246 Chapter 8 production a disadvantage of using continuous fermentation is the chance of contamination by stran variablity other micro-organisms during the long fermentation runs(sometimes several weeks) Ithough this sometimes happens, it should not be overemphasised, since most of the time the fermentation conditions are such that a special niche is created, and only a limited number of other micro-organisms can grow. A more serious problem is the occurrence of variants of the parent production strain by back mutation or loss of genetic elements(plasmids). In addition, phage infections are more likely to occur. Despite the advantages of continuous cultures, the technique has found little application in the fermentation industry. A multi-stage system is the most common continuous fermentation and has been used in the fermentation of glutamic acid. The continuous start-up of a multi-stage continuous system proceeds as follows. Initially batch system fermentation is commenced in each vessel. Fresh medium is introduced in the first vessel, and the outflow from this proceeds into the next vessel. The overall flow rate is then adjusted so that the substrate is completely consumed in the last vessel, and the intended product accumulated. The concentration of cells, products and substrate will hen reach a steady state. The optimum number of vessels and rate of medium input can be calculated from simple batch experiments 8. 4. 4 Problems in the fermentation of amino acids The following problems in the industrial fermentation of amino acids may arise contamination of the culture by other micro-organisms during fermentation; bad fermentation reproducibility due to differences in raw material back mutation or loss of genetic material of the production strain infection of the culture by bacterial viruses(phages hough the first two possibilities can lead to severe problems in the fermentation of amino acids, these problems can be prevented by: using proper plant design maintenance of hygienic conditions throughout the operation; reservation of large batches of raw material with uniform qualities. Much more severe (and much more difficult to control) are the last two possibilities which will now be discussed in more Back mutation and loss of genetic material Back mutation (reversion) of the mutant production strain to the wild type (prototrophic form) can occur. Since auxotrophic mutants and regulatory mutants are widely used in the ahiproduction of amino acids, this can be a severe problem. In nature, mutation ys takes place but this takes some time. However, in fermentation many mutation are enp roduced in a relatively short period of time and the chances of back ∏ Make a list of possible ways of solving the problem of reversion The main ways to solving the problem of reversion are
246 Chapter 8 produclion strain variability multi-stage axltinWUS sysOsm A disadvantage of using continuous fermentation is the chance of contamination by other micmrganisms during the long fermentation runs (sometimes several weeks). Although this sometimes happens, it should not be overemphasised, since most of the time the fermentation conditions are such that a special niche is created, and only a limited number of other micro-organisms can grow. A more serious problem is the occurrence of variants of the parent production strain by back mutation or loss of genetic elements (plasmids). In addition, phage infections are more likely to occur. Despite the advantages of continuous cultures, the technique has found little application in the fermentation industry. A multistage system is the most common continuous fermentation and has been used in the fermentation of glutamic acid. The start-up of a multi-stage continuous system proceeds as follows. Initially, batch fermentation is commenced in each vessel. Fresh medium is introduced in the first vessel, and the outflow from this proceeds into the next vessel. The overall flow rate is then adjusted so that the substrate is completely consumed in the last vessel, and the intended product accumulated. The concentration of cells, products and substrate will then reach a steady state. The optimum number of vessels and rate of medium input can be calculated from simple batch experiments. 8.4.4 Problems in the fermentation of amino acids The following problems in the industrial fermentation of amino acids may arise: 0 contamination of the culture by other microaganisms during fermentation; 0 bad fermentation reproducibility due to differences in raw material; back mutation or loss of genetic material of the production strain; 0 infection of the culture by bacterial viruses (phages). Although the first two possibilities can lead to severe problems in the fermentation of amino acids, these problems can be prevented by: using proper plant design; maintenance of hygienic conditions throughout the operation; reservation of large batches of raw material with uniform qualities. Much more severe (and much more difficult to control) are the last two possibilities which will now be discussed in more detail. Back mutation and loss of genetic material Back mutation (reversion) of the mutant production strain to the wild type (prototrophic form) can occur. Since auxotrophic mutants and regulatory mutants are widely used in the overproduction of amino acids, this can be a severe problem. In ~tuI.e, mutation always takes place but this takes some time. However, in fermentation many generations are produced in a relatively short period of time and the chances of back mutation are enhanced. n Make a list of possible ways of solving the problem of reversion. The main ways to solving the problem of reversion are:
