1

Algae Metabolites in Arizona Surface Waters

Paul Westerhoff , Ph.D.Arizona State University, Civil and Environmental Engineering

David WalkerUniversity of Arizona

Greg BoyerSUNY-College of Environmental Science and Forestry

Milton Sommerfeld & Qiang HuArizona State University, Plant Biology

How can things so small cause such big problems?

• What do you say if a customer asks:– “My water smells, is it safe to drink?”– “Why should I drink tap water if it smells like dirt?”– “If you can’t make it smell and taste good why

should I pay for it?”• What do you say if a WTP operator asks:

– “What do I do to control in-plant algae growth that causes short circuiting and short filter run times now that you convinced me that our previous practice of prechlorinating may kill people?

• What do you say when a utility manager asks:– “What is the best way to monitor and prevent algae

related problems?”

2

All those questions have been asked recently in Central Arizona

WTPs

Presentation Outline

• Methods• Algae Metabolites

– Bulk organic matter– Taste and odors– Cyanotoxins

• Ongoing algae activites– Genetic fingerprinting of culprit algae– Managing nuisance algae

• Conclusions

3

Methods• DOC & DON: Shimadzu TOC-V• DIN: Ion chromatography &

phenate method• T&O: SPME with GC/MS• Algal toxins:

– ELISA– Protein Phosphatase Inhibition

Assay (PPIA)– Anatoxin-a/Saxatoxin – HPLC after

fluorescent derivatization– Cylindrospermopsin – HPLC using

a photodiode array detector• PCR for 16s-RNA genetic

fingerprinting

Bulk Organic Matter Production by Algae

4

Algae release extracellularmaterial (bulk DOC)

0.0

4.0

8.0

12.0

16.0

20.0

0 30 60 90 120 150Time, h

DO

C, m

g/L

Oscillatoria p (10/01/00) Scenedesmus q (10/10/00)

DOC production & DBP formation potential with DISSOLVE metabolites from

Scenedesmus quadricauda

0.0

1.0

2.0

3.0

4.0

5.0

0 1 2 3 4 5 8

Time, d

DO

C, m

g/L

0

100

200

300

400

500

CH

Cl 3

, µg/

L

DOC, mg/L CHCl3

THM formation potential: 118µg CHCl3/mgDOC

5

Many Algae Metabolites Contain Dissolved Organic Nitrogen

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Feb-

02

May

-02

Aug

-02

Oct-0

2

Jan-

03

Apr

-03

Jul-0

3

Oct-0

3

Jan-

04

DON (m

gN/L

)

Lake PleasantBartlett LakeSaguaro Lake

Algae Influence WatershedsProduce NOM (mg/L)

• Laboratory results showed that only 55 to 70% of algae-produced DOC were removable by biodegradation.

• Long-term reservoir storage accumulates refractory algal DOC causing a net gain (~10%) in DOC budgets.

• In an arid region where terrestrial DOC input is low (<0.3 g/m2-yr), controlling reservoir hydraulic retention time (HRT) is key to reducing the amount of DOC production and export to downstream.

6

Western Watersheds Produce T&O (ng/L)

0

20

40

60

80

100

120

1999 2000 2001 2002 2003 2004

MIB

Con

cent

ratio

n (n

g/L)

Lake Pleasant

Bartlett Lake

Saguaro Lake

0

0.05

0.1

0.15

0.2

0.25

00.050.1

0.150.2

0.250.3

0

500

1000

1500

2000

2500

6 12 24 48 1000

500

1000

1500

2000

2500

Light intensity (µmol m-2 s-1)

Geo

smin

ince

lls (u

g/L)

Geo

smin

inM

ediu

m (u

g/L)

Gro

wth

rate

(d-1

)

Geo

smin

inC

ells

/chl

-a

Culprit Algae can be cultured in the lab to study environmental factors

• Increasing light intensity affects biomass production and yield of T&O compounds

• Intracellular MIB and geosminlevels are much higher than concentrations in aqueous media

