PART 1 Introduction
2.3. CELL NUTRIENTS 1. Introduction
The major function of DNA is to carry genetic information in its base sequence. The genetic information in DNA is transcribed by RNA molecules and translated in protein synthesis. The templates for RNA synthesis are DNA molecules, and RNA molecules are the templates for protein synthesis. The formation of RNA molecules from DNA is known as DNA transcription, and the formation of peptides and proteins from RNA is called translation.
Certain RNA molecules function as the genetic information-carrying intermediates in protein synthesis (messenger,m-RNA), whereas other RNA molecules [transfer(t-RNA) and ribosomal (r-RNA)] are part of the machinery of protein synthesis. The ribosomal r-RNA is located in ribosomes which are small particles made of protein and RNA. Ribo- somesare cytoplasmic organelles (usually attached on the inner surfaces of endoplasmic reticulum in eucaryotes) and are the sites of protein synthesis.
RNA is a long, unbranched macromolecule consisting of nucleotides joined by 3¢–5¢
phosphodiester bonds. An RNA molecule may contain from 70 to several thousand nu- cleotides. RNA molecules are usually single stranded, except some viral RNA. However, certain RNA molecules contain regions of double-helical structure, like hairpin loops.
Figure 2.19 describes the cloverleaf structure of t-RNA (transfer RNA). In double-helical regions of t-RNA, A pairs with U and G pairs with C. The RNA content of cells is usually two to six times higher than the DNA content.
Let us summarize the roles of each class of RNA species:
Messenger RNA(m-RNA) is synthesized on the chromosome and carries genetic in- formation from the chromosome for synthesis of a particular protein to the ribosomes.
The m-RNA molecule is a large one with a short half-life.
Transfer RNA(t-RNA) is a relatively small and stable molecule that carries a spe- cific amino acid from the cytoplasm to the site of protein synthesis on ribosomes. t-RNAs contain 70 to 90 nucleotides and have a MW range of 23 to 28 kD. Each one of 20 amino acids has at least one corresponding t-RNA.
Ribosomal RNA(r-RNA) is the major component of ribosomes, constituting nearly 65%. The remainder is various ribosomal proteins. Three distinct types of r-RNAs present in the E. coliribosome are specified as 23S, 16S, and 5S, respectively, on the basis of their sedimentation coefficients (determined in a centrifuge). The symbol S denotes a Svedberg unit. The molecular weights are 35 kD for 5S, 550 kD for 16S, and 1,100 kD for 23S.
These three r-RNAs differ in their base sequences and ratios. Eucaryotic cells have larger ribosomes and four different types of r-RNAs: 5S, 7S, 18S, and 28S. Ribosomal RNAs make up a large fraction of total RNA. In E. coli, about 85%of the total RNA is r-RNA, while t-RNA is about 12%and m-RNA is 2%to 3%.
2.3. CELL NUTRIENTS
fers so greatly in composition from its environment, it must expend energy to maintain itself away from thermodynamic equilibrium. Thermodynamic equilibrium and death are equivalent for a cell.
All organisms except viruses contain large amounts of water (about 80%). About 50%of dry weight of cells is protein, and the proteins are largely enzymes (proteins that act as catalysts). The nucleic acid content (which contains the genetic code and machinery
Figure 2.19. The structure of the transfer RNA (tRNA) molecule and the manner in which the anticodon of tRNA associates with the codon on mRNA by complementary base pairing. The amino acid corresponding to this codon (UUC) is phenylalanine which is bound to the opposite end of the tRNA molecule. Many tRNA molecules contain un- usual bases, such as methyl cytosine (mC) and pseudouridine (y). (With permission, from T. D. Brock, K. M. Brock, and D. M. Ward, Basic Microbiology with Applications, 3d ed., Pearson Education, Upper Saddle River, NJ, 1986, p. 138.)
to make proteins) of cells varies from 10%to 20% of dry weight. However, viruses may contain nucleic acids up to 50%of their dry weight. Typically, the lipid content of most cells varies between 5%to 15%of dry weight. However, some cells accumulate PHB up to 90% of the total mass under certain culture conditions. In general, the intracellular composition of cells varies depending on the type and age of the cells and the composition of the nutrient media. Typical compositions for major groups of organisms are summa- rized in Table 2.7.
