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FERMENTATION BIOTECHNOLOGY
Exam: oral about the first part, written about the second part. The first examination is on appointment. Artificial metabolism: creation of specific metabolism in the bacteria in order to produce something. Why use biotech process?
- The environmental impact is lower.
- Can be very specific due to chemo-, regio- and enantioselectivity
- Several complex reactions might be conducted very rapidly in “one-pot” Fermentation: sensu stricto the fermentation is a metabolic process in which the final acceptor of the electron transport chain is an organic substrate, it is processed by microorganisms, what can be wild type or recombinant cells. But in general, the fermentation can be considered as a productive process based on the use of microbial cells with the aim to produce specific products. These products can organic acids, amino acids, vitamins, antibiotics and others. It is important to assure the optimal conditions to the microorganisms. Biotransformation is a chemical reaction performed by whole cells or isolated (or immobilized) enzymes. The substrate is often synthetic and structurally similar to the product. Requirements: use of defined enzymes, possible exploitation of broad range substrate specificity of enzymes. Also, it’s possible to use the plants ad bio-factory to produce bio-molecules. Different processes to different products.
- Biomass - > baker’s yeast, starters, probiotics
- Anaerobic fermentation - > ethanol, lactic acid
- Primary metabolism - > amino acids, vitamins
- Uncompleted oxidation - > acetic acid, gluconic acid
- Secondary metabolism - > antibiotics
- Recombinant proteins
- Polysaccharides
On the BRENDA site there are 7341 enzymes, these are really low than the genes it’s possible to find, the reason of this low number is that these enzymes are completely characterized (optimal pH, mass, function, gene, position in the genome etc.). Name coding of enzymes is composed by 4 numbers.
- The first number shows the family of the enzyme (oxide-reductase, transferase, ligase etc.).
- The second number: where it processes its reaction
- The third: co-factor required
- The fourth: the substrate of the reaction The number of enzymes is virtually infinite due to all the possible techniques to modify and improve. The direct way to modify the gene were studied and applied in 1956. One of the most requirements is high-throughput activity test, because it needs to screen millions of mutants at the same time. The type of protein must be soluble and active in the expression system. Why microbial cells?
- Show high biodiversity
- High structural variability of produced molecules
- Easy to isolate and cultivate
- High growth rates in short time
- High variety of enzymatic activities (see Brenda-enzymes.org)
- Small dimensions mean possible high specific activities To do the modification: Rational approach (rational design)
- Know everything of the protein: 3D structure, catalytic mechanism, amino acid sequence, its residues, in what condition it’s soluble etc.
- Site-direct mutagenesis
- Cloning-Expression-Purification of the protein
- Activity test------ positive mutant Irrational way, direct evolution
- Casual mutation (error-prone PCR, DNA shuffling), with the error prone PCR the aim is generating new fragments, cloning it in new bacteria and checking the activity till to obtain the desired enzyme. Till to obtain the “perfect” could mean repeat the process many times because the mutation, where it will occur and how it modify the enzyme, is uncontrollable.
- The cultivation conditions
- Operating mode: batch, fed-batch (the final volume is less than the starter), continuous (it is like the grow curve of the bacteria is fixed on the exponential step, there is a continuous addition of substrate). Types of organisms Bacteria : Multitude of strains producing a wide variety of large and small molecule products While E coli is the preferred host for therapeutic proteins, numerous other commercial expression systems are available for small molecule production Streptomyces species are the preferred hosts for secondary metabolites Yeast: Large and small molecules Often hosts are methylotrophic P. pastoris and S. cerevisiae Other hosts include Hansenula polymorpha and Yarrowia lipolytica Fungi: Many different filamentous fungi are used to produce secondary metabolites enzymes, and organic acids at the industrial scale Cyanobacteria : Ancient form of life best known for its edible genus Spirulina grown in open ponds and sold in tablet form as dietary supplement A recombinant strain is used for phototrophic biofuel production Potential producer of cytotoxic molecules. Algae : industrially used for single cell algae (chlorella), β-carotene (Dunaliella), or polyunsaturated fatty acids (PUFAs). Clorella is another genus used as health food, it’s a preferred algal expression host. Plant cells : production of the anticancer plant secondary metabolite paclitaxel in 75 m^2 bioreactors. Other products are the therapeutic enzymes Eleyso TM in recombinant carrot cells or ginseng saponins. As with cyanobacteria and algae, plant cells can be cultivated hetero, photo-, or mixotrophically. To ensure a successful industrial process it needs to consider:
- The microorganism used, its genotype and also assure to the cells the optimal condition to grow and produce the desired molecules.
