MICROBIAL METABOLISM

A.   Basic Concepts of Metabolism

• •Metabolism: Metabolism is a general term for all of the chemical reactions and physical mechanisms of the cell needed to keep the cell alive
• •Anabolism: Smaller molecules are combined to form larger molecules by creating chemical bonds. This process requires energy in the form of ATP
• Catabolism: Larger molecules are broken apart into smaller molecules by breaking chemical bonds. This process releases energy in the form of ATP​

REQUIREMENTS FOR LIFE

• Carbon Source - Cells must consume molecules that have carbon in it. Carbon is needed for Lipids, Amino Acids, Nucleic Acids
• Energy Source - Cells processes require energy - Cells can use ATP to fuel its processes. 

2 GENERAL TYPES OF METABOLIC PROCESSES

1. Anabolic Metabolic Reactions (a.k.a. biosynthetic reactions) - builds up larger molecules from smaller molecules.
2. Catabolic Metabolica Reactions - breaks down large molecules into smaller molecules or components which releases energy. 

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ENZYMES
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• Enzymes are usually proteins, few are with RNA or just RNA(ribosomes)
• Pathways are multi-step chemical reactions
• Enzymes catalyze reactions by decreasing the activate energy needed for the reaction to proceed. In essence, the enzyme speeds up the reaction.
• Enzyme are not considered reactants or products in the chemical reaction. They are not consumed or altered when they catalyze a reaction. For this reason, they are also reusable.
• Enzymes are very specific. They must fit together with the molecule(s) they are acting upon in a lock-and-key fashion. 

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via GIPHY

3 things are needed in order to produce chemical reaction.

• energy in the form of ATP 
• catalyst enzyme 
• ingredients
• Factors that influence enzyme activity
  • shape: (correct temp, ionic conditions, pH, absence of inhibitors, water, chemical modification) 
  • collision rate: (temp, concentration of reactants and enzyme)

The difference between competitive and noncompetitive inhibitors

• competitive inhibitors: binds to the same site as a substrate (-competition for binding)
• noncompetitive inhibitors: bind to a different site from the one the substrate uses. This binding can result in allosteric inhibition by altering the shape of the substrate binding site.

ALL ORGANISMS NEED A SOURCE OF CARBON
​- We can categorize organisms based on how they obtain CARBON from the environment. Organisms are categorized as either autotrophic or heterotrophic based upon how they get their carbon. 

Carbon Source for Humans:
​Humans get their carbon from consuming organic molecules.

• Sugars
• Proteins
• Lipids

Carbon Source for Prokaryotes:
Prokaryotes are more diverse in their metabolism. 

• AUTOTROPHS - organisms that can synthesize their own food from inorganic sources. For example, some prokaryotes can use the carbon in carbon dioxide (CO2) from the air
• HETEROTROPHS - organisms that cannot synthesize their own food. These organisms must consume organic molecules (sugars, proteins or lipids).

ALL ORGANISMS NEED A SOURCE OF ENERGY
​- We can categorize organisms based on how they obtain ENERGY from the environment. Organisms are categorized based upon how they get their energy. 

HOW DO CELLS MAKE ATP?

   ATP is a high energy molecule. In order to make a high energy molecule, you need to use another high energy molecule.

   You can think of energy as the movement of electrons. In order to generate ATP, it must generate something that moves. Then this can be used to generate ATP. The movement of electrons gives the energy needed to make ATP. Need an electron source and an electron acceptor.. 

1) Take a molecule that has high energy electrons and breaks it down, which releases the high energy electrons and releases energy.

2) Electrons move to an electron acceptor molecule. 

Different molecules can be used as electron sources and electron acceptors in the metabolic processes. 

HUMANS:

Electron Sources: Humans must consume proteins, sugars and lipids. 

• Chemoorganotroph - organisms that use organic chemical compounds (proteins, sugars and lipids) as the electron source. 

Lipids

Carbohydrates

Electron Acceptors: Humans are limited to oxygen as their electron acceptor and Pyruvate as their electron acceptor (only occurs in muscle cells when oxygen levels are low).  

