Anaerobic Two-Phase Co-Digestion for Renewable Energy Production: Estimating the Effect of Substrate Pretreatment, Hydraulic Retention Time and Participating Microbial Consortia

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Anaerobic co-digestion of waste biomass in a two-stage anaerobic process was successful, and sequential production of biohydrogen and biomethane was realized, minimizing waste disposal and realising additional energy output for possible replacement of fossil fuels in a green way.

Abstract

Green and sustainable economies have recently become a key issue in long-term growth and well-being. Co-digestion of various waste materials in an eco-friendly way through biogas production has become the preferred method for their utilization and valorization. The possibility of hydrogen and methane yield maximization depends on the most suitable alkali reagent for pretreatment of waste lignocellulosic material, which was revealed in batch tests to determine the hydrogen production potential. The mixture for digestion consisted of pretreated wheat straw mixed with waste algal biomass in a ratio of 80:20 (w/w). The maximum hydrogen yield was achieved after applying sodium hydroxide thermoalkaline pretreatment, with a two-fold higher yield than the untreated control. Hydrogen production was stable and methane was not present in the resultant gas. The influence of the hydraulic retention time (HRT) on the maintenance of cascade installation was studied. The maximum daily concentration of hydrogen was achieved at an HRT of 2 days—42.5% H₂—and the maximum concentration of methane was 56.1% at an HRT of 6 days. Accumulation of volatile fatty acids was registered in the first step and their depletion was noted in the second one. The obtained values of the cellulose content demonstrated that it was utilized by up to 2.75% in the methanogenic bioreactor at the end of the process. Metagenomics analyses revealed the bacteria Thermocaproicibacter melissae (44.9%) and Clostridium cellulosi (41.9%) participated in the consortium, accomplishing substrate hydrolysis and acidogenesis in the first stage. Less in abundance were Thermoanaerobacterium butyriciformans, Calorimonas adulescens, Pseudomonas aeruginosa and Anaerocolumna chitinilytica. Methanogenesis was performed by an archaeon closely related to Bathyarchaeota (99.5%) and Methanobacterium formicicum. The most abundant bacterial strains in the methanogenic fermenter were Abyssalbus ytuae (30%), Proteiniphilum acetatigenes (26%) and Ruficoccus amylovorans (13%).

1. Introduction

Renewable energy sources appear to be more sustainable and environmentally friendly in relation to the environmental impacts caused by fossil fuels as a result of resource reduction due to increased human and industrial activities. Renewable energy resources promote product circularity, applying green technologies towards ecologically safe processes. For sustainable development of resources, energy needs must be adequately fulfilled [1]. Renewables will continue to be expanded in upcoming years. Despite its several advantages, anaerobic digestion of wheat straw is not easy to widely apply owing to its recalcitrant structure and poor hydrolysis. Lignocellulosic biomass, however, is one of the substrates that possesses an abundance of polysaccharides as its main constituent. Wheat straw is a common waste raw material with great availability that can serve as a substrate for anaerobic digestion for green energy production [2]. As a waste from agricultural production, it is an attractive alternative through which both environmental pollution control and energy recovery can be achieved.

Various pretreatment techniques have been investigated for their effect on the digestibility of wheat straw, considering the difficulties due to its high content of lignocellulose and its microbial persistence [3]. Different pretreatment methods are categorized as physical, chemical, biological, or physicochemical. They can be also combined with the aim of increasing their effectiveness, including their operational convenience, and decreasing investment costs [4].

