5m naoh

P. citronellolis NRRL B-2504 and C. necator DSM 428 were co-cultured in a bioreactor with a working volume of 10 L (BioStat B-Plus, Sartorius, Germany), with a starting volume of 9.5 L. The cultivation runs were initiated by inoculating 400 mL of each culture grown in LB medium for 24 h, at 30 °C and 200 rpm, in an orbital shaker.
The temperature and the pH were controlled at 30.0 ± 0.1 °C and 7.00 ± 0.02, respectively. The pH was controlled by the automatic addition of 5 M NaOH (98%, Sigma-Aldrich, USA) or 2 M HCl (37%, Sigma-Aldrich, USA). A constant air flow rate (4 SLPM, standard liters per minute) was kept during the cultivation runs and the dissolved oxygen concentration (DO) was controlled at 30% of the air saturation by the automatic adjustment of the stirring speed (300–800 rpm). Foam formation was suppressed by the automatic addition of Antifoam A (Sigma-Aldrich, USA). The bioreactor was operated under a batch mode during 48 h. Samples (24 mL) were collected from the bioreactor for biomass, PHA, and nutrient quantification.
A bioreactor cultivation was also performed, under similar conditions, with a monoculture of C. necator for comparison.

Biomass of P. taetrolens LMG 2336 (obtained as explained in Section 2.1.1) was inoculated (at 10% (v/v)) in a 500 mL Erlenmeyer flask containing 100 mL of sterile deproteinised sweet whey, and it was incubated for 12 h at 30 °C, with an agitation of 250 rpm. After this time, the culture was again centrifuged (10,000 rpm, 10 min), and the biomass obtained was employed to inoculate the 2 L bioreactor (BioFlo 110; New Brunswick Scientific Co., Edison, NJ, USA), employing again 10% (v/v) of inoculum. The working volume of the bioreactor was 1 L with mechanical agitation. The operating parameters used were 30 °C, 350 rpm, and 1 Lpm aeration, following the conditions optimised by Alonso et al. (2012) [26 (link)] to maximize the production of LBA. Foaming was avoided by automatic addition of diluted (1:10) Y-30 emulsion (Sigma-Aldrich). The bioreactor was equipped with a pH-meter (Mettler Toledo, Greifensee, Switzerland). To maximize the LBA production, pH was left uncontrolled during the exponential growth phase and then with the automatic addition of 5M NaOH (Sigma-Aldrich) and was maintained at a value of 6.5 [26 (link)]. The bioreactor fermentation process lasted a total of 72 h.

The experiments were carried out by cultivating Pseudomonas citronellolis NRRL B-2504, Burkholderia thailandensis E264, and Pseudomonas sp. in 2 L bioreactor runs (BioStat B-Plus, Sartorius, Germany) using Medium E* supplemented with the hydrolysate (at an initial sugar concentration of 31.4 g/L). The bioreactors were inoculated with a 10% (v/v) inoculum (200 mL), prepared as described above, and operated in the batch mode. The pH and temperature were controlled at 7.0 ± 0.1 and 30 ± 0.1 °C, respectively. The pH was controlled by the automatic addition of 2 M HCl and 5 M NaOH (Merck, Darmstadt, Germany). The airflow was kept constant (2 SLPM, standard litres per minute) throughout the runs. The concentration of the dissolved oxygen (DO) decreased from full saturation at the beginning of the runs to 30% of the air saturation, the value at which it was controlled by the automatic adjustment of the stirrer speed between 200 and 2000 rpm. Antifoam A (Sigma-Aldrich) was added automatically to avoid foam formation. Samples (12 mL) were collected from the bioreactor and centrifuged (at 9000× g for 15 min) for cellular separation. The cell-free supernatant was preserved at −20 °C for the sugar, acid, and ammonium quantifications, while the cellular pellets were used for the quantification of the CDW and determination of the PHA content and composition.

For each Lactobacillus/Limosilactobacillus/Lactiplantibacillus strain, 500 μL of a culture grown overnight (10⁸ CFU/mL) was used to inoculate 10 mL of MRS broth, and strains were allowed to grow for different times (7 h, 13 h, and 24 h). E. faecalis BC101, E. faecium BC105, S. aureus SO105, and S. epidermidis SO106, preventively grown on nutrient agar plates, were inoculated into MRS broth and subcultured as described above for lactobacilli.
At the end of each incubation, the supernatant was harvested by centrifugation (10,000 × g for 10 min) (Centrisart G-16C; Sartorius, Göttingen, Germany) and filtered through a 0.22-μm-pore-size filter (polyethersulfone [PES] 0.22-μm syringe filters; VWR International, Milan, Italy) to obtain CFSs at 7 h, 13 h, and 24 h. The final pH (pH50; Vio Lab Geass, Turin, Italy) was adjusted to 6.5 with 5 M NaOH (Merck), and samples were stored at −20°C until their use.