# Microbial Biochemistry

## Carbohydrates

### Learning Objectives

By the end of this section, you will be able to:
1. Give examples of monosaccharides and polysaccharides
2. Describe the function of monosaccharides and polysaccharides within a cell
The most abundant biomolecules on earth are carbohydrates. From a chemical viewpoint, carbohydrates are primarily a combination of carbon and water, and many of them have the empirical formula (CH2O)n, where n is the number of repeated units. This view represents these molecules simply as “hydrated” carbon atom chains in which water molecules attach to each carbon atom, leading to the term “carbohydrates.” Although all carbohydrates contain carbon, hydrogen, and oxygen, there are some that also contain nitrogen, phosphorus, and/or sulfur. Carbohydrates have myriad different functions. They are abundant in terrestrial ecosystems, many forms of which we use as food sources. These molecules are also vital parts of macromolecular structures that store and transmit genetic information (i.e., DNA and RNA). They are the basis of biological polymers that impart strength to various structural components of organisms (e.g., cellulose and chitin), and they are the primary source of energy storage in the form of starch and glycogen.

### Monosaccharides: The Sweet Ones

In biochemistry, carbohydrates are often called saccharides, from the Greek sakcharon, meaning sugar, although not all the saccharides are sweet. The simplest carbohydrates are called monosaccharides, or simple sugars. They are the building blocks (monomers) for the synthesis of polymers or complex carbohydrates, as will be discussed further in this section. Monosaccharides are classified based on the number of carbons in the molecule. General categories are identified using a prefix that indicates the number of carbons and the suffix –ose, which indicates a saccharide; for example, triose (three carbons), tetrose (four carbons), pentose (five carbons), and hexose (six carbons) (). The hexose D-glucose is the most abundant monosaccharide in nature. Other very common and abundant hexose monosaccharides are galactose, used to make the disaccharide milk sugar lactose, and the fruit sugar fructose.
Monosaccharides of four or more carbon atoms are typically more stable when they adopt cyclic, or ring, structures. These ring structures result from a chemical reaction between functional groups on opposite ends of the sugar’s flexible carbon chain, namely the carbonyl group and a relatively distant hydroxyl group. Glucose, for example, forms a six-membered ring ().

### Disaccharides

Two monosaccharide molecules may chemically bond to form a disaccharide. The name given to the covalent bond between the two monosaccharides is a glycosidic bond. Glycosidic bonds form between hydroxyl groups of the two saccharide molecules, an example of the dehydration synthesis described in the previous section of this chapter:
Common disaccharides are the grain sugar maltose, made of two glucose molecules; the milk sugar lactose, made of a galactose and a glucose molecule; and the table sugar sucrose, made of a glucose and a fructose molecule ().

### Polysaccharides

Polysaccharides, also called glycans, are large polymers composed of hundreds of monosaccharide monomers. Unlike mono- and disaccharides, polysaccharides are not sweet and, in general, they are not soluble in water. Like disaccharides, the monomeric units of polysaccharides are linked together by glycosidic bonds.
Polysaccharides are very diverse in their structure. Three of the most biologically important polysaccharides—starch, glycogen, and cellulose—are all composed of repetitive glucose units, although they differ in their structure (). Cellulose consists of a linear chain of glucose molecules and is a common structural component of cell walls in plants and other organisms. Glycogen and starch are branched polymers; glycogen is the primary energy-storage molecule in animals and bacteria, whereas plants primarily store energy in starch. The orientation of the glycosidic linkages in these three polymers is different as well and, as a consequence, linear and branched macromolecules have different properties.
Modified glucose molecules can be fundamental components of other structural polysaccharides. Examples of these types of structural polysaccharides are N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) found in bacterial cell wall peptidoglycan. Polymers of NAG form chitin, which is found in fungal cell walls and in the exoskeleton of insects.

### Key Concepts and Summary

1. Carbohydrates, the most abundant biomolecules on earth, are widely used by organisms for structural and energy-storage purposes.
2. Carbohydrates include individual sugar molecules (monosaccharides) as well as two or more molecules chemically linked by glycosidic bonds. Monosaccharides are classified based on the number of carbons the molecule as trioses (3 C), tetroses (4 C), pentoses (5 C), and hexoses (6 C). They are the building blocks for the synthesis of polymers or complex carbohydrates.
3. Disaccharides such as sucrose, lactose, and maltose are molecules composed of two monosaccharides linked together by a glycosidic bond.
4. Polysaccharides, or glycans, are polymers composed of hundreds of monosaccharide monomers linked together by glycosidic bonds. The energy-storage polymers starch and glycogen are examples of polysaccharides and are all composed of branched chains of glucose molecules.
5. The polysaccharide cellulose is a common structural component of the cell walls of organisms. Other structural polysaccharides, such as N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), incorporate modified glucose molecules and are used in the construction of peptidoglycan or chitin.

### Multiple Choice

### Matching

### Short Answer

### Critical Thinking


# Microbial Biochemistry

## Lipids

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the chemical composition of lipids
2. Describe the unique characteristics and diverse structures of lipids
3. Compare and contrast triacylglycerides (triglycerides) and phospholipids.
4. Describe how phospholipids are used to construct biological membranes.
Although they are composed primarily of carbon and hydrogen, lipid molecules may also contain oxygen, nitrogen, sulfur, and phosphorous. Lipids serve numerous and diverse purposes in the structure and functions of organisms. They can be a source of nutrients, a storage form for carbon, energy-storage molecules, or structural components of membranes and hormones. Lipids comprise a broad class of many chemically distinct compounds, the most common of which are discussed in this section.

### Fatty Acids and Triacylglycerides

The fatty acids are lipids that contain long-chain hydrocarbons terminated with a carboxylic acid functional group. Because the long hydrocarbon chain, fatty acids are hydrophobic (“water fearing”) or nonpolar. Fatty acids with hydrocarbon chains that contain only single bonds are called saturated fatty acids because they have the greatest number of hydrogen atoms possible and are, therefore, “saturated” with hydrogen. Fatty acids with hydrocarbon chains containing at least one double bond are called unsaturated fatty acids because they have fewer hydrogen atoms. Saturated fatty acids have a straight, flexible carbon backbone, whereas unsaturated fatty acids have “kinks” in their carbon skeleton because each double bond causes a rigid bend of the carbon skeleton. These differences in saturated versus unsaturated fatty acid structure result in different properties for the corresponding lipids in which the fatty acids are incorporated. For example, lipids containing saturated fatty acids are solids at room temperature, whereas lipids containing unsaturated fatty acids are liquids.
A triacylglycerol, or triglyceride, is formed when three fatty acids are chemically linked to a glycerol molecule (). Triglycerides are the primary components of adipose tissue (body fat), and are major constituents of sebum (skin oils). They play an important metabolic role, serving as efficient energy-storage molecules that can provide more than double the caloric content of both carbohydrates and proteins.

### Phospholipids and Biological Membranes

Triglycerides are classified as simple lipids because they are formed from just two types of compounds: glycerol and fatty acids. In contrast, complex lipids contain at least one additional component, for example, a phosphate group (phospholipids) or a carbohydrate moiety (glycolipids).  depicts a typical phospholipid composed of two fatty acids linked to glycerol (a diglyceride). The two fatty acid carbon chains may be both saturated, both unsaturated, or one of each. Instead of another fatty acid molecule (as for triglycerides), the third binding position on the glycerol molecule is occupied by a modified phosphate group.
The molecular structure of lipids results in unique behavior in aqueous environments.  depicts the structure of a triglyceride. Because all three substituents on the glycerol backbone are long hydrocarbon chains, these compounds are nonpolar and not significantly attracted to polar water molecules—they are hydrophobic. Conversely, phospholipids such as the one shown in  have a negatively charged phosphate group. Because the phosphate is charged, it is capable of strong attraction to water molecules and thus is hydrophilic, or “water loving.” The hydrophilic portion of the phospholipid is often referred to as a polar “head,” and the long hydrocarbon chains as nonpolar “tails.” A molecule presenting a hydrophobic portion and a hydrophilic moiety is said to be amphipathic. Notice the “R” designation within the hydrophilic head depicted in , indicating that a polar head group can be more complex than a simple phosphate moiety. Glycolipids are examples in which carbohydrates are bonded to the lipids’ head groups.
The amphipathic nature of phospholipids enables them to form uniquely functional structures in aqueous environments. As mentioned, the polar heads of these molecules are strongly attracted to water molecules, and the nonpolar tails are not. Because of their considerable lengths, these tails are, in fact, strongly attracted to one another. As a result, energetically stable, large-scale assemblies of phospholipid molecules are formed in which the hydrophobic tails congregate within enclosed regions, shielded from contact with water by the polar heads (). The simplest of these structures are micelles, spherical assemblies containing a hydrophobic interior of phospholipid tails and an outer surface of polar head groups. Larger and more complex structures are created from lipid-bilayer sheets, or unit membranes, which are large, two-dimensional assemblies of phospholipids congregated tail to tail. The cell membranes of nearly all organisms are made from lipid-bilayer sheets, as are the membranes of many intracellular components. These sheets may also form lipid-bilayer spheres that are the structural basis of vesicles and liposomes, subcellular components that play a role in numerous physiological functions.

