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Chapter Industrial Fermentation Manfred J Mirbach and Bassam El AIi 9.1 Introduction and History 290 9.2 Biochemical and Processing Aspects 292 9.3 9.4 9.5 9.2.1 Overview 292 9.2.2 Microorganisms 293 9.2.3 Culture development 296 9.2.4 Process development 298 9.2.5 Bioreactors 300 9.2.6 Downstream processing 303 9.2.7 Animal and plant cell cultures 304 Food and Feed Treatment by Fermentation 304 9.3.1 Food conservation 304 9.3.2 Feed and agriculture 309 9.3.3 Single cell protein (SCP) 309 Industrial Chemicals by Fermentation 311 9.4.1 Ethanol 311 9.4.2 Other industrial alcohols 312 9.4.3 Organic acids 313 9.4.4 Amino acids 314 9.4.5 Vitamins 316 9.4.6 Industrial enzymes 317 Pharmaceutical Products by Fermentation 318 318 9.5.1 Pharmaceuticals by direct fermentation 9.5.2 Pharmaceuticals via biotransformation 319 9.5.3 Biopolymers 322 9.6 Environmental Biotechnology 9.7 Social and Economic Aspects 323 327 Bibliography 328 9.1 Introduction and History Fermentation can be defined as the alteration or production of products with the help of microorganisms Fermentation has been used to conserve and alter food and feed since ancient times Actually, it was the method of choice to convert fresh agricultural products into durable food items for many thousand years In everyday life, we also know the reverse process, namely the uncontrolled decay of food or organic matter in general Under controlled conditions fermentation is a useful process Yogurt, salami, sauerkraut, soy sauce, vinegar, and kefir are just a few examples of fermented food products that we still know of today Fermentation can be spontaneous or be induced by specifically added microorganisms An everyday example of such an induced fermentation is the addition of baking yeast to flour to make bread or cakes As with bread, fermentation can be done in a normal environment where many different microorganisms are present A more sophisticated way is to exclude unwanted microorganisms by sterilization of the materials before adding a starter culture Since around 1800, the mechanism of fermentation has been studied in a scientific way It started when German scientist Erxleben discovered that yeast induces fermentation Louis Pasteur, a French scientist, made many contributions to microbiology He explained that bacteria produce lactic acid, which then conserves the food Pasteur also noticed that unwanted fermentation can be stopped by heat treatment of the substrate (pasteurization) This technique is still widely applied today to treat milk or fruit juices Actually, the production of neat lactic acid was also the first nonfood industrial application of fermentation The first aseptic fermentation (exclusion of unwanted microorganisms) on an industrial scale was the production of acetone, butanol, and butandiol for rubber production After World War I the production shifted to organic acids, when acetone and butanol became available from other sources An important milestone was the introduction of biological wastewater treatment by fermentation Traditionally, wastewater containing human or animal excrement was sprayed on the fields as fertilizer or simply discharged into rivers and lakes This caused microbial pollution and was the cause of many infectious diseases, like typhus and cholera During the 19th century, modern industrialization started and many people migrated from the agricultural area to the big cities Public hygiene became a major task Therefore, it was a big step forward when public sewage systems and biological wastewater treatment plants were introduced Life in the big cities would be unbearable without wastewater treatment, which is perhaps the most widely used fermentation process, even today Another breakthrough in fermentation and human welfare was the discovery of penicillin It was the first antibiotic and the first really effective medication against bacterial infections It was also the first high-cost product of fermentation and it started the development of high-tech fermentation reactors Amino acid production by fermentation started around 1960 in Japan Initially glutamic acid was the main product It was sold as sodium salt, monosodium glutamate (MSG), a flavor enhancer on oriental cuisine Other amino acids soon followed They are used in food and feed to increase the efficiency of low protein substrates Microbiologically produced enzymes were introduced around 1970 They are used in grain processing, sugar production, fruit juice clarification, and as detergent additives (Table 9.1) Since around 1980 the development of genetic engineering made it possible to tailor microorganisms to perform specific tasks Today it is quite common to alter the DNA of bacteria and to introduce selective genes from other species This allows the production of products with high selectivity and rates that were previously not believed possible Insulin was the first commercial product using genetically engineered bacteria for fermentation Today, many different fermentation processes are applied in industry They range from large-scale low-tech processes, like wastewater treatment to very sophisticated biotechnology processes to produce TABLE 9.1 History of Fermentation Time Event Since >5000 years Since 2500 years Spontaneous fermentation to produce bread, vinegar, soy sauce Fermentation of sugar containing crops to produce wine and beer Commercial use of fermentation processes in Asia (except beer and wine) Commercial use of fermentation in Europe (except beer and wine) Discovery of yeast as the origin of fermentation (Erxleben) Scientific explanation of lactic acid formation (Pasteur) First production of neat lactic acid by industrial fermentation Public wastewater treatment plants Industrial production of butanol and acetone by aseptic fermentation Industrial production of organic acids Discovery of penicillin; commercial production since 1941 Industrial production of amino acids Industrial production of enzymes Introduction