Industrial production of amino acids by fermentation and chemo-enzymatic methods 247 pro, em e .make use of fresh starting material Inoculum) for each run; make use of antibiotics that inhibit growth of the wild type(or revertant) but not the mutant start with mutants which are very stable(many companies spend a lot of money in the isolation of stable mutant strains) Loss of genetic material (for example the constructed plasmid containing the genetic information necessary for overproduction of the amino acid )can also occur. This will happen in situations where there is an energetically favourable advantage for he micro-organism not to have the plasmid in the cell Possible ways to overcome the problem are: construction of a strain in such a way that it is energetically advantageous to overproduce the required amino acid, thus keeping the construct in the ce introduction of a genetic marker into the construct (usually antibiotic resistance encoded by plasmid genes) and operation of the fermentation under selective pressure by adding the relevant antibiotic to the medium. Only the micro-organisms carrying the marker would survive, while others die off. only Phage infection Phage infections sometimes occur in big fermentation plants and cause severe damag stages in Phages are bacterial viruses composed of a nucleic acid core(dNA or RNA)in a protein inction envelope. Infection proceeds in the following way: adsorption onto the bacterial cell followed by introduction of genetic material into the bacterium multiplication within the cell making use of the genetic apparatus of the bacterial liberation of the infective phages by lysis of the cell resulting in the death of the bacterial cell There are several different groups of phages, each with there own characteristics and host range specificity Phages are widely distributed in nature(soil, air, raw materials etc), they are rather stable and can be introduced to the fermentation easily through the air. Phage infections can be recognised if fermentation characteristics change. The slowing down of the process(for example delay in onset of the fermentation and decrease in amino adid production yield are the first signs of a moderate form of phage infection. If lysis of the cells occurs in the growth phase, accompanied by a rise in pH and increase in foaming, this suggests that there is a severe phage infection. Confirmation of a phage infection can be done by electron microscopic observation
Industrial production of amino acids by fermentation and chemo-enzymatic methods 247 soluthm he 0 make use of fresh starting material (inoculum) for each run; problem of 'reversian' 0 make use of antibiotics that inhibit growth of the wild type (or revertant) but not the mutant; 0 start with mutants which are very stable (many companies spend a lot of money in the isolation of stable mutant strains). Loss of genetic material (for example the constructed plasmid containing the genetic information necessary for overproduction of the amino acid) can also occur. This will happen in situations where there is an energetically favourable advantage for the mirro-organism not to have the plasmid in the cell. Possible ways to overcome the problem are: construction of a strain in such a way that it is energetically advantageous to overproduce the requid amino acid, thus keeping the construct in the cell; introduction of a genetic marker into the construct (usually antibiotic resistance encoded by plasmid genes) and operation of the fermentation under selective pressure by adding the relevant antibiotic to the medium. Only the micro-organisms carrying the marker would survive, while others die off. Phage infection Phage infections sometimes occur in big fermentation plants and cause severe damage. Phages are bacterial viruses composed of a nucleic acid core (DNA or RNA) in a protein envelope. Infection proceeds in the following way: adsorption onto the bacterial cell followed by introduction of genetic material into the bacterium; stages in Phage inkdm multiplication within the cell making use of the genetic apparatus of the bacterial cell; liberation of the infective phages by lysis of the cell resulting in the death of the bacterial cell. There are several different groups of phages, each with there own charackristics and host range specifiaty. Phages are widely distributed in nature (soil, air, raw materials etc), they are rather stable and can be introduced to the fermentation easily through the air. Ph infections can be recognised if fermentation characteristics change. The slowing T own of the p'ocess (for example delay in onset of the fermentation) and decmase in amino add production yield are the first signs of a moderate form of phage infection. If lysis of the cells occu~s in the growth phase, accompanied by a rise in pH and increase in fuaming, this suggests that there is a severe phage infection. confirlnation of a phage infection can be done by electron microscopic observation
Chapter 8 latent phage An additional problem is the presence of latent forms of phage infections, in which the nfectons production strain carries the phage genome integrated in the chromosome and only in pecific situations (after induction)releases the phages These may then be changed to e virulent form and infect other micro-organisms present. Prevention of phage infections is difficult and there is no absolute preventative measure. However, phages have their own optimum conditions(pHand temperature)for infection and replication. By choosing the proper conditions it is possible to create an environment that is favourable for bacterial growth but not for phage multiplication Other ways to prevent phage contamination or multiplication are plant hygiene(sterilisation of raw material; disinfection of equipment by or chemical agents; using proper air filters, etc); isolation of phage resistant strains-however, the problem is that after a while other ypes of phages become capable of infecting the strain, inhibition of phage multiplication by prevention of phage adsorption to the cell wall using chemical agents during fermentation(chelating agents capture calcium ions necessary for adsorption; use of surfactants like Tween SAQ 8. 4 Give possible reasons for selecting each of the following operating conditions for a hypothetical amino acid fermentation 1)Fed-batch mode. 2)Monoseptit 3)Antibiotics added to medium 4) Fresh inoculum used for each run 5) Chelating agent added to medium. 8. 5 Recovery of the amino acid from the fermentation broth Methods of product recovery are considered in detail in the BIOTOL text entitled Product Recovery in Bioprocess Technology. In this chapter we will briefly review the methods applicable to recovery of amino acids eral criteria General criteria for the economically feasible recovery of amino acids from the for economic fermentation broth are the recovery should be as simple as possible the recovery yield of the processes should be high process steps that give rise to loss of product should be avoided as much as possible: the process should be easy to scale-up industrially