### Isoprenoids and Sterols

The isoprenoids are branched lipids, also referred to as terpenoids, that are formed by chemical modifications of the isoprene molecule (). These lipids play a wide variety of physiological roles in plants and animals, with many technological uses as pharmaceuticals (capsaicin), pigments (e.g., orange beta carotene, xanthophylls), and fragrances (e.g., menthol, camphor, limonene [lemon fragrance], and pinene [pine fragrance]). Long-chain isoprenoids are also found in hydrophobic oils and waxes. Waxes are typically water resistant and hard at room temperature, but they soften when heated and liquefy if warmed adequately. In humans, the main wax production occurs within the sebaceous glands of hair follicles in the skin, resulting in a secreted material called sebum, which consists mainly of triacylglycerol, wax esters, and the hydrocarbon squalene. There are many bacteria in the microbiota on the skin that feed on these lipids. One of the most prominent bacteria that feed on lipids is Propionibacterium acnes, which uses the skin’s lipids to generate short-chain fatty acids and is involved in the production of acne.
Another type of lipids are steroids, complex, ringed structures that are found in cell membranes; some function as hormones. The most common types of steroids are sterols, which are steroids containing an OH group. These are mainly hydrophobic molecules, but also have hydrophilic hydroxyl groups. The most common sterol found in animal tissues is cholesterol. Its structure consists of four rings with a double bond in one of the rings, and a hydroxyl group at the sterol-defining position. The function of cholesterol is to strengthen cell membranes in eukaryotes and in bacteria without cell walls, such as Mycoplasma. Prokaryotes generally do not produce cholesterol, although bacteria produce similar compounds called hopanoids, which are also multiringed structures that strengthen bacterial membranes (). Fungi and some protozoa produce a similar compound called ergosterol, which strengthens the cell membranes of these organisms.

### Key Concepts and Summary

1. Lipids are composed mainly of carbon and hydrogen, but they can also contain oxygen, nitrogen, sulfur, and phosphorous. They provide nutrients for organisms, store carbon and energy, play structural roles in membranes, and function as hormones, pharmaceuticals, fragrances, and pigments.
2. Fatty acids are long-chain hydrocarbons with a carboxylic acid functional group. Their relatively long nonpolar hydrocarbon chains make them hydrophobic. Fatty acids with no double bonds are saturated; those with double bonds are unsaturated.
3. Fatty acids chemically bond to glycerol to form structurally essential lipids such as triglycerides and phospholipids. Triglycerides comprise three fatty acids bonded to glycerol, yielding a hydrophobic molecule. Phospholipids contain both hydrophobic hydrocarbon chains and polar head groups, making them amphipathic and capable of forming uniquely functional large scale structures.
4. Biological membranes are large-scale structures based on phospholipid bilayers that provide hydrophilic exterior and interior surfaces suitable for aqueous environments, separated by an intervening hydrophobic layer. These bilayers are the structural basis for cell membranes in most organisms, as well as subcellular components such as vesicles.
5. Isoprenoids are lipids derived from isoprene molecules that have many physiological roles and a variety of commercial applications.
6. A wax is a long-chain isoprenoid that is typically water resistant; an example of a wax-containing substance is sebum, produced by sebaceous glands in the skin. Steroids are lipids with complex, ringed structures that function as structural components of cell membranes and as hormones. Sterols are a subclass of steroids containing a hydroxyl group at a specific location on one of the molecule’s rings; one example is cholesterol.
7. Bacteria produce hopanoids, structurally similar to cholesterol, to strengthen bacterial membranes. Fungi and protozoa produce a strengthening agent called ergosterol.

### Multiple Choice

### True/False

### Fill in the Blank

### Critical Thinking

### Short Answer


# Microbial Biochemistry

## Proteins

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the fundamental structure of an amino acid
2. Describe the chemical structures of proteins
3. Summarize the unique characteristics of proteins
At the beginning of this chapter, a famous experiment was described in which scientists synthesized amino acids under conditions simulating those present on earth long before the evolution of life as we know it. These compounds are capable of bonding together in essentially any number, yielding molecules of essentially any size that possess a wide array of physical and chemical properties and perform numerous functions vital to all organisms. The molecules derived from amino acids can function as structural components of cells and subcellular entities, as sources of nutrients, as atom- and energy-storage reservoirs, and as functional species such as hormones, enzymes, receptors, and transport molecules.

### Amino Acids and Peptide Bonds

An amino acid is an organic molecule in which a hydrogen atom, a carboxyl group (–COOH), and an amino group (–NH2) are all bonded to the same carbon atom, the so-called α carbon. The fourth group bonded to the α carbon varies among the different amino acids and is called a residue or a side chain, represented in structural formulas by the letter R. A residue is a monomer that results when two or more amino acids combine and remove water molecules. The primary structure of a protein, a peptide chain, is made of amino acid residues. The unique characteristics of the functional groups and s allow these components of the amino acids to form hydrogen, ionic, and disulfide bonds, along with polar/nonpolar interactions needed to form secondary, tertiary, and quaternary protein structures. These groups are composed primarily of carbon, hydrogen, oxygen, nitrogen, and sulfur, in the form of hydrocarbons, acids, amides, alcohols, and amines. A few examples illustrating these possibilities are provided in .
Amino acids may chemically bond together by reaction of the carboxylic acid group of one molecule with the amine group of another. This reaction forms a peptide bond and a water molecule and is another example of dehydration synthesis (). Molecules formed by chemically linking relatively modest numbers of amino acids (approximately 50 or fewer) are called peptides, and prefixes are often used to specify these numbers: dipeptides (two amino acids), tripeptides (three amino acids), and so forth. More generally, the approximate number of amino acids is designated: oligopeptides are formed by joining up to approximately 20 amino acids, whereas polypeptides are synthesized from up to approximately 50 amino acids. When the number of amino acids linked together becomes very large, or when multiple polypeptides are used as building subunits, the macromolecules that result are called proteins. The continuously variable length (the number of monomers) of these biopolymers, along with the variety of possible s on each amino acid, allows for a nearly unlimited diversity in the types of proteins that may be formed.

### Protein Structure

The size (length) and specific amino acid sequence of a protein are major determinants of its shape, and the shape of a protein is critical to its function. For example, in the process of biological nitrogen fixation (see Biogeochemical Cycles), soil microorganisms collectively known as rhizobia symbiotically interact with roots of legume plants such as soybeans, peanuts, or beans to form a novel structure called a nodule on the plant roots. The plant then produces a carrier protein called leghemoglobin, a protein that carries nitrogen or oxygen. Leghemoglobin binds with a very high affinity to its substrate oxygen at a specific region of the protein where the shape and amino acid sequence are appropriate (the active site). If the shape or chemical environment of the active site is altered, even slightly, the substrate may not be able to bind as strongly, or it may not bind at all. Thus, for the protein to be fully active, it must have the appropriate shape for its function.
Protein structure is categorized in terms of four levels: primary, secondary, tertiary, and quaternary. The primary structure is simply the sequence of amino acids that make up the polypeptide chain.  depicts the primary structure of a protein.
The chain of amino acids that defines a protein’s primary structure is not rigid, but instead is flexible because of the nature of the bonds that hold the amino acids together. When the chain is sufficiently long, hydrogen bonding may occur between amine and carbonyl functional groups within the peptide backbone (excluding the R side group), resulting in localized folding of the polypeptide chain into helices and sheets. These shapes constitute a protein’s secondary structure. The most common secondary structures are the α-helix and β-pleated sheet. In the α-helix structure, the helix is held by hydrogen bonds between the oxygen atom in a carbonyl group of one amino acid and the hydrogen atom of the amino group that is just four amino acid units farther along the chain. In the β-pleated sheet, the pleats are formed by similar hydrogen bonds between continuous sequences of carbonyl and amino groups that are further separated on the backbone of the polypeptide chain ().
The next level of protein organization is the tertiary structure, which is the large-scale three-dimensional shape of a single polypeptide chain. Tertiary structure is determined by interactions between amino acid residues that are far apart in the chain. A variety of interactions give rise to protein tertiary structure, such as disulfide bridges, which are bonds between the sulfhydryl (–SH) functional groups on amino acid side groups; hydrogen bonds; ionic bonds; and hydrophobic interactions between nonpolar side chains. All these interactions, weak and strong, combine to determine the final three-dimensional shape of the protein and its function ().
The process by which a polypeptide chain assumes a large-scale, three-dimensional shape is called protein folding. Folded proteins that are fully functional in their normal biological role are said to possess a native structure. When a protein loses its three-dimensional shape, it may no longer be functional. These unfolded proteins are denatured. Denaturation implies the loss of the secondary structure and tertiary structure (and, if present, the quaternary structure) without the loss of the primary structure.
Some proteins are assemblies of several separate polypeptides, also known as protein subunits. These proteins function adequately only when all subunits are present and appropriately configured. The interactions that hold these subunits together constitute the quaternary structure of the protein. The overall quaternary structure is stabilized by relatively weak interactions. Hemoglobin, for example, has a quaternary structure of four globular protein subunits: two α and two β polypeptides, each one containing an iron-based heme ().
Another important class of proteins is the conjugated proteins that have a nonprotein portion. If the conjugated protein has a carbohydrate attached, it is called a glycoprotein. If it has a lipid attached, it is called a lipoprotein. These proteins are important components of membranes.  summarizes the four levels of protein structure.

### Key Concepts and Summary

1. Amino acids are small molecules essential to all life. Each has an α carbon to which a hydrogen atom, carboxyl group, and amine group are bonded. The fourth bonded group, represented by R, varies in chemical composition, size, polarity, and charge among different amino acids, providing variation in properties.
2. Peptides are polymers formed by the linkage of amino acids via dehydration synthesis. The bonds between the linked amino acids are called peptide bonds. The number of amino acids linked together may vary from a few to many.
3. Proteins are polymers formed by the linkage of a very large number of amino acids. They perform many important functions in a cell, serving as nutrients and enzymes; storage molecules for carbon, nitrogen, and energy; and structural components.
4. The structure of a protein is a critical determinant of its function and is described by a graduated classification: primary, secondary, tertiary, and quaternary. The native structure of a protein may be disrupted by denaturation, resulting in loss of its higher-order structure and its biological function.
5. Some proteins are formed by several separate protein subunits, the interaction of these subunits composing the quaternary structure of the protein complex.
6. Conjugated proteins have a nonpolypeptide portion that can be a carbohydrate (forming a glycoprotein) or a lipid fraction (forming a lipoprotein). These proteins are important components of membranes.

