6.Microbial Growth CHAPTER 6 Microbial Growth Outline 6.1 The PH 123 e12 65 63 The Con 64
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 CHAPTER 6 Microbial Growth Membrane filters are used in counting microorganisms. This membrane has been used to obtain a total bacterial count using an indicator to color colonies for easy counting. 6.1 The Growth Curve 113 Lag Phase 113 Exponential Phase 114 Stationary Phase 114 Death Phase 115 The Mathematics of Growth 115 6.2 Measurement of Microbial Growth 117 Measurement of Cell Numbers 117 Measurement of Cell Mass 119 6.3 The Continuous Culture of Microorganisms 120 The Chemostat 120 The Turbidostat 121 6.4 The Influence of Environmental Factors on Growth 121 Solutes and Water Activity 121 pH 123 Temperature 125 Oxygen Concentration 127 Pressure 129 Radiation 130 6.5 Microbial Growth in Natural Environments 131 Growth Limitation by Environmental Factors 131 Counting Viable But Nonculturable Vegetative Procaryotes 132 Quorum Sensing and Microbial Populations 132 Outline
6.1 The Growth Curve 113 Concepts nary phas When m uch as on.In wide ostnam Time 6.1 The Growth Curve Population growth is studied by analyzing the growth curve of a medium.they usually at is,they are incu bated in a closed culture vessel with hapter 5 emphasizes that microorganisms need access to and the raw st have phospho tions of number of viable cels versus the incubation time.The resulting ses.the most important of which curve has four distinct phases (figure 6.1). n,act and group translocation Lag Phase call on microbial growth completes the chapter zing new components.A lag phase prio The cells may be old and depleted of TP ents.I ean sm was grov ng in pr eviously.Here new enzymes would be progeny of approximat to u bly the r gan causes,eventually the cells ret replicale their DNA.begin to inc lly div derably in length with the tigate the growth and reproduction of in tion of the organism and the nature of the n.Thi owth.mib ated Inocula ation of a culture into he total population number. The cell cycle (pp.87:285-86 chemically different medium also results in a longer lag phase
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 6.1 The Growth Curve Population growth is studied by analyzing the growth curve of a microbial culture. When microorganisms are cultivated in liquid medium, they usually are grown in a batch culture or closed system—that is, they are incubated in a closed culture vessel with a single batch of medium. Because no fresh medium is provided during incubation, nutrient concentrations decline and concentrations of wastes increase. The growth of microorganisms reproducing by binary fission can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases (figure 6.1). Lag Phase When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs, and therefore this period is called the lag phase. Although cell division does not take place right away and there is no net increase in mass, the cell is synthesizing new components. A lag phase prior to the start of cell division can be necessary for a variety of reasons. The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin. The medium may be different from the one the microorganism was growing in previously. Here new enzymes would be needed to use different nutrients. Possibly the microorganisms have been injured and require time to recover. Whatever the causes, eventually the cells retool, replicate their DNA, begin to increase in mass, and finally divide. The lag phase varies considerably in length with the condition of the microorganisms and the nature of the medium. This phase may be quite long if the inoculum is from an old culture or one that has been refrigerated. Inoculation of a culture into a chemically different medium also results in a longer lag phase. 6.1 The Growth Curve 113 Concepts 1. Growth is defined as an increase in cellular constituents and may result in an increase in a microorganism’s size, population number, or both. 2. When microorganisms are grown in a closed system, population growth remains exponential for only a few generations and then enters a stationary phase due to factors such as nutrient limitation and waste accumulation. In an open system with continual nutrient addition and waste removal, the exponential phase can be maintained for long periods. 3. A wide variety of techniques can be used to study microbial growth by following changes in the total cell number, the population of viable microorganisms, or the cell mass. 4. Water availability, pH, temperature, oxygen concentration, pressure, radiation, and a number of other environmental factors influence microbial growth. Yet many microorganisms, and particularly bacteria, have managed to adapt and flourish under environmental extremes that would destroy most higher organisms. 5. In the natural environment, growth is often severely limited by available nutrient supplies and many other environmental factors. 6. Bacteria can communicate with each other and behave cooperatively using population density–dependent signals. The paramount evolutionary accomplishment of bacteria as a group is rapid, efficient cell growth in many environments. —J. L. Ingraham, O. Maaløe, and F. C. Neidhardt Chapter 5 emphasizes that microorganisms need access to a source of energy and the raw materials essential for the construction of cellular components. All organisms must have carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, and a variety of minerals; many also require one or more special growth factors. The cell takes up these substances by membrane transport processes, the most important of which are facilitated diffusion, active transport, and group translocation. Eucaryotic cells also employ endocytosis. Chapter 6 concentrates more directly on the growth. The nature of growth and the ways in which it can be measured are described first, followed by consideration of continuous culture techniques. An account of the influence of environmental factors on microbial growth completes the chapter. Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission. In the latter, individual cells enlarge and divide to yield two progeny of approximately equal size. Growth also results when cells simply become longer or larger. If the microorganism is coenocytic—that is, a multinucleate organism in which nuclear divisions are not accompanied by cell divisions— growth results in an increase in cell size but not cell number. It is usually not convenient to investigate the growth and reproduction of individual microorganisms because of their small size. Therefore, when studying growth, microbiologists normally follow changes in the total population number. The cell cycle (pp. 87; 285–86) Time Lag phase Exponential (log) phase Death phase Stationary phase Log number of viable cells Figure 6.1 Microbial Growth Curve in a Closed System. The four phases of the growth curve are identified on the curve and discussed in the text
