Glycine betaine fortifies microbial metabolism to reclaim methane production in anaerobic digestion under salt stress

Glycine betaine (GB) has been used to resist different environmental stress. However, its effects and mechanisms in mitigating salt stress to recover methane yield in anaerobic digestion (AD) remains unclear. This study focused on investigating the alleviation efficiency and mechanism of salt stress in AD of typical saline organic wastes by GB. GB could facilitate hydrolysis, acidogenesis and methanogenesis steps, resulting in a methane yield increase of 156.3 mL·g⁻¹VS under 10 g·L⁻¹ NaCl. Additionally, GB stimulated extracellular polymeric substances (EPS) secretion by 23% and modulated key enzyme activities (α-GC, F420 and TAOC increased; NP, TBARS, SOD and CAT decreased), thereon restoring metabolic and redox balance. Microbial analysis indicated that GB tightened the microbial network, especially the co-relationship between Methanothrix and other microbes. Metagenomics further revealed upregulation of genes related to methanogenesis (e.g., ftr, mtd and mer), osmoregulation (e.g., proV, kdpA and envZ/ompR), and EPS secretion (e.g., bapA, rfbA and tatA). Furthermore, GB directly participated in methanogenesis pathways. This study not only proposes a broad-spectrum approach to mitigate salt stress of salt-containing AD, but also establishes a universal theoretical framework for designing resilient AD systems from a microbial perspective.