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Aquatic Organic Matter Fluorescence – from phenomenon to applicationsDr Darren Reynolds - Associate Professor in Bio-Sensing ResearchUniversity of the West of England

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Institute of Bio-Sensing Technology

biology for sensors and sensors for biology

Aquatic Organic Fluorescence

from phenomenon to applications

Darren Reynolds

biology for sensors and sensors for biology

17th and 18th Centuries – intellectual

movement - ‘Enlightenment’ the ‘Age

of Reason’.

Newtonian science exerted its greatest

impact on the world

biology for sensors and sensors for biology

Maxwell de Broglie Born

Thompson Wein Rutherford

Faraday Hertz Schrödinger

Stokes Einstein Pauli

Bohr Planck Lewis

Becquerel Crookes Heisenberg

biology for sensors and sensors for biology

Defined as the emission of light by a

substance, where the emitted light

cannot be attributed to incandescence,

i.e. thermal radiation.

biology for sensors and sensors for biology

Sir G. G. Stokes

(1852)

biology for sensors and sensors for biology

biology for sensors and sensors for biology

Observed the fluorescence properties of

humic and fulvic substances and organic

matter in natural waters........... ‘Gelbstoff’

Humic, fulvic, DOC, DOM, CDOM

(Vodacek, Mopper, Blough, Coble)

biology for sensors and sensors for biology

we have and are experiencing a

technological revolution largely driven

by breathtaking advances in;

Applied electro optics

Improvements in data processing and

data handling....

biology for sensors and sensors for biology

Optical Space

www.turnerdesigns.com

biology for sensors and sensors for biology

Optical Space

Wastewater fluorescence

biology for sensors and sensors for biology

Optical Space

Wastewater fluorescence

biology for sensors and sensors for biology

Optical Space

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

biology for sensors and sensors for biology

Optical Space a)

b)

c)

d)

e)

f)

biology for sensors and sensors for biology

T1

Energy

S2

S0

Ground state

λ2

S1

λ2 λ 1 λ 3 λ 4

Fluorescence Absorption Internal

and External

Conversion

Phosphorescence

Vibrational Relaxation

Triplet Excited State

Singlet Excited States

Intersystem Crossing

Internal Conversion

biology for sensors and sensors for biology

biology for sensors and sensors for biology

biology for sensors and sensors for biology

Chlorophyll

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

biology for sensors and sensors for biology

Chlorophyll

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

Whitening Agents

biology for sensors and sensors for biology

Chlorophyll

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

Fluorescent Dyes

biology for sensors and sensors for biology

Chlorophyll

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

Chlorophyll fluorescence

biology for sensors and sensors for biology

Chlorophyll

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

Humic/Fulvic Material

biology for sensors and sensors for biology

Chlorophyll

300 400 500 600 7000

200

400

600

800

1000

Wavelength (nm)

Inte

nsity (

a.u

.)

Wa

ve

le

ng

th

(

nm

)

W a v e l e n g t h ( n m )

2 0 0 . 0 0

2 5 0 . 0 0

3 0 0 . 0 0

3 5 0 . 0 0

4 0 0 . 0 0

4 5 0 . 0 0

5 0 0 . 0 0

5 5 0 . 0 0

6 0 0 . 0 0

3 0 0 .0 0 3 5 0 .0 0 4 0 0 .0 0 4 5 0 .0 0 5 0 0 .0 0 5 5 0 .0 0 6 0 0 .0 0 6 5 0 .0 0 7 0 0 .0 0

9 6 2 . 0 0

8 8 6 . 0 0

8 1 0 . 0 0

7 3 4 . 0 1

6 5 8 . 0 1

5 8 2 . 0 1

5 0 6 . 0 1

4 3 0 . 0 1

3 5 4 . 0 2

2 7 8 . 0 2

2 0 2 . 0 2

1 2 6 . 0 2

5 0 . 0 2

- 2 5 . 9 7

Microbial Processes

biology for sensors and sensors for biology

biology for sensors and sensors for biology

Autochthonous material is created in-situ

through microbial activity – a reflection of the

phys/chem/biol processes

This provides a recycling mechanism for

allochthonous DOM (dissolved organic carbon

fed into the hydrological system from outside).

biology for sensors and sensors for biology

Bacterial origin.

Shelley et al., (1980),

Dalterio et al. (1986)

Determann et al., (1998) Cammack

et al., (2004) and Elliott et al., (2006)

biology for sensors and sensors for biology

Laboratory Field

Num

ber

P

ublis

hed

P

apers

Marine ‘optical map’

(Coble, 1993, Mar.Sci.)

