Bioprocess Flashcards
Basics of microbial stoichiometry, metabolization of substrates, substrate and energy balance
mass and energy balance are used to determine in bioprocess control to determine the stoichiometric requirements of our microbes to form our dseired amount of product, to measure posible presence ofpollutants…etc
Isolation and preservation of microorganisms in the bioindustry.
Strain improvement in the bio-industry
A strain is a genetic variant or subtype of a virus or bacterium
Industrial fermentation media: design and important components.
Oxygen: Added for metabolic functions, an important substrate
Inoculum development, scale-up.
- The inoculum development also includes the preparation of bacterial suspension (either vegetative cells or spores) in sterile tap water, then to the broth. In case of fungi, they hyphae are transferred to the broth
- Vegetative cells: Non-reproductive cells (not involve in the production of gametes), can grow actively and form endospores - Scale-up
- The excercise in scalling up involved several programmed research or steps that must be established so as to predict the final behavior of the final large scale production fermenter. Studies carried out during scale up include
- Inoculum development
- Sterilization establishing the correct sterillization cycle at larger loads
- Environmental parameters: Nutrient availability, pH, tempt ….
Principles of bioreactor design.
Biomass concentration is defined as the concentration of microbial cells in a culture at a given point in time. Biomass monitoring follows the development of the cell concentration over time and characterizes the growth of the cultivated microorganism.
Parts of a bioreactor
1. Reactor vessel: where the reaction occurs
2. Cooling jacket: The reactor is fitted externally with the cooling jacket through which steam (for sterilization) and cooling water (for cooling) are run
3. Agitation system: requires for proper mixing of nutrients and gases for the maximal growth of microorganisms.
Impeller: stirring or mixing the agitation media
Stirrer: Stirring the media
4. Aeration system: introduces air into the medium by sparger, a series of holes in a metal ring –> pump sterile air at the bottom
5. Baffles: metal strips to prevent a vortex and improve aeration efficiency
Batch, fed-batch, continuous and solid-state fermentation systems
Mass transfer operations. Oxygen transfer, the basic context of aeration. Foaming, antifoaming, rheological properties of the medium
Ensuring of fermentation technological parameters: instrumental measurement, sensors, online and offline control, automation
Basic operations of product recovery and purification (downstream processing) I: filtration, centrifugation, sedimentation, electro-precipitation, crystallization
Basic operations of product recovery and purification (downstream processing) II: adsorption, extraction, ultrafiltration, chromatography, lyophilization
1. Adsorption:
- The adhesion of atoms, ions, and molecules from a gas, liquid, or dissolved solid to the solid or liquid surface (rather than accumulate inside)
–> Create a film of the adsorbate (the species that gets adsorbed to the surface) on the surface of the adsorbent (where the adsorption occurs). The molecules of the substance stick on the surface of another substances. We can remove the adsorbate.
- Principle: Due to the vacant space of the adsorbent responsible for stimulating the adhesion of particles onto the available spaces. It depends on the energy between the adsorbate and adsorbent, depends on the types of substances involved (Ex: van der Waals, covalent bonds …)
- A temparature - dependent process: Low temparature favours adsorption.
- The adsorbate is more concentrated on the surface than the other parts of the adsorbent after adsorption.
- Example: Water vapour are adsorbed by sillica gel
2. Extraction:
- The separation of one or more selected components from a mixture, using an extraction solvent.
-Liquid-liquid extraction: Uses 2 immiscible phases (phases that dont mix), usually one aqueous and one organic, to separate the components from 1 phase to another. We use separating funnel. The organic solvent with a higher density than water settle to the bottom to form a lower phase, while those has lower density than water form a layer on the top
- Principle: Based on their relative solubilities in two different immiscible liquids.
- Application: Separate the biomolecules: Penicllin G
3. Ultrafiltration:
- a pressure-driven purification process that separates particulate matter from soluble compounds using an ultrafine membrane media, a semi-permeable membrane.
- Principle: Based on size of the particles. Suspended solids and solutes of high molecular weight are retained in “retentate”, while water and low molecular weight solutes pass through the membrane in the “permeate”.
- The pore size of ultrafiltration membrances ranges from 0.1 to 0.01 microns.
- Applied in cross-flow (The feed flows parallel to the filter membrane) or dead-end ( The feed flows perpendicular to the filter membrane).
- Application: Used in industry and research for purifying and concentrating macromolecular (10^3-10^6 Da) solutions, especially protein solutions.
4. Chromatography:
- A technique in which the components of a test sample are separated by using a flowing phase (mobile phase) in which the sample is introduced, and a non-moving phase (stationary phase) which is in contact with the flowing phase
- Principle: Based on the difference in the strength of the reversible interactions of the sample components with the non-moving phase (their affinity with the stationary phase). When the mobile phase moves along the stationary phase, the component bonds strongly to the stationary phase –> moves slowly, the component bonds weakly to the stationary phase –> Moves faster
- Application: To achieve the desired purity level of bioproducts
- 3 most applied chromatographic techniques:
1. Ion-exchange chromatography: Separates target products from contaminants based on the differences between the overall charges of the compounds in the mixture.
