Distinct Mechanisms between Free Iron Species and Magnetite Addition in Anaerobic Digestion on Alleviating Ammonia Inhibition

Ammonia inhibition often occurs during anaerobic digestion (AD) of the protein-rich substrate. Iron-containing substances were proved to be efficient in alleviating the ammonia stress. However, the mechanisms behind, especially the distinct impacts of different forms of iron materials, are not fully revealed. Here, the alleviating performances of FeCl₃ and Fe₃O₄ on AD systems under ammonia stress were investigated. Moreover, the mechanisms behind these were revealed and compared at the transcriptional level. Results showed that FeCl₃ and Fe₃O₄ additions with an equal amount of iron element content (1.29 mM) led to the increased cumulative biogas and methane yields under an ammonia concentration of 3 g/L. Furthermore, the addition of iron-containing substances alleviated the accumulation of volatile fatty acids (VFAs) and extracellular polymeric substances (soluble carbohydrates and protein) caused by ammonia stress, which also had an obvious positive effect on the electron transfer capability. Microbial analysis demonstrated that the microbes (e.g., orders Methanosarcinales, Clostridiales, and Syntrophobacterales) associated with direct interspecies electron transfer (DIET), syntrophic acetate oxidization, and degradation of organic compounds were enriched. Metatranscriptomic analysis showed that ammonia inhibited the AD process by disrupting cellular redox homeostasis, infecting the ATPase activity, affecting cellular energy supply, inhibiting methane-producing enzyme activity, and suppressing the expression of cell conductive structure genes. Meanwhile, the addition of FeCl₃ and Fe₃O₄ enhanced the cellular basal metabolism and energy supply, as well as microbial electron transfer and enzymic activities on methanogenesis. Metatranscriptomic analysis indicated that the addition of free iron species (FeCl₃) can relieve the ammonia stress on syntrophic propionate and acetate oxidizing bacteria, enhance DIET by stimulating the synthesis of c-type cytochrome, and thus promote methane production. Meanwhile, Fe₃O₄ may promote methane production by stimulating the expression of related genes and facilitating electron transfer in the AD system as a capacitor. Overall, the results demonstrated that ferric chloride and magnetite can alleviate the ammonia inhibition in the AD process of high-nitrogen waste through different mechanisms.