biotechnology, mastering these technologies does not represent a distinct competitive advantage. most reactions can be carried out in standard multipurpose plants. the very versatile organometallic reactions (e.g., conversions with lithium aluminum hydride, boronic acids) may require temperatures as low as -100 °c, which can be achieved only in special cryogenic reaction units, either by using liquefied nitrogen as coolant or by installing a low-temperature unit. other reaction-specific equipment, such as filters for the separation of catalysts, ozone or phosgene generators, can be purchased in many different sizes. the installation of special equipment generally is not a critical path on the overall project for developing an industrial-scale process of a new molecule. since the mid-1990s the commercial importance of single-enantiomer fine chemicals has increased steadily. they constitute about half of both existing and developmental drug apis. in this context, the ability to synthesize chiral molecules has become an important competency. two types of processes are used, namely the physical separation of the enantiomers and the stereo specific synthesis, using chiral catalysts. among the latter, enzymes and synthetic binap (2,2'–bis(diphenylphosphino)–1,1'–binaphthyl) types are used most frequently. large volume (> 103 mtpa) processes using chiral catalysts include the manufacture of the perfume ingredient l-menthol and syngenta's dual (metolachlor) as well as basf's outlook (dimethenamid-p) herbicides. examples of originator drugs, which apply asymmetric technology, are astrazeneca's nexium (esomeprazole) and merck & co's januvia (sitagliptin). the physical separation of chiral mixtures and purification of the desired enantiomer can be achieved either by classical fractional crystallization (having a "low-tech" image but still widely used), carried-out in standard multipurpose equipment or by various types of chromatographical separation, such as standard column, simulated moving-bed (smb) or supercritical fluid (scf) techniques. for peptides three main types of methods are used, namely chemical synthesis, extraction from natural substances, and biosynthesis. chemical synthesis is used for smaller peptides made of up to 30–40 amino acids. one distinguishes between "liquid phase" and "solid phase" synthesis. in the latter, reagents are incorporated in a resin that is contained in a reactor or column. the synthesis sequence starts by attaching the first amino acid to the reactive group of the resin and then adding the remaining amino acids one after the other. in order to ascertain a full selectivity, the amino groups have to be protected in advance. most developmental peptides are synthesized by this method, which lends itself to automation. as the intermediate products resulting from individual synthetic steps cannot be purified, a selectivity of effectively 100% is essential for the synthesis of larger-peptide molecules. even at a selectivity of 99% per reaction step, the purity will drop to less than 75% for a dekapeptide (30 steps). therefore, for industrial quantities of peptides not more than 10–15 amino acid peptides can be made using the solid-phase method. for laboratory quantities, up to 40 are possible. to prepare larger peptides, individual fragments are first produced, purified, and then combined to the final molecule by liquid phase synthesis. thus, for the production of roche's anti-aids drug fuzeon (enfuvirtide), three fragments of 10–12 amino acids are first made by solid-phase synthesis and then linked together by liquid-phase synthesis. the preparation of the whole 35 amino acid peptide requires more than 130 individual steps. microreactor technology (mrt), making part of "process intensification", is a relatively new tool that is being developed at several universities, as well as leading fine chemical companies, such as bayer technology services, germany; clariant, switzerland; evonik-degussa, germany; dsm, the netherlands; lonza, switzerland; pcas, france, and sigma-aldrich, us. the latter company produces about 50 fine chemicals up to multi-kilogram quantities in microreactors. from a technological point of view, mrt, a.k.a. continuous flow reactors, represents the first breakthrough development in reactor design since the introduction of the stirred-tank reactor, which was used by perkin & sons, when they established a factory on the banks of what was then the grand junction canal in london in 1857 to produce mauveïne, the first-ever synthetic purple dye. for a comprehensive coverage of the subject see micro process engineering. examples for reactions that have worked in microreactors include aromatics oxidations, diazomethane conversions, grignards, halogenations, hydrogenations, nitrations, and