Fermentation

In all batch and fed-batch fermentations where cadmium sulphate was added to induce the production of CdS crystallites, it was added as a presterilised concentrated 3 ml solution. The final concentration of cadmium sulphate in the fermentation medium was 1 mM.

2.3.3.1 Batch fermentation

Fermentations were performed at a working volume of 4 L using the 7 L capacity fermenter described in section 2.2.2. The sterilization of fermentation medium (prepared as in section 2.1.3.2.1) containing 0.1 mL.L * polyethylene glycol was carried out in situ at 1 2 rC for 20 minutes with a steamline pressure of 2 bar. Glucose was sterilised separately and added aseptically to the fermenter when cool. Shake flask cultures (25 ml), prepared as in section 2.3.2.2 were used to inoculate the 7 L fermenter, this corresponded to an inoculum size of 0.625% v/v.

All ancillaries of the 7 L fermenter, including air inlet filter line, alkali and antifoam reservoirs and sampling line were separately sterilised in an autoclave at 121°C for 20 minutes. Inlet air was filtered through a 0.2 pm Pall depth - filter and outlet gas filtered through a 0.2 pm Pall cartridge filter (Pall Process Filtration Ltd., U.K.), which was sterilised as part of the fermenter sterilisation.

Fermentation data analysis was carried out as in section 2.2.3, with medium pH being automatically maintained at pH 5.6 with 2 M ammonium hydroxide (N H /O H ). Sterile polypropylene glycol (sterilised at 1 2 rC for 20 minutes) was manually added as an antifoaming agent if required during fermentation.

Samples of culture broth were aseptically removed from the fermenter through a sample line to determine cell biomass by dry weight and optical density and glucose, ethanol, and cadmium concentration of the medium. Intracellular inorganic sulphide concentrations were also determined for the cell content of the sample (see section 2.3.6)

2.3.3.2 Chemostat fermentation

Fermentations were performed at a working volume of 1.5 L using the 2 L capacity fermenter described in section 2.2.2. The sterilization of fermentation medium (prepared as in section 2.1.3.2.1) containing 0.1 mL.L ‘ polyethylene glycol was carried out in situ at 121°C for 20 minutes with a steamline pressure of 2 bar. Glucose was sterilised

separately and added aseptically to the fermenter when cool. Shake flask cultures (10 ml) prepared as in section 23.2.2 were used to inoculate the 2 L fermenter, this corresponded to an inoculum size of 1.49% v/v. All ancillaries of the 2 L fermenter, including air inlet filter line, alkali and antifoam reservoirs and sampling line were separately sterilised in an autoclave at 1 2 rC for 20 minutes. Inlet air was filtered through a 0.2 pm Pall depth - filter and outlet gas filtered through a 0.2 pm Pall cartridge filter (Pall Process Filtration Ltd., U.K.), which was sterilised as part of the fermenter sterilisation.

Fermentation data analysis was carried out as in section 2.2.3, with medium pH being automatically maintained at pH 5.6 with 2 M ammonium hydroxide (N H /O H ). Sterile polypropylene glycol (sterilised at 12TC for 20 minutes) was manually added as an antifoaming agent if required during fermentation.

Samples of culture broth were aseptically removed from the fermenter through a sample line to determine cell biomass by dry weight and optical density, and glucose, and ethanol concentrations in the medium (see section 2.3.6).

The chemostat fermentations were initially grown as batch fermentations to produce biomass and then the chemostat system started. Sterile fermentation medium from a 20 L bell jar was pumped into the chemostat to give the desired dilution rate using a Watson- Marlow pump (Watson-Marlow, Falmouth, U.K.). But this time the fermentation medium (section 2.1.3.2.1) contained 1% (v/v) glucose.

In a chemostat system the value of F/V=D is known as the dilution rate, where:

F = Medium flow rate (L.h^) V = Culture volume (L)

At a steady state, i.e. when there is no change in biomass over time, D = p where: p = Specific growth rate (h^)

2.3.3.3 Fed batch fermentation

A fed-batch fermentation vessel 7 L capacity was used (see section 2.2.2). The sterilization of fermentation medium (prepared as in section 2.1.3.2.2) containing 0.1 mL.L'* polyethylene glycol was carried out. Shake flask cultures (400 ml) obtained as a 2 L shake flask fermentation from section 2.3.2.1 were used to inoculate the 7 L fermenter, this corresponded to an inoculum size of 10% v/v.

All ancillaries of the 7 L fermenter, including air inlet filter line, alkali and antifoam reservoirs and sampling line were separately sterilised in an autoclave at 1 2 rC for 20 minutes. Inlet air was filtered through a 0.2 pm Pall depth-filter and outlet gas filtered through a 0. 2pm Pall cartridge filter (Pall Process Filtration Ltd., U.K.), which was sterilised as part of the fermenter sterilisation.

Fermentation data analysis was carried out as in section 2.2.3, with medium pH being automatically maintained at pH 5.6 with 2 M ammonium hydroxide (N H /O H ). Sterile polypropylene glycol (sterilised at 12 TC for 20 minutes) was manually added as an antifoaming agent if required during fermentation.

Samples of culture broth were aseptically removed from the fermenter through a sample line to determine cell biomass by dry weight and optical density, glucose, ethanol, and cadmium concentration of the medium. Intracellular inorganic sulphide concentrations were also determined for the cell content of the sample (see section 2.3.6.)

The Lab View graphical programming software provided the control algorithm which calculated the exponential glucose feed rate required for a predetermined growth protocol. For the algorithm to control a specified growth protocol, numerical inputs were required: a) Initial biomass estimation (g/L)

b) Culture volume (L) c) Feed concentration (g/L) d) Pump calibration data e) Growth yield on substrate

Growth of an organism at a constant specific growth rate using a fed-batch system requires that nutrient feed rate be pumped into the fed-batch fermenter at an exponential rate. Open loop control when the substrate feed rate is exponential may still allow substrate build up, especially if the original biomass estimate is incorrect. In the case of

S.pombe this would lead to glucose repression and ethanol production. However, the incorporation of closed loop control (an RQ feedback mechanism) helps to combat this by leading to reduction of feed rates etc. The RQ feedback mechanism is discussed in section 3.5.3.1.4.

For a computer controlled (closed loop) system the following equations are required in the operation of the control algorithm:

F = p/Y,.x(t).V(t)

x(t) = x(t-At).e^‘

V(t) = V(t-At)+F/Sf.At

F = substrate feed rate (g.h ‘)

p = specific growth rate required (h ‘)

x(t) = dry biomass concentration (g.L'^) at time t

V = culture volume (L). Here only the addition of substrate was used to modify the calculated volume

Sf = concentration of fed substrate (g.L'^)

Yj = growth yield on substrate (g biomass.g substrate'^)

R = scaling factor (used to adjust substrate feed rates to achieve a desired RQ)

Using the above equations, the substrate solution was fed under the control of the Lab View algorithms by one of two Watson and Marlow peristaltic pumps (Watson-Marlow, Falmouth, U.K.) calibrated for 0-2 rpm and 2-32 rpm. Using either of the pumps, all required feed rates were possible. The required feed rate is sent from Lab View at pre-set time intervals to the pump controller (determines which pump is to be used) as ASCII commands which then controls pump rate by analogue voltage signal.

The fermenter was inoculated and the feed pump rate started for a specific growth rate of O.lh \ The pump feed rate would increase exponentially to maintain this specific growth rate.