ANÁLISIS DEL A.V.A

The shear sensitivity of spheroplasts was investigated by assessing the degree o f cell breakage caused by exposure to shear and the effect o f shearing on centrifugal clarification efficiency.

Spheroplast breakage following exposure to shear was estimated from the release o f total cellular protein and the intracellular enzyme glucose-6-phosphate

dehydrogenase, G-6-PDH. Cells (harvested from HCD run 4) were resuspended to a concentration of 0.14 g mL'1 in periplasmic extraction buffer and incubated overnight at 30°C and 60°C. lmL samples of each suspension were centrifuged in a microfuge (13 000 rpm/~14 OOOg for 5 minutes) and the supernatant collected for the measurement of pre-shear extracellular protein and G-6-PDH concentration. (This method assumes that centrifugation in the microfuge causes minimal damage to the cells. The assumption has previously been experimentally confirmed by N. Murrell, PhD Thesis, 1998).

Samples of the 30°C and 60°C spheroplast suspensions were then sheared (in triplicate) using the rotating disc shear device. The reservoir of the disc device was filled with -1 2 mL suspension (so that it was -2/3 full) and the disc was rotated at 27000 rpm for 15 seconds to produce a shear rate o f l.lxlO6 s'1 with air entrainment. Following shearing, 1 mL samples were centrifuged as before and the supernatant collected to determine the post-shear extracellular protein and G-6-PDH concentration. Total protein and G-6-PDH available for release was measured in homogenised samples of pre-shear suspensions (section 2.4.4.1). Protein and G-6- PDH assays are described in sections 2.2.3.1 and 2.2.4 respectively.

The concentrations measured were used to determine the level o f release due to spheroplast damage that had occurred during shearing. The percentage release was calculated as follows:

P - P

% Release = ~ * 100% (5.8)

M 0 0 " 0

Where Ps = Extracellular protein/ enzyme concentration in sheared sample

P o = Background protein/ enzyme concentration (i.e. extracellular concentration in pre-shear sample)

Pioo = Total enzyme/ protein available for release, measured in a homogenised sample.

The results are given in Tables 5.2.1-5.2.3. G-6-PDH release was only measured in 30°C spheroplast suspensions as incubation at 60°C destroyed all enzyme activity.

Sample Pre-shear extracellular protein (mg mL'1) Post-shear extracellular protein (mg mL'1) Total protein available for release (mg mL'1) % release 1 2.67 2.96 9.35 4.3 2 2.67 2.89 9.35 3.3 3 2.67 2.70 9.35 0.04

Table 5,2.1 Protein release resulting from shearing o f a spheroplast suspension produced by overnight extraction at 30 X . Spheroplasts were sheared at a rate o f

~l . l x l 06 s'1 fo r 15 seconds in a rotating disc shear device.

Sample Pre-shear extracellular G-6-PDH (IUmL'1) Post-shear extracellular G-6-PDH (IUmL'1) Total G-6-PDH available for release (IUmL'1) % release 1 0.11 0.14 1.21 2.5 2 0.11 0.13 1.21 2.3 3 0.11 0.14 1.21 2.4

Table 5.2.2 G-6-PDH release resulting from shearing o f a spheroplast suspension produced by overnight extraction at 30°C. Spheroplasts were sheared at a rate o f

~l.lx lO 6 s'1 fo r 15 seconds in a rotating disc shear device.

Sample Pre-shear extracellular protein (mg mL'1) Post-shear extracellular protein (mg mL'1) Total protein available for release (mg mL'1) % release 1 0.66 0.67 0.95 3.4 2 0.66 0.70 0.95 14.0 3 0.66 0.64 0.95 -7.0

Table 5.2.3 Protein release resulting from shearing o f a spheroplast suspension produced by overnight extraction at 60 XI. Spheroplasts were sheared at a rate o f

~1.1x106 s'1 fo r 15 seconds in a rotating disc shear device.

The results show minimal cell breakage as a result of shearing. The mean protein release for 30°C spheroplasts was 2.5%, which is comparable to the mean G-6-PDH release (2.4%). Values for total protein and G-6PDH release should be similar as G- 6PDH is an entirely cytoplasmic enzyme and approximately 97% of the total protein in the cell is located in the cytoplasm. The mean protein release for the 60°C spheroplasts was 3.5%, again indicating minimal cell breakage. The greater variation

observed in the release figures for 60°C spheroplasts can be attributed to the reduction in the total protein available for release; the difference between ‘0%’ and ‘100%’ release was sufficiently small that errors associated with the protein assay would have had a significant effect on the calculated ‘% release’ value.

