Experimental Study of Two-Stage Anaerobic Co-Digestion of Corn Steep Liquor and Agricultural Wastes for Hydrogen and Methane Production Including Metagenomics

Abstract

This study investigated the dynamics and composition of microbial communities within the bioreactors of a two-stage anaerobic system employed for the bioconversion of corn steep liquor, a food processing byproduct, into hydrogen and methane. The high organic matter content of such wastes positions them as valuable substrates for biotechnological applications. The two-stage anaerobic digestion (AD) process was compartmentalized into a hydrogen-producing bioreactor (3 dm³) and a methane-producing bioreactor (15 dm³), each harboring distinct microbial consortia. The system yielded a maximal hydrogen production of 1.02 L/day and a peak methane production of 24.1 L/day with substrate corn steep liquor and cattle manure in a ratio 1:1. Microbial consortia were recognized as critical drivers of AD performance and biofuel yield. This research demonstrated the efficacy of a two-stage approach, segregating the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis) phases, for optimized energy recovery from the co-digestion of corn steep liquor and cattle manure under controlled conditions. Metagenomic sequencing and a subsequent bioinformatics analysis were utilized to characterize the microbial diversity within each bioreactors. These findings contribute to a deeper understanding of the microbial ecology of AD and hold the potential for broader applications in waste-to-energy bioconversion.

1. Introduction

Contemporary society exhibits heightened interest in the advancement of technologies for sustainable energy generation utilizing renewable resources [1]. AD, specifically the biotransformation of organic waste into biogas, presents a viable pathway within this domain. This process yields multifaceted benefits [2]. Environmentally, biodegradation facilitates a reduction in both the volumetric and organic load of waste materials, thereby mitigating potential ecological perturbations associated with improper waste disposal. Economically, the generation of biogas, a high-calorific renewable fuel, is coupled with a concomitant decrease in expenditures related to waste management and disposal [3]. Amidst escalating climate change concerns, biogas production as a renewable energy source has garnered substantial research attention over the recent decades. Optimizing sustainable hydrogen and methane yields from waste substrates creates some technological hurdles. A two-stage AD strategy, incorporating enhanced hydrolysis to augment the subsequent methanogenesis, has been proposed to maximize the energy recovery from biodegradable organic waste [4]. Hydrogen (H₂), envisioned as a future fuel, offers a high energy density and calorific value, with the added advantage of carbon-neutral combustion [5]. Dark fermentation enables H₂ production from renewable feedstocks. Methane (CH₄), a versatile energy carrier, can be utilized for heat and power generation, as well as being a gaseous vehicular fuel, thereby offering a substitute for natural gas [6].

The AD of diverse organic substrates, encompassing both monosubstrates and heterogeneous blends, has been investigated for biogas generation [7]. The substrate composition significantly modulates the efficiency of the individual digestion stages. Corn steep liquor (CSL), a byproduct of corn wet milling, serves as a versatile industrial feedstock and microbial growth supplement in fermentative processes yielding biofuels, enzymes, and organic acids [8]. The substantial generation of CSL, a readily available waste stream from corn starch production, poses significant environmental challenges [9]. Consequently, research efforts are directed towards its valorization in biotechnological applications, including fructo-oligosaccharide, natamycin, lipid, and docosahexaenoic acid biosynthesis, as well as its utilization as an additive in fresh rice straw silage fermentation [10]. Improved cordycepin production in Cordyceps militaris was achieved through various strategies by specific strain and culture conditions, and utilizing alternative nitrogen sources such as CSL [11]. Corn steep liquor was proven to also be a key ingredient in the manufacture of penicillin [12].

