Nutritional Requirements in Fermentation Processes Wllem H. Kampen 1.0 INTRODUCTION Specific nutritional requirements of microorganisms used in industrial fermentation processes are as complex and varied as the microorganisms in question. Not only are the types of microorganisms diverse (bacteria, molds and yeast, normally), but the species and strains become very specific as to their requirements. Microorganisms obtain energy for support of biosynthe- sis and growth from their environment in a variety of ways. The following quotation is reprinted by permission ofPrentice-Hall, Incorporated, Englewood Cliffs, New Jersey. “The most useful and relatively simple primary classifica- tion of nutritional categories is one that takes into account two parameters: The nature of the energy source and the nature of the principal carbon source, disregarding re- quirements for specific growth factors. Phototrophs use light as an energy source and chemotrophs use chemical energy sources. ” I22 Nutritional Requirements 123 Organisms that use CO, as the principal carbon source are defined as autotrophic; organisms that use organic compounds as the principal carbon source are defined as heterotrophic. A combination ofthese two criteria leads to the establishment of four principal categories: (i) photoautotrophic, (ii) photoheterotrophic, (iii) chemoautotrophic and (iv) chemoheterotrophic organisms. Photoautotrophic organisms are dependent on light as an energy source and employ CO, as the principal carbon source. This category includes higher plants, eucaryotic algae, blue green algae, and certain photosynthetic bacteria (the purple and green sulfur bacteria). Photoheterotrophic organisms are also dependent on the light as an energy source and employ organic compounds as the principal carbon source. The principal representatives of this category are a group of photosynthetic bacteria known as the purple non-sulfur bacteria; a few eucaryotic algae also belong to it. Chemoautotrophic organisms depend on chemical energy sources and employ CO, as a principal carbon source. The use of CO, as a principle carbon source by chemotrophs is always associated with the ability to use reduced inorganic compounds as energy sources. This ability is confined to bacteria and occurs in a number of specialized groups that can use reduced nitrogen compounds (NH,, NO,), ferrous iron, reduced sulfur compounds @I,S, S, S,03,-), or H, as oxidizable energy sources. Chemoheterotrophic organisms are also dependent on chemical energy sources and employ organic compounds as the principle carbon source. It is characteristic of this category that both energy and carbon requirements are supplied at the expense of an organic compound. Its members are numerous and diverse, including fungi and the great majority of the bacteria. The chemoheterotrophs are of great commercial importance. This category may be subdivided into respiratory organisms, which couple the oxidation of organic substrates with the reduction of an inorganic oxidizing agent (electron acceptor, usually O,), and fermentative organisms, in which the energy yielding metabolism of organic substrates is not so coupled. In addition to an energy source and a carbon source, the microorganisms require nutritional factors coupled with essential and trace elements that combine in various ways to form cellular material and products. Since photosynthetic organisms (and chemoautotrophes) are the only net producers of organic matter on earth, it is they that ultimately provide, either directly or indirectly, the organic forms of energy required by all other organisms. [l] I24 Fermentation and Biochemical Engineering Handbook Compounds that serve as energy carriers for the chemotrophs, linking catabolic and biosynthetic phases of metabolism, are adenosine phosphate and reduced pyridine nucleotides (such as nicotinamide dinucleotide or NAD). The structure of adenosine triphosphate (ATP) is shown in Fig. 1. It contains two energy-rich bonds, which upon hydrolysis, yield nearly eight kcaVmole for each bond broken. ATP is thus reduced to the diphosphate (ADP) or the monophosphate (AMP) form. OH OH OH II 0 I NHZ P I \ ADP J -s(adenosinehate I I Figure 1. Chemical structure of ATP, which contains two energy-rich bonds. When ATP yields ADP, the Gibbs free energy change is -7.3 kcaVkg at 37OC and pH 7. Plants and animals can use the conserved energy of ATP and other substances to carry out their energy requiring processes, Le., skeletal muscle contractions, etc. When the energy in ATP is used, a coupled reaction occurs. ATP is thus hydrolyzed. HP Adenoisine -@- 0 -@-Om Adenosine-@&)+ HO-@ + Energy (ADP) hydrolysis (ATP) 0 /I I OH where - is an energy-rich bond and -@ terminally represents - P OH and - P -- internally. 0 II I OH Nutritional Requirements 125 Biochemically, energetic coupling is achieved by the transfer of one or both of the terminal phosphate groups of AMP to an acceptor molecule, most of the bond energy being preserved in the newly formed molecule, e.g., glucose + ATP + glucose-6-phosphate + ADP.['] Mammalian skeleton muscle at rest contains 350-400 mg ATP per 100 g. ATP inhibits enzymatic browning of raw edible plant materials, such as sliced apples, potatoes, etc. 2.0 NUTRITIONAL REQUIREMENTS OF THE CELL Besides a source of energy, organisms require a source ofmaterials for biosynthesis of cellular matter and products in cell operation, maintenance and reproduction. These materials must supply all the elements necessary to accomplish this. Some microorganisms utilize elements in the form of simple compounds, others require more complex compounds, usually related to the form in which they ultimately will be incorporated in the cellular material. The four predominant types of polymeric cell compounds are the lipids (fats), the polysaccharides (starch, cellulose, etc.), the information-encoded polydeoxyribonucleic acid and polyribonucleic acids (DNA and RNA), and proteins. Lipids are essentially insoluble in water and can thus be found in the nonaqueous biological phases, especially the plasma and organelle membranes. Lipids also constitute portions ofmore complex molecules, such as lipoproteins and liposaccharides. Lipids also serve as the polymeric biological fuel storage. Natural membranes are normally impermeable to highly charged chemical species such as phosphorylated compounds. This allows the cell to contain a reservoir of charged nutrients and metabolic intermediates, as well as maintaining a considerable difference between the internal and external concentrations of small cations, such as H', Kf and Na'. Vitamins A, E, K and D are fat-soluble and water-insoluble. Sometimes they are also classified as lipids. DNA contains all the cell's hereditary information. Upon cell division, each new cell receives a complete copy of its parents' DNA. The sequence of the subunit nucleotides along the polymer chain holds this information. Nucleotides are made up of deoxyribose, phosphoric acid, and a purine or pyrimidine nitrogenous base. RNA is a polymer of ribose-containing nucleotides. Of the nitrogenous bases, adenine, guanine, and cytosine are 126 Fermentation and Biochemical Engineering Handbook common to both DNA and RNA. Thymine is found only in DNA and uracil only in RNA.['] Prokaryotes contain one DNA molecule with a molecular weight on the order of 2 x lo9. This one molecule contains all the hereditary information. Eukaryotes contain a nucleus with several larger DNA molecules. The negative charges on DNA are balanced by divalent ions in the case of prokaryotes or basic amino acids in the case of eukaryotes. Messenger RNA-molecules carry messages from DNA to another part of the cell. The message is read in the ribosomes. Transfer RNA is found in the cytoplasm and assists in the translation of the genetic code at the ribosome. Typically 30-70% of the cell's dry weight is protein. All proteins contain C, H, N, and 0. Sulfur contributes to the three-dimensional stabilization of almost all proteins. Proteins show great diversity of biologi- cal knctions. The building blocks of proteins are the amino acids. The predominant chemical elements in living matter are: C, H, 0, and N, and they constitute approximately 99% of the atoms in most organisms. Carbon, an element of prehistoric discovery, is widely distributed in nature. Carbon is unique among the elements in the vast number and variety of compounds it can form. There are upwards of a million or more known carbon compounds, many thousands of which are vital to organic and life processes.