# Microbial Metabolism

## Biogeochemical Cycles

### Learning Objectives

By the end of this section, you will be able to:
1. Define and describe the importance of microorganisms in the biogeochemical cycles of carbon, nitrogen, and sulfur
2. Define and give an example of bioremediation
Energy flows directionally through ecosystems, entering as sunlight for phototrophs or as inorganic molecules for chemoautotrophs. The six most common elements associated with organic molecules—carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur—take a variety of chemical forms and may exist for long periods in the atmosphere, on land, in water, or beneath earth’s surface. Geologic processes, such as erosion, water drainage, the movement of the continental plates, and weathering, all are involved in the cycling of elements on earth. Because geology and chemistry have major roles in the study of this process, the recycling of inorganic matter between living organisms and their nonliving environment is called a biogeochemical cycle. Here, we will focus on the function of microorganisms in these cycles, which play roles at each step, most frequently interconverting oxidized versions of molecules with reduced ones.

### Carbon Cycle

Carbon is one of the most important elements to living organisms, as shown by its abundance and presence in all organic molecules. The carbon cycle exemplifies the connection between organisms in various ecosystems. Carbon is exchanged between heterotrophs and autotrophs within and between ecosystems primarily by way of atmospheric CO2, a fully oxidized version of carbon that serves as the basic building block that autotrophs use to build multicarbon, high-energy organic molecules such as glucose. Photoautotrophs and chemoautotrophs harness energy from the sun and from inorganic chemical compounds, respectively, to covalently bond carbon atoms together into reduced organic compounds whose energy can be later accessed through the processes of respiration and fermentation ().
Overall, there is a constant exchange of CO2 between the heterotrophs (which produce CO2 as a result of respiration or fermentation) and the autotrophs (which use the CO2 for fixation). Autotrophs also respire or ferment, consuming the organic molecules they form; they do not fix carbon for heterotrophs, but rather use it for their own metabolic needs.
Bacteria and archaea that use methane as their carbon source are called methanotrophs. Reduced one-carbon compounds like methane accumulate in certain anaerobic environments when CO2 is used as a terminal electron acceptor in anaerobic respiration by archaea called methanogens. Some methanogens also ferment acetate (two carbons) to produce methane and CO2. Methane accumulation due to methanogenesis occurs in both natural anaerobic soil and aquatic environments; methane accumulation also occurs as a result of animal husbandry because methanogens are members of the normal microbiota of ruminants. Environmental methane accumulation due to methanogenesis is of consequence because it is a strong greenhouse gas, and methanotrophs help to reduce atmospheric methane levels.

### Nitrogen Cycle

Many biological macromolecules, including proteins and nucleic acids, contain nitrogen; however, getting nitrogen into living organisms is difficult. Prokaryotes play essential roles in the nitrogen cycle (), transforming nitrogen between various forms for their own needs, benefiting other organisms indirectly. Plants and phytoplankton cannot incorporate nitrogen from the atmosphere (where it exists as tightly bonded, triple covalent N2), even though this molecule composes approximately 78% of the atmosphere. Nitrogen enters the living world through free-living and symbiotic bacteria, which incorporate nitrogen into their macromolecules through specialized biochemical pathways called nitrogen fixation. Cyanobacteria in aquatic ecosystems fix inorganic nitrogen (from nitrogen gas) into ammonia (NH3) that can be easily incorporated into biological macromolecules.  bacteria () also fix nitrogen and live symbiotically in the root nodules of legumes (such as beans, peanuts, and peas), providing them with needed organic nitrogen while receiving fixed carbon as sugar in exchange. Free-living bacteria, such as members of the genus , are also able to fix nitrogen.
The nitrogen that enters living systems by nitrogen fixation is eventually converted from organic nitrogen back into nitrogen gas by microbes through three steps: ammonification, nitrification, and denitrification. In terrestrial systems, the first step is the ammonification process, in which certain bacteria and fungi convert nitrogenous waste from living animals or from the remains of dead organisms into ammonia (NH3). This ammonia is then oxidized to nitrite  then to nitrate  by nitrifying soil bacteria such as members of the genus , through the process of nitrification. Last, the process of denitrification occurs, whereby soil bacteria, such as members of the genera  and , use nitrate as a terminal electron acceptor in anaerobic respiration, converting it into nitrogen gas that reenters the atmosphere. A similar process occurs in the marine nitrogen cycle, where these three processes are performed by marine bacteria and archaea.
Human activity releases nitrogen into the environment by the use of artificial fertilizers that contain nitrogen and phosphorus compounds, which are then washed into lakes, rivers, and streams by surface runoff. A major effect from fertilizer runoff is saltwater and freshwater eutrophication, in which nutrient runoff causes the overgrowth and subsequent death of aquatic algae, making water sources anaerobic and inhospitable for the survival of aquatic organisms.

### Sulfur Cycle

Sulfur is an essential element for the macromolecules of living organisms. As part of the amino acids cysteine and methionine, it is involved in the formation of proteins. It is also found in several vitamins necessary for the synthesis of important biological molecules like coenzyme A. Several groups of microbes are responsible for carrying out processes involved in the sulfur cycle (). Anoxygenic photosynthetic bacteria as well as chemoautotrophic archaea and bacteria use hydrogen sulfide as an electron donor, oxidizing it first to elemental sulfur (S0), then to sulfate  This leads to stratification of hydrogen sulfide in soil, with levels increasing at deeper, more anaerobic depths.
Many bacteria and plants can use sulfate as a sulfur source. Decomposition dead organisms by fungi and bacteria remove sulfur groups from amino acids, producing hydrogen sulfide, returning inorganic sulfur to the environment.

### Other Biogeochemical Cycles

Beyond their involvement in the carbon, nitrogen, and sulfur cycles, prokaryotes are involved in other biogeochemical cycles as well. Like the carbon, nitrogen, and sulfur cycles, several of these additional biogeochemical cycles, such as the iron (Fe), manganese (Mn), and chromium (Cr) cycles, also involve redox chemistry, with prokaryotes playing roles in both oxidation and reduction. Several other elements undergo chemical cycles that do not involve redox chemistry. Examples of these are phosphorus (P), calcium (Ca), and silica (Si) cycles. The cycling of these elements is particularly important in oceans because large quantities of these elements are incorporated into the exoskeletons of marine organisms. These biogeochemical cycles do not involve redox chemistry but instead involve fluctuations in the solubility of compounds containing calcium, phosphorous, and silica. The overgrowth of naturally occurring microbial communities is typically limited by the availability of nitrogen (as previously mentioned), phosphorus, and iron. Human activities introducing excessive amounts of iron, nitrogen, or phosphorus (typically from detergents) may lead to eutrophication.

