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First day Experiment 1

Objectives To describe the steps to prepare a bioreactor

On our first day of the practical, we were introduced to the bioreactor parts and how it functions. The picture above depicts a 5L bioreactor and a diagram to illustrate it. From this introduction, we understood the required settings and proper techniques to start a run and we prepared the equipment. Next, we prepared a 2L media of LB broth and divided into 2 parts, 1900ml into the bioreactor and 100ml into a culture flask. The broth was then autoclaved together with the bioreactor. During autoclaving, the high temperature will affect the quality of it. Prior to addition of the LB broth, the pH meter was calibrated.
Part of Bioreactor Motor Impeller Sparger Baffles Exhaust Air Filter Inlet Air Filter Purpose To spin the impeller. To stir the culture to provide a homogenized/ uniformed environment for the cells to grow. Provide air (oxygen) for the cell culture. Prevent whirlpool effect in the bioreactor. Provide better and random mixing of culture. Prevent too much release of microorganisms into the air. Filter & Protect the air. Filter the air going into the bioreactor. Filter away micro-organisms.

Condenser RotaMeter Pressure Gauge Temperature Probe Cooling Jacket pH Probe Dissolved Oxygen Probe Level Probe Foam Probe Acid Base Antifoam Sampling Tube Control Panel

Condense water vapour to prevent evaporation to occur in the bioreactor. Measure the airflow rate in the bioreactor. Measure the pressure of air inside the Bioreactor. Measure the temperature of the culture. Pump in water hot/cold to regulate temperature of the bioreactor. Measure pH of broth. Measure amount of dissolved oxygen in the culture. Measure the level of culture. Measure the amount of foam caused by stirring. Regulate pH if it gets too basic. Sulphuric acid will be added to increase the phi Regulate pH when it gets too acidic. Sodium Hydroxide will be added to decrease the phi Prevent foam from forming in the media. Allows us to collect samples from bioreactor aseptically. Main control, Controls the vessels in the bioreactor.

Questions 1. State the differences you would observed between a microbial bioreactor and a mammalian cell bioreactor.

2. Study the work flow on page 1 of your practical guide. Describe the typical activities that are performed for each stage in the fermentation process. Familiarisation with the bioreactor and its operation to familiarize with the bioreactor and recognize its various components, as well as the functions. In addition, we also learn how to set up the bioreactor. Media and Seed Culture Preparation In this experiment, we set up the necessary equipments (the bioreactor etc.), and make the media needed for the fermentor. Seed culture is done where the desired product is cultured and grown to required state. In this case, the desired product is pGLO transformed E.coli (E.coli with glowing genes that allows the bacteria to glow when seen under UV-light). Media preparation is done by mixing the required components and after which, placed in the bioreactor. This media is important as the ingredients used is vital to sustain the growth of cells, serving as nutrients for the cells. Fermentation, Inoculation and Monitoring The inoculated pGLO transformed E.coli was placed in 100ml of broth and left to proliferate. After which, it was placed in the fermenter for further growth through the process of fermentation. This allows various requirements to be achieved so that the cell can grow properly and be used for scale up. When the fermentation is done, samples are collected hourly for 8 times and the sample is monitored by obtaining its absorbance values using a spectrophotometer. The fermentor parameters was then calculated. Harvesting and Turnaround It was harvested into an empty 100ml bottle whereas the remaining is harvested into a 2L bottle. Lastly, the system is shut down and dismantled for cleaning and sterilization for the next set-up.

Experiment 2 : Media Preparation

To prepare the media for seed culture and scale-up fermentation

Preparation of Media 1. 2.0L of Luria Bertani(LB) Medium was prepared. 2. 100ml of the medium was transferred to a shaker flask and the remaining 1900ml was transferred into the 2L bioreactor. 3. The medium was autoclaved at 121oC for 20 minutes and was allowed to be cooled. Media preparation

Questions 1) explain the purpose of each ingredient found in the LB media. Bacto-tryptone 10g This provides the bacterial cells with the necessary peptides and peptones required. Yeast extract 5g This provides the E.coli cells with the necessary vitamins and trace elements required. NaCl 10g This contributes to the osmotic stability and provide essential ions of the bacterial cells. dH2O 1000ml Distilled H2O is used as a solvent. pH 7.5 This provides buffering capacity to the media during bacterial cultivation. 2) What is the purpose of ampicillin and arabinose? The ampicillin allows selection of the plasmid, pGLO. Therefore only bacteria that are ampicillin resistant, pGLO plasmid will grow. Arabinose is an inducer of the expression of the GFP. This is to prevent the slow growth of the GFP that results from over expression of the pGLO plasmid. 3) Why is ampicillin and arabinose added only after autoclaving?
This is because ampicillin and arabinose are heat sensitive products which may be denatured during the process of autoclaving which uses heat. Therefore it has to be added after autoclaving.

