Enhancing Biogas Production: Pre-Treatment of Lignocellulosic Biomass Using Biogas Plant Digestate

Abstract

Pre-treatment of lignocellulosic biomass is a necessary step to improve the degradability of these materials when used as substrates for biogas production, due to their resistance resulting from their complex composition and structural properties. The effect of using digestate for the pre-treatment of maize waste to enhance biogas production in an anaerobic digestion process was assessed through biogas potential tests and long-term operation of an anaerobic reactor model. The biogas potential tests confirmed the positive effect of soaking maize waste in digestate for pre-treatment compared to soaking it in water, as higher specific biogas production rates of 17%, 18%, and 29% were achieved after soaking it in digestate for 1 day, 2 days, and 5 days, respectively. The results from monitoring the long-term operation of the anaerobic reactor demonstrated the suitability of using digestate-soaked maize waste as a co-substrate to maize silage, which may significantly reduce the dependence on maize silage in practical applications. Stable operation of the reactor was also achieved during anaerobic treatment of the pre-treated maize waste itself, with an average specific biogas production of 403 mL/g VS.

1. Introduction

With the increasing demand for energy and the dwindling supply of fossil fuels, the search for renewable energy sources is an urgent task [1,2]. Lignocellulosic biomass represents a raw material with significant potential for bioenergy production and is also one of the most economical and highly renewable natural resources in the world [3]. However, lignocellulosic biomass, as a promising renewable energy source, is characterized by recalcitrance due to its complex compositional and structural organization, which leads to high resistance to biological degradation [4].

The enzymatic degradation of lignocellulosic materials is limited by properties such as lignin content, cellulose crystallinity, and limited surface accessibility. Therefore, the pre-treatment of these materials is essential to improve enzymatic hydrolysis and enhance both biogas and methane yields [5]. To facilitate the anaerobic digestion of lignocellulosic biomass, various pre-treatment technologies are available, including physical, chemical, biological, and combined approaches. Many of these techniques have proven to be highly effective in increasing biogas production.

Physical pre-treatment methods involve mechanical techniques such as chipping, grinding, extrusion, and cavitation [6]. These techniques enhance microbial and enzymatic accessibility during anaerobic digestion and do not generate toxic compounds that might inhibit the process. However, mechanical pre-treatment is generally more costly due to its high energy demands [5]. Biological pre-treatment utilizes fungi, bacteria, enzymes, or microbial consortia to boost biogas production and improve cellulose recovery [7]. Although biological methods are noted for their low capital and operational costs and the absence of chemical additives, they have drawbacks, including long incubation times, low processing rates, and high sensitivity to inhibition [8].

Chemical pre-treatment includes the use of acids, alkalis, various solvents, and oxidizing agents. These methods are considered highly efficient for biomass delignification in a short timeframe [9]. Despite their effectiveness, chemical treatments come with several disadvantages, such as high capital investment, risk of toxic by-product formation, and secondary pollution concerns, especially when large volumes of waste must be treated [8,10].

The study of Jankovičová et al. [11] explored the pre-treatment of lignocellulosic materials using mineral acids and bases. The most favorable results were achieved with 0.05% NaOH. However, even this low concentration required significant reagent use, and neutralization was needed prior to the anaerobic digestion, which increased the salinity of the digestate—a potential issue for soil application [11].

Since many studied pre-treatment methods present challenges such as economic demand, high energy requirements, and the generation of undesirable by-products or waste, for the efficient recovery of lignocellulosic biomass in practice, it is necessary to investigate alternatives that are both economically and ecologically viable. This calls for avoiding the use of chemicals, enzymes, and heat-intensive processes and minimizing the production of secondary waste, which presents additional management challenges—as seen with certain chemical methods.

The aim of this study is to explore an alternative cost-effective and environmentally friendly pre-treatment method for lignocellulosic biomass. In our previous work, we examined a simple method of pre-treatment of lignocellulosic biomass involving soaking it in water, which yielded promising results. Soaking lignocellulosic substrate in water for 1 day showed a positive effect on increasing biogas production compared to the substrate without this pre-treatment [12]. In the current study, we investigated the use of digestate (the liquid part of the fermentation residue) from biogas plants as a simple and advantageous pre-treatment medium.

Digestate is a by-product of anaerobic digestion, consisting primarily of undecomposed organic matter, minerals, water, and microorganisms [13]. Among the main components of the digestate is nitrogen, which is an essential component found in all types of biomass. The nitrogen content of digestate can vary based on the substrates used in biogas production [14].

Since the microorganisms in digestate are well established in the anaerobic digester, we hypothesize that they possess effective capabilities for lignocellulose degradation, and their biochemical potential can be utilized as a biological pre-treatment strategy. The selected pre-treatment method will be assessed through tests of biogas potential and long-term operation of an anaerobic reactor model.