Most of the products formed by organisms are produced as a result of their response to environmental conditions, such as nutrients, growth hormones, and ions. The qualita- tive and quantitative nutritional requirements of cells need to be determined to optimize growth and product formation. Nutrients required by cells can be classified in two categories:
1. Macronutrients are needed in concentrations larger than 10-4 M. Carbon, nitro- gen, oxygen, hydrogen, sulfur, phosphorus, Mg2+, and K+ are major macro- nutrients.
2. Micronutrients are needed in concentrations of less than 10-4 M. Trace elements such as Mo2+, Zn2+, Cu2+, Mn2+, Ca2+, Na+, vitamins, growth hormones, and metabolic precursors are micronutrients.
48 An Overview of Biological Basics Chap. 2
TABLE 2.7 Chemical Analyses, Dry Weights, and the Populations of Different Microorganisms Obtained in Culture
Typical Typical
population dry weight of
Nucleic in culture this culture
Organism Protein acid Lipid (numbers/ml) (g/100 ml) Comments
Viruses 50–90 5–50 <1 108–109 0.0005a Viruses with a lipoprotein sheath may contain 25%lipid.
Bacteria 40–70 13–34 10–15 2 ¥108–2 ¥1011 0.02–2.9 PHB content may reach 90%
Filamentous 10–25 1–3 2–7 3–5 Some Aspergillus and
fungi Penicilliumsp. contain
50%lipid.
Yeast 40–50 4–10 1–6 1–4 ¥108 1–5 Some Rhodotorula and
Candidasp. contain 50%lipid.
Small 10–60 1–5 4–80 4–8 ¥107 0.4–0.9 Figure in ( ) is a com-
unicellular (50) (3) (10) monly found value
algae but the composition
varies with the growth conditions.
aFor a virus of 200 nm diameter.
With permission, from S. Aiba, A. E. Humphrey, and N. F. Millis, Biochemical Engineering, 2d ed., University of Tokyo Press, Tokyo, 1973.
Composition (% dry weight)
2.3.2. Macronutrients
Carboncompounds are major sources of cellular carbon and energy. Microorganisms are classified in two categories on the basis of their carbon source: (1) Heterotrophsuse or- ganic compounds such as carbohydrates, lipids, and hydrocarbons as a carbon and energy source. (2) Autotrophsuse carbon dioxide as a carbon source. Mixotrophs concomitantly grow under both autotrophic and heterotrophic conditions; however, autotrophic growth is stimulated by certain organic compounds. Facultative autotrophs normally grow under au- totrophic conditions; however, they can grow under heterotrophic conditions in the ab- sence of CO2and inorganic energy sources. Chemoautotrophs utilize CO2 as a carbon source and obtain energy from the oxidation of inorganic compounds. Photoautotrophs use CO2as a carbon source and utilize light as an energy source.
The most common carbon sources in industrial fermentations are molasses (sucrose), starch (glucose, dextrin), corn syrup, and waste sulfite liquor (glucose). In laboratory fer- mentations, glucose, sucrose, and fructose are the most common carbon sources. Methanol, ethanol, and methane also constitute cheap carbon sources for some fermentations. In aero- bic fermentations, about 50%of substrate carbon is incorporated into cells and about 50%
of it is used as an energy source. In anaerobic fermentations, a large fraction of substrate car- bon is converted to products and a smaller fraction is converted to cell mass (less than 30%).
Nitrogenconstitutes about 10%to 14%of cell dry weight. The most widely used ni- trogen sources are ammonia or the ammonium salts [NH4Cl, (NH4)2SO4, NH4NO3], pro- teins, peptides, and amino acids. Nitrogen is incorporated into cell mass in the form of proteins and nucleic acids. Some organisms such as Azotobacter sp.and the cyanobacteria fix nitrogen from the atmosphere to form ammonium. Urea may also be used as a nitrogen source by some organisms. Organic nitrogen sources such as yeast extract and peptone are expensive compared to ammonium salts. Some carbon and nitrogen sources utilized by the fermentation industry are summarized in Table 2.8.
Oxygenis present in all organic cell components and cellular water and constitutes about 20%of the dry weight of cells. Molecular oxygen is required as a terminal electron acceptor in the aerobic metabolism of carbon compounds. Gaseous oxygen is introduced into growth media by sparging air or by surface aeration.