### Bioremediation

Microbial bioremediation leverages microbial metabolism to remove xenobiotics or other pollutants. Xenobiotics are compounds synthesized by humans and introduced into the environment in much higher concentrations than would naturally occur. Such environmental contamination may involve adhesives, dyes, flame retardants, lubricants, oil and petroleum products, organic solvents, pesticides, and products of the combustion of gasoline and oil. Many xenobiotics resist breakdown, and some accumulate in the food chain after being consumed or absorbed by fish and wildlife, which, in turn, may be eaten by humans. Of particular concern are contaminants like polycyclic aromatic hydrocarbon (PAH), a carcinogenic xenobiotic found in crude oil, and trichloroethylene (TCE), a common groundwater contaminant.
Bioremediation processes can be categorized as in situ or ex situ. Bioremediation conducted at the site of contamination is called in situ bioremediation and does not involve movement of contaminated material. In contrast, ex situ bioremediation involves the removal of contaminated material from the original site so that it can be treated elsewhere, typically in a large, lined pit where conditions are optimized for degradation of the contaminant.
Some bioremediation processes rely on microorganisms that are indigenous to the contaminated site or material. Enhanced bioremediation techniques, which may be applied to either in situ or ex situ processing, involve the addition of nutrients and/or air to encourage the growth of pollution-degrading microbes; they may also involve the addition of non-native microbes known for their ability to degrade contaminants. For example, certain bacteria of the genera  and  are known for their ability to degrade many environmental contaminants, including aromatic compounds like those found in oil, down to CO2. The genes encoding their degradatory enzymes are commonly found on plasmids. Others, like , produce surfactants that are useful in the solubilization of the hydrophobic molecules found in oil, making them more accessible to other microbes for degradation.

### Key Concepts and Summary

1. The recycling of inorganic matter between living organisms and their nonliving environment is called a biogeochemical cycle. Microbes play significant roles in these cycles.
2. In the carbon cycle, heterotrophs degrade reduced organic molecule to produce carbon dioxide, whereas autotrophs fix carbon dioxide to produce organics. Methanogens typically form methane by using CO2 as a final electron acceptor during anaerobic respiration; methanotrophs oxidize the methane, using it as their carbon source.
3. In the nitrogen cycle, nitrogen-fixing bacteria convert atmospheric nitrogen into ammonia (ammonification). The ammonia can then be oxidized to nitrite and nitrate (nitrification). Nitrates can then be assimilated by plants. Soil bacteria convert nitrate back to nitrogen gas (denitrification).
4. In sulfur cycling, many anoxygenic photosynthesizers and chemoautotrophs use hydrogen sulfide as an electron donor, producing elemental sulfur and then sulfate; sulfate-reducing bacteria and archaea then use sulfate as a final electron acceptor in anaerobic respiration, converting it back to hydrogen sulfide.
5. Human activities that introduce excessive amounts of naturally limited nutrients (like iron, nitrogen, or phosphorus) to aquatic systems may lead to eutrophication.
6. Microbial bioremediation is the use of microbial metabolism to remove or degrade xenobiotics and other environmental contaminants and pollutants. Enhanced bioremediation techniques may involve the introduction of non-native microbes specifically chosen or engineered for their ability to degrade contaminants.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer

### Critical Thinking


# Microbial Growth

## Introduction

We are all familiar with the slimy layer on a pond surface or that makes rocks slippery. These are examples of biofilms—microorganisms embedded in thin layers of matrix material (). Biofilms were long considered random assemblages of cells and had little attention from researchers. Recently, progress in visualization and biochemical methods has revealed that biofilms are an organized ecosystem within which many cells, usually of different species of bacteria, fungi, and algae, interact through cell signaling and coordinated responses. The biofilm provides a protected environment in harsh conditions and aids colonization by microorganisms. Biofilms also have clinical importance. They form on medical devices, resist routine cleaning and sterilization, and cause health-acquired infections. Within the body, biofilms form on the teeth as plaque, in the lungs of patients with cystic fibrosis, and on the cardiac tissue of patients with endocarditis. The slime layer helps protect the cells from host immune defenses and antibiotic treatments.
Studying biofilms requires new approaches. Because of the cells’ adhesion properties, many of the methods for culturing and counting cells that are explored in this chapter are not easily applied to biofilms. This is the beginning of a new era of challenges and rewarding insight into the ways that microorganisms grow and thrive in nature.


# Microbial Growth

## How Microbes Grow

### Learning Objectives

By the end of this section, you will be able to:
1. Define the generation time for growth based on binary fission
2. Identify and describe the activities of microorganisms undergoing typical phases of binary fission (simple cell division) in a growth curve
3. Explain several laboratory methods used to determine viable and total cell counts in populations undergoing exponential growth
4. Describe examples of cell division not involving binary fission, such as budding or fragmentation
5. Describe the formation and characteristics of biofilms
6. Identify health risks associated with biofilms and how they are addressed
7. Describe quorum sensing and its role in cell-to-cell communication and coordination of cellular activities
The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as will be explored in a different chapter. Most bacteria have a single circular chromosome; however, some exceptions exist. For example, , the causative agent of Lyme disease, has a linear chromosome.

### Binary Fission

The most common mechanism of cell replication in bacteria is a process called binary fission, which is depicted in . Before dividing, the cell grows and increases its number of cellular components. Next, the replication of DNA starts at a location on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane. Replication continues in opposite directions along the chromosome until the terminus is reached.
The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis). This process of cytokinesis and cell division is directed by a protein called FtsZ. FtsZ assembles into a Z ring on the cytoplasmic membrane (). The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells. Additional proteins required for cell division are added to the Z ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. The daughter cells are separated by the division septum, where all of the cells’ outer layers (the cell wall and outer membranes, if present) must be remodeled to complete division. For example, we know that specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.

### Generation Time

In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments. Most pathogens grow rapidly, like E. coli, but there are exceptions. For example, , the causative agent of tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s disease (leprosy), grows much more slowly, with a doubling time of 14 days.

### The Growth Curve

Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve. An example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment. The culture density is defined as the number of cells per unit volume. In a closed environment, the culture density is also a measure of the number of cells in the population. Infections of the body do not always follow the growth curve, but correlations can exist depending upon the site and type of infection. When the number of live cells is plotted against time, distinct phases can be observed in the curve ().

### The Lag Phase

The beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth. The initial phase of the growth curve is called the lag phase, during which cells are gearing up for the next phase of growth. The number of cells does not change during the lag phase; however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium. If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase. The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum.

