Tailoring the HHx monomer content of P(HB-co-HHx) by flexible substrate compositions: scale-up from deep-well-plates to laboratory bioreactor cultivations

The enhanced material properties exhibited by the microbially synthetized polyhydroxyalkanoate (PHA) copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] evidence that this naturally biodegrading biopolymer could replace various functionalities of established petrochemical plastics. In fact, the thermal processability, toughness and degradation rate of P(HB-co-HHx) can be tuned by modulating its HHx molar content enabling to manufacture polymers à-la-carte. We have developed a simple batch strategy to precisely control the HHx content of P(HB-co-HHx) to obtain tailor-made PHAs with defined properties. By adjusting the ratio of fructose to canola oil as substrates for the cultivation of recombinant Ralstonia eutropha Re2058/pCB113, the molar fraction of HHx in P(HB-co-HHx) could be adjusted within a range of 2–17 mol% without compromising polymer yields. The chosen strategy proved to be robust from the mL-scale in deep-well-plates to 1-L batch bioreactor cultivations.


Evaluation of the Impact of Gum Arabic as an Emulsifier
To ensure the same canola oil concentration in all replicate wells, the media containing canola oil was emulsified according to the material and methods section. In order to test whether gum arabic could be utilized as a substrate or inhibit growth, mineral salt medium without canola oil and emulsifier was incubated as a negative control for 72 h at 30°C and 225 rpm in 24-deep-well-plates with 3 mL working volume. MSM provided with only gum arabic did not lead significant growth (MSM + GA). A slightly higher CDW in comparison to the negative control is obtained as the gum arabic does not evaporate during lyophilization so that it remains in the sample. Evaluation of the influence of gum arabic compared to cultivations without emulsifier was done in shake flask cultivations with 5 g L -1 canola oil as carbon source. Comparable CDWs of 5.38 ± 0.24 (MSM + GA + CO) and 5.74 ± 0.17 g L -1 Santolin et al.

Supplementary Material
2 (control +) were reached. Results indicated that gum arabic was a suitable emulsifier that did neither inhibit growth nor was utilized as a carbon source ( Figure S1). 2

Carbon Content and C/N ratios calculations
To calculate the carbon content of fructose ( ,0.399 g g -1 ) the molecular weight of fructose ( ) was divided by the number of carbon atoms in the molecule ( ) multiplied by the standard atomic weight of carbon (°( )) (1).
The carbon content of canola oil was approximated at 0.775 g g -1 . For this, the fatty acid average composition in canola oil was withdrawn from (Orsavova et al., 2015) and an average molar mass of a fatty acid was calculated based on this composition. Following, the molar mass of an average triglyceride was calculated considering the molar mass of glycerol as well as the loss of one water molecule at each fatty acid bond. Finally, the averaged molar mass of a triglyceride was divided by the average number of carbon atoms present multiplied by its standard atomic weight (2).
To calculate the nitrogen content of urea ( , 0.466 g g -1 ) the molecular weight of urea ( ) was divided by the number or nitrogen atoms in the molecule ( ) multiplied by the standard atomic weight of nitrogen (°( )) (3).
The C/N (g g -1 ) ratio was calculated as the total carbon (g) represented by the amount of fructose and canola oil in each mixture and dividing it by the total nitrogen (g) supplied as urea (4). with R. eutropha Re2058/pCB113 using fructose and canola oil mixtures as carbon source and urea as nitrogen source. Cell dry weight (CDW; g L -1 ), PHA content of CDW (PHA; wt%), HHx content of PHA (HHx; mol%) and fructose concentration (Fructose; g L -1 ) values are shown over the course of the cultivation for each mixture with a final carbon content of 10 g L -1 and a C/N ratio of 22 g g -1 .