Through the course of this practical series we have gathered the follow learning points about fermentation in general:
Allowed us to understand the true complexity of fermentation and that it actually involves numerous steps and equipment for it to be a success.
Learnt that the different specialised parts of the bioreactor and their functions.
Equipped us with the basic skills of preparing the necessary materials for fermentation, sample collection and extraction and purification of a product.
Allowed us to be more analytical in analysing data such as the OD readings.
Reinforced our theoretical knowledge by applying them in practicals.
Allowed us to understand the various considerations for lab scale fermentation and the methods that were taken to adress this.
We have learnt alot from our practicals and we hope you have picked up something new too by reading our blog. Hope you've enjoyed reading our belong just as we did carrying out our practicals!!! =)
2) GFP has Mr (molecular weight) around 27, 000 kD. Though we were unable to see them, the cell extract also contained hundreds or even thousands of other proteins. Do you think a protein with a Mr of 50, 000 kD would elute in a fraction before or after GFP? Why or why not?
A protein with a Mr of 50, 000 kD would elute in a fraction before GFP, this is because it is bigger than GFP. Therefore larger protein, which fit in fewer pores, takes a more direct path through the column and elute first. The smaller GFP are slower to elute as they meander in and out of pores spending less time in the liquid mobile phase of the column.
To isolate the desired product using enzymes, freezing & thawing and sonication to obtain an extract needed for purification.
To purify the product via gel permeation chromatography and separate impurities by their different sizes.
To analyze the wavelength at which the green fluorescent protein strongly absorbs and give out its usual absorbance by taking absorbance readings using a spectrophotometer set at 476nm.
In this experiment, the Green Fluorescent Protein (GFP) is isolated from the E. coli in the sample by cell disruption techniques (using enzymes, freezing and thawing and sonication) and then purified using gel permeation (a.k.a. size exclusion chromatography). 2 tubes of 10ml broth culture were made to under go the 3 cell disruptions and the purification stages.
Stage 1 – Isolation
The procedure for obtaining a cell pellet from the culture broth is shown below.
1) Firstly, we had to obtain 10ml of culture broth in two test-tube for the rest of the experiments.
2) In order to separate the cells from the liquid broth, we have to centrifuge the culture broth at 10,000 rpm for 5 minutes.
3) A pellet would be formed at the bottom of the tube as the cells are dense whereas the liquid broth is less dense and forms the supernatant.
4) The supernatant was then poured into another tube and both tubes were observed under UV light to confirm that the GFPs are inside the pellet.
The procedures for each of the 3 cell disruption methods are shown below.
Enzymatic Method
1) Firstly, the pellet was resuspended in 500µl of TE buffer of pH 7.5 with a micropipette until there were no visible clumps.
2) Next, 2 drops of lysozyme were added to the resuspended pellet whereby this will initiate the enzymatic digestion of the bacteria cell wall.
3) The enzyme was then allowed to act on the resuspended pellet for 15 minutes.
Freezing and Thawing Method
1) After 15 minutes, the tube was placed in liquid nitrogen until the contents are frozen.
2) Subsequently, the tube was thawed in warm water.
3) The cycle of freezing and thawing was repeated for another 2 times to complete the rupturing of the bacteria cell wall.
4) Freezing and thawing add mechanical stress to the cell wall as the cell water content expands (when frozen) and contracts (when thawed) causing it to rupture.
Sonication Method
1) The cell disruption is completed by the process of Sonication where ultrasonic waves (of higher frequency than sound) are used to implode the bacteria cell wall under the pressure of ultrasonic waves’ vibration.
2) Sonication is done on ice for 4 cycles of 25 seconds with 10 seconds rest in between Sonication cycles so as to prevent denaturation of the GFP protein.
3) Next, the contents of the tube were centrifuged after cell disruption for 20 minutes at 10,000 rpm.
4) The pellet was separated out of the supernatant as done previously during method 1.