4)Show that from the ampicillin stock solution (5mg/ml), 2ml is to be added to the seed culture medium and 38ml is to be added to the fermentation medium. Volume of seed culture medium: 100ml Final concentration: 100g/ml = 10000g/100ml = 10mg/100ml
10-5=2 2ml of ampicillin stock solution (5mg/ml) is needed to attain a final ampicillin concentration of 10mg.

Volume of fermentation medium: 1900ml

Final Concentration: 100g/ml= 190mg/1900ml 190/5=38 38ml of ampicillin stock solution(5mg/ml) is needed to attain a final concentration of 190mg. 5) Show that from the arabinose stock solution (20%), 1ml is to be added to the seed culture medium and 19ml is to be added to the fermentation medium. Seed Culture: M1V1=M2V2 (20%)(Arabinose stock solution to be added)=(0.2)(100ml) V1=100ml Fermentation Medium: M1V1=M2V2 (20%)(Arabinose stock solution to be added)=(190mg)(1900ml) V1=19ml

Experiment 3
Objectives To prepare a bioreactor for fermentation

pH calibration

pH calibration

Manual Calibration did using the control panel.

Pouring of LB broth into bioreactor for Autoclaving

Preparation of Bioreactor 1. The pH electrode was calibrated with the standard buffer solution 2. The pH probe, pO2 probe, foam and level probe was installed into the top plate. (level was checked) 3. The addition agent tubes for acid, base and antifoam was connected. (level was checked) 4. Other accessories such as exhaust condensers, air inlet and exhaust filters and manual sampler unit was installed. (water jacket was checked) 5. Sterilization: All cables except for the temperature probe were disconnected. All silicone tubings except for exhaust filter and female STT coupling of sampling unit was clamped. 6. All filters and sockets were covered with aluminum foil. The setup was autoclaved at 121C for 20 minutes. 7. pO2 electrode was polarized for 6 hours and was calibrated by aerating with nitrogen. 8. Additional lines were connected to peristaltic pumps and the bioreactor was switched to auto/manual. How The Bioreactor Operates. 1. The bioreactor should operate in a way that it pumps in the acid and base automatically into the reactor whenever the system detects that the pH level inside the reactor is not as set. 2. Whenever the Bioreactor control panel detects the environment inside the reactor has insufficient air, the control panel will automatically either o Pump in more air or o Make the impeller stir faster The purpose of making the impeller to operate faster is to disrupt the bigger bubbles of air inside the bioreactor so that the air will be evenly distributed inside the bioreactor. Hence control panel do not have to sparge in air and cells will remain in its optimal condition to proliferate. 3. Stirring causes the Bioreactor to heat up, whenever the Bioreactor detects that the temperature increases, the control panel will pump in cold water into the cooling jacket to avoid the Bioreactor from overheating Questions 1) What is meant by calibration of the pH probe. It is a process to rectify the instrument before using it in our experiment. 2) Why is hydrochloric acid not suitable as correction agent for pH It is because HCl is a corrosive acid, when added as a balancing agent, it may stress the cells and affect the cells performance in the process.

3) What is meant by polarization of the PO2 probe To measure dissolved oxygen in the culture it requires cathode, anode, electrolyte solution and a gas permeable membrane. The material of the membrane is specially selected to allow oxygen to pass through. Oxygen is consumed by the cathode which will create a partial pressure across the membrane and then the oxygen will diffuse into the electrolyte solution. Reduction of oxygen is obtained between 400 to 1200 mV, hence there is a need for a voltage of around 800 mV. Thus, a DO meter actually measures the pressure of oxygen in water and it can be used to measure DO in any medium. 4) What is a peristaltic pump. Peristaltic pump is a type of positive displacement pump used for pumping variety of fluids. The fluid is contained within a flexible hose or tube fitted inside the pump casing. The actual pumping principle, called peristalsis, which is based on alternating compression and relaxation of the hose or tube. It draws content in and propels the product away from the pump. This process makes a peristaltic pump an accurate dosing pump or metering pump. With an equal amount of liquid dosed each time.