Co-digestion refers to the simultaneous anaerobic digestion of several organic wastes in one digester [5]. It is used to enhance energy production from some low-yielding or difficult-to-digest materials [6]. Co-digestion of microalgal biomass with different co-substrates or as a co-substrate has been used in anaerobic digestion processes to increase biogas production, and this process was economically viable with an improved biodegradability [7]. An aquatic ecosystem could become oversaturated with nutrients; then, the algae in water could utilize the excess nitrogen and phosphorus and start growing uncontrollably. This could lead to algal blooms that are undesirable and represent a significant threat to aquatic ecosystems. On the other hand, algal biomass could be a potential substrate for anaerobic digestion for biomethane production due to its high productivity as well as low ash content. Based on their many advantages, microalgae have been deemed a favourable feedstock for energy-generation processes [8]. The advantages include the fact that algal biomass allows for the stability of the process and for improvements in the digestion performance. Separately used or co-digested waste algae biomass could enhance methane production [9]. The use of algae biomass for biofuels and obtaining bioproducts has been researched extensively, including recent studies concerning the digestion of Chlorella vulgaris for biofuel production [10]. Freezing of algal biomass before digestion could be considered as a pretreatment. Some publications mentioned this as a method for algae biomass pretreatment. The advantages of this kind of pretreatment are its easy operation and the possibility for application in places with a naturally cold environment. The high energy consumption in other places (without a naturally cold environment) and thus the high costs of operation and maintenance are the main drawbacks that make this pretreatment process inadequate for industrial applications [11].

Microorganisms play a significant role in anaerobic digestion of organic matter. They are responsible for realising the efficient breakdown of complex organic molecules through a series of consecutive biochemical reactions for energy production. A good understanding of the participating microbial consortia helps to better manage global renewable energy sources [12].

The aim of this work was to reveal the sequential biohydrogen and biomethane production in a cascade of two bioreactors, focusing on the impact of substrates, pretreatment, and HRT and the role of the microbes, to suggest biotechnology for a sensible, effective solution.

2. Materials and Methods

2.1. Raw Materials

Wheat straw was used as the main agricultural lignocellulosic substrate for digestion. It was mechanically chopped, first by a hammer mill, then by a knife mill, until the particle size reached 1–2 mm.

Waste green algal biomass (Chlorella vulgaris and Scenedesmus acutus) was provided by Prof. G. Petkov and was from the collection of the Institute of Plant Phisiology and Genetics—BAS. Chlorella vulgaris and Scenedesmus acutus are species of green microalgae in the division Chlorophyta. They can be found in various natural and engineered freshwater and soil habitats.

The algal biomass (Figure 1) was acquired after it had been stored in a freezer at −18 ÷ −20 °C for months and then applied after thawing.

2.2. Inoculum

The inoculum for the two-phase anaerobic digestion system was acquired from the liquid phase of a working wheat straw anaerobic digestion process for methane production. The digestate from this process was taken and sieved through a 1 mm coarse sieve to remove all residual straw pieces. Thermal pretreatment was applied for inactivation of methanogenic microorganisms when the inoculum was to be used for starting the first phase—hydrogen generation. An aliquot of an already sieved liquid phase was centrifuged at 4500 rpm. The microbial cells collected in the sediment were re-suspended and washed twice with a physiological solution (0.9% NaCl). The microbial cell suspension was heated at 80 °C for 30 min and then it was cooled to room temperature [13]. Inoculum at a concentration of 10% (v/v) was added to the first bioreactor (BR-1) [14]. For the second bioreactor (BR-2)—for methane generation of the cascade—the same liquid phase was used. No pretreatment was applied, and it was introduced into the whole working volume of 15 dm³.

2.3. Pretreatment

Thermo-alkaline pretreatment of wheat straw was conducted in Erlenmeyer flasks (500 mL) with an effective volume of 400 mL. A quantity of 59.5 g of wheat straw was added to each of the flasks. The flasks were sealed with rubber stoppers and kept in a water bath oven operating at 55 °C for 24 h without mixing after addition of the appropriate amount of alkaline reagent (NaOH, Ca(OH)₂ or a mixture of NaOH and KOH)—4% total solids (TSs) of the substrate. An amount of 400 mL of distilled water was added in equal amounts to both flasks to ensure all pretreatments were performed with the same concentration of TS. Upon completion of the thermo-alkaline pretreatment, the flasks were cooled down to ambient temperature (≈20 °C).

The algal biomass before use was subjected to pretreatment by applying a freeze/thawing cycle.

