Long-term effects of cycle time and volume exchange ratio on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from food waste digestate by Haloferax mediterranei cultivated in sequencing batch reactors

Introduction The problem of food waste accumulation, combined with the pollution of petroleum-based plastic, presents a significant environmental challenge (Atiwesh et al., 2021, Paritosh et al., 2017). Converting food waste into bioplastics offers a potential solution to these issues by reducing landfilled food waste and replacing traditional plastics with biodegradable alternatives. Haloferax mediterranei (HM), a halophilic microorganism, is notable for its ability to produce the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) while thriving in high-salinity environments, which inherently reduces contamination risks (Meereboer et al., 2020). Previous studies have demonstrated that HM can produce PHBV from food waste in single-batch reactors, yet these systems are inefficient for large-scale industrialization (Wang and Zhang, 2021). The sequencing batch reactor (SBR) system addresses these limitations by enabling semi-continuous production, thereby enhancing economic feasibility and scalability (Allman, 2024). Despite these advantages, research on the long-term stability and operational optimization of SBRs for PHBV production remains limited. This study aims to fill this knowledge gap by investigating the effects of cycle time and volume exchange ratio (VER) of SBR on PHBV production performance. This work presents an opportunity to address both food waste management and plastic pollution challenges in one approach, contributing to a more sustainable and environmentally friendly future. Materials and Methods The food waste used as a substrate in the experiment was processed into digestate through arrested anaerobic digestion, which breaks down complex molecules into simpler, more digestible forms. The digestate was prepared with controlled composition, including organic acids and other nutrient sources, and was diluted to minimize substrate inhibition. The arrested anaerobic digestion was conducted at 35°C with a hydraulic retention time of 12 days and maintained a consistent organic loading rate of 2.5 g volatile solids (VS)/L-day. The PHBV production process was conducted in a 500 mL SBR, with a working volume of 400 mL (Figure 1). Each cycle included phases of feeding, reaction, and discharging, with the cycle time and VER as key variables. The SBR operated at 37°C with aeration and maintained high salinity (156 g/L NaCl) to support HM's growth while minimizing contamination risks. The cycle time was systematically increased from 1 to 12 days in the initial phase, while the VER remained constant at 0.5 (Figure 2). In the subsequent phase, the VER was adjusted from 0.3 to 0.5, with the cycle time determined by HM growth. The experiment included a 450-day continuous operation, making it one of the longest durations reported for HM fermentation. To assess HM growth, total organic carbon (TOC) reduction, and PHBV production, samples were collected at regular intervals and analyzed using various analytical techniques. Cell density was monitored with OD600nm, and PHBV content was measured using gas chromatography following a series of extraction and purification steps. Results 1.Optimal Dilution and TOC Utilization: HM demonstrated substrate inhibition with undiluted food waste digestate, showing no growth or PHBV production. A dilution factor of 2, with an initial TOC concentration of 7 g/L, achieved optimal PHBV yield, maintaining approximately 70% PHBV content and 6% 3-hydroxyvalerate (HV) fraction in the cellular biomass. This condition was chosen for subsequent SBR operations. 2.Growth and Substrate Utilization: Stable HM growth was observed over the 450 days, with optimal cell concentrations achieved at low VERs due to higher retention of HM cells in the reactor (Figure 3A). Substrate utilization showed a direct correlation with cell growth; however, around 2 g/L TOC remained unutilized, which was hypothesized to be linked to product inhibition (Figure 3B). 3.\PHBV production performance: PHBV cellular content stabilized at around 65% for over 450 days without contamination (Figure 4A). The HV fraction in PHBV remained consistently near 15%, aligning with commercial application standards. PHBV titer followed the same trend as HM growth (Figure 4B). PHBV yield declined as VER decreased, likely due to product inhibition (Figure 4C). PHBV productivity followed organic loading rate (OLR) trends, demonstrating that higher OLR increases PHBV production rate (Figure 4D). Discussion The study demonstrates that SBRs provide a viable solution for long-term PHBV production from food waste, maintaining stable production and minimizing contamination risks due to HM's halophilic nature. However, substrate inhibition from food waste digestate and product inhibition in the SBR pose limitations to production titer and yield. The relationship between PHBV productivity and OLR suggests that increasing OLR can enhance productivity, while controlling VER can mitigate inhibitor accumulation, optimizing yield and titer. These findings underscore the importance of managing cycle time and VER to balance microbial growth, product accumulation, and inhibitory effects. This research offers insights into scaling PHBV production sustainably, aligning with industry goals to reduce reliance on petroleum-based plastics. Conclusion This study successfully demonstrates the feasibility of long-term, stable PHBV production by HM using food waste digestate in SBRs over 450 days. By optimizing operational parameters such as cycle time and VER, the study presents a scalable approach for converting food waste into bioplastics, addressing environmental concerns associated with food waste and plastic pollution. Future research should focus on mitigating substrate and product inhibition to enhance industrial applications of this bioplastic production method, contributing to more sustainable waste management and plastic reduction efforts.