Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Low-quality animal by-product streams for the production of PHA-biopolymers: fats, fat/protein-emulsions and materials with high ash content as low-cost feedstocks.

OBJECTIVE: The rapid accumulation of crude-oil based plastics in the environment is posing a fundamental threat to the future of mankind. The biodegradable and bio-based polyhydroxyalkanoates (PHAs) can replace conventional plastics, however, their current production costs are not competitive and therefore prohibiting PHAs from fulfilling their potential. RESULTS: Different low-quality animal by-products, which were separated by thermal hydrolysis into a fat-, fat/protein-emulsion- and mineral-fat-mixture- (material with high ash content) phase, were successfully screened as carbon sources for the production of PHA. Thereby, Ralstonia eutropha Re2058/pCB113 accumulated the short- and medium-chain-length copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)]. Up to 90 wt% PHA per cell dry weight with HHx-contents of 12-26 mol% were produced in shake flask cultivations. CONCLUSION: In future, the PHA production cost could be lowered by using the described animal by-product streams as feedstock.

Recovery of the PHA Copolymer P(HB-co-HHx) With Non-halogenated Solvents: Influences on Molecular Weight and HHx-Content.

Biodegradable and biocompatible polyhydroxyalkanoates (PHAs) are promising alternatives to conventional plastics. Based on the chain length of their monomers they are classified as short chain length (scl-) or medium chain length (mcl-) PHA polymers. The type of monomers, the composition and the molecular weight (MW) define the polymer properties. To accelerate the use of PHA as a bulk material, the downstream associated costs need to be minimized. This study focuses on the evaluation of non-halogenated solvents, especially acetone as a scl-PHA non-solvent, for the recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) - P(HB-co-HHx) - with an mcl-HHx content >15 mol% and a MW average (M w) < 2 × 105 Da. Solvents and precipitants were chosen regarding zeotrope formation, boiling point differences, and toxicity. Non-halogenated solvent-precipitant pairs were evaluated regarding the MW characteristics (MWCs) of the extracted polymer. Acetone and 2-propanol as a low toxic and zeotropic solvent-precipitant pair was evaluated at different extraction temperatures and multiple extraction times. The extraction process was further evaluated by using impure acetone for the extraction and implementing a multi-stage extraction process. Additionally, P(HB-co-HHx) extracted with three different solvents was characterized by 1H and 13C-APT NMR. The screening of precipitants resulted in a negative influence on the MWCs by ethanol precipitation for extractions with acetone and ethyl acetate, respectively. It was observed, that extractions with acetone at 70°C extracted a higher fraction of PHA from the cells compared to extractions at RT, but the M w was decreased by 9% in average. Acetone with a 2-propanol fraction of up to 30% was still able to extract the polymer 95% as efficient as pure acetone. Additionally, when acetone and ethyl acetate were used in a multi-stage extraction process, a two-stage process was sufficient to extract 98-99% of the polymer from the cells. 1H and 13C-APT NMR analysis confirmed the monomer fraction and structure of the extracted polymers and revealed a random copolymer structure. The presented strategy can be further developed to an ecological and economically feasible PHA downstream process and thus contributes to the commercialization of low-cost PHAs.

Bioconversion of mixed free fatty acids to poly-3-hydroxyalkanoates by Pseudomonas putida BET001 and modeling of its fermentation in shake flasks

Background: The paper reports on the utilization of palm kernel oil (PKO) as a low cost renewable substrate for medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) production by Pseudomonas putida BET001. Investigation on the effects of selected key variables on growth, mixed free fatty acids consumption and mcl-PHA production by the bacterial culture in the shaken flask system were carried out along with its kinetic modeling. Results: The biomass production, fatty acids consumption and mcl-PHA production were found favorable when the strain was cultured in mineral medium at pH 6-7,28°C, aeration surface-to-volume ratio of 0.4 x 10(6) m-1, 250 rpm agitation rate for 48 h. Mcl-PHA production by this strain showed mixed growth and non-growth associated components as described by Luedeking-Piret kinetic model. Conclusion: The findings of this study provided add to the literature on key variables in for achieving good microbial growth and mcl-PHA production in shake flasks culture. In addition, suitable kinetic model to describe cultivation in this system was also presented.

Oxidized Polyethylene Wax as a Potential Carbon Source for PHA Production.

We report on the ability of bacteria to produce biodegradable polyhydroxyalkanoates (PHA) using oxidized polyethylene wax (O-PEW) as a novel carbon source. The O-PEW was obtained in a process that used air or oxygen as an oxidizing agent. R. eutropha H16 was grown for 48 h in either tryptone soya broth (TSB) or basal salts medium (BSM) supplemented with O-PEW and monitored by viable counting. Study revealed that biomass and PHA production was higher in TSB supplemented with O-PEW compared with TSB only. The biopolymers obtained were preliminary characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The detailed structural evaluation at the molecular level was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The study revealed that, when TSB was supplemented with O-PEW, bacteria produced PHA which contained 3-hydroxybutyrate and up to 3 mol % of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units. The ESI-MS/MS enabled the PHA characterization when the content of 3-hydroxybutyrate was high and the appearance of other PHA repeating units was very low.