Preparation of novel and efficient biochar immobilized composite enzymes and its mechanism for promoting methane production in high-solids anaerobic digestion

In this study, cellulase and laccase were firstly co-immobilized on biochar (BC) based on adsorption, covalent binding, and adsorption-crosslinking. The microstructure, physicochemical, and enzymatic properties of biochar immobilized composite enzymes (BICE) and their mechanism to improve high-solids anaerobic digestion (HSAD) performance were investigated. The results showed immobilization did not alter the BC crystal structure, enhanced enzyme tolerance to environment temperature and pH and strengthened the structural stability of enzyme protein. HSAD test results showed immobilization protected enzyme molecules, greatly reduced the risk of enzyme inactivation, and achieved the effect of slow-release of BICE. Additionally, BICE promoted substrate hydrolysis and acidification (λ was shortened by 1.45–3.08-fold), enabled rapid initiation of HSAD, and enhanced system stability, resulting in a 13.5 %–18.5 % improvement in cumulative methane production (CMP) compared to CK (without exogenous additives). Microbiological analyses showed that BICE effectively decomposed the substrate and up-regulated the functional gene expression of lignocellulosic hydrolase through the composite enzyme (maximum cellulase and laccase activities were 215.36 and 1.69 U/g, respectively), which provided sufficient nutrients for acidification. It was also found volatile fatty acids (VFAs) oxidizing bacteria (DMER64, Smithella, norank_f__Synergistaceae) with electroactivity were significantly enriched, methanogenic bacteria (Methanobacterium, Methanosarcina) were notably enriched. The propionate/butyrate metabolic pathway was enhanced, the abundance of functional genes for methanogenesis was up-regulated, direct interspecies electron transfer (DIET) was strengthened, and the methanogenic pathway was shifted to the CO2 type. This study demonstrated the potential of BICE as an exogenous additive using enzymatic hydrolysis synergized with BC to mediate an enhanced syntrophic methanogenesis.