GLG402/502 (MBI402/502) Geomicrobiology Homework 1, Due Tuesday, Feb. 18 1. (10 points) What are some theoretical and practical motivations for the emergence and development of the inter-disciplinary field of Geomicrobiology in the last 20 years? What are some major findings so far? 2. (10 points) Comment on why only a small fraction of subsurface microbes are actually culturable (i.e., compare AODC with CFU numbers). What are possible reasons responsible for this? Try to present as many reasons as possible. If most microorganisms from various environments are not culturable, how do we know that they are very diverse? 3. (5 points) Life-earth co-evolution: what roles did minerals play in the emergence of macromolecules and then life itself? 4. (5 points) Explain the following: E. Coli cells feeding on glucose grow faster when the culture is highly aerated than when the culture is supplied with NO3(NO2- is produced). What will happen if you give fumarate instead? 5. (5 points) The following is a series of coupled electron donors and electron acceptors. Using just the data given in the electron tower, order this series from most energy-yielding to least energy-yielding. H2/Fe3+, H2S/O2, methano/NO3- (producing NO2-), H2/O2, Fe2+/O2, NO2-/Fe3+, and H2S/NO3-. 6. (5 points) Can the plate method be used to isolate a single species from a mixture of two or more? If so, how do you do that? How do you confirm what you get is indeed a pure isolate that you can deposit to a culture bank such as ATCC (www.atcc.com)? 7. (Graduate students only, 10 points) Give one specific example to show the mutual interaction (co-evolution) between the geosphere and biosphere. 1 Lecture 1 (Jan. 28) Intro to Geomicrobiology Dr. Hailiang Dong What is geomicrobiology? Geomicrobiology Where Geology Meets Microbiology Earth Ecosystem Microbial community Species/ taxa Research Significance and Rationale • Origin of life – Did life originate in the subsurface? • Global Biogeochemical cycles – Feedbacks to global climate • Astrobiology – Is there life on Mars and other planets/moons? What about Martian subsurface? • Environmental bioremediation – Subsurface life possesses unique assets, ideal for cleaning up contaminants • Unique products for biotechnology applications – New biotechnology companies are emerging TEM pictures Thermus multireducens Bacillus thermoaureus Alkaliphilus transvaalensis Methods • Imaging • FISH, SEM, TEM • Culturing • Media, glove box, Winogradsky columns • DNA/ RNA sequencing • 1st, 2nd, 3rd generation sequencers • Bioinformatics • Obtaining environmental samples • ROV, field sampling Conditions in Natural Environments • • • • • • Anaerobic (no oxygen) Temperature Pressure Radiation Salinity pH Life in Extreme Environments: An International view • Life in deep freeze - Antarctic adventure • Life in high pressure and boiling water – black smokers and hot springs • Discovery of nanobacteria • Continental deep subsurface microbiology – Discovery of deep subsurface microorganisms in South African gold mines 1. Life in Deep Freeze - Antarctic Adventure Surface of Lake Vostok The borehole beneath Vostok station extends into areas of frozen lake water Ice cores from Lake Vostok 2. Life in high pressure and boiling water Sampling of sediment at the Mariana Trench 11,000 meters depth Growth of barotolerant and barophilic bacteria Black Smokers Black Smokers Tube worms and crabs Yellowstone National Park Microorganisms from Yellowstone boiling water Life on Mars? Martian meteorite Continental deep subsurface microbiology South Africa Deep Gold Mine Project Sampling water in a gold mine Radiation Resistance Found unique microbes and ecosystems