SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 1 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark] 30 Industrial Biotechnology: Discovery to Delivery Gopal K. Chotani*, Timothy C. Dodge*, Alfred L. Gaertner* and Michael V. Arbige* INTRODUCTION acids, vitamins, and natural polymers for Fermentation 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 ethanol production. active proteins, called enzymes, are responsi- Even before knowing about the existence of ble for ethanol fermentation by yeast. During microorganisms, 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 cheese, soy sauces, yogurt, and bread. 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 citric acid long time, it is only now being recognized for fermentation. The process involved fermenta- its potential towards sustainable industrial tion utilizing the mold Aspergillus niger development. whereby sugar 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, acetic acid, oxalic acid, gluconic acid, fumaric acid, and many more. The serendipitous discovery of penicillin in *Genencor International, Danisco Company, 1928 by Alexander Fleming, while research- Palo Alto, CA. ing agents that could be used to combat 1 SVNY337-30[000-000].qxd 15/11/2006 3:04 PM Page 2 sardar g 27B:SVNY337-Kent:Chapter:Ch-30: Techbooks [PPG-Quark] 2 KENT AND RIEGEL’S HANDBOOK OF INDUSTRIAL CHEMISTRY AND BIOTECHNOLOGY bacterial infections, opened the whole new “White Biotechnology” in Europe) was world of antibiotics. 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, Penicillium of materials by biological agents to provide notatum was surface-cultured in flasks. Later, goods and services. Industrial biotechnology a new strain, 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 antibiotic 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-lysine and L-glutamic acid 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 steroids, 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 cell DISCOVERY OF ORGANISMS systems capable of producing new molecules 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, sugars, a nitrogen source, and inorganic salts. and others. Fermentation originally meant cellular activity/ By the end of twentieth century, industrial process without oxygen, 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 microorganism is releasing abundant metabolic energy from growing. Inherent regulatory control processes redox reactions. Depending on the physiolog- allow cells to regulate their enzyme content in ical state of the cell, part of the metabolic direct response to the environment. They pre- energy escapes as heat that must be removed vent the formation of excess endproduct and by the fermentor design. Sucrose, derived superfluous enzymes. In this postgenomic from sugarcane/beet and glucose 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 genes 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, ● Bacteria and actinomycetales enhanced enzymatic activities, or metabolic ● Viruses deficiencies are used to accumulate products. ● Fungi, including yeast Such mutants have been changed so that their ● Protozoa and algae genetic mechanism is no longer sensitive to a The chemical composition of microorganisms particular