- The composition of the medium, during its preparation it’s important to sterilize it and knowing it behavior it’s possible to predict the formation of foam and take the right measures and precautions.
- The grow conditions (pH, T, pO 2 , pCO 2 , mixing time and soon) have to controlled constantly.
- The operating mode: batch, fed-batch, continuous, or perfusion. Large-scale bioprocesses: factors for success
- Genotype of the cell that is controlled and steered by physicochemical environment within the bioreactor, for which a whole arsenal of inline sterilisable sensors are available for control.
- Composition of the culture medium, which is ideally chemically defined and simple. Moreover, when formulating a culture medium recipe, the coalescence characteristics of the medium or the foaming behavior, must be taken into consideration early.
- Cultivation conditions (T, pH, pO 2 , pCO 2 , mixing time, and shear), which are maintained by the bioreactor’s capacity for heat and gas transfer, in most cases, ad hoc hardware changes to industrial bioreactors are limited to changing turbines and impellers only.
- Operating mode such as batch, fed-batch, continuous or perfusion. Types of bioreactors Mechanical stirring (Continuosly Stirred Tank Reactor) Pneumatic stirring (Bubble column and air-lift reactor) Factors for adaptation/modification
- Oxygen: extremely important to assure the grow of the cells if they need it
- heat transfer requirement: is fundamental and the most expensive factor, if it doesn’t work and the temperature becomes too high it could kill all the cells
- sensitivity to shear
- sensitivity to process and culture variations
- sensitivity to local variations within the bioreactor
- current good manufacturing practice (cGMP) requirements
- biosafety requirements (containment levels are normally BLS1 and BLS2)
- specific safety requirements for highly potent pharmaceutical ingredients (HPAPI)
100 mL, the first inoculum has to be gradually adapted, the culture starts from a small to big fermenters, this process could be very expensive. Oxygen is medium component; it has to be specified the quantity. His solubility is 23 mg/L, this is affected by different factors, temperature, pressure. Oxygen transfer rate: the air bubble is wrapped in different strata to reach the microorganism could create problem, it needs also to consider the cells. Antifoam : in case of complex medium it’s possible the formation of foam, due to the medium and bubbles, this is dangerous because can destruct the entire culture. This is possible to control it. 1 using an antifoam (it needs to be not toxic for the cells), has to be used before the scale up (sulphuctant), another way to control it is though the mechanical way, push it to the surface and eliminate it. Cost : power, cost to continue to produce. Problem with oxygen: possible solution: 1 increase the speed way but there is an incensement of heat and pressure. High energy consumption. 2 increase the oxygen air. It’s really important to consider the oxygen transfer rate. Process control PID : control of the pH (affected by temperature, heat, electricity), one way is the addition of basic or acid by a computer which control constantly the pH in the medium, but the addition of acid/basic could be wrong and this can cause a big damage, to avoid some errors as that there is a formula that allows to be more precise, this formula is called PID. The formula considers the proportional, integral and derivate, this is easy to lab scale reactors, very difficult for the industrial. To control the level of foam usually on the top of the reactor there is a piece of metal, an electrode, which in case of touch with the bubble’s foam, sends a signal to the control pc. The concentration of oxygen can be controlled with an electrode. One way to calculate the presence of oxygen is control the consumption rate of it by the bacteria because at the begging the concentration of oxygen is 100%. Another way, but very expensive, is enrich the oxygen concentration in the air that is added to the reactor, another advantage is the possible to control it in remote. There are very big differences in the composition medium between medium for industrial rectors and lab reactor. There exists also the scale down: it is used to optimize the ingredient, their concentration in the medium and other, but having a very small bioreactor, but very similar to the industrial, it’s possible “make research” and try to improve avoiding to spent millions of euros. These reactors are very small /50 mL). it’s possible to buy twenty of this, each is controlled by one pc, control every of them bye one pc and playing the different concentration; one this cost 20.000 euros.