6.Microbial Growth Chapter Micrbial Grwth position.the lag phase will be short or absent. envi nental conditions bial gr limited by the lo Exponential Phase or vit conditions during the xponential phase:that is.the microo The shape of the curve seems to reflect the rate of nutrient uptake not rise further with increasing nutrient concentration. ost un orm in terms of ogical assays (p.99): transport systems (pp.100-4) iological stud Stationary Phase d rowth.That is.all cellulat eases and the growth curve be ults This is growth during which the o not reach such high served in a shift-up experiment in which bacteria are transferred nutrient availability and other factors,as well as the type of mic cells firs ance between cell division and cell death.or the population may hesis.Finally.the expected nha Unbala a人品e网 ulswhen a bacterial population One opu ponents directly from the medium.When shifted to a nutrition very soluble and may be depleted so quickly that ony the surface plicationcontin after the way.Population growt (growing in the abence of ).Fo example. from sugar fermentation that their medium becomes acidic and
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 On the other hand, when a young, vigorously growing exponential phase culture is transferred to fresh medium of the same composition, the lag phase will be short or absent. Exponential Phase During the exponential or log phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing. Their rate of growth is constant during the exponential phase; that is, the microorganisms are dividing and doubling in number at regular intervals. Because each individual divides at a slightly different moment, the growth curve rises smoothly rather than in discrete jumps (figure 6.1). The population is most uniform in terms of chemical and physiological properties during this phase; therefore exponential phase cultures are usually used in biochemical and physiological studies. Exponential growth is balanced growth. That is, all cellular constituents are manufactured at constant rates relative to each other. If nutrient levels or other environmental conditions change, unbalanced growth results. This is growth during which the rates of synthesis of cell components vary relative to one another until a new balanced state is reached. This response is readily observed in a shift-up experiment in which bacteria are transferred from a nutritionally poor medium to a richer one. The cells first construct new ribosomes to enhance their capacity for protein synthesis. This is followed by increases in protein and DNA synthesis. Finally, the expected rise in reproductive rate takes place. Protein and DNA synthesis (sections 11.3 and 12.2) Unbalanced growth also results when a bacterial population is shifted down from a rich medium to a poor one. The organisms may previously have been able to obtain many cell components directly from the medium. When shifted to a nutritionally inadequate medium, they need time to make the enzymes required for the biosynthesis of unavailable nutrients. Consequently cell division and DNA replication continue after the shift-down, but net protein and RNA synthesis slow. The cells become smaller and reorganize themselves metabolically until they are able to grow again. Then balanced growth is resumed and the culture enters the exponential phase. Regulation of nucleic acid synthesis (pp. 275–83) These shift-up and shift-down experiments demonstrate that microbial growth is under precise, coordinated control and responds quickly to changes in environmental conditions. When microbial growth is limited by the low concentration of a required nutrient, the final net growth or yield of cells increases with the initial amount of the limiting nutrient present (figure 6.2a). This is the basis of microbiological assays for vitamins and other growth factors. The rate of growth also increases with nutrient concentration (figure 6.2b), but in a hyperbolic manner much like that seen with many enzymes (see figure 8.17). The shape of the curve seems to reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently high nutrient levels the transport systems are saturated, and the growth rate does not rise further with increasing nutrient concentration. Microbiological assays (p. 99); Nutrient transport systems (pp. 100–4) Stationary Phase Eventually population growth ceases and the growth curve becomes horizontal (figure 6.1). This stationary phase usually is attained by bacteria at a population level of around 109 cells per ml. Other microorganisms normally do not reach such high population densities, protozoan and algal cultures often having maximum concentrations of about 106 cells per ml. Of course final population size depends on nutrient availability and other factors, as well as the type of microorganism being cultured. In the stationary phase the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death, or the population may simply cease to divide though remaining metabolically active. Microbial populations enter the stationary phase for several reasons. One obvious factor is nutrient limitation; if an essential nutrient is severely depleted, population growth will slow. Aerobic organisms often are limited by O2 availability. Oxygen is not very soluble and may be depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth. The cells beneath the surface will not be able to grow unless the culture is shaken or aerated in another way. Population growth also may cease due to the accumulation of toxic waste products. This factor seems to limit the growth of many anaerobic cultures (cultures growing in the absence of O2). For example, streptococci can produce so much lactic acid and other organic acids from sugar fermentation that their medium becomes acidic and 114 Chapter 6 Microbial Growth Growth rate (hr–1 ) Total growth (cells or mg/ml) Nutrient concentration Nutrient concentration Figure 6.2 Nutrient Concentration and Growth. (a) The effect of changes in limiting nutrient concentration on total microbial yield. At sufficiently high concentrations, total growth will plateau. (b) The effect on growth rate. (a) (b)
