BE308 Biomanufacturing Lecture 3 Cell Growth PDF

BE308 Biomanufacturing Lecture 3 GMP Manufacturing Facility https://www.youtube.com/watch?v=nvttgCm6 CNs  Cleanroom Classification https://www.youtube.com/watch?v=IENbOfC4df8 HEPA = high efficiency particulate air [filter] HVAC = Heating, ventilation, and air conditioning Clean...

BE308 Biomanufacturing Lecture 3 GMP Manufacturing Facility https://www.youtube.com/watch?v=nvttgCm6 CNs  Cleanroom Classification https://www.youtube.com/watch?v=IENbOfC4df8 HEPA = high efficiency particulate air [filter] HVAC = Heating, ventilation, and air conditioning Cleanroom Classification  Cryogenic Storage Dewar A specialized type of vacuum flask used for storing cryogens (such as liquid nitrogen or liquid helium), whose boiling points are much lower than room temperature. Named after inventor James Dewar Have walls constructed from two or more layers, with a high vacuum maintained between the layers. This provides very good thermal insulation between the interior and exterior of the dewar, which reduces the rate at which the contents boil away. Precautions are taken in the design of dewars to safely manage the gas which is released as the liquid slowly boils. https://www.tomorrow.bio/post/what-is-a-cryogenic-storage-dewar  https://www.youtube.com/shorts/5l8b kGX6WDM https://www.youtube.com/shorts/FkK M3tuuSLE https://www.coleparmer.com/blog/3-steps-to-protect-liquid-nitrogen-dewar-contents/ Cell Nutrients Macronutrients are needed in concentrations larger than 10-4 M. Carbon, nitrogen, oxygen, hydrogen, sulfur, phosphorus, Mg2+, and K+ Micronutrients are needed in concentrations of less than 10-4 M. Trace elements such as Mo2+, Zn2+, Cu2+, Mn2+, Ca2+, Na+, vitamins, growth hormones, and metabolic precursors are micronutrients. Macronutrients Carbon sources in Industrial fermentations: Molasses (sucrose), starch (glucose, dextrin), corn syrup, and waste sulfite liquor (glucose) Carbon sources in Laboratory fermentations: Glucose, sucrose, and fructose Methanol, ethanol, and methane  Macronutrients Nitrogen 10% to 14% of cell dry weight Nitrogen sources: ammonia or the ammonium salts [NH4Cl, (NH4)2SO4, NH4NO3], proteins, peptides, and amino acids Incorporated into cell mass as proteins and nucleic acids Azotobacter sp. and the cyanobacteria fix nitrogen from the atmosphere to form ammonium Urea may also be used as a nitrogen source Organic nitrogen: yeast extract and peptone Expensive compared to ammonium salts  Macronutrients Oxygen Present in all organic cell components and cellular water Constitutes ~20% of the dry weight of cells Molecular oxygen: Terminal electron acceptor in the aerobic metabolism of carbon compounds Gaseous oxygen is introduced into growth media by sparging air or by surface aeration Hydrogen ~8% of cell dry weight Derived primarily from carbon compounds eg. Carbohydrates Some bacteria such as methanogens can utilize hydrogen as an energy source. Macronutrients Phosphorus Sulfur ~3% of cell dry weight ~1% of cell dry weight Present in nucleic acids and in the cell wall of some Present in proteins and some coenzymes gram-positive bacteria such as teichoic acids Sulfur source Inorganic phosphate salts: KH2PO4 and K2HPO4, are the most common phosphate salts Sulfate salts: (NH4)2SO4 Glycerophosphates can also be used as organic Sulfur containing amino acids phosphate sources. Certain autotrophs utilize S2+ and SO as energy Key element in the regulation of cell metabolism sources. Phosphate level in the media should be less than 1 mM for the formation of many secondary metabolites such as antibiotics  Macronutrients Potassium A cofactor for some enzymes Required in carbohydrate metabolism Cells tend to actively take up K+ and Mg2+ and exclude Na+ and Ca2+ Source: K2HPO4, KH2PO4, and K3PO4 Magnesium A cofactor for some enzymes Present in cell walls and membranes Ribosomes specifically require Mg2+ ions Source: MgSO4.7H2O or MgCl2  Micronutrients Trace elements are essential to microbial nutrition Lack of essential trace elements increases the lag phase (the time from inoculation to active cell replication in batch culture) May decrease the specific growth rate and the yield Most widely needed trace elements are Fe, Zn, and Mn Iron (Fe) present in ferredoxin and cytochrome and is an important cofactor Iron also plays a regulatory role in some fermentation processes Iron deficiency is required for the excretion of riboflavin by Ashbya gosypii Iron concentration regulates penicillin