Lysine Production From Methanol in a 20 Liter Fermenter

Lysine Production From Methanol In A 20L Fermenter

Shown in front of the technician is a 20 liter Chemap fermenter with a minimal salts medium (MS) growing one of the Bacillus methanolicus homoserine auxotrophic mutants MGA3 or NOA3, producing lysine from methanol as sole carbon source.

This is a Fed-batch cultivation of cells carried out at 50 oC in a 20 L Chemap fermenter with a working volume of 14 liters. The agitation rate was 382 cm/s agitator tip speed, an aeration rate of 5.5 liters/min; the pH was maintained at 7.1 by automatic addition of 8 N ammonium hydroxide. Phosphate, magnesium and calcium levels were maintained by automatically feeding a solution of 100:10:1 phosphate-magnesium-calcium 1.0 M KH2PO4, 0.1 M MgCl2, 0.01 M CaCl2. Feeding of this nutrient mix was carried out by connecting the pump to the pH controller, so that the nutrient mix would be added whenever ammonium hydroxide was added to adjust pH. The pump speed was adjusted so that the rate of addition of the nutrient mix was twice the rate of ammonium hydroxide addition to maintain the desired optimal ratio of nitrogen, phosphate, magnesium, and calcium as determined by growth yield experiments previously performed in shake flasks.

Dissolved oxygen was monitored by using a galvanic probe and was maintained at 30% of total saturation by oxygen-enriched aeration. Feeding of oxygen was monitored and controlled by a mass flow controller (Sierra Instruments Inc.) interfaced with a proportional controller (LFE Corp). Foaming was controlled with a liquid level controller (Cole-Parmer) by the automatic addition of a silicon-based anti-foam agent, SAG-471 (Union Carbide).

Inlet and headspace gasses (carbon dioxide, oxygen, nitrogen, argon, methanol, ammonia, and water) were monitored by a Questor mass spectrometer (ABB Extrel Corp.) interfaced to an IBM-AT computer. The inlet and headspace gasses were alternately sampled for 2 min (sample rate, 0.166 s-1) after a 30 s delay to allow the line and ionization chamber to clear. The collected data were averaged every 5 min, stored on the computer, and used in calculations of oxygen uptake rates (OUR) and carbon dioxide evolution rates (CER)(see equations below).

Methanol levels in the reactor were continuously monitored by using an in situ methanol sensor consisting of a silicone tubing (0.062-in. inside diameter by 0.095-in. outside diameter) probe connected to the flame ionization detector of a gas chromatograph (GC). One meter of silicone tubing was used in the probe with an air flow rate of 60 ml/min through the probe. The tubing connecting the probe to the GC was heated to 50 o with self regulating heating tape, and the GC detector temperature was 250 oC. The 0-5 V integrator output signal from the flame ionization detector of the GC was used to monitor and to operate the methanol feed pump (Watsom Marlow) automatically by use of a proportional controller (LFE Corp.) to maintain a methanol concentration of 100 mM in the reactor. A load cell platform was used to determine the amount of methanol that was fed to the reactor. The methanol also contained the required trace metals in the following concentrations: 1.09 g FeSO4 . 7H2O, 0.39 g MnCl2 . 4H2O, 22 mg ZnSO4 . 7H2O, 19 mg CoCl2 . 6H2O, 19 mg Na2MoO4, 19 mg CuSO4 . 5H2O per liter (14, 98, 112, 136, 203, 216, 230).

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Fed-Batch Cultivation of MGA3

Fed-Batch Cultivation of MGA3

Fed-batch fermentations, using an MS medium, resulted in cell densities as high as 50 g/l in the 20-liter reactor. The cells grew exponentially to 50 g of cell dry weight (CDW) per liter with a growth rate of 0.48 h-1 (Fig. 1), and the yield of biomass from methanol remained unchanged (0.48 g of CDW per g of methanol) from that determined by prior shake flask experiments. The methanol level in the reactor remained relatively constant throughout the fermentation as determined by both the methanol probe and the mass spectrometer. The OUR and CER rates indicated that growth was exponential throughout the first 14 hours of the fermentation (Fig. 1) (68, 98, 203, 216).

NOTE: OUR, CER, and RQ can be determined continuously, on-line with CO2 AND O2 analyzers and computer using the equations :

OUR (mmoles/l.h) = FN / VL (( PO[ in ] / Pt - PO[ in ] - PW[ in ] - PC[ in ] ) _ ( PO[out] / Pt - PO[out] - PW[out] - PC[out] ))

CER (mmoles/l.h) = FN / VL (( PC[out] / Pt - PC[out] - PW[out] - PO[out] ) _ ( PC[ in ] / Pt - PC[ in ] - PW[ in ] - PO[ in ] ))


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Craig Bremmon
CEBTech Services
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