**2.1 Ammonia concentration**

Nitrogen is an essential nutrient for microorganisms conducting anaerobic digestion. However, excess amount of nitrogen causes inhibition. According to Rajagopal et al. [19], ammonia concentration between 50 and 200 mg N L<sup>−</sup><sup>1</sup> is beneficial for anaerobic digestion while higher than 1500 mg N L<sup>−</sup><sup>1</sup> inhibits digestion. In the solution, ammonium ion (NH4 + ) is equilibrated with free ammonia (NH3). The equilibrium is governed by pH and temperature [20]. Therefore, higher pH and higher temperature increase NH3 concentration. Free ammonia can diffuse into the cell through the cell membrane and inhibits cell function by disrupting the proton and potassium balance [21]. Therefore, thermophilic (55°C) condition is more sensitive to ammonia inhibition than mesophilic (37°C) condition.

In the dry anaerobic digestion, ammonia inhibition was reported in digestion of high nitrogen-containing biomass or digestion of low nitrogen-containing biomass with inoculum with high nitrogen concentration. Under thermophilic conditions, dry anaerobic digestion of corn stover highly inoculated with wet anaerobic digestion effluent showed smaller amount of biogas production than those with less inoculated one [22]. This was due to high concentration of ammonia in the inoculum. In semi-solid (10% of TS) digestion of chicken manure, 12 and 16 g N L<sup>−</sup><sup>1</sup> of ammonia were accumulated in mesophilic and thermophilic conditions, respectively, and the mesophilic condition showed higher methane production than that of thermophilic one [23]. Zhou et al. also observed low methane yield of thermophilic anaerobic digestion of pig manure, in which NH4 + concentration exceeded 4000 mg N kg<sup>−</sup><sup>1</sup> [17].

In order to overcome ammonia inhibition, several approaches were suggested such as ammonia striping, chemical precipitation, adjusting carbon/nitrogen (C/N) ratio, etc. Ammonia stripping was applied for dry anaerobic digestion of chicken manure. Ammonia in the chicken manure was stripped at high pH with N2 flow after ammonia production by anaerobic fermentation [24]. Ammonia-stripped chicken manure showed 2305 mL kg<sup>−</sup><sup>1</sup> TS of cumulative methane production, which is much higher than the manure without stripping (313 mL kg<sup>−</sup><sup>1</sup> TS) [24]. In anaerobic digestion, C/N ratio of 15–30 is thought to be preferable [25]. A simple way to avoid ammonia inhibition is co-digestion with biomass with low nitrogen content such as crop residue. Co-digestion can dilute ammonia concentration in the reactor and reduce ammonia inhibition. For example, Abouelenien et al. found 1.5–93% increase in methane production in thermophilic co-digestion of chicken manure (C/N ratio of 6) with agricultural waste (coconut, coffee grounds, and cassava; C/N ratio of 17) compared with mono-digestion of chicken manure. Zhou et al. mixed pig manure with rice straw to obtain mixtures with C/N ratio of 10, 20, and 30 and conducted dry thermophilic digestion. The methane yields of C/N ratio of 20 and 30 were 244 and 258 mL g<sup>−</sup><sup>1</sup> VS, while C/N ratio of 10 showed lower and unstable methane production [17].
