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Summary
This video explores the fascinating world of fermentation, a process widely used to create products such as food, beverages, pharmaceuticals, and energy sources. Fermentation involves using specific cells like bacteria, yeast, and mammalian cells to produce chemicals, vitamins, and even proteins. The video delves into how these cells are grown and harvested in industrial settings and provides a detailed guide on producing green fluorescent protein from E. coli bacteria. Through the fermentation process, the video highlights the importance of controlled conditions and rigorous monitoring to ensure successful product creation.
Highlights
Fermentation turns raw materials into useful products like foods and biopharmaceuticals. 🧀🥛
Bacteria and yeast are commonly used cells in fermentation, while mammalian cells are used for vaccines. 🦠🧬
The process requires careful scaling from small seed stocks to large production vessels. 🔬
Key conditions like temperature, pH, and nutrient levels must be strictly controlled. 🌡️⚙️
The GFP fermentation example shows how bacteria are genetically tweaked to produce desired proteins. 🟪🌟
Key Takeaways
Fermentation is used to make diverse products, including food, drinks, and biopharmaceuticals. 🧀🍷💊
Different cells like bacteria, yeast, and mammalian cells are used based on the desired product. 🦠🧬
Scaling up the cell culture is key to the industrial fermentation process. 🔬
Green fluorescent protein (GFP) production illustrates a typical fermentation pathway. 🟢
Precise conditions and monitoring are crucial for successful fermentation. 🌡️⚙️
Overview
Fermentation is a magical process that converts raw materials into things we enjoy daily, from bread and yogurt to energy sources and medicine. It might sound like letting things go bad on purpose, but really it's a brilliant way to grow cells like bacteria and yeast in a controlled environment. Each type of cell, like E. coli, is selected for its superpower to produce a specific product, and then the party begins!
Once the cells are chosen, the magic of scaling up happens. Starting from a mini seed stock, the cells are first grown in small amounts of media until they're ready for the big leagues. Ultimately, these cells take center stage in a bioreactor, producing everything from amino acids to cool glowing proteins! Just like baking a cake, all the ingredients and the conditions need to be just right.
The video highlights the GFP production process involving E. coli engineered to shine bright like a green beacon when a gene is turned on. Throughout, the secret lies in meticulous monitoring—oxygen levels, pH balance, and nutrients are closely watched as the cells grow. The video is a captivating dive into the wonder of fermentation and the precision required to produce the things that enhance our lives.
Bioprocessing Part 1: Fermentation Transcription
00:00 - 00:30 we all know something about fermentation
it's a process used countless times each day to make a variety of dairy products baked
goods and beverages we sometimes think of it as
00:30 - 01:00 letting foods go bad but in a controlled way
with a little help milk becomes yogurt bread Rises and grains decompose creating alcoholic
beverages and alternative fuels but looking at these examples only gives us a clue as to
what's really happening and how we can use the power of fermentation to cost-effectively
create a broad array of biological products so
01:00 - 01:30 what is fermentation a cell can be thought of as a
micro Factory these cells can be bacteria fungi or specific cells from mammals plants or insects in
biotechnology these cells are used to manufacture a product in a process called fermentation for
yogurt butter milk and cheese we use bacteria to
01:30 - 02:00 make breads and alcoholic beverages we use yeast
a fungus and the production of some vaccines require the growth of mammalian cells that are
infected with a specific virus the product the cells manufacture is usually a chemical the
cells contain naturally or a substance that the cells have been genetically altered to
create or even a metabolic waste product of the organisms growth like one of our examples
alcohol there are too many everyday products
02:00 - 02:30 created by commercial-scale fermentation
to even list but some common ones include amino acids biopharmaceuticals dyes enzymes
food products lipids steroids and vitamins fermentation is a reasonably simple process a
cell is selected based on its ability to produce
02:30 - 03:00 the desired product a seed stock of cells is put
into a small amount of media media provides the nutritional products the cell needs to grow
when the population of cells has grown and consumed most of the nutrients it's moved into
a larger vessel with more growth media and the process repeats this scaling up is complete when
the quantity of cells is large and healthy enough
03:00 - 03:30 to transfer into a production vessel often
referred to as a bioreactor or fermenter with plenty of fresh media now available in under
tightly controlled conditions the cells grow and manufacture product when the fermentation is
complete the product is harvested fermentation is known as an upstream biotechnology process
it occurs early in the production flow before recovery purification formulation filling and
packaging to better understand the fermentation
03:30 - 04:00 process we should first find out a little bit
about the cells we use and what they may require to reproduce and stay healthy different cells
have different needs some are aerobic they need oxygen while others are anaerobic and do not
require oxygen all cells require nutrition a properly formulated media contains the necessary
nutrients to allow cells to grow and produce the
04:00 - 04:30 fermenter mixes the cells evenly throughout the
media to suspend the cells and supply the oxygen necessary for growth effective and efficient
fermentation requires rigorous monitoring and control of the environment within the bioreactor
key factors include temperature pressure pH which is a measure of how acidic or alkaline the media
is oxygen usually measured as dissolved oxygen
04:30 - 05:00 within the media and nutrient levels although
the environment and the media are tailored to the needs of specific cells the lifecycle of
almost all batches follows a predictable pattern the growth pattern has four phases lag exponential
or log stationary and death when a cell is first
05:00 - 05:30 introduced to fresh media it has to adapt to its
new environment this creates a lull or lag in the growth timeline after the organism adapts the
batch takes off the cells begin dividing at a constant rate an exponential or logarithmic or log
increase doubling then doubling again and on and on as the nutrients in the media are consumed
