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106 CHAPTER 5 Milestones Box 5.2 Microbial Versatility: Obligate versus Facultative A microorganism that will grow only if provided with CO2 for carbon and light as energy source is an obligate photoautotroph. One that grows only with CO2 and NH4+ or S2– as energy source is an obligate chemoauto...

106 CHAPTER 5 Milestones Box 5.2 Microbial Versatility: Obligate versus Facultative A microorganism that will grow only if provided with CO2 for carbon and light as energy source is an obligate photoautotroph. One that grows only with CO2 and NH4+ or S2– as energy source is an obligate chemoautotroph. Restriction to an obligate lifestyle can be found in the microbial world, but versatility is the more common attribute. An obligate autotroph can actually assimilate a very limited amount of selected organic substrates while growing with CO2 as bulk carbon source and light (for photo-) or inorganics (for chemo-) as energy source. However, many of the organisms that will grow as photo- or chemo- autotrophs can grow as chemoheterotrophs. Such organisms are termed facultative. The term facultative is also used to describe an organism that can grow aerobically if O2 is available or anaerobically in the absence of O2. SUBSTRATE CONSTITUENTS NEEDED FOR GROWTH first six elements listed in Table 5.1. These are discussed here as components of the growth medium. Biochemical analysis of bacterial or archaeal cell mass affirms that their cellular composition is mostly equivalent regardless of the species or the composition of the medium on which they grew. They are composed of the same major components including the monomers that make up the major macromolecules (proteins, nucleic acids, carbohydrates, and lipids). A bacterium is 80% to 90% water. This elemental composition is likewise very similar, with little variance from the percentages of elements shown in Table 5.1. Consequently, a growth medium must provide a microorganism with these elements in one form or another. Requirement for the iron and the trace metals may vary. The macromolecules that are present in all microorganisms are composed of the Carbon Table 5.1 A typical analysis of the elements that may be present in a bacterial or archaeal cell Element Carbon Oxygen Nitrogen Hydrogen Phosphorus Sulfur Sodium Potassium Calcium Magnesium Iron Cu, Zn, Mo, Bo, Se, Cl Ni, Cr, Co, and Wo Percentage of Dry Weight 50 20 14 8 3 1 1 1 0.5 0.5 0.2 0.2 100% The most abundant element in any living cell is carbon. Carbon is the backbone of functional biological molecules. Microbial diversity reflects the ability of organisms to synthesize all of their component molecules from the carbon source that is available. Marked differences exist among microbes in this capacity. For example, a cyanobacterium can synthesize de novo all of its cellular components (protein, nucleic acids) from CO2 if mineral salts are available (Table 5.2). Other organisms Table 5.2 A typical mineral salts medium for the isolation and growth of freeliving bacteria Constituents Amount a (mg/l H2O) NH4Cl NaNO3 Na2HPO4 NaH2PO4 MgSO4 • 7 H2O KCl CaCl2 FeSO4 500 500 210 90 200 40 15 1 Trace Elements µg/l H20 ZnSO4 • 7 H2O H3BO3 MnSO4 • 5 H2O MoO3 CoSO4 CuSO4 • 5 H2O 70 10 10 10 10 5 a It would be necessary to add a carbon source such as glucose at 0.5 to 2.0% ISOLATION, NUTRITION, AND CULTIVATION OF MICROORGANISMS may have a very limited synthetic capacity and require a complex growth medium, such as that shown in Table 5.3. A growth medium with all of the compounds listed in the table would be necessary for the growth of the Table 5.3 A defined medium for the growth of the lactic acid bacterium Streptococcus agalactiae Compound L-alanine L-arginine L-aspartic acid DL-asparagine L-cystine L-cysteine-HCl L-glutamic acid L-glycine L-glutamine L-histidine L-leucine L-lysine L-isoleucine DL-methionine L-phenylalanine L-proline DL-serine L-tryptophan L-tyrosine L-valine Nicotinic acid Ca pantothenate Pyridoxal HCl Thiamine HCl Riboflavin Biotin Folic acid K2HPO4 KH2PO4 NaHCO3 FeSO4 • 7 H2O MnCl2 NaCl ZnSO4 • 7 H2O MgSO4 • 7 H2O Glucose Adenine Guanine Xanthine Uracil Final Concentration (µg/mL) 250 320 500 100 600 600 400 200 100 320 200 320 200 200 200 200 400 200 200 200 10 10 10 0.6 1 0.2 0.02 1,000 1,000 500 10 25 10 10 80 10,000 10 10 10 10 From N. P. Willett and G. E. Morse. 1966. Long-chain fatty acid inhibition of the growth of Streptococcus agalactiae in a chemically defined medium. J. Bact. 91:2245. 