This　study　explored　the　impact　of　different　temperatures　on　the　continuous　production　of　medium-chain　fatty　acids　(MCFAs)　from　waste　activated　sludge　(WAS).　Experimental　results　showed　that　there　was　almost　no　MCFAs　accumulation　at　55　°C.　Both　25　°C　and　37　°C　were　suitable　for　MCFAs　production,　with　25　°C　favoring　high-value　caprylate　production.　The　metagenomic　and　metatranscriptomic　analysis　highlighted　reverse　β-oxidization　as　the　main　chain　elongation　(CE)　cycle.　The　lack　of　CE-related　microorganisms　and　enzymes　at　55　°C　hindered　MCFAs　production,　in　contrast　to　the　heightened　activity　observed　at　25　°C　and　37　°C,　with　peak　activity　at　25　°C　leading　to　increased　longer-chain　MCFAs　synthesis.　37　°C　promoted　hydrolysis　and　acidification,　resulting　in　a　accumulation　of　higher　short-chain　fatty　acids,　but　further　elongation　to　MCFAs　would　be　hindered　by　product　toxicity.　This　research　concludes　that　25　°C　is　the　most　effective　temperature　for　the　production　of　WAS-derived　MCFAs,　offering　significant　economic　advantages.　©　2024　Elsevier　Ltd