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Analysis of Biological System Despite of all their complexity, an understanding of biological system can be simplified by analyzing the system at several different levels: • the cell level: microbiology, cell biology; • the molecular level: biochemistry, molecular biology; • the population level: microbiology, ecology; • the production level: bioprocess. Biochemistry Introduction of the biological system at molecule level. This section is devoted mainly to the structure and functions of biological molecules. Outline of Biochemistry Section Contents-Cell construction • Protein and amino acids • Carbohydrates • Lipids, fats and steroids • Nucleic acids, RNA and DNA Requirements: Understand the basic definitions, characteristics and functions of these biochemicals. Amino acids and proteins Proteins are the most abundant molecules in living cells, constituting 40% - 70% of their dry weight. Proteins are built from α amino acid monomers. Amino acid is any molecule that contains both basic amino and acidic carboxylic acid functional groups. Amino acid H H2N α C COOH R Where "R" represents a side chain specific to each amino acid. Amino acids are usually classified by properties of the side chain into four groups: acidic, basic, hydrophilic (polar), and hydrophobic (nonpolar). α-amino acid are amino acid in which the amino and carboxylate functionalities are attached to the same carbon, the so-called α– carbon. They are the building blacks of proteins. Amino acid Zwitterion is an amino acid having positively and negatively charged groups, a dipolar molecule. H H3N+ C H -H+ COOH H3N+ C +H+ R H -H+ COO- H2N C +H+ R Zwitterion R COO- Amino acid Isoelectric point (IEP) is the pH value at which amino acids have no net charge. IEP varies depending on the R group of amino acids. At IEP, an amino acid does not migrate under the influence of an electric field. Amino acid pH effect on the charge of amino acids We can arbitrarily control the pH of an aqueous solution containing amino acids by adding base or acid. The equilibrium reactions for the simple amino acid (HA) are ] HAH+ = H++AH (1) HA = H++A- (2) Amino acid pH effect on the charge of amino acids The proton dissociation constants are K1, K2 [ A ][ H ] K1 [ HA] (3) [ HA][ H ] K2 [ HAH ] (4) [ ] represents concentration in dilute solution. Amino acid pH effect on the charge of amino acids Taking the logs of equations 3 and 4, yields, [ HA] pH pK1 log [ HAH ] [ A ] pH pK 2 log [ HA] (5) (6) where pH=-log(H+), pK1=-log(K1), and pK2=-log(K2). Amino acid pH effect on the charge of amino acids At Isoelectric point (IEP), [HAH+] = [A-] (7) Equation 5 plus 6 yields pI pH ( pK1 pK2) (8) pI is the pH at the isoelectric point for specific amino acid or protein. If R contains acid or base group, IEP is affected by the such groups. Amino acid pH effect on the charge of amino acids According to amino acid mass balance, the initial amino acid concentration is [HA]0 [HA]0 = [HA] + [A-] + [HAH+] (9) Combining equations 3, 4 and 9, the concentration of amino acids in neutral form [HA], negatively charged form [A-] and positively charged form [HAH+] can be calculated at specific known pH and [HA]0 . Amino acid Isomerism Most amino acids occur in two possible optical isomers, called D and L. •The L amino acids represent the vast majority of amino acids found in proteins. Standard amino acids: there are 20 standard amino acids that are commonly found in proteins. Amino Acids Essential amino acids: An essential amino acid for an organism is an amino acid that cannot be synthesized by the organism from other available resources, and therefore must be supplied as part of its diet. Most of the pants and microorganism cells are able to use inorganic compounds to make amino acids necessary for the normal growth. Eight amino acids are generally regarded as essential for humans: tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine, isoleucine. Two others, histidine and arginine are essential only in children. A good mnemonic device for remembering these is "Private Tim Hall", abbreviated as: PVT TIM HALL: Phenylalanine, Valine, Tryptophan Threonine, Isoleucine, Methionine Histidine, Arginine, Lysine, Leucine limiting amino acid content: the essential amino acid found in the smallest quantity in the foodstuff. Protein source Limiting amino acid Wheat lysine Rice lysine and threonine Maize lysine and tryptophan Pulses methionine Beef methionine and cysteine Whey none Milk none Use of amino acids • Aspartame (aspartyl-phenylalanine-1-methyl ester) is an artificial sweetener. • 5-HTP (5-hydroxytryptophan) has been used to treat neurological problems associated with PKU (phenylketonuria), as well as depression. • L-DOPA (L-dihydroxyphenylalanine) is