Microbial Ecology Test Questions – Flashcards

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Microbial Ecology
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study of biodiversity of microbes in nature; measure of activities & effects on microbes
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Microbial Ecology Organization Levels
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Populations, Guilds, Communities, Ecosystems
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Biogeochemical Cycles
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Most elemental cycles run, controlled & moderated by microbes so not just geochemical cycles
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Enrichment & Isolation
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Not just relying on med microbiology - study microbes out in environment; enrichment highly selective & appropriate inoculum used - can add Organic N, atmospheric N, heterotrophic populations grow, nitrogen-fixing bacteria appear & not overgrown
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Isolation
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separation of individual organisms from the mixed community
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Enrichment cultures
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select for desired organisms through manipulation of medium and incubation conditions; easy to tell bacteria is there, hard to prove that it's not there
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Winogradsky column
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miniature ecosystem w/o homogenous mixture; serves as long-term source of bacteria for enrichment cultures; soil, water ; light; each tube has different environment for different activities
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Enrichment bias
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Microorganisms cultured in lab are frequently only minor compoennts of the natural microbial ecosystem
in lab only able to provide beneficial environments for a few types of microorganims, therefore only these species are benefit
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Pure cultures
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w/ enrichment ; isolation ; manipulation; contain a single kind of microorganism (can be obtained by streak plate, agar dilution or liquid dilution)
they are clones of cells started from an individual cell
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Fluorescent Staining
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Used for enumeration ; mainly for nucleic acids or DNA; view w/ fluorescent microscope; Might have some UV damage; 3 Types: DNA-binding stains, Acridine Orange (orange or green), DAPI
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DAPI ; Acridine Orange (AO)
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Different staining methods:
DAPI and AO fluoresce under UV light, are nonspecific and stain nucleic acids
DAPI stains bright blue; AO stains orange or greenish-orange
*cannot differentiate between live and cells
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Viability Stains (viable counting)
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can differentiate between live and dead cells bcz two dyes are used; based on integrity cell membrane (not intact in dead cells); green cells alive ; red dead
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Fluorescent Antibody (Ab) Staining
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Monochrome staining; can be used as a cell tag; highly specific for the molecule recognized by Ab; used to identify any type of cell for specific Ab; making antibodies is time consuming and expensive
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Green Fluorescent Protein (GFP)
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genetically engineered into cells to track bacteria, if always expressed; can act as a reporter gene, if fused to a promoter of interest; can use fluorescence to see how much bacteria still living
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Nucleic acid probe
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DNA or RNA complimentary to a sequence in a target gene or RNA molecule
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Fluorescent in situ hybridization (FISH)
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phylogenetic probes to find species-specific oligonucleotide signature sequences; highly specific ; simple; multiple probing of single sample; culture not needed when this performed
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FISH Community Analysis
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study unculturable organisms, determine morphology, abundance, microbial associations; probes allow for determination of species that couldn't be differentiated with just simple staining
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Multiple FISH Probes
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Not based on 16s taxonomy; based on physiology & nitrogen cycle; can view different activities of different organisms with supporting metabolisms with different color probes for that specific sig sequence ("Multiplexing"); activities can work off of or support each other in "Consortium"
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FISH Chromosomal Painting
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identify specific genes; locate, count specific metabolic populations; can search for whatever gene you require; can determine specific gene expression & transcription activites by different dyes, physiology & neighboring organisms; find out who's doing what (genes turned on)
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In situ reverse transcriptase (RT) FISH
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Locates specific genes ; detects expression for those genes; needed when RNA is present, not DNA, so RT needed for complementary DNA; can run multiple tests - see specific genes turned on/off
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PCR ; Microbial Community Analysis
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Can determine candidate name for unknown genes until bug isolated ; description correlated
Path 1: Extract total community DNA, amplify with PCR ; fluorescent tagged primers; put on T-RFLP gel; excise bands ; clone 16s rRNA genes; sequence; generate tree; Path 2: extract, amplify with general/restrictive primers (bacteria or endospore-specific); use DGGE gel, excise bands; sequence; generate tree
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Polymerase Chain Reaction (PCR)
