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Industrial : Discovery to Delivery

Gopal K. Chotani*, Timothy C. Dodge*, Alfred L. Gaertner* and Michael V. Arbige*

INTRODUCTION acids, vitamins, and natural polymers for products have penetrated almost food, feed, and other industrial applications every sector of our daily lives. They are used has become well established. Subsequently, in ethical and generic drugs, clinical and microbes have been used as production work- home diagnostics, defense products, nutri- ers in industry. The production of bakers’ tional supplements, personal care products, yeast in deep aerated tanks was developed food and animal feed ingredients, cleaning towards the end of the nineteenth century. The and textile processing, and in industrial German scientist Buchner discovered that applications such as fuel production. active , called , are responsi- Even before knowing about the existence of ble for ethanol fermentation by yeast. During , for thousands of years World War I, Chaim Weizmann used a ancient people routinely used them for mak- microbe to convert maize mash into acetone, ing , soy sauces, yogurt, and . which was essential in the manufacture of the Although humans have used fermentation as explosive cordite. In 1923, Pfizer opened the the method of choice for manufacturing for a world’s first successful plant for long time, it is only now being recognized for fermentation. The process involved fermenta- its potential towards sustainable industrial tion utilizing the mold development. whereby was transformed into citric Since the discovery of fermentative activity acid. Other industrial chemicals produced by of microorganisms in the eighteenth century fermentation were found subsequently, and and its proof by the French scientist Louis the processes were reduced to commercial Pasteur, fermentative production of alcohols, practice. These processes included production amino acids, enzymes (biocatalysts), organic of butanol, , oxalic acid, gluconic acid, fumaric acid, and many more. The serendipitous discovery of in *Genencor International, Danisco Company, 1928 by Alexander Fleming, while research- Palo Alto, CA. ing agents that could be used to combat

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2 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

bacterial infections, opened the whole new “White Biotechnology” in Europe) was world of . American pharmaceuti- deemed the third wave of biotechnology. By cal companies such as Merck, Pfizer, and definition, it is the application of scientific Squibb were the first in mass-producing peni- and engineering principles to the processing cillin in the early 1940s. Initially, of materials by biological agents to provide notatum was surface-cultured in flasks. Later, goods and services. Industrial biotechnology a new , Penicillium chrysogenum, was continues to supply not only unique products, cultured in deep aerated tanks in the presence but is also considered synonymous with sus- of corn steep liquor medium, and gave 200 tainable manufacturing processes. Some dis- times more penicillin than did Fleming’s tinct advantages of industrial biotechnology mold. Streptomycin was next, an include the use of renewable feedstocks (agri- that was particularly effective against the cultural crop materials), generation of less causative organism of tuberculosis. Today, the industrial waste, lower cost for cleanup and list of these antibiotics is long and includes disposal and less pollution. The global market among many others, such important antibi- and fermentation capacity distribution otics as chloramphenicol, the tetracyclines, (Figure 30.1 and 30.2) for industrial biotech- bacitracin, erythromycin, novobiocin, nys- nology products, excluding ethanol, was esti- tatin, and kanamycin. mated in 2002 at $17 billion. McKinsey & Besides the booming antibiotics industry, Company estimates that in the first decade of fermentative syntheses of amino acids such as this century, biotechnology will affect up to L- and L- became billion 20 percent of the worldwide chemical mar- dollar businesses. Despite the long history of ket1. The major volumes of industrial biotech- microbial fermentation processes, under- nology goods such as alcohols, organic acids, standing of the molecular basis of biological amino acids, biopolymers, enzymes, antibi- systems has developed starting from the dis- otics, vitamins, colorants, biopesticides, alka- covery of the double helix DNA only decades loids, surfactants, and , are expected ago. The practice of modern biotechnology to increase; driven by innovative products, started when Boyer and Cohen recombined lower costs, renewable resources, and pollu- DNA, and Boyer and Swanson founded tion reduction. Genentech, the first biotechnology company. Soon, it became clear that novel biotechno- logical manipulations could create new DISCOVERY OF ORGANISMS systems capable of producing new AND MOLECULES and modifying existing products and processes. By the 1980s, a number of new Microbial Diversity biotechnology companies such as Amgen, Microorganisms are chemically similar to Biogen, Cetus, Genencor, and others started higher plant and animal cells; they perform to develop products for healthcare, agricul- many of the same biochemical reactions. tural, and industrial applications. The promise Generally, microorganisms exist as single of biotechnology has been high in terms of cells, and they have much simpler nutrient delivering new products through partnerships requirements than higher life forms. Their with established pharmaceutical, agricultural, requirements for growth usually are limited to and chemical companies such as Eli Lilly, air, a carbon source, generally in the form of Roche, Johnson & Johnson, Monsanto, Shell, , a nitrogen source, and inorganic salts. and others. Fermentation originally meant cellular activity/ By the end of twentieth century, industrial process without , but industrial applications of biotechnology started gaining biotechnology includes both aerobic and momentum and proven laboratory techniques anaerobic fermentation processes. The nutrient started to move into potentially huge markets. source, complex (microbial, plant, or animal The field of industrial biotechnology (called derived) or defined, not only provides carbon, SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 3 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 3

15

12.0

10

5 Billion $/Yr 2.3 2.0 1.4 0.9 0.2 0

EnzymesPolymers Vitamins Antibiotics Amino acids Organic acids Fig. 30.1. Worldwide industrial biotechnology products market (except ethanol).

160 146

120 105

80 69

40 20 16 13 Million Liter 0

Enzymes Polymers Vitamins Antibiotics Amino acids Organicacids Fig. 30.2. Worldwide industrial biotechnology fermentation capacity distribution. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 4 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

4 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

nitrogen, and other elements (P, S, K, Mg, Ca, metabolic pathways that will define the bio- etc.) but also acts as reducing and oxidizing chemical metabolites that can be produced by molecules.2 Of course, in most of the cellular the organism, which usually depends upon the

pathways, O2 from air provides oxidation, environment in which the is releasing abundant metabolic energy from growing. Inherent regulatory control processes redox reactions. Depending on the physiolog- allow cells to regulate their content in ical state of the cell, part of the metabolic direct response to the environment. They pre- energy escapes as that must be removed vent the formation of excess endproduct and by the fermentor design. , derived superfluous enzymes. In this postgenomic from sugarcane/beet and derived era, the sequencing of a genome can be used from starch are often used as the carbon as for metabolic reconstruction of the microbial well as energy sources for fermentation. Use strain. Individual can be identified of other carbon sources such as cellulose, through homology to previously sequenced hemicellulose, fats, etc. might increase in the genes. Transcriptional analysis is another near future. Proteins and fats are more recent tool that allows the simultaneous meas- reduced than saccharides and affect metabo- urement of all actively transcribed genes in a lism in terms of oxidation-reduction fluxes. given organism at any given time. When com- The fluxes also depend on the endproduct(s) bined with genomic information, attempts can being more oxidized or reduced than the start- be made to predict phenotypes or identify the ing substrate(s). organism. Microbes occur in four main groups:3 For industrial processes, microbial strains with faulty regulation, altered permeability, ● and actinomycetales enhanced enzymatic activities, or metabolic ● deficiencies are used to accumulate products. ● Fungi, including yeast Such mutants have been changed so that their ● Protozoa and genetic mechanism is no longer sensitive to a The chemical composition of microorganisms particular controlling metabolite. Modern can be quite varied depending upon such fac- tools typically focus on tors as the composition of the growth creating such strains by making directed medium, the age of the culture, and the cell changes to the DNA of the organism. growth rate. Table 30.1 lists the composition of a typical microbial cell.4 All organisms contain the genetic information to produce a Screening and Selection wide variety of enzymes and hence produce a Product discovery means identifying the great number of chemicals. Individual genes molecular target (enzyme, pathway, metabo- for specific enzymes can be organized into lite) of the intended application. A very early in the discovery of a microbial product involves searching for microorganism(s) that TABLE 30.1 Typical Composition of synthesize the product of interest.5, 23 In the Microbial Cell (About 70% Water) past this meant screening for living microor- ganisms. However, by applying molecular % Dry Cell Weight biology tools,6 today it is possible to screen DNA 3 for the (s) of interest without culturing RNA 20 the organisms. Of course, the rich diversity of 55 Lipid 9 microorganisms in nature is often the starting Liposaccharide 3 point for the screening/selection process. This Peptidoglycan 3 process requires a well-designed growth Glycogen 3 medium, catalyst function, and assay. For Metabolites 3 example, a novel enzyme screening and selec- Metal ions 1 tion strategy is based on an understanding of SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 5 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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the real-world conditions under which the collections. Ironically, a very small number of enzyme must function. Many enzymes are microorganisms that exist in nature have been used in applications, for example, laundry, isolated and those characterized are even , and , that are far removed fewer. There may be many useful reactions in from the natural physiological conditions. organisms yet to be discovered. Faster Such applications expose enzymes to pH, sequencing methods have started to exponen- , and/or chemicals that might tially expand the genomic database. Several inactivate or inhibit them. hundred genomes are in the database and the The classical screening method has its roots tools of are vastly improving in antibiotic discovery. It involves collection for the discovery of genes of interest. of a variety of samples of soils and organic According to J. Craig Venter Institute , and isolation of their microbial popu- (http://www.venterinstitute.org), it should be lation. Being laborious at times (i.e., finding a possible to reduce the cost of sequencing a needle in a haystack), random screening human individual DNA to less than $1000. At methods have been replaced in some cases this low cost and high speed of sequencing, by more efficient selection techniques. These we expect to learn more about human genes techniques subject a microbial source sample by comparing the human genome sequence to to a selection (i.e., pulling a needle genome sequences of other species that have from a haystack selectively with a magnet). For been thoroughly studied. Such diverse infor- example, the growth conditions are selected mation has the potential to aid in the devel- such that only microorganisms expressing the opment of new products, processes, and desired enzyme/pathway/metabolite are able to applications in medicine, agriculture, energy, survive. Therefore, selecting an environment and the environment. (pH, T, growth nutrient) that is optimal for the growth of the desired microorganism increases the success of such a discovery. Cell Engineering Very often, for the source of enzymes, indus- In the earlier days, classical strain develop- trial biotechnology companies look for ment was dependent on the evolutionary microbes called extremophiles that live in process of random . In a normal bac- extreme conditions, because they generally terial population, one mutant arises in about contain enzymes that perform optimally 106 cells. This low rate of spontaneous muta- under extreme conditions. tion is unsatisfactory for strain improvement. In the last decade, tremendous progress has However, mutagens, such as N-methyl-NЈ- been made in sequencing DNA from various nitro-N-nitrosoguanidine (NTG) and Ethyl organisms. It is possible to extract DNA Methane Sulfonate (EMS), are available that directly from the microbial community pres- markedly increase the rate of mutation. ent in a sample taken from the environment. Although it is relatively easy to produce As a result, thousands of gene sequences have mutants, strain improvement requires painstak- been deposited in public databases. This ing effort and ingenuity in devising screening information can be used to screen for pro- tests. Knowledge of microbial physiology is teins/enzymes by searching for similarity at essential in the development of many of these the gene level. Once a homologous gene is screens. Screening for resistance to metabolic identified, it can be transferred to a laboratory analogues is probably the most important host organism such as E. coli or B. subtilis. screening tool for devising means around nor- This allows for the expression of the identi- mal metabolic control mechanisms. Although fied gene product without making the source truly exemplary production organisms for organism overproduce the molecule of inter- important medical and industrial products est. However, application of this technique is were created using this process, the timeframe limited to microorganisms that have been iso- for many of these developments can be meas- lated, characterized, and deposited in culture ured in decades. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 6 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

6 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

In this era of genetic and metabolic engineer- of mathematical models and specifically gen- ing, the timeframe for strain development has erated algorithms that can be used to predict been reduced, although it still is a major effort the right combination of genes that would requiring substantial investment. As in classical enable a cell to overproduce a desired bio- strain development, knowledge of microbial molecule, for example, 1,3-propanediol9, and physiology remains essential to the practice of minimize the synthesis of undesired byprod- metabolic engineering. Understanding the reg- ucts, for example, acetic acid. Using such ulation of genes, in particular the interaction models, scientists have been able to combine of promoters and regulatory proteins, is also in a single host (E. coli), a natural biological essential. The five key strategies for metabolic pathway present in yeast for the production of engineering can be summarized as follows.7, 27 from dihydroxyacetone phosphate, and a pathway present in the bacterium 1. Enhance committed step from central Klebsiella pneumoniae to convert glycerol metabolism or branch point. into 1,3-propanediol, to create a novel and 2. Eliminate transcriptional and allosteric unique process (for details see the section, regulation. Delivery of Products; Figure 30.3). 3. Identify and relieve rate-limiting step(s). Production strains, established through 4. Prevent carbon and energy loss to com- classical mutagenesis and screening to pro- peting pathways. duce metabolites at a commercial level, were 5. Production of foreign enzymes or often started from strains known to overpro- metabolites. duce at least some amount of the desired prod- Sophisticated metabolic models teamed uct. These strains had already overcome some with experimental data are finding wide- of the natural regulation to keep from overpro- spread use in identifying strategic sites ducing metabolites. However, this often led to for metabolic engineering. For example, strains that could overproduce more than one DesignPath® 8 is an approach based on the use endproduct. To maximize production of the

Glucose Acetate

CO2

Acetate

ATP

-- ATP G-3-P Pyr AcetylAcetylCoACoA TCA

DHAPDHAP Gly-3-P Gly 3HPA Propanediol E. coli DHA

Genes recruited from yeast Genes recruited from Klebsiella 1,3 Propanediol (3G) Fig. 30.3. Rational biocatalyst design strategy for 1,3-Propanediol. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 7 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 7

desired endproduct alone, strains were and require access to special expertise; there- selected that could not overproduce a side- fore alternative approaches such as random product. This resulted in the development of mutagenesis and directed evolution have an auxotrophic strain, which required the par- emerged. ticular sideproduct in order to grow. The need Random mutagenesis has been used exten- for specialized growth media often resulted in sively as a tool for increasing the genetic vari- added expense and downstream difficulties as ability and improving microbial strains as the required component(s) were added as described above. However, random mutagen- complex mixtures from animal or plant esis also means less control over the number extracts. By using enzymes from different and types of made. A large number organisms, it is now possible to change gene of clones (called a library) are first generated regulation, prevent carbon loss to competing and then screened or selected for the targeted pathways, and eliminate auxotrophies. properties. Therefore, random mutagenesis The ability to use heterologous enzymes in methods tend to use high throughput or rapid a production host is one of the hallmarks of and efficient screening techniques. modern metabolic engineering. This can be Directed evolution, a term that covers a accomplished by specific directed changes to wide range of techniques designed to quickly the genome or by generating a high level of improve strain performance, uses repetitive diversity. The goal of the effort is to improve rounds of mutagenesis along with screen- a specific phenotype of the strain. This could ing/selection, until a series of mutations accu- be to produce a new metabolite not found in mulate to give an enhanced phenotype. Once the original host or change the energy balance again, the key factors for success are under- in the host or some other critical attribute. In standing of the target gene, the method for any case, deep knowledge of the physiology generating variability, selection, and screen- of the organism is used to develop the appro- ing of the host. The technique makes use of priate screen to find the desired phenotype. the fact that enzymes retain their relevant ter- tiary structures and thereby function. The evolution through a range of acceptable Molecular Engineering molecular structure variations leads to better Despite the diversity of enzymes in nature, enzymes with which the organism can tolerate it is rare that a screened or selected protein/ specific environmental stress. enzyme might have all of the desired charac- Several in vivo as well as in vitro methods of teristics for a targeted application. Therefore, molecular evolution have been developed based it may be necessary to engineer the starting on the recent advances in high-throughput protein by modifying its structure through screening, functional genomics, proteomics, mutation (deletion, insertion, or substitution) and bioinformatics. Molecular evolution tech- of one or more amino acids. Protein engineer- nologies provide efficient tools for creating ing can be carried out by genetic modification DNA libraries (random, directed, recombina- or by chemical means (acetylation, amidation, tional) and processes of selection for a desired oxidation, covalent attachment of ligands). function or characteristic of a target molecule. Compared to the chemical approach, site- For example, “DNA shuffling” is a variation directed genetic manipulation is more pre- of the molecular evolution approach and dictable. The specific changes in the gene involves recombination of closely related sequence are made through the use of recom- DNA sequences, for example, random frag- binant DNA technology. Because the structure mentation and reassembly of the fragments of a protein determines its function, rational into genes. The method is useful to combine protein engineering starts with studying the two or more properties, which have evolved three-dimensional structure of the protein separately, into the target molecule. either by x-ray diffraction, NMR, or molecu- Molecular engineering, optimization, evolu- lar modeling. Often such studies take time tion, and other tools enable the design of SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 8 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

8 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

completely novel biomolecules and proteins status. The complexity of product synthesis for applications in agriculture, medicine, and ranges from a relatively simple and well- industry. understood induction and repression to a very complex regulation. Process development must thus deal with such complexity either by DEVELOPMENT OF A PRODUCTION changing the genetic make-up, or by optimiz- PROCESS ing process conditions. Because classical approaches to strain Strain development are labor intensive and offer lim- Strain development begins with identification ited knowledge, new strain development or isolation of an organism capable of pro- methods now start with a production host that ducing the molecule of interest. Invariably, is capable of rapid genetic manipulation and the initial strain is improved by mutagenesis, efficient product synthesis. Generic hosts and screening, selection, and genetic engineering techniques allow for the construction of mul- to meet the production process and economics tiple strains for multiple products utilizing a requirements. The process requirements baseline fermentation and recovery process. include handling of the strain, fermentation The so-called “toolbox” strategy of building conditions, product recovery, formulation, strains applies to production of enzymes/pro- and application. Not surprisingly, strain and teins as well as biochemicals. Generic host- process development take place in an inte- based strains (denoted GRAS, Generally grated manner. For example, processes that Recognized As Safe) have been proven for are sensitive to the cost of fermentation raw safe use in industrial processes and have been materials look for carbon-efficient strains. approved by regulatory agencies such as the Likewise, processes that aim to achieve high FDA, EPA, and USDA. purity in product need to have Once a desired production strain is devel- that require the use of minimal medium and oped, it can be maintained as a stock culture. strains that produce minimal sideproducts. In maintaining stock cultures, genetic changes New genetic techniques allow the rational must be minimized. This is best achieved by engineering of a production strain. A rational preventing nuclear divisions as most muta- approach means that the genes defining the tions occur as errors during DNA replication. production of a particular metabolite are The method of choice is to store cells or specifically engineered through mutagenesis, spores (if these are produced) in sealed deletion, or over-expression. Controlling ampoules at very low (Յ130ЊC) expression of such genes determines the rate in nitrogen. This method offers the (productivity), yield (carbon conversion effi- great advantage that the culture can be stored ciency), and titer () of the prod- almost indefinitely, thawed, and used immedi- uct. The result is a production organism that is ately as an inoculum without loss of viability optimally tailored for the process and the or diminution in metabolic rate. Cultures kept product. at Ϫ20ЊC to Ϫ60ЊC are satisfactory but less Some bacterial and fungal strains are capa- active than those kept in liquid nitrogen. ble of differentiation (sporulation, filamenta- Although storage at 0–4ЊC allows some tion). This property can affect product growth, this is better than storage at room formation and the physical properties of the temperature. Lyophilization (freeze-) is fermentation broth. Some enzymes are syn- widely used and is very convenient because thesized as secondary products and their freeze-dried cultures retain viability without production does not appear to be growth asso- any genetic changes for years when stored at ciated. Production strains are distinguished on room temperature. It may be noted that all of the basis of their fermentation behavior such these methods are, in effect, techniques to as or recoverability. They can also immobilize intracellular water and yet retain be distinguished by patent or proprietary viability. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 9 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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Fermentation Process The contaminants can also be destroyed by Analysis of the many industrial fermentation heat. Heat sterilization of media is the most processes shows that they are common reac- common method used for sterilization of liq- tions from a chemical, as well as a physical, uid media. This can be accomplished in either viewpoint. Fermentation processes can be clas- a batch or a continuous fashion. sified by the reaction mechanisms involved in Interest in continuous methods for steriliz- converting the raw materials into products. ing media is increasing, but for the successful These include reductions, simple and complex operation of a continuous sterilizer, foaming oxidations, substrate conversions, transforma- of the media must be carefully controlled and tions, polymerizations, hydrolyses, complex the viscosity of the media must be relatively biosyntheses, and the formation of cells. low. The advantages of continuous steriliza- Fermentation processes, except for sterili- tion of media are as follows. zation, have in common many of the familiar 1. Increase in productivity because the unit operations. For short period of exposure to heat mini- example, aerobic fermentations involve the mizes damage to media constituents. “mixing” of three heterogeneous phases: 2. Better control of quality. microorganisms, medium, and air. Other unit 3. Leveling of the demand for process operations include “” of oxygen steam. from the air to the organisms and “heat trans- 4. Suitability for automatic control. fer” from the fermentation medium. Analysis of fermentations by the unit opera- Design and operation of equipment for steril- tion technique has added greatly to the under- izing media are based on the concept of ther- standing of their behavior. Of the operations mal death of microorganisms. Consequently, auxiliary to those in the fermentor, engineers an understanding of the kinetics of the death have made a major contribution towards the of microorganisms is important to the rational design of equipment to provide large volumes design of sterilizers. of sterile medium and air. Close cooperation The destruction of microorganisms by heat between biologists and engineers has resulted implies loss of viability, not necessarily in devising logical methods for screening large destruction in the physical sense. The destruc- numbers of strains, and translating the results tion of organisms by heat at a specific tem- perature follows a monomolecular rate of of shake-flask and pilot-plant experiments to 12,13 production vessels. The scale-up of fermenta- reaction: tions, in some instances, is still empirical dN Ϫ > although sensitive oxygen probes and ϭϪKN ϭϪ(Ae E RT)N, (30.1) dt analysis techniques now available have enabled a rational approach to scaling-up aerated, where K ϭ reaction rate constant with units of Newtonian as well as non-Newtonian, fermen- timeϪ1, N ϭ number of viable organisms/unit tation processes. volume, t ϭ time, T ϭ absolute temperature, E ϭ energy of activation for death, R ϭ gas ϭ Sterilization law constant, and A Arrhenius constant. This equation can be integrated to give the In all fermentation processes, it is necessary design equation: to have contamination-free fermentation