6.Microbial Grow 6.1 The Growth Curve 115 Table 6.1 An Example of Exponential Growth ary ph Time 29 esult from eral fa ors operating in 0= have see 2 well bco ironments hav be a p what in o rall size se linking and ce damaged protein As a result of the and many othe ls become harder to kill and more re osmotic dar edical and industrial microbiology.The e is even evidence tha m more hr pathogcns be Death Phase Detrimental environme ental changes like nutrient deprivation and crobial population,like its growth during the exponential pha otal cell number mains constant because the cels mply fail to no eTowandrprodcc.itisasmcdiocd lead.That is, eath is de Although most of microbial population lydies ina Minutes of incub n,the the po s.For this and other reasons, Figure 6.3 Exp ential Microbial Gr wth.The data from table 6.1 The Mathematics of Growth ity of the log plo y and nat a cu will be ninutes. cells after 40 minutes drngspecific length of mcaed the generation time or dou ting pop ulation increase is exponential or logarithmic (figure 6.3)
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 growth is inhibited. Streptococcal cultures also can enter the stationary phase due to depletion of their sugar supply. Finally, there is some evidence that growth may cease when a critical population level is reached. Thus entrance into the stationary phase may result from several factors operating in concert. As we have seen, bacteria in a batch culture may enter stationary phase in response to starvation. This probably often occurs in nature as well because many environments have quite low nutrient levels. Starvation can be a positive experience for bacteria. Many do not respond with obvious morphological changes such as endospore formation, but only decrease somewhat in overall size, often accompanied by protoplast shrinkage and nucleoid condensation. The more important changes are in gene expression and physiology. Starving bacteria frequently produce a variety of starvation proteins, which make the cell much more resistant to damage in a variety of ways. They increase peptidoglycan cross-linking and cell wall strength. The Dps (DNA-binding protein from starved cells) protein protects DNA. Chaperones prevent protein denaturation and renature damaged proteins. As a result of these and many other mechanisms, the starved cells become harder to kill and more resistant to starvation itself, damaging temperature changes, oxidative and osmotic damage, and toxic chemicals such as chlorine. These changes are so effective that some bacteria can survive starvation for years. Clearly, these considerations are of great practical importance in medical and industrial microbiology. There is even evidence that Salmonella typhimurium and some other bacterial pathogens become more virulent when starved. Death Phase Detrimental environmental changes like nutrient deprivation and the buildup of toxic wastes lead to the decline in the number of viable cells characteristic of the death phase. The death of a microbial population, like its growth during the exponential phase, is usually logarithmic (that is, a constant proportion of cells dies every hour). This pattern in viable cell count holds even when the total cell number remains constant because the cells simply fail to lyse after dying. Often the only way of deciding whether a bacterial cell is viable is by incubating it in fresh medium; if it does not grow and reproduce, it is assumed to be dead. That is, death is defined to be the irreversible loss of the ability to reproduce. Although most of a microbial population usually dies in a logarithmic fashion, the death rate may decrease after the population has been drastically reduced. This is due to the extended survival of particularly resistant cells. For this and other reasons, the death phase curve may be complex. The Mathematics of Growth Knowledge of microbial growth rates during the exponential phase is indispensable to microbiologists. Growth rate studies contribute to basic physiological and ecological research and the solution of applied problems in industry. Therefore the quantitative aspects of exponential phase growth will be discussed. During the exponential phase each microorganism is dividing at constant intervals. Thus the population will double in number during a specific length of time called the generation time or doubling time. This situation can be illustrated with a simple example. Suppose that a culture tube is inoculated with one cell that divides every 20 minutes (table 6.1). The population will be 2 cells after 20 minutes, 4 cells after 40 minutes, and so forth. Because the population is doubling every generation, the increase in population is always 2n where n is the number of generations. The resulting population increase is exponential or logarithmic (figure 6.3). 6.1 The Growth Curve 115 Table 6.1 An Example of Exponential Growth Division Population Timea Number 2n (N0 2n ) log10Nt 0 020 1 1 0.000 20 1 21 2 2 0.301 40 2 22 4 4 0.602 60 3 23 8 8 0.903 80 4 24 16 16 1.204 100 5 25 32 32 1.505 120 6 26 64 64 1.806 a The hypothetical culture begins with one cell having a 20-minute generation time. 90 80 70 60 50 40 30 20 10 0 1.500 1.000 0.500 0.000 Log10 number of cells ( ) Number of cells ( ) 0 20 40 60 80 100 120 Minutes of incubation Figure 6.3 Exponential Microbial Growth. The data from table 6.1 for six generations of growth are plotted directly (•–•) and in the logarithmic form ( °–° ). The growth curve is exponential as shown by the linearity of the log plot.