107 fastidious lactic acid-producing bacterium Streptococcus agalactiae. The carbon source for microbial growth can range from CO or CH4 to any naturally occurring complex organic compound present in the biosphere (such as carbohydrates, peptides, and organic acids). A variety of microbes also exist that can, under appropriate conditions, grow with various synthetic organic compounds as substrate. This is the basis for “bioremediation,” the removal of pollutant chemicals from an environment by use of selected microbes that can utilize these pollutants as a carbon/energy source. Hydrogen Hydrogen plays a number of roles in the life of Bacteria and Archaea—it is a structural atom in organic molecules and is a participant in the complex process of energy generation. A source of hydrogen is essential in autotrophic microorganisms to reduce CO2 to cell material (CH2O). Protons (H+) are involved in the production of ATP via the ATP synthase system in the cell’s membrane of most microorganisms (see Chapter 8). Microorganisms that respire anaerobically (CH4 producers, denitrifiers, and sulfate reducers) gain their energy by transfer of electrons from a substrate to a selected acceptor (Table 5.4). Aerobic respiration involves the transfer of electrons to O2, a factor in establishing a proton gradient for ATP synthesis (Chapter 8). Nitrogen Nitrogen is an integral constituent of amino acids (proteins), nucleotides (nucleic acids), phospholipids, and constituents of the cell wall. A unique property of some Bacteria and Archaea is their ability to obtain cellular nitrogen by fixation of N2 from the atmosphere. These nitrogen fixers reduce N2 to the level of NH4+, which is incorporated into carbon compounds by the synthetic machinery of the cell. The fixation of N2 is not limited to a few species, as was long believed, but occurs in an array of bacterial and archaeal types, as outlined in Table 5.5. Most free-living microorganisms assimilate ammonia from their environment, or they can reduce nitrate. One or both of these are commonly included in a growth medium. A requirement for an organic nitrogen source such as an amino acid is generally confined to microorganisms that evolved in richer environments where various amino acids, nucleic acids, and B-vitamins were readily available. For example, the medium presented in Table 5.3 is a growth medium for a microorganism that has a limited ability to synthesize nitrogen-containing intermediates. The lactic acid bacteria evolved in or on animals or plants where organic nitrogen compounds were available. The growth of most heterotrophic bacteria is stimulated by adding rich nitrogenous material, such as yeast 108 CHAPTER 5 Phosphorus Table 5.4 Types of respiration that occur in Bacteria and Archaea. Examples of organisms that perform this respiration are given Aerobic respiration Oxygen O2 → H2O Anaerobic respiration Iron Fe 3+ → Fe 2+ Nitrate NO3– → NO2–, N2O, N2 Fumarate Fumarate → Succinate Sulfate SO42– → HS– Sulfur So → HS– Carbonate CO2 → CH4 CO2 → CH3COO– Pseudomonas fluorescens Shewanella putrefaciens This element has played a major role in the evolution of life systems on Earth. Phosphorus is a constituent of highenergy compounds, the phospholipids in cell membranes, and nucleic acids. Adenosine triphosphate (ATP) is the principle medium of energy exchange in cellular metabolism and is an indispensable contributor to biosynthetic reactions involved in reproduction and growth. For this reason, phosphates are an integral constituent of culture media. Inorganic phosphates are also an effective buffer at near neutral pHs and at concentrations that are not usually inhibitory to bacterial growth. Phosphate salts are commonly added to culture media to satisfy the phos- Thiobacillus denitrificans Proteus rettgeri Desulfovibrio desulfuricans Desulfurococcus mucosus Methanosarcina barkeri Acetobacter woodii extract (soluble portion of digested yeast cells) at 0.05%, to a basic mineral salts medium. Growth is stimulated because energy need not be expended to synthesize Bvitamins, amino acids, purines, pyrimidines, and other nitrogen-containing compounds. Sulfur Sulfur is a constituent part of two of the amino acids that make up proteins—cysteine and methionine. It is also present in certain B-vitamins (biotin and thiamine) and some other essential constituents of a cell. Sulfur is usually added to a growth medium as a sulfate salt. MgSO4 added to a medium would serve as a source of both sulfur and magnesium. The capacity to reduce sulfate to the sulfide level (SH), the form present in cellular constituents, is a common attribute of bacteria. If an organism cannot reduce sulfate, the addition of the amino acid cysteine will permit growth. Addition of yeast extract or peptone (enzyme digest of protein) in a culture medium meets the need for reduced sulfur compounds in most microorganisms. Reduced inorganic sulfur compounds such as H2S or pyrite (iron sulfide) can serve as the energy source for a group of bacteria called the thiobacilli. Oxidation of sulfides generates sulfate. Sulfur S (inorganic) can