As a sustainable energy source with a high energy yield, hydrogen is a promising alternative to fossil fuels. Co-digestion involves the simultaneous fermentation of multiple substrates and offers opportunities to improve biohydrogen production through synergistic interactions between different waste materials [13]. Hydrogen and methane play a role as energy vectors because both can store, transport, and deliver energy and serve as a medium that allows for the storage and transport of energy from renewable sources, with the aim of further use in various applications like power, transport, or heat [14]. The two-stage AD concept consisting of a hydrogenic process followed by a separate methanogenic process has been suggested as a tool to completely utilize the organic acids produced during dark fermentation and to improve the overall energy conversion efficiency [15]. Most anaerobic reactors do not provide ideal conditions for both acidogenic and methanogenic microorganisms. When the stages are separated, the different microbes receive their favorable environment. The application of two-step processes allows the acidification and methanogenesis phases to be physically separated [4].

This study investigated the sequential biohydrogen and biomethane production within a two-stage bioreactor system. The research focused on elucidating the influence of substrate composition on gaseous product yields, characterizing the temporal dynamics of the microbial community, and, ultimately, developing a biotechnological strategy for the optimized and efficient treatment of targeted waste streams.

2. Materials and Methods

2.1. Preliminary Batch Experiments Set-Up

Batch experiments were conducted using a laboratory installation that consists of four laboratory bottles with total volume of 500 cm³ hermetically enclosed with butyl rubber stoppers. In the rubber stoppers, vents for biogas evacuation were made. The work volume was established at 200 cm³ and the temperature of 35 °C was maintained using water bath, while pH was kept at 5.5. Hydrogen production was followed.

2.2. Two-Stage Bioreactor System

Two continuously stirred-tank bioreactors (CSTRs), with working volumes of 3 dm³ and 15 dm³ (refer to Figure 1), were employed in a sequential anaerobic fermentation system. Both bioreactors were operated in a fed-batch mode with daily substrate replenishment and continuous agitation. Peristaltic pumps facilitated both influent and effluent transfer. Temperature was maintained at 35 ± 0.5 °C in both reactors via an integrated monitoring and control system. The pH of the 3 dm³ bioreactor was controlled at 5.5 through the automated addition of sodium hydroxide or hydrochloric acid solutions, mediated by a pH electrode and regulator connected to peristaltic pumps. A daily volume of 2700 cm³ was transferred from the 15 dm³ bioreactor to a designated effluent reservoir, and an equivalent volume was simultaneously transferred from the 3 dm³ bioreactor to the 15 dm³ bioreactor. A substrate solution, consisting of 75 g of CSL diluted to a final volume of 2700 cm³ with distilled water, was prepared daily and introduced into the 3 dm³ bioreactor to initiate dark fermentation and hydrogen production. The dilution rate (D) was maintained at 0.9 day⁻¹ for the 3 dm³ bioreactor and 0.18 day⁻¹ for the 15 dm³ bioreactor.

2.3. Substrates

ADM^® Corn Steep Liquor 104 (CSL), sourced from “ADM Razgrad EAD”, Razgrad, Bulgaria [16], constituted the primary substrate for the initial experiments. It was fed into the first bioreactor for co-digestion with cattle manure in the continuous process in the system of bioreactors. Its pH was 4.2 ± 0.1; total solids (TS) were determined to be 50.5 ± 0.2% and volatile solids (VS) to be 91 ± 0.3%. Subsequently, the effluent generated by the first bioreactor was utilized as an influent stream for the subsequent methanogenic bioreactor, as depicted in Figure 1. The digestion of corn steep liquor was realized using different agricultural wastes as co-substrates: wastes from potatoes, tomatoes, and cucumbers, and cattle manure. The two substrates were mixed in different ratios: 1:1; 1:2, and 2:1. The investigated organic load values were 5, 10, 15, 20, and 25 g/L. Substrate loading was calculated, normalized to grams of volatile solids (g VS).

