Bioprocessing Part 2: Separation / Recovery

Summary

The video explains the fermentation and recovery processes used in biotechnology, focusing on the production of Green Fluorescent Protein (GFP). It outlines the steps of intracellular fermentation, where the cells are broken open to release the product, and the subsequent recovery process. This involves separating cells from the broth, washing, lysing, and using centrifugation and filtration to isolate the GFP. The clarified lysate containing GFP is then sent for purification. The video highlights the tools and methods used, including centrifuges, homogenizers, and filters, to ensure efficient product recovery.

Takeaways

• 🦠 Many biotechnology companies use fermentation to produce valuable compounds via microorganisms.
• 🔄 Fermentation allows the production of molecules on a large scale using millions of microbial cells.
• 🌱 Two types of production in fermentation: extracellular (product secreted by cells) and intracellular (product kept inside the cells).
• 💧 Extracellular production involves keeping the medium with the product, while intracellular requires cell disruption to release the product.
• ⚙️ The recovery process follows fermentation and involves separating cells from the liquid broth and isolating the molecule of interest.
• 🧬 Tools like centrifuges, homogenizers, and microfiltration are used to extract the product from cells.
• 🔬 Green Fluorescent Protein (GFP) is used as a biological marker to track drug movements in cells.
• 💡 Recovery steps include centrifugation, cell washing, homogenization (cell breaking), and filtration to separate cell debris and collect the product.
• ⚗️ The product (GFP) is purified and concentrated after recovery to ensure stability and remove impurities.
• 📜 The entire process is documented in a Batch Process Record (BPR) for traceability and verification.

Q & A

What is the main purpose of fermentation in biotechnology?

The main purpose of fermentation in biotechnology is to use microorganisms to produce valuable compounds or products on a commercial scale. This allows for the production of large quantities of a molecule of interest by using microorganisms as tiny micro-factories.

What are the two types of production in fermentation, and how do they differ?

The two types of production in fermentation are extracellular and intracellular production. In extracellular production, the product is secreted by the cells and separated from the biomass early in the process. In intracellular production, the product remains inside the cells and is released by disrupting the cells during separation.

What is the role of Recovery in the fermentation process?

Recovery is the step following fermentation, where the cells containing the product of interest are separated from the liquid broth, and the product is extracted from the host cells. It involves separating cells from the broth, breaking them open to release the product, and removing any cell debris.

Why is GFP (Green Fluorescent Protein) used in biotechnology, and what is its role?

GFP is used as a biological marker in biotechnology. When attached to a drug, it provides a visual marker that allows researchers to track where the drug goes in the body. GFP is fluorescent and does not interfere with normal cellular functions, making it ideal for research purposes.

What are the key tools used in the GFP Recovery process?

The key tools used in the GFP Recovery process are a Disc Stack Centrifuge for separating solids from liquids, a Homogenizer to break open E.coli cells, and a 0.22 micron filter to remove any remaining solids from the solution.

What is the purpose of centrifugation in the Recovery process?

The purpose of centrifugation in the Recovery process is to separate solids (the E.coli cells) from the liquid broth. The denser solids are forced to the sides of the centrifuge bowl, while the clarified liquid exits. After the cells are broken open, centrifugation is used again to separate cell debris from the desired product.

What happens during the homogenization step in Recovery?

During homogenization, the cells are forced through a small orifice at high pressure, causing them to rupture and release their internal contents, including the desired product, GFP. The process is repeated to ensure all cells are disrupted.

How is cell debris removed after the cells are lysed in Recovery?

After the cells are lysed, the mixture of cytoplasm and cell contents is centrifuged again to remove the solid cell debris. The liquid, which contains the GFP, is kept, and the remaining solids are discarded.

What is the purpose of filtration in the final step of Recovery?

The purpose of filtration is to remove any remaining small particles or solid materials from the lysate. A 0.22 micron filter is used to ensure the product solution is free of solid contaminants before it moves to the Purification phase.

What happens to the product after the Recovery process is completed?

Once Recovery is complete, the clarified lysate containing the GFP is sent to the Purification step. During Purification, dissolved impurities are removed, and the GFP is concentrated and stabilized for further use.