The polyhydroxybutyrate (PHB) production performance of Bacillus megaterium PNCM 1890 in multiple carbon sources was assessed in a model system of simulated glucose-xylose hydrolysates from several agricultural residues (corn stover, sugarcane bagasse, and banana pseudostem). Factors investigated were the type of nitrogen source, carbon-to-nitrogen (C/N) ratio, carbon-to-phosphorus (C/P) ratio, and amount of trace elements in the fermentation medium. Results show that the type of nitrogen source had a significant effect on bacterial growth and PHB production, with urea as the most preferred nitrogen source. In contrast, the type of simulated hydrolysate had no significant effect on the behavior of the bacteria, implying that the microorganism can potentially utilize hydrolysates from various agricultural residues. However, considering conversion efficiency and operating cost, simulated corn stover hydrolysate was selected as the best carbon source. A fermentation time of 28 h was found sufficient to completely utilize both glucose and xylose from the simulated hydrolysate. PHB was also confirmed as a growth-associated product of the bacteria, with observable patterns of catabolite repression and diauxic growth. Optimum conditions that maximize biomass concentration (7.3 g/L), PHB concentration (3.91 g/L), and substrate consumption (98.77% for glucose; 93.76% for xylose) were 14.3 g/g C/N ratio, 21.4 g/g C/P ratio, and 8 mL of trace elements solution (TES) per L of fermentation medium. Nonetheless, PHB was successfully produced using simulated corn stover hydrolysate and urea as carbon and nitrogen sources, respectively. The study provides baseline information on the use of actual hydrolysate, efficient fermentation, and upscaled production of bioplastics from agricultural residues.

Polyhydroxybutyrate (PHB) is a suitable biodegradable alternative to non-renewable petroleum-based plastics. Readily available agricultural lignocellulosic residues such as banana pseudostem can be utilized as a substrate to reduce the production cost of PHB and improve its feasibility for commercialization. Accordingly, pretreatment of banana pseudostem is needed to efficiently convert the substrate to PHB. In this study, optimization of two pretreatment methods for banana pseudostem – namely, steam explosion and sequential steam explosion – dilute acid pretreatment – were optimized to improve the digestibility of the biomass and consequently increase the production of reducing sugars in the hydrolysate during enzymatic saccharification. Response surface methodology (RSM)-designed experiments showed that among all factors investigated, for both pretreatment methods, the steam explosion temperature had the strongest positive impact on reducing sugar production. Optimum conditions of steam explosion pretreatment were 219.31 °C steam explosion temperature and 10 min of pretreatment time, producing 7.33 g/L (48.87% yield) of reducing sugars in the enzymatic hydrolysate. For sequential pretreatment, optimum conditions were 220 °C steam explosion temperature, 135 °C dilute acid temperature, 44 min of dilute acid reaction time, and 1.57% w/v H2SO4, with a corresponding reducing sugar concentration of 13.02 g/L (86.79% yield). A 90% increase in reducing sugar yield was observed after dilute acid pretreatment of steam-exploded banana pseudostem. Using the hydrolysate from sequentially-pretreated banana pseudostem, PHB (2.64 g/L) was successfully synthesized after 12 h of bacterial fermentation. Hence, sequential pretreatment was proven effective in producing enzymatic hydrolysates from banana pseudostem for PHB production.

In this study, a sustainable protocol for PHA extraction was methodically selected using two multi-criteria decision analysis (MCDA) tools, the analytic hierarchy process (AHP), and grey relational analysis (GRA). AHP was first used to evaluate the proposed criteria categorized into technical, economic, and environmental aspects using a collected survey of pairwise comparisons. Based on the results of AHP, it was identified that both environmental and economic aspects were given higher priorities. Among the criteria, hazards and risks had the highest overall importance, followed by extraction cost and purity. Using GRA, 12 protocol alternatives categorized into solvent extraction and precipitation, non-PHA cell mass (NPCM) digestion, and assisted extraction methods were graded according to the criteria. Overall, the highest priority weights were given to NPCM digestion protocols including sodium hydroxide, sodium hydroxide + sodium dodecyl sulfate (SDS), and ammonia water. The reagents involved in these protocols are ecologically benign and cheaper compared to other solvents, hence the higher grades in the environmental and economic aspects. Sensitivity analysis also proved that these protocols are excellent, particularly if extraction cost is given a higher priority. However, if hazards and risks and purity were given more importance, butyl acetate is preferable to sodium hydroxide. Further investigations such as the validation and optimization of protocols, together with feasibility studies and life cycle analyses, may be integrated with the results of this study to comprehensively determine a sustainable PHA extraction protocol.

Polyhydroxybutyrate (PHB) is considered a potential substitute for conventional, non-biodegradable petroleum-based plastics. However, high production cost has been a major drawback to the commercialization of PHB. The use of low-cost lignocellulosic agricultural residues such as corn stover, together with an effective pretreatment method, can reduce production costs. In this study, optimization of pretreatment methods for corn stover – namely, steam explosion and sequential steam explosion–dilute acid pretreatment – was done to maximize the concentration of reducing sugars in the hydrolysate obtained after enzymatic saccharification. Response surface methodology (RSM)-designed experiments showed that steam explosion temperature had the highest impact on reducing sugar production for both pretreatment methods. Optimum conditions for steam explosion pretreatment were 220.9 °C steam explosion temperature and 11.2 min residence time, yielding a hydrolysate with 9.67 g/L (64.50% yield) of reducing sugars after saccharification. For the sequential pretreatment, 9.14 g/L of reducing sugars (60.93% yield) was produced from the optimum conditions of 224.8 °C steam explosion temperature, 144.2 °C dilute acid temperature, 43.8 min reaction time, and 5% w/v acid concentration. Dilute acid pretreatment significantly decreased the reducing sugar yield after the steam explosion by 5.5%, probably due to the degradation of reducing sugars, making steam explosion sufficient in the pretreatment of corn stover. More importantly, PHB was produced using the hydrolysate from steam-exploded corn stover, with a maximum concentration of 1.81 g/L after 24 h of bacterial fermentation. Therefore, the steam explosion was proven effective in producing sugar-rich hydrolysates from corn stover for PHB production.