of genetically modified microorganisms, production of insulin Since 1500 years Since 500 years 1818 1857 1881 Since around 1900 1910 1925 1928/1929 Since 1960 Since 1970 Since 1980 TABLE 9.2 Overview of Industrial Fermentation Products Category Food Feed Cell mass Organic solvents Organic acids Amino acids Antibiotics Vitamins Enzymes Biopolymers Speciality Pharmaceuticals Environmental Energy Examples Uses or Remarks Sour dough, soy sauce, yogurt, kefir, cheese, pickles, salami, anchovy, sauerkraut, vinegar, beer, wine, cocoa, coffee, tea Silage Conservation of perishable food by the formation of lactic acid and ethanol Yeast, lactic acid bacteria, single cell protein Ethanol, glycerol, acetone, butanediol Lactic, citric, acetic, acrylic, formic acid L-lysine, L-tryptophan, L-phenylalanine, glutamic acid Penicillin, streptomycin, tetramycin, tetracycline B12, biotin, riboflavin Amylase, cellulase, protease, lipase, lab Lanthan, dextran, polyhydroxybutyrate Insulin, interferon, erythropoietin (EPO) Waste and wastewater treatment Ethanol from carbohydrates and methane from organic waste Conservation of green plants by organic acids Used as starter cultures, animal feed Cosmetics, Pharmaceuticals Food, textiles, chemical intermediates Food and feed additives Human and veterinary medicines Food and feed supplements Food processing, tanning, detergents additives Food additives, medical devices, packaging Human medicines Public hygiene Fuel additives or heat generation expensive Pharmaceuticals with genetically modified microorganisms Examples are listed in Table 9.2 9.2 Biochemical and Processing Aspects 9.2.1 Overview Nearly all fermentation processes follow the same principle The central unit is the fermenter in which the microorganisms grow and where they produce the desired products The substrate is the feed of the microorganisms; it also contains any other starting materials that are required for the process The fermentation is started by adding the seed microorganisms, which are present in the starter culture The starter culture is also called inoculum The starter microorganisms are produced in small inoculum fermenters before being added to the main large-scale Starter culture Fermenter Downstream processing Fermentation substrate, sterile Figure 9.1 Schematic flow chart of a fermentation process production fermenters At the end of the fermentation process a complex broth is obtained containing bacteria, products, unconverted substrate, side products, water, and so on It needs further work-up steps for separation and purification before the product is pure enough to be marketed The name downstream processing is used for all the steps that follow the actual biochemical reaction The four parts of a fermentation process are discussed in more detail below (Fig 9.1) 9.2.2 Microorganisms Microorganisms used in fermentation are usually single cells or cell aggregates—often bacteria, sometimes fungi, algae, or cells of plant or animal origin A bacterial cell comprises an outer cell wall lined with a cell membrane that keeps the cell content from leaking out, but allows the transport of nutrients in, and of metabolites out The cell liquid contains everything that the cell needs to live and to proliferate, for instance proteins, enzymes, and vitamins The DNA is the carrier of most of the genetic information Plasmids are DNA units that are independent of the chromosomal DNA They are important for the transfers of genetic information into other cells Chemically, a cell mainly comprises water, protein, and a large number of minor compounds Breaking of the cell wall (lyses) kills the organism and releases the content of the cell into the surrounding medium The energy to keep the cell alive comes from absorption of light or from oxidation of organic or inorganic compounds If the oxidizing agent is oxygen, the microorganisms are called aerobic Anaerobic bacteria survive in an oxygen free environment, because they use chemically bound oxygen from nitrate, sulphate, or carbon dioxide The biomass of the cell mainly comprises the elements carbon, hydrogen, oxygen, sulfur, and nitrogen Therefore, these substrates must be added to enable the cells to grow and multiply Organic substrates are usually the source of carbon, but it can also be carbon dioxide for phototropic species Phototropic microorganisms use the energy of the (sun) light to convert carbon dioxide to organic matter Examples are green algae and bacteria The hydrogen comes from the organic substrates or from water and sometimes from other inorganic hydrogen compounds Sulfur and nitrogen come from organic sources or from inorganic ions, such as sulfate, sulfide, nitrate, or ammonium In addition, a number of minor elements (minerals) are required to support growth Many fermentation processes use sugars as the substrate The principle of the microbial metabolization of glucose is described in Fig 9.2 The first step is the cleavage of the glucose (glucolysis); it is in reality a multistep reaction, which results in the formation of glyceraldehyde3-phosphate A series of complex enzyme-induced reactions leads to pyruvate Depending on the predominating enzymes, pyruvate reacts to L-lactic acid (with lactic dehydrogenase) or acetaldehyde and ethanol (with pyruvic decarboxylase and alcohol dehydrogenase) Primary metabolites During cell growth the nutrients of the substrate are converted to cell mass The chemical compounds produced in this process are called primary metabolites The cell mass itself mainly comprises proteins, but a number of primary waste products are also formed, for instance carbon dioxide, lactic acid, ethanol, and so on Primary metabolites are produced in parallel with the cell mass There are exceptions, however, in which the metabolites are still formed after the cell growth has ceased The most important example of such an exception is the production of citric acid Figure 9.2 Scheme of a bacterial cell Secondary metabolites The formation of secondary metabolites