GROWTH OF Escherichia coli IN A 5 LITRE BATCH FERMENTATION VESSEL

OBJECTIVES
To grow Escherichia coli in submerged culture and to monitor growth, dissolved oxygen, pH, and glucose utilization.

THE EQUIPMENT
A composite glass/stainless steel, with agitator, sparger aeration, sampling line and ports for additions, pH and DO electrodes. There is a water-cooled condenser for cooling the outlet air.

Temperature control is achieved via a wrap around heating jacket, a cooling finger inserted through the lid connected to the water supply through a solenoid valve by tubing and a temperature sensor inserted into a pocket in the top plate. Both sensor and solenoid valve connect to the control unit.

A top drive motor connected to the agitator seal assembly drives the shaft and impellers. This can be controlled manually directly, from the stirrer unit or automatically by the control unit.

Airflow is set by a needle valve and indicated by a ball and tube flow meter. Both exit and inlet gases are sterilised through O.2-micron air filters. A glass steam sterilisation electrode connected to the digital display measures pH. A polargraphic dissolved oxygen electrode measures the dissolved oxygen. Temperature, speed, D02, pH meter outputs and inputs all connect to the control unit, which in turn connect to the computer.

The following will be done for you please watch the video clip before the practical session;

A) Calibration of D02
For the setting of the upper end of the scale 100% saturation is achieved with high agitation rate combined with adequate airflow in the absence of any appreciable oxygen demand. When you are satisfied you have achieved 100% adjust D02 meter to read 100%. For the lower fixed (usually zero) point pass nitrogen gas through the sparger until a satisfactory stable zero is reached.
Change back to air flow and achieve 100% reading by using fermentation agitation rate 600r.p.m. and air flow rate 4L/min (lv/vm). Recheck 100% after sterilization with medium. Why? This calibration should be carried out at 37°C. Why?

B) Calibration of pH electrode
1. Remove pH electrode from vessel carefully and clamp in clamp stand.
2. Connect to meter.
3. Immerse in buffer pH7.
4. Adjust buffer control to 7
5. Washing electrode in between with water, blot dry, repeat with buffer 4 and 10.
6. We adjust zero to 7.Why?

C) Preparation of the media for 4L
Final Concentration Stock Solution
KH2P04 13.6g/L 400ml
(NH4)2S04 2g/L 400ml
MgS04 0.2g/L 40ml

Yeast Extract 5g/L 20g
FeS04 0.0005g/L 4ml
Peptone 40g

Make up to 2 liters; adjust pH to 7 using KOH solution.
Make final volume up to 3200ml.
Separately weigh out 40g of glucose and dissolve in hot tap water. Make volume up to 400ml. Pour into side arm flask provided, plug, foil and sterilize.
Final glucose concentration 10g/L.

C) Setting up of fermenter
Set up vessel in framework, connect air, pH, D02 electrodes, and temperature sensor, assemble motor, fit heating jacket and connect cooling finger. Set airflow rate. Switch on agitation and temperature control. When temperature has reached near set point check D02 and pH.
Connect glucose addition flask using ethanol.
Turn off air and run in glucose
Take a sample as shown-20ml
Inoculate with shaker flasks grown overnight- aseptically adding to side arm flask. Run in. Turn on air.

Initial Set points for Fermentation
TEMPERATURE 37°C
pH 7
Speed 600r.p.m
D02 100% Head Pressure 2-3 p.s.i.
You are required to carry out the following analysis during the laboratory session

Sampling

Take hourly samples.

1. Plate out samples to check for contamination and numbers of cells.
2. Check the optical density (OD) using the spectrophotometer at 650nm with water as a blank.
3. Measure glucose using the glucose meter.
4. Make a note of the pH and D02,
5. Take a 1ml sample and put it in a sterile preweighed eppendorf tube and centrifuge it at 10,000rpm for 10 minutes (do 3 to get an average).
Take off the supernatant and put the tube in the oven overnight at 1050C then reweigh to give dry cell weight.
5) Upload result on Canvas

Laboratory report write up

Length 1000-1200 words
Include diagrams, tables, graphs and calculations.

1. Summarize experimental detail in 100 words or less.
2. Include the following data ; pH, and glucose depletion, D02 from the result that will be given to you on canvas.
3. Plot the growth the curve and calculate the growth rate, mean doubling time and final cell yield.
4. Briefly evaluate results and discuss what they show about the fermentation.
5. How might the fermentation be modified to improve cell yields?
6. Discuss briefly good and bad design points of the equipment
7. Itemize any conclusions you have drawn.

Please note, tables, graphs, diagrams and calculations do not count towards the word count