A combination of palm olein (POl) and fructose was used as carbon source for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by recombinant Cupriavidus necator Re2058/pCB113. Cultures grown using 5g/L PO alone as carbon source produced cell dry weight (CDW) of 5.13g/L, 67% PHA/CDW and accumulated a copolymer containing 27mol% 3HHx in shake flask cultures. When cultures were grown in 5g/L fructose alone as the carbon source they produced CDW of 2.32g/L, 11% PHA/CDW and accumulated only poly(3-hydroxybutyrate) [P(3HB)] homopolymer. When the cells were cultured in 5g/L POl in combination with 7g/L fructose, CDW of 7.41g/L and 80% PHA/CDW was obtained with 17mol% 3HHx monomer fraction. Biosynthesis was carried out using a 13L fermenter to study the accumulation of 3HHx monomer fraction in the bacterial cells at different time point. The molecular weights of P(3HB-co-3HHx) with 4-15mol% 3HHx monomer were in the range between 5.47-6.85×105Da, which were at least two fold higher than previously reported values. Interestingly, the increase in Mwof the copolymer along with the increase in 3HHx molar fraction was observed. The viscoelastic property of the copolymer further confirmed the increase in Mw.
The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has been investigated. Various types and concentrations of plant oils were evaluated for efficient conversion of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5 g/L was found to be most suitable for production of copolymer with a 3HHx content of approximately 70 mol%. The time profile of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA production and PHA synthase activity. (1)H NMR and (13)C NMR analyses confirmed the presence of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and flexible mechanical properties.
Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by 13C NMR analysis. The number average molecular weight (M(n)) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6-3.9.
Polyhydroxyalkanoates (PHAs), a family of biodegradable and renewable biopolymers show a huge potential as an alternative to conventional plastics. Extractive bioconversion (in situ product recovery) is a technique that integrates upstream fermentation and downstream purification. In this study, extractive bioconversion of PHAs from Cupriavidus necator H16 was performed via a thermo-separating aqueous two-phase system to reduce the cost and environmental impacts of PHAs production. Key operating parameters, such as polymer concentration, temperature, and pH, were optimized. The strategy achieved a yield and PF of 97.6% and 1.36-fold, respectively at 5% EOPO 3900 concentration, 30 °C fermentation temperature and pH 6. The PHAs production process was also successfully scaled up in a 2 L bioreactor. To the best of our knowledge, this is the first report on extractive fermentation of PHAs from Cupriavidus necator utilizing a thermo-separation system to achieve a better productivity and purity of the target product.
Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.
This study investigates the effect of strategies on poly(3-hydroxybutyrate) [P(3HB)] production in bioreactor. In the production of P(3HB), urea and glucose feeding streams were developed to characterize the fed-batch culture conditions for new Cupriavidus necator NSDG-GG mutant. Feeding urea in repeated fed-batch stage (RFB-I) at 6, and 12 h in cultivation led to insignificant kinetic effect on the cell dry mass (CDM) and P(3HB) accumulation. Feeding glucose in repeated fed-batch stage (RFB-II) demonstrated that the incremental feeding approach of glucose after urea in fill-and-draw (F/D) mode at 24, 30, 36, 42, and 48 h in fermentation increased CDM and P(3HB) concentration. In the 1st cycle in RFB-II, the cumulative CDM reached the value of 26.22 g/L and then it increased with the successive repeated fed-batches to attain biomass of 145 g/L at the end of 5th cycle of RFB-II. The final cumulative P(3HB) concentration at the end of 5th cycle of RFB-II reached 111 g/L with the overall yield of 0.50 g P(3HB) g gluc- 1; the CDM productivity from the RFB-II cycles was in the range of 0.84-1.3 g/(L·h). The RFB-II of glucose in an increment mode produced nearly 2.2 times more increase in CDM and P(3HB) productivities compared to the decrement RFB-II mode. Repeated cultivation had also the advantage of avoiding extra time required for innoculum preparation, and sterilization of bioreactor during batch, thereby it increased the overall industrial importance of the process.
Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.