### Multiple Choice

### Fill in the Blank

### True/False

### Critical Thinking


# Microbial Biochemistry

## Using Biochemistry to Identify Microorganisms

### Learning Objectives

By the end of this section, you will be able to:
1. Describe examples of biosynthesis products within a cell that can be detected to identify bacteria
Accurate identification of bacterial isolates is essential in a clinical microbiology laboratory because the results often inform decisions about treatment that directly affect patient outcomes. For example, cases of food poisoning require accurate identification of the causative agent so that physicians can prescribe appropriate treatment. Likewise, it is important to accurately identify the causative pathogen during an outbreak of disease so that appropriate strategies can be employed to contain the epidemic.
There are many ways to detect, characterize, and identify microorganisms. Some methods rely on phenotypic biochemical characteristics, while others use genotypic identification. The biochemical characteristics of a bacterium provide many traits that are useful for classification and identification. Analyzing the nutritional and metabolic capabilities of the bacterial isolate is a common approach for determining the genus and the species of the bacterium. Some of the most important metabolic pathways that bacteria use to survive will be discussed in Microbial Metabolism. In this section, we will discuss a few methods that use biochemical characteristics to identify microorganisms.
Some microorganisms store certain compounds as granules within their cytoplasm, and the contents of these granules can be used for identification purposes. For example, poly-β-hydroxybutyrate (PHB) is a carbon- and energy-storage compound found in some nonfluorescent bacteria of the genus . Different species within this genus can be classified by the presence or the absence of PHB and fluorescent pigments. The human pathogen P. aeruginosa and the plant pathogen P. syringae are two examples of fluorescent Pseudomonas species that do not accumulate PHB granules.
Other systems rely on biochemical characteristics to identify microorganisms by their biochemical reactions, such as carbon utilization and other metabolic tests. In small laboratory settings or in teaching laboratories, those assays are carried out using a limited number of test tubes. However, more modern systems, such as the one developed by Biolog, Inc., are based on panels of biochemical reactions performed simultaneously and analyzed by software. Biolog’s system identifies cells based on their ability to metabolize certain biochemicals and on their physiological properties, including pH and chemical sensitivity. It uses all major classes of biochemicals in its analysis. Identifications can be performed manually or with the semi- or fully automated instruments.
Another automated system identifies microorganisms by determining the specimen’s mass spectrum and then comparing it to a database that contains known mass spectra for thousands of microorganisms. This method is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and uses disposable MALDI plates on which the microorganism is mixed with a specialized matrix reagent (). The sample/reagent mixture is irradiated with a high-intensity pulsed ultraviolet laser, resulting in the ejection of gaseous ions generated from the various chemical constituents of the microorganism. These gaseous ions are collected and accelerated through the mass spectrometer, with ions traveling at a velocity determined by their mass-to-charge ratio (m/z), thus, reaching the detector at different times. A plot of detector signal versus m/z yields a mass spectrum for the organism that is uniquely related to its biochemical composition. Comparison of the mass spectrum to a library of reference spectra obtained from identical analyses of known microorganisms permits identification of the unknown microbe.
Microbes can also be identified by measuring their unique lipid profiles. As we have learned, fatty acids of lipids can vary in chain length, presence or absence of double bonds, and number of double bonds, hydroxyl groups, branches, and rings. To identify a microbe by its lipid composition, the fatty acids present in their membranes are analyzed. A common biochemical analysis used for this purpose is a technique used in clinical, public health, and food laboratories. It relies on detecting unique differences in fatty acids and is called fatty acid methyl ester (FAME) analysis. In a FAME analysis, fatty acids are extracted from the membranes of microorganisms, chemically altered to form volatile methyl esters, and analyzed by gas chromatography (GC). The resulting GC chromatogram is compared with reference chromatograms in a database containing data for thousands of bacterial isolates to identify the unknown microorganism ().
A related method for microorganism identification is called phospholipid-derived fatty acids (PLFA) analysis. Membranes are mostly composed of phospholipids, which can be saponified (hydrolyzed with alkali) to release the fatty acids. The resulting fatty acid mixture is then subjected to FAME analysis, and the measured lipid profiles can be compared with those of known microorganisms to identify the unknown microorganism.
Bacterial identification can also be based on the proteins produced under specific growth conditions within the human body. These types of identification procedures are called proteomic analysis. To perform proteomic analysis, proteins from the pathogen are first separated by high-pressure liquid chromatography (HPLC), and the collected fractions are then digested to yield smaller peptide fragments. These peptides are identified by mass spectrometry and compared with those of known microorganisms to identify the unknown microorganism in the original specimen.
Microorganisms can also be identified by the carbohydrates attached to proteins (glycoproteins) in the plasma membrane or cell wall. Antibodies and other carbohydrate-binding proteins can attach to specific carbohydrates on cell surfaces, causing the cells to clump together. Serological tests (e.g., the Lancefield groups tests, which are used for identification of Streptococcus species) are performed to detect the unique carbohydrates located on the surface of the cell.

### Key Concepts and Summary

1. Accurate identification of bacteria is essential in a clinical laboratory for diagnostic and management of epidemics, pandemics, and food poisoning caused by bacterial outbreaks.
2. The phenotypic identification of microorganisms involves using observable traits, including profiles of structural components such as lipids, biosynthetic products such as sugars or amino acids, or storage compounds such as poly-β-hydroxybutyrate.
3. An unknown microbe may be identified from the unique mass spectrum produced when it is analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF).
4. Microbes can be identified by determining their lipid compositions, using fatty acid methyl esters (FAME) or phospholipid-derived fatty acids (PLFA) analysis.
5. Proteomic analysis, the study of all accumulated proteins of an organism; can also be used for bacterial identification.
6. Glycoproteins in the plasma membrane or cell wall structures can bind to lectins or antibodies and can be used for identification.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer


# Microbial Metabolism

## Introduction

Throughout earth’s history, microbial metabolism has been a driving force behind the development and maintenance of the planet’s biosphere. Eukaryotic organisms such as plants and animals typically depend on organic molecules for energy, growth, and reproduction. Prokaryotes, on the other hand, can metabolize a wide range of organic as well as inorganic matter, from complex organic molecules like cellulose to inorganic molecules and ions such as atmospheric nitrogen (N2), molecular hydrogen (H2), sulfide (S2−), manganese (II) ions (Mn2+), ferrous iron (Fe2+), and ferric iron (Fe3+), to name a few. By metabolizing such substances, microbes chemically convert them to other forms. In some cases, microbial metabolism produces chemicals that can be harmful to other organisms; in others, it produces substances that are essential to the metabolism and survival of other life forms ().


# Microbial Metabolism

## Energy, Matter, and Enzymes

### Learning Objectives

By the end of this section, you will be able to:
1. Define and describe metabolism
2. Compare and contrast autotrophs and heterotrophs
3. Describe the importance of oxidation-reduction reactions in metabolism
4. Describe why ATP, FAD, NAD+, and NADP+ are important in a cell
5. Identify the structure and structural components of an enzyme
6. Describe the differences between competitive and noncompetitive enzyme inhibitors
The term used to describe all of the chemical reactions inside a cell is metabolism (). Cellular processes such as the building or breaking down of complex molecules occur through series of stepwise, interconnected chemical reactions called metabolic pathways. Reactions that are spontaneous and release energy are exergonic reactions, whereas endergonic reactions require energy to proceed. The term anabolism refers to those endergonic metabolic pathways involved in biosynthesis, converting simple molecular building blocks into more complex molecules, and fueled by the use of cellular energy. Conversely, the term catabolism refers to exergonic pathways that break down complex molecules into simpler ones. Molecular energy stored in the bonds of complex molecules is released in catabolic pathways and harvested in such a way that it can be used to produce high-energy molecules, which are used to drive anabolic pathways. Thus, in terms of energy and molecules, cells are continually balancing catabolism with anabolism.

### Classification by Carbon and Energy Source

Organisms can be identified according to the source of carbon they use for metabolism as well as their energy source. The prefixes auto- (“self”) and hetero- (“other”) refer to the origins of the carbon sources various organisms can use. Organisms that convert inorganic carbon dioxide (CO2) into organic carbon compounds are autotrophs. Plants and cyanobacteria are well-known examples of autotrophs. Conversely, heterotrophs rely on more complex organic carbon compounds as nutrients; these are provided to them initially by autotrophs. Many organisms, ranging from humans to many prokaryotes, including the well-studied , are heterotrophic.
Organisms can also be identified by the energy source they use. All energy is derived from the transfer of electrons, but the source of electrons differs between various types of organisms. The prefixes photo- (“light”) and chemo- (“chemical”) refer to the energy sources that various organisms use. Those that get their energy for electron transfer from light are phototrophs, whereas chemotrophs obtain energy for electron transfer by breaking chemical bonds. There are two types of chemotrophs: organotrophs and lithotrophs. Organotrophs, including humans, fungi, and many prokaryotes, are chemotrophs that obtain energy from organic compounds. Lithotrophs (“litho” means “rock”) are chemotrophs that get energy from inorganic compounds, including hydrogen sulfide (H2S) and reduced iron. Lithotrophy is unique to the microbial world.
The strategies used to obtain both carbon and energy can be combined for the classification of organisms according to nutritional type. Most organisms are chemoheterotrophs because they use organic molecules as both their electron and carbon sources.  summarizes this and the other classifications.

