Microbial Growth and Its Control PDF

Microbial Growth and Its Control microbiologynow Picking Apart a Microbial Consortium In nature, certain metabolic processes are carried out by microbes that team up to get the job done, a cozy arrangement called a con-...

Microbial Growth and Its Control microbiologynow Picking Apart a Microbial Consortium In nature, certain metabolic processes are carried out by microbes that team up to get the job done, a cozy arrangement called a con- 5 sortium. Such is the case with the oxidation of methane (CH4) linked to the reduction of sulfate (SO42-) in anoxic marine sediments. The overall reaction (CH4 + SO42- S HCO3- + HS- + H2O) is exergonic and the small amount of energy released is shared between two distinct microbes. The methane oxidizer in the consortium is a species of Archaea nicknamed ANME (for anaerobic methanotroph, blue in photo), and its sulfate-reducing partner is a species of Bacteria (brown in photo). The consortium is thought to play a key role in the carbon cycle as a major methane sink, and thus a detailed picture of how it works is important to our understanding of the global carbon economy, climate change, and marine biogeochemistry. Researchers have tried for years to separate the consortium into its components but always found that methane oxidation required both organisms. However, some researchers hypothesized that it might be possible to replace the sulfate reducer with an artificial electron acceptor and that this might unlock the consortium and allow the methanotroph to grow in pure culture. Using an electron acceptor called AQDS, the scientists discovered that they could turn off sulfate reduction in the consortium while maintaining CH4 oxi- dation. During this process, the methanotroph used electrons from CH4 to reduce AQDS rather than passing them on to its sulfate- reducing partner. Several other electron acceptors known to support anaerobic respiration also sustained methane oxidation, giving hope that ANME may eventually be obtained in pure culture. I Cell Division and Population Growth 174 The ability to grow a microbe in pure culture is the “gold standard” for the II Culturing Microbes and Measuring study of its physiology, biochemistry, regulation, and several other aspects of its Their Growth 180 biology. In the case of the ANME–sulfate reducer consortium, several physiologies III Environmental Effects on Growth: were active at once, and resolving these many reactions proved to be a major Temperature 188 scientific challenge. However, if further work shows that ANME can be removed from the consortium and grown in pure culture, detailed aspects of its biology IV Environmental Effects on Growth: can be studied that were not possible when the organism was tightly coupled to pH, Osmolarity, and Oxygen 194 its partner in the consortium (photo). V Controlling Microbial Growth 200 Source: Scheller, S., H. Yu, G.L. Chadwick, S.E. McGlynn, and V.J. Orphan. 2016. Artificial electron acceptors decouple archaeal methane oxidation from sulfate reduction. Science 351: 703–706. 173  174 UNIT 2 MiCrobiAl Grow th And reGul Ation I Cell division and Population Growth Patricia Dominguez-Cuevas I n previous chapters we discussed cell structure and function (Chapter 2) and the principles of microbial nutrition and metabo- lism (Chapter 3). In Chapter 4 we learned the genetic processes that encode the structures and metabolic activities of cells. Here we begin a new unit whose focus is microbial growth and its regulation. (a) (b) (c) In Chapter 5 we lay the groundwork for the entire unit by pre- senting the basic principles of exponential growth, how the envi- Figure 5.2 Septa. The septum that separates dividing cells of the bacterium Bacillus unit 2 ronment affects growth, and some principles of microbial growth subtilis is clearly visible in this series of fluorescent micrographs. (a) DAPI stains the control. Then, after we consider the important topic of microbial entire cell. (b) The green fluorescent protein lights up entire cells. (c) A dye that stains regulation in Chapter 6, we will revisit microbial growth in only the cytoplasmic membrane shows that septa contain membrane (and cell wall) Chapter 7 and reexamine the process from a molecular and regula- material. tory perspective. We finish the unit by delving into the world of viruses and their replication. Although not cells, viruses are criti- The partition that forms between dividing cells is called a septum cally important microbes whose replication shows parallels with and results from the inward growth of the cytoplasmic membrane the growth of microbial cells. and cell wall from opposing directions; septum formation contin- ues until the two daughter cells are pinched off. There are some 5.1 binary Fission, budding, and biofilms variations in this general pattern of binary fission. In some bacte- Growth is the result of cell division and is the ultimate process in ria, such as Bacillus subtilis, a septum forms without cell wall con- the life of a microbial cell. In microbiology, growth is defined as an striction (Figure 5.2), while in the budding bacterium Caulobacter increase in the number of cells. Microbial cells have a finite life span, (see Figure 5.3) constriction occurs but no septum is formed. and a species is maintained only as a result of continued growth of its population. As macromolecules accumulate in the cytoplasm Cell Generations and Generation Time of a cell, they assemble into major cell structures, such as the cell When one cell eventually separates to form two cells, we say that wall, cytoplasmic membrane, flagella, ribosomes, enzyme com- one generation has occurred, and the time required for this process plexes, and so on, eventually leading to the events of cell division is called the generation time (Figure 5.1 and see Figure 5.6). itself. In a growing culture of a rod-shaped bacterium such as During one generation, all cellular constituents increase propor- Escherichia coli, cells elongate to approximately twice their origi- tionally. Each daughter cell receives a copy of the chromosome(s) nal length and then form a partition that constricts the cell into and sufficient copies of ribosomes and all other macromolecular two daughter cells (Figure 5.1). This process is called binary complexes, monomers, and inorganic ions to begin life as an inde- fission (“binary” to indicate that two cells have arisen