Culture-Dependent Unit Lab Project Two Nitrogen Cycle - PowerPoint PPT Presentation

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Culture-Dependent Unit Lab Project Two Nitrogen Cycle

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Culture-Dependent Unit Lab Project Two Nitrogen Cycle Nitrogen Fixation (17.28) N2 fixed to NH3 and NH2 groups - COSTS ATP ONLY done by Prokaryotes - Bacteria and ... – PowerPoint PPT presentation

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Title: Culture-Dependent Unit Lab Project Two Nitrogen Cycle


1
Culture-Dependent UnitLab Project TwoNitrogen
Cycle
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  • Nitrogen Fixation (17.28)
  • N2 fixed to NH3 and NH2 groups - COSTS ATP
  • ONLY done by Prokaryotes - Bacteria and Archaea
  • Conserved O2-intolerant Nitrogenase/Fe-Mo
    cofactor
  • Assayed via acetylene reduction assay -
    understand
  • Acetylene and N2 both share triple bond
    substrates.

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  • Microbial Diversity O2 Strategies - Table 17.10
  • Azotobacter fastest aerobic ETC uses O2,
    capsule
  • Cyanobacteria O2-resistant heterocysts
  • Methanogens anoxygenic phototrophs live
    anoxic
  • Symbionts co-produce O2-binding "leghemoglobin
  • You will view both phototrophs during that lab.

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  • Symbiotic Nitrogen Fixation (19.22)
  • Legumes root hairs secrete inducer molecules
  • Symbionts aerobic soil Rhizobium
    (Proteobacteria)
  • Separate - no fixation together - leghemoglobin
  • Bacteroids misshapen Rhizobium in nodules
  • Non-leguminous Azolla/Anabaena, Alnus/Frankia
    fine line between bacteroids and organelles

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  • Rhizobium Enrichment
  • Dissect, clean, surface-sterilize nodule,
    comparing
  • Antiseptic tissue-safe alcohol, H2O2 (Tables
    20.3-4)
  • Disinfectant not tissue-safe, bleach (Tables
    20.3-4)
  • Media HAS nitrogen and simple sugar - capsule
  • Hospital antiseptics/disinfectants first used by
    Lister, 1976.

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  • Azotobacter Enrichment (12.9, 17.28, and 18.1)
  • Understand Beijerinks enrichment, late 1800s
  • Media lacks nitrogen, has simple sugar - capsule

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Nitrification and Denitrification Prominent in
soil, water, sewage/feces, and GI tract
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  • Nitrification, Aerobic Chemolithotrophy (17.12)
  • Two-step process - ammonia then nitrite oxidation
  • NH3 O2 yields NO2 H2O ATP, Nitrosomonas
  • NO2 O2 yields NO3 ATP, Nitrobacter
  • Both are Proteobacteria (12.3) packed with
    lamellae
  • Soil enrichment using NH3 media with aeration
    measure either NH3 depletion or NO3 appearance.

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  • Anaerobic Nitrate Reduction (17.14)
  • EITHER denitrifying (to N2) or nitrifying (to
    fixed N)
  • NO3 CH2O yields N2 CO2 ATP, Pseudomonas
  • NO3 CH2O yields NH3 CO2 ATP, Enterics
  • Review river lab many other living things also
    nitrify.

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  • Nitrate Reduction Enrichment/Testing - Table 24.3
  • Soil and water inoculation - nitrate reduction
    tube
  • 1 - bubble in Durham N2 (denitrification)
  • 2 - A/B turns red NO2 present (nitrification)
  • 3 (if 2 clear) - Zn turns red no NO3 reduction

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  • Todays Activities
  • Root nodule preparation/plating Azotobacter
    set-up
  • Media preparation and inoculation - nitrification
  • Nitrate reduction inoculation/set-up
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