5) The product (GFP) is now in the supernatant and pressence of GFP can be seen by observing the tubes under UV light.
Stage 2 – Purification
Purification to obtain the GFP protein was done on the supernatant obtained in the isolation steps using size exclusion chromatography. This method of chromatography uses a colum of polymer gel resins (Sephadex G75) which contains small pores as the stationary phase. When the extract flows through the column, smaller molecules interact with the pores of the resin and elutes slower. However, the larger molecules which are too large to interact with the pores will flow through the column faster.
The procedure below shows how this is done.
1) Firstly, eight test tubes were labeled from ‘1’ to ‘8’ and another test tube was labeled as ‘blank’.
2) The blank was then filled with 2.0ml of ammonium bicarbonate. Using this tube as a rough guide, the rest of the test-tubes were marked at the 2.0ml level.
3) The column was next allowed to drain into a waste beaker until the buffer is just above the top of the gel bed.
4) Subsequently, our cell free extract obtained from the isolation steps was transferred into the top of the gel bed by swirling around the inner edge of the column.
5) Next, the stopcock was opened and the sample was allowed to flow into the gel bed and the eluting buffer was collected in the first tube. While the eluant (buffer) flows into the tube, more eluting buffer (ammonium bicarbonate) was added to the top of the column to ensure that it does not run dry.
6) Once the sample has filled the tube to the 2cm mark, the tube was swapped with the next. This was done for the remaining tubes while ensuring that the gel bead does not run dry.
Note: The column was washed with 50ml of ammonium bicarbonate after the experiment so that it could be reused.
Stage 3 – Analysis
After the purification, the solutions from each of the 8 tubes and the blank were transferred to cuvettes and their absorbance readings were taken using a spectrophotometer set at 476nm. This was done for the other set of tubes obtained from the other set cell extract. 476nm is the wavelength at which GFP strongly absorbs light and gives off fluorescence.
The results showing the absorbance readings are shown below.
The graphs show the absorbance readings against their respective fractions of the 2 samples.
Click image to enlarge
Discussion
For discussion purposes, only sample number 2’s graph is used.
Since GFP absorbs light at the wavelength of 476nm, the absorbance obtained is indicative to the amount of GFP present. Hence from observing the absorbance readings and the graph fraction 2 has the highest amount of GFP followed by fraction 1 and fraction 3. Fractions 4 to 8 possess no GFP due to the 0.000 absorbance.
From the 1st fraction to the 2nd fraction, the absorbance increased from 0.385 to 2.745 and from the 2nd fraction to the 3rd fraction it then decreased to 0.054. Hence it can be deduced that the GFP probably had started eluting from the 1st fraction and stopped eluting at the 3rd fraction as a peak is observed spanning the from the 1st to the 3rd fraction as seen from the graph. The 1st fraction is lower in absorbance as the GFP protein requires sometime to elute through the column and would contain a larger amount of ammonium bicarbonate. The 2nd fraction is the highest as this is where most of the GFP is eluted from the column. The 3rd fraction has a low absorbance as almost all the GFP have eluted and only the remaining amounts are collected here.
Hence to obtain the purified GFP product, fractions 1 to 3 should be used and pooled.
Note: There are additional questions to help in better understanding of this experiment. The question and answers are located in the next post.
The answers below adress the questions from our practical manual.
1) Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.
pH – As the bacteria cultured in the fermenter grow, reproduce and carry out metabolic activities, waste materials will be produced. Some of these waste materials that accumulate in the culture broth are acidic and will cause the pH of the broth to decrease. If the pH is allowed to decrease till toxic levels, the bacterial growth will stop or the bacterial cells in the fermenter might die. Depending on the strain and type of the microbe being cultured the waste products might increase or decrease the pH.
Hence to prevent extreme pH deviations from affecting cell growth and survival, acid (for high pH) or bases (for low pH) are pumped into the fermenter via peristaltic pumps to correct the pH back to optimum levels.