[2] Hydrogen is the most abundant of all elements in the universe, and it is thought that the heavier elements were, and still are, being built from hydrogen and helium. It has been estimated that hydrogen makes up more than 90% of all the atoms or three quarters of the mass of the universe.[2] Oxygen makes up 2 1 and nitrogen 78 volume percent of the air. These elements are the smallest ones in the periodic system that can achieve stable electronic configurations by adding one, two, three or four electrons re~pectively.~'][~] This ability to add electrons, by sharing them with other atoms, is the first step in forming chemical bonds, and thus, molecules. Atomic smallness increases the stability of molecular bonds and also enhances the formation of stable multiple bonds. The biological significance of the main chemical elements in microor- ganisms is given in Table 1 Ash composes approximately 5 percent of the dry weight of biomass with phosphorus and sulfur accounting, for respectively 60 and 20 percent. The remainder is usually made up of Mg, K, Na, Cay Fey Mn, Cu, Mo, Coy Zn and Cl.['l Nutritional Requirements 127 Table 1. Physiological functions of the principal Element Symbol Atomic Physiological function Hydrogen Carbon Nitrogen oxygen Sodium Magnesium Phosphorus Sulfur Chlorine Potassium Calcium Manganese Iron Cobalt Copper zinc Molybdenum H C N 0 Na Mg P S c1 K Ca Mn Fe co cu Zn Mo 1 6 7 8 11 12 15 16 17 19 20 25 26 27 29 30 42 Constituent of cellular water and organic cell materials Constituent of organic cell materials Constituent of proteins, nucleic acids and coenzymes Constituent of cellular water and organic materials, as 0, electron acceptor in respiration of aerobes Principal extracellular cation Important divalent cellular cation, inorganic cofactor for many enzymatic reactions, incl. those involving ATP; hctions in binding enzymes to substrates and present in chlorophylls Constituent of phospholipids, coenzymes and nucleic acids Constituent of cysteine, cystine, methionine and proteins as well as some coenzymes as CoA and cocarboxylase Principal intracellular and extracellular anion Principal intracellular cation, cofactor for some enzymes Important cellular cation, cofactor for enzymes as proteinases Inorganic cofactor cation, cofactor for enzymes as proteinases Constituent of cytochromes and other heme or non-heme proteins, cofactor for a number of enzymes Constituent of vitamin B,, and its coenzyme derivatives Inorganic constituents of special enzymes 128 Fermentation and Biochemical Engineering Handbook The predominant atomic constituents of organisms, C, H, N, 0, P, and S, go into making up the molecules of living matter. All living cells on earth contain water as their predominant constituent. The remainder of the cell consists largely of proteins, nucleic acids, lipids, and carbohydrates, along with a few common salts. A few smaller compounds are very ubiquitous and function universally in bioenergetics, e.g., ATP for energy capture and transfer, and NAD in biochemical dehydrogenation. Microorganisms share similar chemical compositions and universal pathways. They all have to accomplish energy transfer and conversion, as well as synthesis of specific and patterned chemical structures.['] The microbial environment is largely determined by the composition of the growth medium. Using pure compounds in precisely defined proportions yields a defined or synthetic medium. This is usually preferred for research- ing specific requirements for growth and product formation by systematically adding or eliminating chemical species from the formulation. Defined media can be easily reproduced, have low foaming tendency, show translucency and allow easy product recovery and purification. Complex or natural media such as molasses, corn steep liquor, meat extracts, etc., are not completely defined chemically, however, they are the media of choice in industrial fermentations. In many cases the complex or natural media have to be supplemented with mainly inorganic nutrients to satisfy the requirements of the fermenting organism. The objective in media formulation is to blend ingredients rich in some nutrients and deficient in others with materials possessing other profiles to achieve the proper chemical balance at the lowest cost and still allow easy processing.r4I Fermentation nutrients are generally classified as: sources of carbon, nitrogen and sulfbr, minerals and vitamins. 3.0 THE CARBON SOURCE Biomass is typically 50% carbon on a dry weight basis, an indication of how important it is. Since organic substances are at the same general oxidation level as organic cell constituents, they do not have to undergo a primary reduction to serve as sources of cell carbon. They also serve as an energy source. Consequently, much of this carbon enters the pathways of energy-yielding metabolism and is eventually secreted from the cell as CO, (the major product of energy-yielding respiratory metabolism or as a mixture of C02 and organic compounds, the typical end-products of fermentation metabolism). Many microorganisms can use a single organic compound to Nutritional Requirements 129 supply both carbon and energy needs. Others need a variable number of additional organic compounds as nutrients. These additional organic nutri- ents are called growth factors and have a purely biosynthetic function, being required as precursors of certain organic cell constituents that the organism is unable to synthesize. Most microorganisms that depend on organic carbon sources also require CO, as a nutrient in very small amounts.['] In the fermentation of beet molasses to ethanol and glycerol, it was found that by manipulating several fermentation parameters, the ethanol yield (90.6%) and concentration (8.5% v/v) remained essentially the same, while the glycerol concentration went from 8.3 gA to 11.9 gA. The CO, formation, however, was reduced! With glycerol levels over 12 gA, the ethanol yield and concentration reduced with the C0,-formation near normal again.[5] In fermentations, the carbon source on a unit of weight basis may be the least expensive raw material, however, quite often represents the largest single cost for raw material due to the levels required. Facultative organisms incorporate roughly 10% of substrate carbon in cell material, when metabolizing anaerobically, but 50-55% of substrate carbon is converted to cells with fully aerobic metabolism. Hence, if 80 grams per liter of dry weight of cells are required in an aerobic fermentation, then the carbon required in that fermen- tation equals (80/2) (100/50) = 80 grams of carbon. Ifthis is supplied as the hexose glucose, with molecular weight 180 and carbon weight 72, then (80) (1 80)/72 = 200 gram per liter of glucose are required. Carbohydrates are excellent sources of carbon, oxygen, hydrogen, and metabolic energy. They are frequently present in the media in concentrations higher than other nutrients and are generally used in the range of 0.2-25%. The availability of the carbohydrate to the microorganism normally depends upon the complexity of the molecule. It generally may be ranked as: hexose > disaccharides > pentoses > polysaccharides Carbohydrates have the chemical structure of either polyhydroxyaldehydes or polyhydroxyketones. In general, they can be divided into three broad classes: monosaccharides, disaccharides and polysaccharides. Carbohy- drates have a central role in biological energetics, the productionofATP. The progressive breakdown of polysaccharides and disaccharides to simpler sugars is a major source of energy-rich compounds.