a drug used to treat Parkinsonism. • Monosodium glutamate is a food additive to enhance flavor. Amino Acid (AA)-Protein Amino acids: basic unit Peptides: amino acid chain, containing 2 or more AA. Polypeptides: containing less than 50 AA. Protein: > 50 AA. Peptides (from the Greek πεπτος, "digestible"), are formed through condensation of amino acids through peptide bonds. Peptide bond: a chemical bond formed between two AA - the carboxyl group of one amino acid reacts with - the amino group of the other amino acid, - releasing a molecule of water (H2O). This is a condensation (also called dehydration synthesis) reaction. Protein • Proteins are the polymers built through the condensation of amino acids. amphoteric, isoelectric point (protein recovery) • Protein constitutes 40-70% dry weight of cell. Its molecular weight is from 6000 to several hundred thousand daltons. Dalton is a unit of mass equivalent to a hydrogen atom, 1 dalton = 1.66053886 × 10−27 kg. • prosthetic groups: organic or inorganic components other than amino acids contained in many proteins. • conjugated proteins: the proteins contain prosthetic groups. Conjugated protein: hemoglobin Prosthetic group: heme in green Amino acid units in red and yellow Heme group Protein Proteins are essential to the structure and function of all living cells and viruses. They can be classified into: - structural proteins: glycoprotein - catalytic proteins: enzymes - transport proteins: hemoglobin - regulatory proteins: hormones (insulin, growth hormone) - protective proteins: antibodies Protein 3-D structure Proteins are amino acid chains that fold into unique 3-dimensional structures. The shape into which a protein naturally folds is known as its native state, which is determined by its sequence of amino acids and interaction of groups. Protein structure The three-dimensional structure can be described at four distinct levels: Primary structure: the amino acid sequence - It is held together by covalent peptide bonds - Each protein has not only a definite amino acids composition, but also a unique sequence. - The amino acid sequence has profound effect on the resulting three-dimensional structure and on the function of protein. Protein structure Secondary structure: highly patterned sub-structures α-helix and β-pleated sheet • It is the way that the polypeptide chain is extended and is a result of hydrogen bonds between protein residues. • Secondary structures are locally defined, meaning that there can be many different secondary motifs present in one single protein molecule. • Two major types of secondary structure are α-helix and β-pleated sheet. Protein structure Secondary structure: α-helix - Formed within the same protein chain. - Hydrogen bonding can occur between - the α-carboxyl group of one residue and - the –NH group of its neighbor four units down the same chain. - The helical structure can be easily disturbed since hydrogen bond is unstable. Protein structure Secondary structure: β-pleated sheet - within the same protein molecule - consists of two or more amino acid sequences that are arranged adjacently and in parallel, but with alternating orientation -Hydrogen bonds can form between the two strands. -Hydrogen bonds established between the N-H groups in the backbone of one strand with the C=O groups in the backbone of the adjacent, parallel strand(s). - The sheet's stability and structural rigidity and integrity are the result of multiple such hydrogen bonds arranged in this way. Protein structure Tertiary structure: the overall shape of a single protein molecule • The tertiary structure is a result of interaction between R groups widely separated along the chain. The folding or bending of an amino acids chain induced by interaction of R groups determines the tertiary structure. • It is held together primarily by hydrophobic interactions but hydrogen bonds, ionic interactions, and disulfide bonds are usually involved too. • The tertiary structure has a profound effect on its function. Protein structure Quaternary structure: the shape or structure that results from the union of more than one protein molecule, which function as part of the larger assembly or protein complex. • Only protein with more than one polypeptide chain has quaternary structure. This structure has an important role in the control of their catalytic activity. • these tertiary or quaternary structures are usually referred to as "conformations," or “folding” and transitions between them are called conformational changes. • The mechanism of protein folding is not entirely understood. Protein Denaturation Protein Denaturation: A protein that is not in its native state and their shape which allows for optimal activity. • Proteins denature when they lose their three-dimensional structure their chemical conformation and thus their characteristic folded structure. • Proteins may be denatured at the secondary, tertiary and quaternary structural levels, but not at the primary structural level. • This change is usually caused by heat, acids, bases, detergents, alcohols, heavy metal salts, reducing agents or certain chemicals such as urea. • The proteins can regain their native state when the denaturing influence is removed. Such denature is reversible. Some other denature is irreversible.