Rapid bacteriological quantification using defined substrate - PowerPoint PPT Presentation

Rapid bacteriological quantification using defined substrate enzymatic activity in municipal wastewater Dr. Pascale Champagne, Ph.D., P .Eng., D.WRE, F .ASCE, F .EWRI David Blair, BASc. 2019 Heraklion, Greece

Introduction Project Objective • Rapid E. coli detection in municipal wastewater treatment systems Field Site • Amherstview WPCP (rated peak capacity: 16,000 m 3 /day) • South Eastern Ontario, Canada Industry Partner & T echnology • TECTA-PDS Inc. (Formerly Veolia Endetec ) 2

Wastewater Treatment Systems in Canada Use of lagoons Compliance Monitoring and T oxicity T esting Requirements Environment Canada . 2016 CCME Council of Ministers . 2009 3

Sampling Locations Secondary Treatment 4 Building

Amherstview Water Pollution Control Plant Secondary Waste Stabilisation Constructed Secondary Waste Stabilisation Constructed T reatment Pond Wetland T reatment Pond Wetland 5

• Chlorophyll-a Known • Turbidity Interferen • Total Suspended ts Solids • Chemical Oxygen Demand Parameters • Total Organic Carbon Organic Material • Dissolved Organic of Interest Matter • Fats, Oils, and Grease • Membrane Filtration • Agar Plates Bacteria • TECTA-PDS Instrument 6

• Chlorophyll-a Known • Turbidity Interferen • Total Suspended ts Solids • Chemical Oxygen Demand Parameters • Total Organic Carbon Organic Material • Dissolved Organic of Interest Matter • Fats, Oils, and Grease • Membrane Filtration • Agar Plates Bacteria • TECTA-PDS Instrument 7

Instrument: TECTA - PDS Enzyme Florescent Fluorophor produced signal e bound by substrate detected bacteria 8

Florescent Spectra in Wastewater • Excitation: single wavelength • Emission: 200-700 nm spectrum • Recorded temporally (0-18 hr.) • Correlative bacteria quantity to fmorescent “time-to-detection” (TTD) Visualization of ideal dual- excitation, spatial and temporal spectrum Li et. al, 2014 9

Instrument Calibration • Correlative bacteria quantity to fmorescent signal • Based on Monod Growth Kinetics – First-order rate constant particular to the exponential growth phase of cultures described – Not subject to substrate inhibition Brown et al. 200 – Concentration of available substrate- binding enzyme considered proportional to the bacterial density • Proprietary trigger/data processing method J. Monod, Annu. Rev. Microbiol ., 3, 371 (1949) 10

Comparison to Reference Method Aeration Basin (Raw Sample) 12 Deviation from Reference Method Aerati 10 EC TC on TECTA Quant. (LOG CFU/100ml) Basin -0.1 ± 8 2.9 ± 0.4 2.3 • E. coli: high variation 6 • T otal coliforms: low variation, high deviation 4 from reference method • Existing method is not 2 E. coli T otal Coliforms viable 0 4 5 6 7 8 Ref. Method (LOG CFU/100ml) 11

Efgects of Sample Pre-treatment Aeration Basin Sample Treatment Dilutio Filtrati E. coli (Avg.) 12 n on Linear (E. coli (Avg.)) T otal Decreases Removes Coliforms 10 TECTA Quant. (LOG CFU/100ml) (Avg.) all water suspende quality d material parameter 8 Decrease s Turbidity Decreases Chlorophyl 6 amount of l-a bacteria 4 2 4 5 6 7 8 Ref. Method (LOG CFU/100ml) 12

Efgects of Sample Pre-treatment Aeration Basin Sample Treatment Avg. E. coli (Avg.) 12 Deviation from Linear (E. Detection Reference Method coli (Avg.)) Aerati Time (hr.) T otal on Coliforms 10 TECTA Quant. (LOG CFU/100ml) Basin (Avg.) EC TC EC TC 8 0.3 ± 4.2 ± Raw 5.06 2.95 2.3 0.4 Filtered -1.8 ± -0.1 ± 7.58 8.13 6 (8 μm) 0.5 0.3 Diluted -3.3 ± 4.9 ± 2.57 3.34 (10:1) 0.4 0.4 4 Filter & -1.8 ± 0.1 ± 8.55 8.88 Dilute 0.3 0.4 2 4 5 6 7 8 Ref. Method (LOG CFU/100ml) 13

