Preparation and performance analysis of microcapsules of methanogenic microbial agents

Low methane content is often required in biogas production from anaerobic digestion, and biomass loss during reactor operation. A microbial immobilization can be utilized to effectively mitigate the microbial escape for the high methane yield. Conventional immobilization approaches include the attachment- and embedding-based techniques. While attachment immobilization is benefits from the conductive materials with the abundant colonization sites, in order to promote the microbial metabolism. However, it can suffer from poor fixation efficacy, leading to the microbial leakage. Alternatively, the embedding immobilization can demonstrate the superior immobilization performance, but with the limited metabolic enhancement. In this work, a high-efficiency microbial capsule system was developed with the synergistic immobilization of hydrogenotrophic methanogens using sodium alginate (SA) and corn stalk biochar (CSB). Specifically, these materials were engineered to optimize the biological CO₂-to-CH₄ conversion. An improved embedding-dripping was utilized to fabricate the capsules. A single-factor experiment was also carried out on the systematic evaluation of their mass transfer efficiency, immobilization capacity, and conversion performance. Optimal SA and CSB concentrations were determined for the subsequent characterization using Fourier transform infrared spectroscopy (FTIR) and cryogenic scanning electron microscopy (cryo-SEM). Coenzyme F₄₂₀ quantification was conducted to clarify the mechanistic basis of the enhanced CO₂ conversion. Results revealed that the microbial capsules were prepared with 4 % SA for a peak methane content of 78%, indicating a 2.8-fold improvement, compared with free-cell controls, alongside optimal immobilization capacity and mass transfer efficiency. FTIR analysis confirmed that the capsules' robust physicochemical stability was achieved under complex operations. Cryo-SEM observations demonstrated that there were the significant variations in the pore size of the porous network structure in the SA-based capsules. The higher SA concentrations (4 %) were produced the smaller pore diameters (15-50 μm), compared with the lower concentrations. The optimal composite formulation was contained 4.0 % SA and 2.0 % CSB, indicating the structural stability, superior immobilization performance, and mass transfer properties. Compared with the CSB-free counterparts, these capsules demonstrated 1.2-2.1 times CO₂ conversion efficiency, with the maximum methane content of 93%, while the mass loss was reduced by approximately 60%. Mechanistic investigations revealed that the three-dimensional porous architecture was provided the extensive microbial colonization sites and favorable microenvironments for the metabolic activity. The CSB incorporation was greatly contributed to the dual functionality: 1) Physical protection of microbial consortia after pore structure optimization; 2) Significant enhancement of coenzyme F₄₂₀ concentration, a critical catalytic component in methanogenesis. The SA's structural stability and CSB's bioelectrochemical properties were combined to facilitate the direct interspecies electron transfer (DIET). An advanced immobilization strategy was integrated into the bothboth the attachment and embedding approaches. Key innovations included: 1) Dual-phase immobilization was combined with the SA's crosslinking matrix with CSB's conductive network; 2) Hierarchical pore architecture was balanced the microbial retention and substrate diffusion; 3) Biochar-mediated metabolic enhancement was achieved after F₄₂₀ upregulation and redox microenvironment modulation. The capsules also demonstrated the exceptional operational stability, maintaining high methane production, indicating their potential to for the industrial-scale biogas upgrading. The sodium alginate biochar microbial capsule was prepared with a rich network porous structure, thus providing the abundant attachment sites for microorganisms. The concentration of sodium alginate was adjusted the pore size of the porous structure. Its stability, immobilization, and mass transfer were improved to adjust the concentration of biochar. The secretion of coenzyme F₄₂₀ was promoted for the survival metabolism of microorganisms during methane production. Moderate CSB was has also promoted the concentration of coenzyme F₄₂₀, thereby increasing methane content. These findings can provide both technical solutions for anaerobic digester optimization and fundamental insights into immobilized microbial ecosystems, particularly for the next-generation bioenergy systems. Future research can also focus on the long-term performance under continuous operation, techno-economic analysis, and field validation in full-scale biogas plants.