Thursday, December 24, 2020

Microbial Growth : Bacterial Growth Curve

All 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.

This chapter 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 Bacterial 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).

Microbial Growth Curve

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.

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. 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.

These shift-up and shift-down experiments demonstrate that microbial growth is under precise, coordinated control and responds quickly to changes in environmental conditions.

Nutrient Concentration and Growth

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.

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 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 or Phase of Decline

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  Microbial 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.

Nutrient Concentration and Growth

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).

Bacterial generation time

Exponential Microbial Growth

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.

Measurement of Microbial Growth

There are many ways to measure microbial growth to determine growth rates and generation times. Either population mass or number may be followed because growth leads to increases in both. The most commonly employed techniques for growth measurement are examined briefly and the advantages and disadvantages of each noted. No single technique is always best; the most appropriate approach will depend on the experimental situation.

Bacterial Generation Time Determination

Measurement of Microbial Cell Numbers

The most obvious way to determine microbial numbers is through direct counting. Using a counting chamber is easy, inexpensive, and relatively quick; it also gives information about the size and morphology of microorganisms. Petroff-Hausser counting chambers can be used for counting procaryotes; hemocytometers can be used for both procaryotes and eucaryotes.

Generation Times for Selected Microorganisms

Procaryotes are more easily counted in these chambers if they are stained, or when a phase-contrast or a fluorescence microscope is employed. These specially designed slides have chambers of known depth with an etched grid on the chamber bottom (figure 6.5). The number of microorganisms in a sample can be calculated by taking into account the chamber’s volume and any sample dilutions required. There are some disadvantages to the technique. The microbial population must be fairly large for accuracy because such a small volume is sampled. It is also difficult to distinguish between living and dead cells in counting chambers without special techniques.

Petroff-Hausser Counting Chamber

Larger microorganisms such as protozoa, algae, and nonfilamentous yeasts can be directly counted with electronic counters such as the Coulter Counter. The microbial suspension is forced through a small hole or orifice. An electrical current flows through the hole, and electrodes placed on both sides of the orifice measure its electrical resistance. Every time a microbial cell passes through the orifice, electrical resistance increases (or the conductivity drops) and the cell is counted. The Coulter Counter gives accurate results with larger cells and is extensively used in hospital laboratories to count red and white blood cells. It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems.

Counting chambers and electronic counters yield counts of all cells, whether alive or dead. There are also several viable counting techniques, procedures specific for cells able to grow and reproduce. In most viable counting procedures, a diluted sample of bacteria or other microorganisms is dispersed over a solid agar surface. Each microorganism or group of microorganisms develops into a distinct colony. The original number of viable microorganisms in the sample can be calculated from the number of colonies formed and the sample dilution. For example, if 1.0 ml of a 1X10-6 dilution yielded 150 colonies, the original sample contained around 1.5X 108 cells per ml. Usually the count is made more accurate by use of a special colony counter.

In this way the spread-plate and pour-plate techniques may be used to find the number of microorganisms in a sample.

Plating techniques are simple, sensitive, and widely used for viable counts of bacteria and other microorganisms in samples of food, water, and soil. Several problems, however, can lead to inaccurate counts. Low counts will result if clumps of cells are not broken up and the microorganisms well dispersed. Because it is not possible to be absolutely certain that each colony arose from an individual cell, the results are often expressed in terms of colony forming units (CFU) rather than the number of microorganisms.

The samples should yield between 30 and 300 colonies for best results. Of course the counts will also be low if the agar medium employed cannot support growth of all the viable microorganisms present.

The hot agar used in the pour-plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates.

Membrane Filtration Procedure to count Colony

Microbial numbers are frequently determined from counts of colonies growing on special membrane filters having pores small enough to trap bacteria. In the membrane filter technique, a sample is drawn through a special membrane filter (figure 6.6). The filter is then placed on an agar medium or on a pad soaked with liquid media and incubated until each cell forms a separate colony. A colony count gives the number of microorganisms in the filtered sample, and special media can be used to select for specific microorganisms (figure 6.7). This technique is especially useful in analyzing aquatic samples.

Colonies on Membrane Filters

Membrane filters also are used to count bacteria directly. The sample is first filtered through a black polycarbonate membrane filter to provide a good background for observing fluorescent objects.

The bacteria then are stained with a fluorescent dye such as acridine orange or DAPI and observed microscopically. Acridine orange–stained microorganisms glow orange or green and are easily counted with an epifluorescence microscope. Usually the counts obtained with this approach are much higher than those from culture techniques because some of the bacteria are dead. Commercial kits that use fluorescent reagents to stain live and dead cells differently are now available. This makes it possible to directly count the number of live and dead microorganisms in a sample (see figure 2.13d).

Measurement of Microbial Cell Mass

Increases in the total cell mass, as well as in cell numbers, accompany population growth. Therefore techniques for measuring changes in cell mass can be used in following growth. The most direct approach is the determination of microbial dry weight.

Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of fungi. It is time consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity.

More rapid, sensitive techniques depend on the fact that microbial cells scatter light striking them. Because microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. When the concentration of bacteria reaches about 10 million cells (107) per ml, the medium appears slightly cloudy or turbid.

Further increases in concentration result in greater turbidity and less light is transmitted through the medium. The extent of light scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentration at low absorbance levels (figure 6.8). Thus population growth can be easily measured spectrophotometrically as long as the population is high enough to give detectable turbidity.