2. Materials and Methods

The experimental part of this study consisted of biogas potential tests and monitoring the long-term operation of a laboratory-scale anaerobic reactor, as outlined in the following diagram (Figure 1).

2.1. Materials

• Substrates

The lignocellulosic substrate used to investigate the effect of pre-treatment for enhanced biogas production was maize waste (MW), as it is among the most commonly produced agricultural residues, especially in Slovakia. Global maize production has steadily increased by around 40% over the last decade [15]. This material includes all post-harvest parts of the maize plant, excluding the grain, such as stems, leaves, husks, and similar components. The plants were harvested at the stage of full physiological maturity and in a dry state, i.e., with a high dry matter content, from agricultural fields in Slovakia. The material was then either manually cut into pieces 1 to 3 cm in size (Figure 2a) or ground to a particle size of 2 mm (Figure 2b) using a mill from KINEMATICA AG, model PX-MFC 90 D (Malters, Switzerland). The maize waste was stored at room temperature throughout the experimental period.

Another substrate used during the long-term operation of the reactor was maize silage (MS), which remains the most commonly utilized feedstock in biogas plants today. The total solids (TS) and volatile solids (VS) contents of the substrates used are presented in

Table 1. Characteristics of the substrates.

Substrate	TS (g/g)	VS (g/g)
maize waste	0.940 ± 0.022	0.866 ± 0.031
maize silage	0.441 ± 0.058	0.427 ± 0.059

.

• Inoculum

Anaerobically stabilized sludge from the wastewater treatment plant in Devínska Nová Ves (Bratislava, Slovakia) was used as the inoculum in the tests of biogas potential. To evaluate the effect of soaking lignocellulosic biomass in digestate compared to using water for soaking, the sludge was diluted 4 times to achieve a suitable volatile solids ratio between inoculum and the substrate, based on the volume capacity of the test bottles. For the long-term operation of an anaerobic reactor, inoculum was sourced from the biogas plant in Hurbanovo (Slovakia). The characteristics of the inocula used are summarized in

Table 2. Characteristics of the inocula.

Inoculum	TS (g/L)	VS (g/L)
tests of biogas potential—impact of digestate	23.63 ± 0.21	17.26 ± 0.23
tests of biogas potential—impact of amount of digestate	17.30 ± 0.21	12.36 ± 0.22
long-term reactor operation	30.54 ± 0.18	20.37 ± 0.19

.

• Digestate

The digestate, representing the liquid part of fermentation residue used for the pre-treatment of lignocellulosic biomass, was sourced from a biogas plant in Hurbanovo (Slovakia). The characteristics of the digestate are presented in

Table 3. Characteristics of the digestate.

Parameter
TS (g/L)	62.39 ± 0.71
VS (g/L)	47.45 ± 0.69
pH	7.91
COD (mg/L)	6300 ± 200
N-NH4+ (mg/L)	1372 ± 6
P-PO43− (mg/L)	22 ± 0.6
VFA (mg/L)	1805 ± 135

. Parameters such as COD (chemical oxygen demand), N-NH₄⁺, P-PO₄³⁻, and VFA (volatile fatty acids) were determined from the filtered digestate sample.

2.2. Analytical Methods

Parameters such as chemical oxygen demand (COD), ammonia nitrogen (N-NH₄⁺), phosphate phosphorus (P-PO₄³⁻), volatile fatty acids (VFA), total solids (TS), and volatile solids (VS) were determined according to the standard methods for the examination of water and wastewater [16]. pH values were measured using a Hach HQ11d pH meter. The biogas composition was analyzed using a GA 2000 Plus gas analyzer (Geotechnical Instruments, Coventry, UK).

2.3. Tests of Biogas Potential

The test of biogas potential was conducted according to method described by Angelidaki et al. [17]. The tests were performed in 310 mL bottles, where the inoculum was mixed with the substrate at a volatile solid ratio of 2:1 (inoculum:substrate). The bottles were then sealed with rubber stoppers and incubated in a thermostat at 37 °C. Biogas volume was measured at specific time intervals. For each substrate variant, the test was performed in triplicate, and the average biogas production was calculated. Blank trials containing only inoculum (without substrate) were also monitored, and their biogas production was subtracted from the corresponding test values during result evaluation. The produced biogas was collected in gas-tight biogas bags for subsequent analysis of its composition.