• Respiration - the process of using an inorganic molecule as the electron acceptor.
  • Aerobic Respiration - oxygen is the electron acceptor specifically
  • Anaerobic Respiration - the inorganic molecule (e.g. pyruvate) is the electron acceptor (occurs in muscles only when oxygen is in short supply)

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PROKARYOTES:

Electron Sources (Energy Source):

• ​Chemoorganotroph - prokaryotic organisms that use organic chemical compounds as the electron source. 
• Chemolithotroph ("litho" means rock) - prokaryotic organisms that use inorganic chemical compounds as the electron source. 
  • Toluene - some prokaryotes can use toluene as their electron source. 
  • Ammonia - some prokaryotes can use ammonia as their electron source. 
  • Arsenic - some prokaryotes can use arsenic as their electron source. 
• Phototroph - Some prokaryotes are able to harness energy of light to energize the electrons from an otherwise low energy source. When the electrons are energized, they are moved (along an electron transport chain) which provides energy for the cell to make ATP.

Electron Acceptors:

• ​Respiration - prokaryotes can use a wide variety of inorganic molecules as electron acceptors in the process of respiration. 2 types of respiration exists, 1) aerobic respiration or 2) anaerobic respiration.
  • Aerobic Respiration - some prokaryotes use the inorganic molecule, oxygen, as the electron acceptor 
  • Anaerobic Respiration - organic molecule used as the electron acceptor IS NOT oxygen 
    • Nitrates - Some prokaryotes can use nitrates as their electron acceptor.
    • Sulfates - Some prokaryotes can use sulfates as their electron acceptor.
    • Iron Compounds - Some prokaryotes can use iron compounds as their electron acceptor.
• Fermentation - Uses organic molecules as the electron acceptor

Humans are limited to using organic molecules as the electron source and oxygen as the only electron acceptor (almost always, since pyruvate can be used in muscle cells).

Organic molecule electron source and oxygen electron acceptor. chemoorganotrophs do this.Aerobic Respiration
​In other words these are aerobically respirating chemoorganotrophs. 

The Generation of ATP in an aerobically respirating chemoorganotroph (like US!) -
Electron source = glucose

Cellular respiration has 3 parts.
1) Glycolysis
2) The Kreb's Cycle / The Citric Acid Cycle
​3) The Electron Transport Chain / Oxidative Phosphorylation

​     Cellular respiration = is the process by which microorganisms obtain the energy available in carbohydrates. They take the carbohydrates into their cytoplasm, and through a complex series of metabolic processes, they break down the carbohydrate and release the energy. The energy is generally not needed immediately, so it is used to combine ADP with phosphate ions to form ATP molecules. During the process of cellular respiration, carbon dioxide is given off as a waste product. This carbon dioxide can be used by photosynthesizing cells to form new carbohydrates. Also in the process of cellular respiration, oxygen gas is required to serve as an acceptor of electrons. This oxygen gas is identical to the oxygen gas given off in photosynthesis.​

B.   Glycolytic Pathways

THREE STAGES OF CATABOLISM
(cellular respiration)

There are 4 division of cellular respiration

• Glycolysis
  • - a glucose molecule is are broken down to form 2 pyruvate molecules
• The Transition Reaction
  
  • ​- pyruvate from glycolysis is transformed into Acetyl CoA before continuing on to the Krebs Cycle 
    
• Krebs Cycle 
  • - pyruvate is further broken down
  • - NADH is created
  • - NADH transports electrons from cellular respiration to the electron transport chain.
• The Electron Transport Chain -  
  • electrons are transported along a series of coenzymes and cytochromes
  • energy is released as the electrons move along the transport chain.
  • chemiosmosis occurs - the energy given off by electrons is used to pump protons across a membrane via a proton pump. 
  • The energy generated from the electrochemical gradient of protons provides the energy for ATP synthesis.

Glycolysis -

   Glycolysis is performed in all living organisms. Glycolysis always takes place in the cytoplasm of the cell. Glycolysis does not require oxygen and can be performed in both aerobic and anaerobic prokaryotes.

Glycolysis can be broken down into 2 phases;
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1.  An energy investment phase -  In the energy investment phase of glycolysis, 2 ATP molecules are used break glucose into 2 glyceraldehyde 3-phosphate (G3P) molecules. 
2.  An energy payoff phase  - In the energy payoff phase, energy is captured from the 2 G3P molecules, and 4 ATP molecules, 2 NADH molecules, and 2 pyruvate molecules are formed. 

