cell culturing and fermentation biotechnology ii
TRANSCRIPT
Cell Culturing and Fermentation
Biotechnology II
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
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, 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
Equipment Required
CO2 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 + 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.
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 CO2
Fermentation reaction in a vessel (flask or syringe) Need to measure volume displaced from CO2 production.
Could be pipet, graduated cylinder, syringe
Chemostat Design Sketches
Fermentation reaction
Water in pipet
Fermentation reaction
Water in cylinder
Record starting volume of water and measure displaced over time
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)
Experimental Design
Need to modify different variables to determine optimum formulation Temperature Type of sugar Type of yeast Qty of sugar and yeast pH
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 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
Fermentation and Cellular respiration
Fermentation yields energy via cellular respiration What is cellular respiration?
It requires O2
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 O2 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 O2?
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
CO2 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 mL displayed for each experiment Could graph fermentation rate vs. sugar concentration