Nutrition and Growth overview.doc

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Microbiology (Biological Sciences)
Brian Lanoil

Microbial Nutrition & Growth (Chapter 4) OVERVIEW The lectures and book chapters describe the basic nature of microbial nutrition and growth. The chapter covers the essential nutrients for growth and a discussion of the growth curve, calculating exponential growth, and several methods for measurement and maintenance of microbial growth are described, including discussions of batch culture versus continuous culture in chemostats. The chapter continues by describing various environmental factors that affect the growth of microorganisms and how prokaryotes and some fungi have managed to adjust their membrane chemistry, internal solute concentrations, and other cellular structures to enable them to withstand and thrive under extremely harsh conditions. A brief discussion of biofilm growth in natural environments is not presented in the book chapter (only in the lecture notes), but establishes important concepts that will be covered in later chapters. OBJECTIVES 1) List the elements that microorganisms require in large amounts (macronutrients) and those they require in trace amounts (micronutrients). Have a general understanding of why they are required. 2) Describe the various types of culture media for microorganisms (complex, defined, selective) and their purpose. 3) Describe the divisome and the role of proteins (FtsZ, MinE, autolysin, bactoprenol) in creating a septum, new cell wall material, and dividing the cell in two. 4) Describe the 4 phases of growth that occur in closed (batch) culture systems, what occurs in each phase, and why the cells enter each phase. 5) Use and apply the exponential microbial growth equation. 6) Describe the three most commonly used methods for measuring cell density and their advantages and limitations. 7) Describe the concept of the chemostat and steady state growth. Understand the application of this principle. 8) Describe the influence of various environmental factors (temperature, pH, water availability, and oxygen concentration) on the growth of microorganisms. 9) Categorize (name) groups of microorganisms according to environmental factors supporting their optimal growth rates. 10)Describe changes in cellular structures and other adaptive mechanisms prokaryotes use to withstand extremes of temperature, pH, water availability and O 2oncentration. 11)Describe how microorganisms adapt to a changing environment, the establishment of biofilm, and the benefits of biofilm to microbial populations (presented in lecture only). CHAPTER OUTLINE I. Common Nutrient Requirements Macroelements or macronutrients (C, H, O, N, P, S, K, Na, Ca, Mg, Fe) are required by microorganisms in relatively large amounts (hence, the name “macro”). The first 6 are the 1 building blocks of the cell: e.g. proteins, nucleic acids, lipids. Cations are important in membrane electron transport (Na, K), in enzymatic functions (Mg) or as structural components (e.g, Fe is a constituent of cytochromes, Mg and Ca are constituents of cell wall polymers). Trace elements or micronutrients (Mn, Zn, Co, Mo, Ni, Cu) are required in trace amounts (“micro”) by most cells and are often adequately supplied in the water used to prepare the media or in the regular media components. They are generally involved as cofactors in enzymatic reactions and may be covalently bound to structural molecules (e.g., cobalt is the central atom of Vitamin B12) Other elements may be needed by particular types of microorganisms: e.g. silicon is needed for cell walls of diatoms (eukaryotic microbes). Siderophores are specific chelating agents that enable cells to chelate Fe(III), an essential macronutrient that is not very soluble at physiological pH. Siderophores are either associated with the cell wall or are excreted into the medium. The chelated iron siderophore binds to a receptor protein on the cell wall. Iron may be transported through the periplasm, complexed to the siderophore, or released and taken up through the cytoplasmic membrane by other carriers that are driven by active transport. Both fungi and bacteria produce siderophores in iron- deficient media. Enterobactin is a siderophore of E. coli that aids in pathogenesis by ensuring that iron limitation does not occur. Aquachelin is a siderophore that forms lipid-soluble micelles that can be transported directly through the cytoplasmic membrane. Growth Factors: organic nutrients (amino acids, vitamins, nucleic acids) Prototrophs synthesize all of their cellular constituents from inorganic N, P and S, and do not require vitamins or other growth factors: e.g. E. coli can grow with glucose as the sole carbon source in media containing inorganic mineral salts. Chemolithotrophs can grow with carbonate as the sole carbon source, and are the ultimate prototrophs. Auxotrophs lack the ability to synthesize an essential growth factor and therefore require it in the medium. Prototroph and auxotroph are used only in reference to organic nutrients, i.e. growth factors DO NOT CONFUSE AUTOTROPH WITH AUXOTROPH: THEY ARE ENTIRELY DIFFERENT! Think of auto as self sufficient, and think of auxo as auxillary (additional). Similarly, do not confuse prototroph with phototroph Certain organic compounds may be required by auxotrophs because they are essential cell components (or precursors of these components) that the cell cannot synthesize. Examples are: Amino acids for protein synthesis Purines and pyrimidines for nucleic acid synthesis Vitamins that function as enzyme cofactors Knowledge of specific growth factor requirements makes possible specific isolation of microorganisms (i.e. defined media). 