### The Log Phase

In the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission and their number increases exponentially. For any given bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate. During the log phase, the relationship between time and number of cells is not linear but exponential; however, the growth curve is often plotted on a semilogarithmic graph, as shown in , which gives the appearance of a linear relationship.
Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.

### Stationary Phase

As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. Waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth. The total number of live cells reaches a plateau referred to as the stationary phase (). In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant. The culture density in a stationary culture is constant. The culture’s carrying capacity, or maximum culture density, depends on the types of microorganisms in the culture and the specific conditions of the culture; however, carrying capacity is constant for a given organism grown under the same conditions.
During the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. Secondary metabolites, including antibiotics, are synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.

### The Death Phase

As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells (). This is the aptly named death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. A few cells, the so-called persisters, are characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.

### Sustaining Microbial Growth

The growth pattern shown in  takes place in a closed environment; nutrients are not added and waste and dead cells are not removed. In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in industries that harvest microbial products. A chemostat () is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.

### Measurement of Bacterial Growth

Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists. The number of bacteria in a clinical sample serves as an indication of the extent of an infection. Quality control of drinking water, food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of disease. Two major approaches are used to measure cell number. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells. Both direct and indirect methods have advantages and disadvantages for specific applications.

### Direct Cell Count

Direct cell count refers to counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample. Let’s look first at a simple and fast method that requires only a specialized slide and a compound microscope.
The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope. The calibrated slide is called a Petroff-Hausser chamber () and is similar to a hemocytometer used to count red blood cells. The central area of the counting chamber is etched into squares of various sizes. A sample of the culture suspension is added to the chamber under a coverslip that is placed at a specific height from the surface of the grid. It is possible to estimate the concentration of cells in the original sample by counting individual cells in a number of squares and determining the volume of the sample observed. The area of the squares and the height at which the coverslip is positioned are specified for the chamber. The concentration must be corrected for dilution if the sample was diluted before enumeration.
Cells in several small squares must be counted and the average taken to obtain a reliable measurement. The advantages of the chamber are that the method is easy to use, relatively fast, and inexpensive. On the downside, the counting chamber does not work well with dilute cultures because there may not be enough cells to count.
Using a counting chamber does not necessarily yield an accurate count of the number of live cells because it is not always possible to distinguish between live cells, dead cells, and debris of the same size under the microscope. However, newly developed fluorescence staining techniques make it possible to distinguish viable and dead bacteria. These viability stains (or live stains) bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane. The primary stain, which fluoresces green, can penetrate intact cytoplasmic membranes, staining both live and dead cells. The secondary stain, which fluoresces red, can stain a cell only if the cytoplasmic membrane is considerably damaged. Thus, live cells fluoresce green because they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids ().
Another technique uses an electronic cell counting device (Coulter counter) to detect and count the changes in electrical resistance in a saline solution. A glass tube with a small opening is immersed in an electrolyte solution. A first electrode is suspended in the glass tube. A second electrode is located outside of the tube. As cells are drawn through the small aperture in the glass tube, they briefly change the resistance measured between the two electrodes and the change is recorded by an electronic sensor (); each resistance change represents a cell. The method is rapid and accurate within a range of concentrations; however, if the culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results. This method also does not differentiate between live and dead cells.
Direct counts provide an estimate of the total number of cells in a sample. However, in many situations, it is important to know the number of live, or viable, cells. Counts of live cells are needed when assessing the extent of an infection, the effectiveness of antimicrobial compounds and medication, or contamination of food and water.

### Plate Count

The viable plate count, or simply plate count, is a count of viable or live cells. It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen. The results are usually expressed as colony-forming units per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the same spot to give rise to a single colony. Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse and a single colony may represent several cells. Some cells are described as viable but nonculturable and will not form colonies on solid media. For all these reasons, the viable plate count is considered a low estimate of the actual number of live cells. These limitations do not detract from the usefulness of the method, which provides estimates of live bacterial numbers.
Microbiologists typically count plates with 30–300 colonies. Samples with too few colonies (<30) do not give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual colonies. Also, counts in this range minimize occurrences of more than one bacterial cell forming a single colony. Thus, the calculated CFU is closer to the true number of live bacteria in the population.
There are two common approaches to inoculating plates for viable counts: the pour plate and the spread plate methods. Although the final inoculation procedure differs between these two methods, they both start with a serial dilution of the culture.

### Serial Dilution

The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method. The goal of the serial dilution process is to obtain plates with CFUs in the range of 30–300, and the process usually involves several dilutions in multiples of 10 to simplify calculation. The number of serial dilutions is chosen according to a preliminary estimate of the culture density.  illustrates the serial dilution method.
A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the first dilution tube solution, which contains 9.0 mL of sterile broth. This step represents a dilution factor of 10, or 1:10, compared with the original culture. From this first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution. The dilution factor is now 1:100 compared with the original culture. This process continues until a series of dilutions is produced that will bracket the desired cell concentration for accurate counting. From each tube, a sample is plated on solid medium using either the pour plate method () or the spread plate method (). The plates are incubated until colonies appear. Two to three plates are usually prepared from each dilution and the numbers of colonies counted on each plate are averaged. In all cases, thorough mixing of samples with the dilution medium (to ensure the cell distribution in the tube is random) is paramount to obtaining reliable results.
The dilution factor is used to calculate the number of cells in the original cell culture. In our example, an average of 50 colonies was counted on the plates obtained from the 1:10,000 dilution. Because only 0.1 mL of suspension was pipetted on the plate, the multiplier required to reconstitute the original concentration is 10 × 10,000. The number of CFU per mL is equal to 50 × 10 × 10,000 = 5,000,000. The number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the 1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights the issue of inaccuracy when colony counts are greater than 300 and more than one bacterial cell grows into a single colony.
A very dilute sample—drinking water, for example—may not contain enough organisms to use either of the plate count methods described. In such cases, the original sample must be concentrated rather than diluted before plating. This can be accomplished using a modification of the plate count technique called the membrane filtration technique. Known volumes are vacuum-filtered aseptically through a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a Petri plate containing an appropriate growth medium. Colonies are counted after incubation. Calculation of the cell density is made by dividing the cell count by the volume of filtered liquid.

