Escherichia coli fermentation

Escherichia coli cells, strain JM107 with plasmid pQR126, developed by French (1993) were grown in batch mode and used for downstream processing studies investigating the recovery of a-am ylase from lysed cells. All chemicals for the fermentation were obtained from Sigma Chemical Company Ltd. (Dorset, U.K.) unless stated otherwise.

An agar solution, 1 L, containing 28 g L'^ Oxoid No. 2 (Oxoid Unipath Ltd., Basingstoke, Hampshire, U.K.) in R.O. water and 1% w/v potato starch was autoclaved and cooled to below 42°C. Kanamycin, 0.02 g L was filter sterilised (0.2 pm, Whatman, Maidstone, U.K.) into the cooled sterile agar solution before the plates were poured. The plates were stored at 4°C for use over a two week period. Plates, 4, were inoculated from -70°C stocks using a sterile loop and incubated at 37°C for ~ 24 h.

An inoculum volume, 200 mL, was prepared in two 2L shake flasks. Each flask contained media parts A and B which were prepared separately until fully dissolved in de-ionised water and then mixed together. Part A constituents were 10 g L^ (NH^)2S0 ^, 2.5 g L'* NaCl, 2.16 g L^ N a2HPO^, 0.64 g L^ KH2PO4 and 1 mL L ^ trace element solution. The composition of the trace element solution was 10 g L*^ CaCl2, 4 g L'^ H3BO3, 2 g L'^ MnCl4.4H2 0, 0.4 g L'^ CUSO4.5H2O, 0.4 g L-i C0CI2.6H2O, 0.2 g L-i NaM o0 4.2H2 0 and 2 g L ^ ZnS0 4.7H2 0. Part B constituents were 0.2 g L'^ FeS0 4.7H2 0 and 0.2 g L'^ citric acid. Part D, containing 0.2 g L‘* M gS0 4.7H2 0 and 4 g L'^ glycerol (BDH, Merck Ltd., Dorset, U.K.), was prepared and dispensed into two bijou bottles and autoclaved with the two shake flasks containing media parts A and B. Part C, containing 0.05 g L'^ thiamine and 0.01 g L'^ kanamycin, was filter sterilised into the shake flasks prior to inoculation from the plates. To inoculate the shake flasks, 3 mL of media from the flasks was pipetted on to the plates and two lines of cells were scraped off and the

media with cells was poured back into the flask. Cells in the shake flasks were grown at 37°C in an orbital incubator at 200 rpm for 23 h.

Fermentations were carried out in 20 L vessels (Model LH20L01, Inceltech, Berkshire, U.K.) with a working volume of 14 L. Parts A and B were made separately and when fully dissolved, added together. Polypropylene glycol, 0.1 mL L'^ PPG (BDH, Merck Ltd., Dorset, U.K.), was added to parts A and B. These components of the media were sterilised in situ in the vessel. Part D, ~ 500 mL, was autoclaved in a side arm flask and part C was filter sterilised into part D. This component of the media was added to the fermenter immediately prior to inoculation. Alkali, 4M NaOH, and antifoam, 25 % v/v PPG, were used as control. The sterile medium was then inoculated with the shake flask contents at the appropriate time. The temperature was controlled at 37°C, the pH was controlled at 7, the stirrer speed was set at 500 rpm and airflow was set at 7 L min k Optical density of fermenter broth was monitored every 2 h to determine growth rates. After 7 h, the stirrer speed was increased to 1100 rpm and the airflow was increased to 12 L min L The fermenter broth was grown to an optical density reading of approximately 30, lasting ~ 24 h. Dissolved oxygen tension levels were maintained above 30% during fermentations.

Fermenter broth, 10 L, was used for downstream processing studies. A tubular bowl centrifuge (Model Sharpies IP, Alfa-Laval Sharpies Ltd., Camberley, U.K.) was used to harvest the cells. The cells were resuspended in supernatant to a final volume of 2 L. Lysis solution, 2 L, at room temperature containing 20% w/v sucrose, 1 mM Na2EDTA and 500 p.g mL'^ hen egg lysozyme was added to the suspension of cells and the solution was gently mixed at room temperature for 10 min. R.O. water, 2 L, ~ 4°C, was then added to the solution and gently mixed at room temperature for a further 10 min. The resulting suspension was used as feed broth for filtration studies.

Filtration studies were conducted on lysed cells as described in section 3.3. Samples of the soluble fraction of lysed cells and whole cells and permeate samples were assayed for total protein levels as described in section 3.3.1.1 and a-am ylase activity as described in section 3.3.1.6. Gel permeation chromatograms were obtained by running samples down a Superose 12 gel filtration column as described in section 3.3.6. Particle size measurements of whole cells and

lysed cells were conducted on the Malvem Series 4700/PCS 100 spectrometer laser sizer as described in section 3.3.2.2. Rheological properties of whole cells and lysed cells were carried out on the RHEOMAT 115 viscometer as described in section 3.3.3.

3.4 Conclusions

The analytical methods described and the models detailed in Chapter 2 will allow the development and experimental verification of bioprocess filtration models. Chapter 4 outlines two approaches to the modelling of filtration systems. The first is based on a statistical approach and requires a high degree of experimentation to develop. The second uses a concentration polarisation model and is based on single experimental measurements of the permeate flux rate for a given membrane pore size. Chapter 5 extends the polarisation modelling approach to a homogenised system. Chapters 6 provides information on an effective strategy for the design and evaluation of membrane filtration systems for use in the bioprocess industries based on experimental and simulation studies.

4 PROCESSING OF WHOLE CELLS