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The net payout for the EMP pathway (glycolysis)

• two ATP molecules
• two NADH molecule, and
• two pyruvate molecules.

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   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. There are different types of glycolysis. The first one we will discuss is the glycolytic pathway used by most microorganisms, the Embden-Meyerhof-Parnas (EMP) pathway. 

Glycolysis via the Embden-Meyerhof-Parnas (EMP) pathway -

• Glucose is the energy source.
• 2 ATP molecules are hydrolyzed into 2 ADP which releases energy, This provides the energy needed to add 2 phosphate molecules (PO4) to glucose.
• The glucose is then split in half.
• 2 more phosphate molecules (PO4) are added. 
• NAD+ is the electron acceptor. It picks up the electrons and delivers them to oxygen in the cell. when the electron is picked up, it also picks up a proton to become NADH The NAD+ will pick up these electrons.
• The phosphate groups are removed from each half of the molecule, generating ATP.

Embden-Meyerhof-Parnas (EMP) Pathway of Glycolysis

   The glycolytic pathway in most prokaryotic cells is called the Embden-Meyerhof-Parnas (EMP) pathway.

DIFFERENT FORMS OF GLYCOLYSIS

    While most prokaryotes undergo glycolysis in the form of the EMP pathway, there are other variations of glycolysis used by some prokaryotes. ​

There are 3 Glycolysis Pathways

1. ​Embden-Meyerhof-Parnas (EMP) pathway.
2. Entner-Doudoroff (ED) pathway
3. ​Pentose Phosphate Pathway (PPP) Also knowns as.... The Phosphogluconate Pathway or The Hexose Monophosphate Shunt. 

Entner-Doudoroff (ED) pathway

   Another glycolytic pathway is the Entner-Doudoroff (ED) pathway.  

   The ED glycolytic pathway is the only form of glycolysis used by the gram-negative pathogen Pseudomonas aeruginosa. Pseudomonas aeruginosa is a common bacteria known for antibiotic resistance. This pathogen causes many of the hospital-acquired infections. 

​    E. coli, also use the ED form of glycolysis, but they also have the ability to use EMP pathway. 

Phagocytosis of P. aeruginosa by neutrophil in patient with bloodstream infection (Gram stain)

​By Paulo Henrique Orlandi Mourao - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=9491587

THE PENTOSE PHOSPHATE PATHWAY

   Another glycolytic pathway that occurs in all cells, which is quite different from the other pathways, is the pentose phosphate pathway (PPP) also called the phosphogluconate pathway or the hexose monophosphate shunt.

​   The PPP creates building blocks (or precursors) for amino acids and nucleotides. This form of glycolysis will take place in the cell when amino acids and nucleotides are needed to form proteins and nucleic acids, respectively (RNA or DNA).

Illustration of the Pentose Phosphate Pathway was provided courtesy of  GdenBesten
​- Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=10472072

THE TRANSITION REACTION
(AFTER GLYCOLYSIS AND BEFORE THE KREB'S CYCLE)

   Before the pyruvate molecules can enter the Kreb's cycle, they must first undergo a transition reaction After glycolysis, each of the three-carbon pyruvate molecules will be decarboxylated to form a two-carbon acetyl group. This produces NADH as well. The acetyl group is attached to a large carrier compound called coenzyme A. 

The Kreb's Cycle
​(The Citric Acid Cycle)

    The Kreb's cycle is step 2 in cellular respiration. Glycolysis has formed pyruvate which still has more electrons that need to be harvested. The next process in microbial metabolism is the Kreb's cycle. This process will release additional electrons from the pyruvate molecules. 

    After the transition step, coenzyme A transports the two-carbon acetyl to the Krebs cycle.  The acetyl group os then oxidized in the cycle producing three NADH molecules, one FADH2, and one ATP by substrate-level phosphorylation, and releasing two CO2 molecules.

   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.
   In prokaryotes, the TCA or citric acid cycle takes place in the cytoplasm. In summary, the Krebs cycle begins by transferring the acetyl group from acetyl CoA to an oxaloacetate molecule to form the citrate molecule. Citrate then undergoes a series of steps that releases 2 carbon dioxide molecules, 3 NADH, 1 FADH2, and 1 ATP or GTP.  