- direct purification processes. Irreversible egg protein denaturation and loss of solubility, caused by the high temperature (while cooking it) Summary of amino acids and protein • Amino acids are basic building blocks of proteins. • They contain acid carboxyl group and base amino group as well as side group R. • They can be neutral, positively or negatively charged. • They are 21 basic amino acid and 10 essential amino acids for human being. Summary of amino acids and protein • Proteins are amino acid chain linked through peptide bond. • They can be classified into structural protein, catalytic protein, transport protein , regulatory and protective proteins in either globular or fibrous forms. Summary of amino acids and protein • Protein has three-dimensional structure at four level. - Primary structure: the sequence of amino acids. - Secondary structure: a way that the polypeptide chain is extended. α-helix and β-pleated sheet formed by hydrogen bond. - Tertiary structure: the overall shape of a protein molecule and the result of interaction between R groups mainly through hydrophobic interaction. - Quaternary: the interaction between different polypeptide chains of protein. This structure is important to the active function of protein especially enzyme. • Protein can be denatured at its three dimensional structure. Protein denature could be reversible or irreversible. Carbohydrates Carbohydrates: • Carbohydrates (monosaccharides) are represented by the general formula (CH2O)n, where n≥3 and are synthesized from carbon dioxide and water through photosynthesis. • Certain carbohydrates are an important storage and transport form of energy in most organisms. • Carbohydrates are classified by the number of sugar units – – – – monosaccharides (such as glucose), disaccharides (such as maltose), Oligosaccharides (fructo-oligosaccharides), and polysaccharides (such as starch, glycogen, cellulose, and chitin). Carbohydrates • Monosaccharides are the simplest form of carbohydrates containing three to nine carbon atom. They consist of one sugar and are usually colorless, water-soluble, crystalline solids. • Monosaccharides are either aldehydes or ketones with many hydroxyl groups added, usually one on each carbon except the functional group. • Imoportant monosaccharides include glucose, ribose and deoxyribose. Glucose Glucose as a straight chain Glc in ring structure Glucose • Glucose (Glc) is one of the main products of photosynthesis and starts cellular respiration. • The cell uses it as a source of energy and metabolic intermediate. Glucose is the source for glycosis and citric acid cycle in metabolic pathway. • The natural form (D-glucose) is also referred to as dextrose, especially in the food industry. D-glucose is in the form of a ring (pyranose) structure. The L-form plays a minor role in biological systems. • Glc is produced commercially via the enzymatic hydrolysis of starch. D-ribose and deoxyribose Ribose and deoxyribose are pentose containing five carbon ring-structure sugar molecules D-ribose deoxyribose D-ribose and Deoxyribose • D-ribose is a component of the ribonucleic acid (RNA) that plays central role for protein synthesis. • Ribose is critical to living creatures. It is also a component of adenosine triphosphate (ATP), and nicotinamide adenine dinucleotide (NAD), that are critical to metabolism. • Deoxyribose is a component of DNA that is important genetic material. Disaccharides Disaccharides are formed by the condensation of two monosaccharides via 1, 4-glycosidic linkage. Maltose Disaccharides Common disaccharides: - sucrose (known as "table sugar", "cane sugar", "saccharose" or "beet sugar") , - lactose (milk sugar) - maltose produced during the malting of barley. Oligosaccharides Oligosaccharides refer to a short chain of sugar molecules - Fructo-oligosaccharides (FOS), which are found in many vegetables, consist of short chains of fructose molecules. - Galacto-oligosaccharides (GOS), which also occur naturally, consist of short chains of galactose molecules. Polysaccharides Polysaccharides are formed by the condensation of more than two monosaccharides by glycosidic bonds. • Polysaccharides have a general formula of Cn(H2O)n-1 where n is usually a large number between 200 and 500. • They are very