Biostat b plus

Fed-batch bioreactor fermentation was conducted in a 5 L bioreactor Biostat B Plus (Sartorius, Germany) with a working volume of 1 L. The optimized medium was inoculated with 10% (v/v) of inoculum. The process conditions were as follows: temperature, 30 °C; agitation, 80 rpm; pH, 7.0. Glycerol supply was coupled to the delivery of KOH—a single solution composed of 20% KOH and 41% glycerol was used for controlling pH. As the fermentation progressed, the automatic pH regulation system controlled the delivery of both the neutralizing agent and glycerol. Thus, the carbon source was simultaneously delivered at a rate synchronized with the rate of acid generation by the bacteria.

M. thermolithotrophicus was continuously grown in a 10-liter fermenter (BIOSTAT B plus; Sartorius, Göttingen, Germany) under diazotrophic conditions with either 10 mM Na₂SO₃ or Na₂SO₄ as sulfur source instead of Na₂S. The final culture volumes were 7 liters for the culture grown with Na₂SO₃ and 6 liters for the culture grown with Na₂SO₄. The culture was continuously sparged with H₂:CO₂ (80%:20%) and N₂ in the ratio of 1:1 and stirred with the speed of 500 rpm, at 65°C. As an inoculum, cultures cultivated in the same media were used in a ratio of 1:10.

To optimize the SSF process according to the RSM, the ranges of process parameters were selected: substrate content 5%–7% w/v, dose of (Flashzyme:Celluclast 1.5L) enzymes 10–30 FPU·g⁻¹ of solid. The SSF process was carried out in bioreactor Biostat B Plus (Sartorius, Göttingen, Germany) in 2 L vessel equipped with pH, temperature, stirring, and foaming controls. The temperature was maintained at 37 °C and stirring at 900 rpm, while pH was controlled at 4.8 by adding 1 M NaOH or 1 M HCl. In the fermentation process, non-hydrated freeze-dried distillery yeast S. cerevisiae (Ethanol Red, Lesaffre, France) at a dose of 1 g·L⁻¹ was used, which corresponded to cell concentration after inoculation of about 1 × 10⁷ cfu/mL. The duration of ethanol fermentation was 96 h. All experiments were performed in triplicate.

Mannitol, arabitol and erythritol production was also analyzed in bioreactor cultures in medium containing: carbon source (GLY1, GLY2)—100 g; (NH₄)₂SO₄—2.7 g; KH₂PO₄—0.2 g; MgSO₄·7H₂O—1 g; yeast extract—1.6 g; in 1 dm³ of tap water. The precultures were grown in 100 cm³ YP medium with 2% of the corresponding glycerol for 72 h in 300 cm³ flasks on a rotary shaker at 28 °C and 140 rpm. Precultures were harvested, the biomass was washed twice with sterile distilled water and the initial optical density (OD₆₀₀) in the bioreactor was adjusted to 0.5. All bioreactor cultures were performed in a 5 dm³ stirred-tank reactor (BIOSTATB-PLUS, Sartorius, Germany) with the working volume of 2.0 dm³ at 28 °C (22 °C for YAAL and YAKE). Aeration and stirring rates were set at 0.8 vvm and 800 min⁻¹, respectively. pH 3 was maintained automatically by additions of 20% (w/v) NaOH solution. All cultures were run until complete exhaustion of the carbon source. A bioreactor with the appropriate medium was sterilized in an autoclave at 121 °C for 20 min. All cultures were conducted in three biological replicates and standard deviations were calculated.

According to the results of the antimicrobial activity assays, H1, H2, and H3 were chosen to be expressed in the 5-l fermentor. A single colony of H1, H2, H3 was incubated in shaking flasks with 10 ml YPD medium at 30 °C (250 rpm). Overnight cultures were inoculated into 200 ml YPD medium and cultivated at 30 °C (250 rpm) to an OD₆₀₀ of 5.0 and then transferred into a 5-l fermentor (BIOSTAT®B plus, Sartorius Stedim Biotech) containing 2 l basal salts medium (50 g/l NH₄H₂PO₄, 20 g/l K₂SO₄, 15 g/l MgSO₄·7H₂O, 6 g/l KH₂PO₄, 0.4 g/l CaSO₄, 1.5 g/l KOH, 45 g/l glucose, and 4.8‰ PMT1). The pH was controlled at 5.5 using H₃PO₄ and NH₄OH, and the temperature was maintained at 29 °C. When the glucose was exhausted, methanol was supplied from 1 to 7 ml/l/h during the first 6 h. Then methanol was supplied to maintain a relative dissolved oxygen (DO) content between 20 and 40% under the speed of 6–8 ml/l/h (Bai et al. 2010 (link)). The fermentation liquid was collected every 24 h to quantify the cell wet weight and the total protein level which was assayed by a Bradford protein assay kit (Tiangen Biotech, Beijing, China). The expression of H1, H2, and H3 was determined by Tricine-SDS-PAGE. The supernatant was purified in the same way as the 1-l shake flasks.

The fermentation studies were conducted in 5-l bioreactors (BIOSTAT®B plus, Sartorius Stedim Biotech). A single colony was incubated in YPD medium at 30°C. An overnight culture was inoculated into 200 ml of fresh YPD medium and cultivated at 29°C (250 rpm) to an OD600 of 4–6. Next, 10% (v/v) inoculum was inoculated into a 5-L fermenter containing 2.0 L modified Med-1 medium (42.9 g/L KH₂PO₄, 14.3 g/l K₂SO₄, 11.7 g/l MgSO₄ 7H₂O, 5 g/l (NH) ₂SO₄, 1.92 g/l citric acid anhydrous, 0.6 g/l CaSO₄, 20.0 g/L yeast extract, 40.0 g/L peptone, 40.0 g/L glucose). The pH value was set to 6.0 at the glucose batch phase and changed to 5.0, 5.5, 6.0, 6.5, or 7.0 in the glucose fed-batch phase using NH₄OH and H₃PO₄. The glucose feeding rate was 6 mL/L/h during the fed-batch culture phase. The dissolved oxygen (DO) level was maintained between 25 and 40% by adjusting the agitation, aeration, and rates. Foaming was controlled via the addition of antifoam. Cultivation broth samples were taken every 12 h for cell wet weight, extracellular protein level and antimicrobial activity analyses after centrifugation at 12,000 rpm for 10 min. The total protein levels, rMP1102 yield and antimicrobial activities were tested.