2.4. Inoculum

The inoculum for the two-stage AD system was derived from the liquid phase of a functional biogas production bioreactor. To prepare the inoculum for the first stage, performed in the hydrogen-producing bioreactor, thermal pretreatment was employed to inactivate methanogenic microorganisms. Initially, the liquid phase underwent sieving to eliminate large, undigested biomass particles, followed by centrifugation at 4500 rpm. The resulting microbial cell pellet was resuspended and washed twice in a 0.9% NaCl physiological solution. This cell suspension was subsequently subjected to thermal inactivation at 75 °C for 30 min, cooled to ambient temperature, and introduced into the first bioreactor at a volumetric ratio of 10–30% [17]. For the second-stage in the methanogenic bioreactor, the same liquid phase from the operational biogas bioreactor was utilized without pretreatment. This inoculum was directly introduced to fill the working volume (15 dm³) of the bioreactor. Glucose was added to the second bioreactor to assess and confirm methanogenic activity.

2.5. Analytical Methods

The quantification of reducing sugars was achieved through a colorimetric assay based on the redox reaction between reducing sugars and sodium dinitrosalicylate, yielding a chromogenic product exhibiting maximum absorbance at 530 nm, measured by UV–Vis spectrophotometer [18]. Protein concentration was determined utilizing the Bradford assay, which relies on the spectral shift of Coomassie Brilliant Blue G-250 upon protein binding, measured at 595 nm [19]. Cellulose content was assessed spectrophotometrically following sequential extraction with an acetic–nitric reagent to eliminate interfering compounds, dissolution in 67% sulfuric acid, and subsequent reaction with anthrone. The resulting chromophore’s absorbance was quantified at 620 nm [20]. Total solids (TS) and volatile solids (VS) were determined gravimetrically [21]. To mitigate volatilization losses during ashing due to high organic content, a controlled, gradual temperature ramp was employed to ensure slow oxidative decomposition. The heating, annealing, and desiccation procedures were iterated until successive mass measurements exhibited negligible variation. Biogas production was measured volumetrically using a water displacement gas holder and graduated cylinder. Methane and carbon dioxide concentrations were determined using a Dräger X-am7000 gas analyzer, (Oslo, Norway) which employs infrared sensors for volumetric percentage measurement. Hydrogen and carbon dioxide concentrations were determined using a Gasboard 3100P gas analyzer, also utilizing infrared sensors for volumetric percentage measurement. Gas yields are based on total mass.

2.6. DNA Extraction

Samples were aseptically collected from each bioreactor and stored at −20 °C. Genomic DNA was subsequently extracted utilizing the GeneMATRIX Environmental DNA and RNA Purification Kit (EURx Ltd., Gdańsk, Poland). DNA quantification was performed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The resulting DNA concentrations and volumes were as follows: Bioreactor 1 (Hydrogen bioreactor)—75 ng/µL and 240 µL; and Bioreactor 2 (Methane bioreactor)—90 ng/µL and 170 µL. The purified genomic DNA was then stored in Elution buffer at −20 °C pending further analysis.

2.7. Metagenome Sequencing and Bioinformatics Analysis

Metagenomic amplicon sequencing, a methodology centered on the targeted sequencing of specific genomic regions, was applied in this study. The construction and subsequent sequencing of metagenomic libraries were outsourced to Macrogen Inc. (Seoul, Republic of Korea). Bacterial metagenomic libraries were generated utilizing primers designed to amplify the V3–V4 hypervariable region of the bacterial 16S rRNA gene (primers 341F–805R). Similarly, archaeal metagenomic libraries were constructed using primers specific to the archaeal 16S rRNA gene (primers 21F–516R). The quantitative and qualitative assessment of DNA within the samples was performed via DNA QC-Picogreen. Library preparation involved the utilization of the LightCycler qPCR system, the JANUS liquid handling system for library pooling, and the Herculase II Fusion DNA Polymerase in conjunction with the Nextera XT Index V2 Kit. Sequencing was conducted on the Illumina MiSeq platform, employing a 301 base-pair paired-end (PE) protocol, targeting approximately 200,000 reads per sample. Raw data statistics revealed the following: the Bioreactor 1 16S sample yielded 641,636 reads, totaling 193.1 Mbp, with a GC content of 53.2% and a Q30 score of 82.7%; and the Bioreactor 2 16S sample produced 635,562 reads, amounting to 191.3 Mbp, with a GC content of 52.9% and a Q30 score of 83.5%. For the Bioreactor 1 Arc sample, a total of 592,956 reads were generated, equating to 178.4 Mbp, with a GC content of 56.4% and a Q30 score of 71.0%. The Bioreactor 2 Arc sample yielded 934,736 reads, totaling 281.3 Mbp, with a GC content of 55.4% and a Q30 score of 72.1%. Downstream analysis involved the generation of Amplicon Sequence Variants (ASVs) for both bacterial and archaeal datasets. ASV generation relies on algorithmic approaches designed to model and mitigate sequencing errors, thereby yielding high-resolution operational taxonomic units that effectively differentiate genuine biological sequences from background noise.

