matter: towards improved in-situ fluorometer development ... · fluorescence spectroscopic...

Fluorescence spectroscopic characterisation of algal organic

matter: towards improved in-situ fluorometer development

Sara I. Khan 1, Arash Zamyadi 1, 2, 3, Narasinga Rao Hanumanth Rao 1, Xiang Li1, 2, Richard M.

Stuetz2, Rita K. Henderson 1, *

1 The bioMASS Lab, School of Chemical Engineering, The University of New South Wales, Sydney,

New South Wales, 2052, Australia

2 UNSW Water Research Centre, School of Civil and Environmental Engineering, The University of

New South Wales, Sydney, New South Wales, 2052, Australia

3 Civil, Mineral and Mining Engineering Department, École Polytechnique de Montreal, Montreal,

Canada

*Corresponding author: [email protected]

Electronic Supplementary Material (ESI) for Environmental Science: Water Research & Technology.This journal is © The Royal Society of Chemistry 2019

Table S1: Characteristics of bloom forming species

Characteristics Dolichospermum circinalis

Cylindrospermopsis raciborskii

Microcystis aeruginosa Chlorella vulgaris

Strain CS-533/02 CS-508 CS-555 CS-554 CS-564 CS-566 CS-42/7Type Cyanobacteria Cyanobacteria Cyanobacteria Cyanobacteria Cyanobacteria Cyanobacteria AlgaeGeometric shape

Filamentous Chained filaments (Trichomes)

Sphere Sphere Sphere Sphere Sphere

Dimensions L = 2-3 µm;W = 2-3 µm

L= 50-300 µm,W = 1.7–4.2 µm

D = 4-6 µm D = 4-6 µm D = 4-6 µm D = 4-6 µm D = 2-10 µm

Fluorescent pigment

PhycocyaninChlorophyll-a






Chlorophyll-a

Strain origin Canning river, Perth

Townsville, North Queensland

Lake burley, Griffin

Irrigation canal, Griffith

St Marys STP, TAS

Werribee, STP, Victoria

Unknown

Toxicity Below detection Non-toxic Toxic below detection

below detection

Toxic Non-toxic

Table S2: Comparisons between individual models versus the unified modelComponent C 1 C 2 C 3 C 4 C 5 C6

MA555 0.99/0.99* 0.97/0.98 0.97/0.99 0.97/0.95 0.99/0.97MA564 0.96/0.98 1.00/0.93 1.00/0.86 0.95/0.94 1.00/0.97MA554 1.00/0.99 0.95/0.94 0.91/0.72 1.00/0.52 0.90/0.70MA566 1.00/0.81 1.00/0.85 0.95/0.94 0.91/0.71 0.99/0.90

DC 0.99/0.89 0.96/0.93 0.96/0.94 0.92/0.91 0.98/0.87 0.97/0.86CV 0.82/0.67 0.95/0.92 0.94/0.92 0.91/0.87 0.97/0.85CR 0.96/0.94 0.97/0.94 0.98/0.96 0.95/0.94 0.95/0.96

* Correspondence is indicated by Tucker correlation coefficients for excitation/emission spectra

Table S3A: Statistical analysis for the AOM properties of algae and cyanobacterial species two-way ANOVA (p value analysis) with α=0.01

Table S3B: Statistical analysis between PARAFAC component and LC-OCD fractions two-way ANOVA (p value analysis) with α=0.01

C1 C2 C3 C4 C5 C6Biopolymers 0.78 0.85 0.002 0.47 0.43 0.0006Proteins 0.63 0.36 0.003 0.24 0.51 0.003Polysaccharides 0.58 0.24 0.41 0.36 0.85 0.14BBlocks 0.64 0.16 0.94 0.79 0.76 0.16LMW acids 0.23 0.54 0.14 0.26 0.18 0.001

DOC DC CR CV MA555 MA554 MA564 MA566C1 0.156 0.42 0.14 0.39 0.23 0.002 0.007 0.004C2 0.0002 0.02 0.004 0.01 0.001 0.35 0.21 0.74C3 0.0008 0.01 0.006 0.003 0.004 0.69 0.35 0.65C4 0.001 0.31 0.61 0.2 0.53 0.013 0.001 0.001C5 0.120 0.26 0.26 0.19 0.31 0.45 0.34 0.48C6 0.004 0.73 0.74 0.23 0.47 0.37 0.26 0.56Normalised C1 0.120 0.21 0.11 0.16 0.19 0.006 0.003 0.002Normalised C2 0.004 0.001 0.003 0.003 0.0006 0.54 0.19 0.39Normalised C3 0.006 0.004 0.002 0.002 0.002 0.32 0.28 0.47Normalised C4 0.005 0.13 0.24 0.14 0.46 0.001 0.0007 0.003Normalised C5 0.07 0.17 0.16 0.16 0.62 0.16 0.46 0.29Normalised C6 0.002 0.32 0.35 0.52 0.37 0.18 0.71 0.34C1: (C2+C4) 0.11 - - - - - - -C2:C4 0.02 - - - - - - -C2:C1 0.004 - - - - - - -C3:C2 0.004 - - - - - - -

Expon

entia

l

DC Station

ary

Decline

Expon

entia

l

CR Station

ary

Decline

Expon

entia

l

CV Station

ary

Decline

Expon

entia

l

MA555

Station

ary

Decline

Expon

entia

l

MA564

Station

ary

Decline

0

5

10

15

20

25

Bioploymers Proteins Polysaccharides HMW Humics LMW Humics and Acids BBLMW neutrals SUVA

DO

C c

once

ntra

tion

(mg/

L a

s C)