- Commonly applied to purify almost all kinds of surface-charged biomolecules (proteins…)
- Using column which consist of charged groups that are covalently linked to the surface of an insoluble matrix. For example: We have negatively charged beads in the column. Positively charged protein binds to negatively charged bead. Negatively charged protein flows through.
2. Gel filtration chromatography (Size exclusion chromatography)
- Based on the molecular size of bioproducts
- Molecules travel through a bed of porous beads with greater or lesser diffusion rates. Smaller biomolecules that can diffuse into the pores of the beads are retained longer and pass through the gel column more slowly. Larger biomolecules which flow through the column interspaces without entering the pores, are eluted rapidly.
3. Affinity chromatography:
- Based on a highly specific binding of the desired product onto the immobilzed ligand attached to an inert matrix (the stationary phase) in the column.
- Application: Receptor-ligand, antigen-antibody
5.Lyophilization or Freeze-drying
- belongs to product polishing steps, where the product in a form that are stable and convenient for transportation and storage.
- A process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase
- Application: used for thermal-sensitive proteins, medicines. Produce high quality products by preserving the native structure and characteristics of active ingredients
Basic processes of enzymatic and microbial bioconversion: oxidation, reduction, hydrolysis, transglycosylation, resolution, isomerization, condensation
Bioconversion: From organic compounds to chemical compound of interest, carried by microorganism
1. Oxidation and reduction: Usually occur in pairs, called redox reaction. Any substance to be oxidized, another substance must be reduced. Electron are conserved in a chemical reaction , not created or destroyed.
- Oxidation is gain of electrons, Reduction is loss of electrons
- Involves a transfer of electrons between 2 species, a change on oxidation state of atoms
- Redox reactions in cells involve reactions between intermediates called “carriers”. Common carrier is the coenzyme NAD+ (nicotinamide-adenine dinucleotide). NAD+ is reduced to NADH and H+. Cell needs only a tiny amount of NAD+ and NADH because they are constantly being recycled.
- Example of reduction: Carboxylic acid—> Primary alcohol (Hydroxyl group is bonded to a primary carbon), catalyzes by LiAlH4 (lithium aluminum anhydride) and acidic environment (H30+)
- At the basis of receiving energy lies the process of biological oxidation. Biological oxidation is the combination of oxidation-reduction transformations of substances in living organisms.
Example: Oxidation of pyruvate — the process involves the transformation of pyruvate obtained in the glycolysis process into an acetyl-CoA. The reaction takes place with the aid of the enzymic complex of pyruvate dehydrogenase. From 1 pyruvate –> 1 acteyl-CoA and 1 NADH and 1 CO2
2. Hydrolysis: The process of breaking a large molecule into smaller molecules by the reaction with water, usually means the cleavage of chemical bonds by the addition of water.
- Example: Proteins –> amino acids (peptide bonds), Polysaccharides –> Monosaccharides (glycosidic linkages)
- Hydrolysis is related to energy metabolism and storage: Energy released from the oxidation of nutrients must be conserved by the cell to drive energy-requiring cell functions. They are chaneled into a special energy-storage molecule: ATP (Adenosine triphosphate), an energy-rich compound. The free energy released from the hydrolysis of the phosphate in ATP is significantly greater than that of the average covalent bond in the cell. When the chemical bonds within ATP are broken, energy is released and can be harnessed for cellular work.
ATP + H20 –> ADP + Pi (inorganic phosphate)
3. Transglycosylation: The transfer of a sugar residue (glycosyl group) from 1 glycoside (a sugar bond to another functional group via a glycosdic bond, through its anomeric carbon) to another by cleaving the glycosidc bond.
- Application: The conversion from pyrimidine to purine.
pyrimidine nucleoside + purine base —> Purine nucleoside
4. Resolution or Optical resolution: The process by which a racemic mixture is separated into its 2 constituent enantiomers
- Racemic mixture: a mixture has equal amount of left- (S) and right- (R) handed enantiomers
- Enantiomers: are stereoisomers that are mirror images of each other that are non-superposable (not identical when rotating –> doesnt completely lined up with other) –> Identical physical characteristics –> Hard to separate.
- Steps: Racemic mixture + Another chiral molecule (acts as a chiral agent) –> Mixture of diastereoisomers (stereoisomers that are not images of each other) –> Separate and Purify –> Separated enantiomers and the recovered agent ( the chiral agent)
5. Isomerization: The process of converting a molecule into its isomers
- Change of structure or configuration
- Can involve either the interconversion of constitutional isomers (same formula, different connectivity) or stereoisomers (same formula, same connectivity, different arrangment)
- In biochemistry, under basic conditions, any carbohydrate can be transformed into its isomer
- Example: D-glucose (An aldose)–> D-fructose (A ketose). Isomerase enzyme catalyses the shifting of a carbonyl group in sugar molecule, between a ketose and an aldose (refers to sugar molecules containing ketone and aldehyde groups, respectively). This conversion is important because D-Fructose is the ingredient to make high-fructose corn syrup, chemicals ….
6. Condensation: The reaction at which small molecules are combined to form a larger molecule, usually with the loss of a small molecule (water, NH3, CH3OH…). If water is lost —> Dehyration
- Covalent bonds form between monomers, allowing them to join together into polymers.