GIF Courtesy of http://www.bartek.ca/malic_krebs.html

Illustration of the Citric Acid Cycle

   The Krebs cycle, is a closed loop. The cycle begins with the two-carbon acetyl molecule and creates 2 CO2, 3 NADH, 1 FADH2, and 1 ATP (or GTP). There are also many intermediates produced by the Kreb's Cycle like amino acids, fatty acids, and nucleotides, that the cell uses as building blocks for biosynthesis. Keep in mind that one glucose molecule will take two turns of the Krebs cycle to process. 

THE ELECTRON TRANSPORT CHAIN

   The electron acceptors (NADH and FADH2) generated in glycolysis, the transition reaction and the Kreb's cycle carries electrons to oxygen in the cell. The electrons go to the oxygen via the electron transport chain that lies within the membrane of the cell. In prokaryotic cells, the electron transport chain lies in its cell membrane.

     The electrons can be delivered to either complex or complex 2 in the electron transport chain. Once at one of these complexes, the electrons gets pulled toward the oxygen molecule as the final electron acceptor. Energy is released as the electrons travel through the electron transport chain and this energy is harnessed to form ATP. 

    The electron transport chain (ETC) exists in the plasma membrane in prokaryotic cells. You may recall that this process occurs in the mitochondria in eukaryotic cells. 

Glucose undergoes glycolysis --> electrons go to NADH --> NADH delivers the electrons to the ETC --->  Energy is released as electrons move through the electron transport chain, and that is used to make ATP.

• - ATP  is an energy-carrying molecule that cells use to power their metabolic processes
• Respiration - process in which cells break down glucose and make ATP for energy​

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    Most of the ATP generated in the electron transport chain, also known as oxidative phosphorylation, which is the final step in cellular respiration.

    What is cellular respiration?

• The NADH and FADH2 generated by glycolysis, the transition reaction and the TCA cycle carry electrons to the electron transport chain (ETC). 
• NADH and FADH2 will then release their electrons into the ETC where the electrons move 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).
  • The ETC is located on the inner part of the cell membrane of prokaryotic 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.

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Aerobic vs. Anaerobic CELLULAR RESPIRATION

   Prokaryotic organisms have a lot of different mechanisms for getting the energy and nutrients they need from the environment. They can obtain energy from organic substances (like animals do), from sunlight, nitrogen, inorganic carbon, or sulfur! Some microbes can switch between 2 modes of metabolism.

   As humans, it may be hard to image life without oxygen, but many microbes are perfectly happy to go without! In fact, some prokaryotes will even die if they are exposed to oxygen.
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• Obligate Aerobes - Prokaryotes that require oxygen (O2) for metabolism (aerobic respiration). Humans are an example of obligate aerobes since we absolutely depend upon the presence of oxygen.

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• Obligate Anaerobes - Prokaryotes that do not need oxygen (or cannot be exposed to oxygen) only undergo anaerobic metabolism (anaerobic respiration) and are classified as obligate anaerobes. For example, C. botulinum, the bacterium that causes botulism, is able to grow inside of canned food in the absence of oxygen.  ​

• Facultative Anaerobes - Prokaryotes that exhibit metabolic flexibility. These microbes have metabolic pathways for when oxygen is present and another set of metabolic pathways for when oxygen is unavailable. For example, the bacteria responsible for staph infections and strep throat are facultative anaerobes.

C.   Fermentation

     Many organisms that do not require oxygen, will undergo a process of fermentation. Fermentation takes place in the absence of oxygen and uses an organic molecule (such as pyruvate) as a final electron acceptor.  Fermenting organisms produce only 2 ATP molecules per glucose molecule. 

There are 2 main types of fermentation:

1. Alcoholic Fermentation
2. Lactic Acid Fermentation

     Alcoholic fermentation produces ethanol which is used to create alcoholic beverages. The alcohol fermentation reaction begins with the decarboxylation of pyruvate. This reaction releases CO2 and produces acetaldehyde. The enzyme alcohol dehydrogenase then transfers an electron from NADH to acetaldehyde to create NAD+.

    The microbe commonly used for alcoholic fermentation, baking and even biofeul production is the yeast Saccharomyces cerevisiae. Yeast are single-celled fungi that belong to the Fungi Kingdom in the Eukarya Domain. 