248 Chapter 8 An additional problem is the presence of latent forms of phage infections, in which the production strain carries the phage genome integrated in the chromosome and only in specific situations (after induction) releases the phages. These may then be changed to the virulent form and infect other micro-organisrns present. Prevention of phage infections is difficult and there is no absolute preventative measure. However, phages have their own optimum conditions (pH and temperature) for infection and replication. By choosing the proper conditions it is possible to create an environment that is favourable for bacterial growth but not for phage multiplication. Other ways to prevent phage contamination or multiplication are: 0 plant hygiene (sterilisation of raw material; disinfection of equipment by temperature or chemical agents; using proper air filters, etc); 0 isolation of phage resistant strains - however, the problem is that after a while other types of phages become capable of infecting the strain; 0 inhibition of phage multiplication by prevention of phage adsorption to the cell wall using chemical agents during fermentation (chelating agents capture calcium ions necessary for adsorption; use of surfactants like Tween). latent phage infections Give possible reasons for selecting each of the following operating conditions for a hypothetical amino acid fermentation. 1) Fed-batch mode. 2) Monoseptic. 3) Antibiotics added to medium. 4) Fresh inoculum used for each run. 5) Chelating agent added to medium. 8.5 Recovery of the amino acid from the fermentation broth Methods of product recovery are considered in detail in the BICKOL text entitled 'Product Recovery in Bioprocess Technology'. In this chapter we will briefly review the methods applicable to recovery of amino acids. generalaisria General criteria for the economically feasible recovery of amino acids from the SaSibiMy f~r~~ic fermentation broth are: the recovery should be as simple as possible; the recovery yield of the processes should be high; process steps that give rise to loss of product should be avoided as much as possible; the process shouid be easy to scaleup industrially
Industrial production of amino acids by fermentation and chemo-enzymatic methods 249 ∏ Amino acid fermentations do have several advantages over other kinds of fermentations, like for example the production of enzymes, in respect of recovery of desired product. Make a list of the advantages that you can think of. The main advantages are: normally the production strain is constructed in such a way that overproduction of the desired amino acid is obtained and no, or only minor concentrations of unwanted contaminants appear optical resolution steps are not necessary(as in the case of most chemical-processes nce only the L-form is synthesised, the required amino acid can be relatively easily separated from cells and protein By making use of the physicochemical properties of the amino acid that is required, it is possible to obtain highly purified and/or concentrated product. This is done by a combination of several processes, the number of which is dictated by the final application for the product and by economical feasibility Specific methods to separate the amino acid required from its contaminant products, cells and proteineous material from the culture broth methods for Following cell and proteineous material removal, one or more specific methods of amino acd amino acid recovery are applied. These are ion exch solvent extraction We will now consider each of these methods in turn 8.5.1 Separation of cells and proteins from the fermentation broth scale ly used, these are centrifugation and filtration.Both methods are used on a large scale and are economically feasible. centrifugation Centrifugation can be operated on a semi-continuous or continuous basis and there are everal different types of centrifuges. Large scale tests have to be performed to choose the proper centrifuge(unloading speed, capacity, separation performance etc) Sometimes poor centrifugation behaviour of cells can be improved by adding flocculation agents. These agents neutralise the anionic charges(carboxyl and phosphate groups)on the surface of the microbial cells. Examples of flocculation agents are alum calcium and ferric salts, tannic acid etc
Industrial production of amino acids by fermentation and chemo-enzymatic methods 249 Amino acid fermentations do have several advantages over other kinds of fermentations, like for example the production of enzymes, in respect of recovery of desired product. Make a list of the advantages that you can think of. n The main advantages are: 0 normally the production strain is constructed in such a way that overproduction of the desired amino acid is obtained and no, or only minor concentrations of, unwanted contaminants appear; optical resolution steps are not necessary (as in the case of most chemical-processes) since only the L-form is synthesised; the required amino acid can be relatively easily separated from cells and protein impurities. By making use of the physicochemical properties of the amino acid that is required, it is possible to obtain highly purified and/or concentrated product. This is done by a combination of several processes, the number of which is dictated by the final application for the product and by economical feasibility. Specific methods to separate the amino acid required from its contaminant products, such as medium components and other amino acids, are plpceeded by removal of the cells and proteineous material from the culture