Turbidity and Microbial Mass Measurement

If the amount of a substance in each cell is constant, the total quantity of that cell constituent is directly related to the total microbial cell mass. For example, a sample of washed cells collected from a known volume of medium can be analyzed for total protein or nitrogen. An increase in the microbial population will be reflected in higher total protein levels. Similarly, chlorophyll determinations can be used to measure algal populations, and the quantity of ATP can be used to estimate the amount of living microbial mass.

The Continuous Culture of Microorganisms

Up to this point the focus has been on closed systems called batch cultures in which nutrient supplies are not renewed nor wastes removed.

Exponential growth lasts for only a few generations and soon the stationary phase is reached. However, it is possible to grow microorganisms in an open system, a system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes.

These conditions are met in the laboratory by a continuous culture system. A microbial population can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods in a continuous culture system.

The Chemostat Continuous Culture System

Two major types of continuous culture systems commonly are used: (1) chemostats and (2) turbidostats.

Continuous Culture System: The Chemostat

A chemostat is constructed so that sterile medium is fed into the culture vessel at the same rate as the media containing microorganisms is removed (figure 6.9). The culture medium for a chemostat possesses an essential nutrient (e.g., an amino acid) in limiting quantities. Because of the presence of a limiting nutrient, the growth rate is determined by the rate at which new medium is fed into the growth chamber, and the final cell density depends on the concentration of the limiting nutrient.

The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture vessel relative to the vessel volume, where f is the flow rate (ml/hr) and V is the vessel volume (ml).

D = f/V

For example, if f is 30 ml/hr and V is 100 ml, the dilution rate is 0.30 hr-1 .

Both the microbial population level and the generation time are related to the dilution rate (figure 6.10). The microbial population density remains unchanged over a wide range of dilution rates. The generation time decreases (i.e., the growth rate rises) as the dilution rate increases.

Chemostat Dilution Rate and Microbial Growth

The limiting nutrient will be almost completely depleted under these balanced conditions. If the dilution rate rises too high, the microorganisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the maximum growth rate. The limiting nutrient concentration rises at higher dilution rates because fewer microorganisms are present to use it.

At very low dilution rates, an increase in D causes a rise in both cell density and the growth rate. This is because of the effect of nutrient concentration on the growth rate, sometimes called the Monod relationship (figure 6.2b). Only a limited supply of nutrient is available at low dilution rates. Much of the available energy must be used for cell maintenance, not for growth and reproduction.

As the dilution rate increases, the amount of nutrients and the resulting cell density rise because energy is available for both maintenance and growth. The growth rate increases when the total available energy exceeds the maintenance energy.

The Turbidostat Continuous Culture System

The second type of continuous culture system, the turbidostat, has a photocell that measures the absorbance or turbidity of the culture in the growth vessel. The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity or cell density. The turbidostat differs from the chemostat in several ways. The dilution rate in a turbidostat varies rather than remaining constant, and its culture medium lacks a limiting nutrient. The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower dilution rates.

Continuous culture systems are very useful because they provide a constant supply of cells in exponential phase and growing at a known rate. They make possible the study of microbial growth at very low nutrient levels, concentrations close to those present in natural environments.

These systems are essential for research in many areas—for example, in studies on interactions between microbial species under environmental conditions resembling those in a freshwater lake or pond. Continuous systems also are used in food and industrial microbiology.

Microbial Responses to Environmental Factors

The Influence of Environmental Factors on Microbial Growth

Microorganisms must be able to respond to variations in nutrient levels, and particularly to nutrient limitation. The growth of microorganisms also is greatly affected by the chemical and physical nature of their surroundings.

An understanding of environmental influences aids in the control of microbial growth and the study of the ecological distribution of microorganisms.

The ability of some microorganisms to adapt to extreme and inhospitable environments is truly remarkable. Procaryotes are present anywhere life can exist. Many habitats in which prokaryotes thrive would kill most other organisms. Procaryotes such as Bacillus infernus even seem able to live over 1.5 miles below the Earth’s surface, without oxygen and at temperatures above 60°C.

Microorganisms that grow in such harsh conditions are often called extremophiles.

In this section we shall briefly review how some of the most important environmental factors affect microbial growth. Major emphasis will be given to solutes and water activity, pH, temperature, oxygen level, pressure, and radiation. Table 6.3 summarizes the way in which microorganisms are categorized in terms of their response to these factors.

Approximate Lower aw Limits for Microbial Growth

The Influence of Solutes and Water Activity on Microbial Growth

Because a selectively permeable plasma membrane separates microorganisms from their environment, they can be affected by changes in the osmotic concentration of their surroundings. If a microorganism is placed in a hypotonic solution (one with a lower osmotic concentration), water will enter the cell and cause it to burst unless something is done to prevent the influx. The osmotic concentration of the cytoplasm can be reduced by use of inclusion bodies. Procaryotes also can contain pressure-sensitive channels that open to allow solute escape when the osmolarity of the environment becomes much lower than that of the cytoplasm.

Most bacteria, algae, and fungi have rigid cell walls that maintain the shape and integrity of the cell. When microorganisms with rigid cell walls are placed in a hypertonic environment, water leaves and the plasma membrane shrinks away from the wall, a process known as plasmolysis. This dehydrates the cell and may damage the plasma membrane; the cell usually becomes metabolically inactive and ceases to grow.

Many microorganisms keep the osmotic concentration of their protoplasm somewhat above that of the habitat by the use of compatible solutes, so that the plasma membrane is always pressed firmly against their cell wall. Compatible solutes are solutes that are compatible with metabolism and growth when at high intracellular concentrations.

