Bioprocessing Part 1: Fermentation

Summary

This 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

00:00

🌐 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.

05:00

🔬 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.

10:14

🌱 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.

Keywords

💡Fermentation

Fermentation is a biological process that involves the conversion of sugars to acids, gases, or alcohol by the action of microorganisms, such as bacteria, yeasts, or fungi. In the context of the video, fermentation is a key biotechnological process used to produce a wide range of products, from dairy items like yogurt to biopharmaceuticals. The script describes how fermentation is controlled and utilized in industrial settings to manufacture products like green fluorescent protein (GFP).

💡Bioreactor

A bioreactor is a container or a device specifically designed for the growth of cells or tissues, where fermentation processes occur. In the video, the bioreactor is central to the fermentation process, providing a controlled environment for cells to grow and produce the desired product. It is equipped with sensors and mechanisms to regulate temperature, pH, and oxygen levels, which are critical for the success of the fermentation.

💡Media

Media in the context of fermentation refers to the nutrient-rich substance in which microorganisms are grown. It provides the necessary nutrients for the cells to grow and produce the desired product. The script mentions that media is formulated to meet the specific needs of the cells being used and is a critical component in the fermentation process.

💡Yeast

Yeast is a type of fungus that is widely used in fermentation processes, particularly for the production of bread and alcoholic beverages. In the video, yeast is mentioned as one of the organisms used in biotechnology to manufacture products through fermentation, highlighting its importance in both food production and industrial applications.

💡Green Fluorescent Protein (GFP)

Green Fluorescent Protein is a protein originally isolated from the jellyfish Aequorea victoria, which exhibits green fluorescence when exposed to light. In the video, GFP is used as an example of a product manufactured through fermentation. The script details the process of inducing GFP production in genetically modified E. coli, showcasing how biotechnology can harness cellular processes for specific outcomes.

💡Aerobic and Anaerobic

These terms refer to the presence or absence of oxygen in the environment required for cellular respiration. Aerobic cells require oxygen, while anaerobic cells do not. The video script explains that different cells have different needs, and the fermentation process must be tailored to whether the cells are aerobic or anaerobic.

💡Lag Phase

The lag phase is an initial phase in the growth of microorganisms where there is little to no increase in population size as the cells adapt to the new environment. In the script, the lag phase is part of the predictable lifecycle of a fermentation batch, where cells first adapt to the fresh media before beginning to grow exponentially.

💡Exponential Phase

The exponential phase, also known as the log phase, is a period of rapid growth in a microbial culture where the population size increases at a constant rate. The video script describes this phase as a period of constant cell division following the adaptation during the lag phase, where cells double in number repeatedly.

💡Stationary Phase

The stationary phase occurs when the growth of microorganisms slows down due to the depletion of nutrients or the accumulation of toxic metabolic waste products. In the video, the stationary phase is reached when the key nutrients are completely consumed, and the number of cells dying equals the number of cells dividing.

💡Death Phase

The death phase is the final stage in the lifecycle of a fermentation batch where more cells die than divide, leading to a decline in the overall population. The script mentions that if fermentation were allowed to continue beyond the stationary phase, the cells would enter the death phase, which is characterized by a logarithmic increase in the death rate.

💡Bioreactor Monitoring

Bioreactor monitoring involves the continuous observation and control of environmental factors within the bioreactor to ensure optimal conditions for cell growth and product formation. The script outlines the use of various instruments to monitor parameters such as temperature, pH, dissolved oxygen, and optical density, which are critical for the successful fermentation process.

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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