A modified Gompertz model was used to describe the kinetics of biogas production as follows:

$$\mathrm{S}\mathrm{B}\mathrm{P}(\mathrm{t})={\mathrm{S}\mathrm{B}\mathrm{P}}_0\ast \mathrm{e}\mathrm{x}\mathrm{p}\left\{-\mathrm{e}\mathrm{x}\mathrm{p}\ast \left[{\displaystyle \frac{{\mathrm{R}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\ast \mathrm{e}}{{\mathrm{S}\mathrm{B}\mathrm{P}}_0}}\ast \left(\mathsf{\lambda }-\mathrm{t}\right)\right]+1\right\}$$

(1)

where SBP(t) is the cumulative specific biogas production (SBP) at time t (mL/g VS), SPB₀ is the total SBP (mL/g VS), R_max is the maximum specific biogas production rate (mL/g VS/d), and λ is the inhibition phase (d). The Solver in MS-Excel was used to determine these kinetic parameters.

In the biogas potential test, the effect of soaking the substrate in digestate compared to soaking it in water, the effect of soaking time, and the effect of substrate particle size on the enhancement of biogas production were assessed. Prior to testing, maize waste was pre-treated under the following conditions:

-

Soaking in digestate at room temperature for 1, 2, or 5 days, using either the cut or ground fraction of maize waste.

-

Soaking in water at room temperature for 1, 2, or 5 days, using the cut fraction of maize waste.

The quantities of inoculum, soaking water, soaking digestate, and maize waste used are listed in

Table 4. Quantities of inoculum, soaking media, and maize waste used.

	Inoculum (mL)	Water (mL)	Digestate (mL)	Cut MW (g)	Ground MW (g)
blank	140	20	-	-	-
blank + digestate	140	18	2	-	-
cut MW soaked in digestate for 1 day	140	18	2	0.238	-
cut MW soaked in digestate for 2 days	140	18	2	0.238	-
cut MW soaked in digestate for 5 days	140	18	2	0.238	-
cut MW soaked in water for 1 day	140	20	-	0.238	-
cut MW soaked in water for 2 days	140	20	-	0.238	-
cut MW soaked in water for 5 days	140	20	-	0.238	-
ground MW soaked in digestate for 1 day	140	18	2	-	0.238
ground MW soaked in digestate for 2 days	140	18	2	-	0.238
ground MW soaked in digestate for 5 days	140	18	2	-	0.238

, with maize waste serving as the substrate in combination with the respective soaking medium. In addition to standard blanks containing only inoculum, the test also included blanks with inoculum and the corresponding volume of digestate used for soaking to account for the endogenous biogas production from both the anaerobic sludge and the digestate alone.

In the biogas potential test aimed at evaluating the effect of the amount of digestate used for pre-treatment, maize waste was pre-treated by soaking it in digestate at room temperature for 1 day in varying volumes of digestate or water. Both the cut and ground fractions of maize waste were used. The quantities of inoculum, soaking water, digestate, and maize waste applied in this test are listed in

Table 5. Quantities of inoculum, soaking media, and maize waste used.

	Inoculum (mL)	Water (mL)	Digestate (mL)	Cut MW (g)
blank	140	20	-	-
blank + digestate	140	-	20	-
cut MW soaked in 5 mL of digestate	140	15	5	0.993
cut MW soaked in 10 mL of digestate	140	10	10	0.993
cut MW soaked in 15 mL of digestate	140	5	15	0.993
cut MW soaked in water	140	20	-	0.993

. As in previous experiments, anaerobic sludge was used as the inoculum. Additionally, blank tests containing the anaerobic sludge along with the corresponding volume of digestate used for soaking were included to account for the endogenous biogas production from both the sludge and the digestate.

2.4. Long-Term Reactor Operation

The long-term operation of the anaerobic reactor model involved daily substrate dosing and biogas production monitoring, with its composition analyzed several times during the operation. Once per week, parameters such as pH, TS, and VS were determined from the collected excess sludge, while parameters such as COD, N-NH₄⁺, P-PO₄³⁻, and VFA were determined in the filtered excess sludge samples. The anaerobic reactor model had a working volume of 15 L and was operated at 37 °C, i.e., under mesophilic conditions. The setup consisted of a sealed reactor vessel with an inlet for substrate dosing, a small outlet connected via tubing to a drum-type laboratory gas meter (Spektrum s.r.o., Skuteč, Czech Republic), a heating device, and a variable-speed stirrer (Hei-TORQUE100, Heidolph, Schwabach, Germany).

At the beginning of operation, the anaerobic reactor was fed with maize silage, and the organic loading rate (OLR) was gradually increased from 0.5 kg VS/m³/d to 1.5 kg VS/m³/d. Subsequently, pre-treated maize waste was added as a co-substrate to the maize silage. The pre-treatment involved soaking the maize waste in digestate for one day at room temperature. The particle size of the maize waste used was 1–3 cm. The volume of digestate used for soaking was adjusted so that the dry matter content of the pre-treated substrate was comparable to that of maize silage. Specifically, 4.8 mL of digestate was used per 1 g of maize waste.