### Oxidation and Reduction in Metabolism

The transfer of electrons between molecules is important because most of the energy stored in atoms and used to fuel cell functions is in the form of high-energy electrons. The transfer of energy in the form of electrons allows the cell to transfer and use energy incrementally; that is, in small packages rather than a single, destructive burst. Reactions that remove electrons from donor molecules, leaving them oxidized, are oxidation reactions; those that add electrons to acceptor molecules, leaving them reduced, are reduction reactions. Because electrons can move from one molecule to another, oxidation and reduction occur in tandem. These pairs of reactions are called oxidation-reduction reactions, or redox reactions.

### Energy Carriers: NAD+, NADP+, FAD, and ATP

The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers we will consider originate from the B vitamin group and are derivatives of nucleotides; they are nicotinamide adenine dinucleotide, nicotine adenine dinucleotide phosphate, and flavin adenine dinucleotide. These compounds can be easily reduced or oxidized. Nicotinamide adenine dinucleotide (NAD) is the most common mobile electron carrier used in catabolism. NAD+ is the oxidized form of the molecule; NADH is the reduced form of the molecule. Nicotine adenine dinucleotide phosphate (NADP), the oxidized form of an NAD+ variant that contains an extra phosphate group, is another important electron carrier; it forms NADPH when reduced. The oxidized form of flavin adenine dinucleotide is FAD, and its reduced form is FADH. Both NAD+/NADH and FAD/FADH2 are extensively used in energy extraction from sugars during catabolism in chemoheterotrophs, whereas NADP+/NADPH plays an important role in anabolic reactions and photosynthesis. Collectively, FADH2, NADH, and NADPH are often referred to as having reducing power due to their ability to donate electrons to various chemical reactions.
A living cell must be able to handle the energy released during catabolism in a way that enables the cell to store energy safely and release it for use only as needed. Living cells accomplish this by using the compound adenosine triphosphate (ATP). ATP is often called the “energy currency” of the cell, and, like currency, this versatile compound can be used to fill any energy need of the cell. At the heart of ATP is a molecule of adenosine monophosphate (AMP), which is composed of an adenine molecule bonded to a ribose molecule and a single phosphate group. Ribose is a five-carbon sugar found in RNA, and AMP is one of the nucleotides in RNA. The addition of a second phosphate group to this core molecule results in the formation of adenosine diphosphate (ADP); the addition of a third phosphate group forms ATP (). Adding a phosphate group to a molecule, a process called phosphorylation, requires energy. Phosphate groups are negatively charged and thus repel one another when they are arranged in series, as they are in ADP and ATP. This repulsion makes the ADP and ATP molecules inherently unstable. Thus, the bonds between phosphate groups (one in ADP and two in ATP) are called high-energy phosphate bonds. When these high-energy bonds are broken to release one phosphate (called inorganic phosphate [P) or two connected phosphate groups (called pyrophosphate [PP) from ATP through a process called dephosphorylation, energy is released to drive endergonic reactions ().

### Enzyme Structure and Function

A substance that helps speed up a chemical reaction is a catalyst. Catalysts are not used or changed during chemical reactions and, therefore, are reusable. Whereas inorganic molecules may serve as catalysts for a wide range of chemical reactions, proteins called enzymes serve as catalysts for biochemical reactions inside cells. Enzymes thus play an important role in controlling cellular metabolism.
An enzyme functions by lowering the activation energy of a chemical reaction inside the cell. Activation energy is the energy needed to form or break chemical bonds and convert reactants to products (). Enzymes lower the activation energy by binding to the reactant molecules and holding them in such a way as to speed up the reaction.
The chemical reactants to which an enzyme binds are called substrates, and the location within the enzyme where the substrate binds is called the enzyme’s active site. The characteristics of the amino acids near the active site create a very specific chemical environment within the active site that induces suitability to binding, albeit briefly, to a specific substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates, enzymes are known for their specificity. In fact, as an enzyme binds to its substrate(s), the enzyme structure changes slightly to find the best fit between the transition state (a structural intermediate between the substrate and product) and the active site, just as a rubber glove molds to a hand inserted into it. This active-site modification in the presence of substrate, along with the simultaneous formation of the transition state, is called induced fit (). Overall, there is a specifically matched enzyme for each substrate and, thus, for each chemical reaction; however, there is some flexibility as well. Some enzymes have the ability to act on several different structurally related substrates.
Enzymes are subject to influences by local environmental conditions such as pH, substrate concentration, and temperature. Although increasing the environmental temperature generally increases reaction rates, enzyme catalyzed or otherwise, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site, making them less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological molecules, to denature, losing their three-dimensional structure and function. Enzymes are also suited to function best within a certain pH range, and, as with temperature, extreme environmental pH values (acidic or basic) can cause enzymes to denature. Active-site amino-acid side chains have their own acidic or basic properties that are optimal for catalysis and, therefore, are sensitive to changes in pH.
Another factor that influences enzyme activity is substrate concentration: Enzyme activity is increased at higher concentrations of substrate until it reaches a saturation point at which the enzyme can bind no additional substrate. Overall, enzymes are optimized to work best under the environmental conditions in which the organisms that produce them live. For example, while microbes that inhabit hot springs have enzymes that work best at high temperatures, human pathogens have enzymes that work best at 37°C. Similarly, while enzymes produced by most organisms work best at a neutral pH, microbes growing in acidic environments make enzymes optimized to low pH conditions, allowing for their growth at those conditions.
Many enzymes do not work optimally, or even at all, unless bound to other specific nonprotein helper molecules, either temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Binding to these molecules promotes optimal conformation and function for their respective enzymes. Two types of helper molecules are cofactors and coenzymes. Cofactors are inorganic ions such as iron (Fe2+) and magnesium (Mg2+) that help stabilize enzyme conformation and function. One example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound zinc ion (Zn2+) to function.
Coenzymes are organic helper molecules that are required for enzyme action. Like enzymes, they are not consumed and, hence, are reusable. The most common sources of coenzymes are dietary vitamins. Some vitamins are precursors to coenzymes and others act directly as coenzymes.
Some cofactors and coenzymes, like coenzyme A (CoA), often bind to the enzyme’s active site, aiding in the chemistry of the transition of a substrate to a product (). In such cases, an enzyme lacking a necessary cofactor or coenzyme is called an apoenzyme and is inactive. Conversely, an enzyme with the necessary associated cofactor or coenzyme is called a holoenzyme and is active. NADH and ATP are also both examples of commonly used coenzymes that provide high-energy electrons or phosphate groups, respectively, which bind to enzymes, thereby activating them.

### Enzyme Inhibitors

Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so (). A competitive inhibitor is a molecule similar enough to a substrate that it can compete with the substrate for binding to the active site by simply blocking the substrate from binding. For a competitive inhibitor to be effective, the inhibitor concentration needs to be approximately equal to the substrate concentration. Sulfa drugs provide a good example of competitive competition. They are used to treat bacterial infections because they bind to the active site of an enzyme within the bacterial folic acid synthesis pathway. When present in a sufficient dose, a sulfa drug prevents folic acid synthesis, and bacteria are unable to grow because they cannot synthesize DNA, RNA, and proteins. Humans are unaffected because we obtain folic acid from our diets.
On the other hand, a noncompetitive (allosteric) inhibitor binds to the enzyme at an allosteric site, a location other than the active site, and still manages to block substrate binding to the active site by inducing a conformational change that reduces the affinity of the enzyme for its substrate (). Because only one inhibitor molecule is needed per enzyme for effective inhibition, the concentration of inhibitors needed for noncompetitive inhibition is typically much lower than the substrate concentration.
In addition to allosteric inhibitors, there are allosteric activators that bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).
Allosteric control is an important mechanism of regulation of metabolic pathways involved in both catabolism and anabolism. In a most efficient and elegant way, cells have evolved also to use the products of their own metabolic reactions for feedback inhibition of enzyme activity. Feedback inhibition involves the use of a pathway product to regulate its own further production. The cell responds to the abundance of specific products by slowing production during anabolic or catabolic reactions ().

### Key Concepts and Summary

1. Metabolism includes chemical reactions that break down complex molecules (catabolism) and those that build complex molecules (anabolism).
2. Organisms may be classified according to their source of carbon. Autotrophs convert inorganic carbon dioxide into organic carbon; heterotrophs use fixed organic carbon compounds.
3. Organisms may also be classified according to their energy source. Phototrophs obtain their energy from light. Chemotrophs get their energy from chemical compounds. Organotrophs use organic molecules, and lithotrophs use inorganic chemicals.
4. Cellular electron carriers accept high-energy electrons from foods and later serve as electron donors in subsequent redox reactions. FAD/FADH, and NADP are important electron carriers.
5. Adenosine triphosphate (ATP) serves as the energy currency of the cell, safely storing chemical energy in its two high-energy phosphate bonds for later use to drive processes requiring energy.
6. Enzymes are biological catalysts that increase the rate of chemical reactions inside cells by lowering the activation energy required for the reaction to proceed.
7. In nature, exergonic reactions do not require energy beyond activation energy to proceed, and they release energy. They may proceed without enzymes, but at a slow rate. Conversely, endergonic reactions require energy beyond activation energy to occur. In cells, endergonic reactions are coupled to exergonic reactions, making the combination energetically favorable.
8. Substrates bind to the enzyme’s active site. This process typically alters the structures of both the active site and the substrate, favoring transition-state formation; this is known as induced fit.
9. Cofactors are inorganic ions that stabilize enzyme conformation and function. Coenzymes are organic molecules required for proper enzyme function and are often derived from vitamins. An enzyme lacking a cofactor or coenzyme is an apoenzyme; an enzyme with a bound cofactor or coenzyme is a holoenzyme.
10. Competitive inhibitors regulate enzymes by binding to an enzyme’s active site, preventing substrate binding. Noncompetitive (allosteric) inhibitors bind to allosteric sites, inducing a conformational change in the enzyme that prevents it from functioning. Feedback inhibition occurs when the product of a metabolic pathway noncompetitively binds to an enzyme early on in the pathway, ultimately preventing the synthesis of the product.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer


# Microbial Metabolism

## Catabolism of Carbohydrates

### Learning Objectives

By the end of this section, you will be able to:
1. Describe why glycolysis is not oxygen dependent
2. Define and describe the net yield of three-carbon molecules, ATP, and NADH from glycolysis
3. Explain how three-carbon pyruvate molecules are converted into two-carbon acetyl groups that can be funneled into the Krebs cycle.
4. Define and describe the net yield of CO2, GTP/ATP, FADH2, and NADH from the Krebs cycle
5. Explain how intermediate carbon molecules of the Krebs cycle can be used in a cell
Extensive enzyme pathways exist for breaking down carbohydrates to capture energy in ATP bonds. In addition, many catabolic pathways produce intermediate molecules that are also used as building blocks for anabolism. Understanding these processes is important for several reasons. First, because the main metabolic processes involved are common to a wide range of chemoheterotrophic organisms, we can learn a great deal about human metabolism by studying metabolism in more easily manipulated bacteria like E. coli. Second, because animal and human pathogens are also chemoheterotrophs, learning about the details of metabolism in these bacteria, including possible differences between bacterial and human pathways, is useful for the diagnosis of pathogens as well as for the discovery of antimicrobial therapies targeting specific pathogens. Last, learning specifically about the pathways involved in chemoheterotrophic metabolism also serves as a basis for comparing other more unusual metabolic strategies used by microbes. Although the chemical source of electrons initiating electron transfer is different between chemoheterorophs and chemoautotrophs, many similar processes are used in both types of organisms.
The typical example used to introduce concepts of metabolism to students is carbohydrate catabolism. For chemoheterotrophs, our examples of metabolism start with the catabolism of polysaccharides such as glycogen, starch, or cellulose. Enzymes such as amylase, which breaks down glycogen or starch, and cellulases, which break down cellulose, can cause the hydrolysis of glycosidic bonds between the glucose monomers in these polymers, releasing glucose for further catabolism.

### Glycolysis

For bacteria, eukaryotes, and most archaea, glycolysis is the most common pathway for the catabolism of glucose; it produces energy, reduced electron carriers, and precursor molecules for cellular metabolism. Every living organism carries out some form of glycolysis, suggesting this mechanism is an ancient universal metabolic process. The process itself does not use oxygen; however, glycolysis can be coupled with additional metabolic processes that are either aerobic or anaerobic. Glycolysis takes place in the cytoplasm of prokaryotic and eukaryotic cells. It begins with a single six-carbon glucose molecule and ends with two molecules of a three-carbon sugar called pyruvate. Pyruvate may be broken down further after glycolysis to harness more energy through aerobic or anaerobic respiration, but many organisms, including many microbes, may be unable to respire; for these organisms, glycolysis may be their only source of generating ATP.
The type of glycolysis found in animals and that is most common in microbes is the Embden-Meyerhof-Parnas (EMP) pathway, named after Gustav Embden (1874–1933), Otto Meyerhof (1884–1951), and Jakub Parnas (1884–1949). Glycolysis using the EMP pathway consists of two distinct phases (). The first part of the pathway, called the energy investment phase, uses energy from two ATP molecules to modify a glucose molecule so that the six-carbon sugar molecule can be split evenly into two phosphorylated three-carbon molecules called glyceraldehyde 3-phosphate (G3P). The second part of the pathway, called the energy payoff phase, extracts energy by oxidizing G3P to pyruvate, producing four ATP molecules and reducing two molecules of NAD+ to two molecules of NADH, using electrons that originated from glucose. (A discussion and illustration of the full EMP pathway with chemical structures and enzyme names appear in Appendix C.)
The ATP molecules produced during the energy payoff phase of glycolysis are formed by substrate-level phosphorylation (), one of two mechanisms for producing ATP. In substrate-level phosphorylation, a phosphate group is removed from an organic molecule and is directly transferred to an available ADP molecule, producing ATP. During glycolysis, high-energy phosphate groups from the intermediate molecules are added to ADP to make ATP.
Overall, in this process of glycolysis, the net gain from the breakdown of a single glucose molecule is:
1. two ATP molecules
2. two NADH molecule, and
3. two pyruvate molecules.

### Other Glycolytic Pathways

When we refer to glycolysis, unless otherwise indicated, we are referring to the EMP pathway used by animals and many bacteria. However, some prokaryotes use alternative glycolytic pathways. One important alternative is the Entner-Doudoroff (ED) pathway, named after its discoverers Nathan Entner and Michael Doudoroff (1911–1975). Although some bacteria, including the opportunistic gram-negative pathogen , contain only the ED pathway for glycolysis, other bacteria, like E. coli, have the ability to use either the ED pathway or the EMP pathway.
A third type of glycolytic pathway that occurs in all cells, which is quite different from the previous two pathways, is the pentose phosphate pathway (PPP) also called the phosphogluconate pathway or the hexose monophosphate shunt. Evidence suggests that the PPP may be the most ancient universal glycolytic pathway. The intermediates from the PPP are used for the biosynthesis of nucleotides and amino acids. Therefore, this glycolytic pathway may be favored when the cell has need for nucleic acid and/or protein synthesis, respectively. A discussion and illustration of the complete ED pathway and PPP with chemical structures and enzyme names appear in Appendix C.

### Transition Reaction, Coenzyme A, and the Krebs Cycle

Glycolysis produces pyruvate, which can be further oxidized to capture more energy. For pyruvate to enter the next oxidative pathway, it must first be decarboxylated by the enzyme complex pyruvate dehydrogenase to a two-carbon acetyl group in the transition reaction, also called the bridge reaction (see Appendix C and ). In the transition reaction, electrons are also transferred to NAD+ to form NADH. To proceed to the next phase of this metabolic process, the comparatively tiny two-carbon acetyl must be attached to a very large carrier compound called coenzyme A (CoA). The transition reaction occurs in the mitochondrial matrix of eukaryotes; in prokaryotes, it occurs in the cytoplasm because prokaryotes lack membrane-enclosed organelles.
The Krebs cycle transfers remaining electrons from the acetyl group produced during the transition reaction to electron carrier molecules, thus reducing them. The Krebs cycle also occurs in the cytoplasm of prokaryotes along with glycolysis and the transition reaction, but it takes place in the mitochondrial matrix of eukaryotic cells where the transition reaction also occurs. The Krebs cycle is named after its discoverer, British scientist Hans Adolf Krebs (1900–1981) and is also called the citric acid cycle, or the tricarboxylic acid cycle (TCA) because citric acid has three carboxyl groups in its structure. Unlike glycolysis, the Krebs cycle is a closed loop: The last part of the pathway regenerates the compound used in the first step (). The eight steps of the cycle are a series of chemical reactions that capture the two-carbon acetyl group (the CoA carrier does not enter the Krebs cycle) from the transition reaction, which is added to a four-carbon intermediate in the Krebs cycle, producing the six-carbon intermediate citric acid (giving the alternate name for this cycle). As one turn of the cycle returns to the starting point of the four-carbon intermediate, the cycle produces two CO2 molecules, one ATP molecule (or an equivalent, such as guanosine triphosphate [GTP]) produced by substrate-level phosphorylation, and three molecules of NADH and one of FADH2. (A discussion and detailed illustration of the full Krebs cycle appear in Appendix C.)
Although many organisms use the Krebs cycle as described as part of glucose metabolism, several of the intermediate compounds in the Krebs cycle can be used in synthesizing a wide variety of important cellular molecules, including amino acids, chlorophylls, fatty acids, and nucleotides; therefore, the cycle is both anabolic and catabolic ().

### Key Concepts and Summary

1. Glycolysis is the first step in the breakdown of glucose, resulting in the formation of ATP, which is produced by substrate-level phosphorylation; NADH; and two pyruvate molecules. Glycolysis does not use oxygen and is not oxygen dependent.
2. After glycolysis, a three-carbon pyruvate is decarboxylated to form a two-carbon acetyl group, coupled with the formation of NADH. The acetyl group is attached to a large carrier compound called coenzyme A.
3. After the transition step, coenzyme A transports the two-carbon acetyl to the Krebs cycle, where the two carbons enter the cycle. Per turn of the cycle, one acetyl group derived from glycolysis is further oxidized, producing three NADH molecules, one FADH2, and one ATP by substrate-level phosphorylation, and releasing two CO2 molecules.
4. The Krebs cycle may be used for other purposes. Many of the intermediates are used to synthesize important cellular molecules, including amino acids, chlorophylls, fatty acids, and nucleotides.

### Multiple Choice

### True/False

### Fill in the Blank

### Short Answer

### Critical Thinking


# Microbial Metabolism

## Cellular Respiration

### Learning Objectives

By the end of this section, you will be able to:
1. Compare and contrast the electron transport system location and function in a prokaryotic cell and a eukaryotic cell
2. Compare and contrast the differences between substrate-level and oxidative phosphorylation
3. Explain the relationship between chemiosmosis and proton motive force
4. Describe the function and location of ATP synthase in a prokaryotic versus eukaryotic cell
5. Compare and contrast aerobic and anaerobic respiration
We have just discussed two pathways in glucose catabolism—glycolysis and the Krebs cycle—that generate ATP by substrate-level phosphorylation. Most ATP, however, is generated during a separate process called oxidative phosphorylation, which occurs during cellular respiration. Cellular respiration begins when electrons are transferred from NADH and FADH2—made in glycolysis, the transition reaction, and the Krebs cycle—through a series of chemical reactions to a final inorganic electron acceptor (either oxygen in aerobic respiration or non-oxygen inorganic molecules in anaerobic respiration). These electron transfers take place on the inner part of the cell membrane of prokaryotic cells or in specialized protein complexes in the inner membrane of the mitochondria of eukaryotic cells. The energy of the electrons is harvested to generate an electrochemical gradient across the membrane, which is used to make ATP by oxidative phosphorylation.

