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Application of pigment Application of pigment analysis and CHEMTAX to analysis and CHEMTAX to field studies of field studies of phytoplankton communities phytoplankton communities Simon Wright Simon Wright Australian Antarctic Division Australian Antarctic Division

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Page 1: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Application of pigment analysis Application of pigment analysis and CHEMTAX to field studies of and CHEMTAX to field studies of

phytoplankton communitiesphytoplankton communities

Simon WrightSimon WrightAustralian Antarctic DivisionAustralian Antarctic Division

Page 2: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

This powerpoint presentation has been cut back considerably to reduce its size from 29MB to somewhat closer to the 2MB requested.

In doing so, I have had to exclude all of my antarctic and shipboard photos (not a great scientific loss), but also a photo sequence on exactly how we filter and extract our samples.

I am placing these separately on a ‘Pigment HPLC’ web site via the Australian Antarctic Division. I will forward the address to the PICODIV site.

I have also annotated some of the slides to make them more stand-alone.

Page 3: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

“Find a simple chemical technique for determining the

abundance of phytoplankton”

This talk will consider how far we have come toward that goal.

The application of pigment analysis to biological oceanography was largely pioneered by Shirley Jeffrey. In one of her first post-docs with George Humphrey, she was given the challenge to:

Page 4: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Outline

Historical perspective – development of CHEMTAX

BROKE 1996 – CHEMTAX at work

Optimising pigment analysis and data

CHEMTAX problems

‘unusual’ algae

choice of inputs

variability of algal pigment content

Modelling pigments in the underwater light field

Current directions

Conclusions

Page 5: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

CLOUD

UV VIS

ICEDMS

UV Pig

Carotenoids Chlorophyll a

Photosynth

Faeces

ZooplanktonBirds Fish

Whales etc

Grazing Food Chain

Marine Snow

Phytoplankton

Viruses Bacteria

Protozoa

Aggregation

Nutrients N, P, Si, Fe

SINKING

DOM

MIXINGMixed Layer

PYCNOCLINE

CO2 O2

WIND DRIVES MIXING

ADVECTION

LIGHT

How do we measure the abundance of phytoplankton in the presence of protozoa, bacteria, detritus and viruses?

Page 6: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Many species can be identified by electron microscopy but cannot be identified by light microscopy [photos omitted]

Even if they could be identified by light microscopy, the statistics of enumeration means that 10000 cells of each type must be counted to ensure 1% precision.

And….“Die numerische Erfassung von Phytoplankton-Arten gleicht einer Danaiden-Arbeit die mit einer Zerstoerung von Koerper und Seele einhergeht” Haeckel, 1890

(roughly =)…….Plankton counting is a task that cannot be achieved without ruin of body and soul

Chlorophylls and carotenoids are useful chemical markers that, in the open ocean, are only found in living phytoplankton. By chromatographically separating them, we can determine the composition and abundance of phytoplankton populations.

Page 7: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Chl a Astaxanthin

Diadinoxanthin

Fucoxanthin

Neofucoxanthin

Phaeophytin a

Carotenes

Chl b

PeridininNeoperidinin

Neoxanthin

Chlorophyllide aChl c

Phaeophorbide aOrigin

TLCJeffrey 1974

Page 8: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Jeffrey 1974

Pigments Algal types or biological processes indicatedChl aChl c Diatoms and / or chrysomonadsFucoxanthinDiadinoxanthin

Chl b Green algaeNeoxanthin

Peridinin DinoflagellatesChlorophyllide a Senescent diatoms (due to chlorophyllase)Phaeophorbide a Faecal pellets of copepodsPhaeophytin a Us. Trace amounts on all c’gramsAstaxanthin Copepods presentHigh chl c:a ratios Senescent phytoplankton or detritus

In earlier times, we thought in terms of individual marker pigments indicating particular algal types or processes.

Page 9: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Abaychi and Riley 1979

Mantoura and Llewellyn 1983

Wright and Shearer 1984

Zapata et al. 1987

Wright et al. 1991

Kraay et al. 1992

Goericke and Repeta 1993

van Heukelem et al. 1994

Barlow et al. 1997

Zapata et al. 2000

HPLC systems development

Steady improvement in HPLC techniques led to recognition of many more pigment markers

Page 10: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Major marker pigments

Ubiquitous Chl a

Unambiguous Alloxanthin Peridinin Prasinoxanthin

Jeffrey and Vesk (1997)

Page 11: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Major marker pigments

Ubiquitous Chl a

Unambiguous Alloxanthin Peridinin Prasinoxanthin

Shared e.g. Fucoxanthin Chl b Zeaxanthin Violaxanthin

Page 12: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Major marker pigments

We can no longer talk in terms of individual marker pigments. Instead we talk of “SUITES” of pigments that may cross conventional taxonomic boundaries.

By the late 80’s it became very apparent that normal interpretation of pigment data amounted to little more than guesswork.