['] During catabolism, glucose, as an example, is converted to carbon dioxide, water and energy. Enzymes catalyze the conversion from complex to simpler sugars. Three major interrelated pathways control carbohydrate metabolism: I30 Fermentation and Biochemical Engineering Handbook - The Embden-Meyerhof pathway (EMP) - - The pentose-phosphate pathway (PPP) The Krebs or tricarboxylic acid cycle (TCA) In the EMP, glucose is anaerobically converted to pyruvic acid and on to either ethanol or lactic acid. From pyruvic acid it may also enter the oxidative TCA pathway. Per mole of glucose broken down, a net gain of 2 moles of ATP is being obtained in the EMP. The EMP is also the entrance for glucose, fructose, and galactose into the aerobic metabolic pathways, such as the TCA-cycle, In cells containing the additional aerobic pathways, the NADH, that forms in the EMP where glyceraldehyde-3-phosphate is converted into 3 -phosphoglyceric acid, enters the oxidative phosphorylation scheme and results in ATP generation.L3I In fermentative organisms the pyruvic acid formed in the EMP pathway may be the precursor to many products, such as ethanol, lactic acid, butyric acid (butanol), acetone and isopropanol.['] The TCA-cycle functions to convert pyruvic and lactic acids, the end products of anaerobic glycolysis (EMP), to CO, and H,O. It also is a common channel for the ultimate oxidation of fatty acids and the carbon skeletons of many amino acids. The overall reaction is: 2C3H403 + 502 + 30 ADP + 30 P; + 6C0, + 4H2O + 30 ATP for pyruvic acid as the starting material.L3I Obviously, the EMP-pathway and TCA-cycle are the major sources of ATP energy, while they also provide intermediates for lipid and amino acid synthesis. The PPP handles pentoses and is important for nucleotide (ribose-5- phosphate) and fatty acid biosynthesis (NADPH,). The Entner-Doudoroff pathway catabolizes glucose into pyruvate and glyceraldehyde-3 -phosphate. It is important primarily in Gram negative The yeast Saccharomyces cerevisiae will ferment glucose, fructose and sucrose without any difficulties, as long as the minimal nutritional requirements of niacin (for NAD), inorganic phosphorus (for phosphate groups in 1 , 3-diphosphoglyceric acid and ATP) and magnesium (catalyzes, with hexokinase and phosphofructokinase, the conversion of glucose to glucose-6-phosphate and fructose-6-phosphate to fructose- 1,6-diphosphate) are met. Table 2 lists some of the important biological molecules involved in catabolism and S. cerevisiae ferments galactose and maltose occasionally, but slowly; inulin very poorly; raffinose only to the extent of one Nutritional Requirements 131 Kinetic energy for biological processes Energy potential third and melibiose and lactose it will not ferment. S. cerevisiae follows the Embden-Meyerhofpathway and produces beside ethanol, 2 moles of ATP per mole of glucose. Table 2. Fundamental Biological Molecules['][3] Simple molecule Constituent Derived macro- atoms molecules CiHiO glycogen, starch, cellulose Glucose (carbohvdrate) proteins fats and oils HSCH2CH(NH2)COOH CiNiHiOiS Cysteine (amino acid) Lv$.!ne2kasic amino acid) NH CH CH(NH2)COOH CrNiHiO CrHiO nucleotides (nucleic acids, DNA and RNA) Adenine (purine) r2 NR \CH I I1 imidine 132 Fermentation and Biochemical Engineering Handbook Catabolite repression, transient repression and catabolite inhibition regulate the utilization of many carbohydrates.['] Catabolite repression is a reduction in the rate of synthesis of certain enzymes in the presence ofglucose or other easily metabolized carbon sources. In addition to this repression during steady-state growth in glucose, a period of more intense repression may occur immediately after the cells have been exposed to very high levels of glucose. This effect may last up to one generation or until glucose levels have been reduced to more acceptable levels. This is transient repression. Catabolite inhibition is a control exerted by glucose on enzyme activity rather than on enzyme formation, analogous to the feedback inhibition in biosyn- thetic pathways. Enzymes involved in the utilization of other carbohydrates are inhibited by glucose. Simple sugars are available in powder or in liquid form and in a variety of purities. Glucose is usually made from corn starch through hydrolysis and sucrose from sugar cane or sugar beets. Sucrose is most often purchased in the form of molasses. Sugar beet molasses is the main by-product of table sugar production. Blackstrap molasses is the remaining by-product of raw sugar production from sugar cane, it is the prevailing type of cane molasses. High test molasses or inverted cane syrup is a by-product of the refineries in which raw sugar is refined into white or table sugar. Both blackstrap and beet molasses are widely used in the fermentation industry. Their approximate composition differs considerably, as indicated in Table 3.151 The data on sugar beet molasses are averages from two (2) samples each of Dutch and Frenchmolasses fromthe 1990 campaign (column A). The US beet molasses data are averages from five (5) factories belonging to American Crystal Sugar over the 1991 season (Column B). The blackstrap molasses data are averages from several samples from Brazil, Dominican Republic and Haiti, over the period 1975-1983. Table 4 is an indication of how complex a medium blackstrap molasses is. Upon diluting to 25%, it was stripped under reduced pressure (40 mm Hg) at 38°C. The distillate was extracted with an ethedpentane (1: 1) mixture. Separation and identification was done with a capillary column and mass spe~trometer.[~] Molasses is produced through nonsugar accumulation during the sugar production process and the accompanying increased solubility of sucrose. Of the non-sugars, the mineral salts have a much greater influence on sucrose solubility than the organic compounds. As a rule of thumb, one gram of mineral salts present in normal molasses will retain five grams of non- crystallizable sucrose. Through chromatographic separation processes, it is possible to recover up to approximately 85 percent of this sucrose. Nutrition a1 Requirements I33 Table 3. Averagecomposition(%)ofEuropean (A) andU.S. (B) beet molasses versus BrazilidCaribbean blackstrap molasses Component Beet Molasses Blackstrap Column A Column B water 16.5 19.2 18.0 sucrose 51.0 48.9 32.0 glucose + fructose 1 .O 0.5 27.0 raffinose 1 .o 1.3 organic non-sugars 19.0 18.0 14.0 ash 11.5 12.1 9.0 - Ash components: Si02 CaO K2O MgO p205 Na,,O J33203 co3 A1203 Sulfates as SO, c1 0.1 3.9 0.26 0.16 0.06 1.3 0.02 0.07 3.5 0.55 - 1.6 11.5 - 6.4 0.21 0.12 0.03 1.6 0.03 - - 0.74 0.8 0.7 3.5 1.9 0.1 0.2 0.4 - 1.8 - 0.4 9.0 Vitamins (md 100 2): Thiamine (B,) 1.3 0.01 8.3 Riboflavin (B,) 0.4 1.1 2.5 Nicotinic acid 51.0 8.0 21.0 Ca-pantothenate (B,) 1.3 0.7 21.4 Folic acid 2.1 0.025 0.04 6.5 1.2 Biotin 0.05 - Pyridoxine-HC1 (B,) 5.4 - The average viscosity of sample B was 1,062 CP at 45°C. 134 Fermentation and Biochemical Engineering Handbook Table 4. Isolation Of Some Volatile Compounds In Blackstrap Molasses[5] methanol methyl formate 2-methyl-furamidon-3 fu firylacetate methylfufiral 2-acetylfuran phenol quaiacol benzaldehyde 2.5 dmethylpyrazine ( 1 , 4) 2,6dimethylpyrazine (1,4) 2-methyl-6-ethylpyrazine (1,4) 2-methyl-5-ethylpyrazine (1,4) trimethylpyrazine (1, 4) syringic acid vanillic acid p-hydroxy benzoic acid p-coumaric acid (trans-) p-coumaric acid (cis-) p-hydroxphenylacetic acid ~~ acetic acid propionic acid isobutync acid n-butyric acid isovaleric acid n-valeric acid isocaproic acid n-caproic acid alanine aspartic acid glutamic acid leucine isoleucine glycine methionine asparagine glutamine valine tyrosine The composition of molasses varies from year to year, since it depends on many factors, such as variety of sugar cane or beet, soil type, climatic conditions (rainfall, sunshine), time of harvesting, process conditions, etc. Beet molasses contains approximately 1.9% N of which roughly 1.2% consists of betaine, 0.6% amine-N and 0.025% ammoniacal-N. Cane molasses does not contain betaine and has less than 50% of the organic nitrogen content of beet molasses. Beet molasses contains a relatively high amount of protein, while cane molasses contains high levels of gums and pectins. Also present are: hemicellulose, reversible and irreversible colloids, pigments, inositol, etc. Both types of molasses also contain trace elements, vitamins and growth factors, however, cane molasses usually contains more than beet molasses. Beet molasses has a characteristic, often unpleasant smell, and a pH around 8.0; while blackstrap molasses usually has a fruity, pleasant, mildly acidic smell and a pH value below 7.0. Nutritional Requirements 135 While sugar alcohols are not common, large scale, they may be used in bioconversions such as from glycerol. Methane, methanol and n-alkanes have been used in biomass production. Fatty acids