2.2. PHB production by bioreactor fermentations

The production of PHB by C. necator ATCC 17697 was carried out by fermentation in a bioreactor using both batch and fed-batch strategies. Fermentations were performed in a 6 l stirred-tank bioreactor (BioFlo 110, New Brunswick Scientific; Edison, NJ), interfaced with Biocommand Bioprocessing software (New Brunswick Scientific) for parameter control and data acquisition. To obtain the bacterial inoculum, a volume of 200 ml of culture medium in a 1 l Erlenmeyer flask was inoculated with single colonies of C. necator ATCC 17697 and incubated at 30 ± 1 °C and 150 rpm for 24 h. This culture was employed to inoculate 2 l of culture medium contained in the bioreactor. The size of the inoculum (10%) is twice that used in the Erlenmeyer flask scale. The pH was measured in situ using a pH electrode (Mettler-Toledo GmbH, Germany). Dissolved oxygen concentration (dO₂, % saturation) was measured with a polarographic probe (InPro6110/320, Mettler-Toledo GmbH) and modulated by controlling the agitation speed and adding filter-sterilized air (0.22 μm). Temperature was maintained at 30 ± 1 °C and foam formation was avoided by the addition of 0.3 % (v/v) antifoam 289 (Sigma-Aldrich; St. Louis, MO). Batch and fed-batch procedures are described below.

At the beginning of the batch fermentation, the dO₂ was set at 100 % saturation. When the dO₂ reached to 20 %, the dO2 regulation was started using a cascade control strategy consisting of varying the agitation speed (800–1000 rpm) and air flow (1–2 l/min) to maintain dO2 saturation between 20 - 40 %.

The pH value was maintained at 7.0 ± 0.2 by the addition of 1 N HCl and 2 N NaOH. Cultures were withdrawn periodically during incubation for the quantification of fructose and ammonium concentration, cell biomass and PHB.

Two fed-batch fermentations were performed applying a three-stage procedure:

Stage 1: Batch culture: Cell growth phase.

Stage 2: Fed-batch culture with carbon and nitrogen supply: Cell growth and PHB accumulation phase.

Stage 3: Fed-batch culture without nitrogen supply: PHB accumulation phase.

In both fermentations, 1N HCl and 25 % (v/v) NH₄OH were used to regulate the pH at 7.0 ± 0.2 from the beginning. The NH₄OH solution also served as a source of nitrogen to avoid its limitation.

However, two fed-batch strategies with different modes of fructose feeding regulation were employed in Stage 2. In the first case (Fed-batch 1), fructose feeding was regulated by dO₂ with a cut-off level of 70 % saturation. In the second case (Fed-batch 2), an exponential fructose feeding strategy was performed applying the exponential feeding equation (Eq. (1))

where F(t) is the substrate solution exponential flow rate (l/h); t denotes the time (h); μ₀ is the specific growth rate (h⁻¹); X₀ and V₀ are the biomass concentration (g/l) and the volume (l) at the beginning of the exponential feeding, respectively; Y_X/S is the biomass yield based on substrate consumption (g_x/g_s) and S_feed is the substrate concentration in the feeding solution (g_s/l) (600 g fructose/l). A series of batch experiments at the bioreactor level were performed with C. necator ATCC 17697 to determine Y_X/S and μ₀, estimated as 0.44 g/g and 0.11 h⁻¹ respectively [53].

The Stage 3, equal for both fermentations, consisted of a fed-batch culture without nitrogen supply. For this purpose, the fructose feeding (600 g/l) was controlled by dO₂, with a cut-off level of 30–20 % saturation and the NH₄OH solution was replaced by 2 N NaOH. Furthermore, in this stage, the agitation was reduced to 800-650 rpm and the aeration to 1 l/min, to improve the PHB production.