Effects of pyrochar/hydrochar on anaerobic digestion of tetracycline contained wastewater at variable temperatures

Anaerobic digestion (AD) plays a significant role in renewable energy recovery from livestock wastewater. However, low temperatures and the presence of antibiotics exacerbate the challenge of wastewater management in cold regions. This study investigated the regulatory mechanisms of biochar (pyrochar and hydrochar) on anaerobic digestion (AD) of wastewater with tetracycline (TC) across temperature gradients (35-15 °C). Lower temperatures (25 °C and 15 °C) reduced methane yield (34.01-94.56%) and delayed startup time (48-120h). Both pyrochar and hydrochar effectively mitigated low-temperature and tetracycline stress, improving methane production by 4.11-41.16%. The further exploration based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS), intermediate spectra and microbial structure analysis revealed the role of different biochars on anaerobic digestion. Hydrochar released organic matter from its incompletely carbonized structure, which directly contributed to the methane yield. In comparison, pyrochar accelerated acetogenesis and methanogenesis via conductive structure-mediated direct interspecies electron transfer (DIET). In addition, this study proposed a dynamic impact of TC under different temperatures. In particular, TC acted as an electron mediator, accelerating methane production by stimulating DIET at 15 °C and 25 °C, while its inhibitory effect to microbes might be a dominant effect at 35 °C. These results were further verified by redundancy analysis and structural equation model that the dual role of biochar in alleviating TC inhibition and enhancing DIET, while TC promoted methanogenesis at low temperatures via DIET activation. These findings highlight temperature-dependent mechanisms of biochar and TC in AD, offering strategies to optimize methane production under the environmental stress of low temperature and antibiotics.