Fatal to humans Discoveries • Thermophilic bacteria (75-122oC) • Extends to ~4-5 km depth • 10-100 millions of bacteria per gram of rock compared to 1 billion of cells/gram in dirt • Most abundant in pore space with water and nutrient (C, N, P and trace metals) • Diversity: most diverse in sedimentary rocks, but igneous rocks (basalts) are also habitable. Mostly autotrophic bacteria • Several millions old (from groundwater dating), dwarf bacteria – 1/1000 of their normal size Discoveries • Metabolic rate: cell division once in a century or even less, compared with minutes, hours, days or at most months • Less than 1 percent of them is culturable • Functions: degrade toxic organic compounds, antibodies, enzymes, novel pigments (Pfizer and ZymoGenetics) Use of microbes for human benefit • CO2 sequestration • Bioremediation • Biotechnology CO2 Sequestration • Colombia River basaltic aquifer • Supercritical CO2 injection • What will the microbes do to the CO2? Bioremediation Challenges - Subsurface Contaminants F F F Surface and groundwater threatened Inadequate technologies Public and environment – at risk F Special cleanup needs Environmental Bioremediation • Degradation of organic contaminants, such as MTBE (Methyl tert-Butyl Ether), TCE (trichloroethane), PCB (polychlorinated biphenyl, vinyl chloride) • Transformation of heavy metals Bacteria - Sediment Interactions - - - - - - -- - - - - - - - -- - Hypothesis Bacteria are capable of dissolving hydroxides and therefore: Mobilize: Cd, Cr, Ni, Pb, Zn Immobilize: U(VI), Mn(IV), Cr(VI), Tc(VII) - - - Biostimulation - - - - - - -- Bioaugmentation Injection of bacteria - -- - - - -- - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - -- - - - - - - Biotechnology • • • • • Multibillion dollar industry Many jobs are available today Laundry detergent Agriculture and food processing Taq Polymerase – Has allowed for DNA fingerprinting – Modern forensics Nanowires? Geomicrobiology Where Geology Meets Microbiology Earth Ecosystem Microbial community Species/ taxa Lecture 2 (Jan. 30) Co-evolution of life and Earth • Early Earth • Origin of life – Panspermia – De novo on Earth – Alternative • Co-evolution of life and earth Early Earth • 4.5 Ga- accretion of Earth • 4.4 Ga- presence of liquid water • 1221L of water delivered from cometary collisions Early Earth • 4.1 Ga- continents and oceans formed Origin of Life • Panspermia – Preformed life arrived on this planet in the form of a spore from another world (Weber and Greenberg, 1985) – Bacillus subtilis enveloped in a mantle of 0.5 mm thickness or greater of composition of H2O, CH4, NH3, and CO in equal parts – The mantle shielded them from UV radiation – Could survive over a period of 4.5-45 Myr to allow them to travel from one solar system to another Origin of Life • Surface Origin Hypothesis – The first membrane-enclosed, self-replicating cells arose out of a primordial soup rich in organic and inorganic compounds in a “warm little pond”. – However, surface conditions were too hostile: • • • • • Dramatic temperature fluctuations Meteorite impacts Dust clouds Storms Highly oxidizing atmosphere Subsurface Origin Hypothesis • Hydrothermal vents on the ocean floor – Abundant energy from disequilibrium between the reduced species from magma (H2, H2S etc.) and cool, more oxidized seawater – Precipitations of pyrite, silicates, carbonates, and Mg-rich clays – These minerals can serve as catalysts to polymerize amino acids, peptides, sugars, and nitrogenous bases, and RNA Early History of Life Co-evolution of life and earth What Is Mineral Evolution? A change over time in: • The diversity of mineral species • The relative abundances of minerals • The compositional ranges of minerals • The grain sizes and shapes of minerals What was the first mineral in the cosmos? Supernovas Diamond & Graphite Diamond Graphite “Ur”-Mineralogy Pre-solar grains contain about a dozen micro- and nano-mineral phases: • • • • • • • • • • • • Diamond/Lonsdaleite Graphite (C) Moissanite (SiC) Osbornite (TiN) Nierite (Si3N4) Rutile (TiO2) Corundum (Al2O3) Spinel (MgAl2O4) Hibbonite (CaAl12O19) Forsterite (Mg2SiO4) Nano-particles of TiC, ZrC, MoC, FeC, Fe-Ni metal within graphite. GEMS (silicate glass with embedded metal and sulfide). Mineral Evolution: How did we get from a dozen minerals (with 10 essential elements) to >5000 minerals (with 72 essential elements) on Earth today? Stage 1: Primary Chondrite Minerals Minerals formed ~4.56 billion years ago in the Solar nebula by melting and cooling. ~60 mineral species Stage 2: Aqueous alteration, metamorphism and differentiation of planetesimals Stage 2: Alteration of planetesimals by heat, water, and impacts ~250 mineral species (4.56-4.55 billion years) • • • • • • Feldspars Quartz Micas Clays Zircon Calcite Stage 3: Planet Formation Stage 3: Formation of a “Dry” Planet ~300 mineral species? Is this the end point of the Moon and Mercury? Stage 3: Formation of a Wet Planet (4.5 to 4.0 billion years ago) ~420 mineral species (hydroxides, clays) Stage 4: Granite Formation (More than 3.5 billion years ago) >1000 mineral species (pegmatites) Partial melting of basalt and/or sediments. Stage 4: Granite Formation (More than 3.5 billion years ago) >1000 mineral species (pegmatites) Pollucite Beryl Tourmaline Spodumene Tantalite Complex pegmatites require multiple cycles of re-melting and element concentration: All known examples are younger than 3.0 Ga. Stage 5: Plate tectonics (More than 3 billion years ago) ~108 km3 of reworking Mayon Volcano, Philippines New modes of volcanism Stage 5: Plate tectonics (More than 3 billion years ago) Massive base metal deposits (sulfides, sulfosalts) Stage 5: Plate tectonics (More than 3 billion years ago) Chalcocite Luzonite & Enargite Covellite & Djurleite Bournonite Geochronite Massive base metal deposits (sulfides, sulfosalts) Stage 5: Plate tectonics (More than 3 billion years ago) 1,500 mineral species Coesite SiO2 Glaucophane, Lawsonite, Jadeite High-pressure metamorphic suites (blueschists; granulites; UHP phases) Stages 3-5: Chemical and physical processes in Earth’s crust and mantle. New geologic processes, especially fluid-rock interactions associated with igneous activity and plate tectonics, led to a greater diversity of geochemical environments and thus new mineral species. ~1500 mineral species Earth’s chemical and physical processes resulted in up to 1500 different mineral species. How did we get to 5000 mineral species on Earth today? The answer is life. Minerals as Protection After Joseph Smyth et al., 1998 Minerals as Catalysis After Jay Brandes et al., 1998 Minerals as Reactants After George Cody et al., 2001 Minerals as Scaffolds After Gustaf Arrhenius et al. (1990, 1994, 1996, etc.) Co-evolution of Life and Rocks The origin of life ~4 billion years ago required some minimal degree of mineral evolution. Sulfides Borates Clays But further mineral evolution depends on life: hence the co-evolution of the geosphere and biosphere. Stage 6: Anoxic Archean biosphere (4.0-2.5 billion years ago) ~1,500 mineral species (BIFs, carbonates) D. Papineau F. Corsetti, USC Stage 7: Paleoproterozoic Oxidation (2.5-1.85 billion years ago) >4,500 mineral species, including perhaps >3,000 new oxides/hydroxides/carbonates Negaunee BIF, ~1.9 Ga Rise of oxygenic photosynthesis. Hypothesis Approximately 2/3rds of all