Most procaryotes increase their internal osmotic concentration in a hypertonic environment through the synthesis or uptake of choline, betaine, proline, glutamic acid, and other amino acids; elevated levels of potassium ions are also involved to some extent. Algae and fungi employ sucrose and polyols—for example, arabitol, glycerol, and mannitol— for the same purpose. Polyols and amino acids are ideal solutes for this function because they normally do not disrupt enzyme structure and function. 

A few procaryotes like Halobacterium salinarium raise their osmotic concentration with potassium ions (sodium ions are also elevated but not as much as potassium). Halobacterium’s enzymes have been altered so that they actually require high salt concentrations for normal activity. Since protozoa do not have a cell wall, they must use contractile vacuoles to eliminate excess water when living in hypotonic environments.

The amount of water available to microorganisms can be reduced by interaction with solute molecules (the osmotic effect) or by adsorption to the surfaces of solids (the matric effect). Because the osmotic concentration of a habitat has such profound effects on microorganisms, it is useful to be able to express quantitatively the degree of water availability.

Microbiologists generally use water activity (aw) for this purpose (water availability also may be expressed as water potential, which is related to aw). The water activity of a solution is 1/100 the relative humidity of the solution (when expressed as a percent). It is also equivalent to the ratio of the solution’s vapor pressure (Psoln) to that of pure water (Pwater).

Aw= Psoln/Pwater

The water activity of a solution or solid can be determined by sealing it in a chamber and measuring the relative humidity after the system has come to equilibrium. Suppose after a sample is treated in this way, the air above it is 95% saturated—that is, the air contains 95% of the moisture it would have when equilibrated at the same temperature with a sample of pure water. The relative humidity would be 95% and the sample’s water activity, 0.95.

Water activity is inversely related to osmotic pressure; if a solution has high osmotic pressure, its aw is low.

Microorganisms differ greatly in their ability to adapt to habitats with low water activity (table 6.4). A microorganism must expend extra effort to grow in a habitat with a low aw value because it must maintain a high internal solute concentration to retain water. Some microorganisms can do this and are osmotolerant; they will grow over wide ranges of water activity or osmotic concentration. 

For example, Staphylococcus aureus can be cultured in media containing any sodium chloride concentration up to about 3 M. It is well adapted for growth on the skin. The yeast Saccharomyces rouxii will grow in sugar solutions with aw values as low as 0.6. The alga Dunaliella viridis tolerates sodium chloride concentrations from 1.7 M to a saturated solution.

Although a few microorganisms are truly osmotolerant, most only grow well at water activities around 0.98 (the approximate aw for seawater) or higher. This is why drying food or adding large quantities of salt and sugar is so effective in preventing food spoilage. As table 6.4 shows, many fungi are osmotolerant and thus particularly important in the spoilage of salted or dried foods.

Halophiles have adapted so completely to hypertonic, saline conditions that they require high levels of sodium chloride to grow, concentrations between about 2.8 M and saturation (about 6.2 M) for extreme halophilic bacteria. The archaeon Halobacterium can be isolated from the Dead Sea (a salt lake between Israel and Jordan and the lowest lake in the world), the Great Salt Lake in Utah, and other aquatic habitats with salt concentrations approaching saturation.

Halobacterium and other extremely halophilic bacteria have significantly modified the structure of their proteins and membranes rather than simply increasing the intracellular concentrations of solutes, the approach used by most osmotolerant microorganisms. These extreme halophiles accumulate enormous quantities of potassium in order to remain hypertonic to their environment; the internal potassium concentration may reach 4 to 7 M. The enzymes, ribosomes, and transport proteins of these bacteria require high levels of potassium for stability and activity. 

In addition, the plasma membrane and cell wall of Halobacterium are stabilized by high concentrations of sodium ion. If the sodium concentration decreases too much, the wall and plasma membrane literally disintegrate. Extreme halophilic bacteria have successfully adapted to environmental conditions that would destroy most organisms. In the process they have become so specialized that they have lost ecological flexibility and can prosper only in a few extreme habitats.

The Influence of pH on Microbial Growth

pH is a measure of the hydrogen ion activity of a solution and is defined as the negative logarithm of the hydrogen ion concentration (expressed in terms of molarity).

pH = -log [H+] = log(1/[H+])

The pH scale extends from pH 0.0 (1.0 M H+) to pH 14.0 (1.0 X 10-14 M H+), and each pH unit represents a tenfold change in hydrogen ion concentration. Figure 6.11 shows that the habitats in which microorganisms grow vary widely—from pH 1 to 2 at the acid end to alkaline lakes and soil that may have pH values between 9 and 10.

pH Scale

It is not surprising that pH dramatically affects microbial growth. Each species has a definite pH growth range and pH growth optimum. Acidophiles have their growth optimum between pH 0 and 5.5; neutrophiles, between pH 5.5 and 8.0; and alkalophiles prefer the pH range of 8.5 to 11.5. Extreme alkalophiles have growth optima at pH 10 or higher. In general, different microbial groups have characteristic pH preferences. 

Most bacteria and protozoa are neutrophiles. Most fungi prefer slightly acid surroundings, about pH 4 to 6; algae also seem to favor slight acidity. There are many exceptions to these generalizations. For example, the alga Cyanidium caldarium and the archaeon Sulfolobus acidocaldarius are common inhabitants of acidic hot springs; both grow well around pH 1 to 3 and at high temperatures.

The Archaea Ferroplasma acidarmanus and Picrophilus oshimae can actually grow at pH 0, or very close to it.

Although microorganisms will often grow over wide ranges of pH and far from their optima, there are limits to their tolerance.

Drastic variations in cytoplasmic pH can harm microorganisms by disrupting the plasma membrane or inhibiting the activity of enzymes and membrane transport proteins. Procaryotes die if the internal pH drops much below 5.0 to 5.5. Changes in the external pH also might alter the ionization of nutrient molecules and thus reduce their availability to the organism.

