increase in intermediate digestion products, such as VFA [40]. Previous studies indicated that methanogens are particularly sensitive to its increased content. However, TAN might also disturb the enzyme reaction, as well as handicap cell functions [41]. The introduction of OW to the feedstock resulted in enhanced TAN content in both T1 and T2. In turn, the supplementation of feedstock with Z led to a minor reduction in the TAN content. However, Z reduced the TAN content by about 14%, as compared with the series without Z application. Previous studies indicated that Z might diminish TAN inhibition as well as enhance digester performance [42]. In AD, the cations present in the crystalline matrix of Z, i.e., Na⁺, Ca²⁺, and Mg²⁺, exchanged with ammonium ions. Another pathway is connected to their high adsorption capacity of 22.9 mg/g related to the unique three-dimensional tetrahedral framework structure [43]. The clinoptilolite applied in this study indicated selective exchange properties with respect to ammonium ions [44].

Phenols are another well-known AD inhibitor. Their negative influence is related to the breakage of cytomembrane, resulting in permeability of the microbe cell wall [24,45]. Generally, phenol inhibition is the main problem that occurs with the AD of lignocellulosic biomass; both OW and BSG are included in this group. As in the case of TAN, zeolites have demonstrated a proven effectiveness in preventing phenol inhibition. In this case, the zeolite accelerated phenol degradation [24], which was confirmed by the significantly lower concentration of phenols for T3 and T4 digestates (Table 2). Previous studies indicated that various types of zeolite have lower adsorption properties than, e.g., activated carbon, such as that by Yousef et al., 2011 [46]. In this study, the applied Z showed a q of 0.36 mg/g, which is a relatively low value. Therefore, its beneficial effect is related to biological mechanisms, rather than a physico-chemical one [24].

D-Limonene is a main essential oil that is typically present in orange peel that might constitute up to 90% of its composition [47]. It is considered one of the most toxic factors in AD related to its antimicrobial properties [14,48]. D-Limonene is particularly harmful to methanogens; therefore, the AD of OW often results in low methane production under both mesophilic and thermophilic conditions [15,28]. Its negative impact is related to handicapping the functionality of cytoplasmic membrane, resulting from the damage of the phospholipid bilayer and connection of the terpenes to membrane proteins [49]. The introduction of Z to the feedstock resulted in reducing the d-limonene content. Compared with the series without Z addition, decreases of 46 and 18% were found in T3 and T4, respectively. This effect was mainly attributed to the adsorption and ion-exchange capabilities of Z [50]. The Z applied in this study indicated a relatively high adsorption capacity of 23.5 mg/g. The obtained results are consistent with those of other studies that indicated the proven efficiency of Z in the removal of various volatile organic compounds [51]. The catalyst function of Z in the decomposition of terpenes might also reduce the d-limonene content [52].

3.2. Methane Production and Its Kinetics

The effect of Z application was also investigated with regard to both biogas and methane production (Figure 3). As compared with SS mono-digestion, the deterioration in both quantities was observed in the presence of OW without Z application. A major reduction in methane production, reaching 19.6 and 12%, was noted in T1 and T2, respectively. Methanogenic archaea are particularly sensitive to d-limonene. This essential oil might also lead to VFA accumulation, and the effect becomes more intense under alkaline conditions [53]. Therefore, many scientists have reported that the effective application of OW should be preceded by an adequate pretreatment strategy to remove d-limonene [54,55,56]. In the study conducted by Martin [54], it was suggested that thermophilic conditions are preferable to mesophilic conditions, as they result in higher methane generation, as well as improved biodegradability; however, in this case, steam distillation was applied prior to AD.

The supplementation of the feedstock in Z resulted in improved biogas and methane production. As compared with the control, methane production was enhanced by 14 and 6% in T3 and T4, respectively. In turn, biogas production was improved by 13 and 12%, respectively. Such a beneficial effect was related to neglecting the d-limonene influence using Z that positively affected methane production. Another aspect that might lead to enhanced methane production is improved microbial activity. Z provides a surface for microbial immobilization that might improve AD efficiency [19]. A similar effect was found in the presence of biochar; this material is also included among the adsorbents commonly applied to eliminate the toxic agents within the AD process [57,58]. Previous studies indicated that the application of biochar to OW resulted in reduced negative influence of d-limonene and, due to its microporous structure, ensured the immobilization of microbes [59]. The increases in methane yield were about 33% and 56% with the application of 10 and 30 g/L biochar, respectively. Calbaro et al. [60] investigated the effect of alkaline pre-treatment of OW with the application of granular activated carbon and zero-valent iron. The results of the AD experiment conducted under mesophilic conditions indicated that the application of this material improved methane production and enhanced process stability at the organic load up to 3 gVS/Ld.