It is important to consider the shear-stress which can have negative effects on the growth rate. To avoid it or reduce it, it’s a good idea study the geometry of the bioreactor, lab and industrial scale, be very careful in the choose of the turbines due to the flux them create inside the bioreactor. It should be considered also the solubility of the oxygen. It’s important because the oxygen is a medium component and the microorganisms assimilate it from the medium and not from the gas phase. There are different factors those affect the its solubility: temperature, pressure, salts, medium components, antifoam, reactor geometry, turbines, speed of the flus, the position of the sparger and also the same microorganisms can influence the oxygen solubility. Comparison of different way to add oxygen CSTR = Continuously stirred tank reactor
Due to the enormous difference of volume between a lab scale fermenter and an industrial there is also a difference in the medium composition. Notes: the cryopreservation might contemplate the use of preservatives (glycerol, DMSO, sucrose…). Dual (or more) – site storage of all cell lines, on-site and off-site in order to avoid loss of the cell line. Increasing the scale of production and hence of the bioreactor working volume up to an appropriate value, in order to meet to market demand Economic factors (market, costa of plant, downstream…) Process (yields and productivity, performances of the downstream process) Before to reach the industrial scale of production is necessary start from another point. The starting point is the lab-scale reactor, this is between 1 and 10 liters, during this step the growth conditions are really optimized. The following/subsequent step there is scale-up between 20-200 liters, this is the pilot-scale. The last step is the industrial production. Notes: it’s possible that between the pilot-scale and the industrial production there are other step those allow to reach the optimum inoculum (10% of the final volume of the bioreactor). It’s important the set-up of scale-dependent parameters mixing, heat and mass transfer (sterilization); nutrients, additions (difference of pH), inoculum, CIP, SIP. Remember that there are important differences in scale-lab medium and the industrial-scale; it is a good idea to use a cheap medium during the first step in order to reduce the costs of production. An example of cephalosporin C production, Acremonium chrysogenum, 1000 ton/y of production
Process improvement: industrial strain
- suitable conditions for best growth and production
- easiness of cultivation on cheap media and large scale
- high yields
- microbiologically and genetically stable
- non pathogen tools to do this: - chemical and physical mutagenesis (long process), - metabolic engineering, protein engineering. Parameters to improve the process
- The volumetric increase (g/L/day), determinate a part of the cost production
- Elimination of undesired pigments of pyrogens, the case one the cellwall of coli (gram
- ) in the case of production of medicine or human protein, this adds another cost.
- Shorter fermentation times
- Less O 2 consumption
- Medium: foaming and viscosity reduction, also cheaper formulation
- Tolerance to high concentration of C and N.
- Phage resistance Identification of the response of the production due to the variation applied on the conditions.
- Placket-burman design: important. After the different experiments and obtained the titer. So, it is calculated when the fermentation is at the lowest and at the highest level, this value x 2 is similar to the value obtained before it was right. Using the statistical tools, it’s possible to calculate the P value which shows if es era right or no, it’s a pure demonstration if the theory is right or wrong. Doesn’t show all the possible interactions.
The same results can be seen by pareto chart.
Statistical interaction: it is defined when the concentration of B its affected by A. Immobilization techniques There are different techniques to immobilize an enzyme or a protein on solid or mobile matrix. Why the biocatalysis? But this can be considered as a green chemistry? Why?
- Prevention: it is better to prevent waste than to treat or clean up waste after it is formed.
- Atom economy: synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product.
- Less hazardous chemical synthesis: whenever practicable, synthetic methodologies should be designed to use and generate substances that possess little or no toxicity to human health and the environment.
- Designing safer chemicals: chemical product should be designed to preserve efficacy of the function while reducing toxicity.
- Safer solvents and auxiliaries: the use of auxiliary substances (solvents, separation agents, etc.) should be made unnecessary whenever possible and, when used, innocuous.
- Design for energy efficiency: energy requirements should be recognized for their environmental and economic impacts and should be minimized. Synthetic methods should be conducted at ambient temperature and pressure.
- Use of renewable feedstock: a raw material re feedstock should be renewable rather than depleting whenever technically and economically practical.
- Reduce derivatives: unnecessary derivatization (blocking group, protection/deprotection, temporary modification of physical/chemical processes) should be avoided whenever possible.
- Catalysis: this process is more precise and superior than the stoichiometric reagents.
- Designed for degradation: chemical products should be designed so that at the end of their function they do not persist in the environment and instead break down into innocuous degradation products.
- Real-time analysis for pollution prevention: analytical methods need to be further developed to allow for real-time in-process monitoring and control prior to the formation of hazardous substances.