Second day
Experiment 4
Objectives To prepare seed culture for scale-up fermentation Background of Green Fluorescent Protein Green Fluorescent protein (GFP) is the most widely studied and exploited protein. It exhibits green fluorescence when exposed under ultraviolet light.GFP is used in almost all organisms and all make our cellular compartments. It can be used as a reporter gene, cell marker or fusion tag. The GFP used as a reporter of expression for the E.coli bacterial cells that is used. Preparation of Seed Culture 1. pGLO transformed Escherichia. Coli was retrieved from -80C freezer. 2. The E.coli was streaked onto a LB/AMP/Ara plate ( ampicillin 100ug/ml and arabinose 0.2%) and incubated for 24 hours. 3. Several colonies of pGLO transformed E.coli was obtained and was transferred to the shaker flask 4. The shaker flask was placed in a shaking incubator and was incubated at 32C for 12 hours. 5. This was then used to inoculate the fermentor for scale-up fermentation.

We prepared a seed culture using cells cultured on a petri dish, the petri dish contain colonies which has the target protein GFP inside. After which, we scraped off a single colony and inoculated into a 100ml culture flask which contains the LB broth(nutrient) After the single cell colony is mixed into the nutrient together with ampicillin and arabinose, it was then incubated for 12 hours for the cells to grow. Ampicillin and arabinose are selective agents that is added to selectively grow cells in the culture that are antibiotic resistant and possesses GFP(Green Fluorescent Protein) properties. Ampicillin and arabinose are heat sensitive so it is added together with the cell-cultured flask after autoclaving the bioreactor. The acid (sulfuric acid) and base(sodium hydroxide) is then connected to the bioreactor aseptically using a rubber tubing through a peristaltic pump connected to a computer system which allows automated balancing of pH without contaminating it.

Acid and base for pH balancing

peristaltic pump

Observing cell growth

The incubated culture flask was observed under UV-light to perceive an estimation of the cells growth. If observation shows a positive result on the growth of the cells, culture would fluorescent under UV-light. This will then allow us to carry on to the next step by transferring it into a bottle which can then be connected to the bioreactor that allows aseptic inoculation. The procedure from cell inoculation to harvesting was recorded and can be referenced at: http://youtu.be/UctHBDyDFTE Questions 1) Describe the sterile techniques used in seed preparation. Seed preparation was done in a laminar fume hood. Hands were washed with soap, donned with gloves and ethanol was sprayed onto the gloves prior before proceeding to work in the hood. In addition, the flask and other required equipments were also swabbed with ethanol and sterile inoculation loops were used. 2) Why do we perform step-wise scale-up instead of transferring directly to the fermenter? Step-wise scale-up allows bacteria to adapt to the culture conditions and optimal growing conditions can be monitored. It also ensures that it is the specific bacteria we want that will produce GFP, without any contamination from other microbes. On the other hand, if the bacteria culture is transferred directly into the fermenter, it may introduce contaminants or other microbes into it.

Third day
Experiment 5
Objective To carry out scale-up fermentation process to increase the yield of desired protein product. Procedure for starting up the Fermentation 1. 2. 3. 4. 5. The medium was brought to cool. Ampicillin was added to a final concentration of 100ug/ml. Arabinose was added to a final concentration of 0.2%. The cooling jacket was completely filled and the air supply was turned on. Using the control panel, the following parameters was set as follow: - Temperature - pH 7.5 - Stirring speed Auto - Oxygen Auto - Airflow 6. A blank sample was collected prior to inoculation. 7. The seed culture was inoculated into the fermenter.

Inserting arabinose

inserting ampicillin

seed culture

Experiment 6
Objective To monitor cell growth and product formation through manual sampling and computer data logging. Procedure 1. 5ml of blank sample was collected. And then 8 fractions of the sample each containing 5ml was collected was every hour. 2. The absorbance reading was obtained for all 8 fractions using a spectrophotometer, using an optical density of 600nm.

