Nutritional Requirements in Fermentation Processes willem i 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, mold and yeast, normally), but the species and strains become very specific as 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 quirements for specific growth factors. Phototrophs use light as an energy source and chemotrophs use chemical energy sources 122
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 biosynthesis 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 classification 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 requirements for specific growth factors. Phototrophs use light as an energy source and chemotrophs use chemical energy sources. ” I22
Nutritional Requirements 123 Organisms that use CO2 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 CO2 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 CO2 as a principal carbon source. The use of CO 2 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(NH3, NO,), ferrous iron, reduced sulfur compounds (HS,S,S2O32), or H, as oxidizable energy sources Chemoheterotrophic organisms are also dependent on chemical energ 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. Thi 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 O2), 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 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
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]
124 Fermentation and Biochemical Engineering Handbook Compounds that serve as energy carriers for the chemotrophs, linkin catabolic and biosynthetic phases of metabolism, are adenosine phosphate and reduced pyridine nucleotides(such as nicotinamide dinucleotide NAD). The structure of adenosine triphosphate(ATP)is shown in Fig. l.It contains two energy-rich bonds, which upon hydrolysis, yield nearly eight kcal/mole for each bond broken. AtP is thus reduced to the diphosphate (ADP)or the monophosphate(AMP)form 六o-P~0-P-0 A ADP L ATP(adenosine triphosphate) Figure 1. Chemical structure of ATP, which contains two energy-rich bonds. When ATP yields ADP, the Gibbs free energy change is- 7.3 kcal/kg at 37.C and pH7. Plants and animals can use the conserved energy of ATP and other substances to carry out their energy requiring processes, i.e., skeletal muscle contractions, etc. When the energy in ATPis used, a coupled reaction occurs AtP is thus hydrolyzed Adenoisine-(-O adenosine.①①+HO⑨+ Energy hydrolysis (ADP where- is an energy-rich bond and-( terminally represents -p OH and P-internally
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 +ATP→ glucose6 phosphate+AD②J the bond energy being preserved in the new ormed molecule, e. g, glucose Mammalian skeleton muscle at rest contains 350-400 mg ATPper 100 g. ATP inhibits enzymatic browning of raw edible plant materials, such as sliced apples 2.0 NUTRITIONAL REQUIREMENTS OF THE CELL Besides a source of energy, organisms require a source of materials for biosynthesis of cellular matter and products in cell operation, maintenance and reproduction. These materials must supply all the elements necessary to ccomplish 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 of more complex molecules, such as lipoproteins and liposaccharides. Lipids also serve as the polymeric biological fuel storag Natural membranes are normally impermeable to highly charged chemical species such as phosphorylated compounds. This allows the cell te contain a reservoir of charged nutrients and metabolic intermediates, as well as maintaining a considerable difference between the internal and extemal concentrations of small cations such as h. K and Nat. Vitamins.EK and D are fat-soluble and water-insoluble. Sometimes they are also classified as lipids DNA contains all the cells 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 cleotides. rogenous base. RNA is a polymer of ribose-containing Of the nitrogenous bases, adenine, guanine, and cytosine are
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. I Prokaryotes contain one DNA molecule with a molecular veight on the order of 2 x 109. 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 o. Sulfur contributes to the three-dimensional stabilization of almost all proteins. Proteins show great diversity of biologi cal functions. The building blocks of proteins are the amino acids. The predominant chemical elements in living matter are: C, H, O, andN, 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. 2I Hydroger is the most abundant of all elements in the universe, and it is thought that th 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 21 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 respectively h13) 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. 1(31 Ash composes approximately 5 percent of the dry weight of biomass with phosphorus and sulfur accounting, for Na, Ca, Fe, Mn, Cu, Mo, Co, Zn and CI. is usually made up of Mg, K respectively 60 and 20 percent. The remainder
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 biological 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 microorganisms 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 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