6.Microbial Growth 6 Chapter 6 Microbial Growth These observations can be equations for the Let No=the initial population number Nthe population at time ho) n=the number of generations in time t Then inspection of the results in table 6.1 will show that N=×2” Solving for n,the number of generations.where all loga rithms are to the base 10. log N,=log No+nlog 2.and -s水-s The time it takes a populatic to double in size thatis the m N=2No Time (hours) k-s2g4.s2+g-s Figu re 6.4 Gen tion Time Determination.The tion tim ve.I re plotted wi h the of c The mean generation time is the reciprocal of the mean growth rate constant The mean generation time ()can be determined directly from semilogarithmic plot of the growth data (figure 6.4)and the times in nature are usually much longer than in culture. 2.Defne ubalanced gwthif-uxrmt alimiting utrient have on the yield 20 gen/r0.5 hr/gen.or 30 min/gen Generation times vary markedly with the species of mi croorganism and environmental conditions.They range from less
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 These observations can be expressed as equations for the generation time. Let N0 the initial population number Nt the population at time t n the number of generations in time t Then inspection of the results in table 6.1 will show that Nt N0 2n . Solving for n, the number of generations, where all logarithms are to the base 10, log Nt log N0 n · log 2, and log Nt log N0 log Nt log N0 n ______________ ______________ log 2 0.301 The rate of growth during the exponential phase in a batch culture can be expressed in terms of the mean growth rate constant (k). This is the number of generations per unit time, often expressed as the generations per hour. n log Nt log N0 k __ ______________ t 0.301t The time it takes a population to double in size—that is, the mean generation time or mean doubling time (g), can now be calculated. If the population doubles (t g), then Nt 2 N0. Substitute 2N0 into the mean growth rate equation and solve for k. log (2N0) log N0 log 2 log N0 log N0 k ________________ _____________________ 0.301g 0.301g 1 k __ g The mean generation time is the reciprocal of the mean growth rate constant. 1 g __ k The mean generation time (g) can be determined directly from a semilogarithmic plot of the growth data (figure 6.4) and the growth rate constant calculated from the g value. The generation time also may be calculated directly from the previous equations. For example, suppose that a bacterial population increases from 103 cells to 109 cells in 10 hours. log 109 log 103 9 3 k _______________ ______ 2.0 generations/hr (0.301)(10 hr) 3.01 hr 1 g _________ 0.5 hr/gen. or 30 min/gen. 2.0 gen./hr Generation times vary markedly with the species of microorganism and environmental conditions. They range from less than 10 minutes (0.17 hours) for a few bacteria to several days with some eucaryotic microorganisms (table 6.2). Generation times in nature are usually much longer than in culture. 1. Define growth. Describe the four phases of the growth curve in a closed system and discuss the causes of each. 2. Define balanced growth, unbalanced growth, shift-up experiment, and shift-down experiment. 3. What effect does increasing a limiting nutrient have on the yield of cells and the growth rate? 4. What are the generation or doubling time and the mean growth rate constant? How can they be determined from growth data? 116 Chapter 6 Microbial Growth Time (hours) Number of cells (×107 ) 1 2 3 45 g 0 0.10 0.50 1.00 2.00 3.00 Lag phase Exponential (log) phase Figure 6.4 Generation Time Determination. The generation time can be determined from a microbial growth curve. The population data are plotted with the logarithmic axis used for the number of cells. The time to double the population number is then read directly from the plot. The log of the population number can also be plotted against time on regular axes.