## 4 Discussion
### Application of the Flocculant to Restore Methanogenesis in Destabilized bioreactors  
PAM-based flocculant was effective for methanogenesis restoration in thermophilic bioreactors destabilized due to accumulation of high VFA concentrations only in non-mixing samples. The most effective recovery of methanogenesis was observed at a concentration of 20 mg/g TS. Methanogenesis was not restored in the samples digested under mixing. In all samples, hydrogen was accumulated in the gas phase, which indicated destabilization of the AD. Most likely, forced flocculation and long-term preservation of floccules was the main reason for the restoration of methanogenesis in destabilized bioreactors. During the experiment, a multidirectional effect of several factors was observed. Based on the results obtained, excessive VFA accumulation is a more significant factor inhibiting the AD process. In this case, the addition of a flocculant has a less inhibitory effect. In previous studies with vigorous mixing and increasing the organic loading rate to 7.6-9.4 kg VS/(m\({}^{3}\) day), an excessive accumulation of VFA was observed, mainly acetate (up to 14 g/L) and propionate (up to 4 g/L) [29]. Minimal mixing promotes the initiation of methanogenic centers [30]. Intense mixing leads to the distribution of the VFA formed in large quantities throughout the entire volume of the digested mass, which can inhibit syntrophic bacteria and methanogenic archaea [29; 30]. In agitated mixtures, floccules break-up was more rapid, and by the end of the experiment 39.2-51.9% were smaller than 100 \(\upmu\)m. This is in accordance with the data of [PERSON] et al., who found that continuous agitation of digested mass promoted more rapid break-up of the flocculant-induced floccules [31]. Thus, to achieve the positive effect of enforced flocculation, mixing of the digested mixture should be stopped or decreased to a minimum in order to extend the duration of floccules existence and to avoid their untimely disintegration. Application of PAM concentrations higher than 20 g/kg TS is probably less efficient due to the inhibitory properties of the flocculant. Our experiments revealed that pH decrease caused by excessive VFA accumulation did not affect flocculation. These results are in agreement with those of [PERSON] et al., who showed that the efficiency of PAM-induced floccules formation did not change at pH decrease [32]. According to [PERSON] et al., the size of PAM-induced floccules depended on an ambient pH: the largest floccules were formed at neutral pH (7.0) and pH decrease to 4.0, while the floccules size decreased at pH above 8.0 [25]. This finding is important for PAM application for the restoration of methanogenesis in reactors destabilized due to VFA accumulation. The larger the floccules size, the higher the probability of the positive effect of the flocculant and the rate of the process restoration. Most of the authors investigating the effect of flocculants on anaerobic digestion agreed that flocculant treatment of SS results in lower mass transfer efficiency and slower hydrolysis [13; 16; 17; 18]. Partial limitation of substrate availability for hydrolysis, which is a result of flocculation, is among the most probable mechanisms of the restorative effect of the flocculant. This decreases the rates of hydrolytic processes and the rate of VFA accumulation in the medium. [PERSON] et al. showed that as a result of PAM addition (3-24 mg/g TS) the concentrations of soluble proteins and polysaccharides decreased by 23-67%, depending on the PAM concentration. It also affected their subsequent release from the floccules [17]. Formation of micronuclees within the floccules is another possible mechanism. The activity of syntrophic bacteria and methanogenic archaea, which are highly sensitive to variations of pH and hydrogen and VFA concentrations in the medium is preserved in these micronuclees. Outer layers of the floccules may act as a protective barrier, which impaired mass transfer of metabolites into its inner zones.