Experiment 3 (Day 3)

Inoculation, Fermentation and Monitoring

Objectives

1. To perform scale-up fermentation process to increase the yield of green fluorescent protein by monitoring and altering the fermentation conditions.
2. To monitor cell growth and product formation through manual sampling for every hour over a period of 10 hours and interpretation of the different phases via computer data logging.

Part I-Procedure for Setting up the Fermentation Process

The steps below outline what was done to prepare the fermenter to start the fermentation process.

1) Firstly, the medium broth in the fermenter had ampicillin added to a final concentration of 100μg/ml and arabinose to a final concentration of 0.2% after it has cooled to below 50oC after autoclaving.

2) The control parameters were set as shown below.

• pH ----------------------- 7.5
• Stirring Speed ----------- Min 0%, Max 90%, Control set to “AUTO”
• pO2 Set Point ------------ Set point 20%, Control set to “AUTO”
• Air Flow ----------------- Min 25%, Max 100%
• Set “Stir” and “AIRFLOW” to “CASC"

Temperature which is supposed to be set at 32oC is not done in this case as the temperature control module of the fermenter was out of order. Hence fermentation was carried out at ambient temperature (room temperature).

3) 100ml of the seed culture (5% of fermenter media volume) was then inoculated into the fermenter using a pump.
4) Fermentation was then allowed to continue for 24hours.

Part II-Procedure for Monitoring the Fermentation Process

The steps below outline what was done to monitor the bacterial growth and various parameters throughout the fermentation process.

1) The parameters such as pH, stirring speed and airflow were monitored by the computer and recorded in a graph. If any of the parameters deviate past the optimum range, automated corrective actions will be carried out by the computer and the various pumps.

2) For every hour within the first 10 hours from the start of the fermentation, samples were drawn from the fermenter and kept in falcon tubes. The steps taken to draw the samples are shown below.

1. Ensure the tubing after sampling tube (tubing placed with one end in 70% ethanol) is clamped tightly.
2. Open the clamp on the tubing before the sampling tube (tubing connected with one end to the fermenter).
3. Draw the culture to approximately 1/3 of the sampling tube by pulling the syringe.
4. Push back the syringe to force the media back into the fermenter (The media that remains in the tubing before the sampling tube.) This step is done so that the next sample we draw will contain the culture in the fermenter and not the one left in the sample drawing tube.
5. Next, clamp the tubing before the sampling tube back tightly.
6. Take out the syringe and pull to fill it with air then place it back.
7. Unclamp the sampling tube and push the syringe to collect the sample in a falcon tube
8. Clamp the tube back and repeat these steps for subsequent sample drawing.

3) The optical density readings of the samples collected during the first 10 hours was taken using a spectrophotometer set at 600nm on another day and analysed.
4) The graph of the parameters through the course of the whole fermentation was also analysed.

The video below shows how samples were taken from the fermenter.

Part III-Monitoring Cell Growth

Results

The results of the Absorbance obtained from the OD600 readings are shown in the table below:

The absorbance of the samples taken within the 10 hour period was measured at 600nm.

Using the formula:

A = ε × C × l

Where A = measured absorbance, 600nm
ε = extinction coefficient
C = concentration of the protein (in mg/mL)
l = length of light path through solution, 1cm cuvette is used

Derived using the equation above,
[Equation 2]: C = A / (ε × l)
[Equation 1]: C0 = A / (ε × l)

Substituting 1 into 2,

Log ( X / X0) = Log ( C / C0)
= {Log [ A/ (ε × l) ] / [ A0 / (ε × l)] }
= Log [ A / A0 ]

Hence,
Log ( X / X0) = Log [ A / (A0) ]

*The calculations for the table are shown here.

The graph of Log (X/X0) against time,

Discussion

The graph above shows log(X/Xo) against time where log(X/Xo) represents the concentration of the E. coli cells in the fermenter.

From inoculation at 0 hours till the 1hour into the fermentation, there was a sharp increase in the cell concentration before falling in concentration from the 1st hour to the 2nd hour of fermentation. This sharp increase could be due to the cells entering the exponential phase almost directly after inoculation and growing at an exponential rate while the decrease from the 1st to the 2nd hour of fermentation could be due to cell death arising from changes in the media conditions (eg. pH was not well maintained). However, this is unlikely to be the case since the media conditions are well regulated by the computer and an absence of a lag phase is highly unlikely. Hence, the likely explanation for this could be that the sample taken at the 1st hour of fermentation was done using incorrect sampling techniques or the OD reading was not taken properly.

Due to this discrepancy, we will take that the portion from 0 to 2 hours, the curve slopes upwards gently to indicate the presence of a lag phase were the cells would be adjusting to the new environment before sloping further to join the original graph at 3rd hour to indicate the start of the exponential phase where the cells would be increasing in numbers at an exponential rate.

From the 3rd hour onwards till the 5th hour, there is an increase in cell concentration at a relatively high rate. This indicates that within this period, the cells are in the exponential phase and growing in numbers at a high rate.

