Anaerobic digestion (AD) is a biotechnological process in which the microorganisms degrade complex organic matter to simpler components under anaerobic conditions to produce biogas and fertilizer. This process has many environmental benefits, such as green energy production, organic waste treatment, environmental protection, and greenhouse gas emissions reduction. It has long been known that the two main species (acidogenic bacteria and methanogenic archaea) in the community of microorganisms in AD differ in many aspects, and the optimal conditions for their growth and development are different. Therefore, if AD is performed in a single bioreactor (single-phase process), the optimal conditions are selected taking into account the slow-growing methanogens at the expense of fast-growing acidogens, affecting the efficiency of the whole process. This has led to the development of two-stage AD (TSAD) in recent years, where the processes are divided into a cascade of two separate bioreactors (BRs). It is known that such division of the processes into two consecutive BRs leads to significantly higher energy yields for the two-phase system (H₂ + CH₄) compared to the traditional single-stage CH₄ production process. This review presents the state of the art, advantages and disadvantages, and some perspectives (based on more than 210 references from 2002 to 2024 and our own studies), including all aspects of TSAD—different parameters’ influences, types of bioreactors, microbiology, mathematical modeling, automatic control, and energetical considerations on TSAD processes.

The main purpose of this study was to identify the microbial communities (bacterial, archaeal and fungal) in a two-stage system of anaerobic bioreactors for the production of hydrogen and methane from the waste substrate—corn steep liquor. Wastes from the food industry are valuable resources with potential in biotechnological production because of their high organic matter contents. In addition, the production of hydrogen and methane, volatile fatty acids, reducing sugars and cellulose content was monitored. Two-stage anaerobic biodegradation processes were performed by microbial populations in the first hydrogen generating bioreactor (working volume of 3 dm³) and in the second methane-generating reactor (working volume of 15 dm³). Cumulative hydrogen yield reached 2000 cm³ or 670 cm³/L a day, while the methane production reached a maximum quantity of 3300 cm³ or 220 cm³/L a day. Microbial consortia in anaerobic digestion systems play an essential role for process optimization and biofuel production enhancement. The obtained results showed the possibility of conducting two separate processes—the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis)—as two stages of anaerobic digestion to favor energy production under controlled conditions with corn steep liquor. The diversity of microorganisms as main participants in the processes in the bioreactors of the two-stage system was followed using metagenome sequencing and bioinformatics analysis. The obtained metagenomic data showed that the most abundant phylum in both bacterial communities was Firmicutes—58.61% and 36.49% in bioreactors 1 and 2, respectively. Phylum Actinobacteria were found in significant quantities (22.91%) in the microbial community in Bioreactor 1, whereas in Bioreactor 2, they were 2.1%. Bacteroidetes are present in both bioreactors. Phylum Euryarchaeota made up 0.4% of the contents in the first bioreactor and 11.4% in the second. As the dominant genera among methanogenic archaea are Methanothrix (8.03%) and Methanosarcina (3.39%), the main fungal representatives were Saccharomyces cerevisiae. New knowledge of anaerobic digestion mediated by novel microbial consortia could be widely used to convert different wastes to green energy.

Experimental studies of two-phase anaerobic digestion of corn steep liquor in semi-continuous automatic and semi-automatic modes of operation of a cascade of two anaerobic bioreactors with monitoring and control systems were performed. Corn steep liquor—a waste product from the process of treating corn grain for starch extraction—was used as a substrate in the process of anaerobic digestion with simultaneous hydrogen and methane production. The daily yields of biohydrogen in bioreactor 1 of the cascade (with a working volume of 8 dm³) are variable. In good operation, they are in the range of 0.7 to 1.0 L of biogas from a 1 dm³ working volume of the bioreactor, and the optimal pH is in the range of 5.0–5.5. The concentration of hydrogen in the biogas from the hydrogen bioreactor 1 is in the range of 14–34.7%. The daily yields of biomethane in bioreactor 2 of the cascade (with a working volume of 80 dm³) vary in the range 0.4 to 0.85 L of biogas from a 1 dm³ working volume of the bioreactor, and the concentration of methane in the biogas from bioreactor 2 is high and remains practically constant (in the range 65–69%). At a dilution rate of 0.4 day⁻¹ and an organic loading rate of 20 gL for bioreactor 1, respectively, and a dilution rate of 0.05 day⁻¹ for bioreactor 2, the best results were obtained. The computer control system is presented. Some energetical considerations were discussed.

As focus of humans has turned to renewable energy, the role of anaerobic digestion has started to become economically viable. Reducing the volume of agro-wastes for the generation of gaseous and liquid fractions with energy carriers and valuable products is an enormous challenge. A two-stage anaerobic digestion process consisting of hydrogenic stage followed by methanogenic stage was studied in a laboratory scale. Five simple nonlinear models of this continuous cascade process were studied in order to determine the optimal ratio of working volumes of bioreactors, in view of maximising energy production. This ratio was reported for all adopted models. The optimal ratio (maximal energy production criterion) depends of the adopted mathematical model. Static characteristics of both bioreactors were obtained using Symbolyc toolbox of Matlab. Numerical experiments concerning dynamics of the main variables of both bioreactors for these models using Simulink of Matlab are performed for different step changes of the dilution rate of the first bioreactor, together with the influence of the substrate (acetate) inhibition for one of the models. The value of the constant of inhibition plays an important role on the admissible interval of the dilution rate. The developed idea could serve for optimally designed experiments of anaerobic digestion for production of hydrogen and methane from lignocelluloses wastes (wheat straw) in two phase process.