from one). pendent entity. Partitioning of the replicated DNA molecule between the two daughter cells depends on the DNA remaining attached to the cytoplasmic membrane during division, with con- striction leading to separation of the chromosomes, one to each daughter cell ( Figure 7.1). The generation time of a given bacterial species is variable and Cell elongation depends on nutritional and genetic factors, and on temperature. Under the best nutritional conditions, the generation time of a laboratory culture of E. coli is about 20 min. A few bacteria can One generation grow even faster than this, but many bacteria grow much slower, Septum with generation times of hours or days being more common. In Septum nature, microbial cells probably grow much slower than their formation maximum rates observed in the laboratory. This is because the conditions and resources necessary for optimal growth in the Completion of laboratory are often not present in a natural habitat, and unlike septum; growth in laboratory pure cultures, microbes in nature coexist formation of with other microbes in microbial communities ( Figure 1.1) and walls; cell separation must compete with their neighbors for resources and space. Budding Cell Division Although cell division in most bacteria occurs by binary fission, in Figure 5.1 binary fission in a rod-shaped bacterium. Cell numbers (and all a few bacteria other forms of growth and cell division occur. Bud- components of the cells) double every generation. ding bacteria are the primary examples here, and these are cells  CHAPTER 5 M i c r o b i a l G r o w t h a n d i t s C o n t r o l 175 that divide as a result of unequal cell growth. In contrast to binary fission that yields two equivalent cells (Figure 5.1), budding division forms a totally new daughter cell, with the mother cell retaining its original identity (Figure 5.3 and Section 7.4). A fundamental difference between budding bacteria and bacteria that divide by binary fission is the formation of new cell wall material from a single point (polar growth) rather than throughout the whole cell (intercalary growth) as in binary fis- Janice Carr, CDC sion. An important consequence of polar growth is that large cyto- plasmic structures, such as internal membrane complexes, are not partitioned during the cell division process and must be formed (a) unit 2 de novo in the developing bud. However, this has an advantage in that more complex internal structures can be formed in budding cells than in cells that divide by binary fission, since the latter cells would have to partition these structures between the two daugh- ter cells. Not coincidentally, many budding bacteria, particularly phototrophic and chemolithotrophic species, contain extensive internal membrane systems that house specific enzymes required to perform their particular metabolic specialties. Some budding bacteria form cytoplasmic extensions such as Michael T. Madigan stalks or hyphae, and classic examples are the genera Caulobacter and Hyphomicrobium (Figure 5.3). These organisms form cellular extrusions from which new cells bud off. Other budding bacteria such as the aquatic bacterium Ancalomicrobium produce multiple appendages that resemble arms extending away from the cell (b) ( Figure 15.57b). The appendages increase the surface-to-volume ratio of the cell ( Section 2.2), which increases its ability to Figure 5.4 biofilms. (a) Scanning electron micrograph of a biofilm of cells of Staphylococcus aureus that formed on an indwelling catheter. (b) A microbial mat of extract nutrients from oligotrophic (very dilute) habitats. Many the purple phototrophic bacterium Thermochromatium tepidum that developed in a budding bacteria also have distinctive life cycles, and we consider small sulfidic hot spring in Yellowstone National Park. these and the group as a whole in Section 15.20. Biofilms Ι# Equal products of cell division: Whether dividing by binary fission (Figure 5.1) or some form of budding (Figure 5.3), microbial cells can grow either in suspension Binary fission: most bacteria or attached to surfaces. The suspended lifestyle, called planktonic growth, is the way many bacteria live in nature, for example, organ- ΙΙ# Unequal products of cell division: isms that inhabit the water column of a lake. However, many other 1. Simple budding: Pirellula, Blastobacter microorganisms show sessile growth, meaning that they grow attached to a surface. These attached cells can then develop into biofilms (Figure 5.4). 2. Budding from hyphae: Hyphomicrobium, Rhodomicrobium, A biofilm is an attached polysaccharide matrix containing Pedomicrobium embedded bacterial cells (Figure 5.4a). Biofilms form in stages, beginning with the attachment of planktonic cells. This is fol- lowed by the production of a sticky matrix and further growth 3. Cell division of stalked organism: Caulobacter and development to form the tenacious and nearly impenetrable mature biofilm. Some biofilms form multilayered sheets with dif- ferent organisms present in the individual layers. These biofilms are called microbial mats; mats composed of various phototrophic and chemotrophic bacteria are common in the outflows of hot springs (Figure 5.4b) and in marine intertidal regions. Biofilms are 4. Polar growth without differentiation of cell size: a common growth form for bacteria in nature because the intensely interwoven nature of the structure prevents harmful Rhodopseudomonas, Nitrobacter, Methylosinus chemicals (for example, antibiotics or other toxic substances) from penetrating. Biofilms are also a barrier to bacterial grazing by protists and prevent cells from being washed away into a poten- Figure 5.3 Cell division in different morphological forms of bacteria. The contrast is shown between cell division in conventional bacteria (cells that divide by tially less favorable habitat. binary fission) and in various budding and stalked bacteria.  176 UNIT 2 MiCrobiAl Grow th And reGul Ation Bacterial biofilms affect many aspects of our lives, including Time Total number Time Total number human health. For example, biofilms have been implicated in dif- (h) of cells (h) of cells ficult-to-treat infections of implanted medical devices, such as 0 1 4 256 (28) artificial heart valves and joints, and indwelling devices, such as 0.5 2 4.5 512 (29) 1 4 5 1,024 (210) catheters (Figure 5.4a). Moreover, symptoms of the disease cystic 1.5 8 5.5 2,048 (211) fibrosis are caused by a tenacious bacterial biofilm that fills the 2 16 6 4,096 (212) lungs and prevents gas exchange. Biofilms also cause fouling and 2.5 32.. 