The pre-treated maize waste was then co-dosed with maize silage daily in a 1:1 ratio according to the VS of the substrates at the following OLRs: 1.50 kg VS/m³/d for 24 days, 1.75 kg VS/m³/d for 28 days, and 2 kg VS/m³/d for 30 days. At an OLR of 2 kg VS/m³/d, the ratio of maize silage to pre-treated maize waste was changed to 1:2 according to the VS of the substrates over 78 days. In the final operational phase, only pre-treated maize waste was processed at an OLR of 1.75 kg VS/m³/d for 85 days.

3. Results and Discussion

3.1. Tests of Biogas Potential

The results of the tests of the biogas potential are presented in the following subsections, including plots of cumulative specific biogas production (SBP). In these, solid lines indicate the experimentally obtained results, while dashed lines represent the values calculated using the modified Gompertz model.

Two blank samples were monitored during the test: inoculum alone and inoculum with added digestate. This was carried out under the hypothesis that higher biogas production might occur when digestate was used to soak the substrate, due to the potential biogas contribution of the digestate itself, given its high total solids content. Based on the cumulative SBP trends observed in these blank experiments (as shown in Figure 3) and the final total SBP values—147.9 mL/g VS for the inoculum alone and 148.5 mL/g VS for the inoculum with digestate—it is evident that the total solids in the digestate are stabilized, similarly to the anaerobic sludge used.

3.1.1. Effect of Using Digestate Versus Water for Pre-Treatment

The effect of soaking of maize waste in digestate was compared to the effect of soaking it in water, as even a one-day water soak has been shown to positively influence biogas production compared to untreated material, as reported by Jankovičová et al. [12].

When the pre-treatment consisted of a one-day soaking period (Figure 4), soaking maize waste in digestate resulted in a 17% increase in SBP compared to soaking it in water. The SBP achieved was 419 mL/g VS for digestate-soaked maize waste and 358 mL/g VS for water-soaked maize waste.

Pre-treatment of the substrate by soaking it for two days (Figure 5) resulted in a total SBP of 494 mL/g VS for digestate-soaked samples and 417 mL/g VS for those soaked in water. This corresponds to an 18% increase in SBP when using digestate was used for pre-treatment compared to water.

For a soaking period of five days (Figure 6), soaking in digestate resulted in an increase up to 29% in SBP compared to soaking in water. The achieved SBP was 458 mL/g VS for digestate-soaked maize waste, while 355 mL/g VS was obtained for water-soaked material.

The results clearly indicate that the pre-treatment of maize waste by soaking it in the liquid fraction of the digestate from the biogas plant had a positive effect on biogas production compared to soaking it in water, as higher SBP values were obtained at each soaking duration when digestate was used.

Table 6. Resulting SPB values and calculated parameters of the modified Gompertz model.

	SBP (Measured) (mL/g VS)	SBP (Model) (mL/g VS)	Rmax (mL/g VS/d)	λ (d)	R2
blank	147.88	142.29	4.21	−4.2823	0.9800
blank + digestate	148.54	143.22	4.56	−3.9645	0.9834
cut MW soaked in digestate for 1 day	419.13	389.14	28.29	−0.2127	0.9743
cut MW soaked in digestate for 2 days	494.51	445.55	38.74	0.7839	0.9801
cut MW soaked in digestate for 5 days	457.96	426.38	44.77	0.7143	0.9856
cut MW soaked in water for 1 day	358.26	338.36	24.01	−0.0583	0.9809
cut MW soaked in water for 2 days	417.31	388.24	32.19	0.0992	0.9832
cut MW soaked in water for 5 days	354.70	334.26	32.38	0.9316	0.9910

represents the resulting values of specific biogas production along with the calculated parameters of the modified Gompertz model for the studied variants. The results of the regression analysis demonstrate a good fit between the model and the experimental data. The small differences between the measured and predicted values, as well as the high coefficient of determination (R²), support the validity of the model. However, the predicted biogas production potential calculated using the modified Gompertz model was slightly lower than the experimentally obtained values.

The positive effect of pre-treating maize waste with liquid digestate is also reported in the study of Hu et al. [18], in which maize waste was pre-treated by soaking it in digestate for four different durations (1, 3, 5, and 7 days) at ambient temperature (20 ± 1 °C). The biomethane potential test showed that all pre-treated samples achieved 65- to 80%-higher methane production compared to the untreated sample. A 3-day soaking period was identified as the optimal pre-treatment method.

