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Transcript
CEE 160L: Introduction to Environmental Engineering and Science Lectures 3 and 4 – Environmental Microbiology Microbiology tutorial • http://classroom.sdmesa.edu/eschmid/Lectur e4-Microbio.htm Outline today’s lecture Different Kinds of Microbes Cell Structure Observing Microbial Growth Different types of microscopy Quantifying Microbial Growth Different Classes of Microbes • • • • • Bacteria Archae Fungi Protists Viruses (NOT MICROBES) Mammalian cells =Bacteria + Archaea Bacteria Bacteria fun facts • Inhabit soil, water, acidic hot springs, radioactive waste, and the deep portions of Earth's crust. • Can live off sunlight and most inorganic and organic substrates • Biomass exceeds that of all plants and animals • Vital in recycling nutrients (e.g nitrogen from the atmosphere) • Important in sewage treatment and the breakdown of oil spills, the production of cheese and yogurt through fermentation, antibiotics manufacture Not fun bacteria facts • People have ~10x more bacterial cells than human cells • Widespread use of antiobiotics has caused bacteria to develop resistant strains • Tuberculosis alone killing about 2 million people a year, mostly in sub-Saharan Africa Deinococcus radiodurans is the most extreme of extremophiles Nicknamed Conan the Bacterium D radiodurans fun facts • First half of name is Greek for “terrible berry” the second half is Latin for “radiation surviving” • Able to withstand DNA damaging radiation does of 5000Gy (exposing 100 people to this does would kill ~50) • Resistance from having multiple copies of its genome and rapid DNA repair mechanisms • Been genetically engineered for use in bioremediation to consume and digest solvents and heavy metals, even in a highly radioactive site 20000mSV= 20Sv=20Gy Sterilization dose: 25kGy Archae “WEIRD BACTERIA” Archaea fun facts • Visually similar to bacteria, but possess genes and several metabolic pathways and structures that are more common to eukaryotes • Widespread like bacteria (including in/on humans). Many in extreme environments. • Important to Earth’s life cycle and human digestion • Do not seem to be pathogenic • Used in biogas production and sewage treatment • Some have very strange shapes Fungi Fungi fun/un-fun facts • • • • Eukaryotes Yeasts. molds, mushrooms Important in decomposition of organic matter Production of antibiotics, and, more recently, various enzymes produced by fungi are used industrially and in detergents • Cause crop losses and food spoilage (matooke blight) • Inconspicuous as filaments except when fruiting (mushrooms, mold) • Can be very toxic (mushroom misidentification) Protists Protist fun facts • Unicellular or multicellular organism without specialized tissues found in plants • Often classified by appearance: "animal-like" protozoa, the "plant-like" protophyta (mostly unicellular algae), and the "fungus-like" slime molds and water molds • Many are photosynthetic and are vital primary producers in ecosystems: algae and plankton • Pathogenic species such as the kinetoplastid Trypanosoma brucei, which causes sleeping sickness and species of the apicomplexan Plasmodium which cause malaria Viruses Virus un-fun facts • Virus is Latin for poison • Not considered alive • Infectious agent that replicates only inside the living cells of other organisms. • Can infect all types of life forms, including bacteria and archaea • Cause of HIV/AIDS and Ebola Two Cell Structures Prokaryotes: Simple Structure Eukaryotes: Complex Structure (membrane-enclosed organelles). Prokaryotic Cell Components Cell Membrane Cell Wall Cytoplasm Capsule Nuclear Region Ribosomes Plasmids Flagella Prokaryotic Cell Structure • Bacteria • Archae Eukaryotic Cell Layout • Fungi • Protists Virus vs. Bacteria What are cells made of? Element C O Source Function Organic compounds, CO2 (sugars, fats, proteins) O2, H2O, organic cmpds, CO2 Main cell constituents H H2, H2O, organic cmpds N NH4, NO2, N2, proteins S SO2-, HS-, S, S2O32-, proteins organic sulfur compounds In Cys, Met, T, biotin CoA P HPO42- In nucleic acids & phospholipids This & next 2 tables based on G. Gottschalk, Bacterial Metabolism, 2nd Ed., p.2-3, 1986 Major Inorganic Elements Element K Source K+ Function Primary inorganic cation, Required in a variety of enzymes. Mg Mg2+ enzymes Required in a variety of Stabilizes cell walls, membranes, Ca2+ in exoenzymes and cell walls, important in spores (heat ribosomes Ca stability) Fe Fe2+, Fe3+ In cytochromes, ferredoxins, Iron-sulfur proteins, cofactor of some enzymes Na Na+ Required by some but not all organisms Cl Cl- Important cellular anion Minor Elements Element Source Function Zn Zn2+ Alcohol