All experiments were conducted with the engineered R. eutropha strain Re2058/pCB113 that produces the copolymer P(HB-co-HHx) when grown on oleaginous feedstocks (Budde, et al., 2011b). The strain was stored in 20% (v v^-1) glycerol at −80°C.

Tryptic soy broth (TSB) media, agar plates and mineral salt media (MSM) compositions have been described previously (Gutschmann et al., 2019). Ralstonia eutropha Re2058/pCB113 was streaked from a cryoculture on a TSB agar plate and incubated for 3–4 days at 30°C. A single colony from the plate was used to inoculate 10 mL TSB using a 125-mL Ultra Yield Flask (Thomson Instrument Company, United States), equipped with an AirOtop membrane (Thomson Instrument Company, United States). TSB was always supplemented with 10 μg mL⁻¹ gentamycin sulfate and 200 μg mL⁻¹ kanamycin sulfate. The preculture was incubated at 30°C and 200 rpm shaking speed for approximately 17 h or until an OD₆₀₀ of 5 was reached. The main cultures in MSM were inoculated to an initial OD₆₀₀ of 0.05. Fructose or canola oil (Edeka Zentrale AG and Co. KG, Germany) were used as carbon sources and urea was used as the sole nitrogen source in the MSM. The explicit amounts are described in the text. All chemicals were purchased from Carl Roth GmbH and Co. KG (Germany) unless stated otherwise.

Specific carbon and nitrogen concentrations (g L⁻¹) and carbon-to-nitrogen ratios [C/N (g g⁻¹)] were used for all experiments. See Supplementary Material for calculations.

24-deep-well-plates with square shape wells and a maximum volume of 11 mL (Duetz-MTPS, Adolf Kühner AG, Switzerland) were used in this study. To ensure identical cultivation conditions for the deep-well-plate replicates, 50 mL of each media with each chosen fructose to canola oil ratio was prepared and inoculated from TSB overnight cultures (see above), and then 3 mL of culture was transferred into each of the wells. To obtain a defined and comparable canola oil concentration in the different wells, the medium was pre-emulsified with gum arabic (GA) before sterilization using an adapted method from Budde et al. (2011a): each medium was prepared by mixing the phosphate buffer, water and K₂SO₄ with the desired amount of canola oil and adding GA to a final concentration 0.3% (w v⁻¹). The mixture was homogenized with an Ultra-Turrax T25 (IKA-Werke GmbH and Co. KG, Germany) for 1 min at 8,000 rpm. After emulsifying the oil, the media was autoclaved, and the remaining media components were added from sterile stocks. GA was chosen as the emulsifier as it has been shown not to support growth of R. eutropha (see Supplementary Figure S1). Plates were incubated for 72 h at 30°C and 225 rpm in an orbital shaker with 50 mm amplitude. Culture volume and incubation conditions were chosen according to manufacturer’s instructions to ensure sufficient oxygen supply. Biological triplicates were performed for each condition.

Four different mixtures of fructose and canola oil were selected from the deep-well-plate cultivations and upscaled to 100 mL cultures, applying the exact same cultivation strategy at this scale. Cultivations were performed in 500-mL DURAN baffled flasks (DWK Life Science GmbH, Germany) sealed with AirOtop membranes to ensure sufficient oxygen supply. These cultivations, performed in biological triplicates, were incubated at 30°C and 200 rpm in an orbital shaker (50 mm amplitude) for 72 h and sampling was performed every 24 h. The cultivations were then repeated doubling the amount of carbon content available to 10 g L⁻¹ but maintaining the C/N ratio by also doubling the urea concentration to 0.92 g L⁻¹.

P(HB-co-HHx) production with fructose and canola oil mixtures was upscaled to 1-L bioreactors using six Multifors2 parallel benchtop bioreactors with two six-blade Rushton impellers (Infors AG, Switzerland). The cultivation temperature was kept constant at 30°C and the pH was maintained at 6.8 ± 0.1 using 1 M H₃PO₄ and 2 M NaOH for pH control. The initial stirring speed was set to 200 rpm, whereas the initial flow rate was set to 0.05 vvm. Via an automatized cascade, aeration was increased up to 0.5 vvm and later stirring was increased up to 1,500 rpm in order to prevent DO values from dropping below 40%. Foam was mechanically broken as described previously (Riedel et al., 2012). Six different mixtures of fructose and canola oil, all yielding a final carbon content of 10 g L⁻¹ and a C/N ratio of 22 (g g⁻¹) were used to produce sufficient amounts of P(HB-co-HHx) copolymers with varying HHx monomer content for polymer characterization.

