Bioreactor Cultivation Conditions

For fed-batch cultivations 1-L Multifors parallel benchtop bioreactors (Infors AG, Switzerland) were used. The cultivation temperature was kept constant at 30°C and the pH maintained at 6.8 ± 0.1 through controlled addition of 1 M H₃PO₄ and 2 M NaOH (or 25% (v v^–1) ammonia for pH-controlled feeding). Stirring was performed using two six-blade Rushton impellers. 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 dissolved oxygen (DO) values from dropping below 40%. Foam was mechanically broken as described previously (Riedel et al., 2012). Additionally, silicon oil was added as antifoam when needed (maximum total amount added of 1 mL). The fed-batch cultivations were always performed in biological duplicates (two independent bioreactor cultivations) and consisted of two stages: a biomass accumulation stage and a PHA production stage.

The biomass accumulation stage was conducted as indicated below with varying concentrations of nitrogen source in the feeding solution: without ammonium chloride, with 1% (w v^–1) ammonium chloride and with 2% (w v^–1) ammonium chloride. The cultivations were run for 48–50 h and the accurate concentration for ensuring availability of nitrogen throughout the complete biomass stage was tested. In the cultivations run afterward, ammonium chloride was replaced by corresponding concentrations or urea.

Initial batch phase until depletion of the 1% (w v^–1) fructose present in the bioreactor followed by the automated start (triggered by sudden DO increase) of exponential feeding with 50% (w v^–1) fructose and 0.56% (w v^–1) urea at the specific growth rate μ_set according to

The initial feed rate (L h^–1) was calculated according to

where Yxs the biomass/substrate yield (calculated from the batch phase), S_i the concentration of the carbon source in the feeding solution, and X₀ and V₀ the biomass concentration (calculated from a correlation between previous OD₅₈₃ and CDW measured values) and bioreactor liquid volume at the end of the batch phase, respectively. During this stage the pH was controlled through addition of 25% ammonia in order to avoid nitrogen limitation. Feeding was performed until the measured OD₅₈₃ exceeded 100 which marked the beginning of the second stage.

PHA accumulation was triggered by nitrogen limitation. Therefore, the pH-control was switched from ammonia to 2 M NaOH. During this stage, a total amount of 135 g L^–1 rapeseed oil were fed to the culture in a constant manner during the first 12 h. The culture was grown for further 32–36 h until the complete oil present in the bioreactor was consumed and the cells had achieved the highest PHA content.

Three complete cycles of biomass accumulation and PHA production were run operating the biomass accumulation bioreactor in a “drain and fill” modus. The biomass accumulation stage was followed as indicated above until OD₅₈₃ >100 and then 90% of the broth was transferred to a second bioreactor for PHA accumulation. To this end external periplasmic pumps were used. The main bioreactor was then refilled with sterile fresh media to a starting volume of 0.5 L and the biomass accumulation stage was repeated. Taking advantage of the high-cell-density achieved during the first stage, the second stage was run without previous sterilization of the bioreactors.