2.8. Data Preprocessing and ASV Generation

After sequencing, Cutadapt (v3.2) [22] was utilized to remove adapter and primer sequences from the raw data. Additionally, forward and reverse reads were trimmed to 250 bp and 200 bp using Cutadapt (v3.2). To generate Amplicon Sequence Variants (ASV) sequences, the reads were performed with error-correction, merging, and denoising processes with DADA2 (v1.18.0) [23]. Sequences with an expected errors of 2 or more were excluded. Erroneous reads were denoised based on an established error model. Following error correction, paired-end reads were merged by overlapping. Chimeric sequences were eliminated using the consensus method with the remove Bimera Denovo function in DADA2. ASVs with lengths shorter than 350 bp were filtered out using R (v4.0.3). Moreover, for microbial community comparison analysis, normalization was performed using QIIME (v1.9) [24]. During this process, subsampling was conducted based on the sample with the lowest read count among all samples to ensure comparability between them. The resulting ASVs were utilized for downstream analysis.

2.9. Taxonomy Analysis and Community Diversity

Each ASV was aligned to the organism with the highest similarity in the corresponding Reference Database (NCBI_16S), using algorithms such as Bayesian classifier (DADA2_v1.18.0, confidence value: 50) [25]. QIIME (v1.9.0) was utilized for downstream ASV analysis. Alpha diversity metrics, such as the Shannon index, Gini–Simpson index, and PD whole tree, were calculated to represent species complexity within individual samples. The observed species (ASVs), which refers to the number of different species, was also provided.

3. Results and Discussion

3.1. Preliminary Batch Anaerobic Co-Digestion of CSL

The process of biohydrogen production needs the proper selection of substrates and also the right conditions to ensure stabilization of the process. The anaerobic co-digestion of CSL and different agricultural wastes was investigated, aiming at the maximization of biohydrogen production in the first stage of the process. The digestion of corn steep liquor was realized using different agricultural wastes in a mixture of co-substrates, and different ratios and organic loads were tested. The dynamics of biohydrogen production was measured at 24, 48, and 72 h from the beginning of the process.

Four experiments, each repeated three times were performed. All results were expressed as means ± standard deviations using Microsoft Excel 2016 and are summarized in

Table 1. Co-substrate variation; organic load = 25 g/L.

Co-Substrates, Ratio	H2 [mL] 24 h	H2 [mL] 48 h	H2 [mL] 72 h
CSL	67 ± 5.3	59 ± 4.5	10 ± 0.5
CSL:potatoes = 1:1	155 ± 9.0	24 ± 1.6	5 ± 0.5
CSL:tomatoes and cucumbers = 1:1	124 ± 8.2	29 ± 2.4	8 ± 1.2
CSL:cattle manure = 1:1	168 ± 8.6	37 ± 2.9	10 ± 2.2

,

Table 2. Ratio variation; organic load = 25 g/L.

