Cell Culturing and Fermentation Biotechnology II Cell Culture
Cell Culturing and Fermentation Biotechnology II
Cell Culture § Definition: the in vitro growth of cells isolated from multicellular 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
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 cells are eukaryotic but still fairly simple to use § Plant cells are found within tissue, more difficult to use, slow growing § Insect cells are closer 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
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, p. H indicator often times included in media to help monitor p. H which is critical to cell survival § The media used depends on the type of cells to culture
Equipment Required § CO 2 incubator: for proper atmospheric conditions § Laminar flow hood: for sterility § Hemocytometer: for cell counting § Inverted Microscope: for viewing cells on hemocytometer
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.
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
Fermentation: Yeast on the Rise
Basic Reaction § Dextrose (glucose) = alcohol + CO 2 § If using maltose as your sugar it first is converted to dextrose and then is broken down. § Quantifying alcohol or CO 2 production are two standard approaches to measuring yeast fermentation.
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
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
Basics of apparatus to measure CO 2 § Fermentation reaction in a vessel (flask or syringe) § Need to measure volume displaced from CO 2 production. Could be pipet, graduated cylinder, syringe
Chemostat Design Sketches Water in cylinder Water in pipet Fermentation reaction Record starting volume of water and measure displaced over time
Formulation Components § Sugar source: glucose, sucrose, maltose, lactose § Water: approx. 10 m. L/g of glucose § Yeast: approx. 8 g/100 m. L § Could make up as a solution (add 1: 1 w/sugar soln. ) § Could add dry powder (approx. 0. 8 gm/10 m. L)
Experimental Design § Need to modify different variables to determine optimum formulation § Temperature § Type of sugar § Type of yeast § Qty of sugar and yeast § p. H
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
Data Analysis § Plot data on graph paper § Record m. L of CO 2 on y axis, time on x axis 0 min 0. 1 m. L 2 min 0. 3 m. L 4 min 2. 0 m. L 5 min 4. 0 m. L time Find the straight part of the curve and calculate the rate of CO 2/min
Fermentation and Cellular respiration § Fermentation yields energy via cellular respiration § What is cellular respiration? § It requires O 2 § Glycolysis § Krebs cycle § Electron transport
Cellular Respiration
So what is fermentation? § An enzymatic process that allows cells to obtain energy from carbs even in the absence of oxygen. § If O 2 is not present cellular respiration is shut down § Glycolysis still occurs if pyruvate is diverted to the fermentation pathway (NADH converted to NAD)
Which is better w/without O 2? § Go back to Candy bar picture.
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 CO 2 and that is why it is commonly used!
Report and Graphs § Final report should include the following: § Hypothesis § Procedure § Results § Discussion § Conclusion Questions § Cited References
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 m. L displayed for each experiment § Could graph fermentation rate vs. sugar concentration
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