Source https://cnx.org/contents/5CvTdmJL@4.9:XjvIvG9J@3/Fermentation

    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:

LACTIC ACID FERMENTATION

​Pyruvate  +  NADH ↔ lactic acid  + NAD+Pyruvate  +  NADH ↔ lactic acid  + NAD+
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• Bacteria that undergo lactic acid fermentation include Lactobacillus, Leuconostoc, and Streptococcus.
• Bacteria that are known to use lactic acid fermentation are called lactic acid bacteria (LAB).
• ,Many LAB are gram-negative and are used in food production.
  • Lactic acid fermentation denatures milk proteins to form yogurt and cheese.
  • Lactobacillus delbrueckii and S. thermophiles are used in yogurt production.
  • There are bacteria that produce more than one fermentation. Such bacteria are said to perform heterolactic fermentation. ,Heterolactic fermentation producing a mixture of lactic acid, ethanol and/or acetic acid, and CO2.  The products of heterolactic fermentation are due to the cell undergoing the branched pentose phosphate pathway of glycolysis as opposed to the EMP pathway for glycolysis. Ab example of a heterolactic fermenter is Leuconostoc mesenteroides, which is used for pickling cucumbers to make pickles and for pickling cabbage to produce sour kraut.

   There are several other fermentation pathways in addition to alcoholic fermentation and lactic acid fermentation. These pathways produce useful compounds such as industrial solvents, cosmetics, pharmaceuticals, as well as foodstuffs like butter, beer wine, yogurt, cheese, and vinegar. Most of these pathways also produce CO2 gas, which is used is baking to make breads and cakes rise.

   Clinically, the API test includes a panel of tests that identify more than 600 species of aerobic and anaerobic bacteria, and about 100 different types of yeasts. The tests produce a metabolic profile for the microbe using pH indicators that can then be matched to a key card to identify the species. 

API Test

CATABOLISM OF LIPIDS AND PROTEINS

• Microbes have the ability to metabolize lipids and proteins.
  
• The catabolic pathways for metabolizing lipids and proteins join with the processes of glycolysis and the Krebs cycle.

• Triglycerides - Enzymes called lipases function to break down glycerol into its smaller fatty acid subunits.
  
• Phospholipids - Enzymes called phospholipases, break apart the phospholipid molecule into its individual fatty acids and phosphorylated head units.
  
• Fatty acids - A process called β-oxidation removes acetyl groups from the ends of fatty acid chains.
• Proteins - Enzymes called proteases break up large proteins into smaller amino acids chains called peptides. 

E.   Photosynthesis

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   Photosynthetic prokaryotes, such as cyanobacteria, use light energy to remove carbon dioxide from the atmosphere and fix it into organic molecules. This is the same basic process carried out by photosynthetic plants.

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Have you ever noticed that the chemical equation for aerobic cellular respiration and photosynthesis are basically the same equation in reverse??

THIS EQUATION IS FOR AEROBIC CELLULAR RESPIRATION.

    Notice that the reactants of cellular respiration are the products of the photosynthesis and the reactants of photosynthesis  are the products of the cellular respiration. Cellular respiration is basically photosynthesis in reverse.

Photosynthetic microbes convert solar energy into chemical energy.. Different photosynthetic organisms use different mixtures of photosynthetic pigments, which increase the range of the wavelengths of light an organism can absorb.

• 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.

• 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.

• 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 photophosphorylationinstead, producing only ATP.

• The light-independent reactions of photosynthesis use the ATP and NADPH from the light-dependent reactions to fix CO2into organic sugar molecules.

• Phototrophs - get energy from the sun.
• Chemotrophs - get energy from chemical compounds.
• Autotrophs - make their own food from inorganic substances.
• Carbon Fixation - recycle nitrogenous compounds in the environment into useful nitrogen-containing compounds like ammonia, that plants need to grow
• Heterotrophs - gets energy by consuming organic compounds of other organisms
• Aerobic - Oxygen is required for metabolism
• Anaerobic - Oxygen is not required for metabolism
• Photoautotroph - Uses light as its source of energy and uses carbon dioxide (or similar compound) as its source of carbon. 
• Photoheterotroph - Uses light as its source of energy and organic compounds as its carbon source
• Chemoautotroph - Uses chemical compounds as its source of energy and carbon dioxide (or similar compounds)
• Chemoheterotroph - Uses organic compounds