Microbial Ecology, Nutrition, and Growth PDF

Elements of Microbial Nutrition, Ecology, and Growth Topics – Microbial Nutrition – Environmental Factors – Microbial Growth 2  Microbial Nutrition Chemical analysis Sources of essential nutrients Transport mechanisms...

Elements of Microbial Nutrition, Ecology, and Growth Topics – Microbial Nutrition – Environmental Factors – Microbial Growth 2  Microbial Nutrition Chemical analysis Sources of essential nutrients Transport mechanisms 3  Microbial nutrition Macronutrients – required in large quantities; play principal roles in cell structure & metabolism – proteins, carbohydrates Micronutrients or trace elements – required in small amounts; involved in enzyme function & maintenance of protein structure – manganese, zinc, nickel 4  Nutrients Inorganic nutrients– atom or molecule that contains a combination of atoms other than carbon and hydrogen – metals and their salts (magnesium sulfate, ferric nitrate, sodium phosphate), gases (oxygen, carbon dioxide) and water Organic nutrients- contain carbon and hydrogen atoms and are usually the products of living things – methane (CH4), carbohydrates, lipids, proteins, and nucleic acids 5  Chemical composition of cytoplasm 70% water proteins 96% of cell is composed of 6 elements – Carbon – Hydrogen – Oxygen – Nitrogen – Phosphorous – Sulfur 6  Obtaining Carbon Heterotroph – an organism that must obtain carbon in an organic form made by other living organisms such as proteins, carbohydrates, lipids and nucleic acids Autotroph - an organism that uses CO2, an inorganic gas as its carbon source – not dependent on other living things 7  Nitrogen Main reservoir is nitrogen gas (N2) 79% of earth’s atmosphere is N2 Nitrogen is part of the structure of proteins, DNA, RNA & ATP – these are the primary source of N for heterotrophs Some bacteria & algae use inorganic N nutrients (NO3-, NO2-, or NH3) Some bacteria can fix N2 Regardless of how N enters the cell, it must be converted to NH3, the only form that can be combined with carbon to synthesize amino acids, etc. 8  Oxygen major component of carbohydrates, lipids and proteins plays an important role in structural & enzymatic functions of cell component of inorganic salts (sulfates, phosphates, nitrates) & water O2 makes up 20% of atmosphere essential to metabolism of many organisms 9  Hydrogen major element in all organic compounds & several inorganic ones (water, salts & gases) gases are produced & used by microbes roles of hydrogen – maintaining pH – forming H bonds between molecules – serving as the source of free energy in oxidation-reduction reactions of respiration10  Phosphorous main inorganic source is phosphate (PO4-3) derived from phosphoric acid (H3PO4) found in rocks & oceanic mineral deposits key component of nucleic acids, essential to genetics serves in energy transfers (ATP) 11  Sulfur widely distributed in environment, rocks, sediments contain sulfate, sulfides, hydrogen sulfide gas and sulfur essential component of some vitamins and the amino acids: methionine & cysteine contributes to stability of proteins by forming disulfide bonds 12  Important mineral ions Potassium Sodium Calcium Magnesium Iron 13 Bacteria are composed of different elements and molecules, with water (70%) and proteins (15%) being the most abundant. Analysis of the chemical composition of an E. coli cell. 14 Sources of essential nutrients Required for metabolism and growth – Carbon source – Energy source 15  Carbon source Heterotroph (depends on other life forms) – Organic molecules – Ex. Sugars, proteins, lipids Autotroph (self-feeders) – Inorganic molecules – Ex. CO2 16  Growth factors Essential organic nutrients Not synthesized by the microbe, and must be supplemented Ex. Amino acids, vitamins 17  Energy source Chemoheterotrophs Photoautotrophs Chemoautotrophs 18  Chemoheterotrophs Derive both carbon and energy from organic compounds – Saprobic decomposers of plant litter, animal matter, and dead microbes – Parasitic Live in or on the body of a host 19 Representation of a saprobe and its mode of action. 20 Extracellular digestion in a saprobe with a cell wall.  Photoautotroph Derive their energy from sunlight Transform light rays into chemical energy Primary producers of organic matter for heterotrophs Primary producers of oxygen Ex. Algae, plants, some bacteria 21  Two types of autotrophs – Chemoorganic autotroph Derives their energy from organic compounds and their carbon source from inorganic compounds – Lithoautotrophs Neither sunlight nor organics used, rather it relies totally on inorganics 22 Methanogens are an example of a chemoautotroph. Methanococcus jannaschii, (motile, inhabits hot vents in the seafloor, uses H gas as E source SEM of a small colony of Methanosarcina Methane-producing archaea 23 Summary of the different nutritional categories based on carbon and energy source. 