Chapter4 Microbial growth Environmental factors on growth Growth in natural environment Effect of environmental factors on growth Water availability, pH, temperature, oxygen concentration, pressure, radiation, and a number of other environmental factors influence microbial growth. Yet many microorganisms, and particularly bacteria, have managed to adapt and flourish under environmental extremes that would destroy most organisms. Effect of temperature on bacterial growth rate Bacteria grow over a range of temperatures; they do not reproduce below the minimum growth temperature nor above the maximum growth temperature. Within the temperature growth range there is an optimum growth temperature at which bacterial reproduction is fastest. Microorganisms are classified as psychrophiles, mesophiles. thermophiles, and extremethemophiles based on their optimal growth temperature. Temperature effect: 1. Psychrophile (0-20oC) 2. Mesophile (45-15oC) 3. Thermophile (40-70oC) 4. Hyperthermophile (65-95oC) 5. Extrem hyperthermophile (80-120oC) Temperature • A most important factor influencing the growth is the temperature sensitivity of enzyme catalyzed reactions. • High temperature: 1. Denature enzymes, transport carries, and other proteins. 2. Microbial membranes are also disrupted by temperature extremes. 3. The lipid bilayer melts and disintegrates. Optimal growth temperature • Mesophiles: – human body temperature * pathogens * opportunists • pyschrophile – close to freezing • thermophile – close to boiling Thermophile and Biotechnology • Enzymes from thermophiles are capable of catalyzing biochemical reactions at high temperature, they are more stable; • DNA polymerase, Taq polymerase, has been isolated from thermophile-Thermus aquaticus, for PCR application; • DNA polymerase, pfu polymerase, isolated from hyperthermophile-Pyrocuccus furiosus, is more stable and useful for PCR application, it also less prone to errors; • Microbial process carried out at high T also eliminate or greatly reduce cooling costs. Molecular basis for growing at extreme temperatures • Molecular adaptations of enzymes to extreme temperatures – Cold-active enzymes are more flexible due to higher portion of a helix and less amount of bsheet – Heat resistant enzymes have different folding patterns because of a few different amino acids in some critical positions from their mesophile counterpart, more salt bridges and more densely packed hydrophobic interior A-helix provides flexibility B-sheet causes more rigidity More b-sheets result in a more rigid structure, which is less cold active More a helixes result in more flexible structure, which is cold resistant Cell membrane must be in a fluidal state in order to be functional Hyperthermophiles have cell membrane as lipid monolayer Bacteria Archaea pH • Many grow best at neutral pH • Some can survive/grow: acid or alkali • Commonly: bacteria and protozoa: neutrophiles algae and fungi: slight acidity. actinomycetes: slight alkaline condition pH effect on cell growth • Organisms that live at low pH are called acidophiles • Fungi tend to be more acid-tolerant than bacteria although their interior pH is close to neutrality • Several bacteria are obligate acidophilic (such as Thiobacillus, Sulfolobus, Thermoplasma) • A few organisms are alkalophilic (pH 10-11), usually found in highly basic habitats such as soda lakes and high carbonate soils. • Microorganisms frequently change the pH of their own habitat by producing acidic or basic metabolic waste products. • Buffers in media: 1. H2PO4-/HPO422. CaCO3 3. Peptides and amino acids Solutes and water activity • Compatible solutes: solutes that are compatible with metabolism and growth when at high intracellular concentrations. • How do microorganisms adapt to hypotonic(低渗) and hypertonic(高渗) environments? 1. The synthesis or uptake of choline(VB复合体), betaine(甜菜碱), proline, glutamic acid, and other amino acids. 2. Elevated levels of potassium ions. 3. Algae and fungi employ sucrose and polyols(多 羟复合物). 4. Protoza use contractile vacuoles. Water activity (aw) • Express quantitatively the degree of water availability of the solution. • P---vapor pressure aw = P solution / P water • aw is inversely related to osmotic pressure; if a solution has high osmotic pressure, its aw is low. Water activity The water activity of a solution is 1/100 the relative humidity of the solution (when expressed as a percent), or it is equivalent to the ratio of the solution's vapor pressure to that of pure water. Approximate lower aw limits for microbial growth: 0.90 – 1.00 for most bacteria, most algae and some fungi as Basidiomycetes, Mucor, Rhizopus. 0.75 for Halobacterium, Aspergillus… 0.60 for some Saccharomyces species Plasmolysis If the concentration of solutes, such as sodium chloride, is higher in the surrounding medium (hypertonic), then water tends to leave the cell. The cell membrane shrinks away from the cell wall (an action called plasmolysis), and cell growth is inhibited. Normal cell Plasmolyzed cell • osmotolerant: grow over wide ranges of water activity or osmotic concentration. • Microorganisms that have a specific requirement for sodium ion are often called halophiles • Organisms able to live in environment high in sugar are called osmophiles • Organisms capable of living in very salty environments are called extreme halophiles • Organisms able to live in very dry environments are called xerophiles Mechanism of Halophiles 1. Modified the structure of their proteins and membranes rather that simply increasing the intracellular concentrations of solutes. 