media and seed cultures. Liquid sterilization ts N0 of the fermentation medium is conducted by In ϭ AΎ ϪE>RT (30.2) 10,11 e dt, two means. Contaminating microorgan- Nf 0 isms can be removed from fluids by . ϭ With improvements in , where No number of contaminating organ- sterile filtration is finding wider use, but can isms in the total fermentation medium to be ϭ only be used with completely soluble media. sterilized, Nf level of contamination that SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 10 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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must be achieved to produce the desired structure, their kinetics is similar to that of ϭ degree of apparent sterility, and ts steriliza- heterogeneous catalyzed chemical reactions. tion time. As discussed in regard to microbial diversity, In estimating the sterilization time for the all microorganisms have basic requirements, medium, one must define the contamination, those being water, a source of energy, carbon, the desired degree of apparent sterility, and nitrogen, salts, and trace metals, and possibly the time–temperature profile of the medium; growth factors. However, the media used to that is, T ϭ f(t). For typical bacterial spore isolate and screen production hosts are not contaminants, the constants used in most necessarily those used in production fermen- designs have the following values. tors. Statistical methods (Plackett–Burman, Box–Benkhen) are used for media screening E ϭ 68,700 cal/g-mol and optimization. The media and conditions R ϭ 1.987 cal/g-mol, K used may change from shake flask through A ϭ 4eϩ87.82,minϪ1 the fermentor stages. The objective in devel- Aerobic fermentation processes also require oping a production medium development is a continuous supply of large quantities of air, to maximize productivity and product qual- typically on the order of one volume of air ity, minimize sideproducts and meet econom- per volume of liquid per minute. Sterilization ics. Generally, large-scale fermentation of this air is mandatory in almost all fermen- media are made up of complex natural mate- tations. Absolute filter cartridges of poly- rials, supplemented with inorganic/organic meric membranes are now used almost salts.15,16 Development of a production exclusively in the fermentation industry. medium combines understanding the physi- Relatively small units have replaced the large ology of growth and product formation depth filters used in the past. Still, water and developed through plate; shake flask, and fer- particulates pose a major problem for filters mentor studies. thus requiring the use of prefilters and traps For example, for deciding on the type of to remove these contaminants before they carbon source, the phenomenon of catabolite reach the absolute filter. Parallel installation repression must be considered. Similarly, reg- of the filters prevents a total shutdown of the ulation of nitrogen and sometimes phospho- fermentation process in the event of filter rus metabolism are important factors to clogging. consider. Sometimes high of salts and free amino acids necessary for high cell density are inhibitory to product forma- Microbial Kinetics tion. One area often overlooked is the elimi- Microbial kinetics14,26 can be separated in nation of unwanted impurities, derived either four distinct levels: at the molecular or from the raw material or produced via metab- enzyme, the macromolecular or cell compo- olism. These impurities represent not only a nent, cellular, and population level. Because waste of the carbon source, but their accumu- each level has its own unique characteristics, lation at high levels may lead to inhibition of different kinetic treatments are needed. growth or even cell death. Moreover, the environment in which these At a population level, a material balance on reactions take place also affects the kinetics. cells growing by binary fission can be For example, reactions at the molecular/ described by: enzyme level involve enzyme-catalyzed dX reactions. When these reactions occur in ϭ m # X, (30.3) , their kinetic behavior is similar to dt that of homogeneous catalyzed chemical where X is the cell concentration and ␮ is the reactions as described in Chapter 31. specific growth rate of the microbial culture. However, when enzymes are attached to inert Monod [17] suggested that cells growing on supports or contained within a solid cell a limiting substrate were controlled by a limiting SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 11 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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enzymatic reaction. Enzymatic reactions had parentheses is often called the specific sub-

been modeled using Michaelis–Menten kinet- strate consumption rate, qs. ics and Monod applied the same methodology Because substrate can also be consumed to microbial growth. He described the spe- to make a microbial product, the concept of cific growth rate by: specific rate can also be used for product formation, m ϭ m # S max ϩ , (30.4) dS Ks S ϭϪ ϩ (qs qp)X, (30.8) ␮ dt where max is the maximum growth rate of the particular strain being studied, s is the where qp is the specific product formation concentration of the limiting substrate, and rate. The simplest expressions relate product

Ks is the so-called affinity constant for the formation to either cell growth or substrate limiting substrate and is equal to the concen- consumption: tration of the substrate resulting in one-half # q ϭ Y > q (30.9a) the maximum growth rate. Combining the p p s s above equations results in the cell balance # q ϭ Y > m. (30.9b) equation: p p x # The last equation is an example of growth- dX ϭ m # S X associated product formation, often observed max ϩ . (30.5) dt Ks S for primary metabolites. However, for many secondary metabolites, much of the product A similar material balance on the limiting formation takes place when growth rate substrate requires knowledge of the relation- slows. This is described as nongrowth-rate ship between cell growth and substrate uti- associated kinetics. Leudeking and Piret18 lization. The simplest relationship would first described a mixture of the two by the fol- assume that a fixed amount of cells could be lowing equation, which takes into account produced from a given amount of substrate, or both growth-rate associated and nongrowth- the yield of on substrate: rate associated forms of production, dX ϭ a # m ϩ b qp . (30.10) dt dX Y ϭ ϭϪ . (30.6) dS dS Ϫ Ideal Types of Fermentors dt There are a large number of different types of The linearity between cell growth and sub- fermentation processes that are used commer- strate consumption does not always hold true, cially, which are selected based on several dif- especially at low specific growth rate. Pirt3 ferent factors.19-21 Depending on the strain to first suggested the concept of maintenance. be used, the fermentation could be aerobic or He described maintenance as the substrate anaerobic, and the desired product could be required to generate energy for cell functions either the biomass itself or a metabolite or independent of growth rate. Examples of cell polymer produced by the biomass. The kinet- functions include maintenance of ion gradi- ics of product formation, whether growth ents and the turnover of macromolecules. The associated or nongrowth associated, also resulting material balance on the limiting sub- influences the process. Often procedures strate is: downstream of the fermentation unit opera- dS m tion have a major control of the overall ϭϪQ ϩ mR X, (30.7) process and determine how the fermentation dt Y max is conducted. where Ymax is the maximum growth yield and Although there are a multitude of possible m is the maintenance coefficient. The term in different fermentation process designs, most SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 12 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

12 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

of them can be lumped under the following constant volume. The only addition after the four,20 shown in Figures 30.4 A to D, start of the fermentation is air, if it is needed, resulting in a constant volume. All compo- • Batch nents are in a constant state of change as the • Fed-batch substrate is consumed and biomass and prod- • Continuous ucts are produced. Figure 30.4A shows the • Continuous with recycle typical batch fermentor characteristic: nutri- The batch process is the simplest fermenta- ent is present from the start of the process; tion to perform. It is a closed system with nothing is fed and no steady state can be

F

V = c or V = c V = c μ = c μ = c X = c X = c

A: Batch Fermentor B: Fed-batch fermentor

X

X

μ μ

time time

F F biomass return

rF, Xr V = c μ = c V = c Concentration X = c Unit

F F

C: Continuous fermentor D: (Partial) Recycling fermentor

X (RF)

X (PRF)

X μ (PRF) μ μ (RF)

time time Fig. 30.4. Characteristics of major fermentor types. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 13 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 13

reached. In other words, characteristic volume can be solved to describe the increase in bio- of culture (V) is constant, denoted by V ϭ c. mass and product concentrations and the But specific growth rate (␮) and cellmass (X) decrease in substrate concentration. The fed- are not constant. Batch fermentations are batch system takes into account the addition often used for highly mutated strains that are of substrate during the fermentation. The developed by classical mutagenesis and selec- mass balance on substrate is described by tion. Such strains are easily taken over by d # ϭ # Ϫ ϩ faster-growing, less-productive strains in pro- (V S) F(t) Sf V(qs qp)X. (30.11) longed cultures. Batch processes are still dt quite common in the antibiotic, organic acid, A total mass balance on the reactor is: and ethanol production industries. d In the fed-batch fermentation, nutrients can (r # V) ϭ r # F(t), (30.12) be added making it an open system for sub- dt F strates, but still a closed system for biomass ␳ and biomass-derived products. As shown in where is the density of the fermentation broth, ␳ is the density of the feed solution Figure 30.4B, in this type of process, the vol- F with S as the substrate concentration, and F(t) ume is not constant. The flow rate of nutrients f can vary during the course of the fermenta- describes the rate of addition of feed as a func- tion. Fed-batch operation is very commonly tion of fermentation time. As in the batch fer- used in industrial processes for the production mentation, these equations can be solved to of baker’s yeast, enzymes, amino acids, and show the concentration-time profiles of sub- many other metabolites. strate, biomass, and product. The last equation In the continuous system, as shown in Figure assumes a negligible change in the reactor 30.4C, by constantly removing contents from mass due to air addition (humidity, oxygen) the fermentor, the process is now open with and gas removal (carbon dioxide, water). Such respect to all components. Typically, the addi- assumptions, in practice, must be checked. tion of feed and the removal of broth are equal, For a typical single-stage continuous fer- resulting in a constant volume. Under these mentor at steady state, a biomass material bal- conditions, a steady state is achieved wherein ance around the reactor yields the following all parameters become constant. Such a sys- relationship, tem is widely used for physiological studies. dX F ϭ 0 ϭϪ X ϩ mX, (30.13) However, industrial uses are typically limited to dt V the production of biomass (single cell protein) for food or feed, and waste treatment plants. from which: The final system, shown in Figure 30.4D, is F the continuous system with a partial (PRF) or m ϭ ϭ D. (30.14) V complete (RF) cell recycle. It is similar to the continuous system, but cells are returned to This means that the dilution rate in the fer- the fermentor by means of a biomass separa- mentor sets the growth rate of the biomass, tion device. Cross-filtration units, , and a change in the dilution rate will cause a and tanks have all been used for bio- change in the growth rate. mass separation.22 In the partial cell recycle Because most single-stage continuous fer- fermentor, a steady state is achieved as in the mentors are used to produce biomass, they are continuous system. This process is typically usually operated to optimize biomass produc- used to increase the productivity of the system tivity. The unit volume biomass productivity and is used commonly in wastewater treatment of such a reactor is defined as DX. This unit and ethanol production type applications. volume productivity can be expressed as The differential equations shown in the K D Microbial Kinetics section are used to ϭ a Ϫ s b DX DY S0 m Ϫ . (30.15) describe the batch fermentation mode. These max D SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 14 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

14 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

By taking the first derivative of the productiv- described above for other substrates, the rela- ity expression with respect to the dilution rate tionship between oxygen concentration and and setting it equal to zero, the dilution rate of growth is of a Michaelis–Menton type and

maximum productivity, Dm, can be found as can be described by a specific rate relation- ship (see Equation 30.5). The K-value for K oxygen typically ranges from 0.5 to 2.0 ppm ϭ m a Ϫ s b Dm max 1 (30.16) for well-dispersed bacteria, yeast, and fungi BK ϩ S s 0 growing at 20–30ºC. For growth at tempera- tures greater than 30oC; the specific oxygen and the maximum productivity, D X,as m uptake increases only slightly with increasing 12 D X ϭ oxygen concentration. m Under steady-state conditions, the oxygen K K D transfer rate must be equal to the oxygen m a Ϫ s b # # a Ϫ s m b max 1 B ϩ Y S0 m Ϫ . uptake rate: Ks S0 max Dm (30.17) ϭ Ϫ QO2X kLa(C* C)mean, (30.20) Biomass recycle as sketched in Figure 30.4D where C* is the concentration of oxygen in is frequently used in fermentors as a way of the liquid that would be in equilibrium with increasing the biomass productivity. The the gas-bubble concentration. kL is the oxygen increased biomass productivity obtained with mass transfer coefficient and a is the bubble a recycle fermentor is a function of the recy- interfacial area. In small, well-mixed systems, cle ratio, r, and the cell concentration factor, the gas-bubble concentration can be assumed ϭ C Xr/X, achieved in the concentrator. to be equal to that in the gas escaping from the Equations expressing the recycle system fermentor. However, in large reactors, the log- behavior are derived from material balances mean average between the inlet and outlet air around the reactor. For the cell biomass bal- concentrations is more appropriate. The oxy- ance at steady state: gen mass transfer coefficient and interfacial

area are typically lumped together as kLa. It is dX F rF F ϭ 0 ϭ (0) ϩ X Ϫ (1 ϩ r)X ϩ mX, not practical to try to separate these two dt V V r V terms. (30.18) Utilizing this relationship, the lumped oxygen mass transfer coefficient can be estimated as from which: Q X ϭ O2 kLa . (30.21) Xr (C* Ϫ C) m ϭ D a 1 ϩ r Ϫ r b ϭ D(1 ϩ r Ϫ rC) mean X (30.19) Scale-Up/Down and Control ϩ Ϫ Ͻ Because (1 r rC) 1, it is possible to There are usually problems scaling up new fer- operate the system at dilution rates greater mentations as well as with translation of than the maximum growth rate. It is this sta- process-improvement data for well-established bility imparted by the cell recycle fermentor fermentations from laboratory operations to system that makes it useful, especially in existing plant equipments.24 In general, fer- waste treatment applications. mentations are scaled up on the basis of achieving similar oxygen transfer capabilities in the plant equipment that proved to be opti- Oxygen Transfer Considerations mal at the bench scale. In aerobic fermentations, oxygen is a basic The oxygen transfer capability required for substrate that must be supplied for growth. As processes can vary greatly. Organisms with a SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 15 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 15

low specific growth rate may require as little Once a plant is built, the conditions of agi- as 25 mmol/L/hr whereas high specific tation, aeration, mass (oxygen) transfer, and growth rate cultures could require ten times become set. Therefore, those that amount. The range of airflow required environmental conditions achievable in plant- can vary from as little as 0.1 VVM to Ͼ1 scale equipment should be scaled down to the VVM. Likewise, mechanical power require- pilot plant and laboratory-type equipment ments can vary from less than 0.5 kW/m3 to (shake flask micro-reactor) to ensure that the Ͼ5 kW/m3. earlier studies are carried out under condi-

There are various correlations between kLa tions that can be duplicated. and power inputs. Some design engineers pre- With the rise of the genomic and post- fer to scale-up/down on the following basis, genomic era, high-throughput gene sequenc- ing, proteomics, metabolomics, and systems P a k a ϭ k a m b (v )b, (30.22) biology have created a wealth of biological L V s data. This has given rise to the need for higher-throughput fermentation technology where Pm is the motor power and vs is the to screen libraries of natural and in vitro gen- ␣ superficial gas velocity. The coefficients erated compounds. Initially screens of new ␤ and are typically in the range of 0.5 for organisms and metabolic pathways were per- large-scale plant equipments. Mixing is one formed in shake flasks and are throughout of the most critical factors of large-scale fer- the last decade increasingly in microtiter mentors and among agitator designs, Rushton plates. Although these methods are straight- turbine type impeller has most often been forward and well proven, they are laborious 25 used and studied. and do not allow complex manipulation of Unaerated mechanical power input can be growth conditions. Furthermore, they do not estimated from: provide a full complement of data for the P r rN 3D5, (30.23) knowledge-based selections of organisms or u desired physiological properties. where N is the rotation speed of the impeller Within the past five years, novel technolo- shaft (rpm) and D is the diameter of the gies have been developed that allow more impeller. directed and controlled experiments to screen For scale-up, however, it is usually the organisms, fermentation, and media condi- removal of heat that causes design problems. tions at small scales. A number of academic With the previously mentioned mechanical and private institutions are developing agitation power inputs, up to 5 kW/m3 of machines that are capable of growing cells energy are needed to remove turbulent heat. at the microliter and sometimes nanoliter The peak metabolic heat load for aerobic scale. They are able to supply aeration, oxy- fermentation of glucose at ~250 mmol gen transfer, a pH control, and online data

O2/liter-hour uptake rate is generally greater output similar to a pilot-scale fermentor. than 35 kW/m3. Even with a fermentation These devices require minute amounts of temperature as high as 37ЊC and cooling cells and media and thus offer time, cost, and water temperature as low as 18ЊC, it is diffi- environmental advantage and ultimately cult to remove the heat in large fermentors enhance productivity. Very small scales have without external heat exchange or extensive been described for cell growth and cell-based cooling coils in the fermentor. Internal spiral assays in microfluidic devices, such as the cooling coils can be undesirable because they compact disk (CD) format technology by could interfere with the mixing patterns. Gyros Microlabs (http://www.gyrosmicro.com). Thus, vertical coils that also act as mixing Intermediate scale microfabricated devices baffles are finding more widespread use. include the bioprocessors platform (PCT Chilling of the cooling water can also be used WO 2002/083852), the Micro Reactor device to increase heat removal. (http://www.gener8.com), and a number of SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 16 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