24  Transport mechanisms Passive transport –do not require energy, substances exist in a gradient and move from areas of higher concentration towards areas of lower concentration – Osmosis - water – Diffusion – Facilitated diffusion – requires a carrier Active transport – require energy and carrier proteins, gradient independent – Carrier-mediated active transport – Group translocation – transported molecule chemically altered – Bulk transport – endocytosis, exocytosis, 25 pinocytosis  Osmosis Diffusion of water through a permeable but selective membrane Water moves toward the higher solute concentrated areas – Isotonic- solute concn of the envt= solute concn of the cell’s internal envt (cytoplasm) ----same rate of water movt == no net charge in cell volume – Most stable – Hypotonic- lower solute concn in the external envt than the cell’s internal envt – Pure water ---most hypotonic envt for cells bec it has no solute – Net direction: fr. Hypotonic soln into the cell ----- if no CW, it will swell and burst – Hypertonic- higher solute concn in the external envt than the cell’s cytoplasm – This will force the water to diffuse out of the cell = high osmotic pressure/potential (growth-limiting effect ---- method of preservation) 26 Representation of the osmosis process (the diffusion of water through a selectively permeable membrane). 27 Cells with- and without cell walls, and their responses to different osmotic conditions (isotonic, hypotonic, hypertonic). 28  Diffusion Net movement of molecules from a high concentrated area to a low concentrated area No energy is expended (passive) Concentration gradient and permeability affect movement 29 A cube of sugar will diffuse from a concentrated area into a more dilute region, until an equilibrium is reached. 30 Diffusion of molecules in aqueous solutions  Facilitated diffusion Transport of polar molecules and ions across the membrane No energy is expended (passive) Carrier protein facilitates the binding and transport – Specificity – bind and transport only a single type of molecule – Saturation – rate of transport is limited by the no. of binding sites of TP – Competition – 2 molecules of similar shape can bind to the same binding site on a CP 31 Representation of the facilitated diffusion process. (Conformational change in the protein) Facilitated diffusion – involves attachment of a molecule to a specific protein-carrier 32  Active transport Transport of molecules against a gradient Requires energy (active) Ex. Permeases and protein pumps transport sugars, amino acids, organic acids, phosphates and metal ions. Ex. Group translocation transports and modifies specific sugars 33  Endocytosis Substances are taken, but are not transported through the membrane. Requires energy (active) Common for eukaryotes Ex. Phagocytosis, pinocytosis 34 Example of the carrier-mediated active transport (permease). The membrane proteins (permease) have attachment sites for esssential nutrient molecules. As these molecules bind to the permease, they are pumped into the cell’s interior through special membrane protein channels. Microbes have these systems for transporting various ions. 35 Active transport Example of group translocation. In this process, the molecule is actively captured, but along the route of transport, it is chemically altered. By coupling transport with synthesis, the cell conserves energy. 36 Example of endocytosis processes. Solid particles are phagocytosed by large cell extensions (pseudopods) and fluids, and/or dissolved substances are pinocytosed into vesicles by very fine cell protrusions (microvilli). Oil droplets fuse with the membrane and are released directly into the cell. 37  38 Brownian movt --- natural tendency of an atom to be in constant random motion  Environmental Factors Temperature Gas pH Osmotic pressure Other factors Microbial association 39  Temperature For optimal growth and metabolism Psychrophile – 0 to 15 °C Mesophile- 20 to 40 °C Thermophile- 45 to 80 °C 40 Growth and metabolism of different ecological groups based on ideal temperatures. Cardinal temp range depends on habitats: typhus rickettsia = 32 – 38; rhinovirus = 33-35; S. aureus = 6-46 E. faecalis = 0 - 44 41 42  Five groups