Several mechanisms for the maintenance of a neutral cytoplasmic pH have been proposed. The plasma membrane may be relatively impermeable to protons. Neutrophiles appear to exchange potassium for protons using an antiport transport system. Extreme alkalophiles like Bacillus alcalophilus maintain their internal pH closer to neutrality by exchanging internal sodium ions for external protons. Internal buffering also may contribute to pH homeostasis.

Microorganisms often must adapt to environmental pH changes to survive. In bacteria, potassium/proton and sodium/proton antiport systems probably correct small variations in pH. If the pH becomes too acidic, other mechanisms come into play. When the pH drops below about 5.5 to 6.0, Salmonella typhimurium and E. coli synthesize an array of new proteins as part of what has been called their acidic tolerance response. 

A proton-translocating ATPase contributes to this protective response, either by making more ATP or by pumping protons out of the cell. If the external pH decreases to 4.5 or lower, chaperones such as acid shock proteins and heat shock proteins are synthesized. Presumably these prevent the acid denaturation of proteins and aid in the refolding of denatured proteins.

Microorganisms frequently change the pH of their own habitat by producing acidic or basic metabolic waste products. Fermentative microorganisms form organic acids from carbohydrates, whereas chemolithotrophs like Thiobacillus oxidize reduced sulfur components to sulfuric acid. Other microorganisms make their environment more alkaline by generating ammonia through amino acid degradation.

Buffers often are included in media to prevent growth inhibition by large pH changes. Phosphate is a commonly used buffer and a good example of buffering by a weak acid (H2PO4) and its conjugate base (HPO4 2–).

If protons are added to the mixture, they combine with the salt form to yield a weak acid. An increase in alkalinity is resisted because the weak acid will neutralize hydroxyl ions through proton donation to give water. Peptides and amino acids in complex media also have a strong buffering effect.

The Influence of Temperature on Microbial Growth

Environmental temperature profoundly affects microorganisms, like all other organisms. Indeed, microorganisms are particularly susceptible because they are usually unicellular and their temperature varies with that of the external environment. For these reasons, microbial cell temperature directly reflects that of the cell’s surroundings. A most important factor influencing the effect of temperature on growth is the temperature sensitivity of enzyme catalyzed reactions.

At low temperatures a temperature rise increases the growth rate because the velocity of an enzyme-catalyzed reaction, like that of any chemical reaction, will roughly double for every 10°C rise in temperature. Because the rate of each reaction increases, metabolism as a whole is more active at higher temperatures, and the microorganism grows faster.

Beyond a certain point further increases actually slow growth, and sufficiently high temperatures are lethal. High temperatures damage microorganisms by denaturing enzymes, transport carriers, and other proteins. Microbial membranes are also disrupted by temperature extremes; the lipid bilayer simply melts and disintegrates.

Thus, although functional enzymes operate more rapidly at higher temperatures, the microorganism may be damaged to such an extent that growth is inhibited because the damage cannot be repaired. At very low temperatures, membranes solidify and enzymes don’t work rapidly. In summary, when organisms are above the optimum temperature, both function and cell structures are affected. If temperatures are very low, function is affected but not necessarily cell chemical composition and structure.

Because of these opposing temperature influences, microbial growth has fairly characteristic temperature dependence with distinct cardinal temperatures—minimum, optimum, and maximum growth temperatures (figure 6.12). Although the shape of the temperature dependence curve can vary, the temperature optimum is always closer to the maximum than to the minimum.

Temperature and Growth

The cardinal temperatures for a particular species are not rigidly fixed but often depend to some extent on other environmental factors such as pH and the available nutrients. For example, Crithidia fasciculata, a flagellated protozoan living in the gut of mosquitos, will grow in a simple medium at 22 to 27°C. However, it cannot be cultured at 33 to 34°C without the addition of extra metals, amino acids, vitamins, and lipids.

The cardinal temperatures vary greatly between microorganisms (table 6.5). Optima normally range from 0°C to as high as 75°C, whereas microbial growth occurs at temperatures extending from -20°C to over 100°C. The major factor determining this growth range seems to be water. Even at the most extreme temperatures, microorganisms need liquid water to grow. 

The growth temperature range for a particular microorganism usually spans about 30 degrees. Some species (e.g., Neisseria gonorrhoeae) have a small range; others, like Enterococcus faecalis, will grow over a wide range of temperatures. The major microbial groups differ from one another regarding their maximum growth temperature.

The upper limit for protozoa is around 50°C. Some algae and fungi can grow at temperatures as high as 55 to 60°C. Procaryotes have been found growing at or close to 100°C, the boiling point of water at sea level (see figure 20.8). Recently strains growing at even higher temperatures have been discovered (Box 6.1). Clearly, prokaryotic organisms can grow at much higher temperatures than eucaryotes.

It has been suggested that eucaryotes are not able to manufacture organellar membranes that are stable and functional at temperatures above 60°C. The photosynthetic apparatus also appears to be relatively unstable because photosynthetic organisms are not found growing at very high temperatures.

Life above 100°C

Microorganisms such as those in table 6.5 can be placed in one of five classes based on their temperature ranges for growth (figure 6.13).

1. Psychrophiles grow well at 0°C and have an optimum growth temperature of 15°C or lower; the maximum is around 20°C. They are readily isolated from Arctic and Antarctic habitats; because 90% of the ocean is 5°C or colder, it constitutes an enormous habitat for psychrophiles. The psychrophilic alga Chlamydomonas nivalis can actually turn a snowfield or glacier pink with its bright red spores. 

