LAB 3 BIOREACTOR

   

  ASSALAMUALAIKUM..AND..HELLO TO ALL READERS

    We are from third year student of Bioprocess Technology in USM. This blog was created as a lab report for our experiment 3 and we dedicate to our lab report to our lovely lecturer  MRS WAN NADIAH BINTI WAN ABDULLAH and MR SHAMAN who was gave a lot information about bioreactor.....THANK YOU VERY MUCHHHH... 

                            HANDSOME MR SHAMAN

BEAUTIFUL MRS WAN NADIAH

Before that, let us introduced our group members

  

LASTLY......

FARAH

Below is our lab report...hope you guys enjoy reading it and get good information...

     INTRODUCTION

    A bioreactor is a vessel in which is carried out a chemical process which involves organisms or biochemically active substances derived from such organisms

   Bioreactors are commonly cylindrical, ranging in size from some liter to cube meters,and are often made of stainless steel.

  Bioreactor design is quite a complex engineering task. Under optimum conditions the microorganisms or cells will reproduce at an astounding rate. The vessel's environmental conditions like gas (i.e., air, oxygen, nitrogen, carbon dioxide) flowrates, temperature, pH and dissolved oxygen levels, and agitation speed need to be closely monitored and controlled.

    

METHODOLOGY

Preparation of media and reagents

1.Yeast extract, peptone and glucose is weighed in the ratio of 3:1:1 and is     added into a flask.

2.300ml of distilled water is added.

3.The solution is stirred until homogeneous solution is obtained.

4.The opening of flask is closed by using cotton wool wrapped with aluminium foil. (figure1)

Figure 1

5.The media is autoclaved at 121^oC with 15 minutes retention time.

6.The flask is stored in an incubator shaker under the conditions of      200rpm and 30^oC overnight.

Setting up bioreactor

1.The bioreactor is cleaned with distilled water to remove any residue from previous usage.

2.Upon cleaning, the orifice of the sparger is inspected for any blockage by running water through the air sparger tubes. (figure 2)

                                                 Figure 2

3.The agitator and agitator driver is lubricated using glysol solution until the lubricant flooded at the top of the drive.

                                                Figure 3

4.The bioreactor is assembled. Glass vessel is connected to the base unit, lifting with the handles of the support frame and the stud is placed at the back of the vessel into the metal fork on the support frame.

5.The drive arm is lowered into the horizontal position.

6.The side shield is replaced to keep it out together with the base unit.

7.The exit gas cooler is fit into a free port and the water connecting tubing is checked for length and connections are made ready for the next day.

8.The pO₂ electrode is located and the green plastic end cap is removed. The bottom metal section is unscrewed and the membrane cartridge inside is checked whether has liquid electrolyte in it. If not, up to half is topped from the bottle provided.

9. The pO₂ electrode is fit into the vessel loosely. The top plastic cap is removed from the electrode.

10. the connection point between the vessel and head plate is smeared with high vaccum grease (figure 4).

 Figure 4

11. All internal part of the bioreactor(vessel, sparger, impeller, agitator and head plate) is sprayed with alcohol solution (figure 5). The head plate and vessel is then connected.

Figure 5

12. Two reagent bottles are prepared (anti-foam and base). Anti-foam is poured into one of the reagent bottles while the one for the base is filled with distilled water prior to autoclave.

13. Yeast extract: Peptone: Glucose (YEPG) medium is prepared with the same ratio of 3:1:1 and distilled water is added until the volume reaches 900ml. The medium is then added into the vessel.

14. All openings, filters and pump are wrapped with aluminium foil and all tubings are clamped before autoclave. (Figure 6)

Figure 6

15. The bioreactor is autoclaved for 15 minutes at temperature of 121^oC. (figure 7)

                                                Figure 7

Inoculation into the media and fermentation.

1. After autoclaving, all aluminium foil is unwrapped and all clamps are removed.

2. Base is filled into the reagent bottles and both the pumps of the reagent bottles are fitted to the correct motors and the tubing connected to the multi-way inlet and clamped closely. (figure 8)

                                              Figure 8

3. Temperature, pH and pO₂ electrode are fitted into the vessel and are calibrated.

4. Inoculum is poured into the vessel under aseptic condition.

5.Speed is adjusted to 300rpm and cascade, pH and pO₂ are all on. Fermentation is run and the sample is collected every 2 hours. (figure 9)

                                              Figure 9

RESULT

                                                               

GRAFT READING

                                                             PART 1

 PART 2

 PART 3



Result for yeast’s OD:

Times	OD
Blank	0.000
3.30pm	0.148
5.30pm	0.793
7.30pm	0.803
9.30pm	0.824
11.30pm	0.811
1.30am	0.940
3.30am	0.934
5.30am	0.901
7.30am	0.324
9.30am	0.171

RESULT OF GLUCOSE READING

TIME	CONDITION	READING (mg/dl)
3.30pm		267
5.30pm		246
7.30pm		226
9.30pm	Diluted	182
11.30pm	Diluted	Low
1.30am	Diluted	Low
3.30am	Diluted	low
5.30am	Diluted	low
7.30am	Diluted	Low
9.30am	Diluted	low

DISCUSSION

     The inoculum that used in the experiment was 150ml of YEPG, 1% yeast extract (10mg/ml), 2% peptone (20mg/ml) and 2% glucose (20mg/ml). While the culture medium used was 10 times of the materials that used to prepare the inoculum.

     

    According to the results of my group obtained, the OD of the yeast (Saccharomyces cerevisiae) was showing increased from 0 hour (OD= 0.148) to 10 hours (OD= 0.940), this indicated the log phase of the yeast cells. After 10 hours growing, the yeast cells were reached the stationary phase from 10 hours (OD= 0.940) until 14 hours (OD= 0.901). After the stationary phase ended, the yeast cells were reached the death phase in which the OD was sharply decrease from 0.901 to 0.321 and finally reached 0.171. The growth curve of any organisms including the yeast cells that we used in this experiment- Saccharomyces cerevisiae are following the sigmoid curve, which start from lag phase, log phase, stationary phase and finally reach the death phase.

      From the results we also can see that the glucose concentration in the culture medium was gradually decreased from 267mg/dl to low concentration (undetectable). This is because the yeast cells required the glucose as the substrate to carry out aerobic fermentation to increase the biomass. Depleted in the glucose will decrease or stop the growth of yeast.

    

     Alkali was used in the experiment because need to regulate and maintain the pH of the medium at pH 6, which is slightly acidic condition. Due to the production of acetic acid during the aerobic fermentation by the yeast, the pH of the medium will dropped to below 6, the unsuitable and too acidic pH will affected the yeast growth, thus the alkali was playing the role in increase the pH of the medium back to pH 6, an optimum pH for the optimum growth of yeast.

     

     Anti foam agent was also been used in the experiment, this is because a lot of foam will be produced during the fermentation process, especially in the aerobic fermentation like we carried out in this experiment. The role of this anti foam agent is to reduce the foam formation in the culture medium. The foam will affect the yeast cells growth and make the medium over flow from the vessel, results in the contamination and waste of the product.

    Because the process of fermentation we carried out was aerobic, which mean that oxygen was required to increase the yeast biomass, so the inlet air was needed to pump into vessel to succeed the fermentation process. 3.3vvm was the level that we used in the experiment in order to pump in the sufficient amount of oxygen into the medium for yeast consumption. The inlet and outlet air were required filter (the pore sizes were 0.2 nanometer) to filter out the microbes normally made up of bacteria and fungus from entering into the vessel which will cause contamination and escaped out to the surrounding air. Exit air cooler was used to condense back the medium which had been evaporated back into the vessel, this is to prevent the excess products loss, even though the efficiency of the cooler is around 95%. Insufficient in oxygen will lead the yeast to carry our anaerobic fermentation, which convert the glucose into bioethanol, no biomass will produce.

      

    The speed of agitation applied during the shake flask culture will influence the maximum growth rate of the yeast when cultured in the bioreactor. The faster the speed of agitation during the shake flask culture (300rpm), the faster the yeast increase in the biomass. If the rpm was at 200 during the shake flask culture, the yeast will show slow growing when cultured in the bioreactor which the speed of the impellers also maintained at 200rpm. This is due to the insufficient obtained in the oxygen and also glucose by the yeast to achieve optimum growth.

    

      The optimum temperature for the yeast to grow is 30 degree celcius. The heat will be produced during the aerobic fermentation, which the temperature of the medium will increase. Unsuitable temperature will affect the yeast growth, so the jacket water inlet is required to regulate and maintain the medium’s temperature at 30 degree celcius.

                                CONCLUSION

The setting up of bioreactor needed to be done carefully to ensure smooth and successful fermentation process. Aseptic technique is vital to ensure maximum yield of product by preventing contamination. At the end of this experiment, better understanding on bioreactor operation was obtained and experiences were gained for the handling of bioreactor during fermentation process.

                                 

 REFERENCE:

1.

 1.Transgalactic Ltd, 2005. What Is Bioreactor?. [online] Available at: <http://www.bionewsonline.com/o/what_is_bioreactor.htm]. [Accessed 5 November 2012 ]

           THANK YOU........