Bioprocessing Part 1: Fermentation
Summary
TLDRThis script delves into the fascinating world of fermentation, a biological process harnessed to produce a myriad of products, from dairy to pharmaceuticals. It explains how cells, be it bacteria or fungi, act as micro-factories to manufacture goods like yogurt, bread, and vaccines. The script outlines the fermentation lifecycle, from the lag phase to death phase, and provides an in-depth look at a green fluorescent protein (GFP) production process, detailing the preparation, monitoring, and harvesting steps within a bioreactor.
Takeaways
- 🍶 Fermentation is a widely used process for producing dairy, baked goods, beverages, and more by controlling the decomposition of food substances.
- 🌱 It involves the use of microorganisms such as bacteria, fungi, or specific cells from mammals, plants, or insects to manufacture products.
- 🧪 The process is initiated by selecting a cell type based on its ability to produce a desired product, then growing it in a nutrient-rich media.
- 🔬 Fermentation is a key upstream biotechnology process that occurs before recovery, purification, and packaging stages.
- 🌡️ Environmental factors like temperature, pressure, pH, and oxygen levels are closely monitored and controlled for optimal cell growth and product production.
- 📈 The growth of cells in fermentation typically follows a pattern with four phases: lag, exponential (log), stationary, and death.
- 🔄 The process involves scaling up from a small seed stock of cells to larger vessels, eventually reaching a production vessel or bioreactor.
- 🧪 Commercial-scale fermentation produces a vast array of products including amino acids, biopharmaceuticals, enzymes, food products, and vitamins.
- 🔬 The production of green fluorescent protein (GFP) is used as an example, where genetically modified E. coli is used to produce the protein as a biological marker.
- 🛠️ Equipment such as bioreactors, spectrophotometers, and glucose analyzers are essential for monitoring and controlling the fermentation process.
- 🔍 The harvested product, or broth, is then moved to downstream processes where the desired product is separated from the media and cells.
Q & A
What is fermentation and how is it used in various industries?
-Fermentation is a biological process used to produce a wide array of products such as dairy, baked goods, and beverages. It involves the controlled breakdown of substances by microorganisms like bacteria, fungi, or specific cells from mammals, plants, or insects. This process is utilized in industries to manufacture products like amino acids, biopharmaceuticals, dyes, enzymes, food products, lipids, steroids, and vitamins.
How does fermentation work at a cellular level?
-At a cellular level, fermentation is a process where cells, which can be bacteria, fungi, or specific cells from other organisms, are used to manufacture a product. These cells act as micro-factories, converting nutrients from the growth media into the desired product, which could be a naturally occurring chemical, a substance genetically altered to produce, or a metabolic waste product.
What are the different types of cells used in biotechnological fermentation?
-In biotechnological fermentation, various types of cells are used, including bacteria for products like yogurt, buttermilk, and cheese; yeast, a fungus, for breads and alcoholic beverages; and mammalian cells for the production of some vaccines.
What is the role of media in fermentation?
-Media in fermentation provides the necessary nutrients for the cells to grow and produce the desired product. It is a carefully formulated mixture that contains elements like nutrients, stabilizers, and sometimes antibiotics to protect the purity of the batch.
How is the fermentation process scaled up in industrial production?
-The fermentation process is scaled up by starting with a small seed stock of cells in a minimal amount of media. As the cells grow and consume nutrients, they are gradually moved into larger vessels with more media. This process repeats until the cells reach a quantity sufficient for transfer into a production vessel, often referred to as a bioreactor or fermenter.
What are the key environmental factors that need to be monitored and controlled during fermentation?
-During fermentation, key environmental factors that need to be monitored and controlled include temperature, pressure, pH, dissolved oxygen levels, and nutrient levels. These factors are critical to ensure optimal growth and product yield from the cells.
What is the typical lifecycle of a fermentation batch?
-The typical lifecycle of a fermentation batch includes four phases: lag, where the cells adapt to the new environment; exponential or log, where cells divide at a constant rate; stationary, where the key nutrients are consumed, and the growth slows; and death, where more cells die than divide if the fermentation continues.
What is the role of IPTG in the GFP fermentation process?
-In the GFP (Green Fluorescent Protein) fermentation process, IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a biochemical inducer used to activate or turn on the expression of the GFP gene in the cells, leading to the production of GFP.
How is the quality of the fermentation process ensured?
-The quality of the fermentation process is ensured through rigorous monitoring, control of the environment within the bioreactor, and adherence to standard operating procedures (SOPs). Sterilization of equipment and media is also crucial to eliminate unwanted microorganisms.
What happens to the fermented product after the fermentation process is complete?
