*3.6. Physical Properties of Produced PHA*

Differential Scanning Calorimetry (DSC) was used to investigate the thermal properties of mcl-PHA, including the glass transition temperature (Tg), melting temperature (Tm), crystallization temperature (Tc), and melting enthalpy (ΔHm). The results obtained for the scl-co-mcl-PHA were Tg = −59.79 ◦C and Tc = 86.8 ◦C with ΔHm = 38.6 J/g, and Tm = 11.239 ◦C with ΔHm = 41.2 J/g (Supplementary Figure S2).

The thermal characteristics of our mcl-PHA differed both in Tm and Tg from those of the previously-reported mcl-PHA from *Pseudomonas* sp. PAMC28620, composed of poly(25.5% 3HO-co-52.1% 3HD-co-5.7% 3HdD-co-16.7% 3HtD) with Tm = 172.8 ◦C, Tg = 3.99 ◦C, and Tc = 54.61 ◦C [44]. The thermal properties also differed from those of the mcl-PHA from *Pseudomonas* sp. MPC6. Composed of poly(89.5% 3HB-co-1.8% 3HHx-co-3.3% 3HO-co-4.4% 3HD-co-1.1% 3HdD) with Tm = 163.5 ◦C, Tg = 2.3 ◦C, and Tc = 46.0 ◦C [45].

As highly crystalline polymers have limited application in industrial and medical fields [46], the mcl-PHA from *P. resinovorans* may have potential in applications that require a sticky and relatively low-temperature modeling polymer, as well as one that is biodegradable.

The molecular weight of the obtained PHA was characterized by Gel Permeation chromatography (GPC), which demonstrated a retention time peak start at 14.07 min, peak maximum at 15.86 min, and peak end at 18.93 min (Supplementary Figure S3). The mcl-PHA had average values, with a number average molecular weight (Mn) of 60,424, weight average molecular weight (Mw) of 114,093, Z-average (Mz) of 177,428, and viscosity average molar mass (Mv) of 105,309. The high polydispersity index value was because of the various monomer unit compositions and breakdown of the polymer in the sample preparation steps. The physical properties of the mcl-PHA produced by *P. resinovorans* differed from PHAs produced by other species [47]. Therefore, we hypothesize that the mcl-PHA has potential applications in the biomedical field or similar industries.

#### *3.7. Mass Balance of Spent Coffee Grounds to Bio-Hydrogen and PHA*

The mass balance was calculated on a carbohydrate basis to confirm the chemical oxygen demand (COD) reduction of SCGs (Figure 7). The 738 g COD of carbohydrates

of the initial SCGs was reduced to 444 g COD in the residue. Then, hydrogen and PHA were produced. Hydrogen was 279 mL/g COD-consumed sugar, which showed higher productivity than the 115 mL–CH4/g COD using SCG hydrolysates reported in another anaerobic fermentation study [48]. In addition, the amount of PHA produced was 14 g/ kg SCG oil, which is a relatively low amount.

**Figure 7.** Mass balance of coffee grounds to bio-hydrogen and PHA.

It is believed that this amount could be increased later through optimization of the culture process and production process. One of the important constituents in coffee is caffeine. Gokulakrishnan et al. reported that caffeine concentrations above 2.5 mg/mL can affect microbial growth [49]. Accordingly, it is necessary to reduce the amount of caffeine in SCGs. As shown by the mass balance, 80% of caffeine was removed in the residues.

However, after dark fermentation for hydrogen production, caffeine in effluent was 0.15 mg/g, which is about 6.5 times higher than the caffeine concentration of 23 mg/L at a sewage treatment plant in Seoul [50]. Caffeine did not cause a decrease in productivity during the hydrogen production process, but the amount of caffeine decomposition was very small. Therefore, it is necessary to establish a caffeine adsorption and removal facility in the post-treatment process when the operation is on an industrial scale.

#### **4. Conclusions**

This study produced eco–friendly energy and biodegradable plastics to induce the valorization of SCGs that are produced in large quantities every year. For bioplastic production, 97% oil was extracted from total oil in the SCGs. OESCGs were hydrolyzed and showed a sugar concentration of 32.26 g/L. OESCGH exhibited a low inhibitor concentration, and the post–treatment process could be omitted. Hydrogen production was 15% higher than that of SCGH. This shows that the process of extracting oil is more advantageous in the next utilization step. The extracted oil was used as a substrate for PHA production. *P. resinovorans*, which can use *n*–hexane remaining in the oil, produced 5.6 g/L of CDW and 29.7% of PHA content at 24 h in a fed–batch culture. In summary, it was shown that waste disposal costs can be reduced by performing both processes to increase the utilization of SCGs that were previously only hydrolyzed or had oil extracted. In addition, hydrogen and PHA were produced as useful resources. These results show that fossil fuel–energy and petroleum–based plastics can be replaced and enable the proposal of an environmental resource circulation model.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym15030681/s1, Figure S1: Qualitative and quantitative analysis of spent coffee grounds oil (A) GC/MS graph of qualitative analysis (B) composition of fatty acids in SCGs oil; Figure S2: Differential scanning calorimetry results of mcl-PHA; Figure S3: Gel permeation chromatography result of mcl-PHA.

**Author Contributions:** Conceptualization, B.-J.K., J.-M.J. and J.-J.Y.; methodology, S.K.B., D.-H.K. and Y.-H.Y.; validation, B.-J.K., J.-M.J., S.J. and J.-J.Y.; investigation, J.-M.J., D.-H.K. and S.J.; data curation, B.-J.K. and J.-M.J.; writing—original draft preparation, B.-J.K. and J.-M.J.; writing—review and editing, S.K.B., D.-H.K. and Y.-H.Y.; supervision, J.-J.Y.; project administration, J.-J.Y.; funding acquisition, J.-J.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT or MSIT; NRF-2020R1A2C2102381) and R&D Program of MOTIE/KEIT [grant number 20018072].

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