Co-Substrates, Ratio	H2 [mL] 24 h	H2 [mL] 48 h	H2 [mL] 72 h
CSL	72 ± 5.3	56 ± 4.5	12 ± 1.2
CSL:cattle manure = 1:1	160 ± 9.4	29 ± 2.4	10 ± 2.6
CSL:cattle manure = 1:2	153 ± 10.2	12 ± 0.8	5 ± 0.8
CSL:cattle manure = 2:1	128 ± 6.5	42 ± 3.3	16 ± 4.8

,

Table 3. Organic load variation; substrates: CSL:cattle manure = 1:2.

Organic Load	H2 [mL] 24 h	H2 [mL] 48 h	H2 [mL] 72 h
5 g/L	98 ± 7.8	63 ± 4.9	7 ± 1.2
10 g/L	152 ± 9.4	90 ± 6.9	26 ± 9.7
15 g/L	145 ± 10.6	21 ± 1.6	5 ± 0.8
20 g/L	130 ± 8.2	86 ± 6.9	32 ± 2.4
25 g/L	149 ± 9.8	8 ± 0.8	5 ± 1.2

and

Table 4. Organic load variation; substrates: CSL:cattle manure = 1:1.

Organic Load	H2 [mL] 24 h	H2 [mL] 48 h	H2 [mL] 72 h
5 g/L	103 ± 7.8	48 ± 3.7	8 ± 0.8
10 g/L	190 ± 9.0	67 ± 4.9	17 ± 5.7
15 g/L	130 ± 9.8	44 ± 3.3	22 ± 7.3
20 g/L	188 ± 8.2	43 ± 2.9	20 ± 1.6
25 g/L	128 ± 8.6	69 ± 4.5	10 ± 2.4

.

The type of biomass is of the utmost importance and is being identified as a key source for hydrogen production in a green manner [26]. The carbon content of agricultural and food wastes affects hydrogen production [27].

The highest hydrogen yield was registered after 24 h when the substrate used was CSL:cattle manure = 1:1 and organic load of 10 g/L.

Biological processes are considered as the most environmentally friendly alternatives for coping with the future demand for hydrogen; namely, biohydrogen production from agricultural waste appears extremely advantageous, as agricultural wastes are abundantly found, cheap, renewable, and highly biodegradable [28].

Figure 2 shows the results of the cumulative yield of hydrogen in the anaerobic co-digestion of CSL and cattle manure in a ratio of 1:1 with a different organic load.

3.2. Two-Stage Anaerobic Co-Digestion of CSL

The potential approach of applying the two-stage anaerobic co-digestion process to utilize waste substrates along with the production of valuable bioenergy carriers was demonstrated. A continuous AD process was accomplished with the substrate that led to the highest hydrogen and methane yields: CSL:cattle manure = 1:1. CSL is rich in soluble carbon and nitrogen nutrients that are readily available for the participating microorganisms’ growth and development. Cattle manure, on its side consists of lignocellulosic material, together with a variety of minerals, including trace quantities of iron, copper, manganese, magnesium, cobalt, and sulphur. Minerals like potassium, nitrogen, calcium, phosphorus, and zinc may be present in higher amounts [29]. It is known for being a suitable substrate for biogas production [30]. The different constituents present in cattle manure help for the proliferation of complex microbial communities [31]. Their combination was proven to be appropriate in the two-stage process for agricultural waste valorization.

The results indicated that the organic matter CSL and cattle manure were digested and the energy carriers hydrogen and methane were obtained at the expense of cellulose and the decrease in reducing sugar content.

Table 5. Continuous process for biohydrogen production in the first bioreactor.