3 64.. plugging of water distribution systems and can form in fuel stor- 3.5 128 10 20 1,048,576 (2 ) age tanks, where they contaminate the fuel by producing souring agents such as H2S. (a) We examine biofilms in more detail elsewhere in this book, unit 2 including Sections 7.9, 20.4, and 20.5. 1000 103 Logarithmic plot MINIQUIz (logarithmic scale) Arithmetic (arithmetic scale) Define the term generation. What is meant by the term Number of cells Number of cells plot 102 generation time? How do binary fission and budding cell division differ? 500 How does the biofilm growth mode differ from that of planktonic cells? Which growth mode better protects the 10 bacterial cells from harm? 100 1 0 1 2 3 4 5 5.2 Quantitative Aspects of Time (h) Microbial Growth (b) During cell division, one cell becomes two. During the time that it Figure 5.5 the rate of growth of a microbial culture. (a) Data for a population takes for this to occur (the generation time), both total cell number that doubles every 30 min. (b) Data plotted on arithmetic (left ordinate) and logarithmic and mass double (Figure 5.1). As we will see, cell numbers in a bac- (right ordinate) scales. terial culture can quickly become very large, and so it is necessary to deal with the topic of microbial growth using quantitative progression of the number 2. As one cell divides to become two methods. cells, we express this as 20 S 21. As two cells become four, we Plotting Growth Data express this as 21 S 22 and so on (Figure 5.5a). A fixed relationship exists between the initial number of cells in a culture and the A growth experiment beginning with a single cell having a genera- number present after a period of exponential growth, and this tion time of 30 min is presented in Figure 5.5. This repetitive pat- relationship is expressed as tern, where the number of cells doubles in a constant time interval, is called exponential growth. When the cell number from such N = N02n an experiment is graphed on arithmetic (linear) coordinates as a where N is the final cell number, N0 is the initial cell number, and function of time, one obtains a curve with a continuously increas- n is the number of generations (a single generation is shown in ing slope (Figure 5.5b). By contrast, when the cell number is Figure 5.1) during the period of exponential growth. The genera- plotted on a logarithmic (log10) scale as a function of time (a tion time (g) of the exponentially growing population is t/n, where semilogarithmic graph), as shown in Figure 5.5b, the points fall on t is the duration of exponential growth expressed in days, hours, a straight line. This straight-line function reflects the fact that the or minutes. From knowledge of the initial and final cell numbers cells are growing exponentially and that the population is dou- in an exponentially growing cell population, it is possible to calcu- bling in a constant time interval. late n, and from n and knowledge of t, the generation time, g. Semilogarithmic graphs are also convenient for estimating the The equation N = N02n can be expressed in terms of n by taking generation time of a culture from actual growth data, since gener- the logarithms of both sides and doing some simple algebra to ation times may be inferred directly from the graph as shown in yield the expression n = [3.3(log N - log N0)]. Using this expres- Figure 5.6. For example, when two points on the curve that repre- sion, it is possible to calculate generation times in terms of mea- sent one cell doubling on the Y axis are selected and vertical lines surable quantities, N and N0 (Sections 5.6–5.8 describe methods drawn from them to intersect the X axis, the time interval mea- for quantifying cell numbers). As an example, consider actual sured on the X axis is the generation time (Figure 5.6b). growth data from the graph in Figure 5.6b, in which N = 108, N0 = The Mathematics of Bacterial Growth 5 * 107, and t = 2: The increase in cell number in an exponentially growing bacterial n = 3.3[log(108) - log(5 * 107)] culture can be expressed mathematically as a geometric = 3.3 (8 - 7.69) = 3.3(0.301) = 1  CHAPTER 5 M i c r o b i a l G r o w t h a n d i t s C o n t r o l 177 Slope = log (4 × 107) – log (2 × 107) constant, abbreviated k. The instantaneous growth rate constant 6 expresses the rate at which the population is growing at any instant = 0.05 (by contrast, g is the mean time required for the cell population to 4 × 107 double); k is expressed in units of reciprocal hours (h-1). The instantaneous rate of growth is a function of the number of cells at a given time (N) multiplied by k, or dN/dt = kN. By integra- Cells/ml tion using natural logarithms (log e), this equation can be 2 × 107 reexpressed as N = N0ekt. Taking the log10 of both sides of this t=6h equation converts natural logs to log10 (so that N can be plotted n=1 Population g = nt = 6 h against t on semilog paper, Figure 5.5b and Figure 5.6) and yields doubles in 6 h the expression log N = kt/2.303 + log N0. The slope of this function unit 2 (k/2.303) is also equal to 0.301/g (Figure 5.6), and thus k can be 0 1 2 3 4 5 6 expressed in terms of g by the expression k = 0.693/g. Time (h) Armed with knowledge of n and t, one can calculate g and k for (a) different microorganisms growing under various conditions. This is often useful for optimizing culture conditions for a newly iso- 1 × 108 Slope = log (108) – log (5 × 107) lated organism and also for testing the positive or negative effect 2 = 0.15 of some treatment on a bacterial culture. For example, comparison 8 × 107 with an unamended control allows factors that stimulate or 6 × 107 inhibit growth to be identified by measuring their effect on the various growth parameters presented here. 4 × 107 Consequences of Exponential Growth Cells/ml During exponential growth, the increase in cell number is ini- 3 × 107 Population tially rather slow but increases at an ever-faster rate. In the later doubles in stages of exponential growth, this results in an explosive increase 2h in cell numbers. For example, in the experiment shown in 2 × 107 2h Figure!5.5, the rate of cell production in the first 30 min of growth t=2 is 1 cell per 30 min. However, between 4 and 4.5 h of growth, the n=1 rate of cell production is 256 