Bioflo 110

Cultivation were performed in a 7-L bioreactor (New Brunswick, BioFlo 110, Edison, N.J., USA) according to Yu et al.^{29 (link)} with some modification. The fermentation process includes three phases: glycerol batch phase, glycerol fed-batch phase and methanol induction phase. Basal Salts Medium (BSM) was used for fed-batch fermentation, composed of 40 g/L glycerol, 22.7 g/L H₃PO₄, 0.93 g/L CaSO₄, 18.2 g/L K₂SO₄, 14.9 g/L MgSO₄ ·7H₂O, 4.13 g/L KOH, 7.0 g/L K₂HPO₄ and 4 mL/L trace solution (6 g/L CuSO₄·5H₂O, 0.08 g/L NaI, 3.0 g/L MnSO₄ · H₂O, 0.2 g/L Na₂MoO₄ ·2H₂O, 0.02 g/L H₃BO₃, 0.5 g/L CoCl₂, 20 g/L ZnCl₂, 65 g/L FeSO₄·7H₂O, 0.2 g/L biotin, and concentrated sulfuric acid, 0.5% (v/v)). The pH of the medium was adjusted and controlled at 5.5 with the addition of 28% (v/v) ammonia solution during the whole cultivation period. In the methanol induction phase, the methanol concentration was controlled at 0.1 ± 0.02% (v/v) by an on-line methanol analyzer (FC2002, Shanghai Super-xinxi, China). The dissolved oxygen concentration was kept above 20% by controlling the stirred speed between 600 and 1200 rpm at a constant airflow of 150 L/h. All reactions were performed in triplicate.

Fed-batch fermentation was carried out using a New Brunswick Bioflo 110 fermenter with a pH meter, a dissolved oxygen electrode, and a temperature electrode. M9 medium (described in “Materials and media” section, 500 mL) was inoculated with an overnight culture of strain BO63L to an initial OD₆₀₀ to 0.08, along with appropriate antibiotics and 0.001% Antifoam 204. The fermentation was initiated with the following settings: Temperature was set to 30 °C, pH was controlled at 7.4 by automatic feeding of 6 N ammonium hydroxide, the airflow rate was kept at 1.5 L/min, and the average stirring rate was maintained at 500 rpm. Gene expression was induced at OD₆₀₀ = 10 by addition of 1 mM IPTG, 0.4% arabinose, and 0.22 μM aTc (final concentration). A glucose stock solution (400 g/L glucose and 12 g/L MgSO₄) was intermittently pulsed into the bioreactor to re-supply glucose, and a yeast extract solution (20%) was intermittently added into the bioreactor. Broth samples (~ 3 mL) were collected at a series of time points to measure cell density and alcohol titer.

Pseudomonas putida KT2440 (ATCC^® 47054™) was grown in a nitrogen-limited mineral medium consisting of (per liter): 2 g Na₂HPO₄·12H₂O, 2 g KCl, 0.3 g Na₂SO₄, 1 g (NH₄)SO₄, 1 g MgSO₄·7H₂O, and 2.5 mL of trace element solution. Each liter of trace element solution contained: 20 g FeCl₃·6H₂O, 10 g CaCl₂·H₂O, 0.03 g CuSO₄·5H₂O, 0.05 g MnCl₂·4H₂O, and 0.1 g ZnSO₄·7H₂O dissolved in 0.5 N HCl. The cultures were supplemented with sodium gluconate or oleic acid as the only substrate in the same carbon concentration (3.8 g/L) in the production media. The cultivation was carried out in a 5 L working volume in a bioreactor (BioFlo 110, New Brunswick Scientific) at 30 °C with an aeration rate of 4 L/min. pH value was maintained at 7 through the modulated addition of concentrated 1 N NaOH and 1 N HCl. The dissolved oxygen was monitored during the whole cycle with O₂ electrode (InPro 6800, Mettler Toledo GmbH, Switzerland) and maintained 50% air saturation by adjusting the agitation rate from 300 to 1000 rpm automatically. A concentrate solution (Sigma-Aldrich, USA) was used as antifoam in response to the antifoam controller. Total fermentation time was 48 h. In addition, parameters such as biomass, mcl-PHA, nitrogen, and phosphorus concentrations were controlled during the experiments.

Fermentation was carried out in a 5 L bioreactor (BioFlo 110, New Brunswick Scientific, Poway, CA, USA). The reactor inoculum was a 48-h fungal pre-culture in a 500 mL medium volume and an initial inoculum of 1.95 · 10⁷ conidia/mL The growth culture medium used had the conditions that optimized biomass production. An air flow enriched with 10% oxygen was used to maximize biomass yield at a rate of 1.5 L/min [19 (link)].

For primary cultures, bacteria were directly taken from the glycerol stock into 5 ml of LB in a 10-ml glass tube and incubated for 6 h at 37°C with shaking (180 rpm). Subsequently, the primary culture was inoculated into 250 ml of LB in a 1-L flask and grown at 37°C, 180 rpm to produce “intermediate cultures”.
Fermentations were performed in 1.5 L of medium (+0.025% antifoam) in a 2-L bioreactor (BioFlo 110, New Brunswick Scientific, Edison, NJ, USA). For 3 h prior to inoculation, the medium was maintained at 37°C, agitated with 600 rpm, and streamed with 1.5 L/min pressurized air. After sampling 1 ml to screen on LB agar plates for growth of microbial contaminants, the medium was inoculated with “intermediate culture” to an initial OD₅₇₈ ≈ 0.05. During the subsequent fermentation aeration (1.5 L/min pressurized air), agitation (600 rpm), temperature (37°C), pH, and dissolved oxygen (20% O₂) were operated by the Primary Control Unit of the BioFlo and continuously documented by the BioCommand software. pH was automatically adjusted to 6.8 ± 0.1 with 0.5 M of H₃PO₄ or 1 M of NaOH.

EPS production was performed in 48 h batch cultures in a 2 L bioreactor (BioFlo 110, New Brunswick Scientific, Paris, France) containing 1 L of S1 cultivation medium with ammonium acetate appropriate concentration. A concentrated solution of glucose was sterilized separately and added to start the culture. Batches were performed under controlled conditions: temperature (30 °C), agitation 300 rpm, fixed aeration rate (0.5 v/v/m) and a regulated pH of 7.2 by automated addition of 1 M NaOH. Bacterial growth was monitored by measuring the turbidity at 620 nm of the culture diluted 5× in 20 g L⁻¹ NaCl (UV-Visible spectrophotometer UV-1800, Shimadzu, Marne-la-Vallée, France). Foaming was avoided by adding pluronic acid oil (BASF, Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France).