16 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

devices by academic laboratories, most promi- the desired product and the instrumentation to nently, from the University of Maryland at detect the regulatory metabolites is necessary. Baltimore (http://www.umbc.edu/cbe/rao.html). To optimally run a fermentation process, it is For instance, the Micro Reactor device is essential to perform the initial fermentation based on a 24-well plate format with several research on fully monitored environmental milliliters fermentation capacity in each well. systems, then correlate the environmental Airflow, pH, temperature, and agitation can observations with existing knowledge of cel- be controlled electronically. The machine pro- lular control mechanisms, and finally repro- vides online readouts for pH, dissolved oxy- duce the desired environmental control gen, and other parameters. Another larger conditions through continuous computer intermediate scale machine offered by DAS- monitoring, analysis, and feedback control of GIP AG, Fedbatch-Pro®, is a modular system the fermentation environment. that delivers a high degree of flexibility to As a result of advances made in sensor devel- perform a number of microbiological applica- opment, today more so than in the past, it is tions (http://www.dasgip.com). It remains to possible to rely on environmental control in be seen if these machines will eventually be order to gain economical fermentation results. capable of replacing the classical scale-up Until recently fermentation control was limited versions of stainless steel fermentors. to that of temperature, pH, and aeration. With the development of numerous sensors and inexpensive computing systems, the engineer Instrumentation and Control can think in terms of sophisticated control sys- In successfully scaling up/down any fermen- tems for fermentation processes. Figure 30.5 tation, knowledge of the regulatory mecha- shows how a highly instrumented fermentor is nisms of metabolic pathways that synthesize designed to secure basic information on almost

Agitation Power Feed Acid/ Base

Gas Pressure Composition

Dissolved Level Oxygen

pH Temperature

Air Cooling Water

Fig. 30.5. Highly instrumented industrial fermentor. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 17 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 17

all the parameters of the fermentation process. feedback control and dynamic optimization of For advanced information, both offline intra- the process.14,28 cellular (e.g., messenger RNA and protein arrays, metabolites) and extracellular (e.g., metabolites, proteins/ by LC/GC) RECOVERY OF FERMENTATION analyses methods are readily available. PRODUCTS One of the most important sensors needed The isolation and purification of fermentation is one that reliably monitors cell density. An products is often collectively referred to as IR fiber-optic cell density probe has been used . The early part of the for this because it can directly monitor cell separation of a bioproduct is the primary growth (without dilution) in high-cell-density recovery process, whereas the elements fur- bacterial fermentations. The ability to do an ther downstream may include purification, online sample filtration through the use of concentration, and formulation. The overall hollow fibers or rotating filters has made pos- goal of downstream processing and formula- sible continuous, online measurement of glu- tion is to recover the product of interest cost cose, lactate, and other metabolites. However, effectively at high yield, purity, and concen- glucose, nitrogen substrate, and phosphate tration, and in a form that is stable, safe, and sensors that can withstand repeated system easy to use in a target application. sterilization are still needed. Fermentation products include the cells Repeated sampling for measurement during themselves, and chemicals, organic a fermentation process can be tedious and and amino acids, antibiotics, , thus indirect measurement via computers lipids, RNA and DNA, vaccines, bulk and appears to be a viable alternative. Combined fine diagnostic and therapeutic proteins, food with other information obtained from sen- and feed ingredients, and enzymes. These sors, these measurements make possible the biomolecules can differ substantially in nature calculation of several fermentation parame- and require a large variety of methods to sep- ters, as shown in Table 30.2. Computer simu- arate and purify them. They are often pro- lation can also be used to indirectly measure a duced in low concentrations in complex given component based on a mass balance media together with many other components. equation of that component for the fermenta- In the development of manufacturing tion process. Besides these uses, the computer processes for the production of biological has applications in fermentation processes molecules, much emphasis is placed on fer- for continuous automated monitoring and mentor design and scale-up, thus one might assume that the recovery processes of fer- mentation products are rather straightforward TABLE 30.2 Gateway Measurements for and relatively simple. Nothing could be fur- Fermentor Monitoring and Control ther from the truth. A point in case is an Parameter Calculation example of an antibiotic production plant. The investment for the recovery facilities is pH Acid/ Base uptake or formation claimed to be about four times greater than that for the fermentor vessels and their auxil- Air flow rate, In & Out O2 uptake rate O2 concentration iary equipment. In organic and amino acid

Air flow rate, In & Out CO2 production rate fermentations, as much as 60 percent of the CO2 concentration fixed costs of fermentation plants is attributa-

O2 uptake rate, Respiratory quotient, ble to the recovery unit operations. CO production rate specific metabolic 2 Figure 30.6 shows a typical recovery process rate for antibiotics, and a schematic overview of a Power input, air velocity O transfer rate 2 typical downstream process in an enzyme Integrated CO2 produced Cumulative metabolic plant is given in Figure 30.7. From these dia- activity grams it is apparent that most recovery SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 18 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

18 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY 1494, 1957. Copyright American Chemical Society and used with the American Chemical Society Copyright 1957. 1494, 49, Ind. Eng. Chem., Basic flow sheet for the recovery of antibiotics. ( Basic flow sheet for the recovery permission of the copyright owner.) Fig. 30.6. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 19 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 19

Fig. 30.7. Typical recovery process for industrial enzymes.

processes involve combinations of the follow- operations can be tailored to the greatest effi- ing procedures. ciency in a cost-effective manner. Possibly a better way to illustrate a downstream process • Fermentor harvest design is shown in Figure 30.8. Here it is • Mechanical separations of cells from fer- demonstrated that recovery is not a linear mentation broth (clarification) sequential process but rather the downstream • Optional disruption of cells process development follows a palette of • of the compound of interest options that can be applied depending on the • Preliminary fractionation procedures required specifications of the end product and • High-resolution separation steps as such, it is a modular approach. • Concentration Several general considerations limit the range • Formulation of practical choices for a process design. The • Optional drying type of production organism (bacteria versus In contrast to fermentation processes, where fungi or mammalian cells) has an impact on typically one format (i.e., a fermentor vessel), the downstream process design. The fermen- is used, a number of widely differing tech- tation media for these organisms can differ niques are necessary to accomplish purifica- substantially and the media themselves can tion and formulation of biological products. play an important role for the recovery strat- The variety of available separation technolo- egy. Defined media often deliver a purer prod- gies is large and the order and permutations of uct but lower in concentration, whereas sequential processing steps are countless. complex media contain more impurities but Although this may hamper standardization of also generate more amount of product. The downstream processes, it also provides the physical, chemical, and biochemical proper- benefit of flexibility because different unit ties of the desired product have an impact on SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 20 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

20 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Fig. 30.8. The downstream processing cascade arranged by final product requirement.

the recovery strategy, as do the microbial cells, or secreted (extracellular). Each of cell’s physiological states and properties. these products requires a different approach Proteins and enzymes require more “gentle” towards the purification strategy. Intracellular conditions to avoid unfolding or chemical and membrane-bound proteins and enzymes denaturation than those required for small are more difficult to recover than the secreted molecules. General parameters to observe are ones, requiring physical or chemical disrup- large swings in or extreme changes in pH val- tion of cells and the challenge of separating ues and temperatures. Both proteins and small the desired compound from viscous or molecules can have hydrophobic, hydrophilic, entraining substances such as nucleic acids or limited properties that need to be and cell-wall debris. Fermentations are typi- considered when devising a recovery and cally not harvested until the cell mass and the purification strategy. product are concentrated, often requiring The principles and equipment discussed some postharvest dilution to avoid entrain- below are mostly focused on proteins and ment losses and low equipment throughput. enzymes. Several other products, such as small Continuous fermentations, on the other hand, molecules, metabolites, vitamins, and acids can generate a more dilute stream with a hydraulic require more specialized methods. However, load that can add significantly to the cost of many of the unit operations described here subsequent concentration. are also used in the recovery of these latter The simplest downstream processes include compounds. direct use of the entire fermentation broth, cells and all. This is suitable for low-cost industrial products. Another cost-effective Separation of Proteins and Peptides method is the lysis of cells in the crude broth Biological products can be inside the cells and direct use of the lysate. Yet another (intracellular), loosely associated with the method that avoids lengthy purification steps SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 21 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 21

is in situ extractive fermentation, whereby control of temperature, pH, oxidants, inhibitors, a product is directly extracted from the and activators is essential at harvest and broth by use of solvents. However, many throughout the recovery process. For exam- industries require more sophisticated purifi- ple, it is important to maintain a molar excess cation methods due to safety and environ- of calcium ion to ensure the thermal stability mental considerations. For instance, it is of certain Bacillus and imperative that genetically modified organ- that contain calcium-binding sites. isms and their DNA are not present in a Industrial products are mostly of the extra- released product. cellular variety. They can be recovered As can be seen from Figure 30.8, the nature directly from the fermentation broth. The pri- of its final application will dictate how pure mary recovery involves removing the cells the product needs to be and which raw mate- from the broth, aptly called cell separation. rials may be used in its manufacture. Three different techniques are commonly Typically, high purity is not required of indus- used to achieve this goal: filtration, microfil- trial enzymes. In contrast, therapeutic pro- tration, and . teins and small drug molecules require the highest level of purification. The purity range Filtration. Filtration can include filter for food and feed compounds is somewhere in presses, rotary drum vacuum filters (RDVF), the middle. Raw materials must meet regula- belt filters, and variations on synthetic mem- tory requirements for the application and brane filtration equipment, such as filter car- must often be tested for their toxicological tridges, pancake filters, or plate and frame properties. Finally, economic considerations filter presses. These processes typically oper- constrain both the range of raw materials and ate in a batch mode: when the filter chamber separations processes that can be used. is filled up or the vacuum drum cake is Although multiple chromatographic steps exhausted, a new batch must be started. This are routinely included in the downstream pro- type of filtration is also called dead-end fil- cessing of human therapeutic proteins, few tration because the only fluid flow is through industrial proteins include even a single chro- the membrane itself. Due to the small size of matographic step.29 Purification, if any, of cells and their compressible nature, typical industrial products is accomplished by less cell cakes have low permeability and filter expensive techniques such as extraction or aids, such as diatomaceous earths, perlite, or . other mined materials are added to overcome this limitation. Moreover, the presence of high Fermentor Harvest and Primary and viscous polymeric fermentation Recovery byproducts can limit filtration fluxes without the use of filter aids. Extracellular Products. In order to prevent In dead-end filtration, a cake forms on the cell lysis, removal of cells from a fermenta- surface of the pad as the filtration proceeds. tion broth is usually started within hours after The cake permeability is the most important harvest. After cell separation, the clarified physical property of a porous medium and the fermentation broth is more stable and can be hydraulic properties of the flow and the spe- stored refrigerated for days. Upon harvest, cific cake resistance are described by Darcy’s most primary recovery steps involve some Law: pretreatment of the fermentation broth, which dV ¢p can range from simple cooling and dilution J ϭ ϭ , (30.24) Adt n (R ϩ R ) for fungal broths to pH adjustments, addition 0 m c

of salts, stabilizers, or agents in where Rm is the resistance of the membrane, ⌬ order to minimize degradation of the desired Rc is the resistance of the filter cake, p is product and facilitate further processing. For the transmembrane pressure, A is the area ␯ labile compounds, such as enzymes, available for filtration, and 0 is the permeate SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 22 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

22 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

viscosity. Cake resistance can be expressed in problems and is now used terms of the specific cake resistance: widely in the recovery of therapeutic proteins and in the food, beverage, dairy, and water ϭ ar V Rc c , (30.25) treatment industries. A Microfiltration units can be configured as ␣ ␳ plate and frame flat sheet equipment, hollow where is the specific cake resistance and c is the mass of dry filter cake per unit volume fiber bundles, or spiral wound modules. The of permeate. The combination of equations membranes are typically made of synthetic 30.24 and 30.25 and integration give: polymers such as Polyethersulfone (PES), Polyamide, Polypropylene, or cellulosic mats. tA r an V n R Alternate materials include ceramics, stain- ϭ c o a b ϩ o m, (30.26) V 2¢p A ¢p less steel, and carbon. Each of these come with its own set of advantages and disadvan- where specific cake resistance can be tages. For instance, ceramic membranes are obtained from the slope of a plot of tA/V ver- often recommended for the filtration of larger sus V/A. particles such as cells because of the wider Fungal fermentations, such as those of lumen of the channels. However, it has been Trichoderma or Aspergillus sp., lend them- shown that spiral wound units can also be selves particularly well to cell separation by used for this purpose, provided appropriate filtration through a rotary drum vacuum filter spacers are used. because of the ease with which the fungal mat Together with related technologies such as can be shaved off by the drum’s knife, renew- , nanofiltration, and reverse ing the filter cake surface to maintain high fil- , microfiltration relies on membranes tration flux. with a defined pore size. The operation mode Bacterial fermentation broths can be is as “cross flow” or “tangential flow” in processed either by filtration or centrifuga- which filtrate is pumped parallel to the mem- tion, but the much smaller size of bacteria brane surface. Cells or cell debris and large generally requires the addition of a polymeric molecules are retained on one side of the flocculant. Most flocculants are cationic and membrane, and the biological product solu- function by bridging the negative surface tion passes through the membrane. The pri- charges on neighboring cells to increase the mary advantage of tangential flow versus particle size and facilitate either sedimenta- dead-end is that membrane only tion rate or filtration flux and clarity. The affects the membrane pores themselves, choice of flocculant and optimization of whereas particulate matter on top of the dosage is a delicate balance among obtaining membrane is continuously swept away. In good separation quality, yield, cost, and mini- that manner, no filter cake is created. mization of residual excess polymer in the Microfiltration is complex and much research product. has been done to describe the theoretical underpinnings of the process. Equipment and Microfiltration. Microfiltration, the use of membrane replacement costs are high. tangential flow anisotropic membranes to per- Capital and operating costs of filters and cen- meate the product of choice while retaining trifuges of equal production capacity can be solids, can be an attractive cell separation more advantageous. Nevertheless, microfil- technique because it does not require the use tration equipment, when designed and oper- of flocculants or filter aids. It is, in principle, ated properly, has the potential to deliver high a more technically sophisticated version throughputs due to its continuous operation, of classic dead-end filtration processes. reduction of process steps and reduction in Microfiltration yields can be low due to pro- the waste generated. It also enables the use of gressive fouling of membranes. Advanced recycle streams to increase product yields and engineering has overcome many of the early decrease the cost of raw materials. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 23 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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Centrifugation. Centrifugation is another separation principles, such as gel filtration, viable method for removing microbes from size exclusion, ion exchange, hydrophobic fermentation broth. Three types of centrifuges interaction, and affinity binding/elution. A are commonly used: (a) disk-stack centrifuges major advance has been the development of (clarifiers), (b) tubular and basket centrifuges, cross-linked pressure-sustaining support matri- and (c) decanter centrifuges that contain solid ces for high volume applications. Continuous removing scrolls. All three can be operated in moving bed technologies, expanded bed a continuous mode for higher throughput. methods, and displacement Less common are tubular centrifuges, which have all contributed to greater versatility and generate the highest centrifugal forces but cost effectiveness of the technique and have have limited throughput. The main challenge greatly expanded the tools available for high- in centrifugation is to identify a suitable floc- resolution separation. More recently, high- culant or process conditions whereby the cell pressure liquid chromatography (HPLC), mass is easily conveyed or discharged from originally an analytical tool, has been refined the bowl without breakup or carry- for preparative separations. over into the centrate. Although centrifuga- Although chromatography offers the great- tion can handle a high concentration of cell est potential and diversity of mechanisms for solids, filtration can provide more complete separation and purification, with some removal of trace solids that can interfere with notable exceptions, it is not cost effective in downstream concentration or purification the manufacture of industrial commodities steps. The presence of flocculants and filter due to the expense, low throughput, and low aids in cell wastes can cause added disposal binding capacity of chromatography resins. costs. Often, the centrate cannot be com- Furthermore, the complexity and control pletely clarified, thus requiring combinations required for reproducible operation can be of a final filtration step and centrifugation to cost prohibitive for very large-scale produc- achieve a nonturbid product. tion systems. It is also uneconomical for con- centration of the product. A large body of Intracellular Products. Intracellular pro- literature exists for the chromatography of duction of bioproducts is less preferable but biological molecules bolstered by its exten- sometimes the only way to produce certain sive use in the industry. compounds in appreciable amounts. In this Purification can also be achieved in other case, cell disruption is required for recovery. ways, through precipitation with salts, crys- High-pressure homogenization, bead mills, tallization, and through aqueous two-phase and chemical or enzymatic disruption of extraction. Some of these methods are associ- the cell wall with lysozyme or similar ated with substantial capital cost, low through- enzymes can be used to achieve cell breakage. put, low yields, or waste issues. Fractional In the case of small molecules, organic sol- precipitation, one of the oldest protein separa- vent extraction has also been described. If cell tion technologies, can be surprisingly effec- debris remains in the centrate, it must be tive to separate a compound of interest from a removed by methods described above, thus complex broth. For instance, the process of adding extra steps to the process. fractional solvent precipitation of blood components has been used since Purification. Purification of the product World War II. can follow or precede the step of product con- It is sometimes necessary to remove an centration. Biological product purification is undesirable side activity from proteins and often and quite mistakenly equated with col- enzymes, particularly when it is not feasible to umn chromatography. Undoubtedly, this tech- delete the side activity genetically. A case in nology is widely applied and has advanced point is the presence of co-secreted proteases. rapidly in recent decades. Chromatography is Even if minor protease contamination from a separation technique that includes various the host organism does not cause significant SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 24 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