may be converted by fungi after hydrolysis by lipase. Other organic acid carbon sources would be oleic, linoleic and linolenic acids. These might also serve as foam control agents. Carbon dioxide is a possible carbon source in nature, but is not practical commercially due to low growth rates. The most economically important and most widely used carbon sources are the carbohydrates. They are commonly found and most are economically priced, 4.0 THE NITROGEN AND SULFUR SOURCE Following the carbon source, the nitrogen source is generally the next most plentiful substance in the fermentation media. A few organisms can also use the nitrogen source as the energy source. Nitrogen and sulfur occur in the organic compounds of the cell, principally in reduced form, as amino and sulfhydryl groups, respectively. Most photosynthetic organisms assimilate these two elements in the oxidized inorganic state, as nitrates and sulfates; their biosynthetic utilization thus involves a preliminary reduction. Many nonphotosynthetic bacteria and fingi can also meet the needs for nitrogen and sulfur from nitrates and sulfates. Some microorganisms are unable to bring about a reduction of one or both of these anions and must be supplied with the elements in the reduced form. In these cases, nitrogen may be supplied as ammonia salts and sulfur as a sulfide or an organic compound like cysteine, which contains a sulfhydryl group, Organic nutrients as amino acids and more complex protein degradation products as peptones may also supply nitrogen and sulfur in the reduced form as well as carbon and energy. Several prokaryotic groups can also utilize the most abundant natural nitrogen source, N,, which is unavailable to eukaryotes. This process of nitrogen assimilation is termed nitrogenjxution and involves a preliminary reduction of N, to ammonia.['] Nitrogen is used for the anabolic synthesis of nitrogen- containing cellular substances, such as amino acids, purines, DNA, and RNA. Many algae and fungi use ammonium nitrate and sodium nitrate as nitrogen sources, however, yeasts and bacteria have problems utilizing nitrogen in this form.['] Few organisms are able to assimilate nitrites. 136 Fermentation and Biochemical Engineering Handbook Organic sources of nitrogen in synthetic media are specific amino acids, purines, pyrimidines, and urea. Urea, depending upon the buffer capacity of the system, will raise the pH-value of the medium. Organic urea is also formed in the urea cycle reaction, starting with ammonia: NH3+ATP + C02+H2NCOOP + ADP Ammonium sulfate produces acidic conditions because the ammonia is rapidly utilized and free acid is then liberated. Many commercial fermentations use complex organic nitrogen sources, which are by-products of the agricultural and food processing industries, such as: corn steep liquor, dried distillers solubles, yeast, fish or bone meal, corn germ or gluten meal, protein peptones, hydrolysates and digests from casein, yeast, cottonseed, milk proteins, etc. These sources of nitrogen provide many other nutrients and are usually reasonably priced. The composition of some of these products is given in Tables 5, 6, and 7. Industrial fermentations are generally more rapid and efficient when these materials are used, since they reduce the number of compounds which the cells would otherwise have to synthesize “de The availability of nitrogen as well as the concentration in the media has to be considered in each case. Proteins can only be assimilated by microorganisms that secrete extracellular proteases, which enzymatically hydrolyze the proteins to amino acids. Microorganisms without this ability require protein hydrolysates, peptones, or digests composed of free amino acids prepared by hydrolyzing proteinaceous materials with acids or enzymes. 5.0 THE SOURCE OF TRACE AND ESSENTIAL ELEMENTS Minerals supply the necessary elements to cells during their cultivation. Typical biological functions of the main elements were listed in Table 1. Table 8 shows the trace element composition in samples of Puerto Rican blackstrap and Dutch beet molasses in the year 1986. Phosphorous occurs principally in the form of sugar-phosphates, such as the nucleotides which compose DNA, RNA, and ATP. Phosphorus is assimilated in its inorganic form where the phosphate ion is esterified. The P-atom does not change in valence and remains as part of a phosphate group. Upon the death of the cell, it is again liberated as inorganic phosphorus through hydrolysis. Sulfur is present to the greatest extent in the amino acids cysteine and methionine. It is also commonly supplied as H2S04 for pH adjustment, and Nutritional Requirements 137 as ammonium sulfate and potassium bisulfate. Many of the other elements are found complexed with enzymes: e.g., Mg2+ with phosphohydrolase and phosphotransferase, K+ with pyruvate phosphokinase (and Mg2+), and Na+ with plasma membrane ATP-ase (and K+ and Mg2+).[l] Table 5. Typical chemical composition of Pharmamedia*;['] a nitro- gen and energy source from the cottonseed embryo; soy bean meal (expeller);f2] meat and bone meal; i31 and peanut meal and hullsr41 [31 [41 dry matter protein, % carbohydrates,% fat, % fiber, % ash, % calcium, % magnesium, % phosphorus, % available P, % potassium, % sulfur, % biotin, mgkg choline, mgkg niacin, mgkg panthotenic acid, mg/kg pyridoxine, mgkg riboflavin, mgkg thiamine, mgkg arginine, % cystine, % glycine, % histidine, % isoleucine, % leucine, % lysine, % methionine, % phenylalanine, % threonine, % tryptophan, % tyrosine, % valine, % *Trademark 99.0 59.2 24.1 4.0 2.6 6.7 0.25 0.74 1.31 0.31 1.72 0.6 1.52 3270 83.3 12.4 16.4 4.82 3.99 12.28 1.52 3.78 2.96 3.29 6.11 4.49 1.52 5.92 3.31 0.95 3.42 4.57 90.0 42.0 29.9 4.0 6.0 6.5 0.25 0.25 0.63 0.16 1.75 0.32 - 2420 30.4 14.1 3.1 2.9 0.62 - - - - - - 2.8 0.59 1.72 0.59 - - - 92.0 50.0 0.0 8.0 3.0 31.0 8.9 1.1 4.4 4.4 1.46 0.26 - 1914 55 8.8 4.4 1.1 4.0 1.4 6.6 0.9 1.7 3.1 3.5 0.7 1.8 1.8 0.2 1.22 2.4 - 90.5 45.0 23.0 5.0 12.0 5.5 0.15 0.32 0.55 0.2 1.12 0.28 - 1672 167.2 48.4 5.3 7.26 4.6 0.7 3.0 1 .o 2.0 3.1 1.3 0.6 2.3 1.4 0.5 2.2 - - 138 Fermentation and Biochemical Engineering Handbook Table 6. Average Composition of Corn Steep Liquor[4] Total solids, Yo ............................................. 54.0 pH ................................................................. 4.2 Ash (oxide), % dry basis ....... Crude protein (N x 6.25) ...... Fat ................................................................. 0.4 Total acids as lactic acid ......... Nitrogen ....................................................... .7.5 Phytic acid .......................... Reducing sugars as glucose ........................... .2.5 Ash constituents. % drv basis: -inorganic phosphorus by difference ......... 1.1 boron (ppm, dry basis) .................................... 2 zinc ............................................ Vitamins, mm drv basis: biotin ............................................................. 0.1 choline ..................................... ........ 5600 ..................................... 0.5 Niacin ................. Thiamine ....................................................... 