Estado de resultados

A co-axial cylinder rheometer (Bohlin VCR system C25, Bohlin Instruments Ltd., Glos., UK) was used to monitor apparent viscosity changes during alkaline lysis of cell suspensions derived from the fermentations (see Figure 2.1). The co-axial cylinder arrangement (see Figure 2.2) consists of a fixed bob, (diameter 25 mm) located in an outer rotating cylinder (diameter 27.5 mm). The default torque bar used was 10.15 gem except where stated otherwise. The rheometer was used as both the lysis mixing vessel and the measuring device, under controlled, laminar conditions as described by Ciccolini et al. (1998). Apparent viscosity was measured every 2 s for 10 min at a fixed shear rate (461 s'^), this having been shown to provide a suitable environment for the study of rheological profiles before (Ciccolini et al, 1998), and the temperature maintained at 20°C.

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2.4.1. Alkaline Lysis Protocol 1: Lysis Step Only

Frozen cell paste was defrosted on ice and then re-suspended in TE buffer (see Table 2.4) at a ratio of 100 mL TE buffer per 12.5 g wet cell weight of cell paste. 6 mL of the re-suspended cell paste was then transferred by pipette into the cup of the co-axial cylinder rheometer. The rheometer was then set­ up for viscosity measurements as described above, and the bob lowered into the cup. The rheometer was started and alkaline lysis initiated immediately by the addition of alkaline lysis solution (see Table 2.4). Apparent viscosity measurements were recorded every 2 s for 10 minutes, after which the lysed cells were disposed of.

2.4.2. Alkaline Lysis Protocol 2: Lysis Step Followed By Neutralisation

Frozen cell paste was defrosted and re-suspended as in protocol 1. 4 mL of the re-suspended cells were transferred to the cup of the co-axial cylinder rheometer, and the rheometer set up for measurement as described in the manual. The rheometer was started and lysis initiated immediately by the addition of 4 mL of alkaline lysis solution. Apparent viscosity was measured as before for a predetermined period, as required by the experiment, and then the reaction was neutralised by the addition of 4 mL of neutralisation buffer (see Table 2.6). Samples were then transferred to labelled universals and stored on ice for subsequent analysis or purification (see section 2.6.4).

2.4.3. Reproducibility of Bohlin Rheometer Data

Apparent viscosity may differ slightly in absolute value at an error of ± 10%.

2.4.4. Rheological Analysis Using Scale-Down Reactor

Rheological analysis was carried out in the scale-down reactor, using a combination of techniques to characterise the viscoelastic properties of the fluid under analysis. The techniques used were shear sweep, strain sweep and shear strain vs. shear rate analysis. The method for each technique is discussed in the following sub-sections, and is supported by the materials and methods section of Chapter 9, where the samples used are listed. The theory

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behind rheological analysis is discussed in Chapter 1, section 1.4. Rheological analysis was only performed on samples from batch fermentations.

Normal practice is to use a cone-and-plate geometry, but initial studies showed this set-up to lack sensitivity, and so the co-axial cylinders geometry was employed for all rheological analysis, with the Bohlin rheometer set at xIO sensitivity.

2.4.4.1. Shear Sweep

12 mL of sample was loaded into the bob, and the rheometer prepared for start, as according to the manufacturer’s instructions. 10.15 gem torque bar was used. The operating shear rate was increased in increments from 1.16 s'^ up to 1160 s'^ with each shear rate held for 7 s. The apparent viscosity was recorded at each shear rate up to the maximum shear rate, after which it retraced back to 0, again recording apparent viscosity.

2.4.4.2. Strain Sweep

Strain sweeps were carried out on 12 mL samples loaded into the cup. Sensitivity was set at 10 times normal rate, and linear sweeps (see Chapter 1, section 1.4.6 for theory) were conducted in fifteen steps between 40% and 60% amplitude. The frequency of oscillation was maintained at 1 Hz, and the storage and loss moduli, as well as the phase angle were recorded. The torque bar selected was the 0.295 gem bar.

2.4.4.3. Shear Stress vs. Shear Rate

This technique was used to generate stress diagrams for each of the fluids under analysis. Samples for rheological analysis were removed from the batch fermenter at early exponential, exponential and stationary growth phases as explained in section 2.3.3. 12 mL of the sample undergoing analysis was then loaded into the cup and the rheometer set up to start as explained in the instruction manual. 10.15 gem torque bar was used. The analysis was started at a very low shear rate (1.16 s'^), before increasing in

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increments to 1160 s'^ (holding each level for 7 s) and the shear stress generated by that shear rate was recorded. Once the final shear rate was reached, the experiment was terminated.

Buffer Preparation

P h o s p h a te B u ffe re d S o lu tio n (P B S )

D isso lve 8 g o f N aCI, 0.2 g o f KCI, 1.44 g o f N a2H P0 4, and 0.24

g o f K H2P O4 in 80 0 m L o f d istille d H2O. A d ju s t pH to 7 .4 and

add w a te r to 1 L. S te rilis e by a u to c la v in g . T E B u ffe r 10 m M T ris -H C L (pH 8.0), Im M E D T A .

P o ta ss iu m A c e ta te (K ao) A dd 11.5 m L g la cia l a c e tic a cid and 2 8 .5 m L H2O to 60 m L 5 M

p o ta s siu m a c e ta te . T h e re s u ltin g so lu tio n is 3 M w ith re s p e c t to p o ta s s iu m and 5 M w ith re s p e c t to a c e ta te .

10% S o d iu m D odecyl S u lp h a te (S D S )

D isso lve 100 g e le c tro p h o re s is g ra d e S D S in 9 00 m L H2O, heat

to 6 8°C to d is s o lv e . A d ju s t to pH 7.2 w ith H C L and a d ju s t

v o lu m e to 1 L.

A lk a lin e Lysis S o lu tio n A d d 9 m L o f 10% S D S to 90 m L o f 2 M S o d iu m H yd ro xid e (B D H ).

Table 2.4: TE, PBS and alkaline lysis buffer ingredients and their production

method (Sambrook et al., 1989).

2.5. Determining Physical Cell Strength Using An Industrial High