suzuki couplings. according to experts in the field, 70% of all chemical reactions could be done in microreactors, however only 10-15% are economically justified. with the exception of some stereospecific reactions, particularly biotechnology, mastering these technologies does not represent a distinct competitive advantage. most reactions can be carried out in standard multipurpose plants. reaction-specific equipment, such as ozone or phosgene generators, is readily available. the installation generally is not a critical path on the overall project for developing an industrial-scale process of a new molecule. whereas the overall demand for outsourced pharmaceutical fine chemicals is expected to increase moderately (see chapter 8), the estimated annual growth rates for the above-mentioned niche technologies are much higher. microreactors and the smb separation technology are expected to grow at a rate of even 50–100% per year. the total size of the accessible market typically does not exceed a few hundred tons per year at best. biotechnology industrial biotechnology, also called "white biotechnology", is increasingly impacting the chemical industry, enabling both the conversion of renewable resources, such as sugar or vegetable oils, and the more efficient transformation of conventional raw materials into a wide range of commodities (e.g., cellulose, ethanol and succinic acid), fine chemicals (e.g. 6-aminopenicillanic acid), and specialties (e.g., food and feed additives). as opposed to green and red biotechnology, which relate to agriculture and medicine, respectively, white biotechnology seeks to improve the economic and sustainable production of existing products, and provide access to new products, especially biopharmaceuticals. it is expected that revenues from white biotechnology will account for 10%, or $250 billion, of the global chemical market of $2,500 billion by 2013. in ten to 15 years it is expected that most amino acids and vitamins and many specialty chemicals will be produced by means of biotechnology. three very different process technologies -biocatalysis, biosynthesis (microbial fermentation), and cell cultures- are used. biocatalysis, a.k.a. biotransformation and bioconversion, makes use of natural or modified isolated enzymes, enzyme extracts, or whole-cell systems for enhancing the production of small molecules. it has much to offer compared to traditional organic synthesis. the syntheses are shorter, less energy intensive and generate less waste, hence are both environmentally and economically more attractive. about 2/3 of chiral products produced on large industrial scale are already made using biocatalysis. in the manufacture of fine chemicals, enzymes represent the single most important technology for radical cost reductions. this is particularly the case in the synthesis of molecules with chiral centres. here, it is possible to substitute the formation of a salt with a chiral compound, e.g., (+)-α-phenylethylamine, crystallization, salt breaking and recycling of the chiral auxiliary, resulting in a theoretical yield of not more than 50%, with a one step, high yield reaction under mild conditions and resulting in a product with a very high enantiomeric excess (ee). an example is astrazeneca's blockbuster drug crestor (rosuvastatin), see chemical / enzymatic synthesis of crestor. chemical / enzymatic synthesis of crestor (rosuvastatin) further examples of modern drugs, where enzymes are used in the synthesis, are pfizer's lipitor (atorvastatin), where the pivotal intermediate r-3-hydroxy-4-cyanobutyrate is now made with a nitrilase, and merck & co.'s singulair (montelukast), where the reduction of a ketone to s-alcohol, which had required stoichiometric amounts of expensive and moisture sensitive "(-)-dip chloride" is now replaced by a ketoreductase enzyme catalyst step. similar rewarding switches from chemical steps to enzymatic ones have also been achieved in steroid synthesis. thus, it has been possible to reduce the number of steps required for the synthesis of dexamethasone from bile from 28 to 15. enzymes differ from chemical catalysts particularly with regard to stereoselectivity, regioselectivity, and chemoselectivity. they can also be modified ("reshuffled") for specific reactions, for use in chemical synthesis. "immobilized enzymes" are those fixed on solid supports. they can be recovered by filtration after completion of the reaction. conventional plant equipment can be used with no, or only modest, adaptations. the international union of biochemistry and molecular biology (iubmb) has developed a classification for enzymes. the main categories are oxidoreductases, transferases, hydrolases, lipases (subcategory), lyases, isomerases and ligases, companies specializing in