production by Penicillium chrysogenum). Cytochrome c with heme c Zinc (Zn) is a cofactor for some enzymes and also regulates some fermentations such as penicillin fermentation. Manganese (Mn) is also an enzyme cofactor and plays a role in the regulation of secondary metabolism and excretion of primary metabolites. Micronutrients Trace elements needed under specific growth Calcium (Ca) is a cofactor for amylases and some conditions are Cu, Co, Mo, Ca, Na, Cl, Ni, and Se proteases Copper (Cu) is present in certain respiratory-chain Present in some bacterial spores and in the cell walls components and enzymes of some cells, such as plant cells Copper deficiency stimulates penicillin and citric Sodium (Na) is needed in trace amounts by some acid production bacteria, especially by methanogens for ion balance. Cobalt (Co) is present in corrinoid compounds such Sodium is important in the transport of charged as vitamin B12 species in eucaryotic cells. Propionic bacteria and certain methanogens require Chloride (Cl) is needed by some halobacteria and cobalt marine microbes, which require Na+ Molybdenum (Mo) is a cofactor of nitrate reductase Nickel (Ni) is required by some methanogens as a and nitrogenase cofactor Required for growth on NO3 and N2 as the sole Selenium (Se) is required in formate metabolism of source of nitrogen. some organisms. Micronutrients Some ions such as Mg2+, Fe3+, and PO3-4 may EDTA (ethylenediaminetetraacetic acid) precipitate in nutrient medium and become unavailable to the cells Polyphosphates Chelating agents are used to form soluble Histidine compounds with the precipitating ions. Tyrosine Ligands: bind to metal ions to form soluble Cysteine complexes Na2 EDTA is the most common chelating agent Major ligands: EDTA may remove some metal ion components of Carboxyl (—COOH) the cell wall, such as Ca2+, Mg2+, and Zn2+ and may aAmine (—NH2) cause cell wall disintegration Mercapto (—SH) groups Citric acid is metabolizable by some bacteria Citric acid Chelating agents are included in media in low concentrations (e.g., 1 mM) Micronutrients Growth factors stimulate the growth and in concentrations from 10-6 M to 10-13 M synthesis of some metabolites Some fatty acids, such as oleic acid and sterols, Vitamins are also needed in small quantities by some organisms. Hormones Amino acids Higher forms of life, such as animal and plant Vitamins usually function as coenzymes cells, require hormones to regulate their Eg. thiamine (B1), riboflavin (B2), pyridoxine metabolism. (B6), biotin, cyanocobalamine (B12), folic acid, Insulin is a common hormone for animal cells lipoic acid, p-amino benzoic acid, and vitamin K Auxin and cytokinins are plant-growth Vitamins are required at a concentration range hormones. of 10-6 M to 10-12 M Depending on the organism, some or all of the amino acids may need to be supplied externally  Growth Media Two major types of growth media are defined and complex media Defined media contain specific amounts of pure chemical compounds with known chemical compositions A medium containing glucose, (NH4) 2SO4, KH2PO4, and MgCl2 is a defined medium Complex media contain natural compounds whose chemical composition is not exactly known A medium containing yeast extracts, peptone, molasses, or corn steep liquor is a complex medium. A complex medium usually can provide the necessary growth factors, vitamins, hormones, and trace elements Advantages of Complex media Often resulting in higher cell yields, compared to the defined medium Complex media are less expensive than defined media Advantages of defined media Results are more reproducible and the operator has better control of the fermentation. Recovery and purification of a product is often easier and cheaper in defined media.  Growth Curve When a liquid nutrient medium is inoculated with a seed culture The organisms selectively take up dissolved nutrients from the medium Convert them into biomass A typical batch growth curve includes the following phases: Lag phase Logarithmic or exponential growth phase Deceleration phase Stationary phase Death phase Lag Phase The lag phase occurs immediately after inoculation and is a period of adaptation of cells to a new environment Microorganisms reorganize their molecular constituents when they are transferred to a new medium Depending on the composition of nutrients, new enzymes are synthesized, the synthesis of some other enzymes is repressed, and the internal machinery of cells is adapted to the new environmental