Microbial Nutrition and Growth

Transcription

1 Microbial Nutrition and Growth Chapter 6 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2 Microbial Nutrition Bacteria require a constant influx of certain substances from their habitat All organisms require a source of elements such as carbon, hydrogen, oxygen, phosphorous, potassium, nitrogen, sulfur, calcium, iron, sodium, chloride, magnesium, and certain other elements Essential nutrient: any substance that must be provided to an organism

3 Microbial Nutrition (cont d) Macronutrients: required in relatively large quantities and play principal roles in cell structure and metabolism - carbon, hydrogen, and oxygen Micronutrients: also known as trace elements - present in much smaller amounts and are involved in enzyme function and maintenance of protein structure - manganese, zinc, nickel

4 Microbial Nutrition (cont d) Inorganic nutrient - an atom or simple molecule that contains a combination of atoms other than carbon and hydrogen - found in the crust of the earth, bodies of water, and the atmosphere Organic nutrients - contain carbon and hydrogen atoms and are the products of living things - simple organic molecules such as methane - large polymers (carbohydrates, lipids, proteins, nucleic acids)

5 Chemical Analysis of the Microbial Cytoplasm Water 70% of all components Proteins Organic compounds 97% of dry cell weight Elements CHONPS 96% of dry cell weight Most chemical elements available to the cell as compounds and not as pure elements Only a few types of nutrients needed to synthesize over 5,000 different compounds

6 Nutritional Categories of Microbes

7 Autotrophs and Their Energy Sources Photoautotrophs - Photosynthetic; capture energy from light rays and transform it to chemical energy - produce organic molecules using CO 2 that can be used by themselves and by heterotrophs

8 Autotrophs and Their Energy Sources (cont d) Chemoautotrophs - chemoorganic autotrophs: use organic compounds for energy and inorganic compounds as a carbon source - lithoautotrophs: rely totally on inorganic minerals and require neither sunlight nor organic nutrients

9 Heterotrophs and Their Energy Sources Chemoheterotrophs - derive both carbon and energy from organic compounds - process these molecules through respiration or fermentation Saprobes - free living organisms that feed on organic detritus from dead organisms - decomposers of plant litter, animal matter, and dead microbes - recycle organic nutrients

10 Heterotrophs and Their Energy Sources (cont d) Parasites - derive nutrients from the cells or tissues of a living host - pathogens: cause damage to tissues or even death - range from viruses to helminths - ectoparasites: live on the body - endoparasites: live in the organs and tissues

11 Heterotrophs and Their Energy Sources (cont d) Parasites (cont d) - intracellular parasites: live within cells such as the leprosy bacillus and the syphilis spirochete - obligate parasites: unable to grow outside of a living host - less strict parasites can be cultured artificially if provided with the correct nutrients and environmental conditions The vast majority of microbes causing human disease are chemoheterotrophs

12 Essential Nutrients Chemicals that are necessary for particular organisms, which they cannot manufacture by themselves Carbon, hydrogen, oxygen, phosphate, and sulfur (CHONPS)

13 Other Important Nutrients Sodium (Na): important for certain types of cell transport Calcium (Ca): stabilizer of cell wall and endospores of bacteria Magnesium (Mg): component of chlorophyll and a stabilizer of membranes and ribosomes Iron (Fe): important component of the cytochrome proteins of cell respiration

14 Other Important Nutrients (cont d) Zinc (Zn): essential regulatory element for eukaryotic genetics - major component of zinc fingers; binding factors that help enzymes adhere to specific sites on DNA Copper, cobalt, nickel, molybdenum, manganese, silicon, iodine, and boron are needed in small amounts by some microbes, but not others Metals can be toxic to microbes The concentration of metal ions can influence the diseases microbes cause

15 How Microbes Eat: Transport Mechanisms Transport of necessary nutrients occurs across the cell membrane, even in organisms with cell walls The driving force of transport is atomic and molecular movement Diffusion: the phenomenon of molecular movement, in which atoms or molecules move in a gradient from an area of higher density or concentration to an area of lower density or concentration

16 Diffusion of Molecules in Aqueous Solutions

17 Diffusion All molecules (solid, liquid, or gas) are in continuous movement As temperature increases, molecular movement becomes faster In any solution, including cytoplasm, these moving molecules cannot travel very far without having collisions with other molecules As a result of these collisions, the directions of colliding molecules are altered and unpredictable