5 Nutritional Requirements I39 Table 7. Analysis of Microbiological Media Prepared Proteins (Difco Manual, Ninth Edition) Constituent Peptone Tryptone Casamino Yeast Acids Extract Ash % 3.53 Soluble Extract % 0.37 Total Nitrogen % 16.16 Ammonia Nitrogen % 0.04 Free Amino Nitrogen % 3.20 Arginine % Aspartic Acid % Cystine % Glutamic Acid % Glycine % Histidine % Isoleucine % Leucine % Lysine % Methionine % Phenylalanine % Threonine % Tryptophan % Tyrosine % Valine % Organic Sulhr % Inorganic Sulhr % Phosphorous % Potassium % Sodium % Magnesium % Calcium % Chloride % Manganese (ma) Zinc (me) Biotin (pg/gm) Riboflavin (pg/gm) Copper (mg/L) Thiamine (PdP) 8.0 5.9 0.22 11.0 23.0 0.96 2.0 3.5 4.3 0.83 2.3 1.6 0.42 2.3 3.2 0.33 0.29 0.07 0.22 1.08 0.056 0.058 0.27 8.6 17.00 18.00 0.32 0.50 4.00 7.28 0.30 13.14 0.02 4.73 3.3 6.4 0.19 18.9 2.4 2.0 4.8 3.5 6.8 2.4 4.1 3.1 1.45 7.1 6.3 0.53 0.04 0.75 0.30 2.69 0.045 0.096 0.29 13.2 16.00 30.00 0.36 0.33 0.18 3.64 11.15 3.8 0.49 5.1 1.1 2.3 4.6 9.9 6.7 2.2 4.0 3.9 0.8 1.9 7.2 0.35 0.88 0.77 0.0032 0.0025 11.2 7.6 10.00 8.00 0.102 0.12 0.03 10.1 9.18 0.78 5.1 6.5 2.4 0.94 2.9 3.6 4.0 0.79 2.2 3.4 0.88 0.60 3.4 0.29 0.04 0.32 0.030 0.040 0.190 7.8 19.00 88.00 1.4 3.2 19.00 140 Fermentation and Biochemical Engineering Handbook Table 8. Average composition of (trace) elements in Puerto Rican blackstrap molasses in 1986 and one European beet molas- ses sample of the same year15] Element Blackstrap Beet Molasses B Ca co cu P Fe Mg Mn Ni Na Pb K Sr Zn 0.041 0.86 0.000054 0.0028 0.071 0.0158 1.14 0.0057 0.000 123 0.058 0.75 2.68 0.01 1 - 0.0003 0.42 0.00006 0.0005 0.012 0.01 15 0.0018 0.083 0.78 3.39 0.004 0.0034 - - Requirements for trace elements may include iron (Fe2+and Fe3+), zinc (Zn2'), manganese (Mn2'), molybdenum (Mo2'), cobalt (Co2'), copper (Cu2'), and calcium (Ca2'). The functions of each vary from serving in coenzyme functions to catalyze many reactions, vitamin synthesis, and cell wall transport. The requirements are generally in very low levels and can sometimes even be supplied from quantities occurring in water or from leachates from equipment. Trace elements may contribute to both primary or secondary metabolite production. Manganese can influence enzyme production. Iron and zinc have been found to influence antibiotic production. Primary metabolite production is usually not very sensitive to trace element concentration, however, this is a different matter for secondary metabolite production. Bacillus licheniformis produces the secondary metabolite bacitracin.L7I A manganese concentration of 0.07 x M is required, but at a concentration of 4.0 x 10" M manganese becomes an inhibitor. Streptomyces griseus produces streptomycin as a secondary metabolite. For Nutritional Requirements 141 maximum growth it requires a concentration of 1 .O x M of iron and 0.3 x 10" zinc, while a zinc concentration of 20 x lom5 M becomes an inhibitor. Aspergillus niger produces citric acid as a primary metabolite. Concentra- tions of 2.0 x loq5 M of zinc, 6.0 x M of manganese act as It produces only citric acid from glucose or sucrose during an iron deficiency and/or proper Cu/Fe ratio in the fermenta- tion media. Raw materials such as molasses may have to be treated to remove iron. Manganese enhances longevity in cultures of Bacillus sp., iron in Escherichia sp., while zinc suppresses longevity of Torulopsis sp.[*I Cells are 80% or more water and in quantitative terms this is the major essential nutrient. Water is the solvent within the cell and it has some unusual properties, like a high dielectric constant, high specific heat and high heat of vaporization. It fUrthermore ionizes into acid and base, and has a propensity for hydrogen bonding. In most fermentations, microorganisms inhabit hypotonic environments in which the concentration of water is higher than it is within the cell. The cell walls are freely permeable to water, but not to many solutes. Water tends to enter the cell to equalize the internal and external water concentrations. Many eukaryotes and nearly all prokaryotes have a rigid wall enclosing the cell, which mechanically prevents it from swelling too much and undergoing osmotic lysis. The product of osmotic pressure and the volume containing one gram-molecule of solute is a constant. Thus, osmotic pressure is directly proportional to the concentration and M of iron and/or 0.02 x P = (0.0821) (T)/V for a substance, where, P is measured in atmospheres, Vin liters, and Tin degrees Kelvin. A solution of 180 g/L of glucose (MW = 180) at 3OoC, contains 1 gram-molecule per liter, Thus, V= 1 liter andK= 303 K. Hence, the osmotic pressure P = 24.9 atm. (366 psi). All the required metallic elements can be supplied as nutrients in the form ofthe cations of inorganic salts. K, Mg, Ca and Fe are normally required in relatively large amounts and should normally always be included as salts in culture media. Table 9 shows which salts are soluble and which are insoluble in water,[g] as well as commonly used inorganic and trace elements and concentration ranges. 142 Fermentation and Biochemical Engineering Handbook Table 9. Solubility of the common salts[5] and commonly used inorganic and trace elements and concentration ranges from Stanbury & Whitaker, 1984) SALT SOLUBLE INSOLUBLE Nitrates Sulfides Chlorides Carbonates Sulfates Phosphates Silicates Acetates Oxalates Source m2p04 Mg S 0,.7H20 KC 1 CaCO, FeS04.4H20 ZnS 0,. 8H20 MnS04.H20 CuS04-5H20 N%MoO4.2H2O A1 1 Na, K, Ca, Ba All others Na, K All others Na, K Na, K All All others All others Ag, Hg, Pb All others Pb, Ca, Ba, Sr All others All others Ca (depends upon concentration) Quantity (g/L) 1 .O-5 .O 0.1-3.0 0.5-12.0 5 .O- 17.2 0.01-0.1 0.1-1.0 0.0 1-0.1 0.003-0.01 0.01-0.1 Nutritional Requirements 143 Oxygen is always provided in water. Some organisms require molecu- lar oxygen as terminal oxidizing agents to fulfill their energetic needs through aerobic respiration. These organisms are obligately aerobic. For obligate anaerobes molecular 0, is a toxic substance. Some organisms are facultative anaerobes and can grow with or without molecular 0,. Lactic acid bacteria have an exclusive fermentative energy-yielding metabolism, but are not sensitive to the presence of Saccharomyces cerevisiae produces ethanol anaerobically and cell mass aerobically, and it can shift from a respiratory to a fermentative mode of metabolism. Sodium and chloride ions are respectively the principal extracellular cations and anions in animals and plants. Potassium is the principal intracellular cation. Candida intermedia, (SCP) single cell protein, grows better on normal alkanes with sources of N, 0, and P, if small amounts of ZnS04.7H,0 are added.[’] Takeda, et al., reported that yeast can be grown by continuously feeding a medium consisting of hydrocarbon fractions boiling at 200° to 360°C, small amounts ofinorganic nitrogen, inorganic salts and organic nitrogen to which the fermentation waste liquor previously used or CSL and ethanol are added.[’] This suggests that SCP production on hydrocarbons may be an outlet for stillage from ethanol-from-beet molasses plants or whey. Table 10 shows the approximate composition of such concentrated stillage from a European producer.[5] Table 10 Composition of a concentrated French Stillage (Ethanol-from-beet molasses) Moisture .................................................... 3 1.4% .................................... 1.2% Lactic acid ................................................... 4.1% .................... 1.3% 1.3% Other organic acids 1.8% 5.4% 0.3% Glucose + fructose ....................................... 0.9% Melibiose ..................................................... 1.4% Ino K+ Na .................................. CaZ+ ............................................................. 0.4% Mgz+ ............................................................ 0.03% Fe3+ ............................................................. 0.02% PZOS ............................................................ 0.34% 144 Fermentation and Biochemical Engineering Handbook The combination of minerals is also important in regulating the electrolytic and osmotic properties of the cell interior. In most cases, the complex industrial carbon and/or nitrogen sources supply sufficient minerals for proper fermentation. 6.0 THE VITAMIN SOURCE AND OTHER GROWTH FACTORS Vitamins are growth factors which fulfill specific catalytic needs in biosynthesis and are required in only small amounts. They are organic compounds that function as coenzymes or parts of coenzymes to catalyze many reactions. Table 11 itemizes the vitamins together with their active forms, catalytic function, molecular precursors, and raw material sources.[''] The vitamins most frequently required are thiamin and biotin. Re- quired in the greatest amounts are usually niacin, pantothenate, riboflavin, and some (folic derivatives, biotin, vitamin B,, and lipoic acid) are required in smaller amounts.[4] In industrial fermentations, the correct vitamin balance can be achieved by the proper blending of complex materials and, if required, through the addition of pure vitamins. A satisfactory growth medium for baker's yeast, for example, can be achieved by mixing cane molasses, rich in biotin, with beet molasses, rich in the B-group vitamins. The production of glutamic acid by Corynebacterium glutamicum is a function ofthe concentration of biotin in the medium, which must be maintained in the range of 2-5 pg/1 .