Psychrophiles are widespread among bacterial taxa and found in such genera as Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter, Moritella, Photobacterium, and Shewanella. The psychrophilic archaeon Methanogenium has recently been isolated from Ace Lake in Antarctica. Psychrophilic microorganisms have adapted to their environment in several ways. Their enzymes, transport systems, and protein synthetic mechanisms function well at low temperatures. 

Temperature Ranges for Microbial Growth

The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and remain semifluid when cold. Indeed, many psychrophiles begin to leak cellular constituents at temperatures higher than 20°C because of cell membrane disruption.

2. Many species can grow at 0 to 7°C even though they have optima between 20 and 30°C, and maxima at about 35°C. These are called psychrotrophs or facultative psychrophiles. Psychrotrophic bacteria and fungi are major factors in the spoilage of refrigerated foods.

3. Mesophiles are microorganisms with growth optima around 20 to 45°C; they often have a temperature minimum of 15 to 20°C. Their maximum is about 45°C or lower. Most microorganisms probably fall within this category. Almost all human pathogens are mesophiles, as might be expected since their environment is a fairly constant 37°C.

Temperature Ranges for Microbial Growth

4. Some microorganisms are thermophiles; they can grow at temperatures of 55°C or higher. Their growth minimum is usually around 45°C and they often have optima between 55 and 65°C. The vast majority are procaryotes although a few algae and fungi are thermophilic (table 6.5). These organisms flourish in many habitats including composts, self-heating hay stacks, hot water lines, and hot springs. 

Thermophiles differ from mesophiles in having much more heat-stable enzymes and protein synthesis systems able to function at high temperatures. Their membrane lipids are also more saturated than those of mesophiles and have higher melting points; therefore thermophile membranes remain intact at higher temperatures.

5. As mentioned previously, a few thermophiles can grow at 90°C or above and some have maxima above 100°C.

Procaryotes that have growth optima between 80°C and about 113°C are called hyperthermophiles. They usually do not grow well below 55°C. Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor.

The Influence of Oxygen Concentration on Microbial Growth

An organism able to grow in the presence of atmospheric O2 is an aerobe, whereas one that can grow in its absence is an anaerobe.

Almost all multicellular organisms are completely dependent on atmospheric O2 for growth—that is, they are obligate aerobes (table 6.3). Oxygen serves as the terminal electron acceptor for the electron- transport chain in aerobic respiration. In addition, aerobic eucaryotes employ O2 in the synthesis of sterols and unsaturated fatty acids. Facultative anaerobes do not require O2 for growth but do grow better in its presence. In the presence of oxygen they will use aerobic respiration.

Aerotolerant anaerobes such as Enterococcus faecalis simply ignore O2 and grow equally well whether it is present or not. In contrast, strict or obligate anaerobes (e.g., Bacteroides, Fusobacterium, Clostridium pasteurianum, Methanococcus) do not tolerate O2 at all and die in its presence. Aerotolerant and strict anaerobes cannot generate energy through respiration and must employ fermentation or anaerobic respiration pathways for this purpose.

Finally, there are aerobes such as Campylobacter called microaerophiles, that are damaged by the normal atmospheric level of O2 (20%) and require O2 levels below the range of 2 to 10% for growth. The nature of bacterial O2 responses can be readily determined by growing the bacteria in culture tubes filled with a solid culture medium or a special medium like thioglycollate broth, which contains a reducing agent to lower O2 levels (figure 6.14).

Oxygen and Bacterial Growth

A microbial group may show more than one type of relationship to O2. All five types are found among the procaryotes and protozoa.

Fungi are normally aerobic, but a number of species—particularly among the yeasts—are facultative anaerobes. Algae are almost always obligate aerobes. It should be noted that the ability to grow in both aerobic and anaerobic environments provides considerable flexibility and is an ecological advantage.

Although strict anaerobes are killed by O2, they may be recovered from habitats that appear to be aerobic. In such cases they associate with facultative anaerobes that use up the available O2 and thus make the growth of strict anaerobes possible. For example, the strict anaerobe Bacteroides gingivalis lives in the mouth where it grows in the anaerobic crevices around the teeth.

These different relationships with O2 appear due to several factors, including the inactivation of proteins and the effect of toxic O2 derivatives. Enzymes can be inactivated when sensitive groups like sulfhydryls are oxidized. A notable example is the nitrogen- fixation enzyme nitrogenase, which is very oxygen sensitive.

Oxygen accepts electrons and is readily reduced because its two outer orbital electrons are unpaired. Flavoproteins, several other cell constituents, and radiation promote oxygen reduction. The result is usually some combination of the reduction products superoxide radical, hydrogen peroxide, and hydroxyl radical.

O2 + e- → O2.– (superoxide radical)

O2.– + e- + 2H+ →H2O2 (hydrogen peroxide)

H2O2 + e- + H+ →H2O + OH- (hydroxyl radical)

These products of oxygen reduction are extremely toxic because they are powerful oxidizing agents and rapidly destroy cellular constituents. A microorganism must be able to protect itself against such oxygen products or it will be killed. Neutrophils and macrophages use these toxic oxygen products to destroy invading pathogens.

Many microorganisms possess enzymes that afford protection against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase, which catalyze the destruction of superoxide radical and hydrogen peroxide, respectively. Peroxidase also can be used to destroy hydrogen peroxide.

Superoxide radical and hydrogen peroxide

Aerotolerant microorganisms may lack catalase but almost always have superoxide dismutase. The aerotolerant Lactobacillus plantarum uses manganous ions instead of superoxide dismutase to destroy the superoxide radical. All strict anaerobes lack both enzymes or have them in very low concentrations and therefore cannot tolerate O2.