TABLE 2.8 Some Carbon and Nitrogen Sources Utilized by the Fermentation Industry
Carbon sources Nitrogen sources
Starch waste (maize and potato) Soya meal Molasses (cane and beet) Yeast extract
Whey Distillers solubles
n-Alkanes Cottonseed extract
Gas oil Dried blood
Sulfite waste liquor Corn steep liquor
Domestic sewage Fish solubles and meal
Cellulose waste Groundnut meal
Carbon bean
With permission, from G. M. Dunn in Comprehensive Biotechnology, M. Moo-Young, ed., Vol. 1, Elsevier Science, 1985.
Hydrogenconstitutes about 8%of cell dry weight and is derived primarily from car- bon compounds, such as carbohydrates. Some bacteria such as methanogens can utilize hydrogen as an energy source.
Phosphorusconstitutes about 3%of cell dry weight and is present in nucleic acids and in the cell wall of some gram-positive bacteria such as teichoic acids. Inorganic phos- phate salts, such as KH2PO4and K2HPO4, are the most common phosphate salts. Glyc- erophosphates can also be used as organic phosphate sources. Phosphorus is a key element in the regulation of cell metabolism. The phosphate level in the media should be less than 1 mM for the formation of many secondary metabolites such as antibiotics.
Sulfurconstitutes nearly 1%of cell dry weight and is present in proteins and some coenzymes. Sulfate salts such as (NH4)2SO4are the most common sulfur source. Sulfur- containing amino acids can also be used as a sulfur source. Certain autotrophs utilize S2+
and S0as energy sources.
Potassiumis a cofactor for some enzymes and is required in carbohydrate metabo- lism. Cells tend to actively take up K+ and Mg2+ and exclude Na+ and Ca2+. The most commonly used potassium salts are K2HPO4, KH2PO4, and K3PO4.
Magnesium is a cofactor for some enzymes and is present in cell walls and mem- branes. Ribosomes specifically require Mg2+ions. Magnesium is usually MgSO47H2O supplied as MgSO47H2O or MgCl2.
Table 2.9 lists the eight major macronutrients and their physiological role.
2.3.3. Micronutrients
Trace elements are essential to microbial nutrition. Lack of essential trace elements in- creases the lag phase (the time from inoculation to active cell replication in batch culture) and may decrease the specific growth rate and the yield. The three major categories of mi- cronutrients are discussed next.
50 An Overview of Biological Basics Chap. 2
TABLE 2.9 The Eight Macronutrient Elements and Some Physiological Functions and Growth Requirements
Required concentration
Element Physiological function (mol l-1)
Carbon Constituent of organic cellular material. Often the energy
source. >10-2
Nitrogen Constituent of proteins, nucleic acids, and coenzymes. 10-3
Hydrogen Organic cellular material and water. —
Oxygen Organic cellular material and water. Required for aerobic
respiration. —
Sulfur Constituent of proteins and certain coenzymes 10-4
Phosphorus Constituent of nucleic acids, phospholipids, nucleotides, and
certain coenzymes 10-4to 10-3
Potassium Principal inorganic cation in the cell and cofactor for some
enzymes. 10-4to 10-3
Magnesium Cofactor for many enzymes and chlorophylls (photosynthetic
microbes) and present in cell walls and membranes. 10-4to 10-3 With permission, from G. M. Dunn in Comprehensive Biotechnology,M. Moo-Young, ed., Vol. I, Elsevier Science, 1985.
1. Most widely needed trace elements are Fe, Zn, and Mn. Iron (Fe) is present in ferredoxin and cytochrome and is an important cofactor. Iron also plays a regulatory role in some fermentation processes (e.g., iron deficiency is required for the excretion of ri- boflavin by Ashbya gosypii and iron concentration regulates penicillin production by Penicillium chrysogenum). Zinc (Zn) is a cofactor for some enzymes and also regulates some fermentations such as penicillin fermentation. Manganese (Mn) is also an enzyme cofactor and plays a role in the regulation of secondary metabolism and excretion of pri- mary metabolites.
2. Trace elements needed under specific growth conditions are Cu, Co, Mo, Ca, Na, Cl, Ni, and Se. Copper (Cu) is present in certain respiratory-chain components and en- zymes. Copper deficiency stimulates penicillin and citric acid production. Cobalt (Co) is present in corrinoid compounds such as vitamin B12. Propionic bacteria and certain methanogens require cobalt. Molybdenum (Mo) is a cofactor of nitrate reductase and ni- trogenase and is required for growth on NO3and N2as the sole source of nitrogen. Cal- cium (Ca) is a cofactor for amylases and some proteases and is also present in some bacterial spores and in the cell walls of some cells, such as plant cells.
