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.

Anaerobic digestion (AD) is a microbially-driven process enabling energy production. Microorganisms are the core of anaerobic digesters and play an important role in the succession of hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes. The diversity of participating microbial communities can provide new information on digester performance for biomass valorization and biofuel production. In this study anaerobic systems were used, operating under mesophilic conditions that realized biodegradation processes of waste wheat straw pretreated with NaOH—a renewable source for hydrogen and methane production. These processes could be managed and optimized for hydrogen and methane separately but combining them in a two-stage system can lead to higher yields and a positive energy balance. The aim of the study was to depict a process of biohydrogen production from lignocellulosic waste followed by a second one leading to the production of biomethane. Archaeal and bacterial consortia in a two-stage system operating with wheat straw were identified for the first time and the role of the most important representatives was elucidated. The mixed cultures were identified by the molecular-biological methods of metagenomics. The results showed that biohydrogen generation is most probably due to the presence of Proteiniphilum saccharofermentans, which was 28.2% to 45.4% of the microbial community in the first and the second bioreactor, respectively. Archaeal representatives belonging to Methanobacterium formicicum (0.71% of the community), Methanosarcina spelaei (0.03%), Methanothrix soehngenii (0.012%), and Methanobacterium beijingense (0.01%) were proven in the methane-generating reactor. The correlation between substrate degradation and biogas accumulation was calculated, together with the profile of fatty acids as intermediates produced during the processes. The hydrogen concentration in the biogas reached 14.43%, and the Methane concentration was 69%. Calculations of the energy yield during the two-stage process showed 1195.89 kWh·t⁻¹ compared to a 361.62 kWh·t⁻¹ cumulative yield of energy carrier for a one-stage process.

Anaerobic digestion (AD) is a widespread biological process treating organic waste for green energy production. In this study, wheat straw and corn stalks without any harsh preliminary treatment were collected as a renewable source to be employed in a laboratory-scale digester to produce biogas/biomethane. Processes parameters of temperature, pH, total solids, volatile solid, concentration of volatile fatty acids (VFA), and cellulose concentration, were followed. The volume of biogas produced was measured. The impact of organic loading was stated, showing that the process at 55 °C tolerated a higher substrate load, up to 45 g/L. Further substrate increase did not lead to biogas accumulation increase, probably due to inhibition or mass transfer limitations. After a 12-day anaerobic digestion process, cumulative volumes of biogas yields were 4.78 L for 1 L of the bioreactor working volume with substrate loading 30 g/L of wheat straw, 7.39 L for 40 g/L and 8.22 L for 45 g/L. The degree of biodegradation was calculated to be 68.9%, 74% and 72%, respectively. A fast, effective process for biogas production was developed from native wheat straw, with the highest quantity of daily biogas production occurring between day 2 and day 5. Biomethane concentration in the biogas was 60%. An analysis of bacterial diversity by metagenomics revealed that more than one third of bacteria belonged to class Clostridia (32.9%), followed by Bacteroidia (21.5%), Betaproteobacteria (11.2%), Gammaproteobacteria (6.1%), and Alphaproteobacteria (5%). The most prominent genera among them were Proteiniphilum, Proteiniborus, and Pseudomonas. Archaeal share was 1.37% of the microflora in the thermophilic bioreactor, as the genera Methanocorpusculum, Methanobacterium, Methanomassiliicoccus, Methanoculleus, and Methanosarcina were the most abundant. A knowledge of the microbiome residing in the anaerobic digester can be further used for the development of more effective processes in conjunction with theidentified consortium.

We propose a new mathematical model describing a biotechnological process of simultaneous production of hydrogen and methane by anaerobic digestion. The process is carried out in two connected continuously stirred bioreactors. The proposed model is developed by adapting and reducing the well known Anaerobic Digester Model No 1 (ADM1). Mathematical analysis of the model is carried out, involving existence and uniqueness of positive and uniformly bounded solutions, computation of equilibrium points, investigation of their local stability with respect to practically important input parameters. Existence of maxima of the input–output static characteristics with respect to hydrogen and methane is established. Numerical simulations using a specially elaborated web-based software environment are presented to demonstrate the dynamic behavior of the model solutions.

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.

In this paper, a new Newton-based extremum-seeking control for dynamic systems is proposed using Kalman filter for gradient and Hessian estimation as well as a stochastic perturbation signal with time-varying amplitude. The obtained Kalman filter based Newton extremum-seeking control (KFNESC) makes it possible to accelerate the convergence to the extremum and attenuate the steady-state oscillations. The convergence and oscillation attenuation properties of the closed-loop system with KFNESC are considered, and the proposed control is applied to a two-stages anaerobic digestion process in order to maximize the hydrogen production rate, which has better robustness and a slower steady-state oscillation with the comparison of Newton-based ESC and sliding mode ESC.