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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
you