known mineral species cannot form in an anoxic environment. Most known minerals are thus a consequence of biological activity. The Rise of Atmospheric Oxygen ? Kump (2008) Nature 451, 277-278. The early rise of oxygen in the ocean Lyons et al., 2014 Copper Minerals log fO2 (oxygen fugacity) 0 Azurite & Malachite -20 -40 Cu2+ Cuprite Cu1+ Native Copper -60 -80 Cu Metal >400 of 650 Cu Minerals Won’t Form Azurite & Malachite Libethenite Aurichalcite Linarite Turquoise Brochthite & Linarite Dioptase Uranium minerals log fO2 (oxygen fugacity) 0 -20 Uranyl (U6+) Lepersonnite, Studtite ,& Curite -40 -60 Uraninite (U4+) -80 Uraninite >220 of 254 U Minerals Won’t Form Autunite Fourmarierite & Becquerelite Boltwoodite Lepersonnite, Studtite ,& Curite Kasolite & Torbernite What was the oxygen level in the Archean Eon? log fO2 (oxygen fugacity) 0 Annabergite -20 Ni3+ -40 -60 -80 Ni2+ Nickel Metal Awaruite >100 of 154 Ni Minerals Won’t Form Annabergite Hellyerite & Zaratite Gillardite on Gaspeite Falcondoite & Willemseite Honessite log fO2 (oxygen fugacity) 0 MnO2 (Mn4+) Manganese Minerals -20 Mn2O3 Rhodocrosite (Mn2+) (Mn3+) -40 -60 Mn3O4 (Mn3+ & Mn2+) MnO (Mn2+) -80 Hollandite, Romanachite, Birnessite (Mn4+) Stages 6-10: Co-evolution of the geosphere and biosphere Changes in Earth’s atmospheric composition at ~2.4 to 2.2 billion years ago represent the single most significant factor in our planet’s mineralogical diversity. >4600 mineral species Stage 8: The “Intermediate Ocean” (1.85-0.85 billion years old) >4600 mineral species (few new species) Oxidized surface ocean; deep-ocean anoxia. Sulfate-reducing microbes. Stage 9: Snowball Earth and Neoproterozoic Oxidation (850 to 542 million years ago) >4600 mineral species (few new species) Skeleton Coast, Namibia Glacial cycles triggered by albedo feedback. Stage 10: Phanerozoic Biomineralization (Less than 542 million years old) >4,900 mineral species (biominerals, clays) Stage 10: Phanerozoic Biomineralization (4,900 mineral species hazenite carbonate silica Stage 10: Phanerozoic Biomineralization Abelsonite—NiC31H32N4 Ravatite—C24H48 Dashkovaite—Mg(HCOO)2.2H2O Evankite—C24H48 Oxammite—(NH4)(C2O4).H2O > 50 Organic Mineral Species Biomineralization—Trilobite Eyes ROOTS: The Rise of the Terrestrial Biosphere Clays Rivers Clouds Fungi Microbes Worms Rhynie Chert (~410 million years old) ROOTS: The Rise of the Terrestrial Biosphere First extensive production of terrestrial clay minerals The Story of Earth CONCLUSIONS Earth has transformed repeatedly, evolving over 4.5 billion years, and it continues to change today. Life and rocks have co-evolved as a consequence of many positive and negative feedbacks. Lecture 3 (2/4/20) Cell biology • Reading: – Chapter 4, Madigan et al. Brock Biology of Microorganisms, 12th edition Cell biology • Microbial diversity – Bacteria – Archaea – Eukarya • Cell physical properties (phenotype) – Morphology (size, shape, flagella, polysaccharide) – Color – Density • Cell chemical properties • Cell structure Cell chemical properties and cell structure • Cell chemical properties – Composition – Chemical reactions with different stains • Reactions with Gram stain • Cell structure – – – – – – Cell wall Cytoplasmic membrane Cytoplasm Ribosomes DNA and RNA Proteins Cell Biology • Microbial diversity – three domains of life: – Bacteria – pathogens, phototrophy – Archaea – common in extreme environments – Eukarya – animals, humans Cell: the single smallest unit of life Three Domains of Life Cell physical properties (phenotype) – Morphology (size, shape, flagella, polysaccharide) – Color – Density Cell physical properties (phenotype) – Morphology • size • shape • flagella, cilia – Color Star shape found from South African Gold mine Typical shapes The Cell Internal Structure Eukarya Bacteria & Archaea Heliobacterium modesticaldum Saccharomyces cerevisiae Chemical composition of cells Structure dictates function • Cell structure – Cytoplasm: a complicated mixture of substances and structures in side the cell – Cytoplasmic membrane: lets nutrients in and wastes out – Cell wall: Supports the cell – DNA: carries the genetic blueprint for the cell – RNA: Converts the blueprint into defined amino acid sequences in protein – Protein: catalyzes reactions and performs cellular functions – Ribosomes: contain RNA and protein and the cell’s protein-synthesizing factories Structure of Cytoplasmic Membrane Phopholipid bi-layer The Major Functions of the Cytoplasmic Membrane Structure dictates function • Cell structure – Cytoplasm: a complicated mixture of substances and structures in side the cell – Cytoplasmic membrane: lets nutrients in and wastes out – Cell wall: Supports the cell – DNA: carries the genetic blueprint for the cell – RNA: Converts the blueprint into defined amino acid sequences in protein – Protein: catalyzes reactions and performs cellular functions – Ribosomes: contain RNA and protein and the cell’s protein-synthesizing factories Cell Wall Structure Gram-negative Leucothrix mucor Gram stain Gram stain Positive Negative Structure dictates function • Cell structure – Cytoplasm: a complicated mixture of substances and structures in side the cell – Cytoplasmic membrane: lets nutrients in and wastes out – Cell wall: Supports the cell – DNA: carries the genetic blueprint for the cell – RNA: Converts the blueprint into defined amino acid sequences in protein – Protein: catalyzes reactions and performs cellular functions – Ribosomes: contain RNA and protein and the cell’s protein-synthesizing factories Nucleotide DNA DNA https://www.youtube.com/watch? v=GIzNlISbCxI https://www.youtube.com/watch?v=o _-6JXLYS-k https://www.achievement.org/achieve r/james-d-watson/ Structure dictates function • Cell structure – Cytoplasm: a complicated mixture of substances and structures in side the cell – Cytoplasmic membrane: lets nutrients in and wastes out – Cell wall: Supports the cell – DNA: carries the genetic blueprint for the cell – RNA: Converts the blueprint into defined amino acid sequences in protein – Protein: catalyzes reactions and performs cellular functions – Ribosomes: contain RNA and protein and the cell’s protein-synthesizing factories Levels of Protein Structure THREONINE VALINE HISTIDINE LEUCINE PROLINE GLUTAMATE GLUTAMATE Levels of Protein Structure Enzyme Example: Cleavage of Sucrose Sucrose (disaccharide substrate) Glucose and fructose (monosaccharide products) Sucrase (enzyme) http://lhs.lps.org/staff/sputnam/Biology/U4Metabolism/enzyme.gif Lecture 4 (2/6/20) Nutrition and Metabolism • Reading: Chapter 5, 6, Madigan Nutrition and Metabolism • • • • Energy class of microorganisms Microbial nutrients Fermentation and respiration Biosynthesis Definitions • Metabolism – All the chemical processes taking place within a cell • Anabolism – The process by which a cell is built up from simple nutrients also called biosynthesis • Catabolism – The process by which chemicals are broken down and energy released Energy Classes • Phototrophs – use light as an energy source • Chemotrophs – use chemicals as an energy source • Chemoorganotrophs – use organic compounds • Chemolithotrophs – use inorganic compounds Energy Classes • Autotrophs: build all of their organic structures from CO2 with energy obtained from either light or inorganic chemicals • Heterotrophs: build their structures from organic C Fig. 