### Electron Transport System

The electron transport system (ETS) is the last component involved in the process of cellular respiration; it comprises a series of membrane-associated protein complexes and associated mobile accessory electron carriers (). Electron transport is a series of chemical reactions that resembles a bucket brigade in that electrons from NADH and FADH2 are passed rapidly from one ETS electron carrier to the next. These carriers can pass electrons along in the ETS because of their redox potential. For a protein or chemical to accept electrons, it must have a more positive redox potential than the electron donor. Therefore, electrons move from electron carriers with more negative redox potential to those with more positive redox potential. The four major classes of electron carriers involved in both eukaryotic and prokaryotic electron transport systems are the cytochromes, flavoproteins, iron-sulfur proteins, and the quinones.
In aerobic respiration, the final electron acceptor (i.e., the one having the most positive redox potential) at the end of the ETS is an oxygen molecule (O2) that becomes reduced to water (H2O) by the final ETS carrier. This electron carrier, cytochrome oxidase, differs between bacterial types and can be used to differentiate closely related bacteria for diagnoses. For example, the gram-negative opportunist  and the gram-negative cholera-causing  use cytochrome c oxidase, which can be detected by the oxidase test, whereas other gram-negative Enterobacteriaceae, like E. coli, are negative for this test because they produce different cytochrome oxidase types.
There are many circumstances under which aerobic respiration is not possible, including any one or more of the following:
1. The cell lacks genes encoding an appropriate cytochrome oxidase for transferring electrons to oxygen at the end of the electron transport system.
2. The cell lacks genes encoding enzymes to minimize the severely damaging effects of dangerous oxygen radicals produced during aerobic respiration, such as hydrogen peroxide (H2O2) or superoxide
3. The cell lacks a sufficient amount of oxygen to carry out aerobic respiration.
One possible alternative to aerobic respiration is anaerobic respiration, using an inorganic molecule other than oxygen as a final electron acceptor. There are many types of anaerobic respiration found in bacteria and archaea. Denitrifiers are important soil bacteria that use nitrate  and nitrite  as final electron acceptors, producing nitrogen gas (N2). Many aerobically respiring bacteria, including E. coli, switch to using nitrate as a final electron acceptor and producing nitrite when oxygen levels have been depleted.
Microbes using anaerobic respiration commonly have an intact Krebs cycle, so these organisms can access the energy of the NADH and FADH2 molecules formed. However, anaerobic respirers use altered ETS carriers encoded by their genomes, including distinct complexes for electron transfer to their final electron acceptors. Smaller electrochemical gradients are generated from these electron transfer systems, so less ATP is formed through anaerobic respiration.

### Chemiosmosis, Proton Motive Force, and Oxidative Phosphorylation

In each transfer of an electron through the ETS, the electron loses energy, but with some transfers, the energy is stored as potential energy by using it to pump hydrogen ions (H+) across a membrane. In prokaryotic cells, H+ is pumped to the outside of the cytoplasmic membrane (called the periplasmic space in gram-negative and gram-positive bacteria), and in eukaryotic cells, they are pumped from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space. There is an uneven distribution of H+ across the membrane that establishes an electrochemical gradient because H+ ions are positively charged (electrical) and there is a higher concentration (chemical) on one side of the membrane. This electrochemical gradient formed by the accumulation of H+ (also known as a proton) on one side of the membrane compared with the other is referred to as the proton motive force (PMF). Because the ions involved are H+, a pH gradient is also established, with the side of the membrane having the higher concentration of H+ being more acidic. Beyond the use of the PMF to make ATP, as discussed in this chapter, the PMF can also be used to drive other energetically unfavorable processes, including nutrient transport and flagella rotation for motility.
The potential energy of this electrochemical gradient generated by the ETS causes the H+ to diffuse across a membrane (the plasma membrane in prokaryotic cells and the inner membrane in mitochondria in eukaryotic cells). This flow of hydrogen ions across the membrane, called chemiosmosis, must occur through a channel in the membrane via a membrane-bound enzyme complex called ATP synthase (). The tendency for movement in this way is much like water accumulated on one side of a dam, moving through the dam when opened. ATP synthase (like a combination of the intake and generator of a hydroelectric dam) is a complex protein that acts as a tiny generator, turning by the force of the H+ diffusing through the enzyme, down their electrochemical gradient from where there are many mutually repelling H+ to where there are fewer H+. In prokaryotic cells, H+ flows from the outside of the cytoplasmic membrane into the cytoplasm, whereas in eukaryotic mitochondria, H+ flows from the intermembrane space to the mitochondrial matrix. The turning of the parts of this molecular machine regenerates ATP from ADP and inorganic phosphate (Pi) by oxidative phosphorylation, a second mechanism for making ATP that harvests the potential energy stored within an electrochemical gradient.
The number of ATP molecules generated from the catabolism of glucose varies. For example, the number of hydrogen ions that the electron transport system complexes can pump through the membrane varies between different species of organisms. In aerobic respiration in mitochondria, the passage of electrons from one molecule of NADH generates enough proton motive force to make three ATP molecules by oxidative phosphorylation, whereas the passage of electrons from one molecule of FADH2 generates enough proton motive force to make only two ATP molecules. Thus, the 10 NADH molecules made per glucose during glycolysis, the transition reaction, and the Krebs cycle carry enough energy to make 30 ATP molecules, whereas the two FADH2 molecules made per glucose during these processes provide enough energy to make four ATP molecules. Overall, the theoretical maximum yield of ATP made during the complete aerobic respiration of glucose is 38 molecules, with four being made by substrate-level phosphorylation and 34 being made by oxidative phosphorylation (). In reality, the total ATP yield is usually less, ranging from one to 34 ATP molecules, depending on whether the cell is using aerobic respiration or anaerobic respiration; in eukaryotic cells, some energy is expended to transport intermediates from the cytoplasm into the mitochondria, affecting ATP yield.
 summarizes the theoretical maximum yields of ATP from various processes during the complete aerobic respiration of one glucose molecule.

### Key Concepts and Summary

1. Most ATP generated during the cellular respiration of glucose is made by oxidative phosphorylation.
2. An electron transport system (ETS) is composed of a series of membrane-associated protein complexes and associated mobile accessory electron carriers. The ETS is embedded in the cytoplasmic membrane of prokaryotes and the inner mitochondrial membrane of eukaryotes.
3. Each ETS complex has a different redox potential, and electrons move from electron carriers with more negative redox potential to those with more positive redox potential.
4. To carry out aerobic respiration, a cell requires oxygen as the final electron acceptor. A cell also needs a complete Krebs cycle, an appropriate cytochrome oxidase, and oxygen detoxification enzymes to prevent the harmful effects of oxygen radicals produced during aerobic respiration.
5. Organisms performing anaerobic respiration use alternative electron transport system carriers for the ultimate transfer of electrons to the final non-oxygen electron acceptors.
6. Microbes show great variation in the composition of their electron transport systems, which can be used for diagnostic purposes to help identify certain pathogens.
7. As electrons are passed from NADH and FADH2 through an ETS, the electron loses energy. This energy is stored through the pumping of H+ across the membrane, generating a proton motive force.
8. The energy of this proton motive force can be harnessed by allowing hydrogen ions to diffuse back through the membrane by chemiosmosis using ATP synthase. As hydrogen ions diffuse through down their electrochemical gradient, components of ATP synthase spin, making ATP from ADP and Pi by oxidative phosphorylation.
9. Aerobic respiration forms more ATP (a maximum of 34 ATP molecules) during oxidative phosphorylation than does anaerobic respiration (between one and 32 ATP molecules).