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new process allows for no TAC polymerase inhibitors of PCR in community; able to amplify PCR
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Environmental Genomics
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Phylogenetic Tree for single genes or total gene pool of community; can identify ; link to phenotypes; Path 1: amplify single gene ; sequence, generate tree; Path 2: restriction enzymes digest all DNA then shotgun sequence or seq directly (w/o cloning); get partial genomes ; can discover new genes, link to phylotypes
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Microbial Activity Measurements
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Microcosms - take sample of soil to lab w/o disturbing environment; label w/ different macromolecules based on metabolic processes wanted; track where things are going, what happens to substrate; Two Types: Radioisotopes ; Microelectrodes
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MAM Types
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Radioisotopes: high sensitivity, turnover rates, fate of substrate ; killed cell control; Microelectrodes: micromanipulator, microbial mats ; multiple electrodes; can look at physical aspects like O2 tension, pH, H2S concentration; can multiplex and look at multiple states at once
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FISH Microautoradiography (MAR)
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14C or 35S used for short term; labeled sugars put on photographic plate; wherever radioactivity is concentrated shows what populations metabolizing
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Microelectrodes
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Test physical parameters ; compounds for specific metabolic activities; can tell where no O2 - orgs fermentative or anaerobic; can do w/o disturbing population
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MAM Stable Isotopes
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Biogeochemical cycles ; food chains: 12C, 13C, etc - stable isotopes that hang around for awhile - used for biological processes; enzymes very selective and will favor one isotope over another; fractionates isotopes by selectively enriching 12CO2 to 12C and diminishes 13CO2 to 13C; shows how much enzymes moderate organisms ; fixes carbon
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Carbon Isotopic Compositions
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Different substrates ; their organic processes ; result - how much carbon; CO2 fixed and fungi breaks down to release carbon into ecosystem
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Populations, Guilds ; Communities
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Community 1: photic zone (oxygenic phototrophs), Comm 2: oxic (O2 loving ; facultative); Comm 3 (sediments): Anoxic (ferm, anaerobic); Guild 1: methanogenic or homoacetogenic bacteria; Guild 2: sulfate- or sulfur-reducing bacteria; Guild 3: denitrifying or ferric iron-reducing bacteria: Guild 4: ferm bacteria (ferm sugars, acids, etc)
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Guilds
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Metabolically related populations of microbes that work together to ferment, depending on environment; each guild makes similar product in own process; each element has own guild to perform their own process; unique to microbial ecology
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Microenvironments
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Prime niche; spatially very small; physical or chemical conditions change rapidly; heterogenous; environments change physiology ; can change (increase/low pH, etc) from physical or chemical; different numbers show levels of O2 for each niche
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Nutrient Levels ; Growth Rate
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Feast or famine - when things get bad, organisms sacrifice self to help rest of population; happens when nutrients low or distribution not uniform; competition; no periods of optimal growth; when nutrients high, metabolism increases exponentially; organisms grow quickly ; die quickly
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Competition
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neighbor gains access to nutrients faster, killing off competition; can also inhibit growth of others by releasing compounds as antibiotics (fungi) or bacteriosis
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Cooperation
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work together instead of fighting; "Syntrophy"; close associations
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Syntrophy
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cooperation, eating together ("commensal") ; working together ("Consortium")
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Consortium
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everyone works together metabolically so can share to attack particular substrate together, better
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Terrestrial Environments (soil)
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Different horizons (layers of environments or microniches); most complex for microbes; each horizon has different nutrients, temps
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Terrestrial Horizons
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O: undecomposed plant materials
A: surface soil (high in organic, dark, tilled for agriculture; plants ; large #'s microbes grow)
B: subsoil (minerals, humus, leached from soil; little organic; low microbial activity)
C: soil base (directly from underlying bedrock; microbial very, very low)
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Soil Aggregate
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Microcolonies form on different soil particulates held together by minerals from organisms or fungi w/ EC polysacch "glue"; each mineral (clay, water, quartz) has different surface properties for different microcolonies to grow & flourish
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Terrestrial Environments (deep subsurface)
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Several to 1000+M; anaerobes that are sluggish chemoorganotrophs & chemolithotrophs (not a lot of organic available); no photosynthesis; divide only once every 100 years
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Aquatic Habitats
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Diverse (oceans, marshes, etc); phototrophic microorganisms are primary producers & drive carbon into ecosystem; phytoplankton bottom dwellers; benthic algae; biologic activity depends on rate of primary production.
