Toxicity of Heavy Metals to P.mendocina, B.subtilis, and P.putida Bacteria

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Toxicity of Heavy Metals to P.mendocina,
B.subtilis, and P.putida Bacteria

• Paul Schramm
• University of Notre Dame
• EMSI REU Summer Program 2003

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Importance

• Metals in the Environment
• Bacteria Environmental Building Blocks
• Bacillus subtilis, Pseudomonas putida, and
  Pseudomonas mendocina
• Gram-positive and gram-negative bacteria
• Extensively studied, especially at Notre Dame

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Aseptic Technique

• All containers, growth mediums, pipette tips and
  other necessary materials autoclaved
• Metal solutions filter sterilized with 0.2?m
  filters
• Bleach used to kill bacteria on dirty glassware
• All transferring done in the clean bench
  UV-light, ethanol, and flame sterilization used

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Procedure

• Metal Stock Solutions made
• Arsenic, Cobalt, Chromium, Copper, Nickel, Zinc,
  Lead

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• Bacteria started in incubator in 5mL growth
  medium
• FEDM/Hepes Growth medium for
  Pseudomonads
• Mixed in a 1L volumetric flask
• 1.0g NH4Cl 0.2g MgSO47H2O
• 0.05g CaCl22H20 5.0g Succinate diNa salt
• 6.5g Glycerol-2-phosphate 0.5g KCl
• 4.77g Hepes 0.125g trace elements
• Trace Elements per100mL DI H2O
• 5mg MnSO47H2O, 6.5mg CoSO47H2O, 2.3mg CuSO4,
  3.3mg ZnSO4, 2.4mg MoO3

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• Experimental stocks of metal made at varying ppm
  (For Example, Cobalt solutions of 2, 4, 8, 12,
  16, and 20ppm made)
• 5mL of each experimental stock placed in 60mL
  autoclaved Nalgene bottles, in triplicate
• 5mL growth medium added to each bottle
• Batch inoculum prepared from growth medium,
  bacteria, and 1.1mL 9mM FeEDTA. Allowed to
  incubate, 5mL inoculum added to each bottle
• No-growth controls prepared from 5mL of stock
  solution and 10mL growth medium
• 50?L 9mM FeEDTA added to each no-growth bottle
• All bottles, each containing 15mL liquid, placed
  in incubator for at least 20 hours

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Variations for B.subtilis

• Different growth medium
• Same as FeDM/Hepes except succinate salt omitted
  and medium autoclaved before adding
• 10mL filter sterilized 50(w/v) D-glucose
• 10mL filter sterilized 5mg/mL L-Tryptophan
• Longer Incubation required

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• After incubation, Absorbance taken with UV-Vis
  Spectrophotometer at 600 nm
• pH checked to assure consistency
• Data graphed, re-trial with adjusted ppm metal
  solutions if necessary
• Samples refrigerated if ICP to be used

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Results Effect of metals on P.Putida
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Arsenic had much less effect on P.Putida than any
of the other metals
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Similar effects of Co on all three bacteria
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Normalization of Cobalt Data
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Copper effects P. p. and P. m. growth similarly
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Cr effects P.p. much more strongly than P.m.
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Results Summary

• Arsenic least toxic and Chromium most toxic to
  P.putida
• Cobalt had similar effects on all bacteria
• Copper effected P.Putida and P.Mendocina
  similarly, while Chromium was much more toxic to
  P.Putida
• P.Putida growth actually encouraged by small
  amounts of Cobalt and Copper

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ICP Inductively Coupled PlasmaDoes adsorption
correlate with toxicity?

• P.mendocina with Copper
• B.subtilis with Cobalt
• Results need to be worked up

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Problems

• Zinc and Lead could not be used precipitation
  problems at high ppm
• Problems with running Copper on the ICP
• Problems with using standards made from powders
  on the ICP bad calibration curves

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What's Next?

• Finish with P.mendocina and B.subtilis bacteria
  for full comparisons for all metals
• Further use of ICP to determine role of
  adsorption in toxicity
• Find ways to stop precipitation of Lead and Zinc
  at high ppm
• Equilibrium modeling of metal speciation to
  ensure no precipitation is occurring
• Try Depleted Uranium
• Complete study in early Sept. and draft
  publication for Geomicrobiology

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Acknowledgements

• Center For Environmental Science and Technology
  (CEST)
• Environmental Molecular Science Institute (EMSI)
• Dr.Patricia Maurice
• Jennifer Forsythe
• Jennifer Schaefer
• Aimee Lavarnway

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Questions?