Slurry acidification is effective for reducing gaseous emissions during slurry storage. However, an alternative to sulfuric acid traditionally used is needed. This study investigated the efficiency of slurry bio-acidification treatment by adding different types and amounts of fermentable substrates to initiate and sustain the fermentation process. The carbon pools in the slurry were quantified to understand the mechanisms involved during the bio-acidification. Substrate addition efficiently reduced slurry pH during storage via lactic acid production. Substrates with a low pH proved beneficial in initiating the fermentation process, but higher glucose dosage did not produce the highest lactic acid concentration. Once the treated slurries reached pH 4.2 during the fermentation process, the production of lactic acid was promoted and provided substrate was still available, the weaker volatile fatty acids were avoided, resulting in lower CH₄ emissions. In conclusion, bio-acidification could replace the sulfuric acid to reduce gaseous emissions during slurry storage.