### The Most Probable Number

The number of microorganisms in dilute samples is usually too low to be detected by the plate count methods described thus far. For these specimens, microbiologists routinely use the most probable number (MPN) method, a statistical procedure for estimating of the number of viable microorganisms in a sample. Often used for water and food samples, the MPN method evaluates detectable growth by observing changes in turbidity or color due to metabolic activity.
A typical application of MPN method is the estimation of the number of coliforms in a sample of pond water. Coliforms are gram-negative rod bacteria that ferment lactose. The presence of coliforms in water is considered a sign of contamination by fecal matter. For the method illustrated in , a series of three dilutions of the water sample is tested by inoculating five lactose broth tubes with 10 mL of sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.1 mL of sample. The lactose broth tubes contain a pH indicator that changes color from red to yellow when the lactose is fermented. After inoculation and incubation, the tubes are examined for an indication of coliform growth by a color change in media from red to yellow. The first set of tubes (10-mL sample) showed growth in all the tubes; the second set of tubes (1 mL) showed growth in two tubes out of five; in the third set of tubes, no growth is observed in any of the tubes (0.1-mL dilution). The numbers 5, 2, and 0 are compared with Figure B1 in Appendix B, which has been constructed using a probability model of the sampling procedure. From our reading of the table, we conclude that 49 is the most probable number of bacteria per 100 mL of pond water.no lo

### Indirect Cell Counts

Besides direct methods of counting cells, other methods, based on an indirect detection of cell density, are commonly used to estimate and compare cell densities in a culture. The foremost approach is to measure the turbidity (cloudiness) of a sample of bacteria in a liquid suspension. The laboratory instrument used to measure turbidity is called a spectrophotometer (). In a spectrophotometer, a light beam is transmitted through a bacterial suspension, the light passing through the suspension is measured by a detector, and the amount of light passing through the sample and reaching the detector is converted to either percent transmission or a logarithmic value called absorbance (optical density). As the numbers of bacteria in a suspension increase, the turbidity also increases and causes less light to reach the detector. The decrease in light passing through the sample and reaching the detector is associated with a decrease in percent transmission and increase in absorbance measured by the spectrophotometer.
Measuring turbidity is a fast method to estimate cell density as long as there are enough cells in a sample to produce turbidity. It is possible to correlate turbidity readings to the actual number of cells by performing a viable plate count of samples taken from cultures having a range of absorbance values. Using these values, a calibration curve is generated by plotting turbidity as a function of cell density. Once the calibration curve has been produced, it can be used to estimate cell counts for all samples obtained or cultured under similar conditions and with densities within the range of values used to construct the curve.
Measuring dry weight of a culture sample is another indirect method of evaluating culture density without directly measuring cell counts. The cell suspension used for weighing must be concentrated by filtration or centrifugation, washed, and then dried before the measurements are taken. The degree of drying must be standardized to account for residual water content. This method is especially useful for filamentous microorganisms, which are difficult to enumerate by direct or viable plate count.
As we have seen, methods to estimate viable cell numbers can be labor intensive and take time because cells must be grown. Recently, indirect ways of measuring live cells have been developed that are both fast and easy to implement. These methods measure cell activity by following the production of metabolic products or disappearance of reactants. Adenosine triphosphate (ATP) formation, biosynthesis of proteins and nucleic acids, and consumption of oxygen can all be monitored to estimate the number of cells.

### Alternative Patterns of Cell Division

Binary fission is the most common pattern of cell division in prokaryotes, but it is not the only one. Other mechanisms usually involve asymmetrical division (as in budding) or production of spores in aerial filaments.
In some cyanobacteria, many nucleoids may accumulate in an enlarged round cell or along a filament, leading to the generation of many new cells at once. The new cells often split from the parent filament and float away in a process called fragmentation (). Fragmentation is commonly observed in the Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found in soil. Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the giant bacterium . Several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells to the environment. Other species may form a long narrow extension at one pole in a process called budding. The tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast (), but it is also observed in prosthecate bacteria and some cyanobacteria.
The soil bacteria  grow in long filaments divided by septa, similar to the mycelia seen in fungi, resulting in long cells with multiple nucleoids. Environmental signals, probably related to low nutrient availability, lead to the formation of aerial filaments. Within these aerial filaments, elongated cells divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give rise to new colonies.

### Biofilms

In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds. Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a biofilm. Microbial mats that float on water, for example, are biofilms that contain large populations of photosynthetic microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species. Regardless of the environment where they occur, biofilms are not random collections of microorganisms; rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.

### Biofilm Structure

Observations using confocal microscopy have shown that environmental conditions influence the overall structure of biofilms. Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-moving fluids. The streamers are anchored to the substrate by a “head” and the “tail” floats downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like shape. The structure of biofilms may also change with other environmental conditions such as nutrient availability.
Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal clusters of microorganisms embedded in a matrix interspersed with open water channels. The extracellular matrix consists of extracellular polymeric substances (EPS) secreted by the organisms in the biofilm. The extracellular matrix represents a large fraction of the biofilm, accounting for 50%–90% of the total dry mass. The properties of the EPS vary according to the resident organisms and environmental conditions.
EPS is a hydrated gel composed primarily of polysaccharides and containing other macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of the biofilm. Channels in the EPS allow movement of nutrients, waste, and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).

### Biofilm Formation

Free-floating microbial cells that live in an aquatic environment are called planktonic cells. The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate, where they become sessile (attached to a surface). This occurs in stages, as depicted in . The first stage involves the attachment of planktonic cells to a surface coated with a conditioning film of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including an extensive matrix and water channels. Appendages such as fimbriae, pili, and flagella interact with the EPS, and microscopy and genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites. This stage is referred to as dispersal.
Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that promote the growth of anaerobes. This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens.
The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum sensing. Quorum sensing—which can occur between cells of different species within a biofilm—enables microorganisms to detect their cell density through the release and binding of small, diffusible molecules called autoinducers. When the cell population reaches a critical threshold (a quorum), these autoinducers initiate a cascade of reactions that activate genes associated with cellular functions that are beneficial only when the population reaches a critical density. For example, in some pathogens, synthesis of virulence factors only begins when enough cells are present to overwhelm the immune defenses of the host. Although mostly studied in bacterial populations, quorum sensing takes place between bacteria and eukaryotes and between eukaryotic cells such as the fungus , a common member of the human microbiota that can cause infections in immunocompromised individuals.
The signaling molecules in quorum sensing belong to two major classes. Gram-negative bacteria communicate mainly using N-acylated homoserine lactones, whereas gram-positive bacteria mostly use small peptides (). In all cases, the first step in quorum sensing consists of the binding of the autoinducer to its specific receptor only when a threshold concentration of signaling molecules is reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in gene expression. The result is the activation of biological responses linked to quorum sensing, notably an increase in the production of signaling molecules themselves, hence the term autoinducer.

