2.3.1 E. coli
1. * Terrific Broth’ (Tartof & Hobbs, 1987). The following reagents were used (g L'^): KH2PO4, 2.3; K2HPO4 3.78; bactotryptone 12 (Difco); yeast extract 24 (Oxoid); kanamycin 0.02 (Sigma); glycerol I 50 g L ’ ; poly propylene glycol (PPG) 1 ; g L * . 2. Defined medium (g L‘^): (NH4)2S0 4, 10; NaCl, 2.5; Na2HP0 4, 2.16; KH2PO4, 0.64;
FeS0 4 7H2O, 0.2, citric acid, 0.2, PPG, 0.1 mL L '\ glycerol, 30, MgSO4.7H20, 0.2, kanamycin, 0.01, thiamine, 0.05, trace elements, 1 mL L"\ The trace elements solution comprises (g L'*) CaCL 10; H3BO3 4; MnCl4.4H20 2; ZnS0 4.7H2 0 2; CUSO4.5H2O 0.4; C0CI26H2O 0.4; NaMo0 4 2H2O (0.2). Citric acid and FeS0 4 7H2O were mixed together first, before adding the other medium components. Glycerol and magnesium salts were autoclaved separately, the kanamycin and thiamine were filter sterilised (0.2pm) into the fermenter. Fed-batch feed medium (g L*^): glycerol, 250; (NH4)2S0 4, 50; Na2HP0 4, 13.5; KH2PO4, 4; FeS0 4.7H2 0, 1, citric Acid, 1, trace elements, 2.5 mL L'% kanamycin, 0.0025, thiamine, 0.25,
3. High Biomass medium, for the cultivation of T4 lysozyme strains (g L'*): (NH4)2S0 4, 7; NaH2PO4.2H20, 6.24; yeast extract, 40; ampicillin, 0.1 (Sigma); glycerol 25 g L '
PPG, 11 gL'^ .
2.3.2 S. lividans
Minimal liquid medium (Hopwood et al, 1985) (g L'*): (NH4)2S0 4, 2; casaminoacids (Difco), 5; MgS0 4.7H2 0, 0.6; PEG6000, 50; NaH2P0 4, 2.16; K2HPO4, 3.58; sodium succinate, 5; thiostrepton, 0.05; trace elements solution, 1 mL L"\ The trace element solution was composed of (g L’^) ZnS0 4 7H2O, 1; FeS0 4 7H2O, 1; MnCl2.4H20, 1; CaCl2, 1.
2.4 Growth conditions
2.4.1 E. coli shake flask cultivation
g-amvlase producing strains: E. coli strains (JM107 and JM83) were cultivated in complex ‘Terrific Broth’ shake flasks for strain selection work (Tartof & Hobbs, 1987). Cells were reactivated fi’om fiozen glycerol stocks by overnight cultivation on nutrient agar plates containing 1% (w/v) insoluble potato starch (Sigma) and 0.02 g L'^ kanamycin (Sigma). A single colony was added to a ‘Terrific Broth’ starter (10 mL) and grown for approximately 14 h. The main culture (0.3 L) was inoculated with 3 mL of the starter culture (1% v/v) and grown at 37°C for 24 h at 200 rpm in an orbital shaking incubator (Adolf Kühner AG, Schweiz, Switzerland). Kanamycin was added at a concentration of 0.01 g L ^ For small scale downstream processing experiments E. coli
JM107 pQR126 cells were reactivated by growing overnight on selective plates. A single colony was suspended in 0.5 L ‘Terrific Broth’ in a shake flask containing 0.01 g L*^ kanamycin and grown for 18 h at 37°C and 200 rpm in an orbital incubator.
T4 Ivsozvme-his producing strains: E. coli JM107 containing pQR752 was reactivated by cultivation on a nutrient agar plate containing ampicillin (0.1 g L'^) at 28°C for 48 h. A single colony was suspended in 33 mL of high biomass medium containing ampicillin, (0.1 g L'^), and grown at 28®C for 13 h at 200 rpm in an orbital shaker (Adolf Kühner AG). The main culture (0.25 L) was inoculated with 2.5 mL of the starter (1% v/v) and grown at 28°C and 200rpm until an ODeoo of 5.0 (8 h) was reached. At this point the culture was induced with 1 mM isopropyl-P-D-thiogalactosidase (IPTG) and grown for a further 3 h before harvest.