Results on Naturalized Treatment Systems Waste Stabilization Pond WSP Calibration Residuals 5 3,00 E. coli (Std.) T otal Coliforms 2,00 (Std.) 1,00 4 TECTA Quant. (LOG CFU/100ml) 0,00 5 - J un - 18 15 - un - J 18 25 - J un - 18 J u l 5- 18 - 15 - J ul - 1 8 25 - J ul 1 8 - 4 - Au g- 1 8 -1,00 -2,00 3 -3,00 -4,00 -5,00 2 Deviation from Reference Method Method EC TC Standard 0.4 ± 0.4 -1.2 ± 0.8 1 1 2 3 4 5 Calibrated 0.0 ± 0.4 0.0 ± 0.8 Ref. Method (LOG CFU/100ml) 14

Results on Naturalized Treatment Systems Constructed Wetland Calibration Residuals 5 3,00 2,00 1,00 4 TECTA Quant. (LOG CFU/100ml) 0,00 5-Jun-18 25-Jun-18 15-Jul-18 4-Aug-18 -1,00 -2,00 3 -3,00 -4,00 -5,00 2 Deviation from Reference Method E. coli (Cal.) Method T otal Coliforms EC TC (Cal.) Standard 0.6 ± 0.5 -0.8 ± 1.3 1 Calibrated 0.0 ± 0.4 -0.1 ± 1.3 1 2 3 4 5 Ref. Method (LOG CFU/100ml) 15

Detection Time by Sampling Point 12 • TTD < 12h for both E. coli and total coliforms across all 10 sampling points • Potential microbial control .) Time to Detect (hr 8 parameter with “near-real time” qualifjcation 6 • Least variance in secondary clarifjer 4 • Less variance in total 2 coliforms than E. coli 0 16

Water Characterization Observed variation in dissolved organic matter Observed variation in fats, oils & grease 35 120 Aerated Surface Aerated Surface Clarifjed Secondary Clarifjed Secondary WSP Efgulent WSP Efgulent 30 Constructed FOG (mg/L) 100 Constructed Wetland Efgulent Wetland Efgulent 25 80 20 60 DOM (mg/L) 15 40 10 20 5 0 0 17

Evaluation of Final Spectra Select Final Signals 50000 1 AE Neat Unfjltered 45000 0, 9 Final Sig CS Neat Unfjltered 40000 0, 8 Final Sig WSP Neat Unfjltered 35000 0, 7 Final Sig 30000 Rel. Intensity 0, 6 25000 0, 5 20000 0, 4 Multivariate statistics show no 15000 0, 3 strong-correlation, but multiple 10000 medium correlations 0, 2 5000 0, 1 Confounded efgects of water 0 0 parameters 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 4 7 0 3 6 0 3 6 9 2 6 9 2 5 8 2 5 2 2 3 3 3 4 4 4 4 5 5 5 6 6 6 7 7 • Prevents statistical isolation in Wavelength (nm) 18 wastewater

Conclusions “Near real-time” • 2-3.5 hrs in secondary treatment bacteria • 5-10 hrs in naturalized systems quantifjcation Method robustness is • FOG levels signifjcantly reduce signal primary dependent on • Insuffjcient signal detection above 75 mg/L water organics • Slope-intercept calibration improves mean but not variance in quantifjcation Custom Calibration • Raw vs. partially treated WW require difgerent calibration method • Levels found in naturalized systems have little Chlorophyll impact on fmorescent attenuation • Seasonal algae blooms is noticeable but Interference insignifjcant in signal attenuation 19

Acknowledgements David Blair Dr. Stephen Brown Eric Marcotte Rami Maassarani Brooke Sanders Ahrani Gnananayakan Champagne Bioresearch Group 20