toxic metabolic waste products build-up cells
05:30 - 06:00 begin to die and growth slows when it reaches the
point that just as many cells are dying as our dividing the batch enters the stationary phase
this is the point at which the key nutrients are completely consumed the fermentation is
stopped and the fermented broth is harvested if the fermentation were allowed to continue the
cells would enter the death phase more cells
06:00 - 06:30 die than divided and similar to the exponential
phase the death rate increases logarithmically now that we have a basic understanding of how
fermentation works let's look at an actual process and see how it all comes together for our
sample process we will look at the production of green fluorescent protein or GFP GFP is broadly
used as a biological marker it's a fluorescent
06:30 - 07:00 dye that's very well tolerated by most cells and
doesn't interfere with normal cellular function in the GFP fermentation process we'll need to add an
antibiotic to protect the purity of the batch and then late in the process a biochemical inducer
to turn on the GFP gene our materials for this
07:00 - 07:30 process will include a bacterial seed stock
in this case e coli that has been genetically enhanced to produce GFP the basic ingredients
for a compatible media which include nutrients stabilizers and antibiotic and an anti foaming
agent and iptg which is the biochemical inducer that switches on the GFP gene the equipment that
we'll be using includes a 300 liter bioreactor a
07:30 - 08:00 uv-vis spectrophotometer to monitor the optical
density which is a measure of the concentration of cells in the bioreactor a glucose analyzer
to measure glucose a key nutrient an offline pH meter to help track the acid-base balance
and adjust online measurements if needed and a broth tank for our final product the bioreactor
is equipped with a water jacket around the vessel
08:00 - 08:30 to regulate temperature and integrated sensors
to monitor key environmental factors including dissolved oxygen pH internal temperature
water jacket temperature and vessel pressure the reactor also has an agitator dedicated ports
for adding seed stock and media ingredients separate ports for acid and base supplement
air filters for supply and exhaust and valves
08:30 - 09:00 for drawing samples and for harvesting most
fermentation and monitoring functions can be managed from the bioreactors dedicated process
controller before the fermentation process can begin the area must be prepared preparation
includes removing equipment and material that won't be used in the process cleaning
and sanitizing the area and equipment and
09:00 - 09:30 sterilizing equipment as required by the SOPs
standard operating procedures sterilization is used to eliminate unwanted microorganisms which
can grow naturally in the fermentation media and process equipment also all required materials and
documentation should be gathered and prepared and all process control software should be loaded
and verified the fermentation batch process
09:30 - 10:00 will be guided and documented with the BPR batch
process record the batch record leads the operator through the process step by step with each step
requiring a sign-off and seperate verification this record also includes spaces for documenting
key times activities and instrument readings the GFP fermentation process really begins with
the expansion of our bacterial seed stock after
10:00 - 10:30 removing the specially modified ecoli from the
freezer and thawing it it's used to inoculate a small amount of fresh media in a shaker
flask after the number of cells has reached the target amount the thriving cells are ready
for fermentation meanwhile in the fermentation
10:30 - 11:00 area operators begin with a complete check of
all critical equipment valves caps and lines are checked hoses are tightened probes are verified
and calibrated and 10 kilograms of hpw high purity water is added to the vessel the bioreactor is
brought up to normal process pressure and held there in order to check for leaks the pressure
is monitored over a 30 minute period if a leak is
11:00 - 11:30 detected the problem is corrected and the test is
run again once the reactor passes the test we are ready to mix the media in the vessel the agitator
is turned on and the ingredients are added yeast extract tryptic soy broth ammonium
chloride sodium by phosphate monopotassium
11:30 - 12:00 phosphate and an anti foam compound once all
the initial ingredients are in another ten kilograms of high purity water is added all
ports and valves are closed all condensate valves are open and the bioreactor begins
an SI p sterilize in place cycle the target
12:00 - 12:30 for sterilization is 121 degrees celsius
for 30 minutes as soon as the temperature climbs to the targeted temperature the
condensate valves are closed and the SI P cycle completes automatically both the vessel
and the media are now sterile and we're ready to add the final ingredients to our media
the glucose hose is attached to the vessel
12:30 - 13:00 the connection is steamed to sterilize it and
the separately sterilized glucose antibiotic solution is pumped into the vessel then
a manual pH reading of the media is taken and the bioreactor is set up for its fermentation
cycle after the inoculation hose is connected to the reactor and steamed for 20 minutes the
expanded seed stock is pumped into the reactor
13:00 - 13:30 containing the media fermentation now begins the
operator takes zero hour readings and begins to regularly monitor batch temperature agitator RPMs
dissolved oxygen levels pH vessel pressure optical density air flow rate and glucose concentrations
optical densities and glucose concentrations are
13:30 - 14:00 of particular interest so they're graphed as
well as documented when the targeted levels of glucose and optical density are achieved it's
time to add iptg to the vessel to activate or turn on the expression of the green fluorescent
protein in the cells after allowing enough time for the cells to produce green fluorescent
protein usually 5 hours more final readings are taken and a sample is drawn to check the
percentage of cell solids the product is now
14:00 - 14:30 referred to as broth the broth which contains
spent media and cells is complete when the key nutrient glucose is mostly consumed and the
batch has reached the desired concentration the batch is then cooled down pumped into a
broth tank and labeled with the batch number volume time and date the fermentation process
is now complete the harvested broth will now
14:30 - 15:00 move downstream to the recovery process where
the cells will be ruptured to free the green fluorescent protein and the protein will be
separated from the other broth components you