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