Bioprocessing Part 1: Fermentation - YouTube

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

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