7

Raw water entering 24th Street WTP from a concrete lined canal

0

20

40

60

80

1999 2000 2001 2002 2003 2004

Con

cent

ratio

n (n

g/L)

MIBGeosminBeta-cyclocitral

8

Powder Activated Carbon can remove T&O compounds within WTPs

0

20

40

60

80

1999 2000 2001 2002 2003 2004

MIB

Con

cent

ratio

n (n

g/L) Raw water

Finished water

Relationships between FPA and GC/MS Analysis

y = 0.1151x + 0.3331R2 = 0.8644

0

1

2

3

4

5

0 20 40 60 80MIB, ng/L

Ear

thy/

Mus

ty F

PA

y = 0.3104x + 0.1524R2 = 0.7126

0

1

2

3

4

5

0 5 10 15 20 25Geosmin, ng/L

Ear

thy/

Mus

ty F

PA

y = 0.1865x + 1.5136R2 = 0.0801

0

1

2

3

4

5

0 2 4 6 8 10 12 14Cyclocitral, ng/L

Ear

thy/

Mus

ty F

PA

9

Predicting Expected FPA Intensity based upon GC/MS data

Earthy Musty FPA Value = 0.800*MIB0.396*Geo-0.110*Cyclocitral0.350

R2 = 0.728

0

1

2

3

4

5

0 1 2 3 4 5

Observed Earthy Musty FPA

Pred

icte

d Ea

rthy

Mus

ty F

PA

Fitted DatapointsData with FPA < 1Outliers

FPA= 2 when MIB=5, Geosmin=3, Cyclocitral=3 ng/L

Cyanotoxins are also present (ng/L to ug/L)

How can we make toxin analysis WTP friendly?

Anabaena, Aphanizomenon, Lyngbya, Cylindrospermopsis

Nerve axons AlkaloidSaxitoxins

Anabaena, Planktothrix, Aphanizomenon, Cylindrospermopsis

Nerve synapseAlkaloidAnatoxin-a, Anatoxin-a (S)

Cylindrospermosis Anabaena AphanizomenonUmezakia

LiverAlkaloidCylindrospermopsin

NodulariaLiverCyclic peptidesNodularin

Microcystis, Anabaena, Oscillatoria, Nostoc

LiverCyclic peptidesMicrocystins

Cyanobacterial generaPrimary target organ in mammals

Chemical Structure

Toxin groups

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ELISA (enzyme-linked immunosorbent assay)

Y Y YY

S tep #1 W ell is co ated w ith ant ibo dy (Y )

Y Y YS tep #2 S am ple is ad ded and m icro cyst in ( ) is bo und by the antibo dy (Y )

Y Y YS tep #3 E nzym e link ed antigen ( ) is add ed and binds to the rem aining uno ccup ied sites o n the antibo dy (Y )

Y Y YS tep # 4 E nzym e su bstra te ( ) is ad ded to p ro d uce co lo r react io n

ELISA Calibration Curve

%Bo = -26.72 Ln [MC] + 40.13R2 = 0.9976

0

20

40

60

80

100

120

0.01 0.1 1 10

Microcystin Concentration [MC] (ug/L)

%B

o =

OD

sam

ple

x 10

0%

O

D n

eg c

ontro

l

11

ELISA Assay Results

Assay No.

Date Kit No.

50%Bo (µg/L)

Slope R2 Acceptable Assay Range (µg/L)

Comments

1 11/27/02 1 0.4 -28.8 0.957 Yes 0.16-1.6 Calibrators less than recommended %Bo ranges. Near expiration date

2 1/22/02 1 0.2 -22.3 0.947 Yes 0.16-1.6

3 2/02/02 2 0.2 -22.7 0.961 Yes 0.16-1.6

4 5/15/02 2 0.2 -24.3 0.947 Yes 0.16-1.6

5 6/25/02 2 0.5 -28.3 0.982 Yes 0.16-1.6

6 8/28/02 3 0.6 -27.0 0.993 Yes 0.16-1.6

7 9/12/02 3 0.6 -24.1 0.975 Yes 0.16-2.5

8 9/19/02 4 0.6 -22.6 0.989 Yes 0.16-2.5

9 10/10/02 5 0.6 -27.6 0.992 Yes 0.16-2.5

10 10/17/02 6 0.6 -26.7 0.997 Yes 0.16-2.5

11 10/25/02 7 0.6 -26.8 0.987 Yes 0.16-2.5

Assay No.