Table 3 shows the state of the art in various strategies that might be applied to boost methane production from various organic wastes. Under thermophilic conditions, major increases in methane production might be found using an appropriately selected substrate i.e., fat-rich or biodegradable wastes. Analyzing various methods, the co-digestion strategy might be considered as a cost-effective and sustainable solution. Another pathway that might be applied in particular to ammonium-rich substrates is the use of adsorption and immobilization additives, such as clays, zeolites, and biochars. Most of these are commonly available natural materials or the materials generated from wastes. Their effect is multi-faceted and related not only to adsorption possibilities, but also their supporting function for microbes [19]. Liu et al. [61] investigated the AcD of SS with food waste with residue biochar application. In this case, the use of 8.0 g/L biochar resulted in improved biogas production by 46% as compared with the control; therein, the methane yield was established at the level of 432.2 mL/gVS. The application of biochar allowed for neutralizing VFA, counteracting the accumulation of free ammonium, accelerating the organic compound decomposition, as well as selectively enriching bacteria and archaea responsible for methane production. A positive effect of Z application was also found in thermophilic AD of slaughterhouse waste; therein, increased biogas production of 700 mL/gVS and enhanced VS removal efficiency was achieved in the presence of Z [29]. In this case, this effect was attributed to the adsorption of ammonia by Z. The thermophilic AcD of pig wastes and Z resulted in significantly improved methane production at doses of 8 and 12 g/L; it was also accompanied by enhanced VS removal [62]. In this case, the application of Z led to a decrease in the negative impact of ammonia and ensured the buffering the capacity in the digester.

Under thermophilic conditions, the most common pre-treatment method is the thermal one. This method applied to SS effectively destroys cell flocs and enhances the solubilization of nutrients, as well as organic matter [16]. However, it is an energy-consuming process; therefore, the energy balance should be evaluated.

Under mesophilic conditions, the common strategy in the case of SS and lignocellulosic biomass is alkaline pretreatment. The main effect of the use of this method is breaking the structures of lignin and hemicelluloses, making them more accessible to AD microbes. The physical pretreatment methods, i.e., microwave or ultrasound, are considered as costly and energy-consuming methods that might also generate toxic intermediates. In turn, biological methods are characterized by low energy consumption and constitute environmentally friendly strategies. However, their implementation on a technical scale might be problematic [63].

Table 3. The strategies for improving methane production from organic wastes.

Main Component	Co-Substrate	Mixing Ratio or Additive Mass	Method for Improving Methane Production %	VS Removal	Improvement of VS Removal (as Compared with Control) %	Methane Yield mLCH4/gVS	Improvement of Methane Yield (as Compared with Control) %	Reference
Thermophilic conditions
WAS	OFMSW	50:50 v/v	-		-	330	233	[64]
SS	FOG	52:48 on the VS basis	-	51	66	670	169	[65]
SS	GW	73:27 on the COD basis	-	45	−10	230	6.5	[66]
WAS	MA	88:12 v/v	-	-	-	388	7	[67]
SS	FW + B	50:50 v/v	Adsorption and immobilization using biochar			432.2	46.2	[61]
Cattle manure	Z	98:2 v/v	Adsorption and immobilization additives using Z	-	-	310	24	[68]
Pig wastes	Z	12 g	Adsorption and immobilization additives using Z	75	198	278	66	[29]
OW	-	-	Steam distillation	-	-	332	-	[54]
SS	-	-	Thermal pre-treatment	39	7	480	47	[69]
SS	-	-	Low-temperature pre-treatment (70 °C)	36.55	10	180	20	[70]
SS	OW + Z OW + BSG + Z	97.9:2.1 VS + 6 g of Z 90.5:1.9:7.6 VS + 3 g of Z	-	61% 62.7%	3 6	420.0 395.0	14 6	This study
Mesophilic conditions
WAS	OFMSW	40:60	Alkaline	67.5	35	337	28	[71]
WAS	FW	2:3	Microwave	56	56	367.6	92	[72]
SS	GT	90:10	Enzyme	-	-	434.3	78	[73]
SS	OW + B	1:1 VS + 30 g of B	Adsorption and immobilization using biochar	-	-	704.10	136	[57]
SS	OW	97.9:2.1 VS	Ultrasound	61.9	4	346	9	[74]