24 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

degradation of the product during processing, Recently developed high-throughput tech- it is likely to damage the product in the final niques for screening crystallization parame- formulation over several months of storage. In ters have lessened the laborious nature of other cases, side activities must be removed identifying appropriate conditions. Both pre- because they interfere in the final application. cipitation and crystallization are excellent Incremental additions of chaotropic salts, methods to concentrate the molecule of such as ammonium sulfate, water-binding choice. A further advantage is that proteins in polymers, such as polyethylene glycol, or crystalline or precipitated form display good organic solvents are used added to a concen- storage stability. Industrial biotechnology trate containing the enzyme, and the relative companies, such as Genencor International, amount of each enzyme versus other protein have developed large-scale crystallization impurities in each fraction is monitored. methods for enzymes.30 Similar to the pooling of chromatographic In the last one to two decades, aqueous two- elution fractions, the fractional “cuts”, which phase extraction has become an attractive purifi- have the most favorable balance of net cation and concentration technique. It provides enzyme purity and recovered yield, are com- the selectivity of classical solvent extraction bined. without its denaturing potential. Utilizing Crystallization is related to precipitation in incompatible two-polymer and polymer–salt that it is governed by a compound’s physico- combinations and adjustments in pH and chemical properties. For example, the second ionic strength, this technique separates pro-

virial coefficient, B22, which characterizes the teins based on differences in hydrophobicity, two-body interactions between protein mole- surface charge, and molecular weight. cules in dilute : Segregation of the separated molecule is gov- ␣ q erned by the partitioning coefficient ( ): 2p C ϭ 2 Ϫ Ϫu(r)>kT a ϭ s B22 2 Ύr (1 e )dr (30.27) , (30.28) M Cw 0 where the Cs is the concentration in the sol- where M is the protein molecular weight, r is vent phase and Cw the concentration in the the intermolecular separation distance, u(r) is water phase. One application of the tech- the interaction potential, k is the Boltzmann nique has been purification of genetically constant, and T is the absolute temperature. engineered chymosin from multiple side The interaction potential u(r), describes the activities produced in the fungal host back- interaction forces between the two protein ground. molecules. An important step in the manufacture of Proteins have long been crystallized for biopharmaceutical injectables is the removal commercial production. This process is of endotoxin and viral contamination. highly scalable, reproducible and can be cost Numerous methods have been described and effective. As is the case with precipitates, include chromatographic steps and the use of can be recovered via centrifugation or tight filter cartridges.31 filtration processes. The purity achieved can Purification of bioproducts from fermenta- be quite high, even though it is often not com- tion, although necessary, comes with a major parable to multiple chromatographic steps drawback. Biological compounds evolved in due to crystalline entrapment of impurities. an environment of complex components are Overall, it often meets the requirements for a adapted to be most stable in these conditions. number of industrial and food applications. A Highly pure forms of bioproducts have a ten- major challenge can be the time-consuming dency towards lower chemical and physical screening of appropriate precipitants, pH stability. For instance, proteases are prone to conditions, and temperatures and the map- autolysis in the absence of impurities that ping of the solubility phase diagrams. inhibit degradation. This is one of the major SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 25 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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challenges faced by the formulator of biolog- problems with their environmental impact in ical products (see Formulation). as much as they require substantial additions of chemicals. Concentration. Clarified filtrates, centrates, or column eluates are usually too dilute for Decolorization and Finishing. Decolor- use in their specific applications, thus, sub- ization is sometimes required for certain stantial amounts of water must be removed. applications, mostly as an aesthetic prefer- This can be achieved by or by ence. It is always desirable to solve these ultrafiltration. Concentration methods used in issues upstream. For example, color can be industrial settings, such as evaporation, which minimized by choice of fermentation medium is done under vacuum, and solvent extraction, components and control of the sterilization may or may not be suitable for dewatering cycle so as to lessen the Maillard reactions proteins because of their potential for thermal between nitrogen and sugars resulting in or chemical denaturation, and due to high caramelization. Color can also be reduced by energy costs associated with evaporation. The treatment with activated carbon, use of benefit of evaporation is that nonvolatile com- antioxidants, and by diafiltration with mem- pounds that may stabilize the proteins are branes. Carbon-impregnated filter pads can retained. be used to combine polish filtration with a Ultrafiltration (UF), which can be a viable decolorization step. alternative, has membrane pore sizes that are “Finishing”, sometimes also called “polish- much smaller than those in microfiltration, ing” refers to one or more filtration steps at allowing only water and small molecules to the end of the downstream process that have travel through the membrane. Thus, proteins, the goal of clarifying and reducing the micro- peptides, and other large molecules can be bial load of the product. Solids are most con- retained as water is forced through the mem- veniently removed using a loaded brane. Typical molecular weight cut-off sizes with cellulosic, synthetic polymer pads, or for UF membranes are 5 to 100 Kilo-Daltons. disposable submicron gauge filter cartridges. This technology has emerged to become one Precoating pads with filter aids prevent pre- of the most common methods for concentra- mature fouling. All these methods can be used tion of large biomolecules. UF is an attractive to remove microbes to the point of sterility method because of its low energy consump- if required. Often, formulation ingredients tion. It can be combined with diafiltration are added to the product concentrates before whereby repeated additions and removal of the final decolorization and finishing steps water lead to a cleaner product. In the case of are performed. smaller molecules, nanofiltration can be used. Nanofiltration membranes have a molecular weight cut-off ranging from 100 to 1000 Formulation Dalton and with such small pore sizes are able Formulation of a product depends on how the to retain molecules such as humic acids and end product will be used and the ability to sat- certain salts. This allows for production of isfy safety requirements. Many bioproducts parasite- and solids-free water without the are supplied in liquid form, as it is convenient need of chemicals. UF fluxes and yields are to meter and use. If the compound is slated for often significantly enhanced by upstream dry products, the concentrate can either be removal or omission of potential membrane stored for later use or applied directly for foulants, such as certain polysaccharides or granulation. In many industries, proteins, antifoams. peptides, and other bioproducts are supplied Precipitation, crystallization, and extraction as encapsulates or granules in order to meet can also be used for concentration, but are safety standards. more typically utilized as purification tech- As with fermentation and downstream niques. The latter methods also may cause processing, precise formulation recipes are SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 26 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

26 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

often kept as closely guarded trade secrets. Product safety is an important consideration Comparatively few publications on new as many bioproducts can cause adverse approaches or new technologies are available responses in humans and many therapeutic in the literature.32 However, the intellectual fermentation products are very potent bioac- property landscape is filled with composition tive molecules. Negative effects can include of matter patents. irritation and allergic reactions, which are due Proteins are large molecules comprised of to the possible immunogenic nature of many tens to hundreds of amino acid subunits. biological compounds. Lyophilization, or Their unique properties are associated with freeze-drying, which is used in therapeutic specific conformations of the amino acid applications results in a powdered product chain. The active conformations are thermo- that is contained in vials as an additional dynamically quite fragile, typically only a safety measure to prevent direct exposure. It 5–15 kcal/mol difference in free energy offers a gentle way of protecting a com- between the stable and the unfolded confor- pound’s fragile nature because the drying mations. Additionally, the amino acid sub- temperature is kept below . However, units that define the protein contain a variety these technologies are expensive. of reactive groups that are subject to chemi- Many of today’s bioproducts are supplied in cal degradation pathways resulting in the a granular form. Granulation is a generic term loss of structure and/or activity. Thus, ensur- for particle size enlargement. Granules in ing the physical and chemical integrity of a consumer products need to provide a tough protein’s native structure is a primary con- barrier to prevent release of the bioactive mol- cern when using proteins or enzymes in ecules in airborne dust, while providing quick applications where its structure is important. release once used in the final application. Many proteins are only soluble up to Enzyme granules contain coatings that are 20–30% (w/w) and therefore considerations designed to withstand the physical impact and for keeping the compounds well solubilized shear forces typically encountered during are of importance. powder processing, maintaining excellent flowability throughout the process. Dry Formulations. Because of the long- Many ways of producing enzyme granules term instability of proteins in aqueous solu- have evolved, with just a handful of methods tion, enzyme producers and formulators have being currently in common use. These include attempted to produce stable solid formula- prilling (spray-chilling), marumerization/ tions since enzymes were first used. Proteases spheronization, drum and high shear granula- tend to degrade via autolysis and can be tion, and fluid-bed coating (Figure 30.9). The incompatible with surfactants. These prob- latter two techniques are superior in produc- lems are easily overcome by storing the ing low dust granules. An overview of the dif- enzyme in the solid state. Initially, commer- ferent technologies is provided in Table 30.3. cially produced enzymes for laundry deter- In general, fluid-bed technology (Figure gents were spray-dried or sprayed onto salt 30.10) is the most flexible approach, giving cores without coating. Spray-drying provides granules with the most uniform appearance a fast and cost-effective way of compartmen- and smooth coatings (Figure 30.11). Other talizing two incompatible ingredients and technologies may have an edge in either cost maintaining enzyme activity over a product’s or throughput, as they are more amenable typical shelf life. However, noncoated gran- to continuous operation. The advantage of ules have led to cases of allergic reactions in the fluidized-bed method is that the entire formulating plant personnel and resulted in a granulation process can be carried out within setback for the enzyme industry in 1970s. a single, contained piece of equipment. The Thus, the technologies used to produce mod- spray-coating process allows the sequential ern dry products have evolved far beyond application of layers of different thick- simple spray-drying. nesses and compositions with almost infinite SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 27 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 27

Fig. 30.9. Common granulation method in enzyme manufacturing.

flexibility. In addition, spray coating allows against deactivation during harsh applica- one-step application of controlled release tions. One example of the latter is the use of coatings. granules to encapsulate enzymes A less commonly used method is spray- against deactivation in the steam-pelleting chilling, whereby the product is incorporated process that is used in producing animal feed into meltable cores. This method is straight- pellets. The use of effective stabilizers and forward and inexpensive but has the drawback coatings can prevent or retard exposure to of being limited by the meltability of its moisture under high heat, yet allow release of ingredients. Moderate to high temperatures the enzyme in the subsequent feed applica- during the shipping or during use of the prod- tion. uct can lead to agglomeration of the capsules. Many therapeutic molecules can be encap- Solid formulations can provide some signif- sulated using other techniques, such as coac- icant advantages, such as enhanced stability, ervation, micro- or nanospheres, microcapsules, delayed or controlled release, and protection or liposomes. Coacervation, often used for SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 28 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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TABLE 30.3 Comparison of Powdered Enzyme Production Methods

Technology Equipment Advantages Disadvantages Prilling (hot melt) Prilling tower Continuous process High product dust High capacity Low point Easy rework Requires predried enzyme Extrusion/spheronization Screw extruder, Inexpensive raw materials Complex multi-step process (marum) Marumerizer, Fluid Continuous process High dust during process bed coater and in product Requires highly concentrated enzyme Drum granulation (high Lödige Mixer High capacity Dusty, multistep process shear) Littleford Mixer Continuous process Requires highly concentrated Fluid bed or Drum Tough granules enzyme Coater Wide particle size distribution Fluid bed (layering/ Fluid bed coater Single, contained reactor Batch process coating) Flexible formulation Difficult rework Tough uniform granules

flavor encapsulation, can be a simple aqueous Other biochemicals, such as flavors, vitamins, phase separation of immiscible droplets, or in or citric acid are sold directly in the - the case of complex coacervation, the use of lized forms and can be incorporated directly opposite electrostatic charges. It is one of the into the final product, such as tablets, cap- basic processes of capsule wall formation. sules, granules, and so on.

Outlet Ai Fan

Fluidized Bed Spray Nozzles

Liquid Enzyme Coated Particles

Plenum Inlet Air

Fig. 30.10. Schematic of a fluidized-bed coater. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 29 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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Fig. 30.11. Commercially available enzyme granules. (Courtesy Genencor International.)

Liquid Formulations. The successful Many catalytically active proteins are application of enzymes in liquid formulations hydrolases and are subject to three principal presents several technical obstacles that are means of deactivation: denaturing or unfold- not encountered in powders and granules. ing, catalytic site inactivation, and proteoly- These problems stem from the fact that liquid sis. Denaturation is best minimized by products are complex aqueous solutions, and controlling temperature and pH and by avoid- physical separation of enzymes from other ing the presence of chemical denaturants. potentially inactivating ingredients is imprac- Catalytic site inactivation is prevented by sup- tical. Necessary ingredients for a viable end- plying sufficient levels of cofactor, typically a product can affect the physical and chemical metal cation, and preventing oxidation of stability of enzymes. In addition, as newer the active site, for example, by formulating types of enzymes are added to formulations with antioxidants. Alternatively, oxidative that already contain proteases, proteolytic resistance can be engineered into the protein degradation of enzymes is a concern. structure. Finally, in the case of proteases, Liquid formulations can be used as pastes proteolysis or autodigestion can be mini- or slurries, dispersions, or emulsions (in mized by reducing water activity, or by addi- aqueous or other solvents) to physically tion of inhibitors. Thus, the final liquid separate the compound to be protected. formulation includes stabilizers, antimicro- Dispersions, pastes, or slurries are not com- bial substances, and osmolytes to reduce the monly used but are a feasible approach when water activity and increase thermodynamic highly concentrated products are needed. For stability. Useful water-sequestering com- instance, many early analytical enzymes were pounds include sugars and other polyols, sold as crystal slurries to preserve stability. such as glycerol, sorbitol, and propylene gly- Dispersions can be prepared from dry materi- col. Useful inhibitors include substrate ana- als. Most of the dispersions are visually logues such as peptides and acid salts. Once cloudy in appearance due to the particle size the formulation excipients are added, the last of the dispersed compounds and are thus step includes a final polish filtration to pro- undesirable in most endproducts. vide a clear liquid. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 30 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

30 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Different industries pose different chal- are “oil in water” or “water in oil” prepara- lenges to the protein formulator. Many feed tions. Proteins, peptides, metabolites, and enzymes are sold as formulated liquid con- other fermentation products can be incorpo- centrates. In this case, the major requirements rated into emulsions. Covalent attachment of for a liquid formulation are enzymatic stabil- hydrophobic compounds, such as fatty acids ity and preservation against microbial growth. or polyethylene glycols, to proteins or pep- It is sometimes not appreciated that the dom- tides lends these hydrophilic molecules to inant factor affecting enzyme stability is the reside in the lipid phase. intrinsic stability of the enzyme itself; formu- lation can do very little to correct for a struc- Whole Cell Recovery turally labile protein. Therefore, it is advisable to make stability an important criterion of the The recovery of whole cells is best explained initial enzyme screening process. by the manufacturing procedure for baker’s An important case is the application of yeast. This process is almost identical to the enzymes in laundry detergents. Market trends early stage of protein recovery, except that the in the United States show that consumers pre- final product is the cell instead of the filtrate. fer to powder detergents by a ratio of After fermentation, the cells are spun out with 2 to 1. These products are stored with no tem- a centrifuge, washed with water, and recen- perature control on shelves in the presence of trifuged to yield a yeast cream with a solids harsh surfactants, such as linear alkylbenzyl concentration of approximately 18 percent. sulfonate (LAS) and require extraordinary Cream yeast can be loaded directly into tanker measures for stabilization. LAS, by its nature as trucks and delivered to customers equipped an effective cleaning agent, causes surfactant- with an appropriate cream yeast handling sys- induced unfolding in proteins. There are tem. Alternatively, the yeast cream can be countless examples of the development of sta- pumped to a plate and frame filter press or a bilization systems in the intellectual property RDVF and dewatered to a cakelike consis- space. A common theme is to reduce the tency with 30–32 percent yeast solids content. water activity and to use borate/glycol stabi- The press cake yeast is crumbled into pieces lizers that bind to the active site of proteases. and packed or spray-dried for dry products. Microbial growth is fairly easy to control After packaging, the yeast is ready for ship- by the addition of food grade antimicrobials ping to retail. such as sodium benzoate, methyl paraben, and various other commercial preparations. In addition, reduction of the water activity Separation of Small Molecules through use of sequestering agents aids in and Metabolites controlling microbial growth. The prevention Metabolites, vitamins, organic and amino of precipitation and hazes is often a highly acids, and specialty chemicals are commonly empirical challenge and one that is very spe- referred to as small molecules, particularly in cific to the enzyme and the specific raw the therapeutic world to differentiate these materials used in the process. It is best to use compounds from proteins or peptides, com- ingredients with high solubility, and to screen monly named or biologi- potential formulations by extended storage at cals. Many of the same unit operations are high and low temperatures. In the case of applied for the recovery and purification of proteins, an additional objective is to keep small molecules as described above. the pH value as far away as possible from the Purification of bulk products has cost con- isoelectric point of the protein, at which the straints, whereas pharmaceuticals are subject protein is least soluble. to strict purity requirements. Emulsions are the most commonly used Possibly the largest class of molecules pro- type of formulation in the food, paint, and per- duced in microorganisms are acids. This group sonal care industries. The typical emulsions includes acetic acid, citric acid, gluconic acid, SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 31 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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lactic acid, and, of course, the amino acids. For the dehydration step, molecular sieves Citric acid is recovered after the cells have made of zeolite can be used. The alcohol is been removed by RDVF. It is then solvent- extracted from the molecular sieve at 99.75 extracted or precipitated as a calcium salt by percent, dehydrated as and captured in

adding hydrated lime [Ca(OH)2]. The precip- condensers. New research focuses on alter- itate is recovered by a second RDVF and then nate aspects of this to acidified with sulfuric acid where it forms cit- improve efficiency such as developing mem- ric acid and calcium sulfate (gypsum). A third branes for ethanol recovery. Low-cost perva- RDVF removes the gypsum. Lactic acid is poration, an energy efficient combination of used in medicines, foods, and beverages and membrane permeation and evaporation, for the production of the biodegradable polymer, ethanol extraction and bioethanol production polylactic acid (PLA). Lactic acid is usually holds much promise. produced as an alkali or earth alkali salt. The liquid left after is subjected Another excellent example is given by to centrifugation, where most of the sus- penicillin recovery, a weak acid. Penicillin pended solids are separated. The clear liquid can be recovered by solvent extraction and its can be recycled by adding it back to the starch partitioning coefficient into solvent can be conversion stage. The moist cake released by steered by the pH value. In the dissociated the centrifuges is mixed with the syrup pro- form it is more hydrophilic, whereas in the duced by the to form a homoge- protonated form it is more hydrophobic. nous mixture and is dewatered in dryers. The Thus, at low pH, penicillin partitions into the dryers produce a Distillers Dried Grains with solvent phase. Solubles (DDGS) meal, which can be formed A number of relatively new methods are into pellets. These are used in many applica- being investigated to improve the recovery of tions, most prominently in animal feed. small molecules. These methods include elec- It is evident from the discussion above that trokinetic separators with bipolar membranes, downstream processing and formulation simulated moving-bed chromatography and depend heavily on the industry and the prod- extraction. The latter is uct. There are countless possibilities on how a practiced for food components. It has also molecule can be recovered, purified, and for- been described for proteins but has not yet mulated. More importantly, the fermentation, found wide acceptance in this field. A fast- recovery, and formulation processes are inti- growing field is the production of bioethanol mately intertwined. Depending on the type of via fermentation processes either from milled production organism and fermentation process corn or from recycled biomass. The fermenta- used; the downstream processing has to adjust tion and saccharification processes can occur accordingly. It is thus imperative that process simultaneously in the fermenting tank by development is highly integrated. In the mod- means of saccharification enzymes (amy- ern fermentation industry, this is usually lases, ). achieved with process development and for- Once the mash is fermented, it is transferred mulation departments working closely to to a distilling unit. The distilling system is deliver the best product at a competitive cost. designed to produce greater than 90 percent Moreover, optimal integration of these depart- combustible bioethanol from fermented ments with the strain development teams is grain mash. The system can be arranged in also seen. Current industry trends also an array of columns: mash columns, alde- include reduction in the number of process hyde-extraction columns, and rectifying steps because each step results in a yield loss. columns. The remaining alcohol-free liquid Sustainable technologies such as reuse of is drawn off from the bottom of the rectify- water, ingredients, and minimal byproduct ing column. This liquid can be blended with generation are also being adopted. Ultimately, the watery meal from the mixing tank before all these efforts are beneficial for improving cooking. the production economics. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 32 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

32 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Regulatory Considerations in production environments to evaluate levels Bioproducts are regulated according to their of protein. This is important for assessing the application, for example, whether enzymes efficiency of containment, respiratory protec- are used in food, feed, detergents, textile pro- tion requirement, and ensuring that environ- cessing, and so on. Regulations also differ ment levels meet occupational exposure country by country. Irrespective of legisla- limits. tion, it is prudent for the product manufac- turer to ensure that the production process, the potential product, and its intended use are safe DELIVERY OF PRODUCTS prior to introduction. Figure 30.12 represents a cell factory model Along with transfer of the strain, fermenta- to produce several categories of products via tion, recovery, and formulation process to the fermentation and processes. manufacturing plant, validated quality control Industrial-scale manufacturing aspects such assays, which are suitable for ensuring prod- as uses, synthesis methods, and costs of some uct quality, must also be provided. The EPA, of these products are reviewed below as well FDA, and other agencies throughout the as in Chapter 32. world are all involved in regulation of prod- ucts derived from naturally occurring organ- Organic Acids and Polymers isms and genetically modified organisms (GMO). Essentially the regulations require: There are many organic acids that can be (1) the use of nonpathogenic organisms, (2) produced by microbial or biochemical the absence of endotoxin or other toxins, (3) a means. However, at present, only acetic acid well-documented and reproducible fermenta- (as vinegar), citric acid, itaconic acid, glu- tion and downstream process, and (4) use of conic acid, 2-keto-gulonic acid, and lactic safe and well-characterized organisms and acid are produced industrially by fermenta- processes. In response to the latter, not only tion. Other organic acids, such as fumaric, are the genetically modified organisms well gallic, malic, and tartaric acids, once pro- characterized, they are designed to barely sur- duced by fermentation or enzyme processes, vive in the general environment. Becasue are now produced commercially, predomi- recombinant organisms can be grown under nantly by the more economic means of contained good large-scale industry practices, chemical synthesis. safety issues relating to environmental release are minimized. Acetic Acid. Acetic acid as a chemical For control and containment of waste, regu- feedstock is manufactured by chemical syn- latory requirements dictate appropriate con- thesis. Acetic acid in the form of vinegar (at tainment that is related to the risk presented least 4% acetic acid by law) is produced by the organism. Most of the processes are largely via the oxidation of ethanol by bacte- contained and hence subject to guidelines for ria of the Acetobacter genus.33 industrial applications of recombinant DNA. Containment may be achieved biologically on O the basis of inherent properties of the organ- isms, for example, their survival in the gen- eral environment is limited. Also, operation H3C OH and design of the manufacturing facility min- imizes potential releases of the recombinant Vinegar is one of the oldest known fermen- organism to the environment. The degree of tation products, predated only by and physical containment is matched to the risk possibly by certain foods from milk. First presented by the organism. The same consid- derived from the spoilage of wine, vinegar has erations apply to products made by recombi- been used as a condiment, food preservative, nant organisms. Air monitoring is performed medicinal agent, primitive antibiotic, and SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 33 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 33

