Bioprocess Tech Notes PDF

What is sterilization? Complete destruction or elimination of all viable organisms - Involves the use of heat, radiation, chemicals and physical removal of cells What would happen if fermentation is invaded by a foreign microbe (contaminant)? 1. Loss of productivity. Medium will be used by the...

What is sterilization? Complete destruction or elimination of all viable organisms - Involves the use of heat, radiation, chemicals and physical removal of cells What would happen if fermentation is invaded by a foreign microbe (contaminant)? 1. Loss of productivity. Medium will be used by the desired microbes and contaminants 2. In continuous fermentation, contaminant may overcome the production of biomass and bioproducts 3. Foreign microbes may contaminate the final desired bioproduct → recovery will be difficult 4. Product from contaminant may cause difficulties in the extraction of final desired bioproduct 5. Contaminant may degrade the desired bioproduct 6. Contaminant with phage in bacteria fermentation result in the lysis of the bacteria culture What are some ways to avoid contamination? 1) Pure inoculum to start the fermentation process 2) Sterilize everything - medium used; fermenter vessel 3) Sterilize all materials (acid/base) to be added to the fermentation process: filtration 4) Maintaining aseptic conditions during sampling of fermentation What are the several sterilization techniques we used during experiments? 1) Moist heat (steam) 2) Dry Heat 3) Chemicals 4) Radiation 5) Filter sterilization Explain the sterilization technique of moist heat (steam) and its drawback. - Autoclaving - 121 degree Celsius for 15-20 mins - Some C’s source or medium components are often autoclaved separately to avoid reactions - Glucose separated with other medium - Most important and widely used - Steam usually used in the sterilization of liquid → medium easy, reliable and economical process - Except for the sterilization of media for animal cell culture → Such media are completely heat sensitive → components filter sterilization Drawbacks: Destruction of the nutrient components that are heat sensitive, like vitamins (biotin), amino acids, growth factors (EGF, NGF) Explain the strerilization technique of dry heat. - Oven heating at 160 degree Celsius for 2 hours or at 170 degree celsius for 1 hour - Sterilization: glassware, metals & objects that does not melt at the temperature Discuss the chemical sterilization techniques used in the lab. - Formaldehyde, phenolics such as Lysol (disinfectant sprays), hypochlorite (bleach), mercuric chloride, ethanol - Must leave no residue that would be toxic to the culture or otherwise adulterate the product - Fast & easy, less effective - Sterilization: apparatus, glassware Explain the radiation sterilization techniques used in the lab. - UV, X-rays, and gamma radiation, electron beam - Gamma radiation has better penetration into materials but is much more dangerous to work with - UV - simplest of the radiations to be used, as it can provided by basic UV light bulbs - UV lamps - installed in culture or clean rooms; effective in killing airborne microbial - UV - sterilize surfaces but cannot penetrate fluids easily Discuss the filter sterilization techniques. - To filter heat sensitive medium or substrate (GFs, vitamins, amino acids, antibiotics) - Involves the removal of microbes rather than destruction according to the size of microbial - Two categories according to where the particles are trapped: - Depth filtration - Membrane/surface filtration How are the filters added in the filtration sterilization technique? 1) When adding a filter of same pore size → throughput (amount of materials passing through the filtration system) increases 2) When adding a filter of differing pore size → separation of multiple microorganisms are possible What are the advantages and limitations of filter sterilization technique? Advantages Disadvantages Absolute sterilization - separates particles Each filter has a specific nominal pore size based on size Used for heat-sensitive media Unable to separate microorganisms that have the same size (deep-end filtration) Removal of multiple particle sizes (depth May require a high differential pressure filtration) Allows for fairly high throughput (cross flow filtration) What is depth filtration? - Comprised of fibres, or fibrous materials, that trap particles as they pass through tortuous path between fibers - The use of porous matrix that is capable of retaining particles from the mobile phase throughout its matrix rather just on its surface What can depth filtration remove? - Remove larger, insoluble contaminants before final filtration through a microfiltration membrane that would otherwise clog relatively quickly - Prefiltration - Depth filters are usually used as pre-filters because they are an economical way to remove 98% of suspended solids and protect elements downstream from fouling or clogging → more economical What are the advantages of depth filtration? - Retain particles in the the tortuous flow channels to a level that size-based screening alone cannot achieve - Cheaper than membrane What important design parameter when it comes to depth filters? - Hydraulic loading - The volumetric flow rate per unit cross sectional area - Typically in the range of 1 - 10 gpm (gallons per minute) What is the backwashing of depth filter? - cleaning a depth filter by reversing the flow of fluid through it To prevent clogging What is membrane filter? - Screen filters, have pores of a certain size that allow only smaller particles to pass through - A special pore-sized membrane to separate microorganisms and suspended particles from process liquid - Able to remove 99.99% of suspended solids and may be used as either prefilters or clarifying filters - Microporous membrane: placed at the last possible point in a filtration system to remove the last remaining traces of microorganisms What is the characteristics of membrane filtration? - Smaller pore size - Claimed to be 100% efficiency in removing microbial - Suitable to filter heat sensitive medium of fermentations - Reliable but expensive During large-scale operations, when are membrane filters used? - Before chromatography step of bioprocessing to ensure the absence of particulates - To remove any residual particulates that might clog subsequent chromatographic steps - To reduce the burden on the downstream chromatographic steps during purification - Downstream checking What are the several types of membrane filtration? 1) Microfiltration 2) Ultrafiltration 3) Nanofiltration 4) Reverse Osmosis filtration Discuss on microfiltration. - Used to separate particles of 0.1 to 10μm, including the removal of microbial cells from the fermentation medium using membrane - Expensive due to the high cost of membranes, but - Quiet operation - Lower energy requirements - The product can be easily washed - Good temperature control is possible, containment is readily achieved Discuss on ultrafiltration and what type of systems do larger-scale operations used? - Have smaller pore sizes (