Substrate-Flexible Two-Stage Fed-Batch Cultivations for the Production of the PHA Copolymer P(HB-co-HHx) With Cupriavidus necator Re2058/pCB113

Abstract

Recent studies of the impact and dimension of plastic pollution have drawn the attention to finding more sustainable alternatives to fossil-based plastics. Microbially produced polyhydroxyalkanoates (PHAs) biopolymers are strong candidates to replace conventional plastic materials, due to their true biodegradability and versatile properties. However, widespread use of these polymers is still hindered by their high cost of production. In the present study, we target high yields of the PHA copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] using a substrate-flexible two-stage fed-batch approach for the cultivation of the recombinant Cupriavidus necator strain Re2058/pCB113. A more substrate-flexible process allows to cope with constant price fluctuations and discontinuous supply of feedstocks on the market. Utilizing fructose for biomass accumulation and rapeseed oil for polymer production resulted in a final biomass concentration of 124 g L–1 with a polymer content of 86 wt% holding 17 mol% of HHx. Productivities were further optimized by operating the biomass accumulation stage in a “drain and fill” modus where 10% of the culture broth was recycled for semi-continuous biomass accumulation, after transferring 90% to a second bioreactor for PHA production. This strategy succeeded in shortening process times rising productivity yields to ∼1.45 g L–1 h–1.

Continuous feeding strategy for polyhydroxyalkanoate production from solid waste animal fat at laboratory‐ and pilot‐scale

“…This phenomenon can be explained by an increased availability of acetyl-CoA for PHA synthesis caused by a shutdown of growth-related metabolism (TCA, anabolism) after nitrogen gets depleted, which gradually increases the molar HB fraction in the polymer. Higher PHA titers than those in this study were achieved in cultivations with R. eutropha to cell densities >100 g L −1 in similar cultivation times for multiple other renewable feedstocks (Arikawa et al, 2017;Arikawa & Matsumoto, 2016;Riedel et al, 2012;Santolin et al, 2021;Sato et al, 2015). Such results are likely to be achieved with WAF as a feedstock by extending the growth phase through the addition of additional nitrogen, but the scope of this study was to establish a possibility to fine-tune the solid WAF feeding profile.…”

Workflow for shake flask and plate cultivations with fats for polyhydroxyalkanoate bioproduction

“…All growth media contained 10 μg mL -1 gentamicin sulfate to prevent contamination. Phosphate-buffered minimal medium, as described previously, was used for all experiments, with an initial pH value of 6.8 (Santolin et al 2021 ). In minimal medium cultivations, nitrogen limitation was applied to trigger maximum PHA accumulation.…”

Polyhydroxyalkanoate production from animal by‐products: Development of a pneumatic feeding system for solid fat/protein‐emulsions

“…For the determination of the CDW and PHA content during screening experiments, the whole shake flask culture was harvested in 50 ml polypropylene test tubes, whereas aliquots of 5–15 ml were sampled in 15‐ml polypropylene test tubes during bioreactor cultivations. The detailed procedures for gravimetric CDW determination by weighting and PHA determination by gas chromatography equipped with a flame ionization detector (GC‐FID) were described previously (Santolin et al, 2021 ). The residual cell dry weight (RCDW) was defined as CDW minus the PHA content in g L −1 .…”