Chapter 3 Bacterial Growth Raina M. Maier 3.1 Growth in Pure Culture in a Flask 3.2 Continuous Culture 3.4 Mass Balance of Growth 3.1.1 The Lag Phase 3.3 Growth in the Environment 3.4.1 Aerobic Conditions 3.1.2 The Exponential Phase 3.3.1 The Lag Phase 3.4.2 Anaerobic Conditions 3.1.3 The Stationary Phase 3.3.2 The Exponential Phase Questions and Problems 3.1.4 The Death Phase 3.3.3 The Stationary and Death References and Recommended 3.1.5 Effect of Substrate Phases Readings Concentration on Growth Bacterial growth is a complex process involving numerous using pure cultures of microorganisms. There are two anabolic (synthesis of cell constituents and metabolites) and approaches to the study of growth under such controlled catabolic (breakdown of cell constituents and metabolites) conditions: batch culture and continuous culture. In a batch reactions. Ultimately, these biosynthetic reactions result in culture the growth of a single organism or a group of organ- cell division as shown in Figure 3.1 . In a homogeneous rich isms, called a consortium, is evaluated using a defined culture medium, under ideal conditions, a cell can divide medium to which a fixed amount of substrate (food) is in as little as 10 minutes. In contrast, it has been suggested added at the outset. In continuous culture there is a steady that cell division may occur as slowly as once every 100 influx of growth medium and substrate such that the amount years in some subsurface terrestrial environments. Such of available substrate remains the same. Growth under both slow growth is the result of a combination of factors batch and continuous culture conditions has been well char- including the fact that most subsurface environments are acterized physiologically and also described mathematically. both nutrient poor and heterogeneous. As a result, cells are This information has been used to optimize the commercial likely to be isolated, cannot share nutrients or protection production of a variety of microbial products including anti- mechanisms, and therefore never achieve a metabolic state biotics, vitamins, amino acids, enzymes, yeast, vinegar, and that is efficient enough to allow exponential growth. alcoholic beverages. These materials are often produced in Most information available concerning the growth of large batches (up to 500,000 liters) also called large-scale microorganisms is the result of controlled laboratory studies fermentations. Membrane Wall DNA FIGURE 3.1 Electron micrograph of Bacillus subtilis, a gram-positive bacterium, dividing. Magnification 31,200 . Reprinted with permission from Madigan et al ., 1997. Environmental Microbiology Copyright © 2000, 2009 by Academic Press. Inc. All rights of reproduction in any form reserved. 37 CCh003-P370519.inddh003-P370519.indd 3377 77/21/2008/21/2008 33:38:34:38:34 PPMM 38 PART | I Review of Basic Microbiological Concepts Unfortunately, it is difficult to extend our knowledge 3.1 GROWTH IN PURE CULTURE of growth under controlled laboratory conditions to an IN A FLASK understanding of growth in natural soil or water environ- ments, where enhanced levels of complexity are encoun- Typically, to understand and define the growth of a particu- tered ( Fig. 3.2 ). This complexity arises from a number of lar microbial isolate, cells are placed in a liquid medium factors, including an array of different types of solid sur- in which the nutrients and environmental conditions are faces, microenvironments that have altered physical and controlled. If the medium supplies all nutrients required chemical properties, a limited nutrient status, and consor- for growth and environmental parameters are optimal, the tia of different microorganisms all competing for the same increase in numbers or bacterial mass can be measured as limited nutrient supply (see Chapter 4). Thus, the current a function of time to obtain a growth curve. Several dis- challenge facing environmental microbiologists is to under- tinct growth phases can be observed within a growth curve stand microbial growth in natural environments. Such an ( Fig. 3.3 ). These include the lag phase, the exponential or understanding would facilitate our ability to predict rates of log phase, the stationary phase, and the death phase. Each nutrient cycling (Chapter 14), microbial response to anthro- of these phases represents a distinct period of growth that pogenic perturbation of the environment (Chapter 17), is associated with typical physiological changes in the cell microbial interaction with organic and metal contaminants culture. As will be seen in the following sections, the rates (Chapters 20 and 21), and survival and growth of pathogens of growth associated with each phase are quite different. in the environment (Chapters 22 and 