conditions During this phase, cell mass may increase a little, without an increase in cell number density. When the inoculum is small and has a low fraction of cells that are viable, there may be a pseudolag phase, which is a result, not of adaptation, but of small inoculum size or poor condition of the inoculum. Low concentration of some nutrients and growth factors may also cause a long lag phase The age of the inoculum culture has a strong effect on the length of lag phase Diauxic growth: Multiple lag phases may be observed when the medium contains more than one carbon source Diauxic growth The length of the relatively unproductive lag phase may be shortened by increasing the inoculum size and ensuring that the growth environment (medium, pH, temperature, etc.) in the bioreactor is the same as the one in which the inoculum was grown  Cell Counting Methods: (1) Optical Density most common methods used in a microbiology lab Optical density at 600 nm (OD600) is typically used to determine the stage of growth of a bacterial culture these measurements help ensure that cells are harvested at an optimum point that corresponds to an appropriate density of live cells. Size and shape as well as dead cells and debris of a cell may add to light scattering. Hence, distinct cell types at same densities may therefore show varying values OD600 when estimated on a similar instrument. place the cell suspension in a cuvette and measure the absorbance in spectrophotometer. To get a relative density measurement, compare to another sample. Otherwise, compare to cell suspensions of known density in order to estimate an absolute cell density https://www.implen.de/od600-diluphotometer/od600/  Cell Counting Methods: (2) Plating Method only works in colony-forming cells such as bacteria. To count cells with plating, the cells are heavily diluted and streaked onto a plate. After given sufficient time for colony growth, the number of colonies are counted. Based on the dilution and the known volume of suspension that was streaked onto the plate, the density of the original suspension can be determined. Plating is only a useful method for microbes, and due to the time required for colony formation it is also the slowest method.  Cell Counting Methods: (3) Counting chambers / Hemocytometers https://www.youtube.com/watch?app=desktop&v=ZukmrTVKCOk Cell Counting Methods: (4) Automated Cell Counter (Image-based) The TC20 automated cell counter uses microscopy with auto-focus that analyzes multiple focal planes to identify the best plane. Without requiring any user input, the sophisticated cell counting algorithm uses the image acquired from the best focal plane to identify cells and exclude debris, thereby calculating the total cell count. https://www.bio-rad.com Cell Counting Methods: (5) Coulter Counters (Impedance-based) https://www.youtube.com/watch?v=bYfZk9PU3XI Cell Counting Methods: (6) Flow cytometer https://www.youtube.com/watch?v=1eIGKh7LaKY Exponential Growth Phase The cells have adjusted to their new environment After this adaptation period cells can multiply rapidly Cell mass and cell number density increase exponentially with time This is a period of balanced growth, in which all components of a cell grow at the same rate Average composition of a single cell remains approximately constant during this phase of growth During balanced growth, the net specific growth rate The deceleration growth phase follows the exponential phase Growth decelerates due to either depletion of one or more essential nutrients or the accumulation of toxic by- products of growth For a typical bacterial culture, these changes occur over a very short period of time The rapidly changing environment results in unbalanced growth During unbalanced growth, cell composition and size will change Stationary Phase The stationary phase starts at the end of the deceleration phase The net growth rate is zero (no cell division) or when the growth rate is equal to the death rate Cells are still metabolically active and produce secondary metabolites Primary metabolites are growth-related products Secondary metabolites are nongrowth-related (e.g., antibiotics, some hormones) Its due to metabolite deregulation Cell lysis may occur and viable cell mass may drop A second growth phase may occur and cells may grow on lysis products of lysed cells (cryptic growth) The death phase (or decline phase) follows the stationary phase A clear demarcation between these two phases is not always possible.