cells per 30 min, and between 5.5 g = n–t = 2 h and 6 h of growth it is 2048 cells per 30 min. Because of this, cell 1 × 107 numbers in laboratory cultures of bacteria can quickly become 0 1 2 3 4 5 very large, with final population sizes of 710 9! cells/ml not Time (h) uncommon. (b) Besides being a theoretical consideration, exponential growth can have implications in everyday life. Consider something we Figure 5.6 Calculating microbial growth parameters. Method of estimating have all experienced, the spoilage of milk. The lactic acid bacteria the generation times (g) of exponentially growing populations with g of (a) 6 h and responsible for the soured flavor of spoiled milk contaminate the (b) 2 h from data plotted on semilogarithmic graphs. The slope of each line is equal to milk during its collection and exist in fresh, pasteurized milk in 0.301/g, and n is the number of generations in the time t. All numbers are expressed in scientific notation; that is, 10,000,000 is 1 * 107, 60,000,000 is 6 * 107, and so on. low numbers; these organisms grow slowly at refrigerator temper- ature (4°C) but much faster at room temperature. If a bottle of fresh milk is left to stand at room temperature overnight, some lac- Thus, in this example, g = t/n = 2/1 = 2 h. If exponential growth tic acid is made, but not enough to affect milk quality. However, if continued for another 2 h, the cell number would be 2 * 108. Two week-old milk, which now contains a week’s worth of slow bacte- hours later the cell number would be 4 * 108, and so on. Besides rial growth (and thus much higher cell numbers), is left standing determining the generation time of an exponentially growing cul- under the same conditions, a huge amount of lactic acid is made, ture by inspection of graphical data (Figure 5.6b), g can be calcu- and spoilage results. lated directly from the slope of the straight-line function obtained in a semilogarithmic plot of exponential growth. The slope of this MINIQUIz line is equal to log(¢N)/¢t and this equals 0.301 n/t (or 0.301/g). In What is a semilogarithmic plot and what information can we the example of Figure 5.6b, the slope would thus be 0.301/2, or derive from it? 0.15. Since g is equal to 0.301/slope, we arrive at the same value of For an exponentially growing culture that increases from 5 * 2 for g. 106 cells/ml to 5 * 108 cells/ml in 8 h, calculate g, n, and k for this culture. The Instantaneous Growth Rate Constant For testing a bacterium’s response to a toxic substance, why Other expressions are often useful in describing exponential would g be useful information? growth, and chief among these is the instantaneous growth rate  178 UNIT 2 MiCrobiAl Grow th And reGul Ation 5.3 the Microbial Growth Cycle state and are thus most desirable for studies of their enzymes or other cell components. Rates of exponential growth vary greatly. The data presented in Figures 5.5 and 5.6 are a reflection of expo- Exponential growth rates are influenced by the growth conditions nentially growing cells. But exponential growth is only part of the an organism is experiencing as well as genetic characteristics of microbial growth cycle. For several reasons, an organism growing in the organism itself. an enclosed vessel, such as a tube or a flask (a batch culture), In general, prokaryotic cells grow faster than eukaryotes, and cannot grow exponentially indefinitely. Instead, a typical growth small eukaryotes tend to grow faster than large ones. But when all curve for the population is obtained, as illustrated in Figure 5.7. The organisms are considered, doubling times for exponential growth growth curve describes the entire growth cycle and is made up of vary enormously, from as little as a few minutes to days or weeks. four phases: lag, exponential, stationary, and death. Organisms living under very stressful conditions, such as those in Earth’s nutrient-poor deep subsurface, may divide every few Lag and Exponential Phases unit 2 months (or even years) ( Sections 20.7 and 20.13). Much plays When a microbial culture is inoculated into fresh growth media into an organism’s exponential growth rate and it is hard to pre- (see Section 5.5), growth begins only after a period of time called dict how fast an organism can grow until it is brought into labora- the lag phase. This interval may be brief or extended depending on tory culture and its ideal growth conditions have been identified. the history of the cells used as inocula and the composition of the growth medium and growth conditions (see Sections 5.9–5.14). If Stationary and Death Phases an exponentially growing culture is transferred into the same In a batch culture, exponential growth cannot be maintained medium under the same conditions of growth, there will be indefinitely. Consider the fact that a single cell of a bacterium essentially no lag and exponential growth will begin immediately. weighing one-trillionth (10-12) of a gram and growing exponen- However, if the inoculum is taken from an old culture, there is tially with a 20-min generation time would produce, if allowed to usually a lag because the cells are depleted of various essential con- grow exponentially in batch culture for 48 h, a population of cells stituents and time is required for their biosynthesis. that weighed 4000 times the weight of planet Earth! Obviously A lag is also observed when a microbial culture is transferred this is impossible, and growth becomes limited in such cultures from a nutrient-rich culture medium to one that is nutrient-poor. because either an essential nutrient in the culture medium is In order to grow, cells must have a complete complement of depleted or the organism’s waste products accumulate. When enzymes for synthesis of the essential metabolites not present in exponential growth ceases for one (or both) of these reasons, the that medium. Hence, when transferred to a nutrient-poor medium, population enters stationary phase (Figure 5.7). time is needed for the biosynthesis of new enzymes and for these In the stationary phase, there is no net increase or decrease in to produce a sufficient pool of required metabolites before growth cell number and thus the growth rate of the population is zero. can actually begin. These events occur during the lag period. Despite growth arrest, energy metabolism and biosynthetic pro- When a growing cell population doubles at regular intervals cesses in stationary phase cells may continue, but typically at a (Section 5.2) the cells are said to be in the exponential phase of greatly reduced rate. Some cells may even divide during stationary growth. Exponential phase cells are typically in their healthiest Growth phases Lag Exponential Stationary Death 10 1.0 Optical density (OD) 0.75 organisms/ml 9 Turbidity Log10 viable (optical density) 0.50 Viable count 8 0.25 7 6 0.1 Time Figure 5.7 typical growth curve for a bacterial population. A viable count measures the cells in the culture that are capable of reproducing. Optical density (turbidity), a quantitative measure of light scattering by a liquid culture (see Figure 5.16), increases with the increase in cell number.  