Bioconversion of citrus waste by long-term DMSO-cryopreserved rumen fluid to volatile fatty acids and biogas is feasible: A microbiome perspective

Preserving rumen fluid as the inoculum for anaerobic digestion of food waste is necessary when access to animal donors or slaughterhouses is limited. This study aims to compare two preservation methods relative to fresh ruminal inoculum: (1) cryoprotected with 5% dimethyl sulfoxide (DMSO) and stored at −20 °C and (2) frozen at −20 °C, both for 6 months. The fermentation activity of different inoculum was evaluated by rumen-based in vitro anaerobic fermentation tests (volatile fatty acids, biomass digestibility, and gas production). Citrus pomace was used as the substrate during a 96-h fermentation. The maximum volatile fatty acids, methane production, and citrus pomace digestibility from fresh rumen fluid were not significantly different from rumen fluid preserved with DMSO. Metagenome analysis revealed a significant difference in the rumen microbial composition and functions between fresh rumen fluid and frozen inoculum without DMSO. Storage of rumen fluid using −20 °C with DMSO demonstrated the less difference compared with fresh rumen fluid in microbial alpha diversity and taxa composition. The hierarchical clustering tree of CAZymes showed that DMSO cryoprotected fluid was clustered much closer to the fresh rumen fluid, showing more similarity in CAZyme profiles than frozen rumen fluid. The abundance of functional genes associated with carbohydrate metabolism and methane metabolism did not differ between fresh rumen fluid and the DMSO–20 °C, whereas the abundance of key functional genes significantly decreased in frozen rumen fluid. These findings suggest that using rumen liquid preserved using DMSO at −20 °C for 180 days is a feasible alternative to fresh rumen fluid. This would reduce the need for laboratories to maintain animal donors and/or reduce the frequency of collecting rumen fluid from slaughterhouses.