pH is the one of the most important factors that should be optimized for dark fermentative hydrogen production. It can affect the hydrogen production yields and also by-product formations. For dark fermentative hydrogen production optimum pH values are said to be between 5 and 6. It is important to stabilize the pH in the system because the acetic and butyric acids produced during hydrogen production will lower the pH to inhibitory acidic values. Since acid and base treatments are the kinds of pre-treatment operations pH changes in the system can change the microbial community too. Before starting continuous or large-scale applications it would be useful to determine the best pH value of the system in small-scale batch experiments [88].

Temperature can affect both microbial community and hydrogen production. Many of the studies on dark fermentative hydrogen production were conducted at mesophilic temperatures but some studies have shown that hydrogen production rates can be higher at thermophilic conditions. But from the view of economical costs thermophilic operation may not be an economically viable solution.