CHAPTER 5 M i c r o b i a l G r o w t h a n d i t s C o n t r o l 179 phase, but no net increase in cell number occurs. This is because consumption and waste production. These limitations can be cir- some cells in the population grow while others die, the two pro- cumvented in a continuous culture device. Unlike a batch culture, cesses balancing each other out (cryptic growth). Eventually, the which is a closed system, a continuous culture is an open system. In population will enter the death phase of the growth cycle, which, the continuous culture growth vessel, a known volume of sterile like the exponential phase, occurs as an exponential function medium is added at a constant rate while an equal volume of (Figure 5.7). Typically, however, the rate of cell death is much spent culture medium (which also contains cells) is removed at slower than the rate of exponential growth and viable cells may the same rate. Once in equilibrium, the culture volume, cell num- remain in a culture for months or even years. ber, and nutrient/waste product status remain constant, and the The phases of bacterial growth shown in Figure 5.7 are reflec- culture attains steady state. tions of the events in a population of cells, not in individual cells. Thus, the terms lag phase, exponential phase, and so on have no The Chemostat and the Concept of Steady State unit 2 meaning with respect to individual cells but only to cell popula- The most common type of continuous culture is the chemostat, tions. Growth of an individual cell (Section 5.1) is a prerequisite a device wherein both specific growth rate (how fast the cells for population growth. But it is population growth that is most grow) and cell density (how many cells per ml are obtained) can relevant to the ecology of microorganisms, because measurable be controlled independently (Figure 5.8). In the chemostat, two microbial activities require microbial populations, not just an factors govern the specific growth rate and cell density, respec- individual microbial cell. tively: (1) the dilution rate (D) which is expressed as F/V, where F is the flow rate (the rate at which fresh medium is pumped in and MINIQUIz spent medium is removed), and V is the culture volume; and In which phase of the growth curve do cells divide in a constant (2)! the concentration of a limiting nutrient, such as a carbon or time period? nitrogen source, present in the sterile medium entering the chemostat vessel. Under what conditions would a lag phase not occur? When a chemostat filled with sterile medium is inoculated, the Why do cells enter stationary phase? cells begin growing and increase in number more rapidly than they are removed in the overflow. As cell numbers increase, the level of the limiting nutrient in the culture decreases. This reduc- 5.4 Continuous Culture tion in the growth-limiting nutrient serves as a feedback loop to reduce the specific growth rate, leading to a decrease in cell den- Up to this point our consideration of microbial population sity as cells are removed in the overflow. However, once the limit- growth has been confined to batch cultures. The environment in a ing nutrient has decreased to a value just sufficient to support a batch culture is constantly changing because of nutrient specific growth rate that compensates for losses of cells through outflow, the che- Fresh Flow-rate mostat reaches steady state, a condition medium from regulator where cell density and substrate concen- reservoir tration do not change over time. In steady state, the specific growth rate Sterile air or other gas of the culture is equal to D; that is, the Gaseous rate of increase in cell numbers due to headspace growth is equal to the rate of decrease in cell numbers due to dilution (outflow). Culture The chemostat steady state is thus a vessel dynamic condition in which cells are continuously growing and continuously being removed. Indeed, this dependency Culture of specific growth rate on substrate concentration (Figure 5.9) drives the feedback loop that allows the chemostat Overflow to be self-regulating and the experi- menter to choose the growth rate of the culture by simply changing the speed of the pump. In a batch culture, the nutri- Hubert Bahl Effluent containing microbial cells ent concentration also affects both growth rate and cell yield, but when the (a) (b) nutrient level exceeds that which sup- ports the maximal growth rate, only cell Figure 5.8 Continuous culture device (chemostat). The population density is controlled by the concentration of a yield is increased by additional substrate limiting nutrient in the reservoir, and the growth rate is controlled by the dilution rate; both parameters are set by the experimenter. (a) Chemostat components. (b) Photo of a chemostat setup. (Figure 5.9).  