For the agricultural wastes that are characterized with high protein and/or urea levels, ammonia toxicity to the bacterial and methanogenic populations in the undefined mixed cultures in anaerobic digestions can hamper reactor performance. (Ammonium is the final digestion product for nitrogen-containing polymers, such as proteins.) Ammonia toxicity in anaerobic digestion, therefore, has been studied in depth over the last 30 years (Velsen 1979; Braun et al. 1981; De Baere et al. 1984; Hashimoto 1986; Koster and Koomen 1988; Koster and Lettinga 1988; Bhattacharya and Parkin 1989; Robbins et al. 1989; Poggi-Varaldo et al. 1991- Troyer 1997- Lay et al. 1998- Lu et al. 2008- . Some of these papers documented maximum concentrations of total ammonium-N (NH — and NH-+) and free ammonia (NH3), which is the inhibiting species, for pure cultures of methanogenic populations (Sprott and Patel 1986; Koster and Koomen 1988; Steinhaus et al. 2007). The general consensus in the literature seems to indicate that hydrogenotrophic methanogens are less sensitive to high free ammonia concentrations compared with acetoclastic methanogens (Sprott and Patel 1986; Angelidaki and Ahring 1993). It is, therefore, not surprising that under high ammonia con­centration an alternative pathway that includes acetate oxidation by bacteria and subsequent hydrogen utilization by methanogens emerges in farm-based digesters without the need for acetoclastic methanogens (Schnhrer et al. 1994, 1996; Angenent et al. 2002b). Angenent et al. (2002b) showed that the relative 16S rRNA levels for hydrogenotrophic methanogens of the order Methanomicrobiales increased from 2.3% to 7% with increasing total ammonia-N concentrations of 2000 to 3600 mg/L, whereas the relative 16S rRNA levels for acetoclastic methanogens in the genus Methanosarcina decreased from 3.8% to 1.2%. Others have observed that acetoclastic methanogens from the family Methanosarcinaceae tend to group themselves in a cluster shape when a relatively high total ammonium-N concentration is present to protect themselves from high free ammonia concentrations (Calli et al. 2005a, b). Thus, several mechanisms to sustain an efficient microbial community function under high free ammonia concentrations may be present, resulting in a diverse methanogenic population even when the total ammonium or free ammonia concentration in the farm-based digester has exceeded the maximum levels reported in the literature.

In a recent study, we have operated four identical ASBR systems-fed swine waste at a VS concentration of 20 g/L for close to a 1000-day operating period (Garcia and Angenent 2009). Several operating changes, including ammonia concentration and temperature increases, were made to study the effect on the interactions between temperature and ammonia inhibition on reactor performance. During period 1 (day 0-378), the methane yield was 0.31L CH4/g VS for all digesters (with no statistical differences among them) at a temperature and total ammonium-N levels of 25°C and ~1200mg ammonium-N/L, respectively. During period 2 (day 379-745), the methane yield at 25°C decreased by 45% when total ammonium-N and free ammonia-N were increased in two of the four ASBRs to levels >4000 mg/L and >80 mg/L, respectively. During period 3 (day 746-988), this relative inhibition was reduced from 45% to 13% compared with the low-ammonia control reactors when the operating temperature was increased from 25°C to 35°C (while the free ammonia levels increased from ~100 to ~250 mg ammonia-N/L because of the temperature increase—chemical equilibrium change). The 10°C increase in temperature doubled the kinetic constant for methanogenesis, which overwhelmed the elevated toxicity effects caused by the increasing concentration of free ammonia, resulting in better reactor performances. This was an unexpected result (for the authors) because the literature predicts that methanogenesis would become severely inhibited with free ammonia concentrations of ~250mg-N/L at a higher (35°C) operating temperature (Koster and Lettinga 1984; Sprott and Patel 1986; Koster and Koomen 1988). Based on the chemical equilibrium change, we had predicted pronounced methanogenic inhibition at tem­peratures higher than 25°C. However, based on a long-term study with methanogenic food webs, it is clear that the farmer/operator may alleviate ammonia toxicity by increasing the operating temperature within the mesophilic range. Even a temperature increase from 30- 35°C to 38-39°C may increase digester performance at elevated ammonia-N concentrations (Garcia and Angenent 2009 ).