- Inherently safer chemistry for accident prevention: substance and the form of a substance used in a chemical process should be chosen to minimize the potential for chemical accidents including releases, explosions and fires. The enzymes are import to accelerate the reactions, it’s very useful due to its selectivity; their selectivity is chemo selectivity, regioselectivity and stereoselectivity; them are really efficient. It’s also really important consider the milder reaction conditions (pH, temperature, pressure…) because them can influence deeply the efficiency of the catalyst. It is possible to use the enzymes inside the cells using the entire cell as a bioreactor. The cells can be used during the growth phase or during the stationary phase (wash them and re-suspend in buffer). Advantage: not need a purification, less cost, only if there no other enzymes those compete with the substrate of the reaction, and the enzyme requires cofactors; for example, the reaction oxido-reductise of a chiton into aldehyde because the catalyst requires NADPH/NADP+. The stoichiometry between the chiton and the cofactors is 1:1. (the cost of 50 mg of NADPH is 100 euro). But using the cells is possible to create a system of the regeneration of NADPH. It is important to consider the solubility/diffusion between the cell walls. Pro:
- Cofactors and systems for their regeneration
- Cost
- Recovery Cons:
- Competing enzymatic activities
- Facilities for fermentation
Immobilized biocatalyst The immobilized catalysts (those can be cells of enzyemes) are attached in or onto the surface of an insoluble support; this support can be a membrane or an inert support. Advantages of immobilized enzymes over the soluble forms of the same enzymes: o Convenience: heterogeneous system workup can be much easier. Upon completion and filtration, reaction mixtures typically contain only solvent and the products of the reactions. o Economical: easily removed from reaction, so while the enzymes are fixed on the support and the reactions go on it’s possible to remove the products from the surface of the support, but the enzymes rest there and are ready for next reaction. o Stability: immobilized enzymes may have greater thermal and operational stability and the corresponding soluble form (lipases). In summery scheme the advantages and disadvantages of homogeneous and heterogeneous catalyst: Before doing the immobilization of the enzyme it’s important to consider some criteria in order to obtain a good result of the process:
- The biological component, the enzyme must have still its catalytic activity and be active
- It must have a long-term stability and durability
- The diffusion phenomena must be favorable for the enzymatic activity, there must be a flow of the reagents. Immobilization methods : adsorption (ionic), covalent bonding, entrapment, encapsulation, self-immobilization/cross-linked. Ionic: discovered in 1916; the first model was an invertase on the activated charcoal (Nelson and Griffin). It is easy to create. To do it it’s necessary to create the right condition of pH, temperature between the enzyme and the support (usually utilized is the aluminum hydroxide) in order to create on the surface of the enzyme an opposite charge of the support (the enzyme and the support have to have opposite charge). Disadvantage: the connection between the two parts is too weak so the leakage is generally a problem, but with a suitable charged matrix it’s possible to promote the creation of ionic interactions. Also, the osmolality is to control. The best-known industrial examples are: the amino acylase (enzyme) on DEAE-Sephadex in the production of amino acids, the other example is the use glucose isomerase in the production of high fructose corn syrup (HFCS). Another example, 20 years ago the industry of milk used 1 delactolyse in column, now it’s cheaper and easier, are used free enzymes. The forces are wan der walls and ionic. Example of adsorption of microbial cells: mammalian cells on micro-carriers and waste/water treatment. This can happen as a natural phenomenon, due to the production of biofilms. It is necessary that the cells can create the biofilm (ex: using iso-poly-saccharide) to attach to the surface so it can be used wood chips into the fermenter to give a place to microorganisms where they can create the biofilm. Entrapment by polymerization: the enzymes may be entrapped within the matrix of a polymeric gel: to do it it’s necessary to incubate the enzymes together the gel monomers, promote the gel polymerization; ex: the gel polyacrylamide. Entrapment by gelation: in this case the whole cells are used as catalysts, them can be mixed with a liquid polymer which may give gelation becoming solid or semi-solid.
- Agar (thermal gelation)
- Alginate (ionic gelation)
- Carrageenan
- Chitosan
- Gelatin
- Collagen The alginate gel, based on the ionic interactions, adding Na+ becomes viscous, adding Ca2+ becomes solid, it is useful to immobilize the cells, especially the big ones. The calcium is added with a needle, the dimension of the needle determinates the dimension of the particles, the gelation is instant, it needs a quite agitation. Gel-fibre entrapment and encapsulation: The gel pore is a crucial factor to ensure that substrate and product molecules can freely pass into and out of the macroscopic structure.
