1 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

2 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 (molecules made by other living things). –Humans and medically important bacteria are heterotrophs.

3 Essentials of Bacterial nutrition Six elements needed in large quantities by all living things: CHONPS –Carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur. H and O are common. Sources of C, N, P, and S must also be provided. Other macronutrients: not as much needed: –Mineral salts such as Ca +2, Fe +3, Mg +2, K + Micronutrients = trace elements; needed in very tiny amounts: things like Zn +2, Mo +2, Mn +2 Elements must be in the correct chemical form! –Diamonds, graphite no good. N 2 used by very few bacteria.

4 Element% dry wgtSource Carbon50organic compounds or CO 2 Oxygen20 H 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 ) Sulfur1 SO 4, H 2 S, S o, organic sulfur compounds Potassium1Potassium salts Magnesium0.5Magnesium salts Calcium0.5Calcium salts Iron0.2Iron salts

5 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, 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.

6 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. Feast or famine: normal is what’s normal for you: Oligotrophs vs. copiotrophs

7 Responses of microbes to nutritional deficiency Siderophores and hemolysins –For collecting iron. (e.g. electron transport systems) Extracellular enzymes: leave cell, go out into medium, break down polymers, release LMW compounds. Genetic regulation: cell uses other nutrient sources. Semi-starvation state: slower metabolism, smaller size. Sporulation: cells form environmentally resistant resting state. Endospores form within cells; others become spores.

8 Responses of microbes to other environmental stresses Compatible solutes: small neutral molecules accumulated in cytoplasm when external environment is hypertonic. Heat shock proteins and other stress proteins –Bacteria express additional genes that code for protective proteins.

9 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.

10 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 Defined Medium for Cytophagas/Flexibacters

11 Physical requirements for growth Prefixes and suffixes: Bacteria require wide ranges of conditions –Optimal conditions implied by “-phile” meaning “love” Some bacteria prefer other conditions, but can tolerate extremes –Suffix “-tolerant”

12 Oxygen: friend or foe? Early atmosphere of Earth had none –First created by cyanobacteria using photosynthesis –Iron everywhere rusted, then collected in atmosphere Strong oxidizing agent Reacts with certain organic molecules, produces free radicals and strong oxidizers : –Singlet oxygen, H 2 O 2 (peroxide), O 3 - (superoxide), and hydroxyl (OH-) radical.

13 Protections of bacteria against oxygen –Bacteria possess protective enzymes, catalase and superoxide dismutase. –Catalase breaks down hydrogen peroxide into water and oxygen gas. –Superoxide dismutase breaks superoxide down into peroxide and oxygen gas. –Anaerobes missing one or both; slow or no growth in the presence of oxygen.

14 Relation to Oxygen Aerobes: use oxygen in metabolism; obligate. Anaerobes: grow without oxygen Microaerophiles: require oxygen, but in small amounts. Capnophiles: require larger amounts of carbon dioxide.

15 Oxygen and microbes continued Classifications vary, but our definitions: –Obligate anaerobes: killed or inhibited by oxygen. –Aerotolerant anaerobes: do not use oxygen, but not killed by it. –Facultative anaerobes: can grow with or without oxygen

16 Effect of temperature Low temperature –Enzymatic reactions too slow; enzymes too stiff –Lipid membranes no longer fluid High temperature –Enzymes denature, lose shape and stop functioning –Lipid membranes get too fluid, leak –DNA denatures As temperature increases, reactions and growth rate speed up; at max, critical enzymes denature.

17 Bacteria and temperature Bacteria have temperature ranges (grow between 2 temperature extremes), and an optimal growth temperature. Both are used to classify bacteria. As temperature increases, so do metabolic rates. At high end of range, critical enzymes begin to denature, work slower. Growth rate drops off rapidly with small increase in temperature.

18 Classification of bacteria based on temperature

19 Terms related to temperature Special cases: –Psychrotrophs: bacteria that grow at “normal” temperature ranges (e.g. room temperature” but can also grow in the refrigerator; responsible for food spoilage. –Thermoduric: more to do with survival than growth; bacteria that can withstand brief heat treatments.

20 pH Effects pH = -log[H+] Lowest = 0 (very acid); highest = 14 (very basic) Neutral is pH 7. Acidophiles/acidotolerant grow at low pH Alkalophiles/alkalotolerant grow at high pH Most bacteria prefer a neutral pH –What is pH of human blood? Some bacteria create their preferred conditions –Lactobacillus creates low pH environment in vagina

21 Low water activity: halophiles, osmophiles, and xerotolerant Water is critical for life; remove some, and things can’t grow. (food preservation: jerky, etc.) Halophiles/halotolerant: relationship to high salt. –Marine bacteria; archaea and really high salt. Osmophiles: can stand hypertonic environments whether salt, sugar, or other dissolved solutes –Fungi very good at this; grandma’s wax over jelly. Xerotolerant: dry. Subject to dessication. Fungi best –Bread, dry rot of wood –Survival of bacterial endospores.

22 Miscellaneous conditions Radiation (solar, UV, gamma) –Can all damage cells; bacteria have pigments to absorb energy and protect themselves. –Endospores are radiation resistant. –Deinococcus radiodurans: extremely radiation resistant Extremely efficient DNA repair, protection against dessication damage to DNA. Barophiles/barotolerant: microbes from deep sea –Baro- means pressure. Actually require high pressure as found in their environment.