[41 Organic growth factors are: vitamins, amino acids, purines and pyrimidines. Some twenty-two amino acids enter into the composition of proteins, so the need for any specific amino acid that the cell is unable to synthesize is obviously not large. The same argument applies to the specific need for a purine or pyrimidine: five different compounds enter into the structure of the nucleic acids. An often cited example of the importance of nutritional quality of natural sources occurred during the early phases of the development ofthe penicillin process.[4] A fivefold improvement in antibiotic yield was obtained when CSL was added to the fermentation medium for Penicillium chrysogenum. It was later found that the CSL contained phenylalanine and phenylethylamine, which are precursors for penicillin G. Today, any one of several nitrogen sources are used in conjunction with continuous additions of another precursor for penicillin G, phenylacetic acid.r4] Nutritional Requirements 145 Table 11. Vitamins: Their Sources and Metabolic Functions[''] (with permission from A. Rhodes and D. L. Fletcher, Principles of Industrial Microbiology, Pergamon, New York, 1966, Ch. 6) iubrmce needed D fulfil metabolic requirement (i) Pyrimidine (ti) 'Ihile (iii) Pyrimidine + Thiazole (iv) Thiamine A-ry growth factor (viumin BI) Thiamin Raw material murcc of growth factor Rice polbhings Wbut aerm Yeut Active form Wiepyro- pbwphuc Riboflavin (vitamin 83 (i) Flavin mono- nucleotide (ii) Flavin adenine dinuclcotidc dydrogen Hydrogen (i) Riboflavin Cereals Cornsteep liquor Cottonsccd flour Pyridoxal phosphate bnho group Md decarboxylation (i) Pyridoxine (ii) Pyridoxamine or pyridoxal (iii) Pyridoxal phosphate Pyridoxal (vitamin Ed Penicillium spent mycelium Yurt Rice polirhinpr Cere& What seeds Maize seeds Cornsteep liquor Cottod flour Penicillium spent mycelium Wheat reeds LiVU Nicotinic acid or nicotina- mide (i) Nicotinamide adenine dinucleotide (ii) Nicotinamide adenine dinucleotide phosphate (i) Nicolinnmidc mononucleotide Hydrogen Hydrogen Hydrogen (I) Nicotinic acid or nicotina- mide (ii) Nucleotides of nicotin- amide PMtolhcnic acid Coenzyme A Acyl group (i) Pantothenic acid Beet molasses Penicillium spcnt mycelium Cornstccp liquor Cottod flour Carboxyl displacement Methyl group synthuia (i) Cyanoco- balamin (ii) Other cobalamins Activated sewage sludge Liver Cow dung Streptomyces grLcur mycelium silage Mat Folic acid Teuah ydrololic acid Formyl group 0) Folic acid (ii) Para-amino benzoic acid Penicillium spent Spinach Liver Cottonseed flour mycelium 146 Fermentation and Biochemical Engineering Handbook Choline Hemins Table 11. (Conf'd) Phosphatides Choline Egg yolk Hops Cell hemins Electrons Hemins Blood Often, cells of a single microbial strain can synthesize more than one member of a chemical family. The final yields of the various members can be shifted by appropriate precursor pressure. The absence or presence of certain growth factors may accomplish this. In the absence of either exogenous phenylalanine or tryptophan, the ratio of tyrocidines A:B:C synthesized by Bacillus brevis is 1:3:7. If either L- or D-phenylalanine is provided, the main component formed is tyrocidine A. If L- or D-tryptophan is furnished, component D predominates; when both phenylalanine and tryptophan are supplied, each of the four components is synthesized.[g] Metabolic modifiers are added to the fermentation media to force the biosynthetic apparatus of the cell in a certain direction.["] Most metabolic blocks of commercial importance, however, are created by genetic manipu- lation. In screening media formulations in a relatively short period of time, banks of shake flask cultivations as well as continuous culture methods are most useful. Nutritional Requirements 147 Betaine or 1 earboxy-N, N, N-trimethylmethanammonium hydroxide inner salt is a relatively new and very interesting compound from a nutritional point of view. Its formula is Betaine is important in both catabolic and anabolic pathways.[12] It is a methyl donor in the body synthesis of such essential compounds as methionine, carnitine and creatinine. Betaine is a very important regulator of osmotic pressure in different plants, bacteria and marine animals. It regulates the osmolality (number ofmoles of solute per 1000 gram of solvent) by acting as a nonpolar salt in the cells by adjusting the concentration of salts and/or loss of water. Betaine is a growth factor in many fermentation processes, stimulating the overproduction of desired metabolites. It fhctions as an osmoprotectant, as a metabolic regulator and as a precursor or intermediate. Betaine or trimethylglycine enhances biochemical reactions, not only in bacteria and fungi, but also in plants and in mammals. Tissue has only a limited capacity for betaine synthesis and during conditions such as stress, the need for additional betaine arises. Cell organs involved in energy metabolism, such as mitochondria and chloroplast, contain high levels of betaine. These are the respective sites of photosynthetic and respiratory func- tion in eukaryotic cells. Sugar beets are high in betaine. It ends up in the molasses during the recovery of sucrose. When fermenting beet molasses to ethanol, the betaine ends up in the stillage. 7.0 PHYSICAL AND IONIC REQUIREMENTS Each reaction that occurs within the cell has its own optimum (range of) conditions. For instance, although a given medium may be suitable for the initiation of growth, the subsequent development of a bacterial strain may be severely limited by chemical changes that are brought about by the growth and metabolism of the microorganisms themselves. In the case of glucose- containing media, organic acids that may be produced as a result of fermentation may become inhibitory to growth. In contrast, the microbial decomposition or utilization of anionic components of a medium tends to 148 Fermentation and Biochemical Engineering Handbook make the medium more The oxidation of a molecule of sodium succinate liberates two sodium ions in the form of the very alkaline salt, sodium carbonate. The decomposition of amino acids and proteins may also make a medium alkaline as a result of ammonia production. To prevent excessive changes in the hydrogen ion concentration, either buffers or insoluble carbonates are often added to the medium. The phosphate buffers, which consist of mixtures of mono-hydrogen and dihydrogen phosphates (e.g., K2HP04 and KH2P04), are the most useful ones. KH2P04 is a weakly acidic salt, whereas KH2HP04 is slightly basic, so that an equimolar solution ofthe two is nearly neutral, having apH of 6.8. Ifa limited amount of strong acid is added to such a solution, part ofthe basic salt is converted to the weakly acidic one: K2HP04 + HCI + KH2P04 + KC1 If, however, a strong base is added, the opposite conversion occurs: KH2P04 + KOH + K2HP04 + H2O Thus, the solution acts as a buffer by resisting radical changes in hydrogen ion concentration (pH) when acid or alkali is produced in the medium. Different ratios of acidic and basic phosphates may be used to obtain pH-values from approximately 6.0-7.6. Good buffering action, however, is obtained only in the range of pH 6.4-7.2 because the capacity of a buffer solution is limited by the amounts of its basic and acidic ingredients. Bacteria and fungi can generally tolerate up to 5 gL of potassium phosphates. When a great deal of acid is produced by a culture, the limited amounts of phosphate buffer that may be used become insufficient for the maintenance of a suitable pH. In such cases, carbonates may be added to media as “reserve alkali” to neutralize the acids as they are formed. By adding finely powdered CaCO, to media, it will react with hydrogen ions to form bicarbonate, which in turn is converted to carbonic acid, which decomposes to CO, and H,O in a sequence of freely reversible reactions.[’] Several fermentations are run on pH control through addition on demand of acid or alkali. The pH of the medium affects the ionic states of the components in the medium and on the cellular exterior surface. Shifts in pH probably affect growth by influencing the activity of permease enzymes in the cytoplasmic membrane or enzymes associated with enzymes in the cell wall.r4] The pH affects solubility; proteins will coagulate and precipitate (salting-out) at their isoelectric point. Nutritional Requirements I49 In the preparation of synthetic media, sometimes precipitates form upon sterilization, particularly if the medium has a relatively high phosphate concentration. The precipitate results from the formation of insoluble complexes between phosphates and mainly calcium and iron cations. By sterilizing the calcium and iron salts separately and then adding them to the sterilized and cooled medium, the problem can be avoided.