Microbiology - Chapter 7 & 8 Microbial Growth – Bacteria reproduce by “binary fission”, a cell divides into two, two to four, four to eight, etc. Cell division can occur quite rapidly depending on nutrient levels, temperature, etc. E. coli can divide every 20 minutes (time to double – generation time) in nutrient media at 37 degrees C. The numbers get so large we express them as the log of the number of cells. Microbiology - Chapter 7 & 8 Microbial Growth is affected by two major factors: Environmental: temperature, pH, Osmotic conditions Chemical: Proper concentrations of C, H, O, N, P, S, some trace elements, and some organic cofactors Microbiology - Chapter 7 & 8 Bacterial Growth Curve: A= Lag phase C= Stationary phase B= Log phase D= Death Phase Know this and be able to explain what is occurring during each phase. Bacteria that produce endospores may not have a death phase. Why? Microbiology - Chapter 7 & 8 Why study such a growth curve? Helps us understand how microbes grow under different conditions Helps us understand how pathogen grow in our body Helps us study the effect of different chemicals, osmotic conditions, even the effect of temperature on bacterial growth. Ex. What would the growth curve for E.coli look like if we incubated at 4 degrees Celsius? At 65? Microbiology - Chapter 7 & 8 How do we measure growth of bacteria in a growth curve? Direct cell count using stained slides that have a grid for counting. (Tedious, a real pain) Indirect: Serial dilution, plates are innocculated - incubated and colonies counted. Number of colonies X dilution factor gives the number of bacteria. Microbiology. Chapter 7 -8 How do we measure growth of bacteria in a growth curve? Direct cell count. Tedious and time consuming Microbiology - Chapter 7 & 8 How do we measure growth of bacteria in a growth curve? Microbiology - Chapter 7 & 8 How do we measure growth of bacteria in a growth curve? Measure cloudiness in a test tube as the number of cells increase (turbidity) using a spectrophotometer. Correlate this data with a standard plate count and now just use the turbidity measurement – look up number from a chart from then on. Saves time and money. Microbiology - Chapter 7 & 8 How do we measure growth of bacteria in a growth curve? Coulter counter. Electronically counts number of bacteria as bacteria pass through a tiny tube. Expensive. Microbiology - Chapter 7 & 8 Physical factors that affect bacterial growth; Mesophiles : grow best moderate temp. 25 – 40 degrees most of our lab microbes Psychrophiles: adapted to survive and grow at cooler temp., even in the frig (below 25 degrees) Listeria (in cheeses and meat) Thermophiles: adapted to and grow at much higher temp. Thermus aquaticus, from oceanic vents, survives at 60 degrees C Leprosy bacilli prefer 30 degrees, most pathogens prefer 37 degrees. Microbiology - Chapter 7 & 8 Physical/Chemical factors that affect bacterial growth; pH: measure acidity and alkalinity of media Bacteria grow best at pH range of 6.5 to 7.5 Fungi grow better at slightly acid condition (5.0 to 5.5) Sabaraud dextrose and Potato dextrose agars One pathogen, Helicobacter pylori, is adapted to and survives in stomach acid (cause of ulcers) Hydrostatic pressure: some bacteria grow really well deep in the ocean at pressures that crush submarines like and “egg” Microbiology - Chapter 7 & 8 Physical/Chemical factors that affect bacterial growth; pH: measure acidity and alkalinity of media Osmotic pressure; relative salt concentrations in water solutions Hypertonic: higher salt concentrations, slows or stops bacterial growth; salt preservative in meat some prefer higher salt: Halophiles some survive and thrive, Vibrio bacteria, V. cholera Hypotonic: lower salt, fresh water, net flow water into cells, bacteria have rigid cell wall resist rupture Isotonic: equal solute (salt) no net flow, preferable Microbiology - Chapter 7 & 8 Chemical factors that affect bacterial growth: Nutrition How microbe acquire nutrients. C, H, O, N, S, P, Ca, Mg, etc Carbon: Autotroph: producers, photosynthetic, use CO2 and H2O, sunlight as energy, make their own food Heterotroph: require preformed food, digestive and absorptive, most microbes Chemoautotroph: unique metabolism, use chemical energy from inorganic molecules, Sulfur and Iron Microbiology - Chapter 7 & 8 Chemical factors that affect bacterial growth: Nutrition How microbe acquire nutrients. C, H, O, N, S, P, Ca, Mg, etc Oxygen: Obligate aerobes: require molecular oxygen (as final electron acceptor in catabolism) Pseudomonas spp. Obligate anaerobes: require atmosphere with no O2 an organic molecule is final electron acceptor in catabolism (like a fermentation pathway) Clostrida - grow in “Brewer Jar” Facultative anaerobes: grow with or without