| Summary: | Raw brewers’ spent grain (BSG), a by-product of beer production and produced at a large scale, presents a composition that has been shown to have potential as feedstock for several biological processes, such as polyhydroxyalkanoates (PHAs) production. Although the high interest in the PHA production from waste, the bioconversion of BSG into PHA using microbial mixed cultures (MMC) has not yet been explored. This study explored the feasibility to produce PHA from BSG through the enrichment of a mixed microbial culture in PHA-storing organisms. The increase in organic loading rate (OLR) was shown to have only a slight influence on the process performance, although a high selectivity in PHA-storing microorganisms accumulation was reached. The culture was enriched on various PHA-storing microorganisms, such as bacteria belonging to the <i>Meganema</i>, <i>Carnobacterium</i>, <i>Leucobacter</i>, and <i>Paracocccus</i> genera. The enrichment process led to specialization of the microbiome, but the high diversity in PHA-storing microorganisms could have contributed to the process stability and efficiency, allowing for achieving a maximum PHA content of 35.2 ± 5.5 wt.% (VSS basis) and a yield of 0.61 ± 0.09 Cmmol<sub>PHA</sub>/Cmmol<sub>VFA</sub> in the accumulation assays. Overall, the production of PHA from fermented BSG is a feasible process confirming the valorization potential of the feedstock through the production of added-value products.
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