[4] Alternatively, a chelating agent such as EDTA (ethylenediamine tetraacetic acid), may be added at a concentration of approximately 0.01%, to form a soluble complex with these metals. when two or more microorgan- isms are placed in a medium, their combined metabolic activities may differ, either quantitatively or qualitatively, from the sum of the activities of the individual members growing in isolation in the same medium. Such phenom- ena result from nutritional or metabolic interactions and are collectively termed synergistic effects.[l3I Typical difficulties in scaleup can occur due to ionic strength. A laboratory fermenter or shake flask sterilized in an autoclave will have a higher nutrient concentration after sterilization due to evaporation. A large fermenter, sterilized in part by direct steam injection, will have a lower concentration, due to steam condensate pickup. These differences usually do not prevent growth, but can certainly alter yield of batches. The range of pH tolerated by most microorganisms can be as broad as 3 to 5 pH units. Rapid growth andor reaction rates are normally in a much more narrow range of 1 pH unit or less. In small scale experiments, it seems to be common to use NaOH for pH control, but it may only contribute problems in scaleup. 8.0 MEDIA DEVELOPMENT Factors that must be considered in developing a medium for large scale fermentations are: 1. The nutrient requirements of the selected microorganism 2. The composition of available industrial nutrients 3. The nutrient properties in relation to storage and handling, pasteurization or sterilization, processing and product purification 4. Cost of the ingredients In the calculation of the cost of the medium, all costs have to be recognized. Thus, in addition to the purchase price, which is obvious, 150 Fermentation and Biochemical Engineering Handbook material handling and storage, labor and analytical requirements must be included. Dilute nutrients require greater storage volumes than concentrated sources. The stability of nutritional requirements is important, refrigeration or heating may be required. High volatility (alcohols), corrosiveness (acids and alkalis) and explosive characteristics (starch powders) pose certain environmental and safety risks. Pretreatment costs for certain raw materials, such as starch liquefaction and saccharification, may be substantial. The rheological properties of the medium may effect such items as mixing, aeration and/or temperature control. The surface tension has an effect on the foaming tendency ofthe broth. Finally, the solids concentration, odor, color, etc., are pivotal in determining the costs of product recovery and purification. Product concentration, yield, and productivity are among the most important process variables in determining conversion costs.[4] The concen- tration ofthe product influences its recovery and refining costs. Raw material costs are affected by the yield. Productivity, or the rate of product formation per unit of process capacity, helps determine the amount of capital, labor, and indirect costs assignable to the product. The influence of the medium on the interplay of these three variables cannot be ignored. Raw material costs in fermentations may vary from 15 to 60% of the total manufacturing cost. Simply trying to cut manufacturing costs by substituting raw materials with cheaper ones may not be the answer. If carbohydrate costs represent, for example, 10% of the total manufacturing cost, it requires a 50% reduction in the carbon source to effect a 5% reduction in manufacturing cost. The question is then how the new raw material effects the multiple interactions of a complex medium. A better approach would be to explore how the impact of a change in raw material would impact the product yield and purity. This could have a far greater influence on the final cost than a cheaper carbohydrate source. Performing multivariable experiments would be the most effective way. Interacting variables used are nutrients, pH, aeration, temperatures, etc. This allows the determination of optimum levels for a given process. As the number of variables increases it becomes impractical to investigate all combinations. An evaluation of five nutrients at only three concentration levels yields 35 or 243 combinations and possible trials. A statistical approach may be taken to deal with the complexity. Several computer programs for statistical experimental design are available. This allows for a three dimensional view of interactions between key variables through re- sponse surface methodology techniques. These techniques not only allow for optimized process conditions, but also lend insight into process requirements Nutritional Requirements 151 that will exceed simple optimization trials. Geiger discusses the statistical approach in greater detail in Ch. 4. Most large scale fermentations, i.e., batch size in excess of 50,000 liters, use inexpensive raw materials in large volume. Here materials such as molasses and corn steep liquor are normally purchased in truck load quantities. The carbohydrate source, because of its large volume, is the only fermentation raw material which has any influence on plant location. In order to maximize production rates one may operate a continuous fermentation process if the product is associated with growth of the organism cells or an intracellular product. Low sensitivity to contamination, such as with a thermophile or production at a reduced pH, are of importance. It is impractical to consider anythmg but batch fermentation when the product is an extracellular metabolite that is sensitive to culture and medium balance. Similarly, batch fermentation is preferred when the culture can undergo mutation at extended operating time and is sensitive to contamination. There are many and varied conditions worldwide which impact on the cost of fermentation raw materials. These can be climatic, e.g., drought or floods, or political, e.g., government subsidies for, or restriction on, farm products or a national ethanol fuel program. These conditions greatly affect the world price of sugar, molasses and corn and are responsible for much of the variability. The rise and fall of sugar prices affects all sources of carbohydrates. To ensure against the effects ofwide swings, aprudent course of action would be to develop processes that permit alternate sources. The demands of the final product may have an important bearing on the selection of the fermentation ingredients. Odor and color on the one hand may play a role, on the other product purity specifications which are very demanding, as is the case for vaccines, require extremely pure nutrients. Some information on industrial protein sources is given in Tables 12, 13 and 14. These were taken from Traders Guide to Fermentation Media Formulations, which can serve as an excellent reference.i4] These materials would primarily be used in large scale fermentations. The economic implications of a fermentation medium on the profitability of a process have to be considered before fermentation process design can be started. Grade and quality information can be obtained for many materials from written sources such as the United States Pharmacopeia, Food Chemical Codex, The Merck Index, and suppliers’ catalogs. 