Upcycling Biodegradable PVA/Starch Film to a Bacterial Biopigment and Biopolymer

Meeting the challenge of circularity for plastics requires amenability to repurposing post-use, as equivalent or upcycled products. In a compelling advancement, complete circularity for a biodegradable polyvinyl alcohol/thermoplastic starch (PVA/TPS) food packaging film was demonstrated by bioconversion to high-market-value biopigments and polyhydroxybutyrate (PHB) polyesters. The PVA/TPS film mechanical properties (tensile strength (σu), 22.2 ± 4.3 MPa; strain at break (εu), 325 ± 73%; and Young’s modulus (E), 53–250 MPa) compared closely with low-density polyethylene (LDPE) grades used for food packaging. Strong solubility of the PVA/TPS film in water was a pertinent feature, facilitating suitability as a carbon source for bioprocessing and microbial degradation. Biodegradability of the film with greater than 50% weight loss occurred within 30 days of incubation at 37 °C in a model compost. Up to 22% of the PVA/TPS film substrate conversion to biomass was achieved using three bacterial strains, Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699), Streptomyces sp. JS520, and Bacillus subtilis ATCC6633. For the first time, production of the valuable biopigment (undecylprodigiosin) by Streptomyces sp. JS520 of 5.3 mg/mL and the production of PHB biopolymer at 7.8% of cell dry weight by Ralstonia eutropha H16 from this substrate were reported. This low-energy, low-carbon post-use PVA/TPS film upcycling model approach to plastic circularity demonstrates marked progress in the quest for sustainable and circular plastic solutions.

Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli

Hydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H2 into protons and electrons. Important achievements have been made over the past two decades in the understanding of these highly complex enzymes. However, most hydrogenases have low production yields requiring many efforts and high costs for cultivation limiting their investigation. Heterologous production of these hydrogenases in a robust and genetically tractable expression host is an attractive strategy to make these enzymes more accessible. In the present study, we chose the oxygen-tolerant H2-sensing regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in Escherichia coli. Using screening experiments in 24 deep-well plates with 3 mL working volume, we investigated relevant cultivation parameters, including inducer concentration, expression temperature, and expression time. The RH yield could be increased from 14 mg/L up to >250 mg/L by switching from a batch to an EnPresso B-based fed-batch like cultivation in shake flasks. This yield exceeds the amount of RH purified from the homologous host R. eutropha by several 100-fold. Additionally, we report the successful overproduction of the RH single subunits HoxB and HoxC, suitable for biochemical and spectroscopic investigations. Even though both RH and HoxC proteins were isolated in an inactive, cofactor free apo-form, the proposed strategy may powerfully accelerate bioprocess development and structural studies for both basic research and applied studies. These results are discussed in the context of the regulation mechanisms governing the assembly of large and small hydrogenase subunits.

Engineering the CBB cycle and hydrogen utilization pathway of Ralstonia eutropha H16 for improved autotrophic growth and PHB production

CO 2 is fixed by all living organisms with an autotrophic metabolism, among which the Calvin-Benson-Bassham ( CBB) cycle is the most important and widespread carbon fixation pathway. Thus, studying and engineering the CBB cycle with the associated energy providing pathways to increase the CO 2 fixation efficiency of cells is an important subject of biological research with significant application potential. In this work, the autotrophic microbe Ralstonia eutropha H16 was selected as a research platform for CBB cycle optimization engineering. By knocking out either CBB operon genes on the operon or mega-plasmid of R. eutropha , we found that both CBB operons were active and contributed almost equally to the carbon fixation process. With similar knock-out experiments, we found while both soluble and membrane-bound hydrogenases (SH and MBH), belonging to the energy providing hydrogenase module, were f unctional d uring autotrophic growth of R. eutropha. And SH played a more significant role. By introducing a heterologous cyanobacterial RuBisCO with the endogenous GroES/EL chaperone system and RbcX, the culture OD 600 of engineered strain increased 89.15% after 72 hours of autotrophic growth, indicating cyanobacterial RuBisCO with a higher activity was functional in R. eutropha and improved upon original CBB pathway. Meanwhile, expression of hydrogenases were optimized by modulating the expression of MBH and SH, which could further increase the R. eutropha H16 culture OD 600 to 93.4% at 72 hours. Moreover, the autotrophic yield of its major industrially relevant product, polyhydroxybutyrate (PHB), was increased by 99.71%. To our best knowledge, this is the first report of successfully engineering the CBB pathway of R. eutropha for improved activity , and is one of only a few cases where the efficiency of CO 2 assimilation pathway was improved. Our work demonstrates that R. eutropha is an extremely useful platform for studying and engineering the CBB for applications in more important organisms, such as agricultural crops, and a potential microbial cell factory to develop industrial biotechnology for sequestrating CO 2 .

2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.

Evaluation of polyhydroxyalkanoate (PHAs) production with a bacterial isolate using cassava flour hydrolysates as an alternative substrate

In the production of polyhydroxyalkanoates (PHA), the costs of the process are mainly associated with the carbon source and most of the production studies are carried out with commercial strains. In this study, PHA production was evaluated using a strain, SB-34, compared with a referenced strain, Ralstonia eutropha H16, growing in cassava flour hydrolysates as an alternative carbon source. SB-34 reached a biomass maximum of 5,49 ± 0,21 g / L, and PHA product of 3,23 ± 0,21 g / L exceeding the maximum values obtained by R. eutropha H16 (3,8 ± 0,45 g / L and 2,42 ± 0,23 g / L, respectively). Analyzes with FTIR indicate that the polymer obtained with SB-34 is like polyhydroxybutyrate P(3-HB) type. Molecular analysis identified SB-34 as Burkholderia sp. with 96 % of similarity.