Because aerobes need O2 and anaerobes are killed by it, radically different approaches must be used when growing the two types of microorganisms. When large volumes of aerobic microorganisms are cultured, either the culture vessel is shaken to aerate the medium or sterile air must be pumped through the culture vessel. Precisely the opposite problem arises with anaerobes; all O2 must be excluded. This can be accomplished in several ways.

(1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used. The medium is boiled during preparation to dissolve its components; boiling also drives off oxygen very effectively. The reducing agents will eliminate any dissolved O2 remaining within the medium so that anaerobes can grow beneath its surface.

Anaerobic Work Chamber and Incubator

(2) Oxygen also may be eliminated from an anaerobic system by removing air with a vacuum pump and flushing out residual O2 with nitrogen gas (figure 6.15). Often CO2 as well as nitrogen is added to the chamber since many anaerobes require a small amount of CO2 for best growth.

(3) One of the most popular ways of culturing small numbers of anaerobes is by use of a Gas-Pak jar (figure 6.16). In this procedure the environment is made anaerobic by using hydrogen and a palladium catalyst to remove O2 through the formation of water. The reducing agents in anaerobic agar also remove oxygen, as mentioned previously.

GasPak Anaerobic System

(4) Plastic bags or pouches make convenient containers when only a few samples are to be incubated anaerobically. These have a catalyst and calcium carbonate to produce an anaerobic, carbon-dioxiderich atmosphere. A special solution is added to the pouch’s reagent compartment; petri dishes or other containers are placed in the pouch; it then is clamped shut and placed in an incubator.

A laboratory may make use of all these techniques since each is best suited for different purposes.

The Influence of Pressure on Microbial Growth

Most organisms spend their lives on land or on the surface of water, always subjected to a pressure of 1 atmosphere (atm), and are never affected significantly by pressure. Yet the deep sea (ocean of 1,000 m or more in depth) is 75% of the total ocean volume. The hydrostatic pressure can reach 600 to 1,100 atm in the deep sea, while the temperature is about 2 to 3°C.

Despite these extremes, bacteria survive and adapt. Many are barotolerant: increased pressure does adversely affect them but not as much as it does nontolerant bacteria. Some bacteria in the gut of deep-sea invertebrates such as amphipods and holothurians are truly barophilic—they grow more rapidly at high pressures.

These gut bacteria may play an important role in nutrient recycling in the deep sea. One barophile has been recovered from the Mariana trench near the Philippines (depth about 10,500 m) that is actually unable to grow at pressures below about 400 to 500 atm when incubated at 2°C. Thus far, barophiles have been found among several bacterial genera (e.g., Photobacterium, Shewanella, Colwellia). Some members of the Archaea are thermobarophiles (e.g., Pyrococcus spp., Methanococcus jannaschii).

Electromagnetic Spectrum

The Influence of Pressure on Microbial Growth

Our world is bombarded with electromagnetic radiation of various types (figure 6.17). This radiation often behaves as if it were composed of waves moving through space like waves traveling on the surface of water. The distance between two wave crests or troughs is the wavelength. As the wavelength of electromagnetic radiation decreases, the energy of the radiation increases—gamma rays and X rays are much more energetic than visible light or infrared waves. Electromagnetic radiation also acts like a stream of energy packets called photons, each photon having a quantum of energy whose value will depend on the wavelength of the radiation.

Sunlight is the major source of radiation on the Earth. It includes visible light, ultraviolet (UV) radiation, infrared rays, and radio waves. Visible light is a most conspicuous and important aspect of our environment: all life is dependent on the ability of photosynthetic organisms to trap the light energy of the sun. Almost 60% of the sun’s radiation is in the infrared region rather than the visible portion of the spectrum.

Infrared is the major source of the Earth’s heat. At sea level, one finds very little ultraviolet radiation below about 290 to 300 nm. UV radiation of wavelengths shorter than 287 nm is absorbed by O2 in the Earth’s atmosphere; this process forms a layer of ozone between 25 and 30 miles above the Earth’s surface.

The ozone layer then absorbs somewhat longer UV rays and reforms O2. This elimination of UV radiation is crucial because it is quite damaging to living systems. The fairly even distribution of sunlight throughout the visible spectrum accounts for the fact that sunlight is generally “white.”

Many forms of electromagnetic radiation are very harmful to microorganisms. This is particularly true of ionizing radiation, radiation of very short wavelength or high energy, which can cause atoms to lose electrons or ionize. Two major forms of ionizing radiation are

(1) X rays, which are artificially produced, and

(2) gamma rays, which are emitted during radioisotope decay.

Low levels of ionizing radiation will produce mutations and may indirectly result in death, whereas higher levels are directly lethal.

Although microorganisms are more resistant to ionizing radiation than larger organisms, they will still be destroyed by a sufficiently large dose. Ionizing radiation can be used to sterilize items. Some procaryotes (e.g., Deinococcus radiodurans) and bacterial endospores can survive large doses of ionizing radiation.  

A variety of changes in cells are due to ionizing radiation; it breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, and polymerizes some molecules. Oxygen enhances these destructive effects, probably through the generation of hydroxyl radicals (OH·). Although many types of constituents can be affected, it is reasonable to suppose that destruction of DNA is the most important cause of death.

Ultraviolet (UV) radiation, mentioned earlier, kills all kinds of microorganisms due to its short wavelength (approximately from 10 to 400 nm) and high energy. The most lethal UV radiation has a wavelength of 260 nm, the wavelength most effectively absorbed by DNA. The primary mechanism of UV damage is the formation of thymine dimers in DNA. Two adjacent thymines in a DNA strand are covalently joined to inhibit DNA replication and function.