2 II. Culture Media Agar petri plates are the basis of the pure culture technique. They allow for a single cell to grow and form a visible colony on the surface of the plate. Not all bacteria grow on agar plates: only 0.001% can be grown this way!! Agar is a polysaccharide used to solidify media. Defined media are those in which all components and their concentrations are known: e.g. glucose-mineral salts medium. Complex media are those that contain some ingredients of unknown composition. Both auxotrophs and prototrophs can grow in these media, but organisms that can not tolerate high levels of organics (chemolithotrophs) can not. Nutrient broth is an example of the most widely used general purpose medium. The medium usually contains: Peptones or protein hydrolysates that provide amino acids for growth Aqueous extracts, usually of beef or yeast, provide vitamins Blood agar or brain-heart infusions enable the growth of fastidious (nutritionally demanding) heterotrophs. Fastidious means fussy or finicky, as derived from Latin. It does not mean fast! If anything, it is the opposite of fast. Selective media favor the growth of particular microorganisms and inhibit the growth of others; Note: all media fall under this category. Differential media distinguish between different of bacteria on the basis of their biochemical reactions: e.g. Staphylococcus aureus ferments mannitol and produces acid: a pH indicator dye will show the color change in the medium from red to yellow. In contrast, S. epidermidis grows on the medium but does not ferment mannitol, hence no color change. Some media can exhibit characteristics of more than one type. For example, blood agar is complex and differential, and distinguishes between hemolytic and nonhemolytic bacteria: the former lyse red blood cells, and show a zone of clearing around the colonies on blood agar plates. Pure cultures can be obtained by streaking with a wire inoculating loop, as shown in the first lecture. The constant streaking of the inoculum across the surface of the plate is a wiping-effect: microorganisms are removed from the wire loop and deposited on the agar, until the loop contains only a few or none. At the end of the wiping process, single cells will be deposited on the agar, and these will grow and multiply to become a visible colony. III. Binary Cell Division Growth is an increase in cellular constituents that may result in an increase in cell size, an increase in cell number, or both. However, for bacterial growth curves, growth is defined as an increase in cell number over time. Replication of the bacterial chromosome takes much longer than cell division, thus multiple rounds of chromosome replication must be initiated at the same time. 3 Prokaryotic cells reproduce by binary fission. The process is controlled by several “Fts” and other proteins, known collectively as the divisome. The FtsZ protein creates a ring around the cell exactly at its midpoint through GTP hydrolysis. Fts means filamentous temperature sensitive, which describes the phenotype of mutated strains that are unable to create Fts proteins. These mutants can not form the protein ring, so the bacteria tend to grow in long filaments without dividing. The MinE protein oscillates from one end of the cell to the other very rapidly and defines the cell mid-point. Without MinE and its rapid oscillation, the FtsZ ring would not form in the center of the cell. Autolysins, enzymes similar to lysozyme, create openings in the cell wall for generation of new membrane and wall constituents for the dividing cells. This is the point where penicillin is the most active against bacterial growth as it interferes with the transpeptidation process and creation of new wall material. Once the membranes and walls are complete, the protein ring contracts to pinch the cell into two new daughter cells. Bactoprenol is a cytoplasmic protein that carries peptidoglycan precursors across cytoplasmic membrane to create new cell wall material during the septation and separation process of daughter cells. IV. Microbial Growth Curve The growth curve usually describes a closed system, i.e. batch culture, which is plotted as the logarithm of cell number versus incubation time since cell division is exponential. Doubling, or generation, times (time required for a cell division) vary markedly (from 10 minutes to several days) with the species of microorganism and environmental conditions. A. The lag phase Exponential growth is only meaningful once cells reach a certain physiological state. However, inoculation of media with a cell suspension does not ensure that the culture will immediately start dividing. In fact, the lag period - a time interval in which cell division does not occur - is generally the rule. There is no single mechanism that explains or characterizes this phase, but it is known that this period is characterized by de novo (new) enzyme synthesis related to gearing the cell up to deal with its new environment. When cells are removed from the exponential growth phase and immediately transferred into fresh media of identical composition and environmental characteristics (e.g. temperature, aeration, pH), there is no lag phase. Thus, the major constraints on cells rapidly entering exponential growth are: 1. Age of culture. The older the culture, the longer the lag phase. Although much of this may be due to a larger fraction of dead cells, older cultures tend to exhibit a viable but non- culturable tendency: i.e. although they are alive, not all cells are readily able to divide and grow in laboratory media. 4 2. Growth substrates. Cultures transferred from rich media to poorer media will always exhibit lag phase, but not necessarily the other way around. Cells initially grown on defined media where they must synthesize vitamins, amino acids, etc. from inorganic N, P, and S easily shift to a richer complex medium having all the ingredients pre-made and available. When grown on rich media and then shifted to defined media, the lag period represents synthesis of enzymes needed to make vitamins and amino acids previously available in the medium. 3. Environmental factors. New enzymes may need to be synthesized to acclimatize the culture to different conditions. For example, facultative aerobes need to gear up for differences in thermodynamic requirements when shifting from aerobic to anaerobic metabolism. B. Exponential growth phase: n N = N 2 o (1) n = t/g (2) N os the initial cell density at the beginning of the exponential growth phase (time zero), N is the cell density at time t, and g is the time required for a cell division (doubling or generation time). n is the number of generations (g) that occur during t, so n = t/g. To derive equation 1 into a usable form, the log of both sides is taken and can be expressed as: log N = log N +on log 2 (3) Solving for n, we get: n = log N – log N = log N – log N (4) o o log 2 0.301 Taking the inverse, we get: n = 3.3 (log N – log N ) o (5) If a culture starts with a single cell, what is the doubling time after 10 h the cell density is 100,000? The correct answer is 36 min. How long will it take for a cell density of 2.0 * 10 cells/ml to be achieved from an initial density 7 of 4.0 * 10 cells/ml if the culture has a doubling time of 35 minutes? The correct answer is 3.3 hr. C. Stationary phase. This aspect of the bacterial growth curve is important for organisms that underg
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