2.4. Experimental Set-Up

Batch experiments were carried out for estimation of the reagent for thermo-alkaline pretreatment based on a batch process of dark fermentation with biohydrogen production. Three variants were pretreated with sodium hydroxide, calcium dihydroxide and a mixture of sodium hydroxide with potassium hydroxide in an equimolar ratio. The fourth flask was the control sample, where the substrate mixture was not thermo-alkaline pretreated. Continuous experiments with feeding were realized in a cascade-integrated system of two bioreactors (Figure 2). Hydrolysis and acidogenesis, accompanied by hydrogen accumulation in the resulting biogas, were carried out in BR-1. This reactor works as a typical continuous-stirring tank reactor with a working volume of 3 dm³. The temperature and stirrer speed were automatically controlled. BR-2 also works as a continuous-stirring tank reactor with a working volume of 15 dm³ and has the same control options as BR-1. For both bioreactors, the appropriate temperature was ensured using blanked heaters, Pt 100 sensors immersed in the working volume of bioreactors and controllers, which allowed us to maintain the temperature at levels of 55 ± 1 °C and 35 ± 1 °C, respectively, in BR-1 and BR-2. The pH in the first bioreactor was maintained at 5.3 ÷ 5.6 using a control system of a pH electrode, a pH controller and two peristaltic pumps—one for hydrochloric acid and the other for sodium hydroxide solutions. After appropriate adjustment of the parameters of the pH controller with hysteresis, the necessary pH stabilization was achieved in the specified optimal range. The pH controller automatically measures the pH level using a built-in probe and sets the doses for the pH corrector in the desired range.

The biogas obtained during the dark fermentation process from both bioreactors was collected in a stainless-steel gasholder, working on the principle of water displacement. Biogas yields were determined visually once a day. The effluent from BR-1 with liquefied products from hydrolysis was transferred to the second bioreactor using a peristaltic pump. A control system of the two-phase process was developed as two separate subsystems for BR-1 and BR-2.

The biogas content was estimated with a “Gasboard 3100P” device (Cubic Sensor and Instrument Co., Ltd., Wuhan, China) equipped with infrared sensors for measuring the relative content of CO₂ and H₂ (in % by volume) or with a Dräger X-am 7000 device equipped with infrared sensors for CH₄ and CO₂ and a catalytic sensor for H₂S (in ppm) measurements.

Cellulose was determined via the spectrophotometric method of Updegraff [15]. Cellulose-containing materials are released from impurities such as lignin, hemicellulose, xylosans and other low-molecular-weight compounds by extraction with an acetate–nitrite mixture. Purified cellulose was dissolved in 67% H₂SO₄, followed by a color reaction with anthrone reagent. The cellulose concentration was determined after measuring the absorbance at 620 nm.

The concentration of volatile fatty acids (VFAs) was determined by a Thermo Scientific gas chromatograph (Focus GC model) equipped with a Split/Splitless injector, a TG-WAXMS A column (length 30 m, diameter 0.25 mm, film thickness 0.25 μm) and a flame ionization detector (FID).

TS and VS were measured using a standard procedure [16]. The pH was measured using a microprocessor pH meter, model: pH210 (HANNA Instruments).

The primary energy obtained during the process from untreated and pretreated biomass (wheat straw), which was expressed as kWh·t⁻¹ raw material, was calculated by the following equation:

E_e = Y × LHV × η_e × (m_p/m_u),

where Y is the yield of the respective energy carrier in m³·t⁻¹; LHV (lower heating value) is assumed to be 9.94 kWh·Nm⁻³ (for methane) and 2.99 kWh·Nm⁻³ (for hydrogen); and m_u and m_p are the sample masses before and after pretreatment [17].

2.5. Library Preparation, Metagenome Sequencing, and Bioinformatics Analyses

Two different libraries were prepared for the needs of the study: a bacterial library (to study the bacterial diversity in the two reactors) and an archaeal library for the methane bioreactor. Total DNA extracted with the GeneMATRIX Bacterial & Yeast Genomic DNA Purification Kit (EURx, Gdańsk, Poland) was used to create the libraries. Preparation of the 16S metagenomic sequencing library for bacteria was performed using a primer pair that targeted the V3-V4 region, while the archaeal metagenomic library was constructed using 21F_Arch and 516R_Arch primers (both from the Macrogen Inc. primer set). Both libraries were created using a Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2. Sequencing was performed by Macrogen Inc. (Seoul, Republic of Korea) with MiSeq–300 PE, which was operated with a reading length of 301 bp and FastQC quality control. The total reads were 629.200 bases; of which 79.6 Mbp belong to the archaeal library and 109.8 belong to the bacterial library. The percentage of Q20 quality reads of the bacterial library was 93.4%, while that of the archaeal library was 81.5%.