18 Diffusion (cont d) If the solute is more concentrated in one area than another, the thermal movement will eventually distribute the molecules evenly Diffusion of molecules across the cell membrane is largely determined by the concentration gradient and permeability of the substance

19 The Movement of Water: Osmosis Osmosis: the diffusion of water through a selectively,or differentially, permeable membrane - has passageways that allow free diffusion of water, but block certain other dissolved molecules - when the membrane is placed between solutions of differing concentrations of solute and the solute cannot pass through the membrane, water will diffuse at a faster rate from the side that has more water to the side that has less water - this will continue until the concentration of water is equalized on both sides of the membrane

20 Model System to Demonstrate Osmosis Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 Inset shows a close-up of the osmotic process. The gradient goes from the outer container (higher concentration of H 2 O) to the sac (lower concentration of H 2 O). Some water will diffuse in the opposite direction but the net gradient favors osmosis into the sac. 2 3 As the H 2 O diffuses into the sac, the volume increases and forces the excess solution into the tube, which will rise continually. Even as the solution outside the sac becomes diluted, there will still be osmosis into the sac. Equilibrium will not occur because the solutions can never become equal. (Why?) Solute Water Glass tube Membrane sac with solution Container with water Pore

21 Osmosis (cont d) Living membranes generally block the entrance and exit of larger molecules and permit the free movement of water Most cells are surrounded by some free water and the amount of water entering or leaving has a major impact on cellular activities and survival This osmotic relationship between cells and their environment is determined by the relative concentrations of the solutions on either side of the cell membrane

22 Cell Responses to Solutions of Differing Osmotic Content Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Osmosis Isotonic solution Hypotonic solution Hypertonic solution Cell wall Cell membrane Cells with cell wall Cell membrane Water concentration is equal inside and outside the cell, thus rates of diffusion are equal in both directions. Net diffusion of water is into the cell; this swells the protoplast and pushes it tightly against the wall; wall usually prevents cell from bursting. Water diffuses out of the cell and shrinks the cell membrane away from the cell wall; process is known as plasmolysis. Cells without cell wall Rates of diffusion are equal in both directions. Net water movement Diffusion of water into the cell causes it to swell, and may burst it if no mechanism exists to remove the water. Water diffusing out of the cell causes it to shrink and become distorted. Solute

23 Osmosis (cont d) Cell membranes and cell walls hinder simple diffusion by adding a physical barrier Simple diffusion is limited to small nonpolar molecules such as oxygen or lipid soluble molecules that may pass through membranes It is imperative that cells move polar molecules and ions across the plasma membrane

24 Transport The process of moving molecules into or out of cells Features of active transport - the transport of nutrients against the diffusion gradient or in the same direction as the natural gradient, but at a rate faster than by diffusion alone - the presence of specific membrane proteins (permeases and pumps) - the expenditure of energy Examples of substances transported actively are monosaccharides, amino acids, organic acids, phosphates, and metal ions

25 Endocytosis: Eating and Drinking by Cells Some eukaryotic cells transport large molecules, particles, liquids, or other cells across the cell membrane requiring the expenditure of energy Endocytosis - cell encloses the substance in its membrane - simultaneously forms a vacuole and engulfs the substance

26 Endocytosis: Eating and Drinking by Cells (cont d) Phagocytosis - accomplished by amoebas and white blood cells - ingest whole cells or large solid matter Pinocytosis: ingestion of liquids such as oils or molecules in solution

27 Transport Processes in Cells Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 6.4 Transport Processes in Cells Examples Description Energy Requirements Passive Simple diffusion A fundamental property of atoms and molecules that exist in a state of random motion None. Substances move on a gradient from higher concentration to lower concentration. Facilitated diffusion Molecule binds to a specific receptor in membrane and is carried to other side. Molecule-specific. Goes both directions. Rate of transport is limited by the number of binding sites on transport proteins. None. Substances move on a gradient from higher concentration to lower concentration. Membrane Protein Extracellular Intracellular Extracellular Intracellular Active Carrier- mediated active transport Atoms or molecules are pumped into or out of the cell by specialized receptors. Driven by ATP or the proton motive force Membrane ATP Protein Extracellular Intracellular Group translocation Molecule is moved across membrane and simultaneously converted to a metabolically useful substance ATP Membrane Protein ATP Extracellular Intracellular Bulk transport Mass transport of large particles, cells, and liquids by engulfment and vesicle formation. Processes generally called endocytosis. Phagocytosis moves solids into cell; pinocytosis moves liquids into cell. ATP Phagocytosis Pseudopods Vacuoles Pinocytosis Microvilli Liquid enclosed by microvilli Oil droplet ATP ATP Vesicle with liquid