Carbohydrates, ExtracellularExtracellula Proteins,Protei Fats, Sugars, Peptides, Organic Acids, ,Am Salts, Air , Vitamins,Vitam Oxygen, Water Enzymes PotentialPote products NucleicN acids, Cellular Pathways Enzymes,Enzym Vitamins,Vitam IntracellulaIntracellular Steroids, DNADNA, RNA AminoAm acids, EnzyEnzymes Organic acids, MetaboliteMetabolites MemMembranes Sugars, PrProteins Alcohols, PePeptides Antibiotics, Cell wall Colorants, Biopolymers,Biopolym Surfactants

Fig. 30.12. A cell factory model.

even today as a household cleansing agent. wood, such as redwood or fir but preferably Today, vinegar is produced almost entirely for cypress. Air is circulated in the generator by a use in foods. Vinegar may be defined as the number of equally spaced inlets. A cir- product of a double fermentation: an alco- culates the ethanol–water–acetic acid mixture holic fermentation of a sugar source (fruits from the collection reservoir up through a and their juices, cereals, syrups) usually by a cooler to the top of the tank. The liquid trick- selected strain of the yeast Saccharomyces les down through the packing and returns to cerevisiae or ellipsoidens and a second fer- the bottom reservoir. The temperature of the mentation to oxidize the alcohol (including generator is about 29ЊC at the top and 35ЊC at synthetic) to acetic acid by a suitable culture the bottom. A portion of the finished vinegar of Acetobacter organisms. The theoretical is periodically withdrawn from the reservoir, maximum yield of acetic acid on glucose by and replaced with the ethanol-containing this route is 67 percent (two moles of acetic charge to maintain ethanol concentration in acid produced from every mole of glucose 0.2–5 percent range. If ethanol is depleted in consumed). A homofermentative culture, the generator, the Acetobacter die, and the Clostridium thermoaceticum, is known to be generator becomes inactive.

capable of fixing CO2 and yielding three The Frings Acetator (produced by the moles of acetic acid from one mole of glucose Heinrich Frings Company of Bonn, Germany) under anaerobic conditions. However, the tech- is a submerged batch fermentor. It consists of nology for this process has not been commer- a stainless steel tank with internal cooling cialized. coils, a high-speed, bottom-entering agitator, Several vinegar-manufacturing processes are and a centrifugal foam breaker. The unique commercially used, including the following. feature of this Acetator is its highly efficient method of supplying air, accomplished by 1. Trickling-bed reactor high-velocity self-aspirating rotor that pulls 2. Submerged cell reactor air in from the room to the bottom of the tank. 3. Tower reactor When the ethanol content falls to 0.2 percent The circulating, trickling generator, which by volume, about 35–40 percent of the fin- is still widely used, is a large tank generally of ished product is removed. Fresh feed is SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 34 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

34 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

pumped in to restore the original level, and pasteurized at 60–65ЊC for 30 min, and then the cycle starts again. Cycle time for 12 per- cooled to 22ЊC. Vinegar can be concentrated cent vinegar is about 35 hr, and the rate of by freezing or by a membrane production can be 10ϫ that of the trickling process. generator. The yield of acetic acid on ethanol The world production (excluding China is higher as well, for example, 94 for the and Russia) of 10 percent vinegar is esti- Acetator compared to 85 percent for the mated to be about 2 billion liters a year or trickling generator. However, much more about 200,000 metric tons of acetic acid. extensive refining equipment is necessary Price depends on the source (fruit, malt, for filtering vinegar produced by the sub- grain, etc). merged process because the mash contains both the vinegar and the bacteria that pro- Citric Acid. Citric acid, whose structure is duced it. shown below, is the most important organic The tower fermentor is a relatively new aer- acid produced by means of fermentation. ation system applied to vinegar production. The fermentor is constructed of polypropy- CO2H lene reinforced with fiberglass. Aeration is

accomplished through a plastic perforated HO2C CO2H plate covering the cross-section of the tower and holding up the liquid. The cost of the OH tower fermentor is said to be approximately half that of a Frings Acetator of equivalent Citric acid is used in soft drinks, candies, productive capacity. It has been reported that , desserts, jellies, jams, as an antioxi- the tower fermentor is satisfactory for produc- dant in frozen fruits and vegetables, and as an ing all types of vinegar. emulsifier in cheese. As the most versatile Since 1993, a two-stage submerged fermen- food acidulant, citric acid accounts for about tation has been used to produce vinegar of 70 percent of the total food acidulant market. more than 20 percent acetic acid. In the first It provides effervescence by combining the fermentor, alcohol is added slowly to a total citric acid with a biocarbonate/carbonate concentration of about 18.5 percent. After the source to form carbon dioxide. Citric acid acetic acid concentration has reached 15 per- and its salts are also used in blood anticoagu- cent, about 30 percent of the liquid from the lants to chelate calcium, block blood clotting, first fermentor is transferred to a second fer- and buffer the blood. Citric acid is contained mentor. In the second fermentor, the fermen- in various cosmetic products such as hair tation is carried out at a reduced temperature shampoos, rinses, lotions, creams, and tooth- of 18–24ЊC and continues until the alcohol is pastes. More recently, citric acid has been almost depleted. used for metal cleaning, substituted for phos- Recombinant strains of Acetobacter aceti, phate in detergents, for secondary oil recov- cloned with either alcohol dehydrogenase or ery, and as a buffer/absorber in stack gas aldehyde dehydrogenase, have been tested desulfurization. The use of sodium citrate in for vinegar production. The bacteria with the heavy-duty liquid laundry detergent formula- aldehyde dehydrogenase gene produced tions has resulted in a rapid increase in the acetic acid more rapidly than those with the use of citric acid. alcohol dehydrogenase, and were more Wehmet first described citric acid as a resistant to high concentrations of acetic product of mold fermentation in 1893. acid. However, it was only in 1919 that commercial Vinegar clarification is accomplished by fermentation processes based on sucrose were filtration, usually with the use of filter aids developed.34 Although many organisms have such as diatomaceous earth or bentonite. After been shown to produce citric acid from carbo- clarification, vinegar is bottled, sealed tightly, hydrates, Aspergillus niger has been the best SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 35 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 35

Fig. 30.13. Pathway leading to citric acid from glucose.

organism for industrial production. Figure Microbiological production of citric acid 30.13 summarizes the reactions leading to can be implemented by three techniques: citric acid from glucose. It is worthwhile to • Solid-state fermentation note that one mole of glucose yields one mole • Liquid surface fermentation of citric acid with no consumption of oxygen. • Submerged culture fermentation The overall reaction is actually energy yield- ing. It yields one mole of ATP and two moles In solid-state, or Koji, fermentation, Aspergillus of NADH2 per mole of citric acid, and mini- niger is grown on moist wheat bran (70–80% mal growth is needed during production. This water) and produces citric acid in 5–8 days. aspect makes fermentative production of cit- This process is practiced only in Japan and ric acid a good candidate for the process of accounts for about one-fifth of Japanese citric cellular immobilization. acid production. In liquid surface, or shallow SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 36 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

36 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

tray, fermentation, beet (containing producer but the theoretical maximum is 112 48–52% sugar) or cane molasses of black- percent on sucrose. The liquid surface fer- strap (containing 52–57% sugar) or high test mentation yield is high (90–95%) but the sub- (containing 70–80% sugar) are introduced merged culture fermentation is a bit lower. into a mixer. Dilute sulfuric acid is added to Reducing the formation of byproducts, adjust the pH to about 6.0. Phosphorus, potas- mainly oxalic acid, has resulted in improve- sium, and nitrogen in the form of acids or ments in the yield of the submerged culture salts are added as nutrients for proper mold process, nearly reaching those of the surface growth and optimal citric acid production. culture process. The mix is then sterilized and finally diluted The fermentation broth from the solid-state, with water to a 15–20 percent sugar concen- surface culture, or submerged culture process tration. The medium flows by gravity into is treated similarly for recovery and refining shallow aluminum pans or trays arranged in of citric acid. Two recovery methods are used: tiers in sterile chambers. Most chambers have precipitation and filtration, and extraction. A provisions for regulation and control of tem- process flowsheet including the fermentation perature, relative humidity, and air circula- and refining section using the first method is tion. Each tray holds about 400 L of solution shown in Figure 30.14. The mycelium, which at a depth of 76 mm. When the medium has is initially filtered out of the fermentation cooled to about 30ЊC, it is inoculated with liquor, may be used as fertilizer after proper spores of Aspergillus niger. The tray fermen- weathering and processing. The clarified tation requires 8 to 12 days. The pH drops to liquor flows to precipitating tanks fitted with about 2 at the end of the fermentation, and the stirrers where it is heated to a temperature of acid content varies from 10 to 20 percent. 80–90ЊC. The oxalic acid present is separated Some oxalic and gluconic acids are also by preferential precipitation through the addi- formed. The temperature is maintained at tion of a small amount of hydrated lime. The 28–32ЊC during the fermentation. Sterile air resulting calcium oxalate is purified sepa- is circulated through the chambers, and the rately in a manner similar to the process relative humidity is controlled between 40 and described for citric acid recovery. 60 percent. Approximately one part of hydrated lime Most newly constructed plants have for every two parts of liquor is added slowly adopted the submerged culture or deep fer- over a one-hour period while the temperature mentation process. The fermentation medium is raised to about 95ЊC. The precipitated cal- consists of sucrose (around 200 g/L) and cium citrate is filtered on a vacuum filter, and salts to provide a balanced supply of the filtrate, free of citrate, is discarded as iron, zinc, copper, magnesium, manganese, waste. The calcium citrate cake is moved to and phosphate. The provision of a suitable acidulation tanks, where it is acidified with culture medium is the most critical factor in dilute sulfuric acid. It is then filtered, and the obtaining a high yield of citric acid. The fer- citric acid mother liquor is decolorized by a mentation is carried out at 25–27ЊC. Oxygen charcoal treatment. The purified liquor is con- is provided by bubbling air at a rate of 0.5–1.5 centrated in a vacuum evaporator, run into a volumes of air/volume of solution/minute crystallizer where, upon cooling, citric acid without mechanical agitation. It is generally crystallizes, generally in the form of the accepted that the formation of pellets between monohydrate. The resulting acid is of USP 1 and 2 mm in diameter in the fermentation grade. mash is most desirable. Pelleting reduces The extraction method treats the filtered broth viscosity, increases oxygen transfer, and fermentation liquor with a highly selective simplifies mycelium separation in the recov- solvent, tri-n-butyl phosphate in kerosene, ery scheme. The submerged fermentation and then recovers free citric acid by coun- has a time cycle of 6–9 days. The yield of cit- terextraction with water. The aqueous solu- ric acid on sugar varies from producer to tion, which is further concentrated and SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 37 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 37

Fig. 30.14. Citric acid flow sheet.

crystallized, yields 92 percent citric acid with The list price of citric acid (anhydrous) has 8 percent soluble impurities. been in the range of 60–90 cents per lb, USP, In the last two decades, the citric acid industry 100-lb bags. has seen some major changes in ownership and expansions. The worldwide production stands at Itaconic Acid. about 1 million ton a year. The major producers include the Swiss-based Jungbunzlauer, La Citique Belge/ Roche in Belgium, Bharat Starch Industries in India, and ADM, Cargill, and Tate HO2C & Lyle in the United States. CO2H SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 38 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

38 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Itaconic acid (methylene succinic acid) is an unsaturated dibasic acid. It is a struc- turally substituted methacrylic acid, and therefore primarily used as a copolymer in acrylic or methacrylic resins. Because pure acrylic fibers are dye-resistant, it is neces- sary to include other components to make the fibers susceptible to dyes. An acrylic resin containing 5 percent itaconic acid offers superior properties in taking and holding printing inks and in bonding. In addition to its main application as a compo- nent of acrylic fibers, itaconic acid is used in detergents, food ingredients, and food shortenings. Previously, itaconic acid was isolated from pyrolytic products of citric acid or produced by converting aconitic acid present in sugar cane juice. It is now produced on a commer- cial basis predominantly by direct fermenta- tion of sugars. The of itaconic acid follows the metabolic sequence shown in Figure 30.15. Although both Aspergillus itaconicus and Aspergillus terreus are known producers of itaconic acid, the latter is superior and is used industrially with either surface (shallow-pan) or submerged (deep-tank) fermentation.35 The medium contains a sugar source, corn steep liquor, ammonium sulfate, and mineral salts of calcium, zinc, magnesium, and copper. The fermentation, similar to that of citric acid, is very sensitive to concentrations of copper and Fig. 30.15. Proposed metabolic sequence for iron. The fermentation is carried out at 39– biosynthesis of itaconic acid. 42ЊC, pH of 2.0–4.0, and under vigorous agi- tation. Moderate, but continuous, aeration is critical. The batch cycle time is 3–6 days. The 4. Filtration to remove carbon highest known product concentration is 5. Evaporation and crystallization 180–200 g/L from a medium containing 30 If a high-purity acid is desired, further purifi- percent sugar. Therefore, the yield of itaconic cation steps such as solvent extraction, ion acid on sugar is typically 50–70 percent. The exchange, and carbon decolorization are itaconic acid recovery scheme involves the used. following. The price of itaconic acid in 2002 was 1. Acidification of itaconic precipitates, if about $2/lb, and the world production is esti- present mated to be about 20,000 tons/year. The 2. Filtration to remove mycelium and other major producers include Cargill (using Pfizer suspended solids technology) in the United States, San Yuan 3. Activated carbon treatment (not neces- in Taiwan, Rhodia in France, and Merck in sary for industrial grade product) Germany. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 39 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 39

Gluconic Acid. Gluconic acid is produced and tofu, baking powders, and effervescent by the oxidation of the aldehyde group of glu- products. cose to a carboxylic acid. Commonly used organisms for gluconic acid fermentation are Aspergillus niger and OH OH Gluconobacter suboxydans. The larger vol- ume production uses the fungal process. Most of the gluconic acid produced from the HO CO H 2 Gluconobacter process is marketed as glu- ␦ OH OH cono- -lactone.

CHO O CO2H OH OH OH HO HO O HO -H2 + H2O OH OH OH OH OH OH OH OH D-Glucose D-Glucono-δ-lactone D-Gluconic acid

Gluconic acid may be prepared from glucose by During gluconic acid fermentation, glucose is oxidation with a hypochlorite solution, by elec- first oxidized (or, more correctly, dehydro- trolysis of a solution of sugar containing a meas- genated) to glucono-␦-lactone. This is carried ured amount of bromine, or by fermentation/ out by glucose oxidase. Hydrogen peroxide is enzymatic conversion (glucose oxidase, cata- also produced in this step, but is decomposed lase) of glucose by fungi or bacteria (U.S. Pat. by . The fermentation can be by either 5,897,995). For economic reasons, the biologi- surface or submerged culture, the latter more cal methods are now preferred. generally practiced in industry. Horizontally Gluconic acid is marketed in the form of a rotating fermentors have also been used. 50 percent aqueous solution, calcium glu- Calcium gluconate fermentation, in which conate, sodium gluconate, and glucono-␦-lac- calcium carbonate is used for neutralization tone. Gluconic acid finds use in metal of the product, is limited to an initial glucose pickling, as an acidulant in foods, as a protein concentration of approximately 15 percent coagulant in tofu (soybean curd) manufacture, because of the low solubility of calcium glu- as a calcium sequestrant in detergent formula- conate in water (4% at 30ЊC). Neutralization tions, in the pharmaceutical area in mineral by sodium hydroxide instead allows the use of (calcium and iron) supplements, and as a up to 35 percent glucose in the medium. cement viscosity modifier in the construction The recovery of calcium gluconate from area. Calcium gluconate is widely used for fermentation broth involves the following. oral and intravenous therapy. Sodium glu- conate, a sequestering agent in neutral or 1. Filtration to remove mycelium and other alkaline solutions, finds use in the cleansing suspended solids of glassware. Glucono-␦-lactone is used as a 2. Carbon treatment for decolorization food flavor and an acidulant in making cheese 3. Filtration to remove carbon SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 40 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

40 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

4. Evaporation to obtain a 15–20 percent the fermentation process and is therefore calcium gluconate solution likely to expand its use. 5. Crystallization at a temperature just above 0ЊC Succinic Acid.

