## 4 Discussion  
### Inactivation of Methanogens in the Inoculum  
[PERSON] et al. [31] and [PERSON] et al. [32] demonstrated that the pretreatment method affects the metabolic pathway and distribution pattern of microbial communities. The authors observed the formation of hydrogen and butyric acid after thermal inactivation and treatment with alkali. In the presented work, the formation of hydrogen was also observed after thermal inactivation of the inoculum, but the content of butyric acid in the medium was less than in the control. It was described that the highest hydrogen production was observed at pH 7.0 [33]. It was also found that acetate is an inhibitor of the hydrogen production process even at neutral pH values. In the aforementioned surveys, the primary inhibition of the biohydrogen production process occurred at an acetate concentration of 50 mM, whereas at an acetate concentration of 200 mM, the formation of hydrogen (36 mL/gVS\({}_{\text{added}}\)) decreased six times. At an acetate concentration of 5-25 mM, the hydrogen yield was 250 mL/gVS\({}_{\text{added}}\).  
The composition of the VFAs formed during DF of CW indicated the predominant process of butyric acid fermentation. This type of metabolism is being performed mainly by _Clostridium_ representatives, such as _C. Pasteurianum_, _C. Buryricum_, _C. Acetobutylcium_, _C. Pectinovorum_[34], as well as the inhabitants of the rumen of the ruminantsenti protists _Butyriibrio_ and bacteria of the human intestinal microflora _Eubacterium_ and _Fusobacterium_[35]. Acetate and butyrate are considered one of the main products of acidogenesis during glucose fermentation [5]. As a result of the decomposition of glucose by the glycolytic pathway, carbon dioxide, hydrogen and acetic acid are formed. This path is the more energy-efficient since four mol of ATP per mol of glucose are produced (two mol in glycolysis reactions, two more--during the formation of acetic acid). However, this pathway has two significant drawbacks: firstly, glucose metabolism along this pathway leads to very sharp acidification of the medium, which was observed in reactors with inoculum 1 and inoculum 2 after acid treatment, as a result of which bacterial growth can be inhibited. Secondly, this pathway does not allow oxidation of reduced electron carriers NADH formed during glycolysis. In the second way, the final product is butyric acid. The formation of butyric acid makes it possible to lessen the decrease in pH, which was observed in control 2. If, as a result of the release of organic acids in the process of butyric acid fermentation, the pH drops below 4.4, a metabolic pathway is activated in bacteria, the end products of which are ethanol, acetone and butanol (in this study were not analyzed).  
Thus, it is impossible to unequivocally answer which of the inactivation methods was the best. However, the work performed allowed us to assume that heat treatment is of low efficiency for a thermophilic inoculum, even though thermophilic methanogens are more sensitive to temperature fluctuations [36]. The mesophilic anaerobic inoculum was sensitive to both acidic and thermal inactivation of methanogens. [PERSON] et al. [37] also compared methods for inactivation of methanogens using a glucose solution as a substrate and found that heat shock treatment promoted the maximum rate of hydrogen production. Consumers of hydrogen in the anaerobic inoculum (lactic acid bacteria, homoacetogens, methanogens and sulfur-reducing bacteria) not only act as competitors for hydrogen producers but also synthesize by-products that can influence the growth of hydrogen-producing bacteria. For example, the release of a protein toxin (bacteriocin) by lactate-producing bacteria acts as a suppressor for hydrogen production and microbial growth [38].  
Thus, when a mixed culture of microorganisms is used as an inoculum, the pretreatment step plays an important role in ensuring the efficiency of hydrogen production from using mixed consortia.