large, often branched, molecules. • They tend to be amorphous, insoluble in water, and have no sweet taste. • When all the constituent monosaccharides are of the same type they are termed homopolysaccharides; when more than one type of monosaccharide is present they are termed heteropolysaccharides. • Examples include storage polysaccharides such as starch and glycogen and structural polysaccharides such as cellulose and chitin. Polysaccharides-starch Starch is a combination of two polysaccharides called amylose and amylopectin. • Amylose is constituted by glucose monomer units joined to one another head-to-tail forming alpha-1,4 linkages. • Amylopectin differs from amylose in that branching occurs, with an alpha-1,6 linkage every 24-30 glucose monomer units. • In general, starches have the formula (C6H10O5)n, where "n" denotes the total number of glucose monomer units. Polysaccharides-starch • Starches are insoluble in water. • They can be digested by hydrolysis, catalyzed by enzymes called amylases, which can break the glycosidic bonds between the 'alpha-glucose' components of the starch. • The four major resources for starch production and consumption in the USA are corn, potatoes, rice, and wheat. • Dietary sources of starch are pasta and bread. Polysaccharides-glycogen • Glycogen is storage form of glucose in animal cells. • Glycogen is a highly branched polymer of 10,000 to 120,000 Glc residues and molecular weight between 106 and 107 daltons. • Most of Glc units are linked by a α-1,4 glycosidic bonds, • approximately 1 in 12 Glc residues also makes a α-1,6 glycosidic bond with a second Glc which results in creating of a branch. Polysaccharide-Cellulose • Cellulose (C6H10O5)n is a long-chain polysaccharide of beta-glucose. • The molecule weight is between 50,000 to 1 million daltons. • The linkage between glucose monomer in cellulose is β-1,4 glycosidic linkage. • It forms the primary structural component of plants and is not digestible by humans. Only a few microorganism can hydrolyze enzyme. Chitin: poly [b - (1, 4) - 2 - acetamido - 2 - deoxi - D glucopyranose ] H H CH2OH O H H HN C O CH3 CH3 C OH HN O H H O H CH2OH O n N-acetylation degree of chitin, i.e. percentage of acetylated amine (amide) 78 10 % Chitin structure •Chitin is important structural polysaccharides in the cell wall of microorganisms and animal shells. •Chitin can be obtained from fungi, insect, lobster, shrimp and krill, but the most important commercial sources are the exoskeletons of crabs obtained as waste from seafood industrial processing. Mangrove crab: Ucides cordatus Steamed Crab Crab Cake Acid washed crab shells (Niu and Volesky, 2000, JCTB). Au Au uptake (mmol/g) 0.25 0.2 Chitin amide: pKa < 3.5 Cl- interference pH 3.4 0.15 pH 2.4 0.1 pH 4.5 0.05 0 0 0.5 1 1.5 2 2.5 3 Equilibrium Au concentration (m m ol/L) Effect of pH (Niu and Volesky, 2003, Hydrometallurgy). Summary of Carbohydrates Carbohydrates are the energy sources for cell living. • Carbohydrates include monosaccharide, disaccharide, and polysaccharides. • Important monosaccharides are glucose and ribose. - Glucose is the energy source for cell metabolism - Ribose is the unit for forming nucleotides and nucleic acid. • Important polysaccharides are storage starch, glycogen, and structural cellulose and chitin. Lipids, fats and steroids Lipids, fats and steroids • Lipids are hydrophobic biological compounds that are insoluble in water, but soluble in nonpolar solvent such as benze, chloroform and ether. • They are present in the nonaqueous biological phase such as plasma membrane. • Cells can alter the mix of lipids in their membrane to compensate for changes in temperature or to increase their tolerance to the presence of chemical agents such as ethanol. Lipids fatty acids : The major component in most lipids made of a straight chain of hydrophobic hydrocarbon group, with a carboxyl group (hydrophilic) at the end. • A typical saturated fatty acid has the form of CH3-(CH2)n –COOH Where n is typically between 12 and 20, such as acetic acid CH3COOH. • A typical unsaturated fatty acid contain double –C=C- , or triple bonds on the hydrocarbon chain, such as Oleic acids: CH3-(CH2)7-HC=CH-(CH2)7-COOH Fats Fats are lipids that are esters of fatty acids with glycerol. glycerol Fatty acids fat Fats • Fats play a vital role in maintaining healthy skin and hair, insulating body organs against shock, maintaining body temperature, and promoting healthy cell function. • They also serve as energy stores for the body and can serve as biological fuel-storage molecules. • In food, there are two types of fats: saturated and unsaturated. • Fats are broken down in the body to release glycerol and free fatty acids. glycerol can be converted to glucose by the liver and thus used as a source