O2, usually are also fermenters, like E. coli Microaerophile: grow best in lower oxygen and higher carbon dioxide, Strep., candle jar Microbiology - Chapter 7 & 8 Problems with oxygen: oxygen can be toxic, it oxidizes and destroys vital cell chemicals; aerobic organisms have enzymes and systems to handle it SOD: superoxide dismutase, enzyme that chemically alters toxic oxygen free radicals and toxic high energy “singlet oxygen” to less toxic hydrogen peroxide Catalase: Converts hydrogen peroxide to oxygen and water Microbiology - Chapter 7 & 8 Nitrogen: Found in all the amino acids, nitrogenous bases of nucleic acids, etc. Hydrogen: found in all biological molecules, Carbs, fats, proteins, nucleic acids, etc Phosphorous: found in nucleic acids, ATP, and phospholipdids of membranes Sulfur: found in 2 or 3 amino acids of microbes Trace elements: inorganic elements needed in very tiny concentrations (manganese, cobalt, Zn, Cr) Microbiology - Chapter 7 & 8 Organic cofactors: Vitamins Required by certain bacteria, “fastidious” hard to grow Coenzymes Many microbes produce their own from scratch, source of our supplements (one a day, GNC) Fastidious organisms may require enriched media to get them to grow (blood, eggs, etc) Some organisms are almost impossible to culture because of their strict parasitic-fastidious nature (syphilis, leprosy) Microbiology chapters 7 - 8 part 2 Metabolism Catabolism Anabolism Both occur simultaneously in cells Catabolism eventually produces the chemical energy (ATP) required for all cellular functions such as anabolism (synthesis), membrane transport, etc. Microbiology chapters 7 - 8 part 2 ATP – Adenosine triphosphate, universal cellular energy Cyclically made and energy is stored and then broken down and the energy is released Microbiology chapters 7 - 8 part 2 Note: ATP is a ribonucleotide, it has ribose, a nitogenous base (adenine), and phosphate. The high energy bond of the terminal of the three phosphates is the one cyclically broken and regenerated. Sugars like glucose can be broken down in a catabolic pathway controlled by many cellular enzymes. Some of the energy released by the breaking of covalent bonds is harvested and stored in the “energy” bonds of ATP. Most any biomolecule can be used for energy; we will focus on the “catabolism” of glucose (a monosaccharide) and later show how the others are involved (lipids, AA, etc) Microbiology chapters 7 - 8 part 2 Quick review on enzymes Organic catalyst (made of carbon, speed up rate of chemical reactions) Made of protein; chains of Amino acids in a specific sequence that fold and coil into specific shapes. Their shape is key to understanding their function. (remember shape determines function) Also, shape is easily affected by changes in temperature. So, heat or cold can cause enzymes to slow down or even stop. An enzyme lowers “activation energy” – energy required for a reaction to begin Microbiology chapters 7 - 8 part 2 Quick review on enzymes Substrates are the material that are acted on by the enzyme Enzymes are often named using the name of the substrate and adding “ase”. Sucrase breaks down sucrose to glucose and galactose. Enzyme driven reactions are often reversible. Microbiology chapters 7 - 8 part 2 Aerobic metabolism; specifically glucose catabolism This stuff is hard “Just do It” Goal: 1. List the three stages of glucose catabolism 2. Know the basic steps of each stage 3. Know how much ATP is made at each stage per molecule of glucose 4. Starting products and end products, other important carriers (NAD) 5. The difference between substrate level phosphorylation and oxidative phosphorylation 6. Theory of chemiosmosis and ATP production at the membrane of the mitochondria Microbiology chapters 7 - 8 part 2 Glucose is a hexose, monosaccharide, C6H12O6 It is systematically broken down through three related “pathways” to Carbon dioxide (CO2) and Water (H2O) Overall Formula:C6H12O6 + ___ O2 CO2 + ___ H2O The three stages: Glycolysis (anaerobic) (in cytoplasm) Krebs cycle (aerobic) (in mitochondria) Electron transport (with chemiosmosis) (aerobic) Microbiology chapters 7 - 8 part 2 Glycolysis: Anaerobic, no oxygen required, linear NZ pathway Begins with ______ End products _________ How much ATP? _______ How many carrier molecules? ____ Name the carrier molecule. ____ Where in the cell? _______ What happens after if the organism Is an aerobe? _____ Facultative? ______ Strict anaerobe? ______ Aerobe deprived of oxygen? ____ Microbiology chapters 7 - 8 part 2 Krebs cycle (TCA, Citric acid cycle) Aerobic stage, Occurs in the fluid of the Matrix Microbiology chapters 7 - 8 part 2 This is a cyclic “pathway” Pyruvic acid is first acted on by an NZ and a coenzyme (COA). The end product is Acetyl-Coa and a