Microbial metabolism consists of the chemical reactions accomplished by cells to support life and growth. Because chemicals called nutrients are changed during these processes, we can follow metabolism of a culture by analyzing the new chemicals created. Lucky for us, the changes are often obvious and easy to detect.
The processes involved in cells and populations living and growing are termed "metabolism", or "physiology". They represent the chemical changes driven by enzymes that are necessary for processing foods, making all molecules necessary for survival, and driving cell division. Since we cannot see most of these things happening inside of tiny cells, we instead look at changes taking place in their growth medium as clues to what is going on. Analyzing these clues is central to identifying bacteria. Which nutrients a microbe can use and the types of metabolic products that are produced are genetically determined and consistent. An unknown organism that uses the same nutrients and produces the same products as E. coli must be E. coli. So by doing metabolic tests we can achieve identification of an unknown microbe.
Growth and metabolism is an ongoing process of using nutrients and materials from the environment to keep cells alive and allow them to reproduce. The overall process can be broken down into several phases.
- Phase 1: Digestion: Think about how your digestive system breaks down foodstuffs so that they can pass through cells of the gut lining and enter the bloodstream. This is the external breakdown of macromolecules to allow their transport into the cell. Proteins and complex carbohydrates are all too large to pass through a membrane - like trying to drive your car through the front door of your home. Instead, cells will dismantle the molecule outside the cell via enzymatic digestion and then transport the parts through the cell where they can be further metabolized. This process is common to all life.
- Phase 2: Glycolysis: The default starting process when discussion metabolism is the use of glucose for producing energy for life - ATP. Glycolysis is a universal process by which sugars are metabolized to produce a 3-carbon molecule called pyruvate (or pyruvic acid). In the process of sugar-to-pyruvate, a cell generates a small amount of energy. Glycolysis is a series of reactions that creates pyruvate. The transformation is done step-by-step in chemical reactions catalyzed by enzymes. Each product of an enzyme in the pathway is an intermediate molecule, some of which are important starting points for biosynthesis. For instance, an intermediate may be used for the next enzymatic step of glycolysis OR for making a particular amino acid. Or, that amino acid could be converted to the intermediate to enter glycolysis. The road between Dallas and Los Angeles has many opportunities for entering or exiting, and intermediate compounds serve to connect the side roads to the interstate. Thus, glycolysis is always going, always important to getting energy and changing molecules to meet cell needs.
- Phase 3: Use of Pyruvate: Pyruvate is the end product of glycolysis and the starting point for a variety of uses, some of which help define the cell's metabolism for identification. Of importance to our discussion here is the use of pyruvate for energy production. One way pyruvate can be used is by a process called fermentation. Energy production and ongoing metabolism require processes of oxidation and reduction - taking electrons out of molecules and putting them in others. Fermentation occurs when the final product of metabolism is created by dumping all those unwanted electrons into an organic molecule (a carbon-based molecule, like pyruvate).
When we think of fermentation we also think of alcohol production. Ethanol is also a product of fermentation, where electrons are dumped into an organic molecule called acetaldehyde. In either of these examples, oxygen is not a factor and the Krebs Cycle (a.k.a Citric Acid Cycle or TCA Cycle) and cytochromes are not involved.
One example of fermentation happens when a human exercises and hits a point where the cardiovascular system cannot supply oxygen fast enough for the muscles to use during exercise. The result is the electrons generated by muscle metabolism are put into pyruvate, making lactic acid. This builds up in muscles and results in fatigue and soreness. As oxygen supply becomes sufficient over the next few hours and days, lactic acid is metabolized via the TCA Cycle and the soreness goes away.
A second means for using pyruvate DOES use the TCA Cycle to completely oxidize pyruvate into CO2 - a molecule without any more electrons to give! The TCA Cycle takes a product of pyruvate (acetyl coA) and adds it to the end product of the cycle to begin the cycle again. The 6-carbon citrate molecule is oxidized, decarboxylated (meaning CO2 is released), oxidized, and decarboxylated again, and then oxidized some more to remove all electrons possible. The product is a 4-carbon molecule that can combine with another acetyl coA to start the cycle all over again.
The electrons represent energy that can be used by the cell to drive ATP production. Little transport molecules called NAD and FAD pick up electrons and carry them to the cytochromes where they are passed along to drive ATP production. So, the bottom line of the TCA Cycle is the complete oxidation of pyruvate to three molecules of carbon dioxide. The electrons removed in the process are transported to the cytochromes for the next phase to be discussed.
- Phase 4: Oxidative Phosphorylation: Oxidative phosphorylation is the process of using electron flow through cytochromes to drive the production of ATP. It consists of two separate but linked activities: electron transport and chemiosmosis. Electron transport draws energy from electrons as they are disposed of via the cytochromes. Chemiosmosis uses a proton gradient to drive ATPase and make ATP.