Date Kit No.

50%Bo (µg/L)

Slope R2 Acceptable Range (µg/L)

Comments

1 11/27/02 1 0.4 -28.8 0.957 Yes 0.16-1.6 Calibrators less than recommended %Bo ranges. Near expiration date

2 1/22/02 1 0.2 -22.3 0.947 Yes 0.16-1.6

3 2/02/02 2 0.2 -22.7 0.961 Yes 0.16-1.6

4 5/15/02 2 0.2 -24.3 0.947 Yes 0.16-1.6

5 6/25/02 2 0.5 -28.3 0.982 Yes 0.16-1.6

6 8/28/02 3 0.6 -27.0 0.993 Yes 0.16-1.6

7 9/12/02 3 0.6 -24.1 0.975 Yes 0.16-2.5

8 9/19/02 4 0.6 -22.6 0.989 Yes 0.16-2.5

9 10/10/02 5 0.6 -27.6 0.992 Yes 0.16-2.5

10 10/17/02 6 0.6 -26.7 0.997 Yes 0.16-2.5

11 10/25/02 7 0.6 -26.8 0.987 Yes 0.16-2.5

PP2A(protein phosphatase-2A assay)

S tep #2 S am ple is added and m icro cyst in ( ) inhibits enzym e

S tep #1 W ell is co ated w ith enzym e P P 1 o r P P 2A ( )

S tep #3 S ubstrate ( ) is added .

O vert im e unihibited enzym e reacts w ith substrate re leasing pho sphate ( ) resu lt ing in yello w co lo r. E nzym es inhibited by m icro cyst in do no t react w ith substrate and pro duce no co lo r.

PO 4P O 4

PO 4PO 4

PO 4

PO 4

PO 4

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Assay No.

Date Enzyme No.

V/Vo= 50% µg/L

Slope R2 Acceptable Assay Range (µg/L)

Comments

1 4/26/02 1 0.15 -3.12 0.9988 Yes 0.05-0.30 2 6/06/02 1 0.15 -3.36 0.9415 Yes 0.05-0.25 3 6/13/02 1 0.15 -3.03 0.9143 Yes 0.05-0.30

Enzyme at room temperature

4 6/20/02 2 0.15 -2.93 0.7110 Yes 0.05-0.25 Poor R2 value 5 6/27/02 2 NA NA 0.2489 No NA 6 6/27/02 2 NA NA 0.3110 No NA 7 7/10/02 2 NA NA NA No NA 8 7/12/02 2 0.6 -0.54 0.9276 Yes 0.2-1.2

New enzyme used, calibrators not in range of Vi/Vo < 1

9 7/12/02 3 0.8 -0.55 0.9741 Yes 0.2-1.2 10 7/24/02 3 0.6 -0.52 0.9656 Yes 0.2-1.2 11 7/24/02 3 0.7 -0.53 0.8232 Yes 0.2-1.5 12 7/24/02 3 0.7 -0.52 0.9626 Yes 0.2-1.5 13 7/24/02 3 0.9 -0.51 0.9306 Yes 0.2-1.5 14 8/02/02 3 NA NA 0.0085 No NA Enzyme was inactive from

overuse at room temperature 15 8/02/02 4 1.1 -0.51 0.9027 Yes 0.2-1.6 16 8/08/02 4 0.6 -1.03 0.9297 Yes 0.2-0.9 17 8/21/02 4 1.4 -0.43 0.9588 Yes 0.3-1.8 18 8/28/02 4 1.0 -0.47 0.9917 Yes 0.2-1.5 19 9/12/02 4 1.0 -0.53 0.9310 Yes 0.4-1.5 20 9/19/02 5 1.5 -0.19 0.7516 No 0.5-2.5 Inconsistent calibrator results 21 9/25/02 5 NA NA NA No NA No color produced 22 9/26/02 4 1.2 -0.36 0.9416 Yes 0.5-2.1 23 10/03/02 4 1.4 -0.35 0.9077 Yes 0.5-2.5