is not directly related to cell growth; rather they are formed because of some other, often unknown, reason They are the side products of bacterial life In nature, they are produced in low concentration, but through laboratory mutation and selection, cells can be optimized to overproduce these metabolites Many antibiotics and vitamins are secondary metabolites The formation of secondary metabolites is not directly proportional to primary metabolism and cell growth Therefore, optimum medium composition and process conditions to maximize the product yield may be different from those which are optimal for cell growth Primary metabolites are often released into the surrounding medium, whereas secondary metabolites tend to remain inside the cell and can be recovered only after lyses of the cell walls Some metabolites are toxic; therefore any fermentation must be monitored for toxins Two types are distinguished: exotoxins are released into the fermentation broth, endotoxins remain inside the cell and are sometimes difficult to detect (Fig 9.3) Glycolysis Pyruvic acid Glucose Glyceraldehydemonophosphate Ethanol Acetic acid Acetaldehyde Lactic acid Figure 9.3 Microbial metabolism (primary) of glucose to lactate or ethanol ADP = adenosine diphosphate; ATP = adenosine triphosphate 9.2.3 Culture development Naturally occurring mixed populations of microorganisms (wild type) not give a satisfactory yield of the target product Improvements are necessary to make a fermentation process economically feasible The first step is the selection of the best culture with respect to selectivity and growth characteristics, such as pH, mechanical stress, and temperature sensitivity This selection is a tedious process based on trial and error screening of a large number of strains Mass screening techniques have been developed for this purpose, for example, agar plates that are doped with specific inhibitors or indicators The primary screening results in several potentially useful isolates, which go into secondary screening Here, false positives are eliminated and the best strains are selected by using a small-scale fermentation technique with shake flasks Although primary and secondary screening yields, hopefully, the best candidate, the best natural (wild type) strain is still not good enough for industrial production Further development is necessary to improve the technical properties of the culture, its stability, and yield The genetic improvement technique induces deliberate mutations in the DNA of the cells Such mutations can be induced chemically, by ultraviolet light, or by ionizing radiation This change is random, that means positive or negative with respect to the intended purpose Therefore, a new selection process is needed to find the improved strains The mutated cells are again screened; the best candidates are selected, again mutated, screened and so on, until a satisfactory strain is obtained Chemical substances induce mutations by reaction with amino acids of the DNA chain Nitrous acid (HNO2), for example, reacts with guanine under deamination leading to xanthene (Eq 9.1) Methylation of the amino groups is also possible, for example, with N-methyl-N'-nitro-N-nitrosoguanidine, a strong mutagen, but without lethal effects A third type of mutation is the insertion of alien molecules between two amino acids, thereby altering the macroscopic structure of the DNA Guanine Xanthene DNA absorbs UV light with a wavelength of kg cells + 1.2 kg CO2 + kg water + 13.2 kcal (9.3) Several types of microorganisms are needed for an optimized continuous process Methylococcus species metabolize the methane; Pseudomonas, Nordica, and Moraxella species are present to convert other hydrocarbons and side-products Next Page Methylomas microorganisms are used to convert methanol to singlecell protein The process conditions are similar to the methane process, but the cells are harvested by electrochemical aggregation and filtration Yeast cells (Candida lipolytica) can convert n-paraffins to SCP The process developed by BP uses a continuous stirred tank reactor under sterile conditions The SCP is harvested by centrifugation and then spraydried The mass balance equation (Eq 9.4) shows that less heat is generated and that a little less oxygen is needed than for the methane process 1.12 kg paraffin + 2.56 kg O2 -> 0.13 kg CO2 + 1.08 kg H2O + kcal 9.4 (9.4) Industrial Chemicals by Fermentation 9.4.1 Ethanol Ethanol is a primary alcohol with many industrial uses It can be produced from sugar containing feedstock by fermentation Alcoholic fermentation is one of the oldest and most important examples of industrial fermentation Traditionally, this process has been used to produce alcoholic beverages, but today it also plays an outstanding role in the chemical and automotive industry The largest potential use of ethanol is as car fuel either neat or as an octane booster and oxygenate in normal gasoline In the United States, it is heavily promoted as a replacement of MTBE (methyl-t-butylether) Ethanol is also an important solvent and starting material for cosmetics and Pharmaceuticals and is also widely used as a disinfectant in medicine Ethanol is produced from carbohydrate materials by yeasts in an extracellular process The overall biochemical reaction is represented by (Eq 9.5) C6H12O6 -> C2H5OH + CO2 + energy (9.5) Sugar containing plant material can be used without chemical pretreatment either directly as mash or after extraction with water Examples are fruits, sugar beets, sugar cane, wheat sorghum, and so on Starch containing agricultural commodities or waste products is pretreated with enzymes Cellulose materials, such as wood, are cooked with acid to break up the polymeric carbohydrate bonds and to produce monomeric or dimeric sugars Feedstock preparation: Sugarcane or sorghum must be crushed to extract their simple sugars Starches are converted to sugars in two stages, liquefaction and saccharification, by adding water, enzymes, and heat (enzymatic hydrolysis)