字幕表 動画を再生する 英語字幕をプリント >>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, buttermilk and cheese, we use bacteria. To make breads and alcoholic beverages we use yeast - a fungus! And the production of some vaccines requires 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 organism's 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. [Music Fades, Nat Sound of Process Establishes, Under For VO] >>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 fermentor. >>With plenty of fresh media now available and 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 fermentor 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 are 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 divide, 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 bacteria 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, an antibiotic and an antifoaming 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 off-line, pH meter to help track the acid/base balance, and adjust on-line 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 bioreactor's 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 separate 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 bacteria seed stock. After removing the specially modified E-coli 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 biphosphate... Monopotassium phosphate... and an Antifoam compound. Once all the initial ingredients are in, another 10 kilograms of High Purity Water is added... all ports and valves are closed... all condensate valves are opened... and the bioreactor begins an SIP - 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 SIP 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. [Bright, Rhythm-Driven Music Establishes, Then Under For VO] 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.
B2 中上級 バイオプロセシング 第1部発酵 (Bioprocessing Part 1: Fermentation) 162 15 kuoyumei に公開 2021 年 01 月 14 日 シェア シェア 保存 報告 動画の中の単語