PP2A Assay Results

Assay Comparison

MC concentration (ug/L)

0

0.5

1

1.5

0 0.5 1 1.5

PP2A

ELI

SA

1:1

13

Assay Comparison

74%100%% acceptable

~$1.00$4.12Cost per well

0.05-0.250.24-2.5

0.147-2.5Range (ug/L)

0.05, 0.240.147MDL (mg/L)

PP2A (toxicity)

ELISA(presence)(measure of…)

Recommend ELISA for monitoring

Intracellular Extraction

Culture Growth = 10 days Culture Growth = 29 daysExtracellular conc. = >2.5µg-MC/L Extracellular conc. = >25 µg-MC/LIntracellular conc. = 56 µg-MC/L Intracellular conc. = 838 µg-MC/L

Sample Number

Microcystis aeruginosa (µg-MC/OD730)24 hrs in MtOH + Beadbeating 24 hrs in MtOH

10 day 29 day 10 day 29 day

1 0.662 1.042 0.437 >1.036

2 0.732 1.076 0.380 >1.036

3 0.634 0.991 0.408 >1.036

4 0.915 0.762 0.775 0.949

5 0.887 0.758 0.648 0.990

6 0.859 0.275 0.592 0.966

7 0.242 1.056 0.680

8 0.275 0.944 0.874

9 0.958 0.829

Mean 0.782 0.926 0.689 0.881

Standard Deviation

0.121 0.154 0.257 0.116

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Microcystin Monitoring

00.10.20.30.40.50.6

Sep

-01

Oct

-01

Nov

-01

Dec

-01

Jan-

02Fe

b-02

Mar

-02

Apr-

02M

ay-0

2Ju

n-02

Jul-0

2A

ug-0

2S

ep-0

2O

ct-0

2MC

con

cent

ratio

n (u

g/L)

Lake PleasantBartlettSaguaro

Intracellular levels always slightly higher than extracellular (dissolved) MC concentrations

MIB and MCLake Pleasant

0

0.05

0.1

0.15

0.2

0.25

0.3

Sep

-01

Oct

-01

Nov

-01

Dec

-01

Jan-

02

Feb-

02

Mar

-02

Apr

-02

May

-02

Jun-

02

Jul-0

2

Aug

-02

Sep

-02

Oct

-02

MC

conc

entra

tion

(ug/

L)

0

5

10

15

20

25

30

35

40

45

MIB

con

cent

ratio

n (n

g/L)

MC MIB

15

MIB and MCBartlett Lake

0

0.1

0.2

0.3

0.4

0.5

0.6

Sep-

01

Oct

-01

Nov

-01

Dec

-01

Jan-

02

Feb-

02

Mar

-02

Apr-

02

May

-02

Jun-

02

Jul-0

2

Aug-

02

Sep-

02

Oct

-02

MC

con

cent

ratio

n (u

g/L)

010

203040

506070

8090

MIB

con

cent

ratio

n (n

g/L)

MC MIB

MIB and MCSaguaro Lake

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

Sep-

01

Oct

-01

Nov-

01

Dec-

01

Jan-

02

Feb-

02

Mar

-02

Apr

-02

May

-02

Jun-

02

Jul-0

2

Aug

-02

Sep-

02

Oct

-02

MC

con

cent

ratio

n (u

g/L)

0

20

40

60

80

100

120

MIB

con

cent

ratio

n (n

g/L)

MC MIB

16

Cyanotoxin Summary• Relatively large background levels of

microcystin found in all wastersheds.• Anatoxin-a only found in fish stomach samples,

however, the likely cause of large fish kills. • Fast degradation rates of anatoxin-a make this

neurotoxin extremely difficult to detect in surface water.

