## 2 Mechanisms of Acidogenic Fermentation
### Technical and Operational Challenges
In dissolved polymer substrates, however, the hydrolysis rate is governed by the concentration of active enzymes, which is affected by sludge concentrations and microbial dynamics [73]. This distinction underscores the importance of adapting operational strategies to the specific substrate type. However, hydrolysis is not always the limiting step. [PERSON] et al. [69] found that acidification could become the primary constraint during protein degradation. In their study with gelatin, acidification rates were slower than hydrolysis rates under both methanogenic and non-methanogenic conditions. This led to amino acid accumulation--up to 40% of the substrate COD in batch experiments and 13% in continuous systems with retention times shorter than 8 d. These findings highlight the importance of addressing both hydrolysis and acidification to optimize VFA production. To overcome these challenges, process optimization must focus on enhancing enzymatic activity, managing microbial community dynamics, and tailoring operational parameters to substrate characteristics. By addressing rate-limiting steps and refining process conditions, the production of VFA from diverse organic feedstocks can achieve greater efficiency and scalability. Substrate characteristics also influence the accessibility and degradation efficiency of biopolymers. For cellulose, properties like polymerization degree and crystallinity impact its susceptibility to cellulases [74]. Proteins are affected by structural traits and amino acid end groups, with highly soluble proteins hydrolyzing more rapidly than fibrous ones [75]. Lipids, due to their hydrophobicity, are less accessible to enzymes and often require emulsification to improve degradation [76]. Additionally, the presence of proteins and lipids in anaerobic reactors can cause operational issues, such as sludge flotation, biomass washout, and reduced mass transfer, further hindering system performance [77]. Strategies like physical or chemical pretreatment, lipid recovery, or the use of extracellular enzymes and Fenton oxidation have been proposed to mitigate these effects and enhance degradation [78]. Interactions among biopolymers further complicate the fermentation process. Research shows that carbohydrates are often prioritized for acidification over proteins and lipids [79]. [PERSON] et al. [73] found that while carbohydrates do not directly inhibit the hydrolysis of dissolved proteins, their rapid degradation under non-methanogenic conditions can suppress protein breakdown due to the accumulation of VFA, particularly acetate. The relationship between substrate types and fermentation products also varies. Carbohydrates are generally associated with higher acetate production, proteins with increased levels of propionate and isovalerate, and lipids with the formation of medium- and long-chain fatty acids like caprotic acid [11]. These complexities highlight the need for tailored strategies to optimize substrate degradation and manage interactions in mixed culture systems. The carbon-to-nitrogen (C/N) ratio, or its equivalent in chemical oxygen demand to total nitrogen (COD/TN), is a key parameter influencing acidification processes in VFA production. This ratio governs microbial activity, substrate degradation, and the resulting VFA composition. Optimal C/N ratios, typically between 15 and 70, enable efficient acidification by balancing carbon and nitrogen availability. Deviations from this range can disrupt the process: high C/N ratios lead to nitrogen deficiency, slowing microbial metabolism and reducing acid yields, while low ratios result in ammonia accumulation, which is toxic to microbes [67]. For instance, increasing the C/N ratio in biowaste fermentation liquid by recovering ammoniacal nitrogen through a gas-permeable membrane contactor demonstrated its potential for improved VFA production. This process maintained high and stable VFA concentrations (37-39 g CODVFA/L), where VFA represented 73-81% of the soluble COD, predominantly acetic, propionic, and butyric acids.