of energy. • The fatty acids are a good source of energy for many tissues, especially heart and skeletal muscle. Phospholipids • Phospholipids such as glycerophospholipids are built on a glycerol core to which are linked two fatty acid-derived "tails" by ester linkages and one "head" group by a phosphate ester linkage. Phospholipids are key components to control the entry or exit of molecules in the cell membrane. Steroids A steroid is a lipid characterized by a carbon skeleton with four fused rings. Different steroids vary in the functional groups attached to these rings. Steroids • Hundreds of distinct steroids have been identified in plants and animals. • Their most important role in most living systems is as hormones-regulate the cell metabolism. • In human physiology and medicine, the most important steroids are cholesterol functioning chiefly as a protective agent in the skin and nerve cells, a detoxifier in the bloodstream, and as a precursor of many steroids. Summary of lipids Lipids are energy storage in cell membrane and regulators of cell metabolism. - fat, phospholipids and steroids. - Important components in cell membrane to compensate for changes in temperature or increase the cell tolerance for some chemicals. Nucleic acids, RNA and DNA Nucleic acid is a complex, high-molecular-weight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are found in all living cells and viruses. Nucleotides Nucleotides are the building blocks of DNA and RNA. • Serve as molecules to store energy and reducing power. • The three major components in all nucleotides are phosphoric acid, pentose (ribose and deoxyribose), and a base (purine or purimidine). • Two major purines present in nucleotides are adenine (A) and guanine (G), and three major purimidines are thymine (T), cytosine (C) and uracil (U). Important Ribonucleotides • Adenosine triphosphate (ATP) and guanosine triphosphate (GTP), which are the major sources of energy for cell work. - The phosphate bonds in ATP and GTP are high-energy bonds. - The formation of phosphate bonds or their hydrolysis is the primary means by which cellular energy is stored or used. • nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The two most common carriers of reducing power for biological oxidation-reduction reactions. Deoxyribonucleic acid (DNA) Deoxyribonucleic acid (DNA) is formed by condensation of deoxyribonucleotides . 3 The nucleotides are linked together between the 3’ and 5’ carbons’ successive pentose rings by phosphodiester bonds 5 Deoxyribonucleic acid (DNA) - DNA is a very large threadlike macromolecule (MW, 2X109 D in E. coli). - DNA contains adenine (A) and guanine (G), thymine (T) and cytosine (C). - DNA molecules are two stranded and have a double-helical three-dimensional structure. DNA double-helical structure Double helical DNA structure The main features of double helical DNA structure are as follows: . - The phosphate and deoxyribose units are on the outer surface, but the bases point toward the chain center. The plane of the bases are perpendicular to the helix axis. - The diameter of the helix is 2 nm, the helical structure repeats after ten residues on each chain, at an interval of 3.4 nm. - The two chains are held together by hydrogen bonding between pairs of bases. Adenine (A) - thymine (T), guanines (G) - cytosine (C). - The sequence of bases along a DNA strand is not restricted in any way and carries genetic information, and sugar and phosphate groups perform a structure role. DNA “Genetic code is the relation between the sequence of bases in DNA (or its RNA transcripts ) and the sequence of amino acids in protein.” (Biochemistry, Lubert Stryer, 1988) - Codon refers to a sequence of three bases on a mRNA. - There are maximum 64 codons. - These codons, when expressed, represent a particular amino acid or “stop” signal for protein synthesis. DNA e.g. -CGCCGCTGC-GCGGCGACG-CGCCGCUGCarg arg sys mRNA DNA - The sequence of the codons determines the sequence of amino acids for a protein synthesis. - Some other combinations of codons regulate when the gene is expressed. Gene: each sequence of codons generating a unique protein. A DNA molecule contains lots of genes. DNA Replication Regeneration of DNA from original DNA segments. DNA Replication - DNA helix unzips and forms two separate strands. - Each strand will form a new double strands. - The two resulting double strands are identical, and each of them consists of one original and one newly synthesized strand. - This is called semiconservative replication. - The base sequences of the new strand are complementary to that of the parent strand. Ribonucleic acid (RNA) • Ribonucleic acid (RNA) is formed by condensation of ribonucleotides. • RNA is a long, unbranched macromolecule and may contain 70 to