PROPIEDADES MECÁNICAS 1 Determinación de la dureza

This section outlines the equipment and instrumentation used for fermentation, on-line data acquisition, cell disruption, HPLC analysis and also the computing equipment used to process data.

2.2.1 F erm en ter equipm ent and in stru m en tation

The fermenter used was a LH 2000, 7L glass fermenter, (LH Fermentation, Reading, England) with a working volume of 5L. The fermenter has three equally spaced six bladed Rushton disc turbines and four tank baffles. Fig.2.2 shows a schematic diagram of the fermenter including the top plate with port allocation.

The following components of the fermenter are identified: a: Top Plate

b: Sample Line

c: One of the three Rushton turbines d: Backing Flange

e: Leg

f: Nut and Washer g: Impeller Shaft

h: Air Sparger (positioned to sparge air below the lowest turbine on the impeller shaft) i: Heat exchanger

j: Bottom Plate k: Nut and Washer

The following ports are identified on the top plate for sampling, inoculation, air input, acid, alkaline and antifoam additions, pH and DOT probes:

1. Phosphoric Acid 2. pH Probe 3. DOT Probe

4. Sterile Air into the Fermenter

5. Three way needle used to expand a single port to three independent ports for introducing (antifoam, 2M NaOH, and sterile medium into the fermenter)

6. Sample port

7. Exit gas into the mass spectrometer through the condenser 8. Inoculum flask

o

Figure 2.2 Diagram of the LH 7L fermenter used for all fermentations in this work. The working volume of 5L was chosen for the fermentations.

The fermenter was instrumented with a TCS 6358, eight loop PID controller (Turnbull Control System Ltd., Worthing, UK.) for converting the analog to digital signals. The TCS unit was communicating with the data monitoring and control system (section 2.2.3) via a serial line.

The pH was measured by a steam sterilisable Ingold 465 (Ingold, Urdorf, Switzerland) probe. The probe was directly connected to the controller. At a given set point, the controller activates the peristaltic pumps for further addition of acid or alkali from the respective reservoirs to the fermenter.

Dissolved oxygen transfer was measured using an Ingold, steam sterilisable polographic DOT probe (Ingold, Urdorf, Switzerland).

Temperature was measured by a resistance thermometer and controlled by a 500W cartridge heater or the cooling water supplied through the heat exchanger for consecutive cooling or heating of the system.

Temperature and pH of the broth, inlet gas into the fermenter and stirrer speed were monitored and controlled under set points (Table 2.5), other variables such as dissolved oxygen tension (DOT), and load cells measuring alkali and acid addition to the broth were measured variables which were only monitored. All these variables were then transmitted to the data logging system (Bio-I or RT-DAS ).

2 .2 .2 E xit gas analysis

The relative amounts of N2, 0 2 , CO2 and Ar in each inlet and outlet gas streams were measured by a 32 channel VG MM8-80, magnetic sector mass spectrometer (VG Ltd., Middlewich, England). The frequency at which analysis was available depended on the number of users on the system at the time. Analysis were usually available every three minutes. Calibration of the mass spectrometer was automatically done every twenty four hours.

Measurements from the mass spectrometer were transfered to an IBM PC which were in turn transferred to the available data logging system (RT-DAS).

2.2.3 D ata acq u isition and p rocessing

Bio-i (BCS Ltd., Maidenhead, Kent, England) and RT-DAS (Real Time Data Acquisition System, Surrey), are the two data monitoring and control systems used for collection, storage and graphical presentation of the on-line data from the TCS unit and the mass spectrometer. The on-line measurements were recorded every three minutes.

On-line data could then be presented numerically or graphically at any time during the fermentation operation. All measurements were then saved on floppy disks to be transferred into a Macintosh computer and/or the Sun workstation for later analysis and correlations of the on-line data with the off-line measurements from fermentation samples.

2.2.4 M icron L ab 40 hom ogeniser

Samples were disrupted within 2 minutes of being removed from the fermenter. A Gaulin Micron Lab 40 homogeniser (APV, Mecklenburger Strasse 223, D-2400 Lubeck 14, Germany) was used to disrupt the cells at 1200 bar by two passes (experimentation showed that these conditions gave maximal protein release irrespective of growth phase). The operating pressure can be set in the range of 100 to 1600 bar by means of a potentiometer which is on the digital display. The sample chamber is maintained at 0°C to minimise sample heating during cell disruption. Adequate cooling to the cell suspension was provided by turning the glycol on to the cooling coils one hour prior to using the homogeniser. The product cylinder was filled up with 40 mL of the cell suspension (section 2.1.6). The cell suspension level must not be below 6mm from the top of the product cylinder to prevent the penetration of solid materials, impurities and air inclusions. Having all parts including the valve housing and the cover centred, the homogenisation step could be started. The process was automated and was started by depressing the Start­ up Automatic operation button. The valve housing and the product cylinder were thoroughly rinsed with deionised water before use. The homogenate was then used to analyse the total protein and the intracellular enzymes of the off-line samples.

2 .2 .5 H igh perform ance liquid ch rom atograp h y (H PL C )

A HPLC system was used for quantitative analysis of ethanol, glucose and pyruvate. Using a LDC Milton Roy isocratic HPLC pump (model Constametric III), the sample was pumped through the autosampler (LDC Milton Roy autosampler, model LC 241) into the column to the UV detector (LDC Milton Roy ultraviolet detector, model spectromonitor III) and then the RI detector (LDC Milton Roy refractive index detector, model RefractoMonitor IV) before discharge to the waste reservoir.

The column used for initial analysis was a 15 cm long fermentation monitoring column (Biorad), protected by a Biorad cation H+ guard column. The mobile phase was 0.004 M H2SO4 (HPLC grade H2SO4 obtained from Fisons). At a flow rate of 0.65 mL.m in'l, the retention times of ethanol, glucose, and pyruvate were 10.6 minutes, 5 minutes, and 5.5 minutes respectively. The operating conditions for both columns were the same. The column was kept in a water bath at 50®C and UV detection was at 210 nm for the pyruvate analysis. Refractive index was used for detection of glucose and ethanol. For a number of fermentations, the observation was made that better resolution was required for ethanol assays (Chapter 3). Therefore an Aminex HPX-87H 30cm column was used. This column gave better resolution between the components, however a longer time was required for analysis of the samples (at a flow rate of 0.7 mL.min"^, retention time was 17 minutes for ethanol and 8 minutes for glucose). A range of known concentrations of ethanol, glucose and pyruvate were used for calibration. The calibration curve for all analytes were linear over the range tested (between 0.5-30 g/L glucose, 0.25-15 g/L ethanol, and 0.033-2 g/L for pyruvate).

The HPLC analysis software used was Perkin Elmer Nelson 2100 which was running on a personal computer, IBM model 55 SX.