serve as a terminal electron acceptor in some bacteria, and many of the Archaea and sulfate serves this purpose in sulfate-reducing Bacteria and Archaea. Table 5.5 Some genera of Bacteria and Archaea that have nitrogen fixation ability Bacteria Heterotrophs Aerobes Azotobacter Klebsiella Beijerinckia Anaerobes Clostridium Bacillus (facultative) Photosynthetics Cyanobacteria Anabaena Oscillatoria Gloeocapsa Purple and green bacteria Chromatium Chlorobium Rhodospirillum Symbiotic Legumes Clover + Rhizobium Soybeans + Rhizobium or Bradyrhizobium Bluebonnets + Rhizobium Nonlegumes Bayberry + Actinomycete Alder + Frankia Archaea Methanogens Anaerobes Methanococcus Methanosarcina Methanobacterium Methanothermus ISOLATION, NUTRITION, AND CULTIVATION OF MICROORGANISMS phate requirement and to provide a buffer to prevent significant changes in pH during growth. Oxygen Table 5.6 109 The function of various elements in bacterial and archaeal nutrition Element Potassium The total number of oxygen atoms present as a cellular component is Sodium equivalent in aerobes and anaerobes. However, molecular oxygen (O2) is toxic to most strictly anaeroMagnesium bic Bacteria and Archaea, so they obtain this element in a combined form from the substrate. As a genIron eral rule, anaerobes utilize growth Cobalt substrates that are in an oxidationCopper, zinc, reduction state equal to or more molybdenum, oxidized than cellular material nickel, tungsten, (CH2O). For example, anaerobes and selenium can utilize sugars, amino acids, or CO2 as carbon source. Aerobic bacteria can grow on reduced substrates (such as methane and propane), when molecular oxygen is available. Aerobic microorganisms use oxygen as a terminal electron acceptor through the electron transport system. A number of aerobes use oxygen also as a reactant to incorporate it into their substrate. Function Utilized in a number of enzymatic reactions as a cofactor and especially in protein synthesis Involved along with chloride in the regulation of osmotic pressure; affects activity of some enzymes; uptake of solutes in some species with Na+ dependent transport systems Integral part of chlorophyll; cation required in enzymatic reactions including those involved in ATP synthesis or hydrolysis Reactive center of heme-containing proteins (cytochromes, catalase, etc.) and component of other proteins Constituent of vitamin B12, complexed to some enzymes Essential components of some enzymes Cations • Vitamins: These organic compounds serve as the prosthetic group (nonprotein catalytic part) of a number of enzymes. Small catalytic amounts of vitamins are required, as they are present in cells in low quantity (varying from a nanogram of vitamin B12 to 250 micrograms/gram dry weight cell of nicotinic acid). The vitamins most frequently required are those less susceptible to destruction by light: thiamine, biotin, and nicotinic acid. The function of the B-vitamins in nutrition is outlined in Table 5.7. Other elements are present in Bacteria and Archaea and are generally involved with enzymatic activity or in cell stability. These are mostly cations and include potassium, sodium, magnesium, iron, and cobalt (Table 5.6). Iron is a constituent of electron transport chains and therefore is an absolute requirement among aerobes. Copper, zinc, and molybdeTable 5.7 The function of the various B-vitamins in nutrition of num are required in small amounts Bacteria and Archaea and are called trace elements. Growth Factors Compound Function Growth factors are specific relatively low molecular weight organic compounds that must be available in the growth medium of some microorganisms because they cannot synthesize them. The substances that generally serve as growth factors are selected amino acids, purines, pyrimidines, and Bvitamins. Microbes do not require fat-soluble vitamins such as A and D, as they are not constituents of microorganisms. The three types of growth factor most often required in bacterial nutrition are as follows: p-Aminobenzoic acid Precursor of folic acid, a coenzyme involved in one carbon unit transfer A coenzyme involved in one carbon unit transfer A prosthetic group for enzymes that act in carboxylation reactions Precursor of NAD and NADP, which are coenzymes involved with hydrogen transfer A component of the flavin mononucleotide (FMN) and dinucleotide (FAD) involved in hydrogen transfer A component of the coenzyme for transaminase and amino acid decarboxylase A coenzyme involved in molecular rearrangements The prosthetic group for a number of decarboxylases, transaldolases, and transketolases A functional part of coenzyme A and the acyl carrier proteins A coenzyme in methane-generating bacteria Folic acid Biotin Nicotinic acid Riboflavin Pyridoxine Vitamin B12 Thiamin Pantothenic acid Coenzyme M