Temperature – There is an optimum temperature for the growth of the cultured microbe and the yield of the product it produces. Hence the temperature of the fermenter must be kept at optimum levels for optimum growth and production of the product of interest (GFP). This is done by the cooling jacket of the fermenter where hot or cold water can be pumped into to correct deviations from the optimum temperature.
Increase in temperature could be due to factors such as microbial respiration, movement of the impeller. Decrease in temperature could be due to factors such as cooling from the surroundings (air-conditioning) or endothermic reactions occuring within the cells.
Dissolved Oxygen – The culture requires an optimum amount of dissolved oxygen to provide a sufficient oxygen supply for cellular respiration. If too little dissolved oxygen is present, the growth rate of the culture might slow down. However, if the dissolved oxygen is too high it may become toxic to the cells as high levels of oxygen might generate reactive oxygen species that could damage the cultured cells’ DNA causing cell death.
To regulate the dissolved oxygen levels in our fermenter, the stirrer speed is varied. If the dissolved oxygen is too low, the stirrer speed will increase to break the air bubbles sparged in by the sparger into smaller bubbles. The smaller bubbles provide a larger interfacial area for oxygen to diffuse out of the bubbles and reach the cells in the culture. The reverse happens for high dissolved oxygen levels.
2) Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600 nm used?
The spectrophotometer can be used to detect substances that are able to absorb light which in this case are cells by emitting light at a particular wavelength (600nm in this case). This emitted light passes through a cuvette which holds the sample and are absorbed by cells found within the sample. The light which is not absorbed is transmitted across the cuvette into the detector which detects the amount of this transmitted light. The reading is translated into absorbance which is the amount of light absorbed by the sample.
By using Beer-Lambert’s law,
A = ε x l x c
Where, A is the absorbance, ε is the extinction coefficient (ml mg-1 cm-1) l is the path length of light (cm), c is the concentration (mg/ml), the cell density can be calculated.
The cell density was measured by using light at wavelength 600nm as it is the best wavelength at which bacterial cells of a certain range of sizes can absorb light. If the wavelength is small, the light might be absorbed by things like proteins and DNA and the spectrophotometer will detect them too. Moreover, if the wavelength is too large, light will not be absorbed by the cells.
Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested?
GFP is a secondary metabolite which is produced during the stationary phase. Since GFP is produced at the stationary phase, the product should be harvested at the stationary phase just before the start of the death phase. The product was actually harvested at the stationary phase.
3) What are some advantages of using a computer control system? From the history chart (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control.
The advantages of using a computer control system are:
Ability to do data logging, data analysis and process control
Allow for automatic adjustment of variables to correct deviations from optimum levels.
Highly efficient in correcting deviations
Allow the use of lesser manpower as no extra labour is required to constantly monitor and make corrects to deviations from optimum levels of variables.
Computer control in our fermentation is relatively efficient. As soon as a deviation from the set point is detected, the respective corrective action is immediately take. For instance, when the dissolved oxygen decreases below the set point, the pO2 probe will detect this change and relay this information to the computer. The computer will then automatically calculate the degree to which the stirrer speed is to be increased to increase the dissolved oxygen levels in the fermenter. It is these kinds of controls that help keep the media and growth conditions as favourable as possible for cell growth and which makes computer control effective.
To perform scale-up fermentation process to increase the yield of green fluorescent protein by monitoring and altering the fermentation conditions.
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.
Ensure the tubing after sampling tube (tubing placed with one end in 70% ethanol) is clamped tightly.
Open the clamp on the tubing before the sampling tube (tubing connected with one end to the fermenter).
Draw the culture to approximately 1/3 of the sampling tube by pulling the syringe.
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.
Next, clamp the tubing before the sampling tube back tightly.
Take out the syringe and pull to fill it with air then place it back.
Unclamp the sampling tube and push the syringe to collect the sample in a falcon tube
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)
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.
The answers below adresses the questions in our lab manual.
(1) On media preparation:
a) Explain the purpose of each ingredient found in the LB media.
