Other ways to make ATP Photosynthesis: light driven ATP synthesis. – Oxygenic and anoxygenic CO 2 used as source of carbon Inorganic molecules can be oxidized producing ATP synthesis by e- transport and chemiosmosis. – Examples: Fe +2 to Fe +3, NH 3 to NO 2 - – Requires O 2 as terminal electron acceptor – Usually CO 2 used as source of carbon 1

Anaerobic metabolism to make ATP Anaerobic respiration: organic compounds oxidized, electrons passed down e- transport chain to some molecule other than oxygen (e.g. NO 3 -, SO 4 -2 ). – Organic molecules like glucose still source of energy – Just like aerobic respiration but w/o O 2 – basis for lab identification test Fermentation: common anaerobic pathway used by many medically important bacteria. – Organic molecules serve as energy sources – Organic molecules serve as electron acceptor. – Electron transport not important in ATP production 2

What’s Fermentation for? 3 Glucose can be oxidized to pyruvic acid with the synthesis of 2ATPs. This alone is enough energy to live on. It depends on the oxidation of NADH to NAD so that NAD is available to accept electrons during the oxidation of glucose.

Why fermentation-2 4

Fermentation: “life without air” Without O 2 as an e- acceptor, NADH cannot be re-oxidized to NAD. Even though aerobic metabolism can produce ~36 ATP from 1 glucose, the 2 ATP from glycolysis is enough. But glycolysis requires that NAD be reduced to NADH; what happens when ALL the NAD becomes NADH with no O 2 to accept the H? Pyruvic acid is reduced, and the product thrown away; NAD restored, glycolysis can be repeated, more ATP made. A variety of ways of solving this problem exist; many types of molecules can be produced from fermentation. 5

Examples for fermentations Lactic acid fermentation – Lactic acid Alcoholic fermentation – Ethanol, carbon dioxide Mixed acid fermentation – Lactic acid, formic acid, succinic acid, ethanol, H 2, CO 2 Propionic acid fermentation – propionic acid, acetic acid, and carbon dioxide 6

Lessons from Fermentation Fermentation is inefficient. If C 6 H 12 O 6 has lots of energy-rich H’s, so does C 3 H 5 O 3 (lactic acid); the product cannot be further metabolized and is thrown away! Only a couple of ATPs are made. Fermentation is quick. Even though few ATPs are made, they are made quickly. Fermentation is wasteful. Large amounts of substrate (e.g. sugar) is used, making large amounts of product (e.g. lactic acid, ethanol, etc.) Variation in products makes fermentation a means for identifying different bacteria. 7

Bacteria and the fragility of existence Bacteria use ATP or the proton motive force to: – Move – Synthesis proteins (lots of them) – Transport molecules into the cell – Synthesize cell materials – Homeostasis Bacteria do not store ATP – Calculations: E. coli has enough ATP to last a few seconds – Thus, cells must keep on making it. Bacteria carefully regulate their use of energy! 8

Bacterial Growth and Nutrition Bacterial nutrition and culture media Chemical and physical factors affecting growth The nature of bacterial growth Methods for measuring population size 9

Matter and energy In order to grow, bacteria need a source of raw materials and energy – Source can be the same (e.g. glucose) or different (e.g. carbon dioxide and sunlight). – Living things can extract energy from matter – Living things can’t turn energy into matter – Living things can use energy to assemble raw materials. – Bacteria can’t grow on nothing! 10

Where do raw materials come from? Bacteria acquire energy from oxidation of organic or inorganic molecules, or from sunlight. Growth requires raw materials: some form of carbon. Autotrophs vs. heterotrophs – Auto=self; hetero=other; troph=feeding. – Autotrophs use carbon dioxide – Heterotrophs use pre-formed organic compounds (organic molecules made by other living things). – Humans and medically important bacteria are heterotrophs. 11

Essentials of Bacterial nutrition Elements needed for life: Macronutrients: needed in larger amounts – Needed in large quantities: CHONPS Carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur. H and O are common. Sources of C, N, P, and S must also be provided. – Macronutrients needed in smaller amounts: Mineral salts such as Ca +2, Fe +3, Mg +2, K + Micronutrients = trace elements; – needed in very tiny amounts; e.g. Zn +2, Mo +2, Mn +2 12

13 Element% dry wgtSource Carbon50organic compounds or CO 2 Oxygen20H 2 O, organic compounds, CO 2, and O 2 Nitrogen14NH 3, NO 3, organic compounds, N 2 Hydrogen8H 2 O, organic compounds, H 2 Phosphorus3inorganic phosphates (PO 4 ) Sulfur1SO 4, H 2 S, S o, organic sulfur compounds Potassium1Potassium salts Magnesium0.5Magnesium salts Calcium0.5Calcium salts Iron0.2Iron salts

Chemical form must be appropriate Not all bacteria can use the same things – Some molecules cannot be transported in – Enzymes for metabolizing it might not be present – Chemical may be used, but more expensive – These differences can be used for identification Some chemicals are inert or physically unusable – Relatively few bacteria (and only bacteria) use N 2 – Diamonds, graphite are carbon, but unusable – P always in the form of phosphate 14

Make it, or eat it? Some bacteria are remarkable, being able to make all the organic compounds needed from a single C source like glucose. For others: – Vitamins, amino acids, blood, etc. added to a culture medium are called growth factors. – Bacteria that require a medium with various growth factors or other components and are hard to grow are referred to as fastidious. 15

Feast or famine: normal is what’s normal for you: Oligotrophs vs. copiotrophs Oligo means few; oligotrophs are adapted to life in environments where nutrients are scarce – For example, rivers, other clean water systems. Copio means abundant, as in “copious” – The more nutrients, the better. – Medically important bacteria are copiotrophs. – Grow rapidly and easily in the lab. 16

Cells frequently face starvation Send molecules out to scavenge nutrients – Molecules collect iron – Enzymes leave cell, break down proteins, starch Cells enter Semi-starvation state: – slower metabolism, smaller size. Sporulation and “resting cells”: – cells have very low metabolic rate – Some cells change shape, develop thick coat – Endospores form within cells; very resistant. – Spores are for survival, triggered by low nutrients 17

Endospore formation 18

Culture Medium Defined vs. Complex – Defined has known amounts of known chemicals. – Complex: hydrolysates, extracts, etc. Exact chemical composition is not known. Selective and differential – Selective media limits the growth of unwanted microbes or allows growth of desired ones. – Differential media enables “differentiation” between different microbes. – A medium can be both. 19

Defined Medium for Cytophagas/Flexibacters 20 Componentgrams K2HPO40.10 KH2PO40.05 MgCl20.36 NaHCO30.05 {CaCl21 ml* {BaCl2.2H2O Na acetate0.01 FeCl.7H2O0.2 ml* RNA0.10 alanine0.15 arginine0.20 aspartic acid 0.30 glutamic acid0.55 glycine0.02 histidine0.20 isoleucine0.30 leucine0.20 lysine0.40 phenylalanine0.30 proline0.50 serine0.30 threonine0.50 valine0.30