This damage is repaired in several ways. In photoreactivation, blue light is used by a photoreactivating enzyme to split thymine dimers. A short sequence containing the thymine dimer can also be excised and replaced. This process occurs in the absence of light and is called dark reactivation. Damage also can be repaired by the recA protein in recombination repair and SOS repair. When UV exposure is too heavy, the damage is so extensive that repair is impossible.

Although very little UV radiation below 290 to 300 nm reaches the earth’s surface, near-UV radiation between 325 and 400 nm can harm microorganisms. Exposure to near-UV radiation induces tryptophan breakdown to toxic photoproducts. It appears that these toxic tryptophan photoproducts plus the near-UV radiation itself produce breaks in DNA strands. The precise mechanism is not known, although it is different from that seen with 260 nm UV.

Visible light is immensely beneficial because it is the source of energy for photosynthesis. Yet even visible light, when present in sufficient intensity, can damage or kill microbial cells.

Usually pigments called photosensitizers and O2 are required. All microorganisms possess pigments like chlorophyll, bacteriochlorophyll, cytochromes, and flavins, which can absorb light energy, become excited or activated, and act as photosensitizers.

The excited photosensitizer (P) transfers its energy to O2 generating singlet oxygen (1O2).

Generation of singlet oxygen

Singlet oxygen is a very reactive, powerful oxidizing agent that will quickly destroy a cell. It is probably the major agent employed by phagocytes to destroy engulfed bacteria.

Many microorganisms that are airborne or live on exposed surfaces use carotenoid pigments for protection against photooxidation.

Carotenoids effectively quench singlet oxygen—that is, they absorb energy from singlet oxygen and convert it back into the unexcited ground state. Both photosynthetic and non-photosynthetic microorganisms employ pigments in this way.

Microbial Growth in Natural Environments

The previous section surveyed the effects on microbial growth of individual environmental factors such as water availability, pH, and temperature. Although microbial ecology will be introduced in more detail at a later point, we will now briefly consider the effect of the environment as a whole on microbial growth.

Microbial Growth Limitation by Environmental Factors

The microbial environment is complex and constantly changing. Characteristically microorganisms in a particular location are exposed to many overlapping gradients of nutrients and various other environmental factors. This is particularly true of microorganisms growing in biofilms. Microorganisms will grow in “microenvironments” until an environmental or nutritional factor limits growth. Liebig’s law of the minimum states that the total biomass of an organism will be determined by the nutrient present in the lowest concentration relative to the organism’s requirements.

This law applies in both the laboratory (figure 6.2) and in terrestrial and aquatic environments. An increase in a limiting essential nutrient such as phosphate will result in an increase in the microbial population until some other nutrient becomes limiting. If a specific nutrient is limiting, changes in other nutrients will have no effect. 

The situation may be even more complex than this. Multiple limiting factors can influence a population over time. Furthermore, as we have seen, factors such as temperature, pH, light, and salinity influence microbial populations and limit growth. Shelford’s law of tolerance states that there are limits to environmental factors below and above which a microorganism cannot survive and grow, regardless of the nutrient supply.

This can readily be seen for temperature in figure 6.13. Each microorganism has a specific temperature range in which it can grow. The same rule applies to other factors such as pH, oxygen level, and hydrostatic pressure in the marine environment. The growth of a microorganism depends on both the nutrient supply and its tolerance of the environmental conditions. Biofilms.

Most microorganisms are confronted with deficiencies that limit their activities except when excess nutrients allow unlimited growth. Such rapid growth will quickly deplete nutrients and possibly result in the release of toxic waste products, which will limit further growth.

In response to low nutrient levels (oligotrophic environments) and intense competition, many microorganisms become more competitive in nutrient capture and exploitation of available resources.

Often the organism’s morphology will change in order to increase its surface area and ability to absorb nutrients. This can involve conversion of rod-shaped procaryotes to “mini” and “ultramicro” cells or changes in the morphology of prosthecate prokaryotes (figure 6.18), in response to starvation. Nutrient deprivation induces many other changes as discussed previously. For example, microorganisms can undergo a step-by-step shutdown of metabolism except for housekeeping maintenance genes.

Morphology and Nutrient Absorption

Many factors can alter nutrient levels in oligotrophic environments. Microorganisms may sequester critical limiting nutrients, such as iron, making them less available to competitors.

The atmosphere can contribute essential nutrients and support microbial growth. This is seen in the laboratory as well as natural environments. Airborne organic substances have been found to stimulate microbial growth in dilute media, and enrichment of growth media by airborne organic matter can allow significant populations of microorganisms to develop. Even distilled water, which already contains traces of organic matter, can absorb one carbon compounds from the atmosphere and grow microorganisms.

The presence of such airborne nutrients and microbial growth, if not detected, can affect experiments in biochemistry and molecular biology, as well as studies of microorganisms growing in oligotrophic environments.

Natural substances also can directly inhibit microbial growth and reproduction in low-nutrient environments. These agents include phenolics, tannins, ammonia, ethylene, and volatile sulfur compounds. This may be a means by which microorganisms avoid expending limited energy reserves until an adequate supply of nutrients becomes available. Such chemicals are also important in plant pathology and may aid in controlling soil-borne microbial diseases.

Counting Viable But Non-culturable Vegetative Procaryotes

In order to study the growth of natural procaryotic populations outside the laboratory, it is essential to determine the number of viable microorganisms present. For most of microbiology’s history, a viable microorganism has been defined as one that is able to grow actively, resulting in the formation of a colony or visible turbidity in a liquid medium.

