Nutritional Requirements 127 Table 1. Physiological functions of the principal elements J (31 Element Symbol Atomic Physiological function Constituent of cellular water C Constituent of organic cell materials Constituent of proteins, nucleic acids and coenzymes Oxygen Constituent of cellular water and electron acceptor in respiration of aerobes Important divalent cellular cation, enzymatic reactions, incl. those involving ATP; functions in binding enzymes to substrates and present in chlorophylls Phosphon P Constituent of phospholipids, 16 Constituent of cysteine, cystine, methionine and proteins CoA and cocarboxylase Chlorine Principal intracellular and extracellular anion Potassium K 19 Principal intracellular cation, cofactor for some enzymes Calcium Important cellular cation, cofactor for enzymes as proteinase Inorganic cofactor Iron Constituent of cytochromes nd other heme or non-heme proteins, cofactor for a number of Cobalt Constituent of vitamin B,2 and Copper Z1 30 Inorganic constituents of Molybdenum Mo special enzymes
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, O 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 [1 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 medi can be easily reproduced, have low foaming tendency, show translucency and allow easy product recovery and purification Complex or natural media such as molasses, corm 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 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 4I Fermentation nutrients are generally classified as: sources of carbon, nitrogen and sulfur, 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 cO2 and organic compounds, the typical end-products of fermentation metabolism). Many microorganisms can use a single organic compound to
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 researching 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 equired as precursors of certain organic cell constituents that the organism is unable to synthesize. Most microorganisms that depend on organic carbon sources also require Co2 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(.5%v/v)remained essentially the same, while the glycerol concentration went from 8.3 g/l to 11.9 g/l. The CO, formation, however, was reduced! With glycerol levels over 12 g/l, the ethanol yield and concentration reduced with the CO2-formation near normal again. 15)In fermentations, the carbon source on a unit of weight basis may be the least expensiveraw material, however, quite often represents the largest single cost forraw material due to the levels required. Facultative organisms incorporate oughly 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. If this is supplied as the hexose glucose, with molecular weight 180 and carbon weight 72, then(80) 180)/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 production of ATP. The progressive breakdown of polysaccharides and disaccharides to simpler sugars is a major source of energy-rich compounds. a 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
Nutritional Requirements 129 supply both carbon and energy needs. Others need a variable number of additional organic compounds as nutrients. These additional organic nutrients 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 fermentation 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. Carbohydrates 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:
130 Fermentation and Biochemical engineering Handbook The Embden-Meyerhof pathway(EMP) he Krebs or tricarboxylic acid cycle(tCa) hosphate pathway(Ppp) In the EMP, glucose is anaerobically converted to pyruvic acid and or 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. 31 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. II The TCA-cycle functions to convert pyruvic and lactic acids, the end products of anaerobic glycolysis(EMP), to CO2 and H2O. 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 2C3H4O3+502+30ADP+30P1→6C02+4H2O+30ATP for pyruvic acid as the starting material. 31 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(nibose-5 phosphate)and fatty acid biosynthesis(NADPH2 ). The Entner-Doudoroff hway catabolizes glucose into pyruvate and glyceraldehyde- 3-phosphate It is important primarily in Gram negative prokaryotes. 161 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 lucose-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 anabolism. 3IS cerevisiae ferments galactose and maltose occasionally, but slowly; inulin very poorly; raffinose only to theextent of one
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 third and melibiose and lactose it will not ferment. S. cerevisiae follows the Embden-Meyerhof pathway and produces besideethanol, 2 moles of ATP per Table 2. Fundamental Biological Molecules!31 simple molecule Constituent Derived macro- oecules. CH-OH c,H,0 ellul grate HSCH2 CH(NH2)Cool C,N,H,O, s steine(amino acid proteins NH2(cH2 )4 CH(NH2)COoH N,H,0 Lysine (asic aming acid CH3(CH2)14cO0H C,H,O fats and ils a C,N,H, O Adenine (purine) and C,N,H,O Carbohydrates, fats, proteins, nucleic ad cids Kinetic energy fo biological processes Energy potential [catabolism anabol⊥sm o2, H2o, simple N, s and P containing compounds
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 macroatoms 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