## 4 Discussion  
### Toxicity of the PAM and its Possible Degradation under Anaerobic Conditions  
In a number of works, low direct toxicity of PAM-based flocculants for anaerobic microorganisms was reported [13; 22]. Our experiments showed that all tested PAM concentrations caused a decrease in the initial methanogenesis rate. However, even the extremely high flocculant concentration of 400 mg/g TS did not inhibit AD completely. This data confirm the results obtained by other researchers, who did not observe the toxicity of PAM flocculants under mesophilic conditions at concentrations of 105 and 415 mg/g TS [13; 20]. Nevertheless, clear patterns of partial inhibition of the anaerobic microbial community by the PAM were found. Increasing the flocculant amount resulted in a decreased specific methane yield per flocculant VS (Figure 1b). This was an indication that the PAM organic matter was not degraded directly to methane. Our data confirm the results by [PERSON] et al., who found that only partial PAM degradation occurred under anaerobic conditions at 35 and 55 \({}^{\circ}\)C, resulting in decreased PAM molecular mass due to partial hydrolysis and in the removal of amide groups. The authors showed that internal C-C bonds in the PAM molecule were to a considerable degree resistant to microbial hydrolysis [23]. The intrinsic inhibitory effect of PAM is associated with the disruption of the processes of biopolymer hydrolysis. PAM limits the availability of waste VS for exposure to microbial enzymes. At elevated PAM concentrations in the medium, hydrolysis of both the waste VS and of the polyacrylamide molecule is probably decelerating [18]. PAM has the most pronounced inhibitory effect on the enzymatic activity of hydrolases [24]. PAM can form a covalent bond and sweep flocculation between cationic PAM and the enzymes, significantly reducing their activity [24]. In addition, some ectoenzymes could be used for degradation of both organics and PAM due to the similar functional groups between them, which can also contribute to a decrease in the efficiency of degradation of the main substrate [24; 25]. The observed decrease in the rates of methanogenesis and the efficiency of the process of anaerobic transformation of VS can also be associated with the toxic effect of PAM decomposition products on anaerobic microorganisms. It is known that the main products of PAM decomposition under anaerobic condition are polyacrylic and acrylic acids, the proportion of which is about 50% of all products formed [17; 18; 25]. While acrylamide and acrylic acid suppress the activity of methanogenic archaea, polyacrylic acid affects both methanogens and hydrolytic and syntrophic bacteria, as well as homoacetogenic bacteria [17; 24]. At the same time, acrylic acid has no significant effect on hydrolytic processes. PAM also has a pronounced inhibitory effect on acidogenesis [24]. Our data show that high concentrations of the PAM had the most significant inhibitory effect on syntrophic bacteria involved in VFA conversion, primarily on the propionate-oxidizing syntrophic bacteria. The concentration of PAM 80-200 g/kg TS led to a decrease in the efficiency of consumption of propionate by 41-49%, while the maximum concentration of PAM completely inhibited the decomposition of propionate. It is well known that the accumulation of propionate is one of the important signs indicating the destabilization of the AD process [26]. We also observed the accumulation and slower degradation of acetate in the samples treated with 80-400 mg PAM/g TS, which indicate the inhibitory effect of PAM or its degradation products on acetate-utilizing methanogens. The sensitivity of acetoclastic methanogens to PAM and its degradation products has been previously shown by [PERSON] et al. [17]. We observed signs of partial degradation of PAM, including an increase in total VFA concentration and total ammonium nitrogen concentration. Formation of VFA, primarily acetate, was observed in all samples, both in the T and B series.