The Sequence Read Archive (SRA) was deposited in the NCBI GeneBank under accession number PRJNA1105838.

2.6. Statistical Analyses

All analyses and tests were performed at least in triplicate. The statistical analyses were carried out using Microsoft Excel 2016. A one-way analysis of variance (ANOVA) test was used to determine the levels of confidence among various results.

All results were expressed as means ± standard deviations, and the p-value was considered to be significant at p < 0.05.

3. Results and Discussion

3.1. Pretreatment

The process of biofuel production is influenced by the type of substrate and its structure, composition and concentration. No less important are the pretreatment methods, inoculum, operational parameters (temperature, pH, HRT), microbial community structure, type of reactors used, etc.

Pretreatment testing showed that the maximum yield of hydrogen was achieved in the variant with sodium hydroxide thermoalkaline pretreatment, which gave about a two-fold higher yield than the untreated control (Figure 3). The applied pretreatments could be reported as effective, as the ester bonds between lignin and polysaccharides are being broken. In this way, both hydrogen and methane production was enhanced. The same statement was true for a variety of lignocellulosic substrates [18].

3.2. Two-Stage Anaerobic Digestion of Wheat Straw and Algal Biomass

Several anaerobic digestion configurations have been suggested so far in order to enhance energy production, including single-stage, leach-bed reactors and hybrid anaerobic digesters [19]. Among them, two-stage digestion was reported to be a promising technology with flexible operation and an enhanced performance [20].

In this study, an improvement in wheat straw anaerobic digestion was shown by adding microalgal biomass. Algal cells are composed of proteins, carbohydrates, pigments, minerals and vitamins [21]. They grow fast and adapt easily, even in waste digestate [22]. The cultivation of microalgae in wastewater gives a solution to save arable land for biomass production, simultaneously utilizing the nutrients available in wastewater [23]. They are a broadly available substrate and appropriate for different applications. Algae are abundantly found and grow uncontrollably when nitrogen and phosphorus are present in excess.

Co-digestion of wheat straw and microalgae biomass was realized for hydrogen and methane production with the advantage of producing two energy carriers. Among the extensively studied methods for obtaining hydrogen, biological hydrogen production is very promising for sustainable clean energy generation. Dark fermentation provides a way of converting organic waste into hydrogen, thus attaining the dual benefits of clean energy production and waste management [24]. The efficiency of biomethane production increases by separating the acidification phase and the methanogenic phase. High methane contents could be reached using two-stage anaerobic digestion. The rate of enzyme degradation of the biomass and its conversion to VFAs is significantly dependent on the type of substrates supplied to the system.

3.2.1. Influence of HRT

Changing the HRT led to variations in the energy carrier yields (Figure 4 and Figure 5).

The maximum daily concentration of hydrogen was achieved at HRT of 2 days, at 42.5% H₂, and the maximum concentration of methane reached was 56.1% at an HRT of 6 days.

The results of the average concentration are comparable to those of the maximum yield, the best being the average yield of hydrogen at an HRT of 2 days—120.9 mL/day/g VS_add—and correspondingly, the best average yield of methane was 581.8 mL/day/g VS_add at HRT = 6 days.

The HRT is a very important parameter during hydrogen and methane production [25]. Short HRTs are associated with hydrogen fermentation. Hydrogen production occurs at lower retention times and depends on the feed flow rate. The hydrogen-producing bacteria that reside in BR-1 are fast growing, but short HRTs or high dilution rates could be applied to eliminate methanogens, which have extremely low growth rates [26]. In BR-2, the HRT describes the average times that certain substrates reside in a digester, which are higher in this reactor. In this case, if the HRT is shorter, the system could stop due to washout of microorganisms.