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Aquatic O2 Relationships
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O2 limited solubility in H2O; O2 depletion when heterotrophs use up oxygen; Stratification of deep lakes: epilimnion & hypolimnion; when frozen, hypo no longer supports water so crashes down below for nutrients & support; might have high levels of organic matter from leaves falling, etc
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Epilimnion
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Upper layer of water
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Hypolimnion
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Lower layer of water - provides nutrients
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Aquatic - Rivers
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highly mixed & high organic levels possible so lower O2 levels
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Biological Oxygen Demand (BOD)
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O2 consuming property of water; measures amt of organic material that can oxidize by microorgs; > BOD = > pollution & > org level; > carbon = < O2
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Effect of high organic levels
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when point source hits, organic carbon and BOD increases while O2 greatly decreases; eventually, carbon taken up and BOD decreases or bacteria moves; levels normalize, move up and down
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Marine Environment
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Inshore - higher nutrient levels, high primary production, high heterotrophic activity, potentially low O2; inorganic materials allow heterotrophs to be vast
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Chlorophyll Distribution
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open ocean depleted of photosynthesizers; high chlorophyll content inland, rivers
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Open Oceans
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limited inorganic nutrients, limited primary production; limited heterotrophs; O2 levels high. Cyanobacteria primary producers O2; 1/2 O2 we breathe from cyanobacteria
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Deep Sea
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Photic zone 0-300M; biologically active lower; Deep >1000M; water relatively inactive biologically; low temp & nutrients, high pressure; physiologically separated microbes based on pressure (baro)
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Barotolerant
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down to 3000M, no growth above 5000M
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Barophilic
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4000-6000m, optimum at 4000M
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Extreme Barophiles
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10000M, opt 7000-8000M, no growth <4000M
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Molecular effects of high pressure
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decrease binding capacity of enzymes, membrane process, use outer membrane porins to bind substrates; limited gene expression
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Hydrothermal vent
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volcanic environment at ocean bottom (deep sea vents); black smoker chimneys very hot, metal sulfides & hydrothermal gradients; metabolism occurs in surrounding areas of sea mounts
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What do the vents release?
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H2S, CO2, NH4+ (no organic material)
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Vent invertebrate communities
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Sessile tubeworms and mollusks that live close to the vent; Receive C source (chemotrophic dependent), few predators; red parts (hemoglobin like) to attract nutrients
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Chemolithotrophs of deep sea
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CO2 fixation from nutrients out of vents, tube worms have trophosomes that live inside tissue of worm; giant clams & methanotrophs that release CO2
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Trophosome
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prokaryotic cell symbionts in tube worms that use hemoglobins to bind O2 and H2S; Houses and delivers H2S and CO2 to chemoautotrophs
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Black smoker chimney worms
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chemolithotrophic bacteria grown on surface of worm bodies to hold nutrients; worms graze hairs & eat; not one homogenous population of bacteria
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Higher temps for microbes
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in situ evidence for colonization, growth at 125-140oC; biological sulfate reduction at 130oC; ATP unstable at 150oC
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Carbon cycle
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carbon reservoirs in land/living plants; a lot of carbon in humus; CO2 transfers; photosynthesis (terrestrial, aquatic); decomp produces methane (CH4) & CO2; both organic aerobic & anaerobic ferments to release carbon back into atmosphere
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Carbon Cycle 2 types
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Main 2 types: CO2 fixation; Carbon mineralization (decomposition)
Other: Methanogenic archaea; Methylotrophs; Carbon monoxide metabolism
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Humus
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mix of phenolic compounds that enzymatically bind together, like a sponge in soil - it holds majority of carbon; microbes must go through humus to get carbon
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methanogenic habits
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swamps, marshes, soils, protozoan endosymbionts, rumen, cows (burp 50L/day)
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Rumen
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digestive area of cow: find bacteria, protozoa, anaerobic fungi, microbial cells digested, microbial protein recovered; strict anaerobic fungi digests cellulose
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Chemoautotrophic benefits
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Maintain access to H2S and CO2
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Chemoautotrophy (sulfur cycle)
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H2S = H+ donor for ETC
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Sulfur-oxidizing chemoautotrophs
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H2S used as e- donor, bacteria fix CO2 to carbohydrate
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Metabolic Diversity
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Presence of specific enzymes dictate metabolic capabilities/habitat distributions
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Biogeochemical cycles
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Chemical transformations by biological organisms; every element has own cycle, almost always moderated by microbes
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Purposes of biogeochemical cycles
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Converts one chemical form to another, maintains essential compounds in biosphere; leaching of ores (copper, gold); heavy metal transforms, biodegrade/biomediate toxins, etc
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Biogeochemical Cycles & Pseudomonas
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pseudomonas altered metabolically and put in toxic environment for cleanup; they don’t compete so eventually die out; fluorescence used to view colonies.