In sodium gluconate fermentation, the medium CO2H also contains corn steep liquor, urea, magne- sium sulfate, and some phosphates. The pH is controlled above 6.5 by addition of sodium hydroxide. One to 1.5 volumes of air per vol- CO2H ume of solution per minute (vvm) are sup- plied for efficient oxygenation and a high Succinic acid, as an intermediate in the chemi- back-pressure, up to 30 psig, is desirable. The cal synthesis of 1,4-butanediol, tetrahydrofu- fermentation cycle is 2–3 days. Continuous ran, and adipic acid, has a very large market fermentation is used in Japan to convert a 35 potential.36 It is a common intermediate in percent glucose solution to sodium gluconate the metabolic pathway of several anaerobic with a yield higher than 95 percent. The con- microorganisms including Anaerobiospirillum tinuous process doubles the productivity of succiniproducens and Actinobacillus succino- the usual batch system. genes. However, succinate is produced by Sodium gluconate can be recovered from mixed-acid fermentations in low yields and con- the fermentation filtrate by concentrating it to centrations along with several byproducts. As 42–45 percent solids, adjusting the pH to 7.5 the sole product of fermentation, it is possible to with sodium hydroxide, followed by drum- produce succinic acid at titers Ͼ100 g/L and drying. In glucono-␦-lactone fermentation, yield of 1.2 moles per mole of glucose. In such Gluconobacter suboxydans converts a 10 per- a process, organisms use the phosphoenolpyru- cent glucose solution to glucono-␦-lactone vate (PEP) carboxykinase pathway to make suc- and free gluconic acid in about 3 days. cinic acid. Other key enzymes include PEP Approximately 40 percent of the gluconic carboxylase, malate dehydrogenase, fumarase, acid is in the form of glucono-␦-lactone. and fumarate dehydrogenase. Carbon dioxide Aqueous solutions of gluconic acid are in concentration has been shown to regulate the equilibrium with glucono-␦-lactone. Crystals levels of these enzymes for production of succi- that separate out of a supersaturated solution nate. Carbon dioxide functions as an electron below 30ЊC are predominantly of free glu- acceptor and modulates the flux of PEP. conic acid; from 30ЊC to 70ЊC, the crystals An E. coli strain has also been engineered are principally glucono-␦-lactone; and above by over-expressing phosphoenolpyruvate car- 70ЊC, they are mainly of ␥-lactone. boxylase. Metabolic engineering of the strain Sodium gluconate sells for 30–70 cents/lb, was also performed in which genes encoding depending on the liquid or dry form. Dry pyruvate:formate lyase and lactate dehydro- sodium gluconate is the main form of glu- genase were inactivated. PEP flux was also conic acid/gluconate consumed in the United increased through inactivation of the glucose- States. The world market of gluconic acids specific phosphotransferase system (PTS) and salts is about 60,000 tons/year. The major system. Ideally, for minimizing salt formation producers include Glucona America (a sub- in an acid fermentation, the pH should be

sidiary of Aveba B.A.) and PMP Fermentation lower than the pKa. Otherwise, the conversion Products (acquired by Fujisawa) in the United of the salt to the free acid adds significant States; Benckister, Jungbunzlauer, and costs in the final purification. Roquette Freres in Europe; and Fujisawa According to http://www.nrel.gov/docs/ and Kyowa Hakko in Japan. An isolated fy04osti/35523.pdf, there is a significant enzyme system (glucose oxidase-catalase) market opportunity for bio-based C4 building has recently become cost-competitive with block diacids. However, in order to be SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 41 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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Anaerobic respiration

O- l Pyruvate accepts C=O electrons from NADH l H-C-OH l

CH3

2 Lactate 2 NAD+ 2NADH

O- l C=O l Glucose C=O l

CH3

2 Pyruvate 2 ADP 2ATP Fig. 30.16. Lactic acid from glucose fermentation.

competitive with petrochemical-derived prod- Lactic Acid Bacteria (LAB) make lactic acid ucts, the fermentation cost needs to be below as their endproduct. As shown in Figure $0.25/pound. Although much progress has 30.16, 2 moles of lactate and 2 moles of ATP been made in the engineering of organisms are formed per mole of glucose used. for succinic acid production, at this point it Although free lactic acid is preferred for most remains commercially unattractive. industrial processes, the anaerobic fermenta- tion operates optimally at pH 5 where the salt Lactic Acid. of the acid is formed. To obtain lactic acid in its free form, the fermentation process must OH be carried out well below its pKa of 3.87. This challenge has been met by the bovine LDH gene into yeast, which has resulted H in 1.19 mole lactate production per mole of H3C glucose during fermentation at low pH. The CO2H fermentor titer reached is Ͼ100 g/l with a productivity of 0.8 g/l/hr. A strain of S. cere- Lactic acid has been used as a food preserva- visiae overexpressing the lactate–proton sym- tive and food-flavoring compound. Recent porter JEN1 resulted in increased titer and attention on lactic acid has been for its use in yield [9]. making polylactic acid (PLA), a biodegrad- Recovery of lactic acid is complicated by able polymer. As a result, the market for lactic the high solubility of its salt. The traditional acid is rapidly growing. Under batch fermen- method of recovering calcium lactate is being tation conditions, homolactic fermentative replaced by membrane, electrodialysis, or SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 42 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

42 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Fig. 30.17. Reactions leading to vitamin C from glucose.

ion-exchange purification methods. Producers cosmetics, it is phosphorylated to prevent its such as Purac, Cargill, ADM, and Jiangxi sell oxidation. lactic acid for $0.50–3.00 per kg depending Figure 30.17 gives the reactions involved in on whether the product is industrial, feed, the combined microbiological and chemical food, or pharma grade. conversions of glucose, via sorbitol, to vita- NatureWorks® LLC has set up a $300 mil- min C, called the Reichstein and Grussner lion plant at Blair, NE, which is capable of process. Sorbitol is made by catalytic hydro- producing about 140,000-tons/year of poly- genation of glucose and L-Sorbose is pro- lactide polymers from corn sugar. It employs duced from sorbitol by the action of several a fermentation process to produce two chiral species of bacteria, the most commonly used isomers of lactic acid from glucose, which are being Acetobacter suboxydans. Because this then cracked to form three lactide isomers. organism is very sensitive to nickel ions, it is The isomers are subsequently polymerized to important that the medium and fermentor be polylactide. free of nickel. The medium normally consists of 100–200 g/L sorbitol, 2.5g/L cornsteep liquor, and an antifoam agent such as soybean 2-Keto-L-Gulonic Acid. oil. The medium is sterilized and cooled to 30–35ЊC, about 2.5 percent inoculum is CO2H added, and the tank is aerated and sometimes stirred. This is the only step based on fermen- O tation, and the step yield is 80–90 percent in HO 20–30 hr. The chemical steps in the conver- sion of sorbose to ascorbic acid involve the OH preparation of the diacetone derivative, which is then oxidized, the acetone groups are HO removed, and the resultant 2-KLG is isomer- ized to the enediol with ring closure. It is OH believed that this currently operates at an overall yield of about 50 percent 2-Keto-L-Gulonic acid (2-KLG) is the pre- vitamin C on glucose. cursor for Vitamin C (L-Ascorbic acid). Groups all around the world have pursued Vitamin C is used on a large scale as an alternative vitamin C production technolo- antioxidant in food, animal feed, beverages, gies. A one-step fermentation process starting pharmaceutical formulations, and cosmetic from glucose to produce 2-keto-L-gluconic applications. About one half of the vitamin C acid was reported to be practiced in China, a is used in vitamin supplements and multivita- microalgae process was developed in the min preparations, one quarter in food addi- United States and a two-stage fermentation tives, 15 percent in beverages, and 10 percent process via the intermediate 2,5-diketo-D- in animal feed. When ascorbic acid is used in gluconic acid (2,5-DKG) was developed in SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 43 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 43

Japan. Genentech37 succeeded in cloning the The major manufacturers include DSM, 2,5-DKG reductase gene from a Corynebacter BASF, and four Chinese producers, viz. sp. and expressing it in an Erwina sp. that Northeast Pharmaceutical, North China naturally produces 2,5-DKG via the fermen- Pharmaceutical, Shijiazhong Pharmaceutical, tative oxidation of glucose. The recombinant and Jiangshan Pharmaceutical. The current Erwina culture is thus capable of carrying out world supply of ascorbic acid is more than the complex oxidation and reduction steps 80 thousand metric tons per year with annual to form 2-KLG in a single fermentation. revenues in excess of US $700 million. Genencor International, in partnership with Eastman Chemical Co. has developed such a single fermentation process using genetically Xanthan Gum.

engineered Pantoea citrea. To maximize the Xanthan gum is an anionic polymer of ␤- product yield on glucose, detailed analysis of (1,4)-D-glucopyranose glucan with side the steps in the formation of 2-KLG was car- chains of 1,3-␣-D-mannopyranose and (1,2)- ried out. In the periplasm of this organism, ␤-D-glucuronic acid residues.38 It is produced glucose is first converted to gluconic acid by naturally by bacteria to enable them to stick to a membrane-bound PQQ-dependent glucose plants. The negatively charged carboxyl dehydrogenase. This is followed by the oxida- groups on the side chains generate viscosity tion of gluconic acid to 2-keto-D-gluconate when the xanthan gum is mixed with water. (2-KDG) by a cytochrome C coupled enzyme, The glucan backbone is protected by the side gluconate dehydrogenase. In a third reaction, chains, thus making it relatively stable to 2-KDG is further oxidized to 2,5-DKG by acids, alkalis, and cellulase enzymes. 2KDG dehydrogenase, another cytochrome C Xanthan gum is used as a thickener, stabilizer, coupled enzyme. Purification, characteriza- emulsifier, and foaming agent in products tion, and determination of the enzyme struc- such as salad dressings, cosmetics, pharma- ture of 2,5-DKG reductase have added ceuticals, paints, lubricants, and ice cream. Its significantly to the understanding and devel- key property is high viscosity at low shear and opment of this production process. thinning character at high shear. Xanthan gum SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 44 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

44 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

is not affected by ionic strength over a wide routes to 1,3-propanediol are known. For pH, shear or temperature range. example, (1) ethylene oxide may be converted Xanthan gum is produced commercially via to 1,3-propanediol over a catalyst in the pres- aerobic submerged fermentation of sugar by ence of phosphine, water, carbon monoxide, Xanthomonas campestris. Different strains or hydrogen, and an acid; (2) a catalytic solution fermentation conditions produce differing phase hydration of acrolein followed by reduc- degrees of actelylation, pyruvylation, and tion; or (3) from glycerol, reacted in the pres- hence functionalities. The production strain is ence of carbon monoxide and hydrogen over grown in medium containing carbohydrate, catalysts having from group VIII of the nitrogen, and salts. The production process of periodic table. Although it is possible to gen- xanthan gum is energy intensive and costly, erate 1,3-propanediol by these methods, they and product titer and productivity are rela- are capital intensive and/or generate waste tively low due to high viscosity. In addition, streams that contain environmental pollutants. the broth is difficult to filter due to cells in the The biological production of 1,3-propane- broth. After fermentation, the broth is steril- diol from glycerol has been known for a num- ized to inactivate cells; the product is precipi- ber of years.9 The use of natural organisms tated or coagulated, separated by centrifuges, (e.g., enteric bacteria such as Klebsiella pneu- dried, and milled. About 20,000 tons of xan- moniae, Citrobacter freundi, and Clostridia, than are produced each year with a market such as C. butyricum), to produce 1,3- value more than US $160 million. propanediol from glycerol has been well stud- ied. Continued optimization of the basic anaerobic glycerol fermentation process has Alcohols produced promising results. A number of The organic chemicals that fall into this cate- batch or fed-batch fermentations using K. gory and can be produced by fermentation pneumoniae or C. butyricum have produced include ethanol, 1,3-propanediol, butanol, titers of 50–75 g/L and a yield of 0.44–0.69 acetone, 2,3-butanediol, and glycerol. mol/mol 1,3-propanediol from glycerol. Butanol, and acetone have been produced However, the complete conversion of glycerol industrially by fermentation, but currently for to 1,3-propanediol is not possible due to the economic reasons, chemical synthesis is the requirement of an additional reducing power manufacturing method of choice. However, as in the form of NAD(P)H. price and availability of ethylene and propy- Neither the chemical nor the biological lene as feedstocks for the synthetic processes methods described above for the production of become subjects of concern, there is a 1,3-propanediol are well suited for industrial renewed interest in examining the fermenta- scale. The chemical processes are energy inten- tion processes. As the cost of crude oil contin- sive and the biological processes require the ues to go up while the price of renewable expensive starting material, glycerol. A more resource such as agricultural crops remains rel- desirable process would be to develop atively stable, there has been an increasing a microorganism that would have the ability to interest in producing chemical feedstocks from convert basic carbon sources such as carbohy- renewable resources by biological means. drates or sugars to the desired 1,3-propanediol. Such a single organism approach developed by 1, 3-Propanediol. Genencor International and DuPont, over- comes this problem by generating reducing HO OH cofactors from the glucose conversion to CO2. The ability to control both carbon and energy 1,3-Propanediol is a monomer with potential flow in the single organism allows for the more utility in the production of polyester fibers efficient use of the input carbon source. and in the manufacture of polyurethanes As shown in Figure 30.18, the conversion and cyclic compounds. A variety of chemical of glucose to 1,3-propanediol requires the SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 45 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 45

Glucose

GA-3-P DHAP glycerol dehydrogenase

GYL-3-P Pyruvate glycerol phosphatase GYL glycerol dehydratase

3-HPA Acetate TMG dehydrogenase

1,3 PDO Fig. 30.18. 1,3-PDO pathway from glucose.

combination of two natural pathways: glucose glycerol are found in the yeast, S. cerevisiae to glycerol and glycerol to 1,3-propanediol. that produces glycerol from the glycolytic The two enzymes involved in the conversion of intermediate dihydroxyacetone-3-phosphate glycerol to 1,3-propanediol have been cloned using two enzymes: dihydroxyacetone-3- and characterized from several organisms, viz. phosphate dehydrogenase and glycerol-3- Klebsiella, Citrobacter, and Clostridium. The phosphate phosphatase. To construct a single first enzyme in the pathway is glycerol dehy- organism to produce 1,3-propanediol from dratase. The dehydratase has been shown to glucose, one could clone the glycerol pathway undergo catalytic inactivation and requires the into a natural 1,3-propanediol producer, or addition of a reactivation complex of two the 1,3-propanediol pathway into a natural additional proteins. The second enzyme in the glycerol producer. Although either of these pathway is a NAD-linked Tri-methylene approaches seems simple and direct, there are Glycol (TMG) dehydrogenase. Both of these problems involving natural regulation of the enzymes have been cloned and expressed in pathways. However, building both pathways E. coli. The pathways for the production of into E. coli is advantageous as it is the most SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 46 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

46 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

completely studied organism. It provides a toiletries, cosmetics, and pharmaceuticals. It rich set of genetic tools (sequenced genome, is also used as an intermediate for manufac- vectors, promoters, well-characterized metab- turing other chemicals such as glycol ethers, olism and physiology) and a large number of ethyl chloride, amines, ethyl acetate, vinegar, metabolic mutants that have been constructed and acetaldehyde. With the ever-increasing and analyzed.39 Moreover, E. coli has been price and dwindling supply of crude oil, used in large-scale fermentation for produc- ethanol fermented from grains and other tion of industrial and healthcare products, renewable organic resources is becoming an does not naturally overproduce glycerol or economical fuel supplement. The U.S. fer- 1,3-propanediol, and thus has a strong likeli- mentation ethanol capacity is now about 4 bil- hood of no natural regulation to overcome. lion gallons a year, a result of efforts by the Modifications of more than 70 E. coli genes ethanol industry to expand its use in fuels, as were analyzed and the final production organ- a replacement for the oxygenate, methyl tert- ism had 18 genes modified. Some of these butyl ether. genes were regulatory elements that indirectly Ethanol is produced by fermentation from affected the function of a few hundred other sugar-containing materials such as grain genes. The optimized strain of E. coli pro- products, fruits, molasses, whey, and sulfite duced 135 g/l of 1,3-propanediol, at 3.5 g/l/h, waste liquor. Yeast, particularly strains of and 51 percent weight yield on glucose. Saccharomyces cerevisiae, is almost exclu- The joint venture between DuPont and Tate & sively used in industrial ethanol fermentation. Lyle has demonstrated the large-scale feasibil- It tolerates ethanol concentrations up to about ity to produce 1, 3-propanediol by fermenta- 20 percent (by volume), and under anaerobic tion (bio-PDOTM). The process uses the conditions converts over 85 percent of the genetically engineered organism developed by available carbohydrates to ethanol and carbon Genencor–DuPont. The joint venture has dioxide. Air or oxygen suppresses the forma- announced that in 2006 they will commercially tion of ethanol from sugar (the Pasteur effect). produce bio- PDO™ from corn to replace Under aerobic conditions, a major portion of petrochemicals in the production of their poly- the carbohydrates goes towards cell growth. mer Sorona®, for many end-use applications, Ethanol is formed via (the including fibers. The bio-PDO™ will be pro- Embden–Meyerhof–Parnes pathway). The duced at a new plant in Loudon, TN, USA. The overall reaction starting from glucose can be bioprocess for making 1,3-propanediol is written as follows, claimed to require 40 percent less capital and C H O S 2C H OH ϩ 2CO ϩ 32 kcal has a 25 percent lower operating cost than the 6 12 6 2 5 2 chemical process for making the same product. The ethanol yield on glucose is thus 51 per- Because of a different impurity profile, the cent by weight. Because the carbohydrate is polymer made from the biological-based 1,3- also used for cell growth and respiration, the propanediol has improved properties and actu- overall yield of ethanol from total carbohy- ally creates a superior product compared to the drate consumed is about 42–46 weight per- chemically based 1,3-propanediol (http://www. cent on a glucose equivalent basis. dupont.com/sorona/faqs.html). Ethanol fermentation can be conducted on any carbohydrate-rich substrate. Molasses, Ethanol. which is the waste mother liquor that remains after the crystallization of sucrose in sugar H H mill operations, is widely used. Blackstrap molasses contains 35–40 percent sucrose and 15–20 percent invert sugars (glucose and H3C OH fructose). High-test molasses contains 22–27 Ethanol is a versatile chemical being used percent sucrose and 50–55 percent invert sug- in industrial solvents, thinners, detergents, ars. Most of the blackstrap molasses do not SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 47 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 47

require the addition of other nutrients for procedures are the same as those using ethanol fermentation. However, high-test molasses as raw materials. molasses requires ammonium sulfate and The grain-based ethanol process, also called other salts such as phosphates. In the dry milling was developed and optimized for molasses process, blackstrap or high-test the beverage industry utilizing starch-based molasses is charged into a mixing tank and feedstock and energy-intensive processes. diluted with warm water to give a sugar con- Such processes tend to produce some nonfer- centration of 15–20 percent. The pH is mentable sugars during cooking of starch. adjusted to 4–5, the mash pasteurized, cooled, Salts added for the needed pH adjustments of and charged into fermentor tanks, and 1–5 the enzymatic treatments. Both of these result percent yeast inoculum is added. The fermen- in extra process costs. A granular starch tation is carried out nonaseptically at hydrolyzing enzyme (GSHE) technology is 23–32ЊC. Antibiotics may be added to control likely to replace the traditional energy inten- possible bacterial contaminations. Because sive process of liquefaction/saccharification the overall reaction is exothermic, cooling is (http://www.biorefineryworkshop.com/ necessary. The fermentation takes 28–72 hr to presentations/Dean.pdf). The GSHE is a new produce an ethanol concentration of 8–10 per- enzyme product to reduce energy usage for cent. Carbon dioxide normally is vented, but the production of inexpensive glucose from if it is to be recovered, then the vent gas is granular starch, which replaces the energy- scrubbed with water to remove entrained intensive liquefaction/saccharification. The ethanol and then purified using activated car- ethanol industry needs continued energy, cost, bon. The bubbling due to carbon dioxide gen- and conversion efficiency improvements. An eration by fermentation could provide sufficient ethanol production process that has the fol- agitation for small tanks. Mechanical agita- lowing key features is very desired. tion may be necessary for large fermentors. • Energy savings—Elimination of jet The fermentation may be conducted batch- cooking wise or continuously, with or without recy- • Capacity increase—High solids and cling yeast. Although continuous fermentation increased ethanol yield and/or cell recycle can significantly improve • Reduction of yeast growth inhibitors— productivity and thus reduce required capital High glucose concentration, Maillard investment, it may have only a limited impact reaction products, and so on. on lowering product costs in as much as a sig- • Operational cost reduction—Labor, time, nificant portion of the total cost comes from and chemicals raw materials. • Value-added co-product, Dried Distiller’s Ethanol can also be produced by fermenta- Grains and Solubles—Higher protein tion of starch, whey, sulfite waste liquor, cel- content lulose, and hemicellulose. Potato or tapioca • Process simplification—Fewer unit oper- can also be converted to ethanol. Grain fer- ations mentations45 require additional pretreatment because yeast cannot metabolize starch GSHE-like novel enzyme-processing tech- directly. The grain (corn, sorghum, barley, nologies with reduced total operating cost wheat, rice, or rye) is ground and heated in an will provide a dramatic acceleration in the aqueous slurry to gelatinize or solubilize the timelines for the establishment of the bio- starch. Starch-liquefying enzymes (thermo- based economy. This developing technology stable alpha ) are added in this step. will also have a major positive impact on the The liquefied starch is then cooled to life cycle costs of bio-based products, and 60–65ЊC. Yeast is then added to carry out fer- address the growing concerns among policy mentation simultaneously with saccharifica- makers and environmentalists. tion by amyloglucosidase (or glucoamylase). The GSHE is a blend of enzymes with syn- The subsequent fermentation and refining ergistic activity on granular starch. The key SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 48 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