152 Fermentation and Biochemical Engineering Handbook Table 12. Composition of Yeast Extract (Standard Grade) Constituent %wlw Moisture >5 as loss on dying Total nitrogen <7.0 Ash 10.1 pH of 2% solution 6.7 i 0.2 at 25" C (autoclaved for 2 min.) Coagulable no precipitate in 5% protein solution, boiling Chloride 0.19 Phosphorus 9.89 Sodium 0.32 Potassium 0.042 Iron 0.028 Calcium 0.04 Magnesium 0.030 Silicon dioxide 0.52 Manganese (ppm) 7.8 I&ad (PPm) 16 Constituent %wlw Arsenic (ppm) 0.1 1 Copper (PPm) 19.0 zinc (PPm) 8.8 AMINO ACIDS Lysine 4.0 Tryptophan 0.88 Phenylalanine Methionine Threonine Leucine Isoleucine Valine Arginine Tyrosine Aspartic Glutamine Glycine Histidine 2.2 0.79 3.4 3.6 2.9 3.4 0.78 0.6 5.1 6.5 2.4 0.94 Nutritional Requirements 153 Table 13. Composition of Various Hydrolyzed Proteins (%) Constituent Blood Meat Meat Casein Cottonseed Peptone Protein Protein Total nitrogen Amino nitrogen (as % of TN- Sorensen) AMINO ACIDS alanine arginine aspartic acid cystine glutamic acid glycine hydroxyproline histidine leucine isoleucine lysine methionine phenylalanine proline serine threonine tyrosine tryptophan valine 10.0 9.5 8.0 8.6 2.3 2.5 2.3 4.3 2.5 3.7 0.5 0.2 2.1 5.5 0.7 3.4 0.5 4.1 4.0 0.4 16.7 2.2 1.5 5.2 2.7 0.6 3.0 1.2 1.5 3.0 0.3 1.7 1.0 2.5 0.8 1.0 0.2 1.8 - - - - Form of material 65% solids 60% solids solution solution 9.7 10.5 - 3.8 3.9 0.4 5.7 - - 1.2 3.8 2.5 4.2 1.1 2.6 - - 2.1 0.2 2.6 60% solids - 13.5 30.0 2.6 3.7 5.7 0.3 20.1 1 .o 2.2 9.4 4.8 6.8 2.8 5.5 9.7 5.6 4.3 4.4 1.2 6.2 - dry solution powder 8.6 3 1.0 2.2 4.0 4.2 0.8 9.9 2.1 1.2 3.2 1.6 1.8 0.9 2.8 2.2 2.3 1.6 1.7 0.3 2.2 dry powder - 154 Fermentation and Biochemical Engineering Handbook Table 14. Typical Nutrient Composition of Distillers Feeds (Corn) From Corn Moisture % Protein % Lipid % Fiber % Ash % AMINO ACIDS mg/g Lysine Methionine Phenylalanine Cystine Histidine Tryptophan Arginine MINERALS K% Na % Ca % Mg % P% Fe, PPm Mn, PPm Zn, ppm cu, ppm 4.5 28.5 9.0 4.0 7.0 0.95 0.50 1.30 0.40 0.63 0.30 1.15 2.10 0.15 0.30 0.60 200.0 60.0 100.0 55.0 1.60 Nutritional Requirements 155 The usual fermentation process classification was in high-volume- low-cost products and low-volume-high-cost products. The first were carried out in fermenters with working volumes of 50,000 liters and up, the latter in fermenters of less than 50,000 liter working volumes. With the advent of biotechnology, the extremely-low-volume-extremely-high-price products entered the market. Vaccines, hormones, specialty enzymes and antibodies fall into this class. A working volume of 500 liters is considered large. Selections of raw materials are based upon these definitions of scale. Commercially prepared microbiological media are available for these fer- mentations. The variability and consistency of these prepared media are, of course, excellent and unlike that of the industrial raw materials. 9.0 EFFECT OF NUTRIENT CONCENTRATION ON GROWTH RATE When inoculating a fresh medium, the cells encounter an environmen- tal shock, which results in a lag phase. The length of this phase depends upon the type of organism, the age and size ofthe inoculum, any changes in nutrient composition, pH and temperature. When presented with a new nutrient the cell adapts itself to its new environment and normally produces the required enzyme. Essentially all nutrients can limit the fermentation rate by being present in concentrations that are either too low or too high. At low concentrations, the growth rate is roughly proportional to concentration, but as the concen- tration increases, the growth rate rises rapidly to a maximum value, which is maintained until the nutrient concentration reaches an inhibitory level, at which point the growth rate begins to fall again. The same type of hyperbolic curve will be obtained for all essential nutrients as the rate-limiting nutrient. The effects ofdifferent nutrients on growth rate can best be compared in terms of the concentrations that support a half-maximal rate of growth, this is the saturation constant (K,).[l3l For carbon and energy sources this concentra- tion is usually on the order of 10” to 1 Om6 M, which corresponds for glucose to a concentration between 20 and 200 ma. In general, K, for respiratory enzymes, those associated with sugar metabolism, is lower than K, for the hydrolytic enzymes, those associated with primary substrate attack. The Monod equation is frequently used to describe the stimulation of growth by the concentration of nutrients as given by: 156 Fermentation and Biochemical Engineering Handbook where, p = specific growth rate, h-' defined as (1/X) (dddt) pmax = maximum value of p, h-' K, = saturation constant, gL' at pmax/2 S = substrate concentration, gl-1 t = time,h The saturation constant K, for Saccharomyces cerevisiae on glucose is 25 ma, for Escherichia on lactose: 20 ma, and for Pseudomonas on methanol: 0.7 ma. Here, pmax is the maximum growth rate achievable when S >> K, and the concentration of all other essential nutrients is unchanged. The saturation constant K, is the approximate division between the lower concentration range where p is essentially linearly related to Sand the higher rate where p becomes independent of S,[gl The effect of excessive nutrient or product concentrations on growth is often expressed empirically as: where, Ki = inhibition, constant, gl-' I = concentration of inhibitor, gl-' Equations 1 and 2 can be combined to illustrate the characteristics common to many substrates: The kinetic models of Eqs. 1,2 and 3 are illustrated in dimensionless form in Fig. 2. It can be seen that the adding of large amounts of substrate to provide high concentrations ofproduct(s) and to overcome the rate-limiting effects of Eq. 1 can result in concentrations that are so high that the Nutritional Requirements 157 fermentation is limited by the effects of Eq. 2. The ideal operating range would be 1 < S/K, < 2 where the growth rate is near its maximum and is relatively insensitive to substrate The concentration ranges which enhance or inhibit fermentation activity vary with each microorgan- ism, chemical species, and growth conditions. 0.8 0.7 yprnax 0.6 0.5 0.4 0.3 0.2 0.1 0 !,no yibitor I' -1 0 1 2 3 4 5 6 7 S/K, for (A); S/K, for (C) with Ki = 1000 K,; I/Ki for (B). Figure 2. (A) Monod Growth Model; (5) model for growth inhibition; and (C) model for substrate activation and inhibition of growth. 158 Fermentation and Biochemical Engineering Handbook Product formation is related to the substrate consumption as follows: where, AP = product concentration - initial product concentration in 81-1. AS = Ygs = product yield, g-P.g-S-' substrate concentration - initial substrate concentration in g1-l This equation is especially useful when the substrate is a precursor for the product. Many other models are available. When calculating a material balance for medium formulation, the choice of models depends upon the data that are available. Ethanol-from-biomass fermentation is an example of product inhibi- tion, while high fermentable carbohydrate concentrations also can become inhibitive. Aiba and Shoda developed a mathematical model for the anaero- bic fermentation, where nine constants are required to describe the model.['3] dS la%' ldP+m +-- - = -- dt Y, dt Y, dt - PO sx ax dt - p K,+S 1+- KP - 90 sx dP dt -_ - - p K,'+S 1+- KP where, S, P, X = concentration of substrate, product and cell mass, g1-l M = maintenance constant, g-S, g-X-' .h-' K,, <, = saturation constants, gl-' Nutritional Requirements 159 Kp , K i, = inhibition constants, gl-L 40 = maximum specific product formation at P = 0, g-P.g-s-1 t = time,h PO = maximum specific growth rate at P = 0, h1 = true growth constant, g-X.g-S-’ YG YP = product yield, g-P.g-X-’ It is doubtful that cellular models of any greater complexity will have much utility in soluble substrate fermentations .[’I However, another level of complexity may be warranted in insoluble substrate fermentations such as hydrocarbons and cellulose. Immobilized microorganisms and enzymes are becoming commer- cially available. One example is co-immobilized yeast and glucoamylase (Gist Brocades NV), which is used to simultaneously saccharify starch dextrins into glucose and ferment this to ethanol in fluidized bed reactors. In these fluidized bed fermenters, the “reaction rate” is controlled by the superficial flow velocity and its effects on the diffusion of substrate from the bulk of the medium to the enzymatically active surface, by the enzymatic reaction at the surface, or by diffusion of the reactant products back into the bulk of the medium being fermented. REFERENCES 1. 2. 3. 4. 5. Stanier, R. Y., Doudoroff, M., and Adelberg, E. A., TheMicrobial World, Third Ed., Prentice Hall, Englewood Cliffs, NJ (1976) West, Robert, C., Handbook of Chemistry and Physics, 60th Ed., CRC Press, Boca Raton, Florida (1 979) Bennett, T. P. and Frieden, E., Modern Topics in Biochemistry, The MacMillan Company, New York (1967) Zabriski, D. 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