- May involve the functional groups of the molecule
- Can occur in basic or acidic conditions or in the presence of a catalyst
- Application: Protein synthesis by amino acids, through peptide bonds. The carboxyl group of 1 aa reacts with the amino group of another aa, to form peptide bond, 1 molecule of H20 is released –> Protein
Aerobic and anaerobic detoxification, nitrification, denitrification, phosphorus and metal removal
Biofuel production technology I.: production of bioalcohol and biodiesel
Biofuel production technology II: biogas production
Biogas is a mixture of gases (mostly methane).
Anaerobic digestion is carried out by human in a variety of reactors called digesters
Bacteria involves:
- Methanogenic bacteria: remove H2 produced by obligate H2-producing bacteria, thereby lowering the H2 partial pressure and enabling the latter to continue producing H2.
- Acetogenic bacteria: remove H2
Substrates for biogas production:
- Industrial and food processing wastes: potato, veggie, cheese …
- Animal and plant wastes
Advantages of biogas
- Cheaper technology
- Helps in clean up of the environment
- Sterile conditions are not required
- Any biodegradable matter can be its substrate
- Easy to generate, transport and store
Disadvantage
- Unattractive commercially and economically
- Produces impurities gas
1. Hydrolysis: Biomass (Proteins, Carbs, Lipids …)+ H20 —> Monomers and Oligomers
Some certain substrates like hemicellulose, cellulose hard to degrade —> Use enzymes
2. Acidogenesis: Monomers and Oligomers —> Smaller organic acids (by acidogenic bacteria)
3. Acetogenesis: —> H20, Co2, Acetate (CH3COOH)
4. Methanogenesis: —> CH4 (main), CO2
Technology of the production of organic acids
- Beet molasses are used because of high sugar content, low cost, low content of trace metals
- Submerged fermentation: mainly used for CA production because of high yield, high productivity, low labor costs. SmF is a method of manufacturing biomolecules in which enzymes and other reactive compounds are submerged into a liquid (alcohol, broth …), which contains nutrients —> Utilize free-flowing liquid substrates (broth, molasses)
- Aeration: enhance yield, reduce fermentation time —> Leads to high amounts of foam —> Can use antifoam to prevent
Other organic acids production:
Gluconic acid –> A. niger
Itaconic acid –> A. terreus
Lactic acid –> Lactobacillus
Technology of the production of amino acids
- Biotin in lysine production: cofactor for pyruvate carboxylase which catalyses pyruvate into oxaloacetate —> Lysine
Technology of the production of antibiotics: beta-lactams, aminoglycoside
Antibiotics: antimicrobial substance active against bacteria, treat or prevent some types of bacterial infection by killing or preventing them from spreading
a) Beta lactam production
- Beta lactam antibiotics: contains a 4-atom beta-lactam ring in their chemical structure ( carbon next to the carbonyl is the alpha carbon, the carbon next to that is the beta carbon, then we hit the nitrogen). The ring contain an amide group (NH-), carbonyl group ( double bond O) in a cyclic form
- Penicillin: treat throat infections, sexually transmitted disease (syphilis, …)
b) Amino glycoside production
- Amino glycoside: contain amino sugar structures
- Streptomycin: treat bacterial infections: rat bite fever, plague
Phase 1: pH increase. After fermentation, add Nh3 and move to phase 2
Phase 3:
- Autolysis: In biology, autolysis, more commonly known as self-digestion, refers to the destruction of a cell through the action of its own enzymes.
Technology of the production of antibiotics: tetracyclines, macrolides
Erythromycin: skin, nose infections, STD (syphilis…)
Technology of the production of vitamins and enzymes
a) Vitamin production:
- Vitamin C (L-ascorbic acid): lower the risk of having disease (heart …), boosts immunity, protects memory …. Human cannot synthesize vitamin C by our body
- KGA: Keto gulonic acid (precursor of vitamin C)
- Toluol = Toluene
- Reichstein process: A combined of chemical and microbial method of the production of vitamin C from D-Glucose.
- Sorbitol = Sugar alcohol
b) Enzyme production
- Production strain: recombinant yeast or some other microbial agent that has been genetically engineered to make a desired compound or product by means of a fermentation process.
=>The production strains grow under very specific conditions to maximize the amount of enzyme that they produce.
- Adv of producing enzymes by fermentation: high productivity, high yield, constant quality, helps to obtain enzymes specifically targeted to perform specific tasks under required conditions (detergent enzymes are active at very low temp)
- Productivity of microorganisms may be improved by natural mutation or genetic modification.
Phases of enzyme production (More details)
1. Selection of enzyme –>Criteria of enzyme: stability, effects of inhibitors, affinity to substrate
5. Optimization of recovery process: When fermentation is complete, the production strain cells are inactivated and removed by centrifugation or filtration, separating the resulting enzyme from its product strain. The enzyme concentrate is then recovered by ultrafiltration, then are purified by ion exchanger or gel filtration
Example: Proteases (enzyme that break the peptide bonds of protein) —> used A.niger as microorganism