48 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

enzymes of the GSHE blend include acid sta- waste from sugarcane to produce up to 30 ble alpha amylases and glucoamylases that million gallons a year of ethanol. According “drill” holes in the starch granules. Thus the to the USDA and DOE, production of 1 bil- GSHE is capable of hydrolyzing insoluble lion ton of biomass per year is possible on a granular (uncooked) starch into fermentable sustainable basis. This scale of biomass has sugars by enabling depolymerization of starch the conversion potential of producing 100 bil- to glucose without the need for the energy- lion gallons of ethanol per year. intensive jet-cooking step, in a Simultaneous In order to prevent the diversion of indus- Saccharification and Fermentation (SSF) trial alcohol to potable uses, it is denatured process for alcohol production. Genencor by the addition of some material that renders International, has achieved high level of expres- the treated alcohol unfit for use as a bever- sion of these enzymes in industrial production age. A fuel mixture such as 10 percent alco- strains resulting in an acceptable cost structure. hol and 90 percent gasoline has become the Research to improve ethanol fermentation most important use of fermentation ethanol. has also focused on the development of a high Ethanol from grain fermentations has been solids, continuous feeding process as well as made competitive as a liquid fuel in the improved yeast strains. Other related develop- United States because of improvements in ments include processes for the use of bio- technology, especially in the area of efficient mass feedstocks, such as cellulosic waste enzymes and energy usage in production material.40-44 The Genencor–NREL collabo- plants, and various government subsidies ration funded by the U.S. DOE has (a) put in designed to reduce the nation’s dependence place the tools required for continual on imported foreign oil. Other countries in improvement of biomass-derived cellulases, Europe as well as Brazil, India, and China (b) built a suite of enzymes with enhanced also have substantial production of fermen- thermostability and improved specific per- tation ethanol for use as fuel. In the United formance at elevated temperatures, (c) devel- States, ADM, Tate & Lyle, and Cargill are oped an improved production strain for the the biggest among a large number of pro- enhanced cellulase components and demon- ducers. (For ethanol recovery, see Recovery strated an enhanced production process. section.) Therefore, plans are in place to provide a developmental product(s) in support of con- Amino Acids tinued industry development. Amino acids, in general, can be represented Developments in genetic engineering have by the formula: made possible the development of new organ- isms (yeasts and bacteria) that can survive in H higher concentrations of ethanol, tolerate RC COOH higher sugar concentrations, grow at higher temperatures, and utilize starch or cellulose NH2 feedstock directly http://www.farmfoundation. Because the amino group is on the ␣-carbon, org/projects/03-18_biofuels.pdf. Ingram’s40 the amino acids with this general formula pathway engineering program has created are known as ␣-amino acids. The ␣-carbon a production organism patented by the becomes asymmetric when R is not an University of Florida, with improved effi- H atom. Naturally occurring amino acids ciency of ethanol production from C5 and C6 have an L-configuration. Amino acids are sugars (xylose, arabinose, galactose, man- the building blocks of proteins, and the nose, glucose). BCI holds the rights to the elementary composition of most proteins is patented engineered bacteria and is building a similar; the approximate percentages are: large-scale biomass-to-ethanol plant in Jennings, LA. The plant is expected to be C ϭ 50 Ϫ 55 N ϭ 15 Ϫ 18 H operational in 2006 and will use bagasse ϭ 6 Ϫ 8 S ϭ 0 Ϫ 4 O ϭ 20 Ϫ 23 SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 49 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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Table 30.4 gives the structure of R, molecular duced by fermentation as the L-. weight, and elementary composition of each Commercially, the most common amino acids of the 20 amino acids commonly found in manufactured are the L-forms of glutamic proteins. acid, mostly Mono-Sodium Glutamate (MSG), Of the natural amino acids, only methionine lysine, phenylalanine, aspartic acid, threo- is manufactured chemically as the racemic nine, , arginine, citrulline, gluta- mixture. All other natural types are pro- mine, histidine, isoleucine, leucine, ornithine,

TABLE 30.4 Twenty Common Amino Acids

Elemental Composition (%wt) Amino Acids R– M.W. C H O N S Alaniue 89 40 8 36 16 0 Arginine 174 41 8 18 32 0

Aspargine 132 36 6 36 21 0

Aspartic acid 133 36 5 48 11 0

Cysteine 121 30 6 26 12 26 Glutamic acid 147 41 6 44 10 0

Glutamine 146 41 7 33 19 0

Glycine 75 32 7 43 19 0 Histidine 155 46 6 21 27 0

Isoleucine 131 55 10 24 11 0

Leucine 131 55 10 24 11 0

Lysine 146 49 10 22 19 0 Methionine 149 40 7 22 9 2 Phenylanine 165 66 7 19 8 0

Proline 115 52 8 27 12 0

Serine 105 34 7 46 13 0 Threonine 119 40 8 40 12 0

Tryptophan 204 65 6 16 14 0

Tyrosine 181 60 6 26 8 0

Valine 117 51 9 12 27 0 SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 50 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

50 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

proline, , and cysteine.46 The biologi- color of preserved foods and suppressing off- cal methods vary (e.g., fermentation, extrac- flavors. Glutamic acid is not an essential tion from natural sources, and enzymatic amino acid. However, it has some pharmaceu- synthesis). Amino acids obtained from puri- tical uses and also improves the growth of fied proteins are derived from chemical or pigs. enzymatic hydrolysis. They can also be iso- A number of glutamic acid-producing bac- lated from industrial byproducts, extracted teria are known, for example, from plant or animal tissues, or synthesized • glutamicum (Synonym by organic, enzymatic, or microbiological Micrococcus glutamicus) means. All these methods yield the L-isomer. • Brevibacterium flavum Alanine and aspartic acid are produced • Brevibacterium divaricatum commercially utilizing enzymes. In the case of alanine, the process of decarboxylation of Among them, Corynebacterium glutamicum aspartic acid by the aspartate decarboxylase is used most commonly in the industry. from Pseudomonas dacunhae is commercial- Kyowa Hakko has recently completed a ized. The annual world production of alanine genome map of its glutamic acid producing is about 200 tons. Aspartic acid is produced organism, and hopes to improve glutamic acid commercially by condensing fumarate and fermentation efficiency and create a next- ammonia using aspartase from Escherichia generation production system through path- coli. This process has been made more con- way engineering. venient with an enzyme immobilization tech- The fermentation medium contains a car- nique. Aspartic acid is used primarily as a raw bon source (glucose or molasses), a nitrogen material with phenylalanine to produce aspar- source (urea, ammonium sulfate, corn steep tame, a noncaloric sweetener. Production and liquor, or soy hydrolyzate), mineral salts to sales of aspartame have increased rapidly supply potassium, phosphorus, magnesium, since its introduction in 1981. Tyrosine, iron, and manganese, and less than 5 micro- valine, leucine, isoleucine, serine, threonine, grams of biotin per liter depending upon the arginine, glutamine, proline, histidine, cit- strain. The biotin requirement is the major rulline, L-dopa, homoserine, ornithine, cys- controlling factor in the fermentation. When teine, tryptophan, and phenylalanine also can too much biotin is supplied for optimal be produced by enzymatic methods. growth, the organism produces lactic acid. The worldwide amino acids market amounts Under conditions of suboptimal growth, glu- to about $5 billion. MSG and the animal feed tamic acid is excreted. The metabolic pathway additives, methionine and lysine, account for involved in the biosynthesis of glutamic acid about 75 percent of this sales value. The other from glucose limits ␣-ketoglutarate dehydro- amino acids are used as precursors in phar- genase activity, which is a notable phenotype maceuticals, food additives, and animal feed. of glutamic acid overproducing microorgan- The worldwide demand for glutamic acid is isms. about 800,000 tons/year, 300,000 tons/year The fermentation is conducted aerobically for methionine, and 500,000 tons/year for in tanks with kLa value in 300 mmol O2/L/ lysine. Other significant amino acids such as hr/atm range. If aeration is not adequate, aspartic acid, phenylalanine, threonine, and lactic acid is produced, and the yield of glu- glycine, each have a worldwide market of tamic acid is poor. If aeration occurs in about 10,000–20,000 tons/ year. Tryptophan excess, even more lactic acid in addition to and cysteine command a global market in the some ␣-ketoglutaric acid is produced. The thousands of tons as well. fermentation temperature is 28–33ЊC, and the optimal pH is 7–8. Continuous feeding of Glutamic Acid. MSG is an important fla- liquid or gaseous ammonia controls pH and vor enhancer for natural and processed foods. supplies ammonium ions to the fermenta- It is also good for protecting the flavor and tion. The fermentation cycle is 24–48 hr, and SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 51 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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the final concentration of glutamic acid is (S. Korea), and Tung Hai Fermentation about 120–150 g/L. The overall yield of glu- Industry, Ve Wong (Taiwan). tamic acid on sugar is about 65 percent on a weight basis. A portion of sugar is used for Lysine. Lysine, biologically active in its L- cellular growth; otherwise glutamic acid configuration, is an essential amino acid in yield on glucose is 86 percent according to human and animal nutrition. The richest the equation: sources of lysine are animal proteins such as ϩ ϩ S meat and poultry, but it is also found in dairy C12H22O11 2NH3 3O2 ϩ ϩ products, eggs, and beans. On the other hand, 2C5H9O4N 2CO2 5H2O cereal grains such as corn, wheat, and rice, are For recovery of glutamic acid after fermen- usually low in lysine. For applications in tation, the broth is clarified by adding acid to human food, lysine in its salt forms can be pH 3.4, heating to 87ЊC, holding for a suffi- added to cookies and bread, and in solution it cient time to coagulate suspended solids, and can be used to soak rice. As most animal feed filtering the coagulated solids. The clarified rations are based on corn and other grains, broth is concentrated by evaporation followed supplementing the feed-stuffs with lysine by glutamic acid crystallization. Other recov- (plus methionine) significantly improves their ery schemes such as membrane filtration or nutritional value for breeding poultry and pigs. ion-exchange can be used. A fermentation process for producing lysine MSG sells for about $2/kg, and glutamic was made possible by using mutants of acid (99.5% pure) for about $4/kg. The major Corynebacterium glutamicum or Brevibacterium manufacturers of these are Ajinomoto, Asahi flavum. Both auxotrophic and regulatory Foods, Kyowa Hakko, Takeda (Japan), Orsan mutants have been obtained for overproduction (France), Biacor (Italy), Cheil Sugar, Mi-Won of lysine. Figure 30.19 shows the biosynthetic

Glucose

Pyruvatee PPEP

CO2

OxaloacetateOxal Glutamate

α-ketoglutarate NH AAspartate 3 Lysine

CO2 Aspartate Phosphate

Dihydrodipicolinate Aspartate Semialdehyde Homoserine

Threonine Methionine Isoleucine Fig. 30.19. Lysine biosynthesis from glucose in Corynebacteria. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 52 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

52 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

pathway of lysine and its metabolic controls protein source in animal feeds, is deficient in Corynebacterium glutamicum. in lysine and other essential amino acids. Molasses was the most common carbon Therefore, it needs to be supplemented with source until being replaced by glucose for lysine for full-value use. reasons of easier downstream processing. Sufficient amounts (over 30 ␮g/L) of biotin Aromatic Amino Acids. The aromatic must be included in the medium to prevent the amino acids, phenylalanine and tryptophan, excretion of glutamic acid. This biotin provide some of the first examples of chem- requirement was previously met by using ical production using microorganisms molasses as the carbon source and must now through the use of pathway engineering.47,48 be added exogenously. The fermentation runs Intermediates in the aromatic amino acid at temperatures of about 28–33ЊC, and pH pathway can also be used as precursors to 6–8. High aeration is desirable. The final other biosyntheses with genes recruited from product concentration is around 100–140 g/L, different organisms. Examples include cate- and the fermentation time is 48–72 hr. The chol, adipic acid, shikimic, and quinic acids. yield of lysine on carbohydrate is about 40–50 In general, the aromatic pathway illustrates percent. The formation of lysine from sucrose the potential of multiple product opportuni- can be represented as follows, ties from one pathway and provides a great leveraging factor with respect to technical and C H O ϩ 2NH ϩ 5O S 12 22 11 3 2 commercial development costs. C H O N ϩ 6CO ϩ 7H O 6 14 2 2 2 2 Initially, work on the aromatic amino acid Ion-exchange resins are used for isolation of pathway of was focused on lysine from fermentation broths. The eluted the construction of a strain for the overpro- lysine is then crystallized from water. The duction of natural amino acids (phenylala- most common commercial form of lysine used nine, tryptophan, tyrosine). These efforts have in animal feed is 98 percent lysine monohy- focused on: drochloride. For lower purity, the entire fer- 1. Cloning and optimization of complete mentation broth may be evaporated and syrup primary aromatic pathway with an or the dried product is used as an animal feed emphasis on removal of transcriptional supplement. and allosteric regulation, that is, removal Standard commercial food-grade lysine of rate-limiting steps (as 98.5% lysine monohydrochloride) is 2. Enhancement of carbon flux to the aro- about $5/kg, and lysine as a feed supple- matic pathway through modification of ment is about $2/kg. The major producers gene activities within central metabo- include Ajinomoto and Kyowa Hakko of lism Japan, Sewon and Chiel Sugar of South Korea, and ADM, BioKyowa (a subsidiary For the aromatic pathway (Figure 30.20), of Kyowa Hakko), Heartland Lysine (a sub- the critical control points are the sidiary of Ajinomoto), and Degussa (for- of phosphoenolpyruvate and erythrose-4- merly joint venture of Cargill and Degussa phosphate to 3-deoxy-D-arabinoheptulosonate in the United States). China’s Global 7-phosphate, DAHP, by DAHP synthase. For Bio–Chem has been increasingly selling tryptophan, the formation of anthranilic acid protein–lysine (protein with enhanced lysine from chorismic acid by anthranilate synthase content), as well. The world demand for is the second critical control point. The tran- lysine is expected to increase continually. In scriptional regulation was overcome through fact, the increasing ethanol fermentation the use of alternative promoters and allosteric capacity for bioethanol production provides regulation was circumvented by the classical an opportunity for increased lysine produc- technique of selection for feedback-resistant tion. Distillers’ Dried Grains (DDG), a mutants using toxic analogues of the repress- byproduct of ethanol fermentation used as a ing compounds. SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 53 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 53 C aro H 2 OCO H 2 H 2 2 CO OH trpB NH CO trpA+ EPSP N H O O OH OH P P HO O HO O OH H N aroA HO IGP Tryptophan H OH 2 CO OH trpC O S-3-P OH P HO O OH O P HO O aroL HO OH H H OH 2 2 H 2 CO N CPDP H CO OH Shikimate H OCO HO 2 CO OH trpC aroE aromatic pathway to tryptophan. aromatic pathway Chorismate OH H 2 P O HO O CO OH DHS E. coli OH O H OOH 2 HO PRA CO N H aroD H 2 Fig. 30.20. CO OH trpD DHQ HO H 2 2 OOH NH CO aroB OH H 2 Anthranilate CO OH DAHP O HO OH OH P O OH P O P HO O O HO O O HO trpE + OH E-4-P PEP C OH 2 HO O aroG SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 54 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

54 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

For tryptophan production in E. coli, the tions, sodium riboflavin-5’-phosphate is natural regulation controlling production of used. tyrosine and phenylalanine was sufficient to keep carbon flowing specifically to trypto- O phan. This eliminated the need for addition of auxotrophic compounds to the growth N NH medium. The major industrial producers of tryptophan are ADM, Kwoya Hakko, and Ajinomoto. N N O The same could not be said for the con- struction of a strain for the overproduction of OH phenylalanine. Here the control mechanisms for tyrosine were not sufficient to keep a significant amount of carbon from being OH diverted. However, instead of using an aux- HO otrophic strain, a technology was developed to keep the gene for chorismate mutase and OH prephenate dehydrogenase present during the growth phase of the fermentation and The first organism employed primarily for then have it excised from the chromosome riboflavin production was Clostridium aceto- during the production phase. Due to the butylicum, the anaerobic bacterium used for sweetener Aspartame, the market for pheny- the microbial production of acetone and lalanine has expanded to 10,000 metric tons butanol. Riboflavin was purely a byproduct per year with an average price of $10 per kg. and was found in the dried stillage residues in The major producers are Nutrasweet and amounts ranging from 40 to 70 ␮g/g of dried Ajinomoto. fermentation solids. Later investigations dis- closed that riboflavin could be produced by yeast such as Candida flareri or C. guillier- Vitamins and Neutraceuticals mondi, and the yield was as high as 200 mg/L. In the commercial market for vitamins, the Other studies on a , Eremothecium most significant are ascorbic acid (Vitamin ashbyii, and a closely related organism known C), the D group (D2, D3, ergosterol), folic as Ashbya gossyii resulted in the production acid, and alpha-tocopherol (Vitamin E). The of much larger amounts of riboflavin. Yields next important group of vitamins is biotin, as high as 10–15 g/L were possible. Then,

cyanocobalamin (B12), inositol, pantothenic major fermentation strain and process acid, and riboflavin (B2). Microorganisms can improvements were made with the Ashbya synthesize many vitamins of medical impor- gossypii strain. The fermentation lasts 8–10

tance. Vitamin B2 (riboflavin) and vitamin days. Cell growth occurs in the first 2 days, B12 (cyanocobalamin) are products of fermen- and enzymes catalyzing riboflavin synthesis tation. Vitamin C (ascorbic acid) precursor, are formed during the growth period. Glycine 2-keto-L-gulonic acid (2-KLG) is produced and edible oil stimulate the formation of microbiologically as well as chemically as riboflavin, but they are not its precursors. The discussed in the Organic Acids and Polymers additions of carbohydrate and oil permit the section. overproduction of riboflavin.49 The riboflavin carbon yield is about 50 percent on carbohy- Riboflavin. Riboflavin is used as a dietary drate, and about 100 percent on oil. supplement in both human food and animal Upon completion of the fermentation, the feed. The yellow-orange riboflavin crystals solids are dried to a crude product for animal are only sparingly soluble in water. To feed supplement or processed to an USP- include riboflavin in water-soluble formula- grade product. In either case, the pH of the SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 55 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 55

fermented medium is adjusted to pH 4.5. For Vitamin B12, cyanocobalamin, is an important the feed-grade product, the broth is concen- biologically active compound. It serves as a trated to about 30 percent solids and dried on hematopoietic factor in mammals and as a double-drum driers. growth factor for many microbial and animal When a crystalline product is required, the fer- species. Its markets are divided into pharma- mented broth is heated for 1 hr at 121oC to sol- ceutical (96–98% pure) and animal feed (80%

ubilize the riboflavin. Insoluble matter is pure) applications. All vitamin B12 is now removed by centrifugation, and riboflavin recov- made commercially by fermentation.50 ered by conversion to the less soluble form. The Spent liquors from streptomycin and other precipitated riboflavin is then dissolved in water, antibiotic fermentations contain appreciable

polar solvents, or an alkaline solution, oxidized amounts of vitamin B12. Bacterial strains pro- by aeration, and recovered by recrystallization ducing high amounts have been specially from the aqueous or polar solvent solution or by selected for commercial production. Today

acidification of the alkaline solution. vitamin B12 is obtained from fermentations The major producers of riboflavin include using selected strains of Propionibacterium DSM and BASF. There have been significant or Pseudomonas cultures. A full chemical

improvements in both of the production synthesis process for vitamin B12 is known. processes. DSM opened a new riboflavin pro- However, it requires some 70 steps and for all duction facility in Grenzach, Germany in practical purposes is of little value. 2000, based on a fermentation process with a The Pseudomonas denitrificans strain is genetically modified Bacillus subtilis. On most often used for commercial production of

the other hand, BASF, working with the vitamin B12 (ref. U.S. pat 3,018,225; U.S. pat University of Salamanca, Spain, engineered 5,955,321). It requires medium containing an improved Ashbya gossipii that produces a sucrose, , and several metallic larger amount of enzymes for riboflavin syn- salts. Dimethylbenzimidazole (10–25 mg/L) thesis, and increased its production capacity and cobaltous nitrate (40–200 mg/L) must be by 20 percent at its Ludwigshafen facility supplemented at the start of the culture in without capital investment. The annual pro- order to enhance vitamin production. Betaine duction of riboflavin by ADM, BASF, and (tri-methyl glycine) stimulates the biosynthe-

