## 2 Materials and Methods
### 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. Figure 1: Algae in the biomass for digestion seen under a light microscope. 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 dm\({}^{3}\). The temperature and stirrer speed were automatically controlled. BR-2 also works as a continuous-stirring tank reactor with a working volume of 15 dm\({}^{3}\) 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 \(\pm\) 1 \({}^{\circ}\)C and 35 \(\pm\) 1 \({}^{\circ}\)C, respectively, in BR-1 and BR-2. The pH in the first bioreactor was maintained at 5.3 \(\div\) 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. Figure 2: Scheme of the experimental setup. 1. Gasholder for hydrogen; 2. peristaltic pump for acid; 3. tank with acid; 4. peristaltic pump for base; 5. tank with base; 6. pH regulator; 7. temperature regulator for BR-1; 8. pH electrode; 9. speed regulator of the electric motor for BR-1; 10. electric motor of the stirrer of BR-1; 11. temperature sensor—thermistor Pt-100 of BR-1; 12. BR-1 (hydrogen generating)—3 L; 13. heating sleeve of BR-1; 14. peristaltic pump for feeding; 15. BR-2 (methane generating)—15 L; 16. electric motor of the stirrer of BR-2; 17. temperature sensor—thermistor Pt-100 of BR-2; 18. heating sleeve of BR-2; 19. speed regulator of the electric motor of BR-2; 20. temperature regulator of BR-2; 21. peristaltic pump for outflow of BR-2; 22. vessel for outflow of BR-2; 23. gasholder for methane. 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 CO\({}_{2}\) and H\({}_{2}\) (in % by volume) or with a Drager X-am 7000 device equipped with infrared sensors for CH\({}_{4}\) and CO\({}_{2}\) and a catalytic sensor for H\({}_{2}\)S (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% H\({}_{2}\)SO\({}_{4}\), followed by a color reaction with anthrone reagent. The cellulose concentration was determined after measuring the absorbance at 620 nm.