SS, sewage sludge; WAS, waste activated sludge, FW, food waste; OFMSW, organic fraction of municipal solid waste; FOG, fat, oil, and grease; GW, grease waste; B, biochar; MA, micro algae; GT, grease trap; OW, orange waste; BSG, brewery spent grain.

Table 3. The strategies for improving methane production from organic wastes.

Main Component	Co-Substrate	Mixing Ratio or Additive Mass	Method for Improving Methane Production %	VS Removal	Improvement of VS Removal (as Compared with Control) %	Methane Yield mLCH4/gVS	Improvement of Methane Yield (as Compared with Control) %	Reference
Thermophilic conditions
WAS	OFMSW	50:50 v/v	-		-	330	233	[64]
SS	FOG	52:48 on the VS basis	-	51	66	670	169	[65]
SS	GW	73:27 on the COD basis	-	45	−10	230	6.5	[66]
WAS	MA	88:12 v/v	-	-	-	388	7	[67]
SS	FW + B	50:50 v/v	Adsorption and immobilization using biochar			432.2	46.2	[61]
Cattle manure	Z	98:2 v/v	Adsorption and immobilization additives using Z	-	-	310	24	[68]
Pig wastes	Z	12 g	Adsorption and immobilization additives using Z	75	198	278	66	[29]
OW	-	-	Steam distillation	-	-	332	-	[54]
SS	-	-	Thermal pre-treatment	39	7	480	47	[69]
SS	-	-	Low-temperature pre-treatment (70 °C)	36.55	10	180	20	[70]
SS	OW + Z OW + BSG + Z	97.9:2.1 VS + 6 g of Z 90.5:1.9:7.6 VS + 3 g of Z	-	61% 62.7%	3 6	420.0 395.0	14 6	This study
Mesophilic conditions
WAS	OFMSW	40:60	Alkaline	67.5	35	337	28	[71]
WAS	FW	2:3	Microwave	56	56	367.6	92	[72]
SS	GT	90:10	Enzyme	-	-	434.3	78	[73]
SS	OW + B	1:1 VS + 30 g of B	Adsorption and immobilization using biochar	-	-	704.10	136	[57]
SS	OW	97.9:2.1 VS	Ultrasound	61.9	4	346	9	[74]

SS, sewage sludge; WAS, waste activated sludge, FW, food waste; OFMSW, organic fraction of municipal solid waste; FOG, fat, oil, and grease; GW, grease waste; B, biochar; MA, micro algae; GT, grease trap; OW, orange waste; BSG, brewery spent grain.

Currently, kinetics is an indispensable tool allowing for the improvement of AD performance. It is widely applied for the operation, designing, prediction, as well as monitoring of the AD process. It describes the metabolism pathways occurring with AD, thus allowing for an understanding of the transformation within the process [75]. The results of kinetics modeling of methane production in corresponding series are presented in

Table 4. The results of kinetic evaluation in experimental series.

Series	Modified Gompertz				Logistic Growth
	MP	Rm	λ	R2	MP	Rm	λ	R2
	mL CH4/gVS	mL CH4/gVS d	d	-	mL CH4/gVS	mL CH4/gVS d	d	-
T0	380.1	17.0	−2.98	0.984	343.1	17.7	−2.30	0.983
T1	335.8	16.8	−0.53	0.993	317.9	17.2	−0.23	0.993
T2	362.3	23.7	−1.06	0.991	320.7	23.5	−0.85	0.989
T3	446.9	25.6	−1.29	0.993	415.5	25.9	−0.93	0.991
T4	443.3	20.2	−2.69	0.988	406.7	20.2	−2.49	0.984

. In this study, two models, i.e., modified Gompertz and logistic growth, indicated the best fit that was confirmed by high R² values. Both models include the maximum methane production rate and lag phase.