DSM is about 4000 metric ton. The feed- sis of vitamin B12, even though the organism grade product sells for about $30/kg and the does not metabolize it. Similarly, choline USP-grade product $50/kg. also has favorable effects, either by activat- ing some biosynthetic steps or altering the Cyanocobalamin. membrane permeability. Glutamic acid, on the other hand, stimulates cellular growth. Because of high betaine and glutamic acid contents, beet molasses (60–120 g/L) is pref- erentially used in industrial fermentations of

vitamin B12. The fermentation is conducted with aeration and agitation. Optimal temper- ature is about 28oC, and pH near 7.0. The yield reported in the literature was 59 mg/L in 1971, using a Pseudomonas strain. A yield of 200 mg/L was reported for vitamin

B12 fermentations using Propionibacteria in 1974. It is believed that yields of vitamin

B12 have been significantly improved since then. About 80 percent of the vitamin produced is outside the cells, and 20 percent inside the SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 56 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

56 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

cells. For recovery, the whole broth is heated harvested by centrifugation, and the concen- at 80–120ЊC for 10–30 min at pH 6.5–8.5, and trated cellmass is spray-dried. The dry cell- treated with cyanide or thiocynate to obtain mass is then broken down into smaller

cyanocobalamin. B12 isolation can then be particles and extracted by a solvent for the oil- accomplished by on a cation- containing DHA. The solvent is then evapo- exchange resin, such as Amberlite IRC 50. rated from the oil, leaving crude oil. The Extraction can also be done by using phenol crude oil is refined, bleached, and deodorized or cresol alone or in a mixture with benzene, to cleanse it of any impurities. The final prod- butanol, carbon tetrachloride, or chloroform; uct is rich in DHA. Likewise, DSM ferments or separation by precipitation or crystalliza- the fungus, and extracts the crude ARA oil by tion upon evaporation with appropriate dilu- the DHA downstream type process. Crude ents such as cresol or tannic acid. Using oils can be extracted with the traditional the extraction method, 98 percent pure hexane extraction method, or with new sol- cyanocobalamin can be obtained with a 75 vents such as isopropanol or supercritical

percent yield. The total world market for CO2. The final cellmass, total fermentation cyanocobalamin is estimated to be in excess time, and the lipid content of the organism are of 10,000 kg/year, and sells for about very important for the economic feasibility, as $25/g. The leading producers include DSM, these factors largely determine the productiv- Wockhardt, and Merck. ity. Media components such as the amount of sugars and nitrogen have a significant effect Docosahexaenoic and Arachidonic Acids. on growth and lipid accumulation. Docosahexaenoic acid, DHA, is a long-chain The market for DHA, ARA, and other polyunsaturated omega-3 (22:6 ␻3) fatty acid essential neutraceuticals used in dairy, bever- whereas arachidonic acid, ARA, is a long- ages, cereals, and , is multibillion dol- chain polyunsaturated omega-6 (20:4 ␻6) lars and growing as the health benefits are fatty acid. Both DHA and ARA are found in determined for prenatal to elderly care. human milk and are important nutrients in infant development, and especially for brain development. DHA is a major structural and Antibiotics functional fatty acid in the gray matter of the Since the early 1940s, an intensive search for brain, the retina of the eye, and the heart-cell new and useful antibiotics has taken place membranes. DHA can be obtained through throughout the world. More than 10,000 the diet in foods such as fatty fish (accumu- antibiotics have been discovered from micro- lated via natural algae) and organ meat. ARA bial sources.51-56 Streptomyces spp., filamen- is the principal omega-6 fatty acid found in tous fungi, nonactinomycete bacteria, and the brain as well. Adults obtain ARA readily from the diet in foods such as meat, eggs, and TABLE 30.5. Examples of Natural milk. The DHA- and ARA-rich oil can also be Antibiotics and Producing Organisms used in supplements and functional foods for older children and adults for improvement in Natural Antibiotic Producing Organism cardiovascular health. Amphotericin B Streptomyces nodosus Martek (http://www.martekbio.com) and Bleomycin Streptomyces verticillus DSM have developed processes to produce Cephalosporin Cephalosporium sp. oils rich in DHA and ARA. The DHA is Erythromycin A Saccharopolyspora erythraea Gentamycin Micromonospora purpurea extracted from fermented microalgae Lovastatin Aspergillus terreus (Cryptecodiunium cohnii) and the ARA is Neomycin Streptomyces fradiae extracted from soil fungus (Mortierelle Penicillin G Pencillium chrysogenum alpina). In the DHA production process, the Spiramycin Streptomyces ambofaciens microalgae are grown in fermentors (80,000 Tetracycline Streptomyces aureofaciens Vancomycin Streptomyces orientalis to 260,000 liters scale). The cellmass is then SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 57 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 57

non-Streptomycete strains of actinomycetales mentation medium to enhance penicillin are major sources of antibiotics, examples of biosynthesis. which are listed in Table 30.5. Of the antibi- Nonpigmented mutants of P. chrysogenum, otics in clinical use, 50 percent are produced for example, Wisconsin 49–133 derivatives, by microbial fermentation, and others by a are universally employed in the industrial combination of microbial synthesis and sub- process. The desired culture is propagated sequent modification. from a laboratory stock in small flasks and transferred to seed tanks. A typical production . medium is as shown below.

H N S CH3 O N CH3 O Phenylacetic acid group CO2H

Beta Lactam ring

Penicillin G

The original mold observed and preserved by Components Grams/Liter Alexander Fleming was a strain of Corn steep liquor 30 Penicillium notatum, a common laboratory Lactose 30 contaminant. Later, cultures of Penicillium Glucose 5.0 chrysogenum were found to be better produc- NaNO3 3.0 MgSO 0.25 ers of penicillin, and the present industrial 4 ZnSO4 0.044 strains have been derived from this species. Phenyl acetamide (precursor) 0.05

The original strains produced the antibiotic CaCO3 3.0 only by surface fermentation methods and in very low yields. Improved media and produc- The medium is sterilized, cooled to 24 ЊC, and tive strains under submerged aerobic fermenta- inoculated. After 24-hr seed growth, larger tion conditions led to dramatic yield increases. fermentors are seeded. Sterile air is sparged Subsequent improvements, principally in through the tank, at about one volume per culture selection and mutation, further volume per minute. The time of the produc- improved yields, reaching 20–30 g/L. tion stage varies from 60 to 200 hr. Table 30.6 gives the structural formulae of For recovery, the broth is clarified by means the “natural penicillins,” which are com- of rotary vacuum filters. The penicillin, being prised of several closely related structures acidic, is extracted from the aqueous phase with aliphatic and aromatic substitutions to into a solvent, such as methyl isobutyl ketone the common nucleus. The early impure or amyl acetate, at a pH of 2.5 by means of a product contained mixtures of these peni- continuous countercurrent extractor, such as a cillins; however, penicillin G has become Podbielniak. The penicillin is then re- the preferred type and the crystalline prod- extracted with an aqueous alkaline solution or uct of commerce. Phenylacetic acid or its a buffer at a pH of 6.5–7.0. About 90 percent derivatives are used as precursors in the fer- recovery yield is typical at this step. The SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 58 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

58 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Table 30.6 Structural Formulae of Natural Penicillins

Formula:

Type of Penicillin Side Chain R Substitutions (G) Benzyl

(X) p-Hydroxybenzyl

(F) 2-Pentenyl

(Dihydro F) n-Pentyl (K) n-Heptyl

(V) Phenoxy

aqueous solution is chilled, acidified, and Division of American Cyanamid Company. extracted again with a solvent, such as ether This antibiotic is produced by Streptomyces or chloroform. Finally, pencillin is re-extracted aureofaciens when grown under submerged into water at a pH of 6.5–7.0 by titration with aerobic conditions on media composed of a solution of base. The base used depends on sugar, corn steep liquor, and mineral salts. which salt of penicillin is desired. The popu- The crystalline compound has a golden yel- lar forms are sodium or potassium salts. A low color, which suggested the trade name. A typical flow sheet for antibiotic recovery is related antibiotic, oxytetracycline (Terramycin), shown in Figure 30.6. a product of Streptomyces rimousus, is pro- duced by Pfizer Inc. It is chemically and Cephalosporins. Microbiological processes biologically similar to chlortetracycline. for production of cephalosporin C resemble in Independent research by both companies many respects those used for penicillin pro- eventually provided the structure of these two duction. Special strains of Cephalosporium important chemotherapeutic agents. Both the have been selected that produce more compounds may be regarded as derivatives of cephalosporin C and less cephalosporin N a nucleus known as tetracycline. Their struc- than the parent culture. The growth of these tures along with those of other clinically strains in certain special fermentation media important tetracyclines are shown in Table has resulted in higher antibiotic titers. Even 30.7. Tetracycline can also be produced by with these improvements in processing, the fermentation of Streptomyces aureofaciens antibiotic concentration, averaging 10–20 under special conditions, such as chloride g/L, are much lower than those reported for starvation or by use of special strains of the the penicillins. organism that fail to halogenate efficiently. Tetracycline possesses many of the chemother- Tetracyclines. In 1948, a broad-spectrum apeutic properties of chlortetracycline and antibiotic, chlortetracycline (Aureomycin), oxytetracycline. Mutations of tetracycline- was introduced by the Lederle Laboratories, a producing organisms have led to other SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 59 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 59

Table 30.7 Structure of Clinically Important Tetracyclines

R1 R2 R3 R4

Tetracycline H OH CH3 H ® 7-Chlortetracycline (Aureomycin ) H OH CH3 Cl ® 5-Oxytetracycline (Terramycin ) OHOHCH3 H 6-Demethyl-7-chlortetracycline (Declomycin®)HOHHCl ® 6-Deoxy-5-oxytetracycline (Vibramycin )OHHCH3 H ® 6-Methylene-6-deoxyl-6-demethyl-5-oxytetracycline (Rondomycin )OHH CH2 H

tetracycline analogues, of which 6-demethyl- as our understanding of disease processes at 7-chlortetracycline (Declomycin) has clinical the biochemical and genetic levels becomes use. Chemical modifications of oxytetracy- more advanced, enzymes or receptors have cline have generated two other useful mem- increasingly served as specific targets for bers of the family, known as Vibramycin and therapeutic intervention. Recombinant DNA Rondomycin. Tetracyclines are active in vivo technology, in particular, has helped to pro- against numerous gram-positive and gram- duce reagents as tools for the development of negative organisms, and some of the patho- practical and high-throughput screening genic rickettsiae and large viruses. methodologies based on mechanism of action. Dramatic price reduction has come with Automation and miniaturization have also improved production. For instance, a million- allowed a rapid increase in the throughput of unit vial of penicillin (1667 units ϭ 1 mg of the screening process. potassium penicillin G) had a wholesale price Several of these bioactive natural products of $200 in 1943. Today, a million units of have been successfully developed as therapeu- potassium penicillin G sell for as little as tics for clinical use. For example, Cyclosporin $0.50 or approximately $20/kg of free acid. A is a fungal decapeptide principally used to Cephalosporins sell for around $250/kg. suppress immune rejection in organ transplant Tetracyclines, used as animal feed supplements, patients. Mevinolin and compatin both control sell for about $60/kg. The antibiotic market is cholesterol synthesis in human. The search for over $12 billion, and volume continues to enzyme- or receptor-targeted microbial prod- increase simultaneous with price drop. ucts does not limit itself to medical use. Cephalosporins, macrolides, penicillins, Several commercially important “antibiotics” quinolones, and tetracyclines account for the such as Nikkomycin and Avermectin have majority of the sales of antibiotics. been found for agricultural applications in In the last twenty years, it has become evi- recent years. dent that the contribution of microorganisms does not have to be limited to the realm of infectious diseases. Metabolites of microor- Biopharmaceuticals ganisms have been found to have many other The term “biopharmaceuticals” has been interesting therapeutic applications. Particularly generally accepted to distinguish the large SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 60 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

60 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY

Table 30.8. Examples of Biopharmaceutical products, Applications and Manufacturers

Biopharma product Application Companies Diabetes Eli Lilly, Novo Nordisk Erythropoetin Anemia Amgen, Johnson & Johnson Human growth hormone Growth deficiency Genentech, Pharmacia ␣ Hepatitis Schering, Roche Interferon ␤ Multiple sclerosis Chiron, Biogen Factor VIII Haemophilia Bayer Tissue Plasminogen Activator Blood clot Genentech Glucocerebroidase Gaucher’s disease Genzyme Therapeutic antibodies Cancer Glaxo, Amgen, Genentech GCSF White blood cell Amgen, Sankyo

molecule pharmaceuticals (mainly proteins) cellmass. Following purification from the that have emerged as a result of the modern inclusion bodies, the C- is removed “biotechnology” from the traditional small from proinsulin using trypsin and car- molecule drugs. boxypeptidase, and the properly folded and Biopharmaceuticals that are industrially cleaved insulin is purified further. Fer- produced by microbial (bacterial or yeast) mentatively or microbially produced recombi- fermentation include insulin, human growth nant proteins can also be expressed as soluble hormone, hepatitis B surface antigen vaccine, proteins inside the cells or secreted outside alpha interferon, beta interferon, gamma the cells. interferon, granulocyte colony stimulating The biopharmaceuticals sales are about $45 factor, and interleukin-2. Table 30.8 lists some billion and represent 10 percent of the total of the major biopharma products, some of pharma market. Today, 25 percent of new which are produced by mammalian cell drugs are biopharmaceuticals. Since the intro- culture. duction of insulin over 25 years ago, 160 bio- Insulin is the first human biopharmaceuti- pharma products, ranging from proteins, cal product that was commercialized using monoclonal antibodies, and nucleic acid- the recombinant DNA technology. Its fermen- based products, have been approved for use. tative expression in E. coli, downstream In 2004 alone, 12 new biopharmaceuticals recovery, and purification serves as a good have been approved, of which only 3 are pro- example for the large-scale production of duced by microbial fermentation, as cell cul- recombinant proteins. Insulin, given its ture is the preferred method for production of rather small size, is expressed as N-terminal biopharma drugs. extended proinsulin using the trp promoter. Drug development, in general, is a slow, The fermentation is carried out at about 37 ЊC lengthy, and expensive process, requiring an and near neutral pH. It is a fast, aerobic fer- average investment of $0.8 billion over 12–15 mentation. The trp operon is turned on when years for discovery, development, preclinical the fermentation runs out of its natural repres- testing, Phase I, II, and III trials, and FDA sor, tryptophan. The chimeric protein as it is approval prior to marketing. Given the com- expressed accumulates very rapidly inside the plications of biopharma drug discovery and cells as insoluble aggregates (inclusion or development, microbial fermentation and bio- reflectile bodies). The formation of inclusion catalysis may aid in introducing rapid and bodies prevents proteolysis and facilitates innovative processes to produce potential product recovery. The recombinant E. coli fer- drug candidates. It may thus be worth mentation usually runs for 18–24 hr. At the exploring the possibility of producing bio- end of fermentation, the inclusion bodies pharmaceuticals by microbial fermentation account for about 10–30 percent of total dry and demonstrating that they can be made SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 61 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

INDUSTRIAL BIOTECHNOLOGY 61

reproducibly to meet the right purity, concen- One of the enzymes that has been used on a tration, and dose at the required market cost large scale is glucose/xylose isomerase, and volume targets. which catalyzes the rearrangement of glucose to fructose, to yield a product with a sweet taste like sucrose. The enzyme is present in Enzymes Streptomyces spp. as well as a few other Microorganisms used for production of organisms. Once again, the native host strain enzymes range from prokaryotic systems has been improved for production of glucose including both the gram-negative and gram- isomerase. positive bacteria to eukaryotic systems such as yeasts and fungi. For most of the history of enzymes, their production has occurred pre- FUTURE: BIOREFINERIES dominantly in strains known to make the Biorefineries integrate bio-based industries/ enzyme of interest. 30, 57-60 Thus, many differ- processing facilities that utilize mostly plant ent types of microorganisms have been materials as feedstocks to produce food, feed, employed to make enzymes for different types fuel, chemicals, materials, and intermediates of uses, as discussed in Chapter 31. for making varieties of products. Fermentative For example, an alkaline protease naturally processes discussed here and biocatalysis dis- secreted by Bacillus licheniformis to break cussed in Chapter 31, can be applied for a down proteinaceous substrates, resulted in wide variety of processes where renewable one of the first commercially produced resource-based materials are used and pro- enzymes, Subtilisin Carlsberg, for use in duced. Bioprocesses are continually becoming detergents. Similarly, ␣-amylase was pro- more efficient through better understanding duced from Bacillus licheniformis because it and controlling of metabolic pathways. Today, naturally secretes a highly thermo-stable ␣- industrial biotechnology companies such as amylase capable of breaking down starch to Genencor International and Novozymes sell more easily digestible oligosaccharides. enzymes produced via microbial fermentation Strains of Bacillus have been some of the processes for a wide variety of bioprocessing workhorse strains for enzyme production for applications. Chemical producers are seeking decades, based mainly upon their ability to new and renewable feedstocks as well as envi- overproduce subtilisin and ␣-amylase. Those ronmentally friendly and sustainable produc- strains and the promoters of these genes are tion processes, in the face of escalating also used to express other enzymes, wild type non-renewable raw material prices. In addi- and engineered. tion, there is a need for development of inter- Glucoamylase, which completely breaks esting new chemistry and products. Major down oligosaccharides to glucose, is produced chemical companies—including Dow Chemical, by strains of the fungal genus, Aspergillus. DuPont, BASF, Degussa, and Celanese—have Strains that overproduce glucoamylase have invested heavily to explore these opportunities, been isolated over the years. Another carbo- often through alliances with small technol- hydrase, an acid cellulase enzyme complex, ogy firms that offer specific expertise found in the fungus Trichoderma, is capable (http://pubs.acs.org/email/cen/html/06080415 of breaking down cellulosic substrates to glu- 0713.html). However, most products made cose, similar to the starch-degrading enzymes. today are relatively low-volume, high-value This particular application has turned out to types, for example, drugs and specialty chem- be much more challenging to commercialize icals. Only a few products, such as ethanol, but has found applications in treatment of tex- are produced by fermentation at a million-ton tiles, feed, and food. New programs initiated scale per year. by the U.S. Dept. of Energy have helped Improvements in fermentation organisms improve the expression and activity of the and processes will have to be achieved in cellulase enzyme complex. order to raise productivity and conversion SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 62 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark]

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efficiency and lower production costs (http:// development, bioprocess design, product www.nrel.gov/docs/fy04osti/35523.pdf). development, and applications. Enzymatic processing is also expected to play a major role in refining biofeedstocks into sugars, protein, oil, and other byproducts. Acknowledgment Such advances alone will enable the vision of The subject of biotechnology first appeared in biorefineries to become a reality. Analogous an earlier edition of this Handbook as a chap- to conventional , progress in three ter titled “industrial Fermentation: Principles, areas will be key for the successful develop- Processes and Product,” written by Dr. Arthur ment of biorefineries: low energy milling of E. Humphrey of Lehigh University. In later biofeedstocks to its components, efficient bio- editions he was joined first by Dr. S. Edward conversion of mixed sugars to products, and Lee and then by Dr. Lewis Ho, both of Pfizer. the utilization of byproducts. These improve- Chapters 30 and 31 in this edition are an out- ments will require integration of all major areas growth of that earlier work, some of which is of industrial biotechnology: novel enzymes and used in the new chapters. Grateful acknowl- microorganisms, functional genomics, pathway edgment of this use is made to the authors of engineering, protein engineering, biomaterial that work.

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