John R. Postgate of the University of Sussex in England was one of the first to note that microorganisms stressed by survival in natural habitats—or in many selective laboratory media—were particularly sensitive to secondary stresses. Such stresses can produce viable microorganisms without the ability to grow on media normally used for their cultivation.

To determine the growth potential of such microorganisms, Postgate developed what is now called the Postgate Microviability Assay, in which microorganisms are cultured in a thin agar film under a coverslip. The ability of a cell to change its morphology, even if it does not grow beyond the single-cell stage, indicates that the microorganism does show “life signs.”

Since that time many workers have developed additional sensitive microscopic and isotopic procedures to evaluate the presence and significance of these viable but nonculturable bacteria in both lab and field. For example, levels of fluorescent antibody and acridine orange–stained cells often are compared with population counts obtained by the most probable number (MPN) method and plate counts using selective and nonselective media.

The release of radioactive-labeled cell materials also is used to monitor stress effects on microorganisms. Despite these advances the estimation of substrate-responsive viable cells by Postgate’s method is still important. These studies show that even when bacteria such as Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Enterobacter aerogenes, and Enterococcus faecalis have lost their ability to grow on conventional laboratory media using standard cultural techniques, they still might be able to play a role in infectious disease.

The situation in natural environments with mixed populations is much more complex. Here, where often only 1 to 10% of observable cells are able to form colonies, the microbiologist is attempting to grow microorganisms that perhaps never have been cultured or characterized. In the future it is possible that media or proper environmental conditions for their growth will be developed.

At present, molecular techniques involving PCR amplification and small subunit ribosomal RNA analysis are increasingly used to analyze the diversity of uncultured microbial populations.

Quorum Sensing and Microbial Populations

For decades microbiologists tended to think of bacterial populations as collections of individuals growing and behaving independently.

More recently it has become clear that many bacteria can communicate with one another and behave cooperatively. A major way in which this cooperation is accomplished is by a process known as quorum sensing or autoinduction. This is a phenomenon in which bacteria monitor their own population density through sensing the levels of signal molecules, sometimes called autoinducers because they can stimulate the cell that releases them.

The concentration of these signal molecules increases along with the bacterial population until it rises to a specific threshold and signals the bacteria that the population density has reached a critical level or quorum. The bacteria then begin expressing sets of quorum-dependent genes. Quorum sensing has been found among both gram-negative and gram-positive bacteria.

Quorum sensing makes good practical sense. Take the production and release of extracellular enzymes as an example. If such enzymes were released by only a few bacteria, they would diffuse away and be rendered ineffective because of dilution.

With control by quorum sensing, the bacteria reach a high population density before they release enzymes, and as a consequence enzyme levels are concentrated enough to have significant effects.

This is an advantage within a host’s body as well as in the soil or an aquatic habitat. If a pathogen can reach high levels at a particular site before producing virulence factors and escaping into surrounding host tissues, it has a much better chance of counteracting host defenses and successfully spreading throughout the host’s body. This explains another pattern in quorum sensing. It seems to be very important in many bacteria that establish symbiotic or parasitic relationships with hosts.

Quorum sensing was first discovered in gram-negative bacteria and is best understood in these microorganisms. The most common signals in gram-negative bacteria are acyl homoserine lactones (HSLs). These are small molecules composed of a 4- to 14-carbon acyl chain attached by an amide bond to homoserine lactone (figure 6.19a). The acyl chain may have a keto group or hydroxyl group on its third carbon. 

Quorum Sensing in Gram-Negative Bacteria

Acyl HSLs diffuse into the target cell (figure 6.19b). Once they reach a sufficiently high level, acyl HSLs bind to special receptor proteins and trigger a conformational change. Usually the activated complexes act as inducers—that is, they bind to target sites on the DNA and stimulate transcription of quorum-sensitive genes. The gene needed to synthesize acyl HSL is also produced frequently, thus amplifying the effect by the production and release of more autoinducer molecules.

Many different processes are sensitive to acyl HSL signals and quorum sensing in gram-negative bacteria. Some well-studied examples are (1) bioluminescence production by Vibrio fischeri, (2) Pseudomonas aeruginosa synthesis and release of virulence factors, (3) conjugal transfer of genetic material by Agrobacterium tumefaciens, and (4) antibiotic production by Erwinia carotovora and Pseudomonas aureofaciens.

Gram-positive bacteria also regulate activities by quorum sensing, often using an oligopeptide signal. Good examples are mating in Enterococcus faecalis, competence induction in Streptococcus pneumoniae, stimulation of sporulation by Bacillus subtilis, and production of many toxins and other virulence factors by Staphylococcus aureus. Quorum sensing even stimulates the development of aerial mycelia and the production of streptomycin by Streptomyces griseus. In this case, the signal seems to be γ-butyrolactone rather than an oligopeptide.

An interesting and important function of quorum sensing is to promote the formation of mature biofilms by the pathogen Pseudomonas aeruginosa, and it may play a role in cystic fibrosis.

Biofilm formation makes sense for the pathogen because biofilms protect against antibiotics and detergents. Quorum sensing should be very effective within biofilms because there will be less dilution and acyl HSL levels will increase rapidly. Under such circumstances, two different bacteria might stimulate each other by releasing similar signals; this appears to be the case in biofilms containing the pathogens P. aeruginosa and Burkholderia cepacia.

Quorum sensing is an example of what might be called multicellular behavior in that many individual cells communicate and coordinate their activities to act as a unit. Other examples of such complex behavior is pattern formation in colonies and fruiting body formation in the myxobacteria.

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