For quantification, the entire 3 mL culture was taken from the deep-well-plates at the end of the cultivations, while for cultivations in shake flasks and bioreactors, 5 mL samples were taken at each sampling point. For cell dry weight (CDW) determination the samples were collected in pre-weighed 15-mL tubes and centrifuged for 15 min and 4°C at 8,000 × g. The pellets were washed with 3.5 mL cold deionized (DI) water and 1.5 mL cold hexane to remove residual oil and then resuspended again in 2 mL DI water, frozen at −80°C and dried for 48 h by lyophilization (Gamma 1–20, Martin Christ Gefriertrocknungsanlagen GmbH, Germany).

The PHA content and composition of the dried cells were determined using a methanolysis protocol and gas chromatography as previously described (Bartels et al., 2020). Residual cell dry weight (RCDW) was defined as CDW minus PHA content in g L⁻¹.

During bioreactor cultivations, fructose and NH₃ concentrations were measured from the supernatant of centrifuged samples. For fructose measurement, 750 µL of supernatant was washed twice by mixing with 750 µL of cold hexane in a 2-mL Eppendorf tube and shaking for 15 min in an overhead shaker (Rotator Drive STR4, StuartScientific, Cole-Parmer, Germany). Centrifugation was performed at 8,000 × g for 2 min and the bottom phase was collected. The washed supernatant was then filtered through an 0.2 µm PES syringe filter and fructose concentration determined via HPLC-RID. Chromatography was performed with 20 µL injection volume at 80°C for 62 min on an Agilent Hi-Plex Ca column. The eluent was DI H₂O with an 0.6 mL min⁻¹ flux. Unfiltered and unwashed supernatant was measured using the Cedex Bio HT Analyzer (Cedex Bio HT Analyzer, Roche Diagnostics International AG, Switzerland) to determine NH₃ consumption.

Molecular weight distribution of the PHA polymers was determined by size exclusion chromatography (SEC) from CDW samples as described previously (Thiele et al., 2021).

The choice of a suitable C/N ratio is crucial for matching the monomer composition of P(HB-co-HHx) to the substrate mixture supplied as, with excess carbon sources, only the preferred substrate will be used so that the effects of different mixing ratios will be negligible. Different C/N ratios, in the range of 5–90 (g g⁻¹) were investigated in deep-well-plates with a working volume of 3 mL and the results are shown in Figure 1.

Increasing final biomass and PHA content values were observed with increasing C/N ratios from 5 to 22 (Figures 1A, B). For fructose, no increase in these values was observed when the C/N ratio was further increased above 22 (Figure 1C), indicating that the added fructose was not consumed. In the case of canola oil (Figure 1D), a stagnation of the achieved biomass was only observed above a C/N ratio of 68 (g g⁻¹). PHA values showed, that cells growing on fructose accumulated only up to about 50 wt% of PHA, while cells growing on canola oil were able to accumulate up to 80 wt% of PHA, which explains why, with canola oil, more carbon source was consumed and final biomass values increased with increasing C/N ratios above 22 (g g⁻¹). As expected, the final CDW values were about four times higher in the first series of experiments (Figures 1A, B), where the added urea concentration was four times higher than in the second series of experiments (Figures 1C, D), with the same C/N ratio.

For the following experiments with fructose and canola oil mixtures, a C/N ratio of 22 (g g⁻¹) was chosen, as both carbon sources were to be completely consumed in order to determine the effects of different mixture ratios on the HHx content.

Seven fructose and canola oil mixtures, all with a total carbon content of 5 g L⁻¹, were used to test the effect of varying substrate ratios on the molar compositions of P(HB-co-HHx) in 24-deep-well-plates with a working volume of 3-mL (see Figure 2). Depending on the amount of canola oil, the HHx content increased linearly from 0 mol% when no oleaginous feedstock was available to 16 mol% with canola oil as the sole carbon source (see Supplementary Figure S2 for linear correlation). Comparable final biomass values over 4 g L^-1 with 65–90 wt% PHA were observed with all mixtures except when fructose was supplied as the sole carbon source. With a maximum accumulation of 57 wt% PHA, the final biomass values of pure fructose cultures showed the lowest CDW accumulation.