180 UNIT 2 MiCrobiAl Grow th And reGul Ation Rate and periods—weeks or even months. Exponential phase cells are usu- Only yield affected yield affected ally most desirable for physiological experiments. Such cells are available at any time in the chemostat, and the vessel can be ) repeatedly sampled. Chemostat cultures have been used to study ) the growth and physiology of cells at submaximal growth rates, Growth rate ( and from such studies, several tenets of microbial physiology have Growth yield ( emerged; these include the fact that the ribosome content of cells increases in proportion to their specific growth rate and that nutrient concentration controls both specific growth rate and cell yield (Figure 5.9). The chemostat has also been used in studies of microbial ecol- unit 2 ogy and evolution. For example, because the chemostat can mimic 0 0.1 0.2 0.3 0.4 0.5 the low substrate concentrations that are often found in nature, it Nutrient concentration (mg/ ml) is possible to ask which organisms in mixed cultures of known composition compete best at various specific growth rates or when Figure 5.9 the effect of nutrients on growth. Relationship between nutrient concentration, growth rate (green curve), and growth yield (red curve). Only at low particular nutrients are limiting. This can be done by monitoring nutrient concentrations are both growth rate and growth yield affected. changes in the diversity of the microbial community as a function of variations in D or the limiting nutrient. One can study the evo- lution of a pure culture in the chemostat by subjecting the culture The effects of varying D in a chemostat culture are shown sche- to a growth or nutrient challenge and asking whether these condi- matically in Figure 5.10. As seen, there are rather wide limits over tions more rapidly select for particular spontaneous mutants dis- which D controls growth rate, although at both very low and very playing new physiological properties than in batch cultures where high D, the desired steady state with actively growing cells breaks all nutrients are in excess. down. In steady state, if the concentration of the limiting nutrient Chemostats have also been used for the direct enrichment and in the inflowing medium is increased at a constant D, cell density isolation of bacteria from nature. From a natural sample, one can will increase but the growth rate will remain the same. Thus, by select a stable population under the chosen conditions of nutrient varying D or the concentration of the growth-limiting nutrient, concentration and D and then slowly increase D until a single one can establish dilute (for example, 105 cells/ml), moderate (for organism remains. In this way, microbiologists studying the example, 107 cells/ml), or dense (for example, 109 cells/ml) cell growth rates of various soil bacteria isolated a bacterium with a populations growing at any specific growth rate. 6-min doubling time—the fastest-growing bacterium known! Experimental Uses of the Chemostat MINIQUIz A practical advantage of the chemostat is that a cell population can be maintained in the exponential growth phase for long How do microorganisms in a chemostat differ from microorganisms in a batch culture? What happens in a chemostat if the dilution rate exceeds the maximal specific growth rate of the organism? 10 5 Bacterial concentration Do pure cultures have to be used in a chemostat? Limiting nutrient concentration (g/l) Bacterial concentration (g/l) 6 8 4 II Culturing Microbes and Doubling time (h) 6 3 4 Measuring their Growth 4 2 2 1 Doub ling t ime 2 I n the next few sections we consider how microbes are grown in laboratory culture and how microbial growth is measured. Culturing microbes and assessing their growth are common Limiting nutrient events in the daily routine of many microbiologists and microbi- 0 0 0 ology laboratories. 0 0.25 0.5 0.75 1.0 Dilution rate (h–1) Washout 5.5 Growth Media and laboratory Culture Figure 5.10 Steady-state relationships in the chemostat. The dilution rate Laboratory cultures of microorganisms are grown in culture (D) is determined from the flow rate and the volume of the culture vessel. Note that at media, nutrient solutions tailored to the particular organism to high D, growth cannot balance dilution, and the population washes out. Note also that be grown. Because laboratory culture is required for the detailed although both the population density remains constant and the concentration of the study of any microorganism, careful attention must be paid to the growth-limiting nutrient remains near zero during steady state, the specific growth rate selection and preparation of media for laboratory culture to be (as reflected in the doubling time) can vary over a wide range.  CHAPTER 5 M i c r o b i a l G r o w t h a n d i t s C o n t r o l 181 successful. Culture media must be sterilized before use, and steril- (tryptic soy broth), yeast cells (yeast extract), or any of a number of ization is typically achieved by heating the medium under pres- other highly nutritious substances (Table 5.1). Such digests are sure in an autoclave. We discuss the operation and principles of the commercially available in dehydrated form and need only be autoclave in Section 5.15, along with other methods for sterilizing hydrated with distilled water to form a culture medium. However, culture media and laboratory devices. the disadvantage of a complex medium is that its exact nutritional composition is unknown. An enriched medium, used for the culture Classes of Culture Media of nutritionally demanding (fastidious) microbes, many of which Two broad classes of culture media are used in microbiology: are pathogens, is a complex medium to which additional highly defined media and complex media. Defined media are prepared nutritious substances (such as serum or blood) are added. by adding precise amounts of pure inorganic or organic chemi- Culture media are sometimes prepared that are selective or dif- cals to distilled water. Therefore, the exact composition of a ferential (or both), especially media used in diagnostic microbi- unit 2 defined medium (in both a qualitative and quantitative sense) is ology. A selective medium contains compounds that inhibit the known. Of major importance in any culture medium is the car- growth of some microorganisms but not others. For example, bon source because all cells need large amounts of carbon to selective media are available for the isolation of certain patho- make new cell material ( Section 3.1). The particular carbon gens, such as Salmonella or those strains of E. coli that cause food- source and its concentration depend on the organism to be cul- borne illnesses. A differential medium is one to which an indicator tured. table 5.1 lists recipes for four different culture media. Some (typically a dye) is added, which reveals by a color change defined media, such as the one listed for Escherichia coli, are con- whether a particular metabolic reaction has occurred during sidered “simple” because they contain only a single carbon growth. Differential media are useful for distinguishing bacteria source. In such a medium, E. coli must biosynthesize all organic and are widely used in clinical diagnostics and microbial taxon- molecules from glucose. omy. Differential and selective media are further discussed in For culturing