There was an urgent need for objective computational methods for determining the phytoplankton community composition from pigment data.

Page 13: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Computational methods in pigment analysis

Page 14: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

1. Simple or multiple linear regression

e.g. Gieskes and Kraay 1983

Statistically sound

Does not distinguish algal groups with

shared marker pigments

Computation methods

Page 15: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

1. Simple or multiple linear regression

2. Multiple simultaneous equations

Everitt et al. 1990

Letelier et al.1993

Peekin 1997

van Leeuwe et al. 1998

Computation methods

Page 16: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

1. Simple or multiple linear regression

2. Multiple simultaneous equations

Letelier et al. 1993

[Chla]Prochl = 0.91([Chlb] - 2.5[Prasino])

[Chla]Cyano = 2.1{[zeax] -0.07([Chlb] - 2.5[Prasino])}

[Chla]Chrys = 0.9[19’-but] Chrys

[Chla]Prym = 1.3[19’-hex] Prym

[Chla]Bacill = 0.8{[fuco] - (0.02[19’-hex] Prym +

0.14[19’-but] Chrys)}

[Chla]Dino = 1.5[perid]

[Chla]Pras = 2.1[prasino]

Computation methods

Page 17: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

1. Simple or multiple linear regression

2. Multiple simultaneous equations

Allowed shared marker pigments

Difficult to set up

Computation methods

Page 18: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

1. Simple or multiple linear regression

2. Multiple simultaneous equations

3. Matrix factorization

Computation methods

Page 19: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

1. Simple or multiple linear regression

2. Multiple simultaneous equations

3. Matrix factorization

CHEMTAX (Mackey et al. 1996, Wright et al. 1996)

Computation methods

Page 20: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

uses a table of concentration ratios of all pigments for each algal group

Algal Class PigmentChl c3 Peridinin 19’-but Fucox 19’-hex Prasinox

Diatom - - - 0.75 - -Hapto3 0.045 - - - 1.7 -Hapto4 0.048 - 0.25 0.58 0.54 -Cryptophyte - - - - - -Prasinophyte - - - - - 0.32Chlorophyte - - - - - -Dinoflagellate - 1.06 - - - -Cyanobacteria - - - - - -

Each ratio is iteratively modified to minimize the difference between observed and calculated total pigment concentration

Matrix factorization

(Half of table only)

Page 21: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Currently based on a MATLAB platform

Can distinguish algal groups with qualitatively

identical pigment compositions using differences

in pigment ratios (Wright et al, 1996)

Requires the user to enter the expected mix of

algal components which the software then

optimises

Microscopic examination of the samples is thus

essential

CHEMTAX software

Page 22: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Changes in pigment ratios with depth

It is essential to split samples into a series of depth strata that are computed independently (Mackey et al., 1998, Higgins and Mackey, 2000, Wright and van den Enden, 2000)

Hapto4 Pigment Ratios vs Depth

0

0.5

1

1.5

2

2.5

0 100 200

Depth (m)

Pig

men

t:C

hl

a R

atio

chlc3/chla

19butfu/chla

fucox/chla

19hexfu/chla

diadinox/chla

1004 Samples were split into 8 depth layers. Samples from each layer were computed independently. Graph at left shows the computed ratios for type 4 haptophytes (e.g. Phaeocystis spp.) vs. depth.The smooth change with depth suggests that CHEMTAX is measuring something real.

Page 23: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Phytoplankton community structure and stocks in the East Antarctic marginal ice

zone (BROKE survey, January - March 1996) determined by CHEMTAX analysis of HPLC

pigment signatures

S. W. Wright and R. L. van den Enden (2000) Deep-Sea Research II, 47, 2363 - 2400

Does CHEMTAX work?

An example where it worked well to map phytoplankton communities in the Southern Ocean

Page 24: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Study Area

Chlorophyll by satellite

Page 25: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

67891011121314

15

1617

18

19

20

2122 2930

31

32

33

34353637383940

41

42

43

484950515253

54

5556

57

58

59

6061

65

66

67686970717273

74

75

76

7778

8283

84858687888990

91

92

93

94

9596

100

101102103104105106

107

108

109

110

111

112113

119120

121

122

123

124

125126127128129130131132

136137

138

139

140

141

142

143144

145146147

7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0

Longitude (deg)

-67

-66

-65

-64

-63

-62L

atit

ud

e (d

eg)

BROKE CTD sites

6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0- 7 5

- 6 5

- 5 5

- 4 5

- 3 5Location Map AUSTRALIA

ANTARCTICA

STUDY AREA1 4 7 9

11 1316

18

Ice shelf

Ice shelf

1000m

Page 26: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.8 -65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

TEMP112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

-2 .0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.01.21.41.61.8

ice

Antarctic Slope Front

Tmin

Pycnocline

Page 27: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Total Chlorophyll a 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.1