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Plant-Microorg Interactions
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Ways to put Nitrogen into ecosystem; plants (legumes/nonlegumes), Gram (-), N2 fixing bacteria, root/stem nodule bacteria; Plants reliable - bacteria nutrient rich & gets N2 from plant; high species specificity (only 1 species per plant)
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Nitrogen Cycling (what is it required for)
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Protein synthesis; Nucleic acid synthesis; other cell components; Major reservoir of N2 is atmostphere
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What benefits are there with nitrogen fixation?
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Symbiotic relationships with plants (Rhizobium sp. and legumes)
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Non-symbiotic bacteria able to fix nitrogen
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Cyanobacteria; Azotobacter sp.; Clostridium sp.
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CO2 fixation
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CO2 -> Organic carbon; CO2 is a major reservoir of carbon; CO2 fixation is done by Primary Producers (autotrophs)
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Carbon Mineralization (Decomposition)
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Organic carbon -> CO2; Sugar/protein/lipid decomposition; Organic carbon is often trapped in complex molecules (cellulose & lignin)
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Carbon mineralization (what happens?)
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Complex molecules not readily decomposed, so accumulation occurs; Many bacteria and fungi responsible for decomposition
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CH4 production from CO2
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Anaerobic process done by methanogenic Archaea
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The sulfur cycle (2 types)
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Sulfur oxidation & reduction
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Sulfur oxidation
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elemental sulfur
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Sulfur Reduction
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Process called dissimilatory sulfate reduction
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What can SO4^2- be used for?
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As a sulfur source converted to -SH groups in proteins; This is called assimilatory reduction
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Ecosystem
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sum total of all organisms in specified environment
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Habitat
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portion of ecosystem suited to a specific population
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Eutrophic lake, eutrophication
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buildup of inorganic/organic material (nutrients) = eutrophication = fish die
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Microenvironments
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different oxygen levels, physical & chemical changes can change environment rapidly
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Aquatic habitats depend on what?
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rate of primary production (carbon)
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Primary producers of aquatic?
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phytoplankton & benthic algae
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PCR gel T-RLFP
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restriction enzymes cut DNA at specific sequences; identifies higher level of phylogenetic tree (more general)
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PCR gel DGGE
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separates down to the molecules of DNA, even if 1 base pair differs; identifies lower levels of tree (more specific)
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Winogradsky column
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it is selective for isolation & different gradients; microbial diversity in artificial environments
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Chromosomal staining
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ISRT: dyes nucleic acids; gene expression
FISH: dyes DNA, gene transcription
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In situ?
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experiment that uses intact tissue (no harm to sample bcz not individual cells)
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Acridine Orange
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DNA staining, also use DAPI
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gene expression staining
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ISRT
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gene transcription staining
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FISH chromosomal painting
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Why are Gram (-) more dangerous as endotoxin?
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because Lipid A layer of LPS has toxins
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