The first part of this process is disposal of electrons via the cytochromes. Oxygen is important to cells because it gives a cell something to dispose of electrons into during Ox-Phos respiration. Think back to your earlier biology courses and you will recall that the oxygen is used by the cytochromes as the dumping ground for electrons, combining oxygen and electrons with protons to create molecules of water. So, electrons plus protons plus oxygen yields water. Where do the electrons come from? Pyruvate is completely oxidized to CO2 by the enzymes of the TCA Cycle. Remember the little NAD and FAD trucks that carried electrons to the cytochromes as the TCA cycle rolled on. They were taking electrons to the cytochromes where they (sometimes called the electron transport chain) transported the electrons to combine them with protons and oxygen to make water.
Starting to sound familiar? What we need to understand when talking about bacteria, is that oxygen isn't the only compound that can accept electrons for some bacteria. When oxygen is the final electron receptor of respiration, it is termed aerobic respiration. When another molecule takes those electrons instead, it is termed anaerobic respiration. So, picture the cytochromes passing electrons along and making ATP, but then giving those electron to nitrate or carbonate or sulfate instead. Because some microbes CAN do this, we can test an unknown microbe for the ability to do anaerobic respiration and in doing so get one step closer to identification. To recap, oxidative phosphorylation begins with the process of electron transport for disposal in an inorganic molecule. When that molecule is oxygen, the process is aerobic respiration. When the molecule is not oxygen, it is termed anaerobic respiration.
So that is half the Ox-Phos story: disposal of electrons. What is not mentioned in this is the fact that NAD and FAD are delivering both electrons AND protons, but the cytochromes cannot deal with the protons in the same way. So, the protons get pushed OUT through the membrane during the electron transfer process of the cytochromes, and in doing so, the cell creates a proton gradient - much higher proton concentrations on one side of the membrane when compared with the concentration on the other. This gradient represent a source of power as those protons force their way back through a proton channel connected to ATPase. Like water held back by a hydroelectric dam, when it is allowed to rush through a duct the force of the water can turn a turbine in the duct and thus create useable energy - electricity. In the same way, the pressure of the high concentration of protons outside the membrane can drive the ATPase enzyme to make ATP as the protons flood through a membrane channel back into the cell. So, the proton gradient provides the energy to drive ATP production, just as water provides the energy to drive electrical production at the dam.
So, electron transport via the cytochromes disposes of the electrons from metabolism and creates the proton gradient, while chemiosmosis uses the proton gradient to generate ATP.
- Phase 5: Making Building Blocks for Growth: Most teaching of cell metabolism focuses on glycolysis, TCA Cycle, and oxidative phosphorylation. This is important, because these processes keep the cell supplied with energy for all its processes. However, the assumption is that cells get a ready supply of glucose to start glycolysis, and this discussion overlooks the fact that cells have to generate all of the building blocks needed for growth. The missing component is something often called intermediary metabolism - the pathways and reactions that connect everything together. For instance, we have already discussed the fact that intermediates can serve as entry points for non-sugars into glycolysis. The same thing is true for the TCA Cycle. Key intermediates that we call precursors serve unique roles as intermediates used for energy production in glycolysis and TCA Cycle and also as the starting points for making all those building blocks for growth. From the 12 precursor molecules, all amino acids, nucleotides, fats, vitamins, etc. can be made by some microbes.
Other microbes have lost some of these pathways and must obtain missing nutrients from their environment or eukaryotic host. Each of these 12 precursors serves as a decision point - is it more important to use the intermediate for energy production or making an amino acid, for example. These decisions are made by allowing or inhibiting the biosynthetic pathway (the one making the building block) to use the intermediate. Plenty of glycine available to the cell? Then use that intermediate for energy metabolism instead of making unneeded glycine! This process of feedback inhibition insures efficient use of glycolysis and TCA Cycle intermediates for the need at the moment. With a ready supply of all building blocks and a regulated process to insure sufficient supplies are provided from nutrients the cell is processing, the cell is now staged for growth.
- Phase 6: Macromolecular Biosynthesis: Once the supply of energy and raw materials is established, a cell is ready to make macromolecules. Energy is used to combine the building blocks to produce proteins, nucleic acids, complex carbohydrates, lipids, and other essential cell compounds. These can be enzymes or structural molecules that allow a cell to function properly, repair faulty structures, and maximize its operations.
- Phase 7: Cell Division: The ultimate goal of the cell is to divide. The macromolecular biosynthesis enables this. Often, some of the macromolecules produced serve as signal molecules to take stock of the environmental conditions and signals, and the state of metabolism to determine whether it is time to initiate cell division.
The next section takes a look at this process from the perspective of bacterial growth. Follow your instructor's guidance on how to proceed. You may be asked to complete the quiz at this link before moving on.