Fresh/waste ‘optical map’

(Baker, 2001, ES&T)

Rapid Technological

improvements

Aquatic Fluorescence Research

Wastewater

Fluorescence

biology for sensors and sensors for biology

– Water recycling/nano filtration

– Drinking water treatment processes -

chlorination

– Urban watersheds quality monitoring

– Catchment water quality monitoring

– Wastewater quality monitoring

biology for sensors and sensors for biology

Real-time monitoring of water and

wastewater quality using a fluorescence

technique

Optical Spectroscopy in the Aquatic

Environment

Elsholt Works, Yorkshire Water (May,1998)

biology for sensors and sensors for biology

biology for sensors and sensors for biology

The characterisation of sewage using

fluorescence

Effluent and Sewage Network Management

Inst. Mech. Engineers (February 2000)

biology for sensors and sensors for biology

biology for sensors and sensors for biology

Field based fluorescence devices for

urban/fresh/drinking/waste water

systems have been limited;

– Low knowledge base

– Technological challenges

– Lack of appropriate field trails

biology for sensors and sensors for biology

Real-time monitoring of river water quality

using in-line continuous acquisition of

fluorescence excitation and emission

matrices.

Future Water Sensing Technologies

Warrington, (February, 2010)

biology for sensors and sensors for biology

biology for sensors and sensors for biology

Samples Correlation (peak/parameter/Pearson's r

unless stated)

References

Raw settled/treated sewage from 3 different treatment works

(n=129)

T1 BOD5 0.960

0.970

0.960

Reynolds & Ahmad (1997)

Raw settled/treated sewage (n=25) T1–T2 BOD5 0.980 Ahmad & Reynolds (1999)

Synthetic sewage treated via a rotating bio-disc contactor (n =45)

FTotal = Total fluorescence intensity

Settled and treated sewage samples over a 3 month period (n=56)

FTotal = Total fluorescence intensity

FTotal

T1

FTotal–T1

FTotal

T1

FTotal–T1

BOD5

COD

TOC

BOD5

COD

TOC

COD-BOD

BOD5

COD

TOC

BOD5

COD

TOC

COD-BOD

0.890

0.920

0.910

0.980

0.980

0.980

0.840

0.790

0.820

0.800

0.930

0.940

0.930

0.710

Reynolds (2002)

Filtered raw sewage T1 COD

TOC

Nk

NH4-N

COD

TOC

Nk

NH4-N

0.420

0.410

0.690

0.650

0.560a

0.530a

0.760a

0.840a

Vasel & Praet (2002)

Treated effluent samples (over a 3 month period) T1 COD 0.900 Lee and Ahn (2004)

Wastewater samples (96 in total) using CODDissolved values T1 CODDissolved.

COD

0.370

0.510

Wu et al., (2006)

Sewage effluents (n=16) C1 DOC 0.140 Cumberland & Baker (2007)

Wastewater effluents (223 samples - sewage, trade and pollution

incidents)

T1

T2

C2

A

BOD5

TOC

BOD5

TOC

BOD5

TOC

BOD5

TOC

0.906b

0.876b

0.848b

0.802b

0.771b

0.870b

0.720b

0.808b

Hudson et al., (2008)

biology for sensors and sensors for biology

a)

b)

c)

d)

e)

f)

I

II

III

IV

V

VI

VII

biology for sensors and sensors for biology

A concerted effort to tackle the

approaching data Tsunami is necessary

Application driven technology needs to be

developed, tested and evaluated in the

field

biology for sensors and sensors for biology

Fluoro-sensor Development

Tryptophan-like fluorescence

Laboratory assessment

Field deployment

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Preliminary Field Trials

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• Aquatic fluorescence is not new

• Fluorescence sensing has history

• Technology Readiness Level is high

• Clear Identified Applications

biology for sensors and sensors for biology

• Application-led field studies/trials

– Sensor performance

– Generation of data sets for evaluation

– Development of appropriate data

management tools (application driven)

biology for sensors and sensors for biology

Hudson, N., Baker, A., and Reynolds,

D. (2007). Fluorescence analysis of

dissolved organic matter in natural,

waste and polluted waters – a review.

River Research Applications, 23, 631-

649.

biology for sensors and sensors for biology

Henderson, R.K. et al. (2009).

Fluorescence as a potential

monitoring tool for recycled water

systems: A review. Water Research,

43, 863-881.

biology for sensors and sensors for biology

Paula G. Coble, Andy Baker, Jamie

Lead, Robert M. Spencer, Darren M.

Reynolds.

2013 Cambridge University Press

biology for sensors and sensors for biology

John Attridge

Chelsea Technologies Group, UK.

Robin Thorn & Gareth Robinson

Centre for Research in Biosciences, UWE, UK.

Elfrida Carstea

National Institute of R&D for Optoelectronics, Romania.

Andy Baker

Water Research Centre, UNSW, Australia.

biology for sensors and sensors for biology

darren.reynolds@uwe.ac.uk

http://www.biosensingtech.co.uk/

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