Improving Bioplastic Production: Enhanced P(3HB-co-3HHx) Synthesis from Glucose by Using Mutant Cupriavidus necator.

Abstract

Background: Biodegradable polyhydroxyalkanoates (PHAs) hold promises for various applications in industries ranging from packaging to biomedical engineering, highlighting the importance of this pioneering research in sustainable materials synthesis.

Objectives: The objective of this investigation was to present the successful production of polyhydroxyalkanoate (PHA) copolymer P(3HB-co-3HHx) from glucose utilizing a newly mutated strain of Cupriavidus necator. This mutant strain carries the pBPP-ccr_MeJ_Ac-emd plasmid which harbors a short-chain-length-specific PhaJ enzyme. The primary aim is to demonstrate the enhanced production efficiency and specificity of P(3HB-co-3HHx) through genetic manipulation and enzyme engineering, thereby advancing the feasibility and sustainability of PHA-based bioplastic production.

Materials and methods: To design the inputs conditions,a central composite factorial design (CCFD) based on a one-variable-at-a-time (OVAT) experiment was conducted. This experiment aimed to identify key chemical factors and their operational ranges affecting PHBHHx production by the mutant strain. Later, batch and repeated fed-batch (RFB) culture were run in a stirred tank bioreactor (STBR) with a working volume of 2 L which was inoculated by 200 ml (10% v/v) of freshly grown seed culture (18 h). This methodology ensured controlled exploration of individual variables, facilitating the selection of optimal conditions for PHBHHx production. Total glucose concentrations during fermentation were assessed through the phenol-sulfuric acid assay.

Results: The study demonstrates the effectiveness of the designed model in predicting PHBHHx production during fermentation runs with predicted values closely aligning with experimental results. This underscores the model satisfactory fitness with the experimental design. Additionally, a surprising enhancement was observed in the fermentation process with repeated fed-batch (RFB) leading to a substantial increase in cell dry weight (CDW), PHBHHX concentration, and 3HHx fraction, approximately 7 times, 7 times and 4.5 times, respectively. Confirmation of copolymer production was further validated through analytical techniques including FTIR spectroscopy, NMR, and TEM analysis. These findings collectively highlight the promising potential of RFB as a method to significantly improve PHBHHx production covering the way for further advancements in biopolymer manufacturing processes.

Conclusions: Our study reveals the potential of newly engineered C. necator NSDG-GGΔB1/pBPP-ccr_MeJ_Ac-emd mutant strain for efficient PHBHHx copolymer production. Process parameters such as glucose and urea concentration, and agitation rate significantly influenced PHBHHx yield. This research stands out by utilizing a novel strain for PHBHHx synthesis. Characterization confirmed high-quality polymer production. Our findings offer a sustainable approach for converting inexpensive carbon sources into valuable PHBHHx though further optimization for scale-up is warranted.