• While numbers of C. raciborskii have, at times, dominated the phytoplankton cylindrospermopsin has been found in very low levels and only in concentrated samples (plankton tows).

Early warning indicators for culprit algae

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General strategy

Band excision

Field sample

DNA extraction &purification

genomic DNA

PCR amplificationof 16S rDNA fragments

Field PCR products

DGGE separation

PCR amplification

AGCTCCATTAGGGACTCGAGGTAATCCGTG

DNA sequencing

Species identification

Databasematching

cyanobacterial fingerprintsDNA extractionfrom the gel

DNA from an individual species

0

5

10

15

20

25

30

Time of the year (2002)

MIB

(ng

L-1)

A

B

IV

DNAmarker4/15 5/15 7/29 8/12 8/26 9/30 10/14 10/28 11/12 11/25

5/15 7/29 8/12 8/26 9/30 10/14 10/28 11/12 11/25

Relation between DNA profiles and MIB throughout the seasons

18

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8

Sampling sites

MIB

(ng

L-1)

Pima Rd through 29th Ave (8/28/02)

IV

DNAmarkerPima Scotts

Rd Rd56th

ST24th

ST16th

STCentral

Ave19th

Ave29th

Ave

1 2 3 4 5 6 7 8

A

B

Relation between DNA profiles and MIB at selected sites

0

10

20

30

40

50

60

70

80

90

24th ST 16th ST CentralAve

19th Ave 29th Ave

Sampling sites

MIB

(ng

L-1)

C

A

B

Before Cu2+ treatment

After Cu2+ treatment

After Cu2+ treatment Before Cu2+ treatment

DNAmarker 24th ST 16th ST Central 19th Ave 29th Ave

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1: Anabaena TAC4262: Saguaro lake sample (6/15/04)3: Nodularia strain 5754: Cylindrospermopsis AWT2055: Plankothrix PCC78116: Microcystis LE-37: Nostoc PCC731028: Saguaro lake sample (6/22/04)9: Aphanizomenon strain Zayi10. No DNA sample

100 bp DNA ladders used at both sides of the sample lanes.

Detection of Cylindrospermopsis in Saguaro Lake samples using a specific gene probe

1 2 3 4 5 6 7 8 9 10

Summary• Algae metabolites include:

– Bulk OM (mg/L) that produces THMs and is enriched in organic nitrogen

– T&O terpenoids (ng/L) with high intracellular levels. Produced by cyanobacteria with specific genetic fingerprints, but which represent a small percentage of the total algae biomass

– Cyanotoxins (ug/L) are present, have lead to fish kills (makes news headlines), and has raised concern by many WTPs

– 16S rDNA primers have resolved 16 different cyanobacterialsequences from the AZ Canal by PCR and DGGE techniques

• Variable hydrologic conditions, reservoir management, and water conveyance will affect magnitude for the significance of algae metabolites in Arizona

• T&O and cyanotoxins can be removed in WTPs, but provide added motivation for WTPs to install GAC

• #1 problem reported by each WTP related to algae: how to control inplant algae growth without prechlorination. So this is our challenge for 2005

• Arizona watersheds are representative of many others throughout the western US

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Paul Westerhoff, Ph.D.Paul Westerhoff, Ph.D.Arizona State University Arizona State University

Associate ProfessorAssociate ProfessorDepartment of Civil & Environmental Engineering Department of Civil & Environmental Engineering

http://ceaspub.eas.asu.edu/pwest/http://ceaspub.eas.asu.edu/pwest/

Acknowledgements:Acknowledgements:AwwaRFAwwaRF

City of PhoenixCity of PhoenixCity of TempeCity of TempeCity of PeoriaCity of Peoria

Salt River ProjectSalt River ProjectCentral Arizona ProjectCentral Arizona Project

Michelle Cummings, Michelle Cummings, MyLinhMyLinh Nguyen, Mario EsparzaNguyen, Mario Esparza--SotoSotoASU ASU –– NSF Water Quality CenterNSF Water Quality Center