*Article* **Two-Stage Bio-Hydrogen and Polyhydroxyalkanoate Production: Upcycling of Spent Coffee Grounds**

**Beom-Jung Kang 1,†, Jong-Min Jeon 1,† , Shashi Kant Bhatia <sup>2</sup> , Do-Hyung Kim 3, Yung-Hun Yang 2, Sangwon Jung <sup>4</sup> and Jeong-Jun Yoon 1,\***


**Abstract:** Coffee waste is an abundant biomass that can be converted into high value chemical products, and is used in various renewable biological processes. In this study, oil was extracted from spent coffee grounds (SCGs) and used for polyhydroxyalkanoate (PHA) production through *Pseudomonas resinovorans*. The oil–extracted SCGs (OESCGs) were hydrolyzed and used for biohydrogen production through *Clostridium butyricum* DSM10702. The oil extraction yield through *n*–hexane was 14.4%, which accounted for 97% of the oil present in the SCGs. OESCG hydrolysate (OESCGH) had a sugar concentration of 32.26 g/L, which was 15.4% higher than that of the SCG hydrolysate (SCGH) (27.96 g/L). Hydrogen production using these substrates was 181.19 mL and 136.58 mL in OESCGH and SCGH media, respectively. The consumed sugar concentration was 6.77 g/L in OESCGH and 5.09 g/L in SCGH media. VFA production with OESCGH (3.58 g/L) increased by 40.9% compared with SCGH (2.54 g/L). In addition, in a fed–batch culture using the extracted oil, cell dry weight was 5.4 g/L, PHA was 1.6 g/L, and PHA contents were 29.5% at 24 h.

**Keywords:** bio-hydrogen; polyhydroxyalkanoate; mcl-PHA; *Pseudomonas resinovorans*