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer


# Microbial Metabolism

## Fermentation

### Learning Objectives

By the end of this section, you will be able to:
1. Define fermentation and explain why it does not require oxygen
2. Describe the fermentation pathways and their end products and give examples of microorganisms that use these pathways
3. Compare and contrast fermentation and anaerobic respiration
Many cells are unable to carry out respiration because of one or more of the following circumstances:
1. The cell lacks a sufficient amount of any appropriate, inorganic, final electron acceptor to carry out cellular respiration.
2. The cell lacks genes to make appropriate complexes and electron carriers in the electron transport system.
3. The cell lacks genes to make one or more enzymes in the Krebs cycle.
Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, the other two conditions are genetically determined. Thus, many prokaryotes, including members of the clinically important genus Streptococcus, are permanently incapable of respiration, even in the presence of oxygen. Conversely, many prokaryotes are facultative, meaning that, should the environmental conditions change to provide an appropriate inorganic final electron acceptor for respiration, organisms containing all the genes required to do so will switch to cellular respiration for glucose metabolism because respiration allows for much greater ATP production per glucose molecule.
If respiration does not occur, NADH must be reoxidized to NAD+ for reuse as an electron carrier for glycolysis, the cell’s only mechanism for producing any ATP, to continue. Some living systems use an organic molecule (commonly pyruvate) as a final electron acceptor through a process called fermentation. Fermentation does not involve an electron transport system and does not directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms carrying out fermentation, called fermenters, produce a maximum of two ATP molecules per glucose during glycolysis.  compares the final electron acceptors and methods of ATP synthesis in aerobic respiration, anaerobic respiration, and fermentation. Note that the number of ATP molecules shown for glycolysis assumes the Embden-Meyerhof-Parnas pathway. The number of ATP molecules made by substrate-level phosphorylation (SLP) versus oxidative phosphorylation (OP) are indicated.
Microbial fermentation processes have been manipulated by humans and are used extensively in the production of various foods and other commercial products, including pharmaceuticals. Microbial fermentation can also be useful for identifying microbes for diagnostic purposes.
Fermentation by some bacteria, like those in yogurt and other soured food products, and by animals in muscles during oxygen depletion, is lactic acid fermentation. The chemical reaction of lactic acid fermentation is as follows:
Bacteria of several gram-positive genera, including , , and , are collectively known as the lactic acid bacteria (LAB), and various strains are important in food production. During yogurt and cheese production, the highly acidic environment generated by lactic acid fermentation denatures proteins contained in milk, causing it to solidify. When lactic acid is the only fermentation product, the process is said to be homolactic fermentation; such is the case for Lactobacillus delbrueckii and S. thermophiles used in yogurt production. However, many bacteria perform heterolactic fermentation, producing a mixture of lactic acid, ethanol and/or acetic acid, and CO2 as a result, because of their use of the branched pentose phosphate pathway instead of the EMP pathway for glycolysis. One important heterolactic fermenter is Leuconostoc mesenteroides, which is used for souring vegetables like cucumbers and cabbage, producing pickles and sauerkraut, respectively.
Lactic acid bacteria are also important medically. The production of low pH environments within the body inhibits the establishment and growth of pathogens in these areas. For example, the vaginal microbiota is composed largely of lactic acid bacteria, but when these bacteria are reduced, yeast can proliferate, causing a yeast infection. Additionally, lactic acid bacteria are important in maintaining the health of the gastrointestinal tract and, as such, are the primary component of probiotics.
Another familiar fermentation process is alcohol fermentation, which produces ethanol. The ethanol fermentation reaction is shown in . In the first reaction, the enzyme pyruvate decarboxylase removes a carboxyl group from pyruvate, releasing CO2 gas while producing the two-carbon molecule acetaldehyde. The second reaction, catalyzed by the enzyme alcohol dehydrogenase, transfers an electron from NADH to acetaldehyde, producing ethanol and NAD+. The ethanol fermentation of pyruvate by the yeast  is used in the production of alcoholic beverages and also makes bread products rise due to CO2 production. Outside of the food industry, ethanol fermentation of plant products is important in biofuel production.
Beyond lactic acid fermentation and alcohol fermentation, many other fermentation methods occur in prokaryotes, all for the purpose of ensuring an adequate supply of NAD+ for glycolysis (). Without these pathways, glycolysis would not occur and no ATP would be harvested from the breakdown of glucose. It should be noted that most forms of fermentation besides homolactic fermentation produce gas, commonly CO2 and/or hydrogen gas. Many of these different types of fermentation pathways are also used in food production and each results in the production of different organic acids, contributing to the unique flavor of a particular fermented food product. The propionic acid produced during propionic acid fermentation contributes to the distinctive flavor of Swiss cheese, for example.
Several fermentation products are important commercially outside of the food industry. For example, chemical solvents such as acetone and butanol are produced during acetone-butanol-ethanol fermentation. Complex organic pharmaceutical compounds used in antibiotics (e.g., penicillin), vaccines, and vitamins are produced through mixed acid fermentation. Fermentation products are used in the laboratory to differentiate various bacteria for diagnostic purposes. For example, enteric bacteria are known for their ability to perform mixed acid fermentation, reducing the pH, which can be detected using a pH indicator. Similarly, the bacterial production of acetoin during butanediol fermentation can also be detected. Gas production from fermentation can also be seen in an inverted Durham tube that traps produced gas in a broth culture.
Microbes can also be differentiated according to the substrates they can ferment. For example, E. coli can ferment lactose, forming gas, whereas some of its close gram-negative relatives cannot. The ability to ferment the sugar alcohol sorbitol is used to identify the pathogenic enterohemorrhagic O157:H7 strain of E. coli because, unlike other E. coli strains, it is unable to ferment sorbitol. Last, mannitol fermentation differentiates the mannitol-fermenting  from other non–mannitol-fermenting staphylococci.

### Key Concepts and Summary

1. Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD+ from NADH so that glycolysis can continue.
2. Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly. Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis.
3. Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of microbes.
4. During lactic acid fermentation, pyruvate accepts electrons from NADH and is reduced to lactic acid. Microbes performing homolactic fermentation produce only lactic acid as the fermentation product; microbes performing heterolactic fermentation produce a mixture of lactic acid, ethanol and/or acetic acid, and CO2.
5. Lactic acid production by the normal microbiota prevents growth of pathogens in certain body regions and is important for the health of the gastrointestinal tract.
6. During ethanol fermentation, pyruvate is first decarboxylated (releasing CO2) to acetaldehyde, which then accepts electrons from NADH, reducing acetaldehyde to ethanol. Ethanol fermentation is used for the production of alcoholic beverages, for making bread products rise, and for biofuel production.
7. Fermentation products of pathways (e.g., propionic acid fermentation) provide distinctive flavors to food products. Fermentation is used to produce chemical solvents (acetone-butanol-ethanol fermentation) and pharmaceuticals (mixed acid fermentation).
8. Specific types of microbes may be distinguished by their fermentation pathways and products. Microbes may also be differentiated according to the substrates they are able to ferment.

### Multiple Choice

### Fill in the Blank

### Matching

### Short Answer

### Critical Thinking


# Microbial Metabolism

## Catabolism of Lipids and Proteins

### Learning Objectives

By the end of this section, you will be able to:
1. Describe how lipids are catabolized
2. Describe how lipid catabolism can be used to identify microbes
3. Describe how proteins are catabolized
4. Describe how protein catabolism can be used to identify bacteria
Previous sections have discussed the catabolism of glucose, which provides energy to living cells, as well as how polysaccharides like glycogen, starch, and cellulose are degraded to glucose monomers. But microbes consume more than just carbohydrates for food. In fact, the microbial world is known for its ability to degrade a wide range of molecules, both naturally occurring and those made by human processes, for use as carbon sources. In this section, we will see that the pathways for both lipid and protein catabolism connect to those used for carbohydrate catabolism, eventually leading into glycolysis, the transition reaction, and the Krebs cycle pathways. Metabolic pathways should be considered to be porous—that is, substances enter from other pathways, and intermediates leave for other pathways. These pathways are not closed systems. Many of the substrates, intermediates, and products in a particular pathway are reactants in other pathways.

### Lipid Catabolism

Triglycerides are a form of long-term energy storage in animals. They are made of glycerol and three fatty acids (see ). Phospholipids compose the cell and organelle membranes of all organisms except the archaea. Phospholipid structure is similar to triglycerides except that one of the fatty acids is replaced by a phosphorylated head group (see ). Triglycerides and phospholipids are broken down first by releasing fatty acid chains (and/or the phosphorylated head group, in the case of phospholipids) from the three-carbon glycerol backbone. The reactions breaking down triglycerides are catalyzed by lipases and those involving phospholipids are catalyzed by phospholipases. These enzymes contribute to the virulence of certain microbes, such as the bacterium  and the fungus . These microbes use phospholipases to destroy lipids and phospholipids in host cells and then use the catabolic products for energy (see Virulence Factors of Bacterial and Viral Pathogens).
The resulting products of lipid catabolism, glycerol and fatty acids, can be further degraded. Glycerol can be phosphorylated to glycerol-3-phosphate and easily converted to glyceraldehyde 3-phosphate, which continues through glycolysis. The released fatty acids are catabolized in a process called β-oxidation, which sequentially removes two-carbon acetyl groups from the ends of fatty acid chains, reducing NAD+ and FAD to produce NADH and FADH2, respectively, whose electrons can be used to make ATP by oxidative phosphorylation. The acetyl groups produced during β-oxidation are carried by coenzyme A to the Krebs cycle, and their movement through this cycle results in their degradation to CO2, producing ATP by substrate-level phosphorylation and additional NADH and FADH2 molecules (see Appendix C for a detailed illustration of β-oxidation).
Other types of lipids can also be degraded by certain microbes. For example, the ability of certain pathogens, like , to degrade cholesterol contributes to their virulence. The side chains of cholesterol can be easily removed enzymatically, but degradation of the remaining fused rings is more problematic. The four fused rings are sequentially broken in a multistep process facilitated by specific enzymes, and the resulting products, including pyruvate, can be further catabolized in the Krebs cycle.

### Protein Catabolism

Proteins are degraded through the concerted action of a variety of microbial protease enzymes. Extracellular proteases cut proteins internally at specific amino acid sequences, breaking them down into smaller peptides that can then be taken up by cells. Some clinically important pathogens can be identified by their ability to produce a specific type of extracellular protease. For example, the production of the extracellular protease gelatinase by members of the genera  and  can be used to distinguish them from other gram-negative enteric bacteria. Following inoculation and growth of microbes in gelatin broth, degradation of the gelatin protein due to gelatinase production prevents solidification of gelatin when refrigerated. Other pathogens can be distinguished by their ability to degrade casein, the main protein found in milk. When grown on skim milk agar, production of the extracellular protease caseinase causes degradation of casein, which appears as a zone of clearing around the microbial growth. Caseinase production by the opportunist pathogen  can be used to distinguish it from other related gram-negative bacteria.
After extracellular protease degradation and uptake of peptides in the cell, the peptides can then be broken down further into individual amino acids by additional intracellular proteases, and each amino acid can be enzymatically deaminated to remove the amino group. The remaining molecules can then enter the transition reaction or the Krebs cycle.