2.4.2 S. lividans shake flask cultivation
Spores were maintained in 20% (v/v) glycerol at -70°C. An aliquot (5 pL) of the spore solution was streaked out on a 14 cm diameter petri dish (Bibby Sterilin Ltd, Stone, Staffs, UK) containing % strength Tryptone Soya Broth (Oxoid) solidified with 2% (w/v) bacteriological agar (Gibco) and supplemented with 1% (w/v) insoluble potato starch (Sigma) and 0.05 g L'* Thiostrepton (Squibb Institute). This plate was then incubated at 28°C for 7 to 11 days until sporulation occurred. The spores were then harvested by
pouring 7 mL of sterile Revese Osmosis (RO) water onto the plate. The spores were scraped off into the liquid using a sterile loop. A 2 L flask (baffled with a stainless steel spring) containing 0.5 L of minimal liquid medium (Hopwood et al, 1985) supplemented with 5 g L'^ sodium succinate and 0.05 g L’* Thiostrepton was inoculated with 1.5 mL of harvested spore suspension and incubated at 28°C in an orbital incubator at 300 rpm for 97 h.
2.4.3 Complex medium batch fermentation
Cells were reactivated as before and a seed culture was prepared by suspending 2-3 colonies in 0.5 L Terrific Broth medium in a 2 L side arm flask. The culture was grown in an orbital shaker at 37°C and 200rpm for 12 h. Batch cultivation was carried out using either 14 L (Chemap, Alfa Laval Engineering Ltd, Middlesex, UK), or 42 L (LH Inceltech, Pangboume, UK) fermenters. The fermenter was seeded with a 2% (v/v) inoculum of stationary phase (12 h) E. coli cells. Fermentation samples were taken regularly to determine at-line biomass, optical density, a-amylase titres and plasmid stabilities. Parameters, such as dissolved oxygen and pH, were monitored using Ingold electrodes (Mettler Toledo Ltd, Leicester, UK), and parameters such as pH, antifoam and temperature were controlled using TCS instrumentation (Turnbull Control Systems Ltd, Worthing, UK). The pH was maintained at pH 7 using 2M phosphoric acid and 4M NaOH, with air supply and agitator speed were set at sufficiently high levels to prevent DOT dropping below 30% (1 w m and 1000 rpm). The temperature was set to 37°C. Cultivation parameters and exhaust gas values were monitored using a real time data acquisition system (RT-DAS, Acquisition systems, Reading, UK). Exhaust gas composition was measured using a mass spectrometer (Prima 600, Fisons Scientific instruments, UK).
2.4.4 Defined medium batch fermentation
E. coli JM107 pQR126 were reactivated as before. Sterile cultivation medium was aliquoted onto a nutrient agar plate, the cells were scraped off the surface of the agar plate with a sterile loop, and the resulting cell suspension aseptically transferred into a 2 L side arm shake flask containing 0.5 L of medium. The culture was grown at 37°C for 18-24 h in an orbital shaker at 200 rpm. The fermenter (20 L LH Inceltech) was
inoculated with a 6% (v/v) inoculum from a defined medium shake flask culture. The cells were grown at 37°C and pH 7, controlled with 4M NaOH, and the DOT maintained above 40% (1-1.5 w m and 1000 rpm). Large scale fermentation (300 L) was carried out, in collaboration with C Turner and E. Fischer, in the same way as the smaller scale fermentation but seeded using 15 L (5%) of defined medium broth grown in a 20 L fermenter (LH Inceltech). The fermentation was monitored in the same way as the complex medium fermentation and stopped when the carbon source had been exhausted (ODgoo ~25).
2.4.5 Defined medium fed-batch fermentation
Fed-batch fermentations were carried out in collaboration with C. Turner. The fermenter containing defined medium was seeded with a 6% (v/v) inoculum and cultivated under the same conditions as the defined batch fermentation. Glycerol and other nutrients (section 2.3 .1) were fed by pump under the control of a feed algorithm at a growth rate of 0.2 h'* (Gregory & Turner, 1993). When the culture reached a DOT of approximately 20%, the feed was added at a constant rate to give a declining specific growth rate. This maintained constant DOT and prevented oxygen limitation, enabling higher cell densities to be achieved.