5-1 Microbial Nutrients Group 1 2 4 3 5 6 7 8 9 10 11 12 Period 1 2 3 4 5 6 Key: Essential for all microorganisms Essential cations and anions for most microorganisms Trace metals, some essential for some microororganisms Used for special functions Unessential, but metabolized Unessential, not metabolized 13 14 15 16 17 18 Microbial Nutrients • C – 50% of dry weight of a typical cell, major element in all classes of macromolecules – Get C from CO2, or organic C • N: – 12% dry weight, a major element in proteins, nucleic acids, and several other constituents – NH3, NO3, and N2 as a source Microbial Nutrients • P, S, K, Mg, Ca, Na, Fe – P: required in the cell for synthesis of nucleic acids and phospholipids, available in PO4 – S: required by several amino acids, available in SO4 or HS– K: required by all organisms, enzymes. available in K ions – Mg: functions to stabilize ribosomes, cell membranes, and nucleic acids and required for the activity of many enzymes. Microbial Nutrients • P, S, K, Mg, Ca, Na, Fe – Ca: helps stabilize the bacterial cell wall – Na: non-essential, seawater organisms need it for its habitat – Fe: Play a major role in cellular respiration, being a key component of Fe-S proteins involved in electron transport – Fe-binding agent called siderophores which solubilize Fe and transport it into the cell Table 5-1 Micronutrients and Growth Factors • Micronutrients – Cr, Co, Cu, Mn, Mo, Ni, Se, W, V, Zn, Fe • Growth factors – Vitamins, amino acids, purines and pyrimidines • Culture media – Chemically defined – Undefined Table 5-2 Table 5-3 Table 5-4 Fig. 5-5 Isolated colonies at end of streak Confluent growth at beginning of streak Energetics • • • • • • A+BC+D+G Exergonic: releases energy Endergonic: adsorbs energy Concept of activation energy Concept of enzymes Oxidation-reduction reactions Concept of Activation Energy Fig. 5-6 Free energy Activation energy— no enzyme Substrates (A  B) ∆G0 = Gf0(C  D) Gf0(A  B) Activation energy with enzyme Products (C  D) Progress of the reaction E0 (V) Redox couple -0.60 -0.50 -0.40 Oxidation-reduction reaction and electron tower H2  2e- + -0.30 (1) -0.20 -0.10 0.0 2H+ +0.10 (2) +0.20 1/2O2 + 2e-  +0.30 O2- +0.40 +0.50 2H+ +O 2- +0.60  H2 O +0.70 (3) 2H+ + 2e-  H2 +0.80 +0.90 (1) H2  fumarate2 (2) H2  NO3 E0 = -0.421 V (3) H2  1 2  O2 2 succinate NO2 H2O  + H2O ∆G0  = –86 kJ ∆G0  = –163 kJ ∆G0  = –237 kJ Metabolic Pathway • Fermentation – Redox reactions occur in the absence of any added terminal electron acceptors • Respiration – Molecular oxygen or other oxidant serves as the terminal electron acceptor Fig. 5-15 STAGE I: PREPARATORY REACTIONS Glucose Hexokinase Isomerase Glucose-6- Fructose-6- Phosphofructokinase Fructose-1,6Aldolase STAGE II: MAKING ATP AND PYRUVATE Glyceraldehyde-3- 2 Glyceraldehyde-3-P dehydrogenase 2 1,3-Bisphosphoglycerate 2 2 NAD+ Electrons 2 NADH To Stage III Phosphoglycerokinase 2 3-Phosphoglycerate 2 2-Phosphoglycerate Enolase 2 Phosphoenolpyruvate STAGE III: MAKING FERMENTATION PRODUCTS Pyruvate kinase 2 Pyruvate NADH To Stage II NAD+ Lactate Pyruvate dehydrogenase decarboxylase Pyruvate:Formate lyase Acetate formate Lactate Acetaldehyde Alcohol dehydrogenase Formate hydrogenlyase H2  CO2 NADH NAD+ Ethanol CO2 To Stage II Respiration • Electron acceptor – O2, NO3, Fe3+, SO4, CO3 • Electron donor: – Organic carbon, H2, Fe2+, H2S, NH3 Biosynthesis • • • • Sugars Amino acids Nucleotides Fatty acids Microbial Growth • • • • • • Overview of growth Growth cycle of population Measurement of growth Effect of temperature on growth Microbial growth at low or high pH O2 as a factor in microbial growth Microbial Growth • Overview of growth – Binary fission – Population growth: defined as an increase in the number of microbial cells in a population – Growth rate: the change in cell number or cell mass per unit time – Exponential growth N = N02n Fig. 6-1 One generation Cell elongation Septum Septum formation Completion of septum; formation of walls; cell separation Number of cells (arithmetic scale) Logarithmic Arithmetic 102 500 10 100 0 1 2 3 Time (h) 4 5 1 Number of cells (logarithmic scale) Fig. 6-8 1000 103 Fig. 6-10 Growth phases Exponential Stationary Death 1.0 10 Log10 viable organisms/ml 0.75 9 8 Turbidity (optical density) 0.50 Viable count 0.25 7 6 0.1 Time Optical density (OD) Lag Growth Cycle of Population • Lag phase – Time required for the cells to reactivate • Exponential phase – Healthiest state, desirable for studying enzymes or other components – The rate is influenced by environmental conditions • Stationary phase – Generation time of 20 min for 48 hours would produce biomass of 4000 times the weight of the Earth – Two effects inhibits growth and stationary phase • Wastes built up • Essential nutrients used up • Death phase Measurements of Growth • Total cell count • Viable count – plate count • Turbidimetric counts: optical density in a photometer or spectrophotometer Fig. 6-14 Direct Count To calculate number per milliliter of sample: 12 cells  25 large squares  50  103 Ridges that support coverslip Coverslip Number /mm2 (3  102) Sample added here; care must be taken not to allow overflow; space between coverslip and slide is 0.02 mm 1 ( 50 mm). Whole grid has 25 large squares, a total area of 1 mm2 and a total volume of 0.02 mm3. Microscopic observation; all cells are counted in large square (16 small squares): 12 cells (in practice, several large squares are counted and the numbers averaged.) Number /mm3 (1.5  104) Number /cm3 (ml) (1.5  107) Fig. 6-15 Viable Cell – Plate Count Spread-plate method Surface colonies Incubation Sample is pipetted onto surface of agar plate (0.1 ml or less) Sample is spread evenly over surface of agar using sterile glass spreader Typical spread-plate results Pour-plate method Surface colonies Solidification and incubation Sample is pipetted into sterile plate Sterile medium is added and mixed well with inoculum Subsurface colonies Typical pour-plate results Fig. 6-16 Sample to be counted 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 9-ml broth 1/10 Total dilution (10–1) 1/100 (10–2) 1/103 (10–3) 1/104 (10–4) 1/105 (10–5) 1/106 (10–6) Plate 1-ml samples 159 17 2 0 Too many colonies colonies colonies colonies colonies to count = 159  103 Plate Dilution count factor 1.59  105 Cells (colony-forming units) per milliliter of original sample Effect of Temperature on Growth • Cardinal temeprature – Minimum temp: freezing of the cytoplasmic membrane – Optimum temp: rapid growth – Maximum temp: inactivation of one or more key proteins in the cell – Characteristic of each type of organism, but not completely fixed. Depending on environmental conditions Fig. 6-18 Enzymatic reactions occurring at maximal possible rate Growth rate Optimum Enzymatic reactions occurring at increasingly rapid rates Minimum Maximum Temperature Membrane gelling; transport processes so slow that growth cannot occur Protein denaturation; collapse of the cytoplasmic membrane; thermal lysis Fig. 6-19 Thermophile Mesophile Growth rate Example: Escherichia coli Psychrophile Hyperthermophile Example: Geobacillus stearothermophilus Example: Thermococcus celer Hyperthermophile Example: Pyrolobus fumarii 60° 106° 88° 39° Example: Polaromonas vacuolata 4° 0 10 20 30 40 50 60 Temperature (°C) 70 80 90 100 110 120 Temperature Classes of Organisms • Psychrophiles – In