based on optimum growth temperature 1.Psychrophiles: cold-loving 2.Psychrotrophs: Grow between 0 C and 20 to 30 C; Cause food spoilage 3.Mesophiles: moderate-temperature-loving 4.Thermophiles: Optimum growth temperature of 50 to 60 C; Found in hot springs and organic compost 5.Hyperthermophiles: Optimum growth temperature >80 C Effect of amount of food on its cooling rate Example of a psychrophilic photosynthetic Red snow organism. Early summer snowbank provides a perfect habitat for psychrophilic, photosynthetic organisms like Chlamydomonas nivalis 46  Addtl terms Psychrotrophs (facultative psychrophiles) – grow slowly in cold but have an opt. temp above 20°C ---ex. L. monocytogenes and S. aureus Thermoduric microbes – survives short exposure to high temp but are normally mesophiles --- contaminants of heated or pasteurized foods (ex. Giardia, Bacillus and Clostridium 47  Gas Two gases that most influence microbial growth – Oxygen Respiration Oxidizing agent – Carbon dioxide 48  Oxidizing agent Oxygen metabolites are toxic These toxic metabolites must be neutralized for growth Three categories of bacteria – Obligate aerobe – Facultative anaerobe – Obligate anaerobe 49  Obligate aerobe Requires oxygen for metabolism Possess enzymes that can neutralize the toxic oxygen metabolites – Superoxide dismutase and catalase Ex. Most fungi, protozoa, and bacteria (Micrococcus and Bacillus) 50 Toxic Forms of Oxygen Singlet oxygen: Oxygen boosted to a higher-energy state and is reactive Superoxide free radicals: O2– Peroxide anion: O22– Hydroxyl radical (OH)  Facultative anaerobe Does not require oxygen for metabolism, but can grow in its presence During minus oxygen states, anaerobic respiration or fermentation occurs Possess superoxide dismutase and catalase Ex. Gram negative pathogens (intestinal bact and staphylococci) 52  Obligate anaerobes Cannot use oxygen for metabolism Do not possess superoxide dismutase and catalase The presence of oxygen is toxic to the cell 53 Anaerobes must grow in an oxygen-minus environment, because toxic oxygen metabolites cannot be neutralized. Anaerobic or CO2 indicator system 54 Culturing technique for anaerobes Thioglycollate broth enables the identification of aerobes, facultative anaerobes, and obligate anaerobes. P. aeruginosa S. aureus E. coli C. butyricum Use of thioglycollate broth to demonstrate oxygen requirements. 56 Oxygen requirements 57 The Effect of Oxygen on the Growth of Various Types of Bacteria  pH Cells grow best between pH 6-8 (6.5 and 7.5: Neutrophils Exceptions would be acidophiles (pH 0), and alkalinophiles (pH 10). Some bacteria are very tolerant of acidity or thrive in it: Acidophiles(preferred pH range 1 to 5) Molds and yeasts grow best between pH 5 and 6 Euglena mutabilis – grows in acid pools with a pH bet 0-1.0 Thermoplasma – lives in hot, coal piles at a pH of 1-2 Molds – tolerates moderate acid --- common spoilage of pickled foods Proteus spp. – pH10 59  Osmotic pressure Hypertonic environments (increased salt or sugar; higher in solutes than inside the cell ) cause plasmolysis due to high osmotic pressure Obligate (extreme) halophiles vs. facultative halophiles Halophiles Requires high salt concentrations (high osmotic pressure Withstands hypertonic conditions Obligates: opt = 25%, 9% min. Ex. Halobacterium and Halococcus Facultative halophiles (osmotolerant= tolerate high osmotic pressure) – Can survive high salt conditions but is not required; 0.1%- 20% – Ex. Staphylococcus aureus 60  Other factors Radiation- UV, x-rays, cosmic rays Barophiles – withstand high pressures Spores and cysts- can survive dry habitats/ extreme drying --- enzymes are inactivated 62  Ecological association Influence microorganisms have on other microbes – Symbiotic relationship – Non-symbiotic relationship 63  Symbiotic Organisms that live in close nutritional relationship Types – Mutualism – both organism benefit ex. Termite & protozoa; bacteria and protozoa (in ruminants) – Commensalism – one organisms benefits – Parasitism – host/microbe relationship 64 An example of commensalism, where Staphylococcus aureus provides vitamins and amino acids to Haemophilus influenzae. Satellitism, a type of 65 commensalism  Non-symbiotic Organisms are free-living, and do not rely on each other for survival Types – Synergism – shared metabolism ---soil bacteria(provide the plant with minerals) and plant roots(provides growth factors) – members cooperate and share nutrients – Antagonism- competition between microorganisms ---- production of antibiotics (e.g. bacteriocin) – some members are inhibited or destroyed by others 66  Biofilms ✓ Microbial communities form slime or hydrogels ✓ Starts via attachment of planktonic bacterium to surface structure. ✓ Bacteria communicate by chemicals via quorum sensing ✓ Share nutrients; Sheltered from harmful factors (disinfectants etc.) ✓ Cause of most nosocomial infections Clinical Focus: Delayed Bloodstream Infection Following Catheterization  Microbial Associations Non-symbiotic Symbiotic Orgs.are free- Orgs. live in close living; nutritional relationships relationship are not required for survival mutualism commensalism parasitism synergism antagonism 69  Interrelationships between microbes and humans Human body = rich habitat for symbiotic bacteria (e.g. skin and alimentary tract) Can be commensal, parasitic, and synergistic Ex. E. coli in the intestine Lactobacillus in vagina 70  Microbial Growth “Growth” = the acquisition of biomass leading to cell division, or reproduction Reqmnts: nutrients and proper environmental factors 2 levels: 1)synthesis of new cell components and inc. in size, 2) inc. in the no. of cells 71  Microbial Growth Binary fission Generation time Growth curve Enumeration of bacteria 72  Binary fission The division of a bacterial cell Parental cell enlarges and duplicates its DNA Septum formation divides the cell into two separate chambers Complete division results in two identical cells 73 Representation of the steps in binary fission of a rod-shaped bacterium. 74  Binary Division 1 to 2 to 4 to 8 to ?  Generation time The time required for a complete division cycle (= doubling time) Length of the generation time is a measure of the growth rate Exponentials are used to define the numbers of bacteria after growth vary markedly with the species of microorganism and environmental conditions; --- can range from 10 minutes for a few bacteria to several days with some eucaryotic microorganisms; (20 min (E. coli) to > 24h (M. tuberculosis) 76  Mean Generation Time and Growth Rate The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes; time required for cell to divide 1 Growth rate (min-1) = mean generation time Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation Mean Generation Time and Growth Rate Representation of how a single bacterium doubles after a complete division, and how this can be plotted using exponentials. 79 The mathematics of population growth Bacterial Growth Curve: Arithmetic vs. Exponential Plotting  Growth curve Shows population growth of the culture Illustrates the dynamics of growth The standard bacterial growth curve describes various stages of growth a pure culture of bacteria will go through, beginning with the addition of cells to sterile media and ending with the death of all of the cells present. Usually analyzed in a closed system called a batch culture; plotted as the logarithm of cell number versus the incubation time. 83  Phases of Growth (in growth curve) Lag phase Log phase Stationary phase Death phase 84  Lag phase Cells are adjusting, enlarging, and synthesizing critical proteins and metabolites Not doubling at their maximum growth rate “flat” period of adjustment, enlargement; little growth associated with a physiological adaptation to the new environment varies considerably in length depending upon the condition of the microorganisms and the nature of the medium 85  Log phase Maximum exponential growth rate of cell division Adequate nutrients Favorable environment === active growth 86  Stationary phase Survival mode – depletion in nutrients, released waste can inhibit growth When the number of cells that stop dividing equal the number of cells that continue to divide ------ rate of cell growth equals rate of cell death cause by depleted nutrients & O2, excretion of organic acids & pollutants ==Cell death may result from Nutrient limitation & Toxic waste accumulation (e.g. acid buildup from fermentation); as well as O2 depletion, critical population level reached 87  Death phase A majority of cells begin to die exponentially due to lack of nutrients A chemostat will provide a continuous supply of nutrients, thereby the death phase is never achieved. as limiting factors intensify, cells die exponentially in their own wastes 88 The four main phases of growth in a bacterial culture. The growth curve in a bacterial culture. 