many microorganisms, knowledge of the exact Chapter 28. composition of a medium is not essential. In these instances com- plex media may suffice and may even be advantageous. Complex Nutritional Requirements and Biosynthetic Capacity media are made from digests of microbial, animal, or plant prod- Of the four culture medium recipes in Table 5.1, three are defined ucts, such as milk protein (casein), beef (beef extract), soybeans and one is complex. The complex medium is easiest to prepare and TAblE 5.1 Examples of culture media for microorganisms with simple and demanding nutritional requirementsa Defined culture medium Defined culture medium for Complex culture medium for either Defined culture medium for for Escherichia coli Leuconostoc mesenteroides E. coli or L. mesenteroides Thiobacillus thioparus K2HPO4 7 g K2HPO4 0.6 g Glucose 15 g KH2PO4 0.5 g KH2PO4 2 g KH2PO4 0.6 g Yeast extract 5 g NH4Cl 0.5 g (NH4)2SO4 1 g NH4Cl 3 g Peptone 5 g MgSO4 0.1 g MgSO4 0.1 g MgSO4 0.1 g KH2PO4 2 g CaCl2 0.05 g CaCl2 0.02 g Glucose 25 g Distilled water 1000 ml KCl 0.5 g Glucose 4–10 g Sodium acetate 25 g pH 7 Na2S2O3 2 g Trace elements (Fe, Co, Amino acids (alanine, arginine, asparagine, Trace elements (as in first Mn, Zn, Cu, Ni, Mo) aspartate, cysteine, glutamate, glutamine, column) 2–10 μg each glycine, histidine, isoleucine, leucine, lysine, 2–10 μg each methionine, phenylalanine, proline, serine, Distilled water 1000 ml Distilled water 1000 ml threonine, tryptophan, tyrosine, valine) pH 7 100–200 μg of each pH 7 Purines and pyrimidines (adenine, guanine, Carbon source: CO2 from air uracil, xanthine) 10 mg of each Vitamins (biotin, folate, nicotinic acid, pyridoxal, pyridoxamine, pyridoxine, riboflavin, thiamine, pantothenate, p- aminobenzoic acid) 0.01–1 mg of each Trace elements (as in first column) 2–10 μg each Distilled water 1000 ml pH 7 (a) (b) a The photos are tubes of (a) the defined medium described, and (b) the complex medium described. Note how the complex medium is colored from the various organic extracts and digests that it contains. Photo credits: Cheryl L. Broadie and John Vercillo, Southern Illinois University at Carbondale.  182 UNIT 2 MiCrobiAl Grow th And reGul Ation supports growth of both Escherichia coli and Leuconostoc mesenteroi- des, the examples used in Table 5.1. By contrast, the simple defined medium supports growth of E. coli but not of L. mesenteroides. Growth of the latter in a defined medium requires the addition of!several nutrients not needed by E. coli. The nutritional needs of L. mesenteroides can be satisfied by preparing either a highly supplemented defined medium, a rather laborious undertaking because of all the individual nutrients that need to be added (Table! 5.1), or by preparing a complex medium, a much less demanding operation. The fourth medium listed in Table 5.1 supports growth of the James A. Shapiro, University of Chicago unit 2 bacterium Thiobacillus thioparus but would not support the growth of any of the other organisms. This is because T. thioparus is both a chemolithotroph and an autotroph ( Section 3.3) and thus has no organic carbon requirements. T. thioparus derives all of its car- bon from CO2 and conserves energy from the oxidation of the sul- fur compound thiosulfate (Na2S2O3). Thus, T. thioparus has the greatest biosynthetic capacity of all the organisms listed in the table, surpassing even E. coli in this regard. (a) The take-home lesson from Table 5.1 is that different microor- ganisms may have vastly different nutritional requirements. For successful cultivation, it is necessary to understand an organism’s physiology and nutritional requirements and then supply it with University of Chicago the nutrients it needs in both the proper form and amount. James A. Shapiro, Laboratory Culture Once a sterile culture medium has been prepared, microbes can be inoculated and the culture can be incubated under conditions (b) that will support growth. In a laboratory situation, inoculation will typically be with a pure culture and into a culture medium that is either liquid (Table 5.1) or solid (Figure 5.11). Liquid culture media are solidified with agar, an algal polysaccharide. Solid James A. Shapiro, University of Chicago James A. Shapiro, University of Chicago media immobilize cells, allowing them to grow and form visible, isolated masses called colonies (Figure 5.11). Microbial colonies are of various shapes and sizes depending on the organism, the cul- ture conditions, the nutrient supply, and other physiological parameters. Some microorganisms produce pigments that cause the colony to be colored (Figure 5.11). Colonies permit the micro- biologist to visualize the composition and presumptive purity of a culture. Plates inoculated from a mixed culture (such as a natural (c) (d) sample, Figure 5.11e) or from a contaminated pure culture will usually contain more than one colony type. Once a sterile culture medium has been prepared, it is ready for inoculation. This requires aseptic technique (Figure 5.12), a series of steps to prevent contamination during manipulations of cultures and sterile culture media, both liquid and solid. With liquid medium, the goal is to transfer a culture while protecting the tube or bottle rim from air currents or contact with nonsterile surfaces (Figure 5.12a). With agar Petri plates, the plan is basically the same but with a greater emphasis on keeping the surface of Figure 5.11 bacterial colonies. Colonies are visible masses of cells formed from the division of one or a few cells and can contain over a billion (109) individual cells. Paul V. Dunlap (a) Serratia marcescens, grown on MacConkey agar. (b) Close-up of colonies outlined in part a. (c) Pseudomonas aeruginosa, grown on trypticase soy agar. (d) Shigella flexneri, grown on MacConkey agar. (e) An agar plate containing many different bacterial (e) colonies that developed from plating a dilution of seawater.  CHAPTER 5 M i c r o b i a l G r o w t h a n d i t s C o n t r o l 183 1. Flaming the loop 2. Tube cap is removed. 3. Flaming the tube tip 4. Only sterilized 5. The tube is reflamed. 6. Tube is recapped sterilizes it. sterilizes the surface. portion of loop and then steps 2–6 unit 2 enters tube. repeated with tube of fresh medium. (a) Transfering a liquid culture Loop is then resterilized in the Subsequent streaks flame. are at angles to the first streak. 