Regulating the molar fraction of 4-hydroxybutyrate in Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by biological fermentation and enzymatic degradation

Abstract

The regulation of 4-hydroxybutyrate (4HB) molar fraction in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] of a local isolate Cupriavidus sp. USMAA1020 was attempted by employing a feeding strategy through fed-batch fermentation in 100-L fermenter. The growth of Cupriavidus sp. USMAA1020 was enhanced by frequently feeding carbon and nitrogen at a ratio of 5 (C/N 5) using a DO-stat with cascade mode at 20% (v/v) dissolved oxygen (DO). The feeding of C/N 5 and the use of the DOstat mode were able to regulate the 4HB composition from 0-67 mol% by sequential feeding of c-butyrolactone and supplementing oleic acid. A high 4HB molar fraction of 67 mol% with a PHA concentration of 5.2 g/L was successfully obtained by employing this feeding strategy.Notably, enzymatic degradation carried out enhanced the 4HB composition of the copolymer synthesized. PHB depolymerase enzyme from Acidovorax sp. was used to degrade this P(3HB-co-70-mol%4HB) copolymer and the 4HB composition could be increased up to 83 mol%. The degradation process was observed by monitoring the timedependent change in the weight loss of copolymer films. The percentage of weight loss of solvent-cast film increased proportionally up to 19% within 3 h, whereas salt-leached films showed 90% of weight loss within 3 h of incubation and were completely degraded by 4 h. The molecular weight (M n ) of the films treated with enzyme demonstrated a slight decrease. SEM observation exhibited a rough surface morphology of the copolymer degraded with depolymerase enzyme.

Tailoring the surface architecture of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) scaffolds

“…Sequential feeding strategy is also known to result in improved production of copolymer and higher accumulation of secondary monomer 24. By employing this modified two‐stage cultivation for P(3HB‐ co ‐4HB) biosynthesis, the regular time consuming process of enrichment followed by aseptic transfer of bacterial cells into nitrogen‐free MM containing carbon source could be resolved.…”

Designing Novel Interfaces via Surface Functionalization of Short-Chain-Length Polyhydroxyalkanoates

“…Modifications of surfaces Mechanical modifications (1) Creating physical micro-rough, grooves or pores by enzyme degradation [63], alkali hydrolysis [64], particulate leaching or freeze-drying methods [65].…”

A Process Engineering Approach to Improve Production of P(3HB) byCupriavidus necatorfrom Used Cooking Oil

“…This strategy has been reported in some studies for the production of different PHA polymers. Faezah et al [11] reported the use of the DO-stat mode to feed oleic acid and/or γ-butyrolactone to Cupriavidus sp. USMAA1020, as a strategy to regulate the molar fraction of 4-hydroxybutyrate in the P(3HB-co-4HB) polymer.…”

“…However, the process set-up requires the selection of a suitable substrate feeding strategy to properly control the concentration of the carbon source throughout the fed-batch phase. Several strategies are available to feed the cultures and have been tested for PHA production from different substrates, including, among others, pulse feeding [4,5], continuous feeding with defined feed rate [6,7], exponential feeding [8,9], control of nutrient feed through dissolved oxygen (DO) concentration (DO-stat mode) [10][11][12], and pH control (pH-stat mode) [13,14].…”

“…USMAA2-4 [20], and Cupriavidus sp. DSM19416 [11]. A strategy combining DO-and pH-stat modes was also reported to feed oleic acid to Aeromonas hydrophila [21] and Pseudomonas putida KT2442 [22] to overcome the fact that, for high cell densities, the DO concentration did not respond to substrate depletion.…”