oceans, Antarctic – Optimal: 15oC, Max: 20oC, Min: 0oC or lower – Molecular adaptations: different structures of enzymes. They function optimally in the cold, and are inactivated at even moderate temp. Microorganisms from Antarctic sea ice Temperature Classes of Organisms • Thermophiles – Soils at mid day 50-70oC – Optimal temp: > 45oC – Three conclusions • Prokaryotic organisms able to grow at higher temp than eukaryotes • The most thermophilic of all prokaryotes are certain species of archaea • Nonphototrophic organisms able to grow at higher temp than phototrophic forms Table 6-1 Temperature Classes of Organisms • Hyperthermophiles – Hot Springs, boiling water, Yellowstone National Park – Hydrothermal vents in the bottom of the oceans: > 350oC – Optimal temp: > 80oC Growth of hyperthermophiles in boiling water Temperature Classes of Organisms • Molecular adaptations to thermophily – Enzymes and other proteins are stable to heat, and function optimally at high temp. – Ribosomes, other constituents, cytoplasmic membrane are heat stable, saturated fatty acids – Protein-synthesizing machinery itself needs to be heat stable Microbial Growth at Low or High pH • Acidophiles: pH < 5, some at < 0.7 • Alkaliphiles: pH ~10-11, most of them are archaea – Intracellular pH is still neutral • Neutrophiles Fig. 6-24 pH Example Moles per liter of: 0 7 Volcanic soils, waters Gastric fluids Lemon juice Acid mine drainage Vinegar Rhubarb Peaches Acid soil Tomatoes American cheese Cabbage Peas Corn, Salmon, Shrimp Pure water 8 Seawater Acidophiles 1 2 Increasing acidity 3 4 5 6 Alkaliphiles Neutrality 9 10 Increasing alkalinity 11 12 13 14 Very alkaline natural soil Alkaline lakes Soap solutions Household ammonia Extremely alkaline soda lakes Lime (saturated solution) H+ OH– Fig. 6-25 Halophile Example: Staphylococcus aureus Example: Alivibrio fischeri Growth rate Halotolerant Nonhalophile Example: Escherichia coli 0 NaCl (%) Extreme halophile Example: Halobacterium salinarum Fig. 6-27 Oxic zone Anoxic zone Table 6-4 1. (10 points) What are some theoretical and practical motivations for the emergence and development of the inter-disciplinary field of Geomicrobiology in the last 20 years? What are some major findings so far? 2. (10 points) Comment on why only a small fraction of subsurface microbes are actually culturable (i.e., compare AODC with CFU numbers). What are possible reasons responsible for this? Try to present as many reasons as possible. If most microorganisms from various environments are not culturable, how do we know that they are very diverse? 3. (5 points) Life-earth co-evolution: what roles did minerals play in the emergence of macromolecules and then life itself? 4. (5 points) Explain the following: E. Coli cells feeding on glucose grow faster when the culture is highly aerated than when the culture is supplied with NO3(NO2- is produced). What will happen if you give fumarate instead? 5. (5 points) The following is a series of coupled electron donors and electron acceptors. Using just the data given in the electron tower, order this series from most energy-yielding to least energy-yielding. H2/Fe3+, H2S/O2, methano/NO3- (producing NO2-), H2/O2, Fe2+/O2, NO2-/Fe3+, and H2S/NO3-. 6. (5 points) Can the plate method be used to isolate a single species from a mixture of two or more? If so, how do you do that? How do you confirm what you get is indeed a pure isolate that you can deposit to a culture bank such as ATCC (www.atcc.com)? 7. (Graduate students only, 10 points) Give one specific example to show the mutual interaction (co-evolution) between the geosphere and biosphere. 1
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