Studies on Microbial Production and Extraction of poly β-hydroxybutyrate using Bacillus Subtilis

Abstract

Poly-β-hydroxybutyrate (PHB) has wide applications in industry as it has properties similar to that of conventional plastics. Most important properties of PHB are its biodegradable nature, thermo-stability, and biocompatibility, durability, non-toxicity and water insolubility. Bacillus subtilis is a well-known bacterium that has been used for the industrial production of several proteins and biochemicals. Bacillus subtilis (ATCC®6051™) was used for PHB production. PHB was quantified using UV-VIS spectrophotometer and High-Pressure Liquid Chromatography (HPLC). The PHB yield of B. subtilis (ATCC®6051™) was estimated in batch cultivation by HPLC and GC methods. In this study, B. subtilis was employed for intracellular PHB Production in batch and fed-batch cultivation. The batch kinetic study of B. subtilis culture was conducted in an optimized medium in stirred tank bioreactor. The B. subtilis culture showed maximum biomass formation (1.78±0.1 g/l) and PHB accumulation (1.18±0.04 g/l) with 66.29 % PHB content of dry cell weight (DCW) at 28 h in optimized media using shake flask cultivation. The overall productivity was found to be 0.039 g/l/h. B. subtilis culture showed maximum biomass (1.79±0.026 g/l) and PHB accumulation (1.23±0.024 g/l) with the PHB content of 68.72% of DCW after 24 h in a bioreactor. The overall productivity was found to be 0.051 g/l/h. Substrate inhibition reduces the overall production in microbial production of PHB. Therefore, substrate (glucose, phosphate, and ammonium) inhibition studies were performed in shake flask conditions. The complete inhibition of growth of bacterial culture was seen at glucose, phosphate, and ammonium concentration of 120 g/l, 80 g/l, and 100 g/l, respectively. The optimum value of kinetic parameters was estimated to be: 0.376 h-1 for μm, 4.162 g/l for KSG, 1.611 g/l for KSN, 0.427 g/l for KSP, 123.6 g/l for KIG, 103 g/l for KIN, 82.4 g/l for KIP, 1.646 g/g for 1/Y(x/SG), 0.0923 g/g/h for mSG, 0.688 g/g for k1, 0.0041 for k2 and 9.671 for n. Initial values of biomass (𝑥=0.05 𝑔/𝑙), substrate (𝑆𝐺=7.3 𝑔/𝑙,) and PHB concentration (P = 0.012 g/l) and optimum parameters values were utilized to get a modified Monod model for PHB production. Inhibition kinetics data was also utilized for the prediction of inhibition kinetics parameters in the batch model. The productivity was found to be 0.041 g/l/h. The batch model was utilized for the prediction of the best fed-batch strategy. In constant feed rate fed-batch cultivation in shake flask, B. subtilis culture showed maximum biomass (1.91±0.05 g/l) and PHB production (1.37±0.08 g/l) with PHB content of 71.73% of dry cell weight at 30 h. The productivity was found to be 0.046 g/l/h. PHB production was enhanced 1.16 folds in constant feed rate fed-batch conditions, as compared to batch cultivation of B. subtilis. In this study, batch kinetic parameters for biomass production, substrate utilization and PHB production were estimated for B. subtilis culture. The resulting data was utilized for the development of mathematical model for production of biomass and PHB. This constant feed rate fed-batch cultivation was performed in a bioreactor using statistically optimized media. The maximum biomass obtained was 1.66±0.050 g/l and PHB was 1.42±0.05 g/l with the PHB content of 85.54% of DCW at 30 h cultivation. Two-stage cultivation of B. subtilis was performed using two medium of different composition for growth and production in bioreactor using constant fed-batch strategy. In bioreactor, in which maximum biomass (1.95±0.045 g/l) and PHB production (1.396±0.017 g/l) were obtained in 8th h of production with a PHB content of 93.33% of DCW. This is the highest reported PHB yield by fermentation to the best of our knowledge. The maximum biomass (1.953±0.045 g/l) at 4 h and PHB production (1.396±0.017 g/l) at 8 h were obtained during mass production of PHB in a bioreactor. In this study, several off-line feeding strategies were examined for enhanced biomass and PHB production by B. subtilis. After shake flask trials it was established that enhanced PHB production took place by constant feed rate fed-batch cultivation strategy. The successful model based feeding strategy can be applied for the production of PHB on commercial scale. A new extraction process with non-toxic and cheaper solvent was developed. In this study, different parameters like acetone percentage, and solvent pH, temperature, and incubation period were optimized using statistical tools such as Plackett-Burman (PB) design and Response Surface Methodology (RSM). An attempt was made to develop a robust process for the extraction of PHB from cells by liquid-liquid extraction. Different parameters like acetone percentage (30 % to 70 %), and pH (5 to 9), temperature (30 °C to 70 °C), and incubation periods (30 to 70 min) were chosen for optimization using Central Composite Design (CCD). The most effective factors predicted by PB design were cell biomass (t-value of 0.0404), incubation temperature (t-value of 0.0057), solvent pH (t-value of 0.0039), solvent percentage (t-value of 0.0023), incubation period (t-value of 0.0009). The agitation speed (t-value of -0.0094) did not show a significant effect on PHB recovery. The optimum conditions for the enhanced PHB recovery and purity were found to be solvent pH 7, extraction temperature - 43 °C, incubation time - 70 min, and percentage acetone – 30 % by Central Composite Design (CCD). The experimentaly recovered PHB was 0.87 g/g with a purity of 95.02% which is higher than the model predicted value of PHB recovery of 0.84 g/g of biomass with a purity of 97.23 %. In this study, PHB extraction process parameters were optimized for PHB recovery (g/g) and purity (%) using PB design and RSM statistical tools. The cheaper and green solvent acetone was employed for extraction of PHB. PHB extracted from this process is less toxic as compared to the PHB extracted using the halogenated solvents (chloroform, di-chloromethane, dichloroethane and sodium hypochlorite). The extracted PHB can be utilized in food industries, pharmaceutical and tissue engineering applications.