2. Accumulate enormous quantities of potassium for stability and activity of the enzymes, ribosomes and transport proteins. Effects of Oxygen on Cell Growth Terms used to describe O2 relationships of microorganisms: • Aerobes: – Obligate aerobes: oxygen presence necessary – Facultative aerobes: oxygen not necessary, but better with it – Microaerophilic aerobes: required O2 at low level than atmospheric • Anaerobes: – Aerotolerant: O2 not required, and growth no better if O2 present – Obligate (strict) anaerobes: Oxygen harmful or lethal 1. Obligate aerobes • grow in presence of oxygen; • no fermentation; • oxidative phosphorylation 2. Obligate anaerobes • • • • no oxidative phosphorylation fermentation killed by oxygen lack certain enzymes: superoxide dismutase: O2-+2H+ to H2O2 catalase: H2O2 to H20 + O2 peroxidase: H2O2 to H20 /NAD to NADH) 3. Aerotolerant anaerobes • respire anaerobically • not killed by oxygen 4. Facultative anaerobes • fermentation • aerobic respiration • survive in oxygen 5. Microaerophilic bacteria • Grow low oxygen • Killed high oxygen Aerobic, anaerobic, facultative, microaerophilic and aerotolerant anaerobe growth A small amount of agar has been added to keep the liquid from becoming disturbed and the redox dye, resazurin, which is pink when oxidized and colorless when reduced, is added as a redox indicator. The above results were obtained after a reducing agent such as thioglycolate (巯基醋酸盐) was added to the medium. Resazurin (刃天青)，a redox indicator dye, can be used to detect oxygen in the medium. Different features (a) Obligate aerobes-growth occurs only in the short distance to which the oxygen diffuses into the medium. (b) Facultative anaerobes growth is best near the surface, where oxygen is available, but occurs throughout the tube. (c) Obligate anaerobes-oxygen is toxic, and there is no growth near the surface. (d) Aerotolerant anaerobes-growth occurs evenly throughout the tube but is not better at the surface because the organisms do not use oxygen. (e) Microaerophiles, aerobic organisms that do not tolerate atmospheric concentrations of oxygen-growth occurs only in a narrow band of optimal oxygen concentration. How to culture • Aerobic: culture vessel is shaken to aerate the medium or sterile air must be pumped through the culture vessel. • Anaerobes: 1. Media contain reducing agents such as thioglycollate or crysteine . Boiling 2. Oxygen also may be eliminated from an anaerobic system by removed air with a vacuum pump and flushing out residual O2 with nitrogen gas. 3. GasPak jar: by using hydrogen and a palladium(钯) catalyst to remove O2 through the formatin of water. 4. Plastic bags or pouchs: only a few samples pouch: catalyst + calcium carbonateCO2 Toxic Forms of Oxygen protein inactivation • Singlet oxygen • Superoxide anion (O2 ) • Hydrogen peroxide (H2O2) . • Hydroxyl radical (OH ) Enzymes that destroy toxic oxygen (a) H2O2+H2O2 (b) Catalase 过氧化氢酶 H2O2+NADH+H+ (c) O2-+O2-+2H+ 2H2O+O2 Peroxidase 过氧化氢酶 2H2O+NAD+ Superoxide dismutase 过氧化氢歧化酶 H2O2 +O2 (d) 4O2-+4H+ Catalase+Superoxide dismutase 2H2O +3O2 How do you know a microbial cell contains catalase? CATALASE TEST Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or superoxide. Both of these molecules are toxic to bacteria. Some bacteria, however, possess a defense mechanism which can minimize the harm done by the two compounds. These resistant bacteria use two enzymes to catalyze the conversion of hydrogen peroxide and superoxide back into diatomic oxygen and water. One of these enzymes is catalase and its presence can be detected by a simple test. The catalase test involves adding hydrogen peroxide to a culture sample or agar slant. If the bacteria in question produce catalase, they will convert the hydrogen peroxide and oxygen gas will be evolved. The evolution of gas causes bubbles to form and is indicative of a positive test. • Pressure (deep sea): nutrient recycling barotolerant barophilic • Radiation: sunlight: visible light: photosynthetic uv radiation: 260nm TT dimer Infrared rays: heat Radio waves: ionizing: X-ray, gamma rays, spore mutation Microbial Growth in natural enviroments • Viable but nonculturable vegetative procaryotes • Only 1-10% of observable cells are able to form colonies. • PCR • Small subunit ribosomal RNA analysis • Sensitive microscopic and isotopic procedures Quorum sensing and microbial populations • Quorum sensing: a phenomenon in which bacteria monitor their own population density through sensing the levels of signal molecules, sometimes called autoinducers because they can stimulate the cells that releases them. • The bacteria reach a high population density before they release enzymes. This is an advantage within a host’s body as well as in the soil or an aquatic habitat. eg. A pathogen G- figure 6.19 P133 G+: an oligopeptide signal • competence induction in Streptococcus pneumoniae • stimulation of sporulation by Bacillus subtilis. Others: • promote the formation of nature biofilms • two different bacteria might stimulate each other by releasing similar signals Questions •What environmental factors affect growth of microorganisms? •How to name microorganisms that grow best at a temperature? •How do you name microorganisms having various O2 tolerance? •What enzymes are involved in detoxifying toxic active oxygen? • Quorum sensing and microbial populations?