Sodium (Na) is needed in trace amounts by some bacteria, especially by methanogens for ion balance. Sodium is important in the transport of charged species in eucaryotic cells. Chloride (Cl-) is needed by some halobacteria and marine microbes, which require Na+, too. Nickel (Ni) is required by some methanogens as a cofactor and Selenium (Se) is required in formate metabolism of some organisms.
3. Trace elements that are 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 10-6Mand are toxic at high concentrations, such as 10-4M.
Some ions such as Mg2+, Fe3+, and PO3-4 may precipitate in nutrient medium and be- come unavailable to the cells. Chelating agentsare used to form soluble compounds with the precipitating ions. Chelating agents have certain groups termed ligandsthat bind to metal ions to form soluble complexes. Major ligands are carboxyl (—COOH), amine (—NH2), and mercapto (—SH) groups. Citric acid, EDTA (ethylenediaminetetraacetic acid), polyphosphates, histidine, tyrosine, and cysteine are the most commonly used chelating agents. Na2EDTA is the most common chelating agent. EDTA may remove some metal ion components of the cell wall, such as Ca2+, Mg2+, and Zn2+and may cause cell wall disintegration. Citric acid is metabolizable by some bacteria. Chelating agents are included in media in low concentrations (e.g., 1 mM).
Growth factorsstimulate the growth and synthesis of some metabolites. Vitamins, hormones, and amino acids are major growth factors. Vitamins usually function as coen- zymes. Some commonly required vitamins are thiamine (B1), riboflavin (B2), pyridoxine (B6), biotin, cyanocobalamine (B12), folic acid, lipoic acid, p-amino benzoic acid, and vita- min K. Vitamins are required at a concentration range of 10-6Mto 10-12M. Depending on the organism, some or all of the amino acids may need to be supplied externally in con- centrations from 10-6M to 10-13M. Some fatty acids, such as oleic acid and sterols, are also needed in small quantities by some organisms. Higher forms of life, such as animal and plant cells, require hormones to regulate their metabolism. Insulin is a common hor- mone for animal cells, and auxin and cytokinins are plant-growth hormones.
2.3.4. Growth Media
Two major types of growth media are defined and complex media. Defined mediacontain specific amounts of pure chemical compounds with known chemical compositions. A medium containing glucose, (NH4) 2SO4, KH2PO4, and MgCl2is a defined medium. Com- plex mediacontain natural compounds whose chemical composition is not exactly known.
A medium containing yeast extracts, peptone, molasses, or corn steep liquor is a complex medium. A complex medium usually can provide the necessary growth factors, vitamins, hormones, and trace elements, often resulting in higher cell yields, compared to the de- fined medium. Often, complex media are less expensive than defined media. The primary advantage of defined media is that the results are more reproducible and the operator has better control of the fermentation. Further, recovery and purification of a product is often easier and cheaper in defined media. Table 2.10 summarizes typical defined and complex media.
52 An Overview of Biological Basics Chap. 2
TABLE 2.10 Compositions of Typical Defined and Complex Media
Defined medium
Constituent Purpose Concn (g/liter)
Group A
Glucose C, energy 30
KH2PO4 K, P 1.5
MgSO4◊7H2O Mg, S 0.6
CaCl2 Ca 0.05
Fe2(SO4)3 Fe 15 ¥10-4
ZnSO4◊7H2O Zn 6 ¥10-4
CuSO4◊5H2O Cu 6 ¥10-4
MnSO4◊ H2O Mn 6 ¥10-4
Group B
(NH4)2HPO4 N 6
(NH4)H2PO4 N 5
Group C
C6H5Na3O7◊2H2O Chelator 4
Group D
Na2HPO4 Buffer 20
KH2PO4 Buffer 10
Complex medium used in a penicillin fermentation
Glucose or molasses (by continuous feed) 10%of total
Corn steep liquor 1–5%of total
Phenylacetic acid (by continuous feed) 0.5–0.8%of total Lard oil (or vegetable oil) antifoam by
continuous addition 0.5%of total
pH to 6.5 to 7.5 by acid or alkali addition