-After the fermentation process is complete, the product, referred to as broth, is harvested. This broth contains spent media and cells. It is then cooled, pumped into a broth tank, and labeled with relevant information. The broth will move downstream to the recovery process, where the cells are ruptured to release the product, which is then separated from other components.
Outlines
🌐 Introduction to Fermentation
Fermentation is a biological process used to produce a wide array of products, including dairy, baked goods, and beverages. It involves the controlled breakdown of substances by microorganisms such as bacteria, fungi, and specific cells from various organisms. In biotechnology, these organisms are utilized to manufacture products like yogurt, bread, cheese, and alcoholic beverages. The script explains that fermentation is not just about letting food spoil but doing so in a controlled manner to create useful products. It also introduces the concept of cells as micro-factories and the role of different types of cells in the fermentation process, including bacteria, yeast, and mammalian cells. The script further elaborates on the types of products made through fermentation, such as amino acids, biopharmaceuticals, dyes, enzymes, food products, lipids, steroids, and vitamins.
🔬 The Fermentation Process
The script delves into the technical aspects of fermentation, describing it as a simple yet controlled process. It involves selecting a specific cell type based on its ability to produce a desired product. The process begins with a small seed stock of cells that are grown in a nutrient-rich media. As the cells multiply and consume nutrients, they are transferred to larger vessels with more media, eventually reaching a production vessel or bioreactor. The script emphasizes the importance of tightly controlled conditions within the bioreactor, including temperature, pressure, pH, and oxygen levels. It also outlines the typical lifecycle of a fermentation batch, which includes four phases: lag, exponential or log, stationary, and death. The script provides an overview of the requirements for different cells, such as aerobic and anaerobic needs, and the necessity for proper nutrition and environmental control.
🌱 Production of Green Fluorescent Protein (GFP) via Fermentation
This section of the script focuses on a specific example of fermentation: the production of green fluorescent protein (GFP), a biological marker used in various applications due to its fluorescent properties and non-interference with cellular functions. The process involves using a genetically modified E. coli bacterial seed stock to produce GFP. The script outlines the materials needed, such as the bacterial seed stock, compatible media ingredients, an antibiotic, an anti-foaming agent, and IPTG, a biochemical inducer. It also details the equipment used, including a 300-liter bioreactor, a UV-Vis spectrophotometer, a glucose analyzer, and offline pH and broth tanks. The script describes the preparation of the fermentation area, sterilization procedures, and the step-by-step process of setting up the bioreactor, including the addition of media ingredients, sterilization, inoculation, and the monitoring of various parameters during the fermentation cycle. The process culminates in the addition of IPTG to activate GFP production and the eventual harvesting of the fermented broth containing GFP.
Mindmap
Keywords
💡Fermentation
💡Bioreactor
💡Media
💡Yeast
💡Green Fluorescent Protein (GFP)
💡Aerobic and Anaerobic
💡Lag Phase
💡Exponential Phase
💡Stationary Phase
💡Death Phase
💡Bioreactor Monitoring
Highlights
Fermentation is a process used to make dairy products, baked goods, and beverages.
Fermentation involves controlled decomposition of foods by microorganisms.
Cells used in biotechnology for fermentation can be bacteria, fungi, or specific cells from various organisms.
Fermentation is used to manufacture a wide range of products including amino acids, biopharmaceuticals, and vitamins.
The fermentation process involves selecting a cell type, growing it in media, and scaling up in a bioreactor.
Different cells have different requirements for growth, such as aerobic or anaerobic conditions.
Effective fermentation requires monitoring and control of temperature, pressure, pH, and nutrient levels.
The growth pattern of cells in fermentation typically follows a predictable pattern of lag, exponential, stationary, and death phases.
The production of green fluorescent protein (GFP) is an example of a specific fermentation process.
GFP is used as a biological marker and is well-tolerated by most cells.
The GFP fermentation process includes the use of a genetically enhanced E. coli seed stock.
Media for GFP fermentation includes nutrients, stabilizers, antibiotic, and an anti-foaming agent.
Equipment for the GFP process includes a bioreactor, spectrophotometer, glucose analyzer, and pH meter.
The bioreactor is equipped with a water jacket, sensors, agitator, and ports for adding ingredients and sampling.
Preparation for fermentation involves cleaning, sanitizing, and sterilizing the area and equipment.
The fermentation batch process is guided and documented with a batch process record.
The GFP process begins with the expansion of the bacterial seed stock in a shaker flask.
The bioreactor is checked for leaks and sterilized before the media is mixed and ingredients are added.
The fermentation cycle is monitored for key parameters such as temperature, pH, and glucose concentration.
IPGT is added to the vessel to activate the expression of GFP in the cells.
The harvested broth containing GFP moves downstream for recovery, where the protein is separated from other components.
Transcripts
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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