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

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Introduction

• Rapid E. coli detection in municipal wastewater treatment

systems Project Objective

• Amherstview WPCP (rated peak capacity: 16,000 m3/day)
• South Eastern Ontario, Canada

Field Site

• TECTA-PDS Inc. (Formerly Veolia Endetec)

Industry Partner & T echnology

Wastewater Treatment Systems in Canada

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Use of lagoons

Environment Canada. 2016

Compliance Monitoring and T

• xicity T

esting Requirements

CCME Council of Ministers. 2009

Sampling Locations

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Secondary Treatment Building

Amherstview Water Pollution Control Plant

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Secondary T reatment Secondary T reatment Waste Stabilisation Pond Waste Stabilisation Pond Constructed Wetland Constructed Wetland

Known Interferen ts

• Chlorophyll-a
• Turbidity
• Total Suspended

Solids Organic Material

• Chemical Oxygen

Demand

• Total Organic Carbon
• Dissolved Organic

Matter

• Fats, Oils, and Grease

Bacteria

• Membrane Filtration
• Agar Plates
• TECTA-PDS

Instrument

Parameters

• f Interest

6

Known Interferen ts

• Chlorophyll-a
• Turbidity
• Total Suspended

Solids Organic Material

• Chemical Oxygen

Demand

• Total Organic Carbon
• Dissolved Organic

Matter

• Fats, Oils, and Grease

Bacteria

• Membrane Filtration
• Agar Plates
• TECTA-PDS

Instrument

Parameters

• f Interest

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Instrument: TECTA - PDS

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Enzyme produced by bacteria

Fluorophor e bound substrate

Florescent signal detected

Florescent Spectra in Wastewater

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• 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

Instrument Calibration

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• 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 – 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)

Brown et al. 200

Comparison to Reference Method

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Aerati

• n

Basin Deviation from Reference Method EC TC

• 0.1 ±

2.3 2.9 ± 0.4

4 5 6 7 8 2 4 6 8 10 12

Aeration Basin (Raw Sample)

• E. coli

T

• tal Coliforms
• Ref. Method (LOG CFU/100ml)

TECTA Quant. (LOG CFU/100ml)

• E. coli: high variation
• T
• tal coliforms: low

variation, high deviation from reference method

• Existing method is not

viable

4 5 6 7 8 2 4 6 8 10 12

Aeration Basin Sample Treatment

• E. coli (Avg.)

Linear (E. coli (Avg.)) T

• tal

Coliforms (Avg.)

• Ref. Method (LOG CFU/100ml)

TECTA Quant. (LOG CFU/100ml)

Efgects of Sample Pre-treatment

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Decreases all water quality parameter s Decreases amount of bacteria

Dilutio n

Removes suspende d material Decrease Turbidity Chlorophyl l-a

Filtrati

• n

4 5 6 7 8 2 4 6 8 10 12

Aeration Basin Sample Treatment

• E. coli (Avg.)

Linear (E. coli (Avg.)) T

• tal

Coliforms (Avg.)

• Ref. Method (LOG CFU/100ml)

TECTA Quant. (LOG CFU/100ml)

Efgects of Sample Pre-treatment

13

Aerati

• n

Basin Deviation from Reference Method Avg. Detection Time (hr.) EC TC EC TC Raw 0.3 ± 2.3 4.2 ± 0.4 5.06 2.95 Filtered (8 μm)

• 1.8 ±

0.5

• 0.1 ±

0.3 7.58 8.13 Diluted (10:1)

• 3.3 ±

0.4 4.9 ± 0.4 2.57 3.34 Filter & Dilute

• 1.8 ±

0.3 0.1 ± 0.4 8.55 8.88

Results on Naturalized Treatment Systems

14 Method Deviation from Reference Method EC TC Standard 0.4 ± 0.4

• 1.2 ± 0.8

Calibrated 0.0 ± 0.4 0.0 ± 0.8

• 18

• 1 8

• 1 8

• 5,00
• 4,00
• 3,00
• 2,00
• 1,00

0,00 1,00 2,00 3,00

WSP Calibration Residuals

1 2 3 4 5 1 2 3 4 5

Waste Stabilization Pond

• E. coli (Std.)

T

• tal Coliforms

(Std.)

• Ref. Method (LOG CFU/100ml)

TECTA Quant. (LOG CFU/100ml)

1 2 3 4 5 1 2 3 4 5

Constructed Wetland

• E. coli (Cal.)

T

• tal Coliforms

(Cal.)

• Ref. Method (LOG CFU/100ml)

TECTA Quant. (LOG CFU/100ml)

Results on Naturalized Treatment Systems

15 Method Deviation from Reference Method EC TC Standard 0.6 ± 0.5

• 0.8 ± 1.3

Calibrated 0.0 ± 0.4

• 0.1 ± 1.3

5-Jun-18 25-Jun-18 15-Jul-18 4-Aug-18

• 5,00
• 4,00
• 3,00
• 2,00
• 1,00

0,00 1,00 2,00 3,00

Calibration Residuals

Detection Time by Sampling Point

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2 4 6 8 10 12

Time to Detect (hr .)

• TTD < 12h for both E. coli

and total coliforms across all sampling points

• Potential microbial control

parameter with “near-real time” qualifjcation

• Least variance in secondary

clarifjer

• Less variance in total

coliforms than E. coli

Water Characterization

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20 40 60 80 100 120

Observed variation in fats, oils & grease

Aerated Surface Clarifjed Secondary WSP Efgulent Constructed Wetland Efgulent

FOG (mg/L)

5 10 15 20 25 30 35

Observed variation in dissolved organic matter

Aerated Surface Clarifjed Secondary WSP Efgulent Constructed Wetland Efgulent

DOM (mg/L)

Evaluation of Final Spectra

18 2 4 1 2 7 3 3 5 3 3 7 3 6 9 4 1 4 3 3 4 6 5 4 9 7 5 2 9 5 6 1 5 9 3 6 2 5 6 5 7 6 8 9 7 2 1 7 5 3 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000

Select Final Signals

AE Neat Unfjltered Final Sig CS Neat Unfjltered Final Sig WSP Neat Unfjltered Final Sig

Wavelength (nm)

• Rel. Intensity

Multivariate statistics show no strong-correlation, but multiple medium correlations Confounded efgects of water parameters

• Prevents statistical isolation in

wastewater

Conclusions

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• 2-3.5 hrs in secondary treatment
• 5-10 hrs in naturalized systems

“Near real-time” bacteria quantifjcation

• FOG levels signifjcantly reduce signal
• Insuffjcient signal detection above 75 mg/L

Method robustness is primary dependent on water organics

• Slope-intercept calibration improves mean but not

variance in quantifjcation

• Raw vs. partially treated WW require difgerent

calibration method

Custom Calibration

• Levels found in naturalized systems have little

impact on fmorescent attenuation

• Seasonal algae blooms is noticeable but

insignifjcant in signal attenuation

Chlorophyll Interference

Acknowledgements

David Blair

• Dr. Stephen Brown

Eric Marcotte Rami Maassarani Brooke Sanders Ahrani Gnananayakan Champagne Bioresearch Group

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