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, 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
Water in cylinder
Water in pipet
Fermentation reaction
Fermentation
reaction
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
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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