several thousand nucleotides. RNA molecule is usually single stranded. • RNA contains adenine (A), guanine (G), cytosine (C) and uracial (U). A-U, G-C in some double helical regions of t-RNA. Classification of RNA According to the function of RNA, it can be classified as: • Messenger RNA: (m-RNA) synthesized on chromosome and carries genetic information to the ribosomes for protein synthesis. It has short half-life. • Transfer RNA (t-RNA) is a relatively small and stable molecule that carries a specific amino acid from the cytoplasm to the site of protein synthesis on ribosomes. • Ribosomal RNA (r-RNA) is the major component of ribosomes, constituting nearly 65%. r-RNA is responsible for protein synthesis. • Ribozymes are RNA molecules that have catalytic properties. Summary of Cell Construction Cells contain biologically important chemicals such as protein, carbohydrates, lipid and nucleic acids. Protein • Proteins are amino acid chain linked through peptide bond. • They can be classified into structural protein, catalytic protein, transport protein and protective proteins in either globular or fibrous forms. Summary of Cell Construction Protein • Protein has three-dimensional structure at four level. - The primary structure is determined by the sequence of amino acids. It is held together by peptide bonds. - The secondary structure is a way that the polypeptide chain is extended, including α-helix and β-pleated sheet formed by hydrogen bonds. - The tertiary structure is the overall shape of a protein molecule, formed by the hydrophobic interaction of R chain. - Interaction between different polypeptide chains. Only protein with more than one polypeptide chain has quaternary structure. • Protein can be denatured at its three dimensional structure. Protein denature could be reversible or irreversible. Summary of Cell Construction Carbohydrates are the energy sources for cell living. • Carbohydrates include monosaccharide, disaccharide, and polysaccharides. • Important monosaccharides are glucose and ribose. - Glucose is the energy source for cell metabolism - Ribose is the unit for forming nucleotides and nucleic acid. • Polysaccharides are made of monosaccharides through glycosidic bonds. Summary of Cell Construction Lipids: fats, phospholipids and steroids • Lipids are hydrophobic biological compounds that are insoluble in water. • They are present in the nonaqueous biological phase such as plasma membrane. • Cells can alter the mix of lipids in their membrane to compensate for changes in temperature or to increase their tolerance to the presence of chemical agents. • Steroids are regulators. Summary of Cell Construction Nucleotides are basic units of nucleic acids DNA and RNA. • Nucleotides include pentose, base and phosphoric acid. • Bases include purine or pyrimidine. • Two major purines present in nucleotides are adenine (A) and guanine (G), and three major pyrimidines are thymine (T), cytosine (C) and uracil (U). • Ribonucleotides - adenine triphosphate (ATP) stores energy. - NAD and NADP are important carriers of reducing power. Summary of Cell Construction DNA • DNA contains genetic information. • DNA contains adenine (A) and guanine (G), and thymine (T), and cytosine (C). A-T G-C • DNA has a double helical structure. • The bases in DNA carry the genetic information. Summary of Cell Construction RNA • RNA functions as genetic information-carrying intermediates in protein synthesis. • It contains adenine (A) and guanine (G), and cytosine (C) and uracil (U). • m-RNA carries genetic information from DNA to the ribosomes for protein synthesis. • t-RNA transfers amino acid to the site of protein synthesis • r-RNA is for protein synthesis. Cell Nutrients Nutrients required by cells can be classified in two categories: - Macronutrients are needed in concentrations larger than 10-4 M. C, N, O, H, S, P, Mg 2+, and K+. - Micronutrients are needed in concentrations less than 10-4 M. Mo, Zn, Cu, Mn, Ca, Na, vitamins, growth hormones and metabolic precursors. Cell Nutrients- Macronutrients Carbon compounds are the major sources of cellular carbon and energy. • Heterotrophs use organic carbon sources such as carbohydrates, lipid, hydrocarbon as a carbon source. • Autotrophs can use carbon dioxide as a carbon source. They can form carbohydrate through light or chemical oxidation. • In aerobic fermentations, about 50% of substrate carbon is incorporated into cell mass and about 50% of it is used as energy sources. • In anaerobic fermentation, a large fraction of substrate carbon is converted to products and a smaller fraction is converted to cell mass (less than 30%). Cell Nutrients- Macronutrients Carbon sources: - In industrial fermentation, the most common carbon sources are molasses (sucrose), starch (glucose, dextrin), corn