In the study by Liu et al., liquid digestate with a high N-NH₄⁺ concentration (3750 mg/L) was used to pre-treat wheat straw, which improved substrate degradability and increased methane yield. Wheat straw was pre-treated by soaking it in liquid digestate for 3, 5, and 7 days at 38 °C. The specific methane production for untreated wheat straw was 162 mL/g VS, while values of 217 mL/g VS (34% increase), 243 mL/g VS (50% increase), and 247 mL/g VS (53% increase) were achieved after pre-treatment for 3, 5, and 7 days, respectively. This study also compared the liquid digestate pre-treatment with ammonia solution pre-treatment at the same N-NH₄⁺ concentration, and found that both methods yielded similar biogas production levels. This finding confirms that ammonolysis plays a major role in the synergism action of various substances in liquid digestate and is the key factor contributing to the increase in biogas production. However, unlike the ammonia solution pre-treatment method, pre-treatment using liquid digestate with a high concentration of N-NH₄⁺ does not require additional chemical reagents, thereby significantly reducing the cost of pre-treatment. Therefore, high-N-NH₄⁺ liquid digestate is recommended for the pre-treatment of lignocellulosic material [19].

3.1.2. Effect of Substrate Particle Size

To evaluate the effect of substrate particle size on biogas production during pre-treatment by soaking in digestate at three different durations, the performance of cut maize waste with a particle size of 1–3 cm was compared with that of ground maize waste with a particle size of 2 mm. The cumulative specific biogas production profiles for both cut and ground fractions pre-treated by soaking them in digestate are shown in Figure 7 (soaking time: 1 day), Figure 8 (soaking time: 2 days), and Figure 9 (soaking time: 5 days).

The positive effect of reducing substrate particle size on biogas production was evident only at the one-day soaking duration (Figure 7). At this pre-treatment time, a total SBP of 498 mL/g VS was achieved using the ground fraction (2 mm), compared to 419 mL/g VS for cut fraction (1–3 cm) of the substrate. This represents a 19%-higher biogas yield with the smaller particle size fraction. Physical pre-treatment by particle size reduction increases the substrate’s surface area, enhancing enzyme accessibility [20,21].

Similarly, Menardo et al. investigated the effect of particle size on methane yield from lignocellulosic substrates such as wheat and barley straw. When the particle size of wheat straw was reduced from 5 cm to 0.2 cm, the specific methane yield increased by 17%, from 285 to 334 mL CH4/g VS. In the case of barley straw, the reduction from 5 cm to 0.2 cm led to an even greater increase of 29%, from 286 to 370 mL CH4/g VS [22].

Nevertheless, at soaking times of two and five days, particle size reduction of the substrate did not prove effective, as lower biogas yields were observed when using the ground maize waste fraction. At a soaking time of 2 days (Figure 8), the total SBP was 449 mL/g VS for the ground substrate and 495 mL/g VS for the cut substrate. At a soaking time of 5 days (Figure 9), the total SBP was 428 mL/g VS for the ground maize waste and 458 mL/g VS for the cut fraction. Similarly, in the study by De la Rubia et al., the highest methane yields were observed for the largest particle size fraction (1.4–2.0 mm) compared to smaller particle size fractions (0.36–0.55 mm and 0.71–1.0 mm) [23]. A reduction in particle size can accelerate hydrolysis, leading to the accumulation of volatile fatty acids and excessive acidification and thus process failure [24].

However, when using the ground fraction of the substrate, we observed a slightly higher methane content in the biogas produced compared to the cut fraction. At a one-day soaking time, the methane content was 40.1% for the ground fraction and 37.5% for the cut fraction. At a soaking time of 2 days, these values were 40.8% for the ground fraction and 39.8% for the cut fraction. At a soaking time of 5 days, the measured methane content was 41.0% for the ground fraction and 40.0% for the cut fraction.

The positive effect of MW particle disintegration during digestate pre-treatment—where biochemical potential, including microbial activity, can be exploited—may be most pronounced for ground MW at shorter soaking durations. This is due to the enhanced access of the digestate to the lignocellulosic structure, provided by the larger specific surface area of smaller particles. At longer soaking times, digestate components penetrate deeper into the larger particles of the substrate.

Table 7. Resulting SPB values and calculated parameters of the modified Gompertz model.

	SBP (Measured) (mL/g VS)	SBP (Model) (mL/g VS)	Rmax (mL/g VS/d)	λ (d)	R2
cut MW soaked in digestate for 1 day	419.13	389.14	28.29	−0.2127	0.9743
cut MW soaked in digestate for 2 days	494.51	445.55	38.74	0.7839	0.9801
cut MW soaked in digestate for 5 days	457.96	426.38	44.77	0.7143	0.9856
ground MW soaked in digestate for 1 day	498.25	449.69	41.86	0.6135	0.9716
ground MW soaked in digestate for 2 days	449.03	409.35	39.67	0.9160	0.9755
ground MW soaked in digestate for 5 days	428.20	408.35	40.46	0.9291	0.9846

summarizes the resulting values of specific biogas production and the calculated parameters of the modified Gompertz model for cut and ground maize waste soaked in digestate at all tested durations. As shown by the experimental and modeled SBP values in the figures, the modified Gompertz model describes the anaerobic decomposition of the substrates well, as indicated by high coefficients of determination.