27). In this chapter, we begin with a review of growth under pure culture condi- 3.1.1 The Lag Phase tions and then discuss how this is related to growth in the environment. The first phase observed under batch conditions is the lag phase in which the growth rate is essentially zero. When an inoculum is placed into fresh medium, growth begins after a period of time called the lag phase. The lag phase is defined to transition to the exponential phase after the ini- tial population has doubled ( Yates and Smotzer, 2007 ). The lag phase is thought to be due to the physiological adapta- tion of the cell to the culture conditions. This may involve a time requirement for induction of specific messenger vs. RNA (mRNA) and protein synthesis to meet new culture requirements. The lag phase may also be due to low initial densities of organisms that result in dilution of exoenzymes (enzymes released from the cell) and of nutrients that leak from growing cells. Normally, such materials are shared by cells in close proximity. But when cell density is low, these FIGURE 3.2 Compare the complexity of growth in a flask and growth materials are diluted and not as easily taken up. As a result, in a soil environment. Although we understand growth in a flask quite initiation of cell growth and division and the transition to well, we still cannot always predict growth in the environment. exponential phase may be slowed. 9.0 Turbidity (optical density) 1.0 0.75 8.0 Stationary 0.50 7.0 Death CFU/ml 10 6.0 Log 0.25 Optical density 5.0 Exponential 4.0 0.1 Lag Time FIGURE 3.3 A typical growth curve for a bacterial population. Compare the difference in the shape of the curves in the death phase (colony-forming units versus optical density). CCh003-P370519.inddh003-P370519.indd 3388 77/21/2008/21/2008 33:38:38:38:38 PPMM Chapter | 3 Bacterial Growth 39 The lag phase usually lasts from minutes to several placed into a medium other than TSB, for example, a min- hours. The length of the lag phase can be controlled to eral salts medium with glucose as the sole carbon source, some extent because it is dependent on the type of medium a lag phase will be observed while the cells reorganize and as well as on the initial inoculum size. For example, if an shift physiologically to synthesize the appropriate enzymes inoculum is taken from an exponential phase culture in for glucose catabolism. trypticase soy broth (TSB) and is placed into fresh TSB Finally, if the inoculum size is small, for example, medium at a concentration of 10 6 cells/ml under the same 10 4 cells/ml, and one is measuring activity, such as disap- growth conditions (temperature, shaking speed), there will pearance of substrate, a lag phase will be observed until be no noticeable lag phase. If the inoculum is taken from the population reaches approximately 10 6 cells/ml. This is a stationary phase culture, however, there will be a lag illustrated in Figure 3.4 , which compares the degradation phase as the stationary phase cells adjust to the new condi- of phenanthrene in cultures inoculated with 10 7 and with tions and shift physiologically from stationary phase cells 10 4 colony-forming units (CFU) per milliliter. Although the to exponential phase cells. Similarly, if the inoculum is degradation rate achieved is similar in both cases (compare the slope of each curve), the lag phase was 1.5 days when a 4 100 500 mg/l phenanthrene low inoculum size was used (10 CFU/ml) in contrast to only 105 mg/l cyclodextrin 0.5 day when the higher inoculum was used (10 7 CFU/ml). 80 Inoculum ϭ 104 cells/ml 60 3.1.2 The Exponential Phase 40 Remaining The second phase of growth observed in a batch system is Inoculum ϭ 107 cells/ml phenanthrene (%) 20 the exponential phase. The exponential phase is character- 0 ized by a period of the exponential growth—the most rapid growth possible under the conditions present in the batch sys- 012345678tem. During exponential growth the rate of increase of cells Time (days) in the culture is proportional to the number of cells present FIGURE 3.4 Effect of inoculum size on the lag phase during degrada- at any particular time. There are several ways to express this tion of a polyaromatic hydrocarbon, phenanthrene. Because phenanthrene concept both theoretically and mathematically. One way is is only slightly soluble in water and is therefore not readily available for cell uptake and degradation, a solubilizing agent called cyclodextrin was to imagine that during exponential growth the number of 0 1 2 3 added to the system. The microbes in this study were not able to utilize cells increases in the geometric progression 2 , 2 , 2 , 2 cyclodextrin as a source of carbon or energy. Courtesy E. M. Marlowe. until, after n divisions, the number of cells is 2 n ( Fig.