syrup, and waste sulfite liquor (glucose). - In laboratory fermentations, glucose, sucrose and fructose are the most common carbon sources. Ethanol, methanol and methane also constitute cheap carbon sources. Cell Nutrients- Macronutrients Nitrogen compounds are important sources for synthesizing protein, nucleic acid. • Nitrogen constitutes 10% to 14% of cell dry weight. • The most commonly used nitrogen sources are ammonia or ammonium salts such as ammonium chloride, sulfate and nitrate, protein, peptides, and amino acids. Urea can be cheap source. • In industrial fermentation, nitrogen sources commonly used are soya meal, yeast extract, distillers solubles, dry blood and corn steep liquor. Cell Nutrients- Macronutrients Oxygen constitutes about 20% of the cell dry weight. - Molecular oxygen is required as terminal electron acceptor in the aerobic metabolism of carbon compounds. - Gaseous oxygen is introduced into growth media by sparging air or by surface aeration. - Improving the mass transfer of oxygen in a bioreactor is a challenge in reactor control. Cell Nutrients- Macronutrients Hydrogen: 8% of dry cell weight source: carbohydrates. Phosphorus: 3% of cell dry weight - present in nucleic acids and in the cell wall of some gram-positive bacteria. - a key element in the regulation of cell metabolism. - sources: Inorganic phosphates. The phosphate level should be less than 1 mM for the formation of many secondary metabolites such as antibiotics. Cell Nutrients- Macronutrients • Sulfur: 1% of cell dry weight - present in protein and some coenzymes. - source: Ammonium sulfate, Sulfur containing amino acids such as cysteine some autotrophs can use S0 and S2+ as energy sources. • Potassium: a cofactor for some enzyme and is required in carbohydrate metabolism. cofactor: any of various substances necessary to the function of an enzyme, such as metal ions. - source: potassium phosphates. • Magnesium: a cofactor for some enzyme and is present in cell walls and membranes. Ribosomes specifically requires Mg2+ . - sources: Magnesium sulfate or chloride Cell Nutrients- Micronutrients Micronutrients could be classified into the following categories (required less than 10-4 M): - most widely needed elements. - trace elements needed under specific growth conditions . - Trace elements rarely require. - Growth factor. Cell Nutrients- Micronutrients Micronutrients could be classified into the following categories: - most widely needed elements are Fe, Zn and Mn. Such elements are cofactors for some enzyme and regulate the metabolism. - trace elements needed under specific growth conditions are Cu, Co, Mo, Ca, Na, Cl, Ni, and Se. For example, copper is present in certain respiratory-chain components and enzymes. Cell Nutrients- Micronutrients -Trace elements rarely required are B, Al, Si, Cr, V, Sn, Be, F, Ti, Ga, Ge, Br, Zr, W, Li and I. These elements are required in concentrations of less than 10-6M and are toxic at high concentration. - Growth factor is also micronutrient. Growth factor stimulates the growth and synthesis of some metabolites. e.g. Vitamin, hormones and amino acids. They are required less than 10-6M. Nutrients for S. cerevisia ethanol production glucose (40g/L), NH4Cl (1.32 g/L), MgS04.7H2O (0.11 g/L), CaCl2.2H2O (0.08 g/L), K2HPO4 (2.0 g/L). Growth medium There are two types of growth medium: defined medium and complex medium. Defined medium contains specific amounts of pure chemical compounds with known chemical compositions. glucose (40g/L), NH4Cl (1.32 g/L), MgS04.7H2O (0.11 g/L), CaCl2.2H2O (0.08 g/L), K2HPO4 (2.0 g/L). Defined medium - the results are more reproducible and the operator has better control of the fermentation. - the recovery and purification processes are easier and cheaper. Growth medium Complex medium contains natural compounds whose chemical composition is not exactly known. - yeast extract, peptone, molasses or corn steep. - high yields: providing necessary growth factor vitamins, hormones and trace metals. - Complex media is less expensive than defined media. glucose (40g/L), yeast extract, NH4Cl (1.32 g/L), MgS04.7H2O (0.11 g/L), CaCl2.2H2O (0.08 g/L), K2HPO4 (2.0 g/L). Summary of Cell Nutrients Nutrients that required by cell living can be categorized into macronutrient that are required higher than 10-4M, micronutrients that less than 10-4M. Macronutrients include N, C, O, H, S, P, K and Mg. They are major components in cell dry weight. Micronutrients are classified into most widely needed elements, needed under specific conditions and rarely needed one. Growth medium can be either defined or complex. Summary of cell construction Biopolymers subunit bonds for subunit linkage functions Characteristic three-D structure protein Carbohydrates (polysaccharides) DNA RNA lipids