From the 5th to the 7th hour, the cell concentration dropped before having a sharp increase again till the 8th hour. The decrease in cell concentration could be due to cell death as the E. coli cells proceed into the death phase. This could have arisen due to accumulation of toxic waste products or depletion of cellular reserves of energy. The sharp increase in cell concentration on the 7th hour could be due to cell death in the 5th to 7th hour. When the cells die some of them will lyse releasing their intracellular contents which can be used by other live cells as nutrients. Due to this increase in nutrients due to cell lysis, the living E. coli cells will use these nutrients to grow and enter a secondary growth phase. It is the sharp increase in the 7th to 8th hour that the secondary growth phase’s exponential phase occurs as seen by the large sudden increase in cell concentration.

From the 8th to 10th hour there is a very slow increase in cell concentration as the rate of increase of cell concentration begins to slow down. This period is most likely the beginning stationary phase of the secondary growth curve where the cell growth rate is almost equal to the cell death rate and is usually caused by the exhaustion of a critical nutrient or accumulation of toxic waste products. It is at this phase where maintenance of the cells occurs, though there is increase in cell concentration, metabolism still occurs. Secondary metabolites such as our protein of interest, GFP is produced at this stage and accumulated intracellularly.

Part IV-Part Monitoring Parameters

Results

The graphs below shows the data obtained from the computer regarding the various parameters that were controlled by it over the course of the fermentation. This graph was not that of our experiment but that of a different class from previous years’.

Click Image to Enlarge

Discussion

The graph above shows the history plot of the different variables (pH, pO2, stirring speed and temperature) measured by probes and plotted by the computer during the course of the fermentation.

pH

The pH was held rather constant throughout the course of the fermentation beginning at a pH of 7.40 and held constant at 7.40 until 6.25 hours where it increased to pH of 7.55 and held constant at 7.55 for the rest of the fermentation. The small increase could be due to the fact that the bacteria might be growing exponentially at that portion and probably is in the middle of its exponential growth phase, releasing an increased amount of metabolic waste products that could have caused a slight increase in pH. The region before was maintained at a lower pH level of 7.40 as the bacteria could be in the lag or early exponential phase and hence not produce as much metabolic waste to significantly affect the pH as much.

The pH was kept rather constant throughout the fermentation run is it was controlled and monitored by the computer which will correct any deviation from optimum pH if they occur by adding base to increase the pH if it falls below optimum or by adding acid to decrease the pH if it increases above optimum.

pO2

The dissolved oxygen drops initially from about 80% to about 15% during the first 3.75 hours of fermentation. This drop is due to the cells utilising the oxygen for their metabolism. Even though the cells are usually in the lag or very early exponential phase during this time and the cells are not increasing much in cell concentration, metabolism still occurs to allow the cells to survive hence the drop in pO2 levels are seen. The pO2 levels are not kept steady and constant here by the computer as the set point is set at 20% which means it will start to correct deviations only if it falls below this set point.

After 3.75 hours many spikes are seen till the approximately 9th hour of fermentation, this is probably when the cells are undergoing their exponential growth phase and are experiencing their highest growth rate and metabolism and is where oxygen utilization is at its highest. Hence as the dissolved oxygen oxygen is rapidly utilised and falls below the set point, the computer actively corrects this deviation by increasing the dissolved oxygen by increasing the stirrer speed. This process occurs many times causing numerous spikes to appear on the graph.

From the 9th hour of fermentation till approximately 18.75 hours the dissolved oxygen level remained relatively constant at 20%. This is probably when the cells are within the stationary phase to the death phase where the cells are no longer growing or dying and is a period of maintenance, hence there is minimal usage of oxygen for metabolism hence not much dissolved oxygen is used. Hence the graph shows an almost linear line without spikes as the computer is not correcting any deviations of pO2.

From the 18.75 hours till 21.5 hours many spikes are seen as the cells undergo a secondary growth curve and begin to enter the exponential phase of that curve. The reasons behind the spikes are the same of that from 3.75 – 9 hours.

After the 21.5 hours the pO2 levels begin to increase. This could be due to cells undergoing death phase or harvesting when the oxygen utilisation by the cells decreases and rate of the surrounding oxygen dissolving in the media surpasses the oxygen utilisation of the cells causing an increase of pO2 beyond the set point.

Stirrer speed

The stirrer speed corresponds directly to the dissolved oxygen level where it increases when the dissolved oxygen is low and decreases when the dissolved oxygen is high and hence matches the pO¬2 graph. It remains constant during the first hours of fermentation before rising and producing spikes, followed by a decrease, a constant level of stirring, an increase followed by spikes and finally a decrease.

Stirrer speed affects the dissolved oxygen level as by increasing the stirrer speed, it breaks up the bubbles from the sparger into smaller bubbles which have larger interfacial surface for more efficient diffusion of oxygen. This increased diffusion increase the dissolved oxygen levels in the media.

Notice that the stirrer speed never falls below 200rpm as a certain level of agitation is required to ensure that the cells do not settle to the bottom of the fermenter.

Temperature

The temperature in the fermenter was kept constant at 32oC throughout the whole fermentation run. The small spikes occur due to the computer trying to control deviations by adding warm water into the cooling jacket to increase the temperature if it falls below the set point and vice versa.

Note:
The numerous large spikes seen on the graph could be due to the high sensitivity of the probes or since this fermentation is done on a small scale with a low volume, any small changes may be reflected by big changes when plotted on a graph.
This graph also does not correspond to our growth curve that we plotted as this history plot belongs to another group’s.

Attention: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.