Adjusting the appropriate operational parameters of bioreactors, such as increasing the HRTs and organic loading rates, is a way to increase biodegradation efficiencies [27]. The recalcitrant nature of lignocellulosic biomass hinders its efficient exploitation for energy production [28]. However, lignocellulose has been exploited as a substrate for biogas production due to its high energy potential. The cellulose concentration was determined to deplete during the process (2.75% in the methanogenic bioreactor at the end of the process).

The H₂S concentration in biogas varied from 100 to 10,000 ppmv depending on the feedstock’s sulphur content. Our measurements showed values from 200 to 283 ppmv. No methanogenesis inhibition was registered. There is also a threshold concentration of H₂S in biogas which will impact further applications, so technologies are being developed for H₂S removal from biogas [29].

Utilization of algal biomass for the production of gaseous biofuels using methane as an energy carrier supports a circular economy. In the digestion of algae, all cell compounds—sugars, fats, nucleic acids and proteins—could be involved. While protein in algal cells can result in undesirable ammonia formation, other industrial waste that is rich in carbon but poor in nitrogen compounds, like wheat straw, could be added [30]. The moisture and nutrient content of microalgae can favour lignocellulosic waste digestion.

Anaerobic digestion of algal biomass implies costs reductions via the elimination of drying wet biomass and extraction before processing, as they become unnecessary [31]. The increase in cellulase activity might be favourable for the biodegradation of algal biomass and straw, which could supply the bioreactors with nutrients and finally increase the methane production rate.

3.2.2. Volatile Fatty Acid Formation

Cellulose and hemicellulose are degraded and fermented by anaerobic microbes to produce volatile fatty acids, the main intermediates, which are transferred to the second bioreactor. VFAs are generally produced through anaerobic digestion. In fermentative hydrogen production, metabolic end products are known to affect the hydrogen yield. Figure 6 represents VFA formation in BR-1 and Figure 7—their further utilization during methane formation.

In BR-1, the main VFAs present were acetate and butyrate acids, with propionate and caproate acids present in lower quantities. The influence of the HRT on VFA production was not strong. In BR-2, the quantity of propionate exceeded that of acetate and the other VFAs registered. At HRT = 7.5, a whole spectrum of VFAs was detected. At HRT = 10, only acetate and propionate were found in small quantities, which is probably due to the better utilization of the VFAs. This result is in relation to the highest methane yield at the same HRT.

The VFA content could be altered according to the type of substrate. In the present study, the organic loading was greater. With these high loads and the presence of easily digestible organic matter (algal biomass), it is possible to obtain only the basic fatty acids in BR-1. In BR-2, the loads from the residual organic matter from the hydrogenic BR-1 were also high, and the used retention time allows for a more complete utilization of different classes of biochemical compounds. Hence, a greater variety of fatty acids was obtained.

During the consecutive stages of this biochemical process, e.g., hydrolysis, acidogenesis, acetogenesis and methanogenesis, complex organic substrates are converted into various short-chain volatile fatty acids such as acetic, propionic, lactic and butyric acid by acidogenic bacteria in the hydrolysis and acidogenesis stages [32]. In the later stages, acetogenic microbes oxidize VFAs into acetate, hydrogen and carbon dioxide, which are the main substrates for methane production by methanogens.

The anaerobic processes in the two-stage energy carrier production rely on differences in the activities of the participating acidogens and methanogens that reside in the two bioreactors and in their different physiological needs, bearing in mind the high physiological specialization and growth rates. In the first stage in BR-1, acidogens transform the substrate to obtain H₂, CO₂, volatile fatty acids, lactic acid and alcohols at optimal pHs of 5–6. In the second stage in BR-2, the remaining volatile fatty acids, lactic acid, and alcohols in the first stage are converted to CH₄ and CO₂ by methanogens at optimal pHs of 7–8. The two-stage system permits a higher organic loading at lower retention times. However, the retention time should be sufficient for the microorganisms to have enough time to degrade the substrate [33].