### Biofilms and Human Health

The human body harbors many types of biofilms, some beneficial and some harmful. For example, the layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off infections by pathogens. However, other biofilms in the body can have a detrimental effect on health. For example, the plaque that forms on teeth is a biofilm that can contribute to dental and periodontal disease. Biofilms can also form in wounds, sometimes causing serious infections that can spread. The bacterium  often colonizes biofilms in the airways of patients with cystic fibrosis, causing chronic and sometimes fatal infections of the lungs. Biofilms can also form on medical devices used in or on the body, causing infections in patients with in-dwelling catheters, artificial joints, or contact lenses.
Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating counterparts. Several hypotheses have been proposed to explain why. Cells in the deep layers of a biofilm are metabolically inactive and may be less susceptible to the action of antibiotics that disrupt metabolic activities. The EPS may also slow the diffusion of antibiotics and antiseptics, preventing them from reaching cells in the deeper layers of the biofilm. Phenotypic changes may also contribute to the increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of efflux pumps, membrane-embedded proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an important mechanism of antibiotic resistance among biofilm-associated bacteria. Finally, biofilms provide an ideal environment for the exchange of extrachromosomal DNA, which often includes genes that confer antibiotic resistance.

### Key Concepts and Summary

1. Most bacterial cells divide by binary fission. Generation time in bacterial growth is defined as the doubling time of the population.
2. Cells in a closed system follow a pattern of growth with four phases: lag, logarithmic (exponential), stationary, and death.
3. Cells can be counted by direct viable cell count. The pour plate and spread plate methods are used to plate serial dilutions into or onto, respectively, agar to allow counting of viable cells that give rise to colony-forming units. Membrane filtration is used to count live cells in dilute solutions. The most probable cell number (MPN) method allows estimation of cell numbers in cultures without using solid media.
4. Indirect methods can be used to estimate culture density by measuring turbidity of a culture or live cell density by measuring metabolic activity.
5. Other patterns of cell division include multiple nucleoid formation in cells; asymmetric division, as in budding; and the formation of hyphae and terminal spores.
6. Biofilms are communities of microorganisms enmeshed in a matrix of extracellular polymeric substance. The formation of a biofilm occurs when planktonic cells attach to a substrate and become sessile. Cells in biofilms coordinate their activity by communicating through quorum sensing.
7. Biofilms are commonly found on surfaces in nature and in the human body, where they may be beneficial or cause severe infections. Pathogens associated with biofilms are often more resistant to antibiotics and disinfectants.

### Multiple Choice

### Fill in the Blank

### Matching

### Short Answer

### Critical Thinking


# Microbial Growth

## Oxygen Requirements for Microbial Growth

### Learning Objectives

By the end of this section, you will be able to:
1. Interpret visual data demonstrating minimum, optimum, and maximum oxygen or carbon dioxide requirements for growth
2. Identify and describe different categories of microbes with requirements for growth with or without oxygen: obligate aerobe, obligate anaerobe, facultative anaerobe, aerotolerant anaerobe, microaerophile, and capnophile
3. Give examples of microorganisms for each category of growth requirements
Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would argue about the need for water, but what about oxygen? Can there be life without oxygen?
The answer is that molecular oxygen (O2) is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent. Only after cyanobacteria started releasing oxygen as a byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did oxygen levels increase in the atmosphere. This event, often referred to as the Great Oxygenation Event or the Oxygen Revolution, caused a massive extinction. Most organisms could not survive the powerful oxidative properties of reactive oxygen species (ROS), highly unstable ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with which they come in contact. Singlet oxygen (O2•), superoxide  peroxides (H2O2), hydroxyl radical (OH•), and hypochlorite ion (OCl−), the active ingredient of household bleach, are all examples of ROS. The organisms that were able to detoxify reactive oxygen species harnessed the high electronegativity of oxygen to produce free energy for their metabolism and thrived in the new environment.

### Oxygen Requirements of Microorganisms

Many ecosystems are still free of molecular oxygen. Some are found in extreme locations, such as deep in the ocean or in earth’s crust; others are part of our everyday landscape, such as marshes, bogs, and sewers. Within the bodies of humans and other animals, regions with little or no oxygen provide an anaerobic environment for microorganisms. ().
We can easily observe different requirements for molecular oxygen by growing bacteria in thioglycolate tube cultures. A test-tube culture starts with autoclaved thioglycolate medium containing a low percentage of agar to allow motile bacteria to move throughout the medium. Thioglycolate has strong reducing properties and autoclaving flushes out most of the oxygen. The tubes are inoculated with the bacterial cultures to be tested and incubated at an appropriate temperature. Over time, oxygen slowly diffuses throughout the thioglycolate tube culture from the top. Bacterial density increases in the area where oxygen concentration is best suited for the growth of that particular organism.
The growth of bacteria with varying oxygen requirements in thioglycolate tubes is illustrated in . In tube A, all the growth is seen at the top of the tube. The bacteria are obligate (strict) aerobes that cannot grow without an abundant supply of oxygen. Tube B looks like the opposite of tube A. Bacteria grow at the bottom of tube B. Those are obligate anaerobes, which are killed by oxygen. Tube C shows heavy growth at the top of the tube and growth throughout the tube, a typical result with facultative anaerobes. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration. The aerotolerant anaerobes in tube D are indifferent to the presence of oxygen. They do not use oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate anaerobes are. Tube E on the right shows a “Goldilocks” culture. The oxygen level has to be just right for growth, not too much and not too little. These microaerophiles are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below the 21% found in the atmosphere.
Examples of obligate aerobes are , the causative agent of tuberculosis and , a gram-positive bacterium that colonizes the skin. , the causative agent of severe bacterial meningitis, and N. gonorrhoeae, the causative agent of sexually transmitted gonorrhea, are also obligate aerobes.
Many obligate anaerobes are found in the environment where anaerobic conditions exist, such as in deep sediments of soil, still waters, and at the bottom of the deep ocean where there is no photosynthetic life. Anaerobic conditions also exist naturally in the intestinal tract of animals. Obligate anaerobes, mainly , represent a large fraction of the microbes in the human gut. Transient anaerobic conditions exist when tissues are not supplied with blood circulation; they die and become an ideal breeding ground for obligate anaerobes. Another type of obligate anaerobe encountered in the human body is the gram-positive, rod-shaped  spp. Their ability to form endospores allows them to survive in the presence of oxygen. One of the major causes of health-acquired infections is C. difficile, known as C. diff. Prolonged use of antibiotics for other infections increases the probability of a patient developing a secondary C. difficile infection. Antibiotic treatment disrupts the balance of microorganisms in the intestine and allows the colonization of the gut by C. difficile, causing a significant inflammation of the colon.
Other clostridia responsible for serious infections include C. tetani, the agent of tetanus, and C. perfringens, which causes gas gangrene. In both cases, the infection starts in necrotic tissue (dead tissue that is not supplied with oxygen by blood circulation). This is the reason that deep puncture wounds are associated with tetanus. When tissue death is accompanied by lack of circulation, gangrene is always a danger.
The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of oxygen. The most common approach is culture in an anaerobic jar (). Anaerobic jars include chemical packs that remove oxygen and release carbon dioxide (CO2). An anaerobic chamber is an enclosed box from which all oxygen is removed. Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air ().
Staphylococci and Enterobacteriaceae are examples of facultative anaerobes. Staphylococci are found on the skin and upper respiratory tract. Enterobacteriaceae are found primarily in the gut and upper respiratory tract but can sometimes spread to the urinary tract, where they are capable of causing infections. It is not unusual to see mixed bacterial infections in which the facultative anaerobes use up the oxygen, creating an environment for the obligate anaerobes to flourish.
Examples of aerotolerant anaerobes include lactobacilli and streptococci, both found in the oral microbiota. , which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.
The optimum oxygen concentration, as the name implies, is the ideal concentration of oxygen for a particular microorganism. The lowest concentration of oxygen that allows growth is called the minimum permissive oxygen concentration. The highest tolerated concentration of oxygen is the maximum permissive oxygen concentration. The organism will not grow outside the range of oxygen levels found between the minimum and maximum permissive oxygen concentrations.