89  Measurement of bacterial growth Turbidity Direct cell count Automated devices – Coulter counter – Flow cytometer – Real-time PCR 90 1)Turbidity = Absorbance method Based on the diffraction or “scattering” of light by bacteria in a broth culture Light scattering is measured as optical absorbance in a spectrophotometer Optical absorbance is directly proportional to the concentration of bacteria in the suspension = Light scattered is proportional to number of cells. Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm. 91 1)Turbidity = Absorbance method Accurate measure of cells when concentration not too high. Easy and quick to measure (can measure a sample in less than a minute). Measurement of cell mass may be used to approximate the number of microorganisms if a suitable parameter proportional to the number of microorganisms present is used. ---- Suitable parameters may be dry weight, light scattering in liquid solutions, or biochemical determinations of specific cellular constituents such as protein, DNA, or ATP 92 The greater the turbidity, the larger the population size. 93 Turbidity measurements as indicators of growth  Direct cell count method Microscopic cell counts – Calibrated “Petroff-Hausser counting chamber,” similar to hemacytometer, w/c can also be used – Generally very difficult for bacteria since cells tend to move in and out of counting field – Can be useful for organisms that cannot be cultured – Special stains (e.g. serological stains or stains for viable cells) can be used for specific purposes  Direct cell count method Microscopic cell counts may be accomplished by direct microscopic observation on specially etched slides (such as Petroff-Hausser chambers or hemocytometers) or by using electronic counters like Coulter Counters, count microorganisms as they flow through a small hole or orifice. Direct cell count methods do not distinguish between living and dead cells. The direct cell method counts the total dead and live cells in a special microscopic slide containing a premeasured grid. Direct microscopic count of bacteria. 97 A Coulter counter uses an electronic sensor to detect and count the number of cells. 98 Coulter counter  Direct cell count method Serial dilution and colony counting – Also know as “viable cell counts” – Concentrated samples are diluted by serial dilution – involve plating diluted samples (using a pour plate or spread plate) onto suitable growth media and monitoring colony formation; this type of method counts only those cells that are reproductively active 99  Direct cell count method Serial dilution and colony counting – typically carried out by Colony Forming Units (CFU) assay. 100 Measurement of Microbial Growth Serial dilution (cont.) – Diluted samples are spread onto media in petri dishes and incubated – Colonies are counted. The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual). – A simple calculation, with a single plate falling into the statistically valid range, is given below: CFU # colonies counted in original sample = ml (dilution factor)(volume plated, in ml)  Measurement of Microbial Growth Serial dilution (cont.) – If there is more than one plate in the statistically valid range of 25 – 250 colonies, the viable cell count is determined by the following formula:  Measurement of Microbial Growth CFU =  C ml [(1* n1) + (0.1* n 2) +...] * d1 * V Where: C = Sum of all colonies on all plates between 25 - 250 n1= number of plates counted at dilution 1 (least diluted plate counted) n2= number of plates counted at dilution 2 (dilution 2 = 0.1 of dilution 1) d1= dilution factor of dilution 1 V= Volume plated per plate Standard Plate Count 105  Measuring Growth MPN (Most Probable Number) – Put 10, 1, and 0.1 ml into 10-mls broth Repeat 5 times per volume – Statistical accurate sampling – Public Health Standards are written for MPN Chapter 6 Other Measurements of Microbial Growth Membrane filtration Used for samples with low microbial count/concentration A measured volume (usually 1 to 100 ml) of sample is filtered through a membrane filter (typically with a 0.45 μm pore size) The filter is placed on a nutrient agar medium and incubated Colonies grow on the filter and can be counted Standards for Public Health: 0 E.coli / 100 ml of water Direct microscopic count: Counting chambers (slides) for microscope Measurement of Microbial Growth Mass determination – Cells are removed from a broth culture by centrifugation and weighed to determine the “wet mass.” – The cells can be dried out and weighed to determine the “dry mass.” Measurement of enzymatic activity or other cell components Measuring Microbial Growth Direct Methods Plate counts Filtration MPN Direct microscopic count Indirect Methods Turbidity Metabolic activity Dry weight