Cell Culturing and Fermentation

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Cell Culturing and Fermentation

• Biotechnology II

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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

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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

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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

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Equipment Required

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

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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.

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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

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Fermentation Yeast on the Rise
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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.

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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

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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

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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

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Chemostat Design Sketches
Water in pipet
Water in cylinder
Fermentation reaction

Fermentation reaction
Record starting volume of water and measure
displaced over time
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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 11 w/sugar
  soln.)
• Could add dry powder (approx. 0.8gm/10mL)

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Experimental Design

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

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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

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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
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Fermentation and Cellular respiration

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

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Cellular Respiration
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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)

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Which is better w/without O2?

• Go back to Candy bar picture.

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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!

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Report and Graphs

• Final report should include the following
• Hypothesis
• Procedure
• Results
• Discussion
• Conclusion Questions
• Cited References

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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