### Detoxification of Reactive Oxygen Species

Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified. Even organisms that do not use aerobic respiration need some way to break down some of the ROS that may form from atmospheric oxygen. Three main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and catalase. Each one catalyzes a different reaction. Reactions of type seen in Reaction 1 are catalyzed by peroxidases.
In these reactions, an electron donor (reduced compound; e.g., reduced nicotinamide adenine dinucleotide [NADH]) oxidizes hydrogen peroxide, or other peroxides, to water. The enzymes play an important role by limiting the damage caused by peroxidation of membrane lipids. Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the powerful superoxide anions generated by aerobic metabolism:
The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.
Obligate anaerobes usually lack all three enzymes. Aerotolerant anaerobes do have SOD but no catalase. Reaction 3, shown occurring in , is the basis of a useful and rapid test to distinguish streptococci, which are aerotolerant and do not possess catalase, from staphylococci, which are facultative anaerobes. A sample of culture rapidly mixed in a drop of 3% hydrogen peroxide will release bubbles if the culture is catalase positive.
Bacteria that grow best in a higher concentration of CO2 and a lower concentration of oxygen than present in the atmosphere are called capnophiles. One common approach to grow capnophiles is to use a candle jar. A candle jar consists of a jar with a tight-fitting lid that can accommodate the cultures and a candle. After the cultures are added to the jar, the candle is lit and the lid closed. As the candle burns, it consumes most of the oxygen present and releases CO2.

### Key Concepts and Summary

1. Aerobic and anaerobic environments can be found in diverse niches throughout nature, including different sites within and on the human body.
2. Microorganisms vary in their requirements for molecular oxygen. Obligate aerobes depend on aerobic respiration and use oxygen as a terminal electron acceptor. They cannot grow without oxygen.
3. Obligate anaerobes cannot grow in the presence of oxygen. They depend on fermentation and anaerobic respiration using a final electron acceptor other than oxygen.
4. Facultative anaerobes show better growth in the presence of oxygen but will also grow without it.
5. Although aerotolerant anaerobes do not perform aerobic respiration, they can grow in the presence of oxygen. Most aerotolerant anaerobes test negative for the enzyme catalase.
6. Microaerophiles need oxygen to grow, albeit at a lower concentration than 21% oxygen in air.
7. Optimum oxygen concentration for an organism is the oxygen level that promotes the fastest growth rate. The minimum permissive oxygen concentration and the maximum permissive oxygen concentration are, respectively, the lowest and the highest oxygen levels that the organism will tolerate.
8. Peroxidase, superoxide dismutase, and catalase are the main enzymes involved in the detoxification of the reactive oxygen species. Superoxide dismutase is usually present in a cell that can tolerate oxygen. All three enzymes are usually detectable in cells that perform aerobic respiration and produce more ROS.
9. A capnophile is an organism that requires a higher than atmospheric concentration of CO2 to grow.

### Multiple Choice

### Matching

### Short Answer

### Critical Thinking


# Microbial Growth

## The Effects of pH on Microbial Growth

### Learning Objectives

By the end of this section, you will be able to:
1. Illustrate and briefly describe minimum, optimum, and maximum pH requirements for growth
2. Identify and describe the different categories of microbes with pH requirements for growth: acidophiles, neutrophiles, and alkaliphiles
3. Give examples of microorganisms for each category of pH requirement
Yogurt, pickles, sauerkraut, and lime-seasoned dishes all owe their tangy taste to a high acid content (). Recall that acidity is a function of the concentration of hydrogen ions [H+] and is measured as pH. Environments with pH values below 7.0 are considered acidic, whereas those with pH values above 7.0 are considered basic. Extreme pH affects the structure of all macromolecules. The hydrogen bonds holding together strands of DNA break up at high pH. Lipids are hydrolyzed by an extremely basic pH. The proton motive force responsible for production of ATP in cellular respiration depends on the concentration gradient of H+ across the plasma membrane (see Cellular Respiration). If H+ ions are neutralized by hydroxide ions, the concentration gradient collapses and impairs energy production. But the component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the folding of the molecule, promoting denaturation and destroying activity.
The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that an organism can tolerate is called the minimum growth pH and the highest pH is the maximum growth pH. These values can cover a wide range, which is important for the preservation of food and to microorganisms’ survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0–7.5, but the minimum growth pH is closer to 4.2.
Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7 (see ). Most familiar bacteria, like , staphylococci, and  spp. are neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0–6.0.
Microorganisms that grow optimally at pH less than 5.55 are called acidophiles. For example, the sulfur-oxidizing  spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH values of 2.5–3.5. Species of the archaean genus  live in acid mine drainage at pH values of 0–2.9.  bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8 and also contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic production of lactic acid. The vagina’s acidity plays an important role in inhibiting other microbes that are less tolerant of acidity. Acidophilic microorganisms display a number of adaptations to survive in strong acidic environments. For example, proteins show increased negative surface charge that stabilizes them at low pH. Pumps actively eject H+ ions out of the cells. The changes in the composition of membrane phospholipids probably reflect the need to maintain membrane fluidity at low pH.
At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and 10.5. , the pathogenic agent of cholera, grows best at the slightly basic pH of 8.0; it can survive pH values of 11.0 but is inactivated by the acid of the stomach. When it comes to survival at high pH, the bright pink archaean , found in the soda lakes of the African Rift Valley, may hold the record at a pH of 10.5 (). Extreme alkaliphiles have adapted to their harsh environment through evolutionary modification of lipid and protein structure and compensatory mechanisms to maintain the proton motive force in an alkaline environment. For example, the alkaliphile  derives the energy for transport reactions and motility from a Na+ ion gradient rather than a proton motive force. Many enzymes from alkaliphiles have a higher isoelectric point, due to an increase in the number of basic amino acids, than homologous enzymes from neutrophiles.