Day	Biogas Yield, L/day	Hydrogen Yield, %	Total Solids, %	Volatile Solids, %	Glucose, g/L	Cellulose, g/L	Protein, g/L
1	0.00	0.00	46.1	75.4	0.27	0.19	0.12
2	4.90	25.45	43.7	74.2	0.36	0.16	0.14
3	5.28	19.29	43.7	75.0	0.31	0.16	0.11
4	5.28	18.59	34.3	73.6	0.30	0.21	0.14
5	5.59	16.80	38.2	73.2	0.25	0.19	0.15
6	5.59	12.16	40.3	72.7	0.24	0.20	0.17
7	4.43	16.60	42.9	73.5	0.23	0.18	0.18
8	4.64	13.64	35.0	73.0	0.19	0.18	0.24
9	4.95	12.60	35.1	72.9	0.18	0.17	0.23

and

Table 6. Continuous process for biomethane production in the second bioreactor.

Day	Biogas Yield, L/day	Methane Yield, %	Total Solids, %	Volatile Solids, %	Glucose, g/L	Cellulose, g/L	Protein, g/L
1	27.96	69.00	27.4	72.4	0.18	0.11	0.33
2	25.56	73.23	30.2	73.0	0.12	0.15	0.31
3	22.44	72.47	29.5	74.0	0.13	0.12	0.34
4	25.24	71.30	31.5	74.6	0.20	0.16	0.36
5	23.28	72.80	32.7	73.1	0.14	0.15	0.33
6	34.70	69.45	31.3	72.7	0.12	0.16	0.27
7	20.01	77.52	34.3	73.0	0.15	0.11	0.39
8	21.32	71.19	39.4	74.0	0.14	0.11	0.37
9	20.86	77.37	38.5	74.0	0.12	0.10	0.36

contain the measured values for the hydrogen or methane yield, total solids and volatile solids, glucose, cellulose, and protein and their changes during the continuous processes in the hydrogen and in the methane bioreactors. The values presented vary as, every day, the process is being fed.

The graphics in Figure 3 show the daily hydrogen (a) and methane (b) yields from the co-digestion, in comparison with a single digestion of corn steep liquor. The overall hydrogen yield from co-digestion in 2.29 dm³ biogas/L working volume versus 1.55 from the single digestion performed. The overall methane yielded from co-digestion is 10.68 dm³ biogas/L working volume versus 1.45 from the single digestion. It can be seen that the yields from co-digestion are greater than those from the single digestion. In addition, the overall hydrogen and methane yields are greater for the whole process with the mixture of two substrates.

In the report of Nindhia et al. (2025), they also established that the biogas production with only cattle dung as a substrate had an almost linear increase, while accomplishing a co-digestion by the addition of cabbage waste made the production of biogas higher [32]. According to Mata-Alvarez et al. (2000), the digestion of more than one substrate in the same digester can establish positive synergism and the additional nutrients introduced could support microbial growth [33].

3.3. Comparative Analysis of the Taxonomic Composition at a Phylum Level in the Two Bioreactors for Anaerobic Waste Degradation with Hydrogen and Methane Production

The microbial composition of bioreactors plays a critical role for the process efficiency. The microbial community in the two bioreactors was analyzed and their structure at the phylum, genus, and species levels was compared.

The microbial composition analysis was performed using 16S rRNA sequencing on the MiSeq Illumina platform—300 PE. The data were processed using bioinformatics methods for the identification and quantitative analysis of the predominant taxonomic groups.

In Bioreactor 1, the predominant phyla were Bacillota (synonym Firmicutes) (46.78%) and Bacteroidota (44.54%). In Bioreactor 2, the dominant phyla were Bacteroidota (40.66%) and Bacillota (19.79%). Significantly, higher proportions in Bioreactor 2 were observed for Chloroflexota (8.08%), Verrucomicrobiota (3.49%), and Synergistota (3.18%), which were present at less than 1% in Bioreactor 1 (Figure 4).

At the genus level, Bioreactor 1 was characterized by a high relative abundance of the genera Hallella (44.22%) and Limosilactobacillus (28.43%), while, in Bioreactor 2, these genera were represented at 11.01% and 0.03%, respectively. In Bioreactor 2, a significant proportion of other bacterial genera belonging to Bacteroidota (18.78%), as well as Levilinea (7.05%) and Fontisphaera (2.39%), were observed, which were weakly represented or absent in Bioreactor 1 (Figure 5). Among the dominant species in Bioreactor 1 were Hallella multisaccharivorax (44.22%), Limosilactobacillus mucosae (28.43%), Bifidobacterium pseudolongum (7.27%), and Caproicibacterium lactatifermentans (5%).