For the operation of two bioreactors in a coupled system, we have assumed that the synchronization of the flows of waste raw material (substrate) in BR-1 and BR-2 is important. When implementing this process, it is good to synchronize the volumes of the bioreactors, so that both processes can take place without inhibition. In this study, the chosen ratio between the working volumes of the two reactors was 1:5. This is why the HRT of the second bioreactor is always a multiple of the HRT of the first reactor. The organic loading at all HRTs of BR-1 remains the same. Therefore, in

Table 1. Comparison of the energy efficiency of the system depending on the HRT in the first bioreactor.

HRT, Day	Energy Carrier	Total Yield, dm3	Yield, m3/t VS	Lower Heating Value, kWt.h/cm3	Total Energy for the Whole System, kWt.h/t
1.2	Hydrogen	17.87	115.29	2.99	2441
	Methane	71.06	579.18	9.94
1.5	Hydrogen	10.39	105.76	2.99	1877
	Methane	68.90	440.31	9.94
2	Hydrogen	16.92	120.85	2.99	2458
	Methane	81.44	581.75	9.94
3	Hydrogen	8.23	58.80	2.99	1274
	Methane	42.37	302.67	9.94

, reflecting the total energy that could be obtained from the combustion of the produced hydrogen and methane, only the HRT of the first reactor is indicated in the first column.

3.2.3. Energy

From an energetic point of view, a residence time of 2 days led to better results. With a further increase in the residence time to an HRT of 3 days, the production of hydrogen was mainly negatively affected. The lower amounts of hydrogen produced, together with low concentrations of VFAs, are indicative of delayed hydrolysis, although the contact time is longer between the microorganisms and the feedstock. On the other hand, a shorter retention time could lead to an increase in the number of actively dividing microbial cells and an acceleration of the metabolic processes.

3.2.4. Microbial Communities’ Composition

The bioconversion of lignocellulose into hydrogen, methane and carbon dioxide was carried out with the participation of specific microbial communities. The microbial community composition was characterized and so the biodiversity in the hydrogen- and methane-producing bioreactors was determined.

A metagenomics analysis of the communities performing anaerobic digestion and gas production showed that completely different species were present in the two bioreactors. The reason for this is that the acidogenic and acetogenic microbes differ fundamentally from methanogenic microbes in their physiological needs and surrounding condition requirements. Bacterial species diversities in BR-1 and BR-2 are presented and compared in Figure 8. Remarkably, the hydrogen-producing fermenter contained exclusively Clostridia (99.69%) with two predominant species in the community: Thermocaproicibacter melissae (44.86%) and Clostridium cellulosi (41.94%). Two other clostridial species (each of them close to 4% of the community) were Thermoanaerobacterium butyriciformans (3.90%) and Calorimonas adulescens (3.77%). Less abundant clostridial genera were Anaerocolumna of the family Lachnospiraceae (1.70%) and Caldanaerobius of the family Thermoanaerobacteraceae (1.16%), followed by other minor Clostridia such as Biomaibacter (of the family Tepidanaerobacteraceae—0.18%) and Syntrophomonas zehnderi, Ruminococcus champanellensis, Saccharofermentans acetigenes, Eubacterium coprostanoligenes and Ruminococcus gnavus, as the last five species sharing totally 0.05% of the community. The aerobic/microaerophilic bacilli were very limited: Caldibacillus thermoamylovorans (0.08%) and Niallia endozanthoxylica (0.03%). Pseudomonads were about 2%, represented by Pseudomonas aeruginosa (Figure 9).

The presence of Thermocaproicibacter melissae (genus Oscillospiraceae) was determined, as the first bioreactor operates at 55 °C and the strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37–60 °C, with an optimum production at 50–55 °C and at pHs of 5.0–7.0. This is the most probable producer of caproic acid in BR-1. A very similar thermophilic chain-elongating bacterium MDTJ8 producing n-caproate from polymeric saccharides starch and hemicellulose was recently isolated from a thermophilic acidogenic anaerobic digester by Van Nguyen et al., producing n-caproate from human waste (2023) [34]. However, the authors identified the bacterium at the genus level only.

