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β-oxidation is a well-known pathway for fatty acid (FA) degradation. However, the wide range of feedstocks, their intermediates, and complex microbial networks involved in anaerobic digestion (AD) make β-oxidation unclear during lipid digestion having a variety of long-chain fatty acids (LCFAs). Here, we demonstrated the detailed metabolic pathway of major bacteria and enzymes responsible for the β-oxidation of individual saturated FAs (C160 and C18 and unsaturated FAs (C181 and C182). C160 showed no negative impact on AD. T