## 3 Results and Discussion
### Chemical Changes during Hydrolysis  
Chemical oxygen demand was measured over the course of the hydrolysis experiment to examine the amount of soluble COD released due to hydrolysis and for further AD (Figure 3). Soluble COD concentrations for corn silage treatments trended upwards over time but did not significantly differ pre- and post-hydrolysis (Figure 3a). Similarly, soluble COD concentration did not increase in common reed (Figure 3b), regardless of treatment (\(p>0.05\)), and was approximately half the value compared to corn silage. The higher initial COD concentration in corn silage compared to common reed was likely due to conversion of some corn biomass to soluble fermentation products during the ensiling process. Figure 1: Cumulative hydrogen yield in the bioaugmentation of three different anaerobic fungal species: (**a**) corn silage (_Zea mays_ L.); (**b**) common reed (_Phragmites australis_). Error bars show standard deviation. Figure 2: Cumulative carbon dioxide yield in the bioaugmentation of three different anaerobic fungal species: (**a**) corn silage (_Zea mays_ L.); (**b**) common reed (_Phragmites australis_). Error bars show standard deviation. The main VFA produced during hydrolysis of corn silage were acetic, propionic, and butyric acids. Total VFA concentration trended upward over the course of the hydrolysis experiment for corn silage in all treatments; however, these increases were not statistically significant (Figure 4). In comparison, VFA production during hydrolysis of common reed was limited, with no significant difference in concentration observed over the course of the experiment for any treatments. It is unlikely that the lack of VFA accumulation could have been attributed to microbial conversion, as minimal gas volume was produced, suggesting an overall lack of microbial activity. The absence of a functional methanogenic phase in the experimental system could have been inhibitory. The inhibitory concentration of VFA for the specific fungal species investigated here is unknown, but it is possible that the concentrations observed were detrimental to continued fungal growth. The pH of the digestate in corn silage flasks decreased from approximately 7.0 on day 1 to 5.6-5.8 on day 11 for all treatments (Figure 5a). The pH of digestate in common reed flasks ranged between 7.2-8.0 on day 1 (Figure 5b), which was not statistically different from corn silage (\(p>0.05\)). At the end of hydrolysis, the pH values of common reed decreased to 6.1-6.9, which again was not statistically different from corn silage (\(p>0.05\)). Figure 4: Volatile fatty acids (VFA) production 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. Figure 3: Soluble COD production in the bioaugmentation of three different anaerobic fungal species: (**a**) corn silage (_Zea mays_ L.); (**b**) common reed (_Phragmites australis_). Error bars show standard deviation. The optimum pH range for the growth of AF is between 6.0 and 7.0 [33], so reduction of pH to <6.5 due to VFA accumulation may have contributed to conditions unfavorable for anaerobic fungal activity. In the rumen, there is both a constant supply of buffering capacity as well as organic acid removal through the production of saliva and the symbiotic activities of the host animal and microbial consortium, respectively. The complexity of the rumen system is difficult to mimic in vitro in the laboratory, but a better approximation of the conditions which are favorable for AF to survive and be active will be essential for future success in this area of research. Ammonia is known to inhibit hydrolysis during AD at >200 mg L\({}^{-1}\)[34], but that threshold was not exceeded in our study (Table 2). Similarly, metals can inhibit biological hydrolysis processes when present in sufficient concentration.