The lag phase is the time required for bacteria to adapt to methane production. During this period, microorganisms are adapting to the new environment, synthesizing essential enzymes, and preparing for active growth [76]. In both models, λ was negative, indicating that methane production was initiated from the beginning of the experiment, and thus indicating suitable conditions for the growth of microorganisms. A similar trend was also achieved in other studies [77]. In turn, lag phase prolongation might indicate a delay in methane production caused by the presence of inhibitors or the application of hardly biodegradable substrates [78]. Regarding the methane production rate, the highest value was found in the presence of OW and Z (T3), indicating the beneficial influence of Z application. As compared with both T0 and T1, an enhancement above 50% was noted. Generally, the use of zeolites led to a significant improvement in the methane production rate depending on the adopted dose that is related to enhancing microbial activity, improving the degradation of organic matter, and addressing inhibitory factors in the system.

In (T4) supplied by OW, BSG, and Z, the deterioration of the methane production rate occurred, in contrast with the trial without Z application (T2). This fact might be related to the adopted lower dose of zeolite, as in the case of T3. However, increasing the dose of zeolite may not bring a favorable result due to the reduction in free water and disturbing nutrient transport in the digester. This effect was observed in AS of piggery waste, where the deterioration of kinetics was found at the highest Z dose [79]. In addition, Z might adsorb various organic compounds present in BSG, making them less effective. It should be noticed that the lowest R_m was found in T1, also confirming the inhibition phenomenon.

The predicted methane production (MP) achieved in this study corresponded to the experimental data. However, in both models, the highest values were achieved, as compared with experimental data. Diminished MP was found in T1 and T2. In turn, the application of Z resulted in the improvement of MP in both models. Major growths were found for T3 supplied by SS, OW, and Z, reaching 18 and 21% in the modified Gompertz and logistic growth models, respectively, as compared with the reference trial (without Z use).

3.3. Energy Balance Evaluation

One of the crucial factors within the successful implementation of this strategy is the evaluation of energy balance. This fact is particularly important considering the thermophilic conditions for AD that require additional energy to heat the feedstock of the digester. Figure 4 shows that, in all cases, the thermal energy obtained as a result of methane combustion will completely cover the total energy demand, which consists of heating the feedstock to thermophilic conditions as well as covering the heat losses through the digester walls. It should be noticed that, in T1 and T2 with diminished methane production, the profit of thermal energy was reduced by 27 and 7% as compared with T0, where mono-digestion of SS was conducted, thus excluding the use of this strategy in WWTPs. In T3 and T4, the profit of thermal energy was improved by 67 and 69% as compared with T0. In turn, with reference to the series without Z application (T1 and T2), the profits were enhanced by 94 and 76% in T3 and T4, respectively. Therefore, the proposed technology might be considered as a profitable solution.

4. Conclusions

In this study, the application of Z was proposed as a solution allowing for overcoming the difficulties of the thermophilic co-digestion of sewage sludge and beverage industry wastes represented by OW and BSG. The introduction of Z resulted in significantly improved stability parameters as well as reduced inhibitors, i.e., VFA, d-limonene, and phenols. Moreover, enhanced biogas and methane production was achieved in the presence of Z. As compared with SS mono-digestion, methane production was enhanced by 14 and 6% in the OW and SS (T3) and OW, BSG, and SS (T4) series, respectively. The average values were 420.2 and 395.2 mLCH₄/gVS in T3 and T4, respectively. It was accompanied by an improved methane production rate, also revealing a beneficial effect of Z application. The energy balance evaluation indicated that, in the presence of Z, the achieved gain of thermal energy was enhanced by 67 and 69% as compared with SS mono-digestion, making it a profitable solution for WWTPs. The obtained results might indicate the pathway in the case of the effective application of other organic wastes containing inhibitors in the AD process conducted under thermophilic conditions.

However, it should be noted that the conducted research is at the preliminary stage and presents some shortcomings. The future perspective should include the following issues:

• The experiment should be continued in the continuous mode that involves the influence of operational parameters, i.e., hydraulic retention time and organic loading rate, on AD performance;
• The identification of the microbial community should be conducted; it would indicate the metabolic pathways with the anaerobic bioconversion of selected substrates and allow the full understanding of the impact of Z.