Cell Culturing and Fermentation

1. Cell Culturing and Fermentation Biotechnology II

2. Cell Culture • Definition: the in vitro growth of cells isolated from multi-cellular organisms • Process: Cells will continue dividing until they fill up the container; cell to cell contact stops cell division • Uses: vaccines, research of all kinds including stem cell, recombinant DNA, production of antibodies

3. Types of Cell used • Bacterial cells were first used to make genetically engineered products and they reproduce very quickly. Being prokaryotes makes them simple • Yeast cellsare eukaryotic but still fairly simple to use • Plant cellsarefound within tissue, more difficult to use, slow growing • Insect cellsarecloser to humans but not as fragile as mammalian cells • Mammalian cells are commonly used but they do grow slower and are more fragile than bacterial cells. Also have more complex nutrient requirements

4. Cell Culture Process • Contaminating the cell culture is area of concern • To avoid contamination mfg. techs do the following: • Sterilize all containers used • Change the media frequently • Media is the source of nutrition, pH indicator often times included in media to help monitor pH which is critical to cell survival • The media used depends on the type of cells to culture

5. Equipment Required • CO2 incubator: for proper atmospheric conditions • Laminar flow hood: for sterility • Hemocytometer: for cell counting • Inverted Microscope: for viewing cells on hemocytometer

6. Cell Culture Process • Count cells • Using a hemocytometer the cells are counted to determine if they are ready to harvest • Can stain cells with trypan blue to determine if they are still alive. All cells which exclude the dye are viable. All stained cells are dead.

7. Cell Culture Process • View cells • Inverted microscope is used to determine cell growth and activity • Adherent cells are ones which are stuck to the flask or vessel • Cell counting video for cell culture

8. Fermentation: Yeast on the Rise

9. Basic Reaction • Dextrose (glucose) = alcohol + CO2 • If using maltose as your sugar it first is converted to dextrose and then is broken down. • Quantifying alcohol or CO2 production are two standard approaches to measuring yeast fermentation.

10. Biofuel • Ethanol is referred to as a biofuel as it is manufactured by fermenting grains, plant biomass, or other microorganisms such as yeast. • Can be used directly as fuel although most often mixed with gasoline (gasohol). • Billions of gallons of ethanol are produced each year

11. Bioethics of Biofuels • Should grains be used as an energy source? • Each bushel (56 lbs.) yields 2.5 gallons • Amt. of farmed land has declined and population has grown 50% over last 25 years. • 6% of US corn crop is used for ethanol production • There are environmental benefits of ethanol compared to petroleum

12. Basics of apparatus to measure CO2 • Fermentation reaction in a vessel (flask or syringe) • Need to measure volume displaced from CO2 production. Could be pipet, graduated cylinder, syringe

13. Chemostat Design Sketches Water in pipet Water in cylinder Fermentation reaction Fermentation reaction Record starting volume of water and measure displaced over time

14. Formulation Components • Sugar source: glucose, sucrose, maltose, lactose • Water: approx. 10mL/g of glucose • Yeast: approx. 8g/100 mL • Could make up as a solution (add 1:1 w/sugar soln.) • Could add dry powder (approx. 0.8gm/10mL)

15. Experimental Design • Need to modify different variables to determine optimum formulation • Temperature • Type of sugar • Type of yeast • Qty of sugar and yeast • pH

16. Experimental Design • Need to run controlled experiments • Ensure apparatus is working consistently • Record all data • Only modify one variable at a time • Analyze data and determine next experiment

17. Data Analysis • Plot data on graph paper • Record mL of CO2 on y axis, time on x axis 0 min 0.1 mL 2 min 0.3 mL 4 min 2.0 mL 5 min 4.0 mL time Find the straight part of the curve and calculate the rate of CO2/min

18. Fermentation and Cellular respiration • Fermentation yields energy via cellular respiration • What is cellular respiration? • It requires O2 • Glycolysis • Krebs cycle • Electron transport

19. Cellular Respiration

20. So what is fermentation? • An enzymatic process that allows cells to obtain energy from carbs even in the absence of oxygen. • If O2 is not present cellular respiration is shut down • Glycolysis still occurs if pyruvate is diverted to the fermentation pathway (NADH converted to NAD)

21. Which is better w/without O2? • Go back to Candy bar picture.

22. So, how do yeast cells live in a fermenter? • It sounds like there is too little ATP to keep us alive in an anaerobic environment? • For higher organisms this is true. But what do you know about yeast? • It is prokaryotic and uses a small amount energy • Yeast can import and metabolize carbs very rapidly which compensates for small production of ATP • Because yeast metabolizes carbs so quickly it is a great generator of CO2 and that is why it is commonly used!

23. Report and Graphs • Final report should include the following: • Hypothesis • Procedure • Results • Discussion • Conclusion Questions • Cited References

24. Report and Graphs • Graph some of your data such as the difference in fermentation rates between the different sugars, yeast, temp., etc. • Data table of time and mL displayed for each experiment • Could graph fermentation rate vs. sugar concentration