## 3 Results and Discussion
### Chemical Changes during Hydrolysis
Alkaline metals, such as Na\({}^{+}\), Mg\({}^{2+}\), K\({}^{+}\), and Ca\({}^{2+}\), up to a range of 400 mg L\({}^{-1}\) help maintain alkalinity and pH in AD. However, higher concentrations would cause toxicity and inhibit AD processes [34]. In our study, Na\({}^{+}\) and Mg\({}^{2+}\) concentrations were very low (<1 mg L\({}^{-1}\)) in all treatments and well below reported inhibitory levels, i.e., <750 mg L\({}^{-1}\) for Mg\({}^{2+}\)[35] and 3500-5500 mg L\({}^{-1}\) for Na\({}^{+}\)[36]. Concentration of K\({}^{+}\) was 1-3 mg L\({}^{-1}\) for all treatments, again below the inhibitory concentration of 400 mg L\({}^{-1}\)[34]. Similarly, for Ca\({}^{2+}\) the values were <1 mg L\({}^{-1}\) for all treatments and below inhibitory concentrations (>7000 mg L\({}^{-1}\)). Based on this, the low degree of hydrolysis observed in all treatments was not likely caused by inhibition from ammonia or metals. When considering our overall results, we can conclude (1) that there was limited hydrolytic activity in any of the reactors, regardless of fungal species or substrate type, and (2) the activity that was present was likely due to background microflora, including the bacteria that are present on the feedstock and not the AF. The most likely explanation for these results is that the AF were unable to survive, or at least be active, in the environment provided in this study. Low activity and survival of anaerobic fungi when applied to non-rumen environments has been reported by others [17; 18; 20]. Figure 5: Changes in pH in the bioaugmentation of three different anaerobic fungal species with and without activation: (**a**) corn silage (_Zea mays_ L.); (**b**) common reed (_Phragmites australis_). Error bars show standard deviation. An anaerobic fungi are known for having a close symbiotic activity and interspecies H\({}_{2}\) transfer with other microbes in the rumen [37; 38]. The absence of these relationships, or the lack of time for such relationships to develop using the experimental design of this study, may also account for the poor hydrolytic activity observed. Coculturing AF with other rumen hydrolytic bacteria, such as _Fibroacter succinogenes_, could be a potential approach to take in the future to increase viability [39]. [PERSON] et al. [40] inoculated F. _succinogenes_ together with methanogenic occultures of _Caecomyes/M. smithii_ grown on rye grass. They found that there was an increase in stem degradation and attributed this to complementary fibrolytic activities between the two species. By comparison, our study utilized only AF to help degrade the substrate. The heat sterilization used to eliminate background microflora from anaerobic digestae may have limited potential for symbiotic relationships to develop between AF and bacteria. Conversely, antibiosis has been reported between ruminal bacteria and AF in laboratory studies [41], which highlights our current poor understanding of AF ecology. [PERSON] et al. [42] recently reported up to a 60% increase in biogas yield from animal manures bioaugmented anaerobic fungi. In that study, the authors used an undefined mixture of AF isolated from a cow's rumen, resulting in an AF community composed of >6 groups (including _Anaeromyces_ spp., _Neocallimastix_ spp., and _Pironyces_ spp. used in our study) [43]. It is possible that the mixed AF culture approach is a better strategy for ensuring AF activity and survival when used in bioaugmentation compared to single species inoculations, as we have described here. The benefit of a mixed AF culture could be due simply to higher diversity increasing the chances for survival under artificial conditions, and/or could be due to interactions between the different community members. The combination of our results and those by [42] strongly suggest that successful outcomes from the addition of AF to hydrolysis and/or anaerobic digestion may require use of mixed complex communities.