### Key Concepts and Summary

1. Collectively, microbes have the ability to degrade a wide variety of carbon sources besides carbohydrates, including lipids and proteins. The catabolic pathways for all of these molecules eventually connect into glycolysis and the Krebs cycle.
2. Several types of lipids can be microbially degraded. Triglycerides are degraded by extracellular lipases, releasing fatty acids from the glycerol backbone. Phospholipids are degraded by phospholipases, releasing fatty acids and the phosphorylated head group from the glycerol backbone. Lipases and phospholipases act as virulence factors for certain pathogenic microbes.
3. Fatty acids can be further degraded inside the cell through β-oxidation, which sequentially removes two-carbon acetyl groups from the ends of fatty acid chains.
4. Protein degradation involves extracellular proteases that degrade large proteins into smaller peptides. Detection of the extracellular proteases gelatinase and caseinase can be used to differentiate clinically relevant bacteria.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Microbial Metabolism

## Photosynthesis

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the function and locations of photosynthetic pigments in eukaryotes and prokaryotes
2. Describe the major products of the light-dependent and light-independent reactions
3. Describe the reactions that produce glucose in a photosynthetic cell
4. Compare and contrast cyclic and noncyclic photophosphorylation
Heterotrophic organisms ranging from E. coli to humans rely on the chemical energy found mainly in carbohydrate molecules. Many of these carbohydrates are produced by photosynthesis, the biochemical process by which phototrophic organisms convert solar energy (sunlight) into chemical energy. Although photosynthesis is most commonly associated with plants, microbial photosynthesis is also a significant supplier of chemical energy, fueling many diverse ecosystems. In this section, we will focus on microbial photosynthesis.
Photosynthesis takes place in two sequential stages: the light-dependent reactions and the light-independent reactions (). In the light-dependent reactions, energy from sunlight is absorbed by pigment molecules in photosynthetic membranes and converted into stored chemical energy. In the light-independent reactions, the chemical energy produced by the light-dependent reactions is used to drive the assembly of sugar molecules using CO2; however, these reactions are still light dependent because the products of the light-dependent reactions necessary for driving them are short-lived. The light-dependent reactions produce ATP and either NADPH or NADH to temporarily store energy. These energy carriers are used in the light-independent reactions to drive the energetically unfavorable process of “fixing” inorganic CO2 in an organic form, sugar.

### Photosynthetic Structures in Eukaryotes and Prokaryotes

In all phototrophic eukaryotes, photosynthesis takes place inside a chloroplast, an organelle that arose in eukaryotes by endosymbiosis of a photosynthetic bacterium (see Unique Characteristics of Eukaryotic Cells). These chloroplasts are enclosed by a double membrane with inner and outer layers. Within the chloroplast is a third membrane that forms stacked, disc-shaped photosynthetic structures called thylakoids (). A stack of thylakoids is called a granum, and the space surrounding the granum within the chloroplast is called stroma.
Photosynthetic membranes in prokaryotes, by contrast, are not organized into distinct membrane-enclosed organelles; rather, they are infolded regions of the plasma membrane. In cyanobacteria, for example, these infolded regions are also referred to as thylakoids. In either case, embedded within the thylakoid membranes or other photosynthetic bacterial membranes are photosynthetic pigment molecules organized into one or more photosystems, where light energy is actually converted into chemical energy.
Photosynthetic pigments within the photosynthetic membranes are organized into photosystems, each of which is composed of a light-harvesting (antennae) complex and a reaction center. The light-harvesting complex consists of multiple proteins and associated pigments that each may absorb light energy and, thus, become excited. This energy is transferred from one pigment molecule to another until eventually (after about a millionth of a second) it is delivered to the reaction center. Up to this point, only energy—not electrons—has been transferred between molecules. The reaction center contains a pigment molecule that can undergo oxidation upon excitation, actually giving up an electron. It is at this step in photosynthesis that light energy is converted into an excited electron.
Different kinds of light-harvesting pigments absorb unique patterns of wavelengths (colors) of visible light. Pigments reflect or transmit the wavelengths they cannot absorb, making them appear the corresponding color. Examples of photosynthetic pigments (molecules used to absorb solar energy) are bacteriochlorophylls (green, purple, or red), carotenoids (orange, red, or yellow), chlorophylls (green), phycocyanins (blue), and phycoerythrins (red). By having mixtures of pigments, an organism can absorb energy from more wavelengths. Because photosynthetic bacteria commonly grow in competition for sunlight, each type of photosynthetic bacteria is optimized for harvesting the wavelengths of light to which it is commonly exposed, leading to stratification of microbial communities in aquatic and soil ecosystems by light quality and penetration.
Once the light harvesting complex transfers the energy to the reaction center, the reaction center delivers its high-energy electrons, one by one, to an electron carrier in an electron transport system, and electron transfer through the ETS is initiated. The ETS is similar to that used in cellular respiration and is embedded within the photosynthetic membrane. Ultimately, the electron is used to produce NADH or NADPH. The electrochemical gradient that forms across the photosynthetic membrane is used to generate ATP by chemiosmosis through the process of photophosphorylation, another example of oxidative phosphorylation ().

### Oxygenic and Anoxygenic Photosynthesis

For photosynthesis to continue, the electron lost from the reaction center pigment must be replaced. The source of this electron (H2A) differentiates the oxygenic photosynthesis of plants and cyanobacteria from anoxygenic photosynthesis carried out by other types of bacterial phototrophs (). In oxygenic photosynthesis, H2O is split and supplies the electron to the reaction center. Because oxygen is generated as a byproduct and is released, this type of photosynthesis is referred to as oxygenic photosynthesis. However, when other reduced compounds serve as the electron donor, oxygen is not generated; these types of photosynthesis are called anoxygenic photosynthesis. Hydrogen sulfide (H2S) or thiosulfate  can serve as the electron donor, generating elemental sulfur and sulfate  ions, respectively, as a result.
Photosystems have been classified into two types: photosystem I (PSI) and photosystem II (PSII) (). Cyanobacteria and plant chloroplasts have both photosystems, whereas anoxygenic photosynthetic bacteria use only one of the photosystems. Both photosystems are excited by light energy simultaneously. If the cell requires both ATP and NADPH for biosynthesis, then it will carry out noncyclic photophosphorylation. Upon passing of the PSII reaction center electron to the ETS that connects PSII and PSI, the lost electron from the PSII reaction center is replaced by the splitting of water. The excited PSI reaction center electron is used to reduce NADP+ to NADPH and is replaced by the electron exiting the ETS. The flow of electrons in this way is called the Z-scheme.
If a cell’s need for ATP is significantly greater than its need for NADPH, it may bypass the production of reducing power through cyclic photophosphorylation. Only PSI is used during cyclic photophosphorylation; the high-energy electron of the PSI reaction center is passed to an ETS carrier and then ultimately returns to the oxidized PSI reaction center pigment, thereby reducing it.

### Light-Independent Reactions

After the energy from the sun is converted into chemical energy and temporarily stored in ATP and NADPH molecules (having lifespans of millionths of a second), photoautotrophs have the fuel needed to build multicarbon carbohydrate molecules, which can survive for hundreds of millions of years, for long-term energy storage. The carbon comes from CO2, the gas that is a waste product of cellular respiration.
The Calvin-Benson cycle (named for Melvin Calvin [1911–1997] and Andrew Benson [1917–2015]), the biochemical pathway used for fixation of CO2, is located within the cytoplasm of photosynthetic bacteria and in the stroma of eukaryotic chloroplasts. The light-independent reactions of the Calvin cycle can be organized into three basic stages: fixation, reduction, and regeneration (see Appendix C for a detailed illustration of the Calvin cycle).
1. Fixation: The enzyme ribulose bisphosphate carboxylase (RuBisCO) catalyzes the addition of a CO2 to ribulose bisphosphate (RuBP). This results in the production of 3-phosphoglycerate (3-PGA).
2. Reduction: Six molecules of both ATP and NADPH (from the light-dependent reactions) are used to convert 3-PGA into glyceraldehyde 3-phosphate (G3P). Some G3P is then used to build glucose.
3. Regeneration: The remaining G3P not used to synthesize glucose is used to regenerate RuBP, enabling the system to continue CO2 fixation. Three more molecules of ATP are used in these regeneration reactions.
The Calvin cycle is used extensively by plants and photoautotrophic bacteria, and the enzyme RuBisCO is said to be the most plentiful enzyme on earth, composing 30%–50% of the total soluble protein in plant chloroplasts.A. Dhingra et al. “Enhanced Translation of a Chloroplast-Expressed  However, besides its prevalent use in photoautotrophs, the Calvin cycle is also used by many nonphotosynthetic chemoautotrophs to fix CO2. Additionally, other bacteria and archaea use alternative systems for CO2 fixation. Although most bacteria using Calvin cycle alternatives are chemoautotrophic, certain green sulfur photoautotrophic bacteria have been also shown to use an alternative CO2 fixation pathway.

### Key Concepts and Summary

1. Heterotrophs depend on the carbohydrates produced by autotrophs, many of which are photosynthetic, converting solar energy into chemical energy.
2. Different photosynthetic organisms use different mixtures of photosynthetic pigments, which increase the range of the wavelengths of light an organism can absorb.
3. Photosystems (PSI and PSII) each contain a light-harvesting complex, composed of multiple proteins and associated pigments that absorb light energy. The light-dependent reactions of photosynthesis convert solar energy into chemical energy, producing ATP and NADPH or NADH to temporarily store this energy.
4. In oxygenic photosynthesis, H2O serves as the electron donor to replace the reaction center electron, and oxygen is formed as a byproduct. In anoxygenic photosynthesis, other reduced molecules like H2S or thiosulfate may be used as the electron donor; as such, oxygen is not formed as a byproduct.
5. Noncyclic photophosphorylation is used in oxygenic photosynthesis when there is a need for both ATP and NADPH production. If a cell’s needs for ATP outweigh its needs for NADPH, then it may carry out cyclic photophosphorylation instead, producing only ATP.
6. The light-independent reactions of photosynthesis use the ATP and NADPH from the light-dependent reactions to fix CO2 into organic sugar molecules.

### Multiple Choice

### True/False

### Fill in the Blank

### Short Answer

### Critical Thinking