It appears from the results that the spheroplast integrity is unaffected by shear rates up to l.lxlO6 s'1 and, in addition, increasing the extraction temperature has no appreciable effect on shear sensitivity. Spheroplasts are thought to be relatively flaccid and able to deform when subjected to a shearing force which would explain their insensitivity to shear. The results are in agreement with those of E. Fischer (PhD Thesis, 1996) and N. Murrell (PhD Thesis, 1998), both of whom observed minimal breakage of E. coli spheroplasts when exposed to shear in laboratory equipment and during centrifugation.

Following analysis of spheroplast breakage, the effect of shearing on centrifugal clarification efficiency was examined. Spheroplast suspensions were produced by overnight periplasmic extraction at 30°C and 60°C. Half o f each suspension (-250 mL) was sheared using the rotating disk shear device. Suspensions were again sheared for 15 seconds at 27 000 rpm, in the presence of air-liquid interfaces. A centrifuge spin test, described in section 2.4.2.2, was performed on both sheared and non-sheared suspensions. 10 mL samples of each suspension were spun in the Beckman J2-M1 centrifuge using the JS 13.1 spin-out rotor at 6720 rpm for various time intervals between 3.5 and 47 minutes. (The centrifuge was operated at 6720 rpm to maintain consistency with later experiments). The clarification efficiency obtained at each run time was calculated based on optical density at 600nm (section 2.4.2.3). The equivalent Q/Z corresponding to each run time has been calculated in Appendix 8, and the relationship between clarification efficiency and equivalent Q/Z is shown in Figures 5.2.2 and 5.2.3.

The graphs indicate that exposure to shear resulted in a reduction in clarification efficiency during the centrifugation of spheroplasts produced at both extraction temperatures. The percentage reduction in clarification increased with increasing equivalent Q/Z (i.e. as spin time decreased). For 30°C spheroplasts, the reduction in

clarification resulting from exposure to shear ranged from 0.2% at low Q/Z to 7.0% at high Q/Z. For 60°C spheroplasts, the reduction in clarification was slightly lower, ranging from 0.3% at low Q/Z to 4.3% at high Q/Z.

99.99 * 99.9 * 99 95 90 75 60 10'9 Equivalent Q /z (ms*1)

Figure 5.2.2 Relationship between clarification efficiency and equivalent Q /Z fo r sheared (O) and non-sheared (B) spheroplasts produced by overnight periplasmic extraction at 30X1. Spheroplasts were recovered in the laboratory J2-M1 centrifuge operated at 6720 rpm (RCF = 3720g) using the JS 13.1 rotor. Spheroplasts were sheared at a rate o f - l .l x l O 6 s'1 fo r 15 seconds in a rotating disc shear device. Error bars represent the standard deviation fo r triplicate spin tests using the same feed material. Results show a reduction in clarification efficiency fo r sheared spheroplasts compared to non-sheared spheroplasts. The reduction in clarification ranges from 0.2% at low Q /Z to 7.0% at high Q/Z.

99.99 99.9 JS O 99 95 90 80 10‘ JZ 10 10‘ Equivalent Q/L (m s ')

Figure 5,2.3 Relationship between clarification efficiency and equivalent Q /Z fo r sheared (O) and non-sheared (B) spheroplasts produced by overnight periplasmic extraction at 60 Spheroplasts were recovered in the laboratory J2-M1 centrifuge operated at 6720 rpm (RCF = 3720g) using the JS 13.1 rotor. Spheroplasts were sheared at a rate o f - l.l x l O 6 s’1 fo r 15 seconds in a rotating disc shear device. Error bars represent the standard deviation fo r triplicate spin tests using the same feed material. Results show a reduction in clarification efficiency fo r sheared spheroplasts compared to non-sheared spheroplasts. The reduction in clarification ranges from 0.3% at low Q /Z to 4.3% at high Q/Z.

The reduction in clarification is likely to result from a change in the particle size distribution of sheared spheroplast suspensions, with an increase in the proportion of smaller particulates which require longer centrifugation time for sedimentation. Reduction in the average particle size also provides an explanation for the greater reduction in clarification efficiency observed at higher Q/Z. Hewitt et al., (1998), studied the effect of fluid mechanical stress on E. coli strain W3110 during continuous cultivation in an agitated bioreactor. Their results showed that exposure of continuous cultures to high aeration and agitation rates (3 w m and 1200 rpm respectively) had no effect on cell physiology, cell size or cell integrity. However, exposure to agitation rates of 1200 rpm for 7 hours did result in the stripping away of the outer polysaccharide layer of the cell. (Experiments were performed in a 5L

cylindrical glass bioreactor (162 mm diameter x 300 mm total height) using a working volume of 4L and employing two 82 mm six-bladed paddle type impellers). Hence, the exposure of spheroplasts to much higher rates of agitation within the shear device may have resulted in damage to the outer membrane, releasing submicron sized particles that are more difficult to clarify by centrifugation. Alternatively, shearing may change the particle size distribution simply by breaking up cell clumps or aggregates.

5.2.3 Clarification of spheroplast suspensions using a tubular bowl and a disk