1 2 Isolated colonies Confluent growth at 5 at end of streak beginning of streak 3 4 James A. Shapiro, University of Chicago 1. Loop is sterilized and a loopful of 2. Initial streak is worked in well 3. Appearance of well-streaked plate after inoculum is removed from tube. in one corner of the agar plate. incubation shows colonies of the bacterium Micrococcus luteus on a blood agar plate. (b) Streaking a Petri plate Figure 5.12 Aseptic transfer. (a) Liquid media: After the tube is recapped at the end, the loop is resterilized. (b) Solid media: making a streak plate to obtain pure cultures. The plate cover is opened just enough to permit streaking manipulations. In streaking a plate, the microbial cells are separated by the streaking process to yield widely separated single cells that then grow and divide to form colonies. the!agar protected from aerosols or particulate matter that could MINIQUIz drop in (Figure 5.12b). Why would a complex culture medium for Leuconostoc A mastery of aseptic technique is required for maintaining pure mesenteroides be easier to prepare than a chemically defined cultures, as airborne contaminants are virtually everywhere, even medium? in what may appear to be a very clean microbiology laboratory. In which medium shown in Table 5.1, defined or complex, do you Picking an isolated colony and restreaking it is the main method think Escherichia coli would grow the fastest? Why? E. coli will not for obtaining pure cultures from mixed liquid or plate (Figure!5.11e) grow in the medium described for Thiobacillus thioparus. Why? cultures and is a common procedure in the microbiology labora- What is meant by the word sterile? Why is aseptic technique tory. Other techniques for obtaining pure cultures have been necessary for successful maintenance of pure cultures in the developed that are especially suited for particular groups of bacte- laboratory? ria with unusual growth requirements and these will be discussed How many cells could be present in a single bacterial colony? in Sections 19.2 and 19.3.  184 UNIT 2 MiCrobiAl Grow th And reGul Ation 5.6 Microscopic Counts of Microbial Microscopic Cell Counts in Microbial Ecology Despite its many potential caveats, microbial ecologists often use Cell numbers microscopic cell counts on natural samples. But they do so using Assessing cell numbers gives quantitative information on the stains to visualize the cells, often stains that yield phylogenetic or state of a microbial culture or community. Several methods for other key information about the cells, such as their metabolic enumerating a microbial population have been developed, each properties. having their own strengths and caveats. We begin with the classic There are many stains that can be used in a general way. For “total count” carried out by microscopic examination of a culture example, the stain DAPI ( Section 1.6 and Figure 1.20e) stains or natural sample. all cells because it reacts with DNA. Other general fluorescent stains can differentiate live from dead cells by detecting whether Total Cell Count the cytoplasmic membrane is intact or not. By contrast, fluores- unit 2 Total counts of microbial numbers in a culture or natural sample cent stains that are highly specific for certain organisms or can be done by simply observing and enumerating the cells pres- groups of related organisms can be prepared by attaching fluo- ent by a microscopic cell count. Microscopic counts can be per- rescent dyes to specific nucleic acid probes. For example, formed either on samples dried on slides or on liquid samples. phylogenetic stains that stain only species of Bacteria or only spe- Dried samples can be stained to increase contrast between cells cies of Archaea can be used in combination with nonspecific and their background ( Sections 1.6 and 19.3). With liquid sam- stains to determine the proportion of each domain present in a ples, counting chambers consisting of a grid with squares of sample ( Section 19.4). Other fluorescent probes have been known area etched on the surface of a glass slide are used developed that target genes encoding enzymes that catalyze spe- (Figure 5.13). When the coverslip is placed on the chamber, each cific metabolic processes; if a cell is stained by one of these square on the grid has a precise volume. The number of cells per probes, a metabolism can be inferred that may reveal the cell’s unit area of grid can be counted under the microscope, giving a ecological role in the microbial community. In all of these cases, measure of the number of cells per small chamber volume. The if cells in!the sample are present in only low numbers, for exam- number of cells per milliliter of suspension is calculated by ple in a sample of ocean water, this limitation can be overcome employing a conversion factor based on the volume of the cham- by first! concentrating the cells on a filter and then counting ber sample (Figure 5.13). them after staining. Microscopic counting is a quick and easy way of estimating Because they are easy to do and often yield useful baseline infor- microbial cell numbers. However, it has several limitations that mation, microscopic cell counts are common in ecological studies restrict its usefulness. For example, without special staining tech- of natural microbial environments. We pursue this theme in more niques ( Section 19.3), dead cells cannot be distinguished from detail in Chapter 19. live cells, and precision is difficult to achieve, even when replicate counts are made. Moreover, small cells are often difficult to see MINIQUIz under the microscope, which can lead to erroneous counts, and What are some of the problems that can arise when unstained cell suspensions of low density (less than about 106 cells/milliliter) preparations are enumerated in microscopic counts? will have few if any cells in a microscope field unless the sample is first concentrated and resuspended in a small volume. Finally, Using microscopic techniques, how could you tell whether Archaea were present in an alpine lake where total cell numbers motile cells must be killed (usually with formaldehyde) or other- were only 105/ml? wise immobilized before counting, and debris in the sample may easily be mistaken for microbial cells. Ridges that support coverslip To calculate number per milliliter of sample: Coverslip 12 cells × 25 large squares × 50 × 103 Number/mm2 (3 × 102) Sample added here. Care must be taken not to allow overflow; space Microsc