28 Environmental Factors That Influence Microbes Microbes are exposed to a wide variety of factors in addition to nutrients Environmental factors affect the function of metabolic enzymes Survival in a changing environment is largely a matter of whether the enzyme systems of microorganisms can adapt to alterations in their habitat

29 Ecological Groups by Temperature of Adaptation Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Growth Rate of Minimum Maximum Optimum Psychrophile Psychrotroph Mesophile Thermophile Extreme thermophile Temperature C

30 Environmental Factor: Gases The atmospheric gases that influence microbial growth are O 2 and CO 2 - O 2 has the greatest impact on microbial growth - O 2 is an important respiratory gas and a powerful oxidizing agent Microbes fall into one of three categories - those that use oxygen and detoxify it - those that can neither use oxygen nor detoxify it - those that do not use oxygen but can detoxify it

31 How Microbes Process Oxygen As oxygen enters cellular reactions, it is transformed into several toxic products - singlet oxygen (O): an extremely reactive molecule that can damage and destroy a cell by the oxidation of membrane lipids - superoxide ion (O 2- ): highly reactive - hydrogen peroxide (H 2 O 2 ): toxic to cells and used as a disinfectant - hydroxyl radicals (OH - ): also highly reactive

32 How Microbes Process Oxygen (cont d) Most cells have developed enzymes that scavenge and neutralize reactive oxygen byproducts Two-step process requires two enzymes Superoxide ion is converted into hydrogen peroxide by superoxide dismutase Hydrogen peroxide is converted into harmless water and oxygen by catalase

33 Carbon Dioxide Capnophiles: organisms that grow best at a higher CO 2 tension than is normally present in the atmosphere Important in the initial isolation of the following organisms from clinical specimens - Neisseria(gonorrhea, meningitis) - Brucella (undulant fever) - Streptococcus pneumoniae

34 Environmental Factor: Osmotic Pressure Osmophiles: live in habitats with high solute concentration Halophiles: prefer high concentration of salt - Obligate halophiles Halobacterium and Halococcusgrow optimally at solutions of 25% NaCl but require at least 9% NaCl - Facultative halophiles: remarkably resistant to salt, even though they do not normally reside in high salt environments - Staphylococcus aureus can grow on NaCl media ranging from 0.1% to 20%

35 Associations Between Organisms Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Associations Between Organisms Symbiotic Organisms live in close nutritional relationships; required by one or both members. Non symbiotic Organisms are free-living; relationships not required for survival. Mutualism Obligatory, dependent; both members benefit. Commensalism The commensal benefits; other member not harmed. Parasitism Parasite is dependent and benefits; host harmed. Synergism Members cooperate and share nutrients. Antagonism Some members are inhibited or destroyed by others.

36 Strong Partnerships: Symbioses Symbiosis: a general term to denote a situation in which two organisms live together in a close partnership - symbionts: members of a symbiosis Three main types of symbiosis occur - Mutualism: organisms live in an obligatory but mutually beneficial relationship - Commensalism: the partner called the commensal receives benefits, while its partner is neither harmed nor benefitted - Parasitism: a relationship in which the host organism provides the parasitic microbe with nutrients and a habitat; parasite usually harms the host to some extent

37 Associations but Not Partnerships: Antagonism and Synergism Antagonism: an association between free-living species that arises when members of a community compete Synergism: - an interrelationship between two organisms that benefits them but is not necessary for survival

38 Biofilms: The Epitome of Synergy Biofilms - mixed communities of bacteria and other microbes that are attached to a surface and each other - form a multilayer conglomerate of cells and intracellular material