broth. Following cell and proteineous material removal, one or more specific methods of amino acid recovery are applied. These are: aystallisation; 0 ionexchange; 0 electrodialysis; 0 solvent extraction; 0 decolorisation; 0 evaporation. We will now consider each of these methods in turn. rnelhodsbr amino acid r-ery 8.5.1 Separation of cells and proteins from the fermentation broth Two methods are commonly used, these are centrifugation and filtration. Both methods are used on a large scale and are economically feasible. Centrifugation can be operated on a semi-continuous or continuous basis and there are several different types of centrifuges. Large scale tests have to be performed to choose the proper centrifuge (unloading speed, capacity, separation performance etch centrifugation Sometimes poor centrifugation behaviour of cells can be improved by adding flocculation agents. These agents neutralise the anionic charges (carboxyl and phosphate groups) on the surface of the microbial cells. Examples of flocculation agents are alum, calcium and ferric salts, tannic acid etc
Chapter 8 filtration Filtration is another possibility to remove the cells from the amino acid containing broth factors to be considered are: properties of the filtrate(solid/liquid ratio); nature of the solid particles(type of micro-organism); adequate pressure(or vacuum) to obtain adequate flow rates; negative effects of antifoaming agents on filtration. teraids Sometimes filtration can be improved by using filteraids. These filteraids, which are a faster flow rate. Before filtration a thin layer is us by of a resulting filter cake leading to standard filters). After that a mix is made with the harvest broth and filtration is started 8.5.2 Crystallisation Crystallisation is often used as a method to recover the amino acid. Because of the pH amphoter ic character f contain both adif and asio groups)d of amino acids their the isoelectric point(zero net charge temperature Since temperature also influences the solubility of amino acids and their salts, lowering the temperature can be used in advance as a means of obtaining the required product lts Precipitation of amino acids with salts, like ammonium and calcium salts, and with metals like zinc are also commonly used. This is followed by acid (or alkali)treatment to obtain the free or acid form of the amino acid 8.5.3 lon exchange lon exchange resins have been widely used for the extraction and purification of amino acids from the fermentation broth The adsorption of amino acids by ion exchange resins is strongly affected by the ph of soluton ant nd by the presence of f contaminant ions. There are two types of ion exchange resins; cation exchange resins and anion exchange resins. Cation exchange resins bind positively charged amino acids( this is in the situation where the ph of the exchange resins bind negatively charged amino acids (pHof the solution is higher than IEP). Elution of the bound amino acid (s)is done by introducing a solution containing he counterion of the resin Anion exchange resins are in their exchange capacity and durability than cation exchange resins yeer used for industrial separation. In general, ion exchange as a tool for sep on is only used when other steps fail, because of its tedious operation, small capacity and high costs Examples given in literature are 1)strongly basic anion exchange(Oh)for the separation of: L-glutamic acid from broth
250 Chapter 8 fihhll filleraids PH Oemp8fatUEl Salts cation and anion exchange resins Filtration is another possibility to remove the cells from the amino acid containing broth. Factors to be considered are: 0 properties of the filtrate (solid/liquid ratio); 0 nature of the solid particles (type of micr~rgani~m); 0 adequate pressure (or vacuum) to obtain adequate flow rates; negative effects of antifoaming agents on filtration. Sometimes filtration can be improved by using filteraids. These filteraids, which are based on diatomaceous earth, improve the porosity of a resulting filter cake leading to a faster flow rate. Before filtration a thin layer is used as a precoat of the filter (normally standard filters). After that a mix is made with the harvest broth and filtration is started. 8.5.2 Crystallisation Crystallisation is often used as a method to recover the amino acid. Because of the amphoteric character (contains both acidic and basic groups) of amino acids, their solubility is greatly influenced by the pH of the solution and usually show minima at the isoelectric point (zero net charge). Since temperature also influences the solubility of amino acids and their salts, lowering the temperature can be used in advance as a means of obtaining the requved product. Precipitation of amino acids with salts, like ammonium and calcium salts, and with metals like zinc are also commonly used. This is followed by acid (or alkali) treatment to obtain the free or acid form of the amino acid. 8.5.3 Ion exchange Ion exchange resins have been widely used for the extraction and purification of amino acids from the fermentation broth. The adsorption of amino acids by ion exchange resins is strongly affected by the pH of the solution and by the presence of contaminant ions. There are two types of ion exchange resins; cation exchange resins and anion exchange resins. Cation exchange resins bind positively charged amino acids (this is in the situation where the pH of the solution is lower then the isoelectric point (IEP) of the amino acid), whereas anion exchange resins bind negatively charged amino acids (pH of the solution is higher than IEP). Elution of the bound amino acid(s) is done by introducing a solution containing the counterion of the resin. Anion exchange resins are generally lower in their exchange capacity and durability than cation exchange resins and are seldom used for industrial separation. In general, ion exchange as a tool for separation is only used when other steps fail, because of its tedious operation, small capacity and high costs. Examples given in literature are: 1) strongly basic anion exchange (OH) for the separation of 0 L-glutamic acid from broth;