CO2 molecule. Remember this occurs twice for each glucose molecule. (One glucose is split into two pyruvic acid molecules.) Microbiology chapters 7 - 8 part 2 The acetyl-COA reacts with an enzyme and another substrate (a 4-C molecule called oxaloacetic acid) to produce Citric Acid, a 6 carbon tri-carboxylic acid; 3 carboxyl groups Several enzymes systematically oxidize the citric acid into a 5-C acid, then a 4-C acid and eventually back to the original oxaloacetic acid – thus a cycle. Each time the terminal carboxyl group is removed a CO2 molecule is produced. Thus, one glucose, causes the cycle to turn twice, each turn produces 3 CO2 (one at Acetyl COA step and two in the cycle) Now for the hard part. Understanding that an oxidation reduction reaction is going on at each step. (Here **Krebs**), at glycolysis, and even electron transport) Let’s first review oxidation- reduction (aka: redox) Microbiology chapters 7 - 8 part 2 Oxidation – Reduction Organic molecules like glucose have covalent bonds between C-C, C-H, C-O, O-H C6H12O6 When the molecule is broken down -, the covalent bonds are broken – electrons are removed and transferred to carrier molecules. Oxidation is the removal of electrons and/or adding Oxygen In Glycolysis the glucose is broken into two Pyruvates, The electrons and a H+ are transferred to a carrier, NAD. NAD gains the electron (and Hydrogen too), it is reduced to NADH, thus oxidation and reduction go together. Microbiology chapters 7 - 8 part 2 Oxidation – Reduction Look again at glycolysis. Glucose is oxidized and the carrier NAD is reduced. For every glucose, two Carriers are produced 2 NADH (what happens to them, they have to be regenerated – oxidized back to NAD) Aerobes eventually produce CO2 and H2O Thus oxygen is the final electron acceptor( producing Water). Anaerobes use a different set of enzymes, a Fermentative pathway that generates other acids, alcohols or gasses (lactic acid, ethanol, CO2) ** electron acceptor is an “organic molecule”** If no regeneration of NAD, the glycolysis pathway shuts down and the organism dies – no ATP Microbiology chapters 7 - 8 part 2 Glycolysis, no oxygen, fermentation, only 2 ATP per molecule of glucose Glycolysis, with oxygen, followed by Krebs and electron transport, can generate much more ATP (sometimes as much as 36). Aerobic mechanisms are much more energy efficient. In the Krebs cycle many more carrier molecules like NADH are generated and thus lead to more ATP. (Other carriers FAD, NADP – we just use NAD as a representative type of carrier). The constantly turning of the cycle produces a steady stream of reduced carriers (NADH) which pass the electrons to a set of carrier-processor molecules imbedded in the membrane of the Mitochondria. These carriers are called the “electron” transport chain. Microbiology chapters 7 - 8 part 2 Return to Krebs and show how it works with electron transport chain. Note how glycolysis and Krebs are working together. Note that each produces reduced carriers that need to be processed. Microbiology chapters 7 - 8 part 2 The electrons are passed down the chain and end up being added to oxygen. The Hydrogen ion (H+) is pumped out (proton pump) and flows back in at special sites to be added to the Oxygen and electron to form Water. Energy is conserved (harvested; stored) in the bonds of ATP Microbiology chapters 7 - 8 part 2 Theory of Chemiosmosis: Proton pump, increased H+ ion concentration, flow through ATP synthase related channel, energy is harvested in high energy bonds of ATP. Enough to generate 34 more ATP. Microbiology chapters 7 - 8 part 2 Fermentation: Many microbes ferment sugars and other substrates to make ATP without oxygen See pg 234 in text: NADH reduces pyruvate and ethanol and carbon dioxide are produced Other end products are seen: lactic acid, acetic, acid We use biochemical tests and the end products of sugar fermentation to ID bacteria (charts in Bergey’s) ** later in lab, particularly with unknown 2 Some bacteria, like E. coli and Bacillus use nitrogen electron acceptors to regenerate NAD. Nitrate and Nitrite reduction are examples. Pg 233 in text. The enzyme system is called Nitrate reductase Microbiology chapters 7 - 8 part 2 Other fuels Proteins: digested to amino acids Amino acids are : ‘deaminated’ : amino group removed, the reulting ‘acid’ can be further metabolized, more ATP decarboxylated: carboxyl group removed, the end products then enter glycolysis or Krebs to make ATP Microbiology chapters 7 - 8 part 2 • Lipids are catabolized to Glyerol and Fatty acids • Glycerol easily enters glycolysis and Krebs just like pyruvate • Fatty acids are chopped into 2 or 3 acid fragments that are broken downt to carbondioxide • Even nucleic acids – OH SO MUCH MORE!!! Take biochem.