3.1.3. Effect of Soaking Time in Digestate

For the digestate soaking pre-treatment method, the effect of soaking duration on biogas production enhancement was also evaluated. A comparison of the SBP profiles at different soaking times for cut MW is presented in Figure 10. The results indicate that increasing the soaking time in digestate had a positive effect on biogas production. Compared to a one-day soaking period, SBP increased by 18% at a soaking time of 2 days and by 9% at a soaking time of 5 days. A similar trend was observed in the methane content of the biogas. The methane content was measured to be 37.5%, 39.8%, and 40.0% at soaking times of 1, 2, and 5 days, respectively.

However, extending the soaking time beyond 2 days proved ineffective, as an 8%-higher SBP was achieved for a soaking time of 2 days compared to a soaking time of 5 days. A comparable observation was reported by Zheng et al. [25] who found the highest methane yield when straw was pre-treated with biogas slurry for 4 days, with a decline in performance observed at 6 and 8 days of pre-treatment. This may be attributed to the consumption of volatile fatty acids (VFAs) by methanogens during the pre-treatment phase, which reduces their availability for subsequent anaerobic digestion, thereby negatively affecting overall methane production [25].

In the case of the ground substrate fraction, the reduction in pre-treatment efficiency with longer soaking times was more pronounced. When using the ground MW fraction (Figure 11), increasing the soaking time in the digestate to 2 days resulted in a 10% decrease in SBP and increasing the soaking time to 5 days led to a 14% decrease compared to a soaking time of 1 day. Prolonged pre-treatment times may lead to excessive cellulose and hemicellulose decomposition, thereby reducing the availability of fermentable material for the subsequent anaerobic digestion process [26].

The decrease in biogas production with longer soaking durations is likely due to the degradation of readily fermentable compounds during the pre-treatment phase. As a result, the amount of available substrate for anaerobic microorganisms during digestion was reduced, leading to lower biogas yields after extended soaking times.

The difference between the modeled and experimental values from Figure 10, especially in the case of cut MW soaked for 1 day, may be attributed to measurement inaccuracies, which were also reflected by minor fluctuations in biogas production that were not captured by the mathematical model. Nevertheless, the modified Gompertz model is still considered to describe the anaerobic decomposition of substrates well, as evidenced by high coefficient of determination values ranging from 0.97 to 0.99, as shown in Table 7.

3.1.4. Effect of the Amount of Digestate

The effect of the amount of digestate used for pre-treatment of maize waste was also investigated. Various combinations of digestate and water amounts were compared, with water alone serving as a control. The results of the biogas potential test using the cut MW fraction are shown by the course of the SBP in Figure 12.

The results obtained confirmed that increasing the amount of digestate in the soaking medium during pre-treatment enhanced biogas production, with the highest SBP values obtained when the highest amount of digestate was used (15 mL digestate + 0 mL water). This positive effect of a higher concentration of pre-treatment medium also corresponds with the results of other works dealing with the pre-treatment of lignocellulosic biomass.

In the study by Zheng et al., swine manure digested effluent containing high concentrations of ammonia nitrogen (1000, 1500, and 2000 mg/L) were used to pre-treat rice straw. The highest methane yield was achieved when using the highest concentration of ammonia nitrogen (2000 mg/L) for rice straw pre-treatment. In addition, monitoring changes in the surface structure showed that the trial containing 2000 mg/L ammonia had the most intensive destruction of lignocelluloses of the rice straw [25]. Similarly, in the study by Dong et al. [27], different biogas slurry–water ratios were tested for maize stalk pre-treatment. A 4:1 volume ratio resulted in greater lignin removal compared to a 3:2 ratio, indicating that the increased amount of biogas slurry contributed to enhanced lignocellulose breakdown [27].

Table 8. Resulting SPB values and calculated parameters of the modified Gompertz model.

	SBP (Measured) (mL/g VS)	SBP (Model) (mL/g VS)	Rmax (mL/g VS/d)	λ (d)	R2
cut MW soaked in water	659.44	649.11	27.89	−2.4512	0.9912
cut MW soaked in 5 mL of digestate	739.69	727.36	29.77	−2.3241	0.9921
cut MW soaked in 10 mL of digestate	725.12	712.13	26.69	−2.7209	0.9925
cut MW soaked in 15 mL of digestate	786.74	762.38	30.75	−2.4189	0.9884

shows the resulting values of specific biogas production and the calculated parameters of the modified Gompertz model for the tested variants using cut and ground maize waste. The high value of the coefficient of determination further confirms that the modified Gompertz model provides a reliable description of the anaerobic degradation process.