Measuring OD using mass spectrophotometer

Sample reference 1 2 3 4 5 6 7 8

Hours 0 1 2 3 4 5 6 7 8

OD600 0.286 0.476 0.581 0.851 1.095 1.407 1.625 1.76 1.76

ln (x(x0) 0 0.509426 0.708759 1.09042 1.342518 1.593223 1.737271 1.817077 1.817077

Questions 1) Explain the control philosophy for pH, temperature and dissolved oxygen as as used in the fermentation process. The pH, temperature and dissolved oxygen levels are controlled by an automated system which is connected to the control panel of the vessel. It reads the condition of the vessel and adjust these factors accordingly to suit the set conditions. pH level is decrease by adding acid- sulfuric acid and increase by addition of base-sodium hydroxide. Similarly, oxygen level is increased by sparging in air or stirring the vessel whilst temperature is regulated through the water jacket which surrounds the cell culture circumference. 2) Describe the principle of the spectrophotometer which was used to determine the cell density (OD600), Why was 600nm used? This is because, at 600nm wavelength, the cells which produces the GFP which we are interested in, absorbs strongly at this wavelength. Therefore it can give a more accurate qualitative analysis of our product. 3) Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested. It is a primary metabolite. It should be harvested during the exponential phase. It was harvested during the exponential phase, therefore it can be observed that the OD increases as the sample was taken longer after the inoculant. 4) What are some advantages of using 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 computer controlled system is able to substitute humans supervision and it can accurately adjust the condition of the vessel by reading the different factors such as pH, temperature and dissolved oxygen and balancing with connected equipments. It is a very effective system whereby supervision labour is reduced and it can adjust conditions of the vessel faster and more accurately.

Experiment 7
Objective To harvest the fermentation broth for downstream processing and clean the equipment for the next batch. Procedure for Harvesting & Turnover 1. Inoculum in the flask was transferred into a sterilized bottle in the hood 2. Inoculum inside the bottle was transferred into the bioreactor using the peristaltic pump 3. After adjusting the bioreactor system, and setting the system to the Manual mode, Bioreactor was ready to operate 4. Sample was collected at 0 hour at first then every 1 hour 8 fractions of the sample was collected. All the 8 fractions were viewed under the UV light for GFP detection.

sample collecting

On the third day, we transferred the inoculum from the shaker incubator to a sterilized bottle in the hood. The transferred inoculum was then added into the bioreactor using the peristaltic pump, the peristaltic pump allows the inoculum to enter the bioreactor in a aseptic form. A Probe for dissolved oxygen (DO) can be calibrated as the bioreactor requires a 100% dissolved oxygen level environment, when the DO level is low, air is sparged into the bioreactor and to ensure that there is adequate DO supply for the cells. Before starting the bioreactor, we have to check the pH level inside the bioreactor, by doing so it is made sure that there is adequate amount of acid(sulphuric acid) and base( Sodium Hydroxide) solution. When the Bioreactor is operating the Acid and base is pumped into the bioreactor whenever the system detects that the bioreactor pH is not as set. The first fraction was collected at 0 hours, and subsequently every one hour, a total of 8 fractions are collected after the entire experiment. The last fraction should contain the most GFP cells as compared to the first fraction, hence when viewed under the UV light, the longer the waiting time for collection of the fraction the brighter the cells would be.

Results and Discussion on Fermentation

2.5 2 ln (x(x0) 1.5 1

Growth rate of cells at intervals

y = 0.2292x + 0.2628

gradient= 0.2292
0.5 0 0 2 4 Time (h) 6 8 10

Exponential phase equation:

Graph is plotted

[y-axis], to time(t) [x-axis].

Gradient of graph

Therefore, from the gradient we can obtain the maximum specific growth rate of the cells.

8th 1st
Final fractions collect 1~7 8th and 1st fraction

After collecting the 8 fractions of sample for the experiment, it is observed that the fluorescence intensity increases as the time increase. The first fraction did not show much fluorescence intensity because it was collected immediately after adding the culture to the bioreactor. The last fraction had the highest fluorescence intensity. Because as the time increases, the cell growth in the bioreactor increases hence producing more cells. Hence, it is concluded that cell growth increases as the time increases, so overall the last fraction has the strongest fluorescence intensity due to its highest concentration of cells.

Personal Learning Points about the fermentation

We have attained technical skills to operations of the bioreactor. In addition, we have a clearer understanding on how the upstream process of fermentation works. During the process of sample taking, unexpected situations whereby the pressure in the bioreactor cause the culture to flow out into the sample tube, before we could even draw it out from the syringe, which could have caused contamination to the entire culture. Fortunately we manage to stop the flow-out and collect our sample in time without any issues.