- Bacto-trptone is used to provide essential amino acids for the growing bacteria. - Yeast extract is used to provide plethora of organic compounds required for bacteria growth. - Sodium chloride provides sodium ions to E. coli for transport and osmotic balance.
b) What is the purpose of ampicillin?
It is to inhibit the growth of microbial contaminants.
c) Why is ampicillin added only after autoclaving?
If it is added before autoclaving, the ampicillin would be degraded when the autoclaving is done and render it ineffective.
(2) On equipment preparation:
a) What is meant by calibration of the pH probe?
The calibration process correlates the voltage produced by the probe. The calibration of the pH probe is performed with at least two standard calibration buffer solutions that are based on the range of pH values to be measured. Such calibration buffer solutions are available commercially of pH of 4.01, 7.00 and 10.00.
The probe must be calibrated at a zero point and a span point. Typically a probe will be calibrated at a pH of 7.0 and 10.0 or 4.01. The 7.0 calibration buffer solution is the Zero point calibration and the 10.0 or 4.01 calibration buffer is used for the Span adjustment. If you plan to measure pH of acidic solutions, calibration buffer pH 4.01 should be used and if you plan to measure pH of basic solutions, calibration buffer of 10.00 is used instead.
Calibration is done here by first placing the probe into the pH 7.00 buffer and allowing the pH reading to stabilize. For older pH meters, turn the calibration knob till pH 7.00 is shown, for modern pH meters just set it to calibration mode instead.
After calibrating at pH 7.00, rinse the probe with distilled water and do the same thing with the calibration buffers of pH 4.01 and/or pH 10.00. The pH probe should be rinsed with distilled water when moving from one buffer to the next.
b) Why is hydrochloric acid not suitable as a correction agent for pH?
It is not suitable as a correction agent for pH because depending on temperature and agitation, hydrochloric acid at a concentration above 10% will produce a hydrogen chloride vapor that is very corrosive when combined with water vapour in the air. This corrosive vapour can corrode the metals in the fermenter. If hydrochloric acid is used, there must proper ventilation where the gases can be easily dissipated.
c) What is meant by polarization of the pO2 probe? The pO2 probes contain 2 electrodes. They are the anode and the cathode which are both in contact with an electrolyte solution. The anode and the cathode are separated from the solution being measured, by an oxygen permeable membrane in which oxygen can diffuse through.
As oxygen passes by the membrane, reduction of oxygen occurs at the surface cathode which is exposed at the tip of the electrode. Oxygen molecules diffuse through the semi-permeable membrane and combine with the KCl electrolyte solution. The current produced is a result of the following reduction of oxygen at the cathode.
The pO2 probe measures oxygen tension amperometrically. The pO2 electrodes produce a current at a constant polarizing voltage which is directly proportional to the partial pressure of oxygen diffusing to the reactive surface of the electrode.
It is a type of positive displacement pump used for pumping a variety of fluid. It is used to remove liquid from the fermenter or to add acid or base, antifoaming reagent and nutrients to the culture gradually.
(3) On seed preparation:
a) What is the purpose of arabinose?
Arabinose is used as an inducer for the production of green fluorescent protein.
b) Describe the sterile techniques used in seed preparation.
The lab bench top was disinfected with 70% ethanol before the seed preparation was done. The bunsen burner was ignited to produce a flame for sterilizing of the cap of the cryovial (containing the bacteria culture) before opening it and to provide an aseptic zone to work in. A sterile plastic inoculating loop was used to inoculate the bacteria on to the agar plate. The agar plate cover was left half open when streaking was done instead of fully open to reduce the chance of contaminants from falling into the agar plate.
c) Why do we perform step wise scale up instead of transferring directly to the fermenter?
We perform step wise scale up to allow the cells to adapt to the culture conditions and grow to a large enough number to seed the fermenter. If we transfer the cell culture from the cryovial directly into the fermenter, there would be too little cells and the cells would not be able to grow or might have a very long lag phase.