3.2. Monitoring of Long-Term Reactor Operation

During the long-term operation of the anaerobic reactor, the effect of the pre-treatment of soaking in the liquid part of the fermentation residue from the biogas plant for 1 day at room temperature on biogas production was investigated. The pre-treated maize waste was used as a co-substrate with maize silage at a ratio of 1:1 (based on VS of the substrates) under three different organic loading rates (OLRs): 1.5 kg VS/m³/d, 1.75 kg VS/m³/d, and 2 kg VS/m³/d. At the highest OLR of 2 kg VS/m³/d, the long-term operation of the anaerobic reactor was further used to assess the possibility of replacing a larger proportion of the maize silage with pre-treated maize waste, i.e., at a ratio of maize silage:maize waste = 1:2 (based on VS of the substrates). In the final phase, the reactor was operated at 1.75 kg VS/m³/day using only pre-treated maize waste as the substrate, without any maize silage. This operational mode is marked as MS:MW = 0:1 in the subsequent figures. The effect of the method used for the pre-treatment of maize waste and the ratio of the dosed substrates on biogas production are shown in Figure 13, which presents the specific biogas production profile over the course of reactor operation.

At a 1:1 dosage ratio of co-substrates (on a VS basis), the anaerobic treatment was characterized by stable reactor operation, even with a gradual increase in the OLR. At an OLR of 1.5 kg VS/m³/d, the average daily biogas production reached 10.8 L/d, with a specific biogas production of 479 mL/g VS and a methane content of 49%. When the OLR was increased to 1.75 kg VS/m³/d, an increase in specific biogas production was observed, with an average SPB value of 495 mL/g VS. The average value of daily biogas production was 13.0 L/d, and the measured methane content during this phase was 47%.

Even at a higher OLR of 2 kg VS/m³/d, the system maintained relatively high performance, with an average SBP of 467 mL/g VS, a daily biogas production of 14.0 L/d, and a methane content of 49%. A temporary drop in production was observed between days 71 and 83 due to the interruption of substrate dosing. However, reactor performance returned to a steady state immediately after dosing resumed.

These results confirm the suitability of using digestate-soaked maize waste as a co-substrate for maize silage at a 1:1 ratio (on a VS basis).

After 86 days of operation at an OLR of 2 kg VS/m³/d, the ratio of the dosed pre-treated maize waste to maize silage was increased to assess whether the application of the studied method of maize waste pre-treatment could substitute an even more substantial proportion of maize silage in practical applications. At the applied co-substrate dosage ratio, maize silage:maize waste = 1:2 (on a VS basis), the reactor maintained stable operation, with an average daily biogas production of 13.5 L/d and an SBP of 451 mL/g VS. These findings support the feasibility of using higher proportions of pre-treated maize waste as co-substrate in practice.

When only pre-treated maize waste was used as the substrate, a decrease in biogas production was observed. The achieved average daily biogas production was 10.9 L/d, the SBP was 403 mL/g VS, and the methane content remained at 49%. These biogas production values, although lower, still confirm the suitability of using maize waste pre-treated by soaking it in digestate as a substrate for biogas production. This is also confirmed by the stability of the reactor operation at the given substrate dosing conditions.

Throughout the long-term reactor operation, changes in the characteristics of the sludge and filtered sludge water were monitored. The course of changes in the pH parameter is shown in Figure 14. For proper operation of the process, the optimum operating pH range for the maximum growth of methanogens is above 6.8 [28,29]. During operation, this range was maintained, and the pH did not exceed 7.3 as long as both co-substrates were processed.

In contrast, Jankovičová et al. reported a significant pH drop and process inhibition when maize waste soaked in water was used at higher OLRs, likely due to low buffering capacity [12]. In the present study, the use of digestate helped maintain process stability and even caused a slight pH increase, particularly when maize waste was used alone. This effect can be attributed to the higher content of ammoniacal nitrogen in the digestate, both in the liquid phase and suspended solids, which contributed to increased alkalinity and thereby stabilized the anaerobic digestion process.

When monitoring the VFA concentration (Figure 15), a slight effect of increasing the OLR was observed. At OLRs of 1.5 kg VS/m³/d and 1.75 kg VS/m³/d, the VFA values remained within the range of 1200–2100 mg/L. However, at an OLR of 2 kg VS/m³/d, an increase in the VFA concentration to a concentration of almost 2800 mg/L was observed. After this initial rise, the VFA concentration stabilized in the range of 2300–2800 mg/L, and no further significant accumulation of VFAs was observed in the reactor, indicating that the system maintained process balance even at the higher loading rate.