Clostridium species are of utmost importance among other microorganisms for fermentative H₂ production. Clostridium cellulosi is known to convert cellulose into ethanol, acetate and hydrogen at the optimal temperature of 60 °C [35]. Considering the role of Thermoanaerobacterium butyriciformans, this species was reported to produce hydrogen from lignocellulose sugars [36], similar to another newly isolated Thermoanaerobacterium sp. strain MJ1. High hydrogen yields have been obtained by MJ1 using acid-pretreated sugarcane bagasse hydrolysate [37]. Another species with a relatively high share in BR-1 was Calorimonas adulescens (Figure 9). This is a thermophilic species that produces ethanol in addition to acetate and CO₂ during growth on complex substrates, which is also typical for Caldanaerobius and Thermoanaerobacterium genera [38].

Differing from BR-1, the methane-generating BR-2 contains a bacterial community dominated by representatives of Flavobacteriaia (30.28%) and Bacteroidia (27.81%), as well as Opitutae (13.13%), Synergistia (2.43%), Desulfuromonadia (2.22%), and, to a lesser extent, Negativicutes (0.97%), Anaerolineae (0.51%), Verrucomicrobiae (0.41%), Syntrophia and Tissierellia (0.24% each), followed by Syntrophobacteria (0.08%), Erysipelotrichia (0.04%), and Ignavibacteria (0.02%), and Actinomycetes, Myxococcia, and Spirochaetia (with one-tenth of a percent each) (Figure 10). Clostridia constituted 6.03% of the community, in contrast to the hydrogen-producing fermenter.

An analysis of the archaeal community showed the presence of four species, with one of them completely conquering BR-2. The most abundant archaeal representative possessed a high similarity (99.2% 16S rRNA homology) with the phylum Bathyarchaeota and comprised 99.5% of the archaeal community. Other archaea in BR-2 were Methanobacterium formicicum, Methanoplasma termitum, and an unidentified species of the genus Thermoproteota (Figure 11).

Methanobacterium formicicum is a representative of the phylum Euryarchaeota and is often found in anaerobic biogas-producing systems [39]. However, the presence of Bathyarchaeota and Methanoplasma in the anaerobic digester is quite unusual. Both methanogens belong to Proteoarchaeota, of the phylum Thermoproteota, a lineage of hyperthermophilic Crenarchaeota, which is abundant in deep marine subsurface sediments [40]. Although Bathyarchaeota has not yet been cultivated in pure cultures, there are data on their metabolism, thanks to reconstructed genomes. According to Evans et al. (2015) [41], these archaea are methylotrophic methanogens, feeding on a wide variety of methylated compounds. They break down peptides, glucose, fatty acids, and glucose. The identification of key genes related to methanogenesis reveals mechanisms close to those of Euryarchaeota methanogens.

Crenarchaeota is one of the most abundant thermophilic archaeal phyla in terrestrial environments. However, nonthermophilic Crenarchaeota can be found in soils and marine freshwater sediments. New discoveries are anticipated, because archaea have become one of the most exciting current topics of microbial aquatic research [42]. The natural abundances of uncultured archaea are substantial; thus, for elucidating metabolic pathways and fully understanding archaea, combinations of new cultivation strategies with high-resolution molecular technologies and bioinformatics tools must be used. The dominant development of Crenarchaeotes in our case can be attributed to the high temperatures during the process. A spontaneous initial inoculation of BR-2 with Crenarchaeota, along with the algal part of the substrate, probably occurred during operation of the two-stage system.

4. Conclusions

This study presents a promising green technology for the utilization of waste wheat straw from agriculture and waste algal biomass as renewable sources for biohydrogen and biomethane production. This was accomplished in two steps with a suitable HRT and residing microbial and archaeal consortia, identified using metagenomics.

The hydrogen yield was slightly decreased when the HRT in BR-1 was increased due to possible lowering of the bacterial number and the need for time for development of new hydrogen-producing bacteria. The methane yield in BR-2 increased when the HRT was increased to 6 days. The overall energy obtained in the system at an HRT of 2 days exceeds the energy yield at an HRT of 3 days by 50%; thus, operating the process at the most appropriate HRT will lead to a more effective process.

Metagenomics analyses could allow the use of a defined consortium for each hydrogenic and methanogenic bioreactor of the system.