Four of the previously tested mixtures, namely, 5:1, 1:1, 0.5:1, and 0:1 [carbon ratio fructose to canola oil (g g⁻¹)], were scaled up, following the same cultivation strategy (C/N ratio and total carbon content), to 100 mL working volume in shake flask cultivations. When utilizing the same final carbon content as in deep-well-plates cultivations (5 g L⁻¹), comparable biomass values were obtained (see Supplementary Figure S3). Nevertheless, in the three mixtures with the higher canola oil contents, a decrease in the PHA content between 48–72 h was observed showing also an increased HHx content at the end of the cultivation in comparison with the previous deep-well-plate experiments. To avoid premature degradation of the PHA granules and to achieve higher final yields, it was decided to double the used carbon content to 10 g L⁻¹ while maintaining the C/N ratio of 22 (g g⁻¹) (see Figure 3). With this approach, comparable results were achieved in terms of PHA content and composition as with the deep-well-plate cultivations, while the final biomass yield was approximately doubled (see Table 1).

To demonstrate the scalability of our approach, 1-L bioreactor cultivations were carried out transferring the cultivation strategy from shake-flask cultivations (constant C/N ratio and total carbon content). Again, a linear correlation was found between the amount of canola oil and fructose used and the HHx content obtained (see Supplementary Figure S2). With final biomass values between 9–13 g L⁻¹ and PHA contents between 60–88 wt%, the final yields were slightly better than in the previous shake flask cultures (Figure 4). The data showed that fructose was not consumed at least during the first 24 h of cultivation, suggesting that canola oil was consumed first, and the cells later switched to fructose as the non-preferred carbon source (Figure 5). In the three cultures with the highest fructose ratios, residual fructose concentrations were measured between 3–5 g L^-1, indicating that the cells would have reached slightly higher PHA contents with slightly lower HHx molar contents if the cultivations had been operated for a longer period. In these three cultures nitrogen depletion also set in later than in cultures with higher canola oil ratios (see Supplementary Figure S4).

When comparing the final CDW values at all scales investigated, a slight increase in biomass yields was observed when moving from deep-well-plates to shake flasks and from shake flasks to bioreactor cultivations (see Table 1). Accordingly, when twice the total amount of carbon was added in the second shake flask run, the final CDWs approximately doubled. Within each scale, biomass values were comparable throughout all mixtures, whereby the values were slightly higher with higher canola oil contents. PHA values were comparable at all scales, with a general increase in polymer accumulation (from 60%–90%) measured with increasing canola oil contents. A linear correlation between the canola oil content in each mixture and the final HHx molar content was observed at all scales (see Supplementary Figure S2) showing that it was possible to tailor P(HB-co-HHx) by only applying different fructose and canola oil mixtures. When higher polymer yields were obtained (shake flask with 10 g L⁻¹ CC vs. shake flask with 5 g L⁻¹ CC and bioreactor vs. shake flask with 10 g L⁻¹ CC), slightly lower molar HHx contents were observed.

Molecular weight characteristics of the produced copolymers after 72 h of cultivation in shake flask and bioreactor scale were determined by size exclusion chromatography. A decrease of the molecular weight with increasing scale was noticeable, whereas no marked decrease in M_w was observed with increasing HHx contents (Table 2). Only the shake flask cultivations containing 5 g L⁻¹ carbon as substrate showed a slight decrease in the M_w of about 10% with increasing HHx molar fraction. When the final carbon content was doubled from 5–10 g L⁻¹ in shake flask cultivations, a clear decrease in the final M_w, up 35% was observed. In bioreactor cultivations, where higher PHA contents per CDW were reached compared to shake flask cultivations, the lowest molecular weights around 3.5 × 10⁵ Da were obtained. The polydispersity index Đ, around 2.5, was comparable along the scales. During bioreactor cultivations no clear decrease in the M_w was observed over time (see Supplementary Table S1).