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Sharon C. Long

Emerging Diagnostics - A Sneak Peak at How Tomorrow’s Technologies are Being Used at the University of Wisconsin Today. Sharon C. Long. Today’s Objective. To get you thinking about new tools and approaches to diagnosing the biology in your processes Activated sludge Biosolids Source water

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Sharon C. Long

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  1. Emerging Diagnostics - A Sneak Peak at How Tomorrow’s Technologies are Being Used at the University of Wisconsin Today Sharon C. Long

  2. Today’s Objective To get you thinking about new tools and approaches to diagnosing the biology in your processes • Activated sludge • Biosolids • Source water • Distribution systems • Etc.

  3. “Omics” • Metabolomics – aims to compare differences between biological samples based on their metabolic profiles • Genomics – using sequences of DNA and RNA to develop useful biological knowledge • Proteomics –used to relate microbial activities to the identity of organisms in multispecies communities

  4. Today’s Examples • Using ATP profiles to evaluate overall microbial community changes • Recognition of short sequences of DNA to evaluate presence of certain target microorganism - FISH • Measuring the presence of short sequences of DNA to evaluate presence of certain target microorganism - PCR • Sequencing the DNA of environmental samples to assess community structure

  5. ATP • Transports chemical energy within cells for metabolism • Present in all living cells • Amount in a sample is roughly proportional to the number of cells present • Measure the amount of light produced using firefly enzymes

  6. Study of Microbial Occurrence in Wells 370 ME/ml 45,300 ME/ml 12,600 ME/ml 860 ME/ml 460 ME/ml 1,912,000 ME/ml 801,000 ME/ml 241,000 ME/ml 8,620 ME/ml 5,200 ME/ml From: Andrew Jacque, PhD, PE, Water Quality Investigations LLC and Town & Country Engineering

  7. 370 ME/ml Well Study 45,300 ME/ml 12,600 ME/ml 860 ME/ml From: Andrew Jacque, PhD, PE, Water Quality Investigations LLC and Town & Country Engineering

  8. Fluorescent in situ Hybridization (FISH) • Use short sequences of single stranded DNA complimentary to a sequence unique or general to the target organism(s) • Attach a fluorescent dye • Prepare a sample • Permeation • Hybridization • Quantify using microscopy

  9. Applications of FISH • Diagnosis of foaming in biosolids anaerobic digesters Klare Keadle, Zachary Carroll, Daniel R. Noguera, and Sharon C. Long

  10. Foaming and FISH Results • Provided qualitative confirmation of the presence and relative density of target filaments • M. parvicella was confirmed to be numerous • Other known filamentous Archeae also present in digesters

  11. Applications of FISH 2 • Characterize microorganisms in aerated anoxic Enhanced Biological Phosphorus Removal Ramesh K. Goel, Patricia Sanhueza and Daniel R. Noguera

  12. 120 Run II ( 0.3 % O ) 2 100 80 60 120 % Run III ( 0.6 % O ) 2 40 100 % P removal 20 % Rhodocyclus related 80 120 Run IV ( 1.2 % O ) 2 0 0 10 20 30 40 50 60 100 60 Time (days) % 80 40 % P removal 60 20 % % Rhodocyclus related 40 0 % P removal 0 5 10 15 20 25 30 20 % Rhodocyclus related Time (days) 0 0 10 20 30 40 Time (days) EBPR FISH Results More than 90 % Population was Rhodocyclus related

  13. Polymerase Chain Reaction (PCR) • Use of Taq polymerase • Target short (~100 to 400 bp), unique sequences of DNA or RNA for species identification • Target broadly occurring sequences of DNA for organism family identification • Products may also be sequenced to yield further information Exponential Replication 1 2 3 4 5

  14. Measure Presence of STEC in Wastewater (A) E. coli O157:H7 (B) Non-STEC E. coli or Shigella (C) E. coli absent (D) Toxigenic organism, not STEC, not Shigella (E) Non-O157:H7 STEC or Shigella India Mansour, Mark Walter, and Sharon C. Long

  15. DNA Sequencing Trevor Ghylin

  16. Database of Wastewater Microbes (Proprietary)

  17. EBPR Genomic Study

  18. Acknowledgements

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