dehydrogenase, alkaline phosphotase, aldolase, RNA and DNA polymerase Mn Mn2+ Superoxide dismutase, photosystem II, cofactor of PEP carboxykinase Mo MoO42- Nitrate reductase, nitrogenase, xanthine dehydrogenase, formate dehydrogenase Se SeO32- Glycine reductase, formate dehydrogenase Co Co2+ Coenzyme B12 enzymes Cu Cu2+ Cytochrome oxidase, nitrite reductase, Ni Ni2+ Urease, hydrogenase, factor F430 W WO42- Some formate dehydrogenases What are cells made of? Component % wt mol. wt # per cell # of kinds H2O 70 18 4 x 1010 1 Inorg. ions 1 40 2.5 x 108 20 Carbohydrates 3 150 2 x 108 200 Amino acids 0.4 120 3 x 107 100 Nucleotides 0.4 300 1.2 x 107 200 Lipids 2 750 2.5 x 107 50 Other small mol.0.2 150 1.5 x 107 200 Proteins 15 40,000 106 2-3000 DNA 1 2,500,000,000 4 1 RNA: 6 rRNA 0.5-1,000,000 6 x 104 2 tRNA 25,000 4 x 105 40 mRNA 1,000,000 103 1000 J.D. Watson, Molecular Biology of the Gene, Table 3.3, 1970 Macromolecules: 95% of dry weight Macromolecule wt % protein 52.4 (50 to 80%) polysaccharide 16.6 (12 to 28%) lipid 9.4 (8%) RNA 15.7 DNA 3.2 15 to 25% A.H. Stouthamer, Antonie van Leeuwenhoek 39, 545-565 (1973) Empirical Formulas • For Organic Portion • C5H7O2N = CH1.4O0.4N0.2 • CH1.9O0.3N0.24P0.02Na0.01S0.007K0.006Mg0.005 Cl0.003Fe0.001 reported for E. coli Metabolic Characterization of Microorganisms • Carbon Source – Organic carbon - heterotrophs – Inorganic carbon (CO2) - autotrophs • Energy Source – Organic chemicals – chemoorganotrophs • Carbon based compounds other than CO2: Sugars (glucose), Gasoline, etc. – Inorganic chemicals – chemolithotroph • NH3, Fe2+, CO2, H2, S0 – Light (photosynthesis) - phototrophs How do cells get their energy? Electrons are transferred through a series of redox reactions that creates a “proton motive force” utilized to generate ATP (adenosine triphosphate). ATP is the primary energy form in cells. Metabolic Characterization • Electron Acceptor (electrons are shuttled via electron carriers to the terminal electron acceptor) – Aerobic – Require Oxygen – Anaerobic – Use something other than oxygen or carry out fermentation Examples: NO3-, SO4-2, CO2 CH3COOH CO2 + CH4 Metabolic Characterization • Electron Acceptor – Obligate Anaerobes – require complete absence of O2 because it is toxic to them Example: methanogens – Facultative – can grow without oxygen or in low oxygen environments • Will use oxygen if present; otherwise another electron acceptor is used. Metabolic Characterization Examples Aerobic conventional heterotroph Carbon & Energy Source: Organic Carbon Electron Acceptor: Oxygen Facultative conventional heterotroph Carbon & Energy Source: Organic Carbon Electron Acceptor: Oxygen or another electron acceptor Metabolic Characterization Examples Aerobic chemolithotroph Energy Source: Inorganics Carbon Source: Inorganics Electron Acceptor: Oxygen How to observe microbes? • Light Microscopy – Bright Field, Dark Field and Phase Contrast • Electron Microscopy – Transmission Electron Microscopy – Scanning Electron Microscopy Light Microscopy 3-D Imaging – Interference contrast and Atomic Force Microscopy Confocal Microscopy Electron Microscopy Shapes of Bacteria • Coccus – Chain = Streptoccus – Cluster = Staphylococcus • Bacillus – Chain = Streptobacillus • • • • Coccobacillus Vibrio = curved Spirillum Spirochete Chapter 4 Cell Growth Binary Fission (Prokaryotes) • Budding (Fungi) http://mpf.biol.vt.edu/research/budding_yeast_model/p p/intro.php Measuring Microbial Growth Direct Observation or Direct Counting Observe or Count cells in known volume of sample microscopically Cells often hard to see, so some sort of staining procedure commonly used Two classes of stain: Specifically stain nucleic acids or proteins by adsorption onto these materials Stains that react (fluoresce) as a result of metabolic activity in the cell Viable Counts • Spread a known volume of diluted sample on an agar plate and count colonies after incubation. – Assumes each colony resulted from a single cell • Reported as colony forming units (CFU)/volume (e.g., mL) • Major limitation: Selectivity! – Although sometimes an advantage (e.g., specific organism tests) • For Environmental samples, microscopic stain counts are usually 102 to 104 times the viable count. Biochemical Methods • • • • • • Measure Measure Measure Measure Measure Measure Total Cell Protein Nucleic Acids ATP Chlorophyll (use for algae) enzyme activity respiration: – Oxygen uptake Microbial Biomass • Dry weight of cells (e.g., VSS) • Turbidity – Rapid measurement that can be correlated in some cases to other techniques (e.g., dry weight) Questions • Who is the audience? • What is the premise of the study? Why do it? • What was their approach? • What did they observe?