### Key Concepts and Summary

1. Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0.
2. Acidophiles grow optimally at a pH near 3.0. Alkaliphiles are organisms that grow optimally between a pH of 8 and 10.5. Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH.
3. Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH and above the maximum growth pH.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Microbial Growth

## Temperature and Microbial Growth

### Learning Objectives

By the end of this section, you will be able to:
1. Illustrate and briefly describe minimum, optimum, and maximum temperature requirements for growth
2. Identify and describe different categories of microbes with temperature requirements for growth: psychrophile, psychrotrophs, mesophile, thermophile, hyperthermophile
3. Give examples of microorganisms in each category of temperature tolerance
When the exploration of Lake Whillans started in Antarctica, researchers did not expect to find much life. Constant subzero temperatures and lack of obvious sources of nutrients did not seem to be conditions that would support a thriving ecosystem. To their surprise, the samples retrieved from the lake showed abundant microbial life. In a different but equally harsh setting, bacteria grow at the bottom of the ocean in sea vents (), where temperatures can reach 340 °C (700 °F).
Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest at the optimum growth temperature for the organism. The lowest temperature at which the organism can survive and replicate is its minimum growth temperature. The highest temperature at which growth can occur is its maximum growth temperature. The following ranges of permissive growth temperatures are approximate only and can vary according to other environmental factors.
Organisms categorized as mesophiles (“middle loving”) are adapted to moderate temperatures, with optimal growth temperatures ranging from room temperature (about 20 °C) to about 45 °C. As would be expected from the core temperature of the human body, 37 °C (98.6 °F), normal human microbiota and pathogens (e.g., E. coli, Salmonella spp., and Lactobacillus spp.) are mesophiles.
Organisms called psychrotrophs, also known as psychrotolerant, prefer cooler environments, from a high temperature of 25 °C to refrigeration temperature about 4 °C. They are found in many natural environments in temperate climates. They are also responsible for the spoilage of refrigerated food.
The organisms retrieved from arctic lakes such as Lake Whillans are considered extreme psychrophiles (cold loving). Psychrophiles are microorganisms that can grow at 0 °C and below, have an optimum growth temperature close to15 °C, and usually do not survive at temperatures above 20 °C. They are found in permanently cold environments such as the deep waters of the oceans. Because they are active at low temperature, psychrophiles and psychrotrophs are important decomposers in cold climates.
Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called thermophiles (“heat loving”). They do not multiply at room temperature. Thermophiles are widely distributed in hot springs, geothermal soils, and manmade environments such as garden compost piles where the microbes break down kitchen scraps and vegetal material. Examples of thermophiles include  and  spp. Higher up on the extreme temperature scale we find the hyperthermophiles, which are characterized by growth ranges from 80 °C to a maximum of 110 °C, with some extreme examples that survive temperatures above 121 °C, the average temperature of an autoclave. The hydrothermal vents at the bottom of the ocean are a prime example of extreme environments, with temperatures reaching an estimated 340 °C (). Microbes isolated from the vents achieve optimal growth at temperatures higher than 100 °C. Noteworthy examples are  and , archaea that grow at 105 °C and survive autoclaving.  shows the typical skewed curves of temperature-dependent growth for the categories of microorganisms we have discussed.
Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes. Very low temperatures affect cells in many ways. Membranes lose their fluidity and are damaged by ice crystal formation. Chemical reactions and diffusion slow considerably. Proteins become too rigid to catalyze reactions and may undergo denaturation. At the opposite end of the temperature spectrum, heat denatures proteins and nucleic acids. Increased fluidity impairs metabolic processes in membranes. Some of the practical applications of the destructive effects of heat on microbes are sterilization by steam, pasteurization, and incineration of inoculating loops. Proteins in psychrophiles are, in general, rich in hydrophobic residues, display an increase in flexibility, and have a lower number of secondary stabilizing bonds when compared with homologous proteins from mesophiles. Antifreeze proteins and solutes that decrease the freezing temperature of the cytoplasm are common. The lipids in the membranes tend to be unsaturated to increase fluidity. Growth rates are much slower than those encountered at moderate temperatures. Under appropriate conditions, mesophiles and even thermophiles can survive freezing. Liquid cultures of bacteria are mixed with sterile glycerol solutions and frozen to −80 °C for long-term storage as stocks. Cultures can withstand freeze drying (lyophilization) and then be stored as powders in sealed ampules to be reconstituted with broth when needed.
Macromolecules in thermophiles and hyperthermophiles show some notable structural differences from what is observed in the mesophiles. The ratio of saturated to polyunsaturated lipids increases to limit the fluidity of the cell membranes. Their DNA sequences show a higher proportion of guanine–cytosine nitrogenous bases, which are held together by three hydrogen bonds in contrast to adenine and thymine, which are connected in the double helix by two hydrogen bonds. Additional secondary structures, ionic and covalent bonds, as well as the replacement of key amino acids to stabilize folding, contribute to the resistance of proteins to denaturation. The so-called thermoenzymes purified from thermophiles have important practical applications. For example, amplification of nucleic acids in the polymerase chain reaction (PCR) depends on the thermal stability of , an enzyme isolated from T. aquaticus. Degradation enzymes from thermophiles are added as ingredients in hot-water detergents, increasing their effectiveness.

### Key Concepts and Summary

1. Microorganisms thrive at a wide range of temperatures; they have colonized different natural environments and have adapted to extreme temperatures. Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes.
2. Psychrophiles grow best in the temperature range of 0–15 °C whereas psychrotrophs thrive between 4°C and 25 °C.
3. Mesophiles grow best at moderate temperatures in the range of 20 °C to about 45 °C. Pathogens are usually mesophiles.
4. Thermophiles and hyperthemophiles are adapted to life at temperatures above 50 °C.
5. Adaptations to cold and hot temperatures require changes in the composition of membrane lipids and proteins.

### Multiple Choice

### Matching

### Short Answer

### Critical Thinking


# Microbial Growth

## Other Environmental Conditions that Affect Growth

### Learning Objectives

By the end of this section, you will be able to:
1. Identify and describe different categories of microbes with specific growth requirements other than oxygen, pH, and temperature, such as altered barometric pressure, osmotic pressure, humidity, and light
2. Give at least one example microorganism for each category of growth requirement
Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels, although these factors require significant adaptations. We also find microorganisms adapted to varying levels of salinity, barometric pressure, humidity, and light.

