Potentiometric titrations of glycoamylase treated bacteria: implications for extracellular polysacch

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Potentiometric titrations of glycoamylase
treated bacteria implications for extracellular
polysaccharide substances (EPS)
Advisor Dr. Jeremy Fein Mentor Brian Ginn Lisa
Vukovits EMSI Symposium University of Notre
Dame August 3, 2006
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Introduction

• Does extracellular polysaccharide (EPS) affect
  the binding ability of Pseudomonas putida?
• Remove the EPS from the bacterial surface to
  determine if there is a change in the binding
  ability (adsorption) to the functional groups on
  the cell wall.

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Extracellular Polysaccharide (EPS)

• Sugar- Based
• Purpose not fully understood
• One of its roles could be to
• ensure a moist environment
• around the cells, where
• nutrients can diffuse
• through the slime.
• Glucoamylase used to remove EPS

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Pseudomonas putida

• Why use P. putida?
• Ubiquitous soil bacterium
• Well- studied
• High- levels of EPS
• Known metal- binding ability
• (adsorption)
• 5. Bioremediation

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Potentiometric Titrations

• Potentiometric titrations performed to determine
  the concentration of binding sites and their
  protonation constants.
• P. putida has charged functional groups, number
  available varies based on pH.
• How many sites are protonated at a given time,
  are calculated using K values
• Lower the pH ?reduced available binding sites
• Increase pH ?increase available binding sites.

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Non- Treated P. putida

• P. putida cultured in TSB/ yeast agar for 24 hrs.
• Cultures washed six times.
• Samples suspended in nitrogen- treated NaClO4,
  mass measured.
• Samples titrated.

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Glucoamylase-Treated P. putida

• P. putida cultured in TSB/yeast broth for 24
  hours
• Cultures washed 4 times
• 1.2 -1.5g of putida suspended in 500 mL of NaClO4
  with 300 µl of enzyme.
• Incubated for four hours.
• Culture washed 6 times times.
• Samples suspended in nitrogen- treated NaClO4,
  mass measured.
• Culture titrated.

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Results
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Conclusions

• The results comparing the enzyme- treated vs. not
  treated bacteria is not significant.
• EPS does not affect the adsorption behavior of
  the bacteria.

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Future Research

• Metal binding research
• Use EXAFS to determine bacterial functional
  groups
• Calculate the K values for each binding site
• Detailed knowledge of the physiology and
  biodegradation capabilities of this organism is
  essential for providing the scientific
  foundations for the emerging bioremediation
  industry.

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Acknowledgements

• Mr. Brian Ginn
• Dr. Valli Svwarvensen
• EMSI Staff