39 Steps in the Formation of a Biofilm Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 Pioneer bacteria colonize a surface. 2 Pioneers secrete extracellular material that helps keep them on the surface and serves as attachment point for later colonizers. Quorum sensing chemicals (red dots) are released by bacteria. Bacteria Gauze fiber Extracellular matrix 3 In many (but not all) biofilms, other species join and may contribute to the extracellular matrix and/or participate in quorum sensing with their own chemicals or the ones released by other species. 4 Biofilms serve as a constant source of bacteria that can escape and become free-living again. Courtesy of Ellen Swogger and Garth James, Center for BiofilmEngineering, Montana State University

40 Biofilms: The Epitome of Synergy (cont d) Quorum sensing: used by bacteria to interact with members of the same species as well as members of other species that are close by Structure of the biofilm - large, complex communities form with different physical and biological characteristics - the bottom may have very different ph and oxygen conditions than the surface - partnership among multiple microbial inhabitants - cannot be eradicated by traditional methods

41 The Study of Bacterial Growth Binary fission - one cell becomes two - parent cell enlarges - duplicates its chromosome - starts to pull its cell envelope together to the center of the cell - cell wall eventually forms a complete central septum

42 Miremos en detalle!!

43 How do microorganisms grow?

44 The Population Growth Curve In closed systems or batch cultures, numerous factors prevent cells from continuously dividing at their maximum rate Growth curve:a predicable pattern of a bacterial population growth in a closed system can be measured by

45 Crecimiento Poblacional Bacteriano El ciclo de la curva de crecimiento Fase Lag: periodo de aclimatación a condiciones de crecimiento, síntesis de RNA, duplicación DNA Fase Estacionaria: se agotan nutrientes, se acumulan desperdicios, procesos de división celular y muerte están en balance Fase Exponencial: número de células se duplica a intervalos regulares de tiempo, ocurre bajo condiciones ideales de crecimiento (Ej..abundancia de nutrientes) Fase de Muerte: las condiciones prevalecientes no pueden sostener más crecimiento y las células mueren

46 Crecimiento Poblacional Bacteriano crecimiento exponencial: número de células se duplica a intervalos regulares de tiempo datos de una población que se duplica cada 30 minutos

47 Analyzing Population Size Without Culturing Turbidity/turbidometry - a clear nutrient solution becomes turbidor cloudy as microbes grow in it - the greater the turbidity, the larger the population size Counting - direct cell count: measured microscopically - Coulter counter: electronically scans a fluid as it passes through a tiny pipette - flow cytometer: works similarly to a Coulter counter, but can measure cell size and differentiate between live and dead cells

48 Medidas de Crecimiento Microbiano Enumeración Indirecta: turbidez/espectrofotómetro 600nm incident light detected light se miden lecturas de absorbancia a 600nm absorbancia a 600nm aumenta según aumenta el # de células

49 Steps in a Viable Plate Count Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 500 ml inoculated flask Equally spaced time intervals 60 min 120 min 180 min 240 min 300 min 360 min 420 min 480 min 540 min 0.1 ml sample added to tube 600 min Sample is diluted in liquid agar medium and poured or spread over surface of solidified medium Plates are incubated, colonies are counted None Number of colonies (CFU) per 0.1 ml <1* Total estimated cell population in flask <5,000 10,000 20,000 35,000 65, , , , ,000 1,150,000 *Only means that too few cells are present to be assayed.

50 The Rate of Population Growth Generation timeordoubling time: the time required for a complete fission cycle, from parent cell to two daughter cells - generation:increases the population by a factor of two - as long as the environment remains favorable, the doubling effect can continue at a constant rate

51 The Rate of Population Growth (cont d) The length of the generation time is a measure of the growth rate of an organism - average generation time is minutes - shortest generation times can be minutes - Mycobacterium lepraehas a generation time of days - environmental bacteria have generation times measured in months - most pathogens have relatively short generation times

52 Medidas de Crecimiento Microbiano Enumeración Indirecta: turbidez/espectrofotómetro turbidez se mide a intervalos regulares log Ab bsorbance nm se determina el número de células asociado a cada lectura de absorbancia (curva estándar) minutes log bacteria al numbers/ml Direct count

53 Matemática de crecimiento microbiano (una vez se obtiene crecimiento exponencial) g (tiempo de generación): = t(a f )-t(a i ) =30 min k= velocidad de crecimiento, número de generaciones por unidad de tiempo k = (logn f log N o ) / t log Absorban nce nm log bacte erial numbers/ml N o =número de células inicial N f = No=número de células final min

54 Can we prevent the death phase? How?