Hallella multisaccharivorax is the new name for Prevotella multisaccharivorax, a mesophilic Gram-negative bacterium [34,35]. There is no direct evidence indicating that this species produces hydrogen. This species, is known for fermenting various sugars into acids such as acetic and succinic acids, but hydrogen production has not been reported in its metabolic profile. However, other species within the Prevotella genus have been associated with hydrogen production in anaerobic digestion processes. For instance, members of the Prevotella genus have been observed in hydrogen-producing granules, suggesting a role in hydrogen production within mixed microbial communities [36]. Recent research [37] demonstrated that supplementing anaerobic digestion (AD) systems with Limosilactobacillus strains enhanced the co-production of hydrogen and methane. These bacteria facilitated the hydrolysis of lignocellulosic substrates, leading to increased volatile fatty acid (VFA) production and hydrogen accumulation.

Notably, the upregulation of the hydrogenase gene hydC was observed, indicating active hydrogen production pathways. The ability of Limosilactobacillus strains to enhance hydrogen production in AD systems suggests their potential utility in co-digestion processes involving substrates like corn steep liquor and agricultural wastes. By improving the hydrolysis stage and facilitating hydrogen accumulation, these bacteria could contribute to more efficient bioenergy production. Another new study [38] showed that, in a semi-continuous process, lactic acid fermentation (LA) in the acidogenic reactor significantly enhanced the two-stage anaerobic digestion of food waste, increasing biomethane production by 17.02% and organic matter removal by 4.84%. LA fermentation improved substrate utilization and shifted microbial communities in the methanogenic reactor, notably increasing the Methanosarcina abundance while decreasing the Methanosaeta abundance. This microbial shift lowered the hydrogen pressure and favored the conversion of LA to acetic acid, enhancing the methanogenesis-related enzyme activity and overall biogas quality. Clostridium sp. (provided with 2.13% in Bioreactor 1) are the key bacteria producing hydrogen as a byproduct of their metabolism. It is likely that the interaction between the bacterial-biofilm-forming community (Hallella) and the hydrogen-producing community is of paramount importance for the development of a stable microbial consortium for sustainable hydrogen production.

In Bioreactor 2, the dominant species were Hallella multisaccharivorax (11%), Levilinea saccharolytica (7.05%), Fontisphaera persica (2.39%), and Aminivibrio pyruvatiphilus (2.23%). Bioreactor 2 contained a more diverse microbial community with a higher proportion of Chloroflexota and Synergistota, which are associated with methanogenesis and the secondary degradation of organic matter.

Bacillota play a key role in carbohydrate fermentation to hydrogen, while Bacteroidota and Synergistota participate in syntrophic interactions supporting methanogenesis. According to Li et al. (2018), [39], a high content of Levilinea saccharolytica is associated with increased methane production in anaerobic environments. Similar results were reported by Zhang et al. (2019) [40], where Bacteroidota dominated methanogenic bioreactors.

In our previous study [41], where only corn steep liquor was used as a substrate, the dominant phylum in both bioreactors was Firmicutes (now renamed Bacillota), comprising 58.61% in Bioreactor 1 and 36.49% in Bioreactor 2. In the current study, this phylum is represented with 46.78% in Bioreactor 1 and relatively less in Bioreactor 2. Another significant similarity was the higher content of the genus Bifidobacterium in the first bioreactor and its significantly lower values in the second one—22.91% with only corn steep liquor as a substrate compared to 7.84% in the current study.