### Osmotic and Barometric Pressure

Most natural environments tend to have lower solute concentrations than the cytoplasm of most microorganisms. Rigid cell walls protect the cells from bursting in a dilute environment. Not much protection is available against high osmotic pressure. In this case, water, following its concentration gradient, flows out of the cell. This results in plasmolysis (the shrinking of the protoplasm away from the intact cell wall) and cell death. This fact explains why brines and layering meat and fish in salt are time-honored methods of preserving food. Microorganisms called halophiles (“salt loving”) actually require high salt concentrations for growth. These organisms are found in marine environments where salt concentrations hover at 3.5%. Extreme halophilic microorganisms, such as the red alga  and the archaeal species  in , grow in hypersaline lakes such as the Great Salt Lake, which is 3.5–8 times saltier than the ocean, and the Dead Sea, which is 10 times saltier than the ocean.
Dunaliella spp. counters the tremendous osmotic pressure of the environment with a high cytoplasmic concentration of glycerol and by actively pumping out salt ions. Halobacterium spp. accumulates large concentrations of K+ and other ions in its cytoplasm. Its proteins are designed for high salt concentrations and lose activity at salt concentrations below 1–2 M. Although most halotolerant organisms, for example  spp. in salt marshes, do not need high concentrations of salt for growth, they will survive and divide in the presence of high salt. Not surprisingly, the staphylococci, micrococci, and corynebacteria that colonize our skin tolerate salt in their environment. Halotolerant pathogens are an important cause of food-borne illnesses because they survive and multiply in salty food. For example, the halotolerant bacteria S. aureus, Bacillus cereus, and V. cholerae produce dangerous enterotoxins and are major causes of food poisoning.
Microorganisms depend on available water to grow. Available moisture is measured as water activity (a, which is the ratio of the vapor pressure of the medium of interest to the vapor pressure of pure distilled water; therefore, the aw of water is equal to 1.0. Bacteria require high aw (0.97–0.99), whereas fungi can tolerate drier environments; for example, the range of aw for growth of  spp. is 0.8–0.75. Decreasing the water content of foods by drying, as in jerky, or through freeze-drying or by increasing osmotic pressure, as in brine and jams, are common methods of preventing spoilage.
Microorganisms that require high atmospheric pressure for growth are called barophiles. The bacteria that live at the bottom of the ocean must be able to withstand great pressures. Because it is difficult to retrieve intact specimens and reproduce such growth conditions in the laboratory, the characteristics of these microorganisms are largely unknown.

### Light

Photoautotrophs, such as cyanobacteria or green sulfur bacteria, and photoheterotrophs, such as purple nonsulfur bacteria, depend on sufficient light intensity at the wavelengths absorbed by their pigments to grow and multiply. Energy from light is captured by pigments and converted into chemical energy that drives carbon fixation and other metabolic processes. The portion of the electromagnetic spectrum that is absorbed by these organisms is defined as photosynthetically active radiation (PAR). It lies within the visible light spectrum ranging from 400 to 700 nanometers (nm) and extends in the near infrared for some photosynthetic bacteria. A number of accessory pigments, such as fucoxanthin in brown algae and phycobilins in cyanobacteria, widen the useful range of wavelengths for photosynthesis and compensate for the low light levels available at greater depths of water. Other microorganisms, such as the archaea of the class Halobacteria, use light energy to drive their proton and sodium pumps. The light is absorbed by a pigment protein complex called bacteriorhodopsin, which is similar to the eye pigment rhodopsin. Photosynthetic bacteria are present not only in aquatic environments but also in soil and in symbiosis with fungi in lichens. The peculiar watermelon snow is caused by a microalga , a green alga rich in a secondary red carotenoid pigment (astaxanthin) which gives the pink hue to the snow where the alga grows.

### Key Concepts and Summary

1. Halophiles require high salt concentration in the medium, whereas halotolerant organisms can grow and multiply in the presence of high salt but do not require it for growth.
2. Halotolerant pathogens are an important source of foodborne illnesses because they contaminate foods preserved in salt.
3. Photosynthetic bacteria depend on visible light for energy.
4. Most bacteria, with few exceptions, require high moisture to grow.

### Multiple Choice

### Fill in the Blank

### Short Answer


# Microbial Growth

## Media Used for Bacterial Growth

### Learning Objectives

By the end of this section, you will be able to:
1. Identify and describe culture media for the growth of bacteria, including examples of all-purpose media, enriched, selective, differential, defined, and enrichment media
The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.

### Nutritional Requirements

The number of available media to grow bacteria is considerable. Some media are considered general all-purpose media and support growth of a large variety of organisms. A prime example of an all-purpose medium is tryptic soy broth (TSB). Specialized media are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. One type, enriched media, contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, organisms that cannot make certain nutrients and require them to be added to the medium. When the complete chemical composition of a medium is known, it is called a chemically defined medium. For example, in EZ medium, all individual chemical components are identified and the exact amounts of each is known. In complex media, which contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion, are all examples of complex media.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media. An example of a selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae. These species are commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts. The enrichment cultures foster the preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria. The differential media make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium. In , the differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.

### Key Concepts and Summary

1. Chemically defined media contain only chemically known components.
2. Selective media favor the growth of some microorganisms while inhibiting others.
3. Enriched media contain added essential nutrients a specific organism needs to grow
4. Differential media help distinguish bacteria by the color of the colonies or the change in the medium.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Biochemistry of the Genome

## Introduction

Children inherit some characteristics from each parent. Siblings typically look similar to each other, but not exactly the same—except in the case of identical twins. How can we explain these phenomena? The answers lie in heredity (the transmission of traits from one generation to the next) and genetics (the science of heredity). Because humans reproduce sexually, 50% of a child’s genes come from the mother’s egg cell and the remaining 50% from the father’s sperm cell. Sperm and egg are formed through the process of meiosis, where DNA recombination occurs. Thus, there is no predictable pattern as to which 50% comes from which parent. Thus, siblings have only some genes, and their associated characteristics, in common. Identical twins are the exception, because they are genetically identical.
Genetic differences among related microbes also dictate many observed biochemical and virulence differences. For example, some strains of the bacterium  are harmless members of the normal microbiota in the human gastrointestinal tract. Other strains of the same species have genes that give them the ability to cause disease. In bacteria, such genes are not inherited via sexual reproduction, as in humans. Often, they are transferred via plasmids, small circular pieces of double-stranded DNA that can be exchanged between prokaryotes.