A similar trend was observed in monitoring the COD parameter (Figure 16). There was a significant increase due to the increase in the OLR to 2 kg VS/m³/d. However, from the 92nd day of operation, COD values stabilized to levels comparable to those observed at lower OLRs, confirming the reactor’s ability to adapt to increased substrate input without long-term destabilization.

The effect of increasing the OLR was not evident when monitoring changes in N-NH₄⁺ concentration during reactor operation (Figure 17). The N-NH₄⁺ values varied between 400 and 600 mg/L. However, larger fluctuations in N-NH₄⁺ concentration values were observed when the co-substrate dosing ratio was increased.

The P-PO₄³⁻ values (Figure 18) were in the range of 10–30 mg/L, with a notable increase observed following the increase in the OLR to 2 kg VS/m³/d. Both N-NH₄⁺ and P-PO₄³⁻ concentrations showed a rising trend when only pre-treated maize waste was used as a substrate. Based on the N and p values determined during operation, it can be concluded that their concentrations were sufficient to ensure an appropriate COD:N:P ratio for anaerobic digestion, considering the optimal COD:N:P ratio of approximately 300:5:1 and also the reported minimum PO₄³⁻ concentration required by anaerobic microorganisms of 5–7 mg/L [30]. Furthermore, the concentrations of N-NH₄⁺ and P-PO₄³⁻ observed throughout operation did not reach levels associated with inhibitory effects on biogas production.

No significant fluctuations or increasing trends were observed in the TS and VS contents of the anaerobic sludge in the reactor (Figure 19), which suggests that accumulation of undecomposed material in the reactor did not occur. The TS and VS values were approximately 70–90 g/L and 55–70 g/L, respectively.

From the presented results of anaerobic reactor operation, either from the achieved values of biogas production or the monitored parameters of sludge and sludge water, it is possible to establish the suitability of using digestate from biogas plants for the pre-treatment of maize waste. The positive effect of using digestate for pre-treatment is found not only in its biochemical potential to degrade the lignocellulosic structure but also in its sufficient nitrogen content, which also ensures necessary alkalinity of the system.

This positive effect of the higher nitrogen content in digestate used for the pre-treatment of lignocellulosic biomass is further supported by comparison with the work of Jankovičová et al., where the method of pre-treating maize waste by soaking it only in water at room temperature for one day was investigated. In that study, during long-term reactor operation, co-digestion with maize silage eventually led to process failure, preceded by a sharp pH drop and VFA accumulation. Thus, when only water was used for the pre-treatment of maize waste, the system did not have sufficient buffering capacity compared to the use of digestate for pre-treatment [12].

The use of digestate to pre-treat lignocellulosic biomass can therefore serve to establish an alkaline environment for subsequent anaerobic digestion, enhance system alkalinity, improve buffering capacity, and prevent acid accumulation during fermentation [26].

Many of the studies published so far dealing with the pre-treatment of maize waste (either straw or stalks) by conventional chemical or physical methods report results with significant increases in biogas yield [31,32,33]. As reported in the study by Venturin et al., the pre-treatment of maize stalk with H₂O₂ resulted in a biogas biochemical potential of 644 mL/g VS, an increase of 54% compared to our results when using one-day soaking in digestate (419 mL/g VS) [34]. Li et al. [35] obtained a maximum biogas value of 427 mL/g VS by pre-treating maize waste with ammonia at a 4% concentration. In a study by Lizasoain et al. [36], a biogas yield of 585 mL/g VS was obtained when maize waste was pre-treated by the steam explosion method at 160 °C for 2 min. Compared to conventional methods such as acid or alkaline hydrolysis, steam explosion, or organosolvent pre-treatment, which may show higher pre-treatment efficiencies, the use of digestate offers several advantages. It is an environmentally friendly and economically viable alternative that does not require expensive chemicals or high energy consumption. Due to its properties mentioned in the previous discussion, digestate may reduce operating costs and promote sustainable biomass processing within a circular bioeconomy.

4. Conclusions

The effect of soaking maize waste in digestate as a pre-treatment method for increasing biogas production was assessed. Results from the biogas potential test revealed that soaking maize waste in digestate had a positive effect on increasing biogas production compared to soaking it in water, as higher SBP values were obtained at each soaking time when soaking it in digestate. The pre-treatment of maize waste by soaking it in digestate demonstrated the biochemical potential of this soaking medium, likely due to the presence of microorganisms capable of breaking down lignocellulosic structures. The long-term monitoring of the anaerobic reactor confirmed the suitability of using digestate-pre-treated maize waste as both a co-substrate with maize silage and as a standalone substrate. The stable reactor performance during all phases was most likely enabled by the high ammoniacal nitrogen content of digestate, which increased the alkalinity of the substrate and improved process stability.