3.4. Archaeal Community in Both Bioreactors

Archaea play a significant role in anaerobic degradation, particularly in methanogenesis. In Bioreactor 1, the genus Methanocorpusculum (21.92%) was predominant, whereas its presence in Bioreactor 2 was significantly reduced (0.47%). On the other hand, the genera Methanothrix (16.56%) and Methanosarcina (4.15%) dominated in Bioreactor 2, indicating an active methanogenic process (Figure 6). The genus Methanomassiliicoccus (5.86%) was also identified in Bioreactor 2, consistent with the studies of Bassani et al. (2015) [42], which report its role in the degradation of methyl compounds. Notably, a high abundance of archaea from the class Thermoprotei (50.81%) was observed in Bioreactor 2, which is due to the specific thermophilic and acidophilic environment.

Previously [41], archaea from the phylum Euryarchaeota represented 11.4% of the microbial community in Bioreactor 2, whereas, in the present study, Methanothrix and Methanosarcina together accounted for approximately 20.7%. This suggests that the methanogenic process described here may have higher activity or a different profile of methanogenic pathways, potentially related to the use of different substrates. A study by Regueiro et al. (2012) [43] found that Methanosarcina dominated anaerobic bioreactors using industrial waste, which is similar to our findings for Bioreactor 2.

Except the biotechnology we described, other technologies were also applied. Bioelectrochemical-system-anaerobic digestion enhances the electron transfer activity between microorganisms and their resistance to toxic and hazardous substances by means of electrical currents, making the process stable and faster. It is applied as a strategy for enhancing the performance of anaerobic digestion [44]. However, it is a complex process to accomplish, considering the reactor size and applied voltage, external electric fields, etc. Pyrolysis is applied according to the substrate used to convert plastic waste into valuable products like fuel oil, solid residue, and combustible gas. Various types of plastic waste can be processed [45]. Methane pyrolysis technologies for hydrogen production requires high temperatures to overcome the activation energy barriers [46]. The technology can be chosen in relation to the substrates, conditions, microbial consortia, bioreactors construction, performance, and cost-effectiveness.

The advantage of the TSAD system is that it produces hydrogen, together with methane. We have in mind that biohythane is a gaseous mixture of 10–30% H₂ and 70–90% CH₄, whose physicochemical properties are similar to natural gas. Biohythane has superior characteristics compared to other biofuels. When addressing vehicles powered by methane, the use of hythane (with 30% H₂) can achieve a CO₂ emission reduction of about 70 gkm⁻¹. In order to obtain high-quality hythane for less carbon dioxide emissions, the ratio between the volumes of the bioreactors must be properly selected, together with other characteristics [47]. Finally, biohythane has a high fuel efficiency and pollution control than petrol and diesel, and can be obtained by the biotransformation of organic wastes.

4. Conclusions

The potential application of the two-stage anaerobic co-digestion process for the utilization of waste substrates along with the production of valuable bioenergy carriers was demonstrated. This study revealed the significant differences in the microbial composition of the two anaerobic bioreactors from the two-stage system. Bioreactor 1 was dominated by Bacillota, which is associated with a higher carbohydrate fermentation and hydrogen production. In Bioreactor 2, Bacteroidota and Chloroflexota were predominant, supporting methanogenesis. The archaeal analysis showed that Methanothrix and Methanosarcina prevailed in the second bioreactor, correlating with a more efficient methanogenic process. The obtained results highlight the opportunities for realizing anaerobic degradation processes with an optimized microbial community structure in relation to the substrate for an enhanced desired product production. We have chosen the most appropriate substrate mixture and ratio for maximizing the hydrogen and methane production in a continuous process. It was accomplished in a cascade system of bioreactors with definite volumes and specific microbial consortia, developed by us, based on wastes from agriculture utilization under the concept of circular economy. The created system of two bioreactors could help for the solvation of two problems, decreasing waste disposal and obtaining two energy carriers, hydrogen and methane, as fuels of the future in one process, addressing climate change by replacing fossil fuels with renewable energy sources. Renewables can create significant environmental, social, and economic benefits.