Figure S1: AOM composition of (i) DC (ii) CR (iii) CV (iv) MA555) and (v) MA564 at different growth phases

Bio

poly

mer

s

Bui

ldin

g bl

ocks

LMW

Aci

ds a

nd H

S

LMW Neutrals

Nitrate

Ammonium

Byp

ass

0 10 20 30 40 50 60 70 80 90 1000

2

4

6

8

10

12

Retention Time in Minutes

Rel

. Sig

nal R

espo

nse

-- OCD × 0.5

Exponential phase

Stationary phase

Decline phase

(A)

Bio

poly

mer

s

Bui

ldin

g B

lock

s

LMW Neutrals

Nitrate

Ammonium

Byp

ass

0 10 20 30 40 50 60 70 80 90 1000

2

4

6

8


Rel

. Sig

nal R

espo

nse

-- OCD × 1-- UVD × 1-- OND × 0.5

Exponential phase

Stationary phase

Decline phase

(B)

Biop

olym

ers

Build

ing

Bloc

ks

LMW

Aci

ds a

nd H

S

LMW Neutrals

Nitrate

Ammonium

Bypa

ss

0 20 40 60 80 1000

2

4

6

8

10

12


Rel

. Sig

nal R

espo

nse

-- OCD × 0.5-- UVD × 1

Exponential phase

Stationary

Decline phase

(C)

Bio

poly

mer

s

Bui

ldin

g B

lock

s

LMW

Aci

ds a

nd H

S

LMW Neutrals

Nitrate

Ammonium

Byp

ass

0 20 40 60 80 1000

2

4

6

8

10

12


Rel

. Sig

nal R

espo

nse

-- OCD × 0.5-- UVD × 1

Exponential Phase

Stationary phase

Decline

(D)

Figure S2: LC-OCD chromatograms of AOM from (A) MA555 (B) CR (C) CV and (D) DC at different growth phase

Figure S3: Contour plots of the six AOM components identified from the complete F-EEMs dataset

Figure S4: Normalized fluorescence intensity of observed AOM components along the growth phase of species

DC CR CV MA555 MA554 MA564 MA566

Exponential Stationary0

1

2

3

4

5C1 C2 C4 C6 C3

Fmax


0.1

0.2

0.3

0.4

0.5

C1 C2 C4 C6C3 C5

Fmax

/DO

CFigure S5: Fmax and Fmax/DOC for DC for observed AOM components


0.4

0.8

1.2

1.6C1 C2 C4 C6

Fmax


0.1

0.2

0.3

0.4

0.5C1 C2 C4 C6 C3C5

Fmax

/DO

C

Figure S6: Fmax and Fmax/DOC for CR for observed AOM components


20

40

60

80

100

120

C1 C2 C4 C6C3 C5

Fmax


2

4

6

8

10

C1 C2 C4 C6C3 C5

Fmax

/DO

CFigure S7: Fmax and Fmax/DOC for CV for observed AOM components


5

10

15

20

25

C1 C2 C4 C6C3 C5

Fmax

Exponential Stationary

0

0.2

0.4

0.6

0.8

1

C1 C2 C4 C6 C3C5

Fmax

/DO

C

Figure S8: Fmax and Fmax/DOC for MA555 for observed AOM components


0.4

0.8

1.2

1.6

2C1 C2 C4 C6

Fmax

0.143695361

0.078764963


0.1

0.2

0.3

0.4

0.5

C1 C2 C4 C6 C3C5

Fmax

/DO

C



0.4

0.8

1.2

1.6

2

C1 C2 C4 C6C3 C5

Fmax


0

0.1

0.2

0.3

0.4

0.5

C1 C2 C4 C6C3 C5

Fmax

/DO

C



0.4

0.8

1.2

1.6C1 C2 C4 C6 C3C5

Fmax


0

0.1

0.2

0.3

0.4

0.5

C1 C2 C4 C6 C3C5

Fmax

/DO

C


C1 C2 C40

1

2

3

4

5

6

7

DC CR CV MA555 MA554 MA564 MA566Fm

ax (R

U)

(A)

bB bA bA

aB

abBabA

aA

bA

cA cA

aA

cB cB cB cB cAbcA cC

abA

cAB

aA

C3 C5 C60

2

4

6

8

10

12

Fmax

(RU

)

(B)

cA

dA

aA

bA

dA dAdA

bB bA abCaB abA abA abA

bB bA

aB

bB bA bA bA

Figure S12 (A & B): Interaction averages of observed fluorophores during exponential phase

For statistical comparison of means, same upper-case letters indicate no significant effect of changes in AOM fluorophores for a same species, and same lower-case letters indicate no significant difference among species for a particular AOM component

C1 C2 C40

2

4

6

8

10

DC CR CV MA555 MA554 MA564 MA566Fa

mx

(RU

)

abB

bA

abBabB

abAabA aA

bA

cA

bA

aA

cB cA cB bB bA

aA

bB

bAbA

bA

(A)

C3 C5 C60

20

40

60

80

100

120

DC CR CV MA555 MA554 MA564 MA566

Fmax

(RU

)

cA cA

aA

bA

aA aAaB

aB aB aB

(B)

Figure S13 (A & B): Interaction averages of observed AOM components during stationary phase

For statistical comparison of means, same upper-case letters indicate no significant effect of changes in AOM fluorophores for a same species, and same lower-case letters indicate no significant difference among species for a particular AOM component

C1 C2 C3 C4 C5 C60

5

10

15

20

25Exponential

StationaryFm

ax (R

U)

Figure S14: Single factor averages of observed AOM components (C1-C6) across exponential and stationary phase

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