making enzymes are novozymes, danisco (genencor). codexis is the leader in modifying enzymes to specific chemical reactions. the highest-volume chemicals made by biocatalysis are bio-ethanol (70 million metric tons), high-fructose corn syrup (2 million metric tons); acrylamide, 6-aminopenicillanic acid (apa), l-lysine and other amino acids, citric acid and niacinamide (all more than 10,000 metric tons). biosynthesis i.e. the conversion of organic materials into fine chemicals by microorganisms, is used for the production of both small molecules (using enzymes in whole cell systems) and less complex, non-glycosylated big molecules, including peptides and simpler proteins. the technology has been used for 10,000 years to produce food products, like alcoholic beverages, cheese, yogurt, and vinegar. in contrast to biocatalysis, a biosynthetic process does not depend on chemicals as starting materials, but only on cheap natural feedstock, such as glucose, to serve as nutrient for the cells. the enzyme systems triggered in the particular microorganism strain lead to the excretion of the desired product into the medium, or, in the case of hmw peptides and proteins, to the accumulation within so-called inclusion bodies in the cells. the key elements of fermentation development are strain selection and optimization, as well as media and process development. dedicated plants are used for large-scale industrial production. as the volume productivity is low, the bioreactors, called fermenters, are large, with volumes that can exceed 250 m3. product isolation was previously based on large-volume extraction of the medium containing the product. modern isolation and membrane technologies, like reverse osmosis, ultra- and nano-filtration, or affinity chromatography can help to remove salts and by-products, and to concentrate the solution efficiently and in an environmentally friendly manner under mild conditions. the final purification is often achieved by conventional chemical crystallization processes. in contrast to the isolation of small molecules, the isolation and purification of microbial proteins is tedious and often involves a number of expensive large-scale chromatographic operations. examples of large-volume lmw products made by modern industrial microbial biosynthetic processes are monosodium glutamate (msg), vitamin b2 (riboflavin), and vitamin c (ascorbic acid). in vitamin b2, riboflavin, the original six- to eight-step synthetic process starting from barbituric acid has been substituted completely by a microbial one-step process, allowing a 95% waste reduction and an approximately 50% manufacturing cost reduction. in ascorbic acid, the five-step process (yield ≈ 85%) starting from d-glucose, originally invented by tadeus reichstein in 1933, is being gradually substituted by a more straightforward fermentative process with 2-ketogluconic acid as pivotal intermediate. after the discovery of penicillin in 1928 by sir alexander fleming from colonies of the bacterium staphylococcus aureus, it took more than a decade before a powdery form of the medicine was developed. since then, many more antibiotics and other secondary metabolites have been isolated and manufactured by microbial fermentation on a large scale. some important antibiotics besides penicillin are cephalosporins, azithromycin, bacitracin, gentamicin, rifamycin, streptomycin, tetracycline, and vancomycin. cell cultures animal or plant cells, removed from tissues, will continue to grow if cultivated under the appropriate nutrients and conditions. when carried out outside the natural habitat, the process is called cell culture. mammalian cell culture fermentation, also known as recombinant dna technology, is used mainly for the production of complex big molecule therapeutic proteins, a.k.a. biopharmaceuticals. the first products made were interferon (discovered in 1957), insulin, and somatropin. commonly used cell lines are chinese hamster ovary (cho) cells or plant cell cultures. the production volumes are very small. they exceed 100 kg per year for only three products: rituxan (roche-genentech), enbrel (amgen and merck & co. ), and remicade (johnson & johnson). fine chemical production by mammalian cell culture is a much more demanding operation than conventional biocatalysis and –synthesis. the bioreactor batch requires more stringent controls of operating parameters, since mammalian cells are heat and shear sensitive. in addition, the growth rate of mammalian cells is very slow, lasting from days to several months. while there are substantial differences between microbial and mammalian technologies (e.g., the volume / value relationships are 10 $/kg and 100 tons for microbial, 1,000,000 $/kg and 10 kilograms for mammalian technology; the cycle times are