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

ice

ASF

Tmin

Pycnocline

Page 28: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Diatoms 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

ice

ASF

Tmin

Pycnocline

Page 29: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Haptophyte Type 3 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.00.10.10.20.20.30.30.30.40.40.50.60.60.60.70.80.80.90.90.91.0

ice

ASF

Tmin

Pycnocline

Page 30: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Haptophytes Type4 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

ice

ASF

Tmin

Pycnocline

Page 31: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Prasinophytes 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

0.14

ice

ASF

Tmin

Pycnocline

Page 32: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Chlorophytes 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.000.010.020.030.040.050.060.070.080.090.100.110.120.130.140.150.160.170.180.190.200.21

ice

ASF

Tmin

Pycnocline

Page 33: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Cryptophytes 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

ice

ASF

Tmin

Pycnocline

Page 34: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.8 -65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Phaeophytin a 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.000.010.020.030.060.090.120.150.180.210.240.270.300.330.360.390.420.450.480.510.540.570.60

ice

ASF

Tmin

Pycnocline

Page 35: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

-65.6 -65.4 -65.2 -65.0 -64.8 -64.6 -64.4 -64.2 -64.0 -63.8 -63.6 -63.4 -63.2 -63.0

Latitude

Prasinophytes 112E

-200

-180

-160

-140

-120

-100

-80

-60

-40

-20

0

Dep

th (

m)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

0.14

ice

ASF

Tmin

Pycnocline

Page 36: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

BROKE conclusions

1. Effect of StratificationMIXED STRATIFIED

Chl a (µg.L-1) 0.4 2.0 Diatoms Pycnocline Pycnocline Prasinophytes Tmin Pycnocline Hapto4s Tmin Pycnocline

2. ‘Hole’ in algal distribution at the ice edge, except for Cryptophytes

3. Generally uniform to pycnocline under ice

4. Importance of frontal features - downwelling tongue from Tmin layer

These observations could not have been obtained using microscopy or any other method currently available.

Page 37: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Optimizing pigment data

Page 38: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Aim: Sensitivity – maximum peak height

Accuracy

Integrity – lack of pigment degradation

Reproducibility of retention times

Data reliability

These aims require care at each step of the process

Optimising pigment data

Page 39: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Field sampling

It is important to realise that the pigment composition of the sample starts changing from the moment it is enclosed in a dark Niskin bottle.

For maximum reproducibility of pigment ratios, all samples should be subjected to the same time delay from collection to end of filtration, and all should be handled in the same light regime (preferably very dim).

For example, in our cruises, it is normally 40 minutes before we can sample the Niskin bottles after the physical and chemical oceanographers have collected their samples. Thus we never see diatoxanthin. It has all been converted to diadinoxanthin in the dark.

Page 40: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Sample filtration

Dim light, cool lab

Fluorescence check (each sample is measured in a Turner fluorometer - double checks HPLC result)

Small filter (13mm GF/F, extractable in 1.5 ml solvent)

Removal of water from filter reproducibly

Double label cryotubes (black pen & engraver)

Freeze in liq. N2 – directly into 45 L Dewar

Page 41: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Sonication in methanol

Small volumes (1.5ml)

Internal standard

140 ng β–apo-8’-carotenal (Fluka)

Precision

Accounts for volume changes

Checks injection status

Straight to refrigerated (-10°C) autoinjector stage

Extraction

Page 42: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

More on the internal standard and data reliability

As well as improving analytical precision, the internal standard provides data reliability.

Thus if you have a sample with no chlorophyll but a good internal standard peak, then you know that the injection and the chromatogram are OK.

The filtration may have been faulty (e.g. holed filter). This is where you go back to check the fluorescence measurement you made while filtering.

The fluorescence check has also saved us when fatigued shipboard workers have labelled two sets of cryotubes with the same numbers!

Page 43: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

A photographic sequence of pigment extraction has been omitted here, to reduce the size of the file. It will be posted on the Australian Antarctic Division web site. The address will be forwarded to the PICODIV site.

Page 44: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Our extraction procedure

Extraction is performed in 2.5ml plastic syringes, with a leur lock tap.Add 1.5 ml cold methanolAdd 25 ul internal standard solution with ~140ng apo-8’-carotenal (Fluka)(add these first to avoid delays once filter is thawed)Remove frozen filter from cryotubeWhile still frozen, cut 1 x lengthways, 4 x sideways into small pieces, with small scissors. Pieces fall into the syringe and thaw.Extract with a probe sonicator (4mm diameter, 50 W, 60 seconds), moving the syringe up and down to ensure that no pieces of filter avoid the sonic beam.The filter is completely disrupted into a slurry. It gets quite warm.Immediately put a plunger into the syringe, attach a 3 mm dia. leur lock filter (0.45 um, nylon, Advantec MFS Inc) and a needle, and squirt the extract into an amber autosampler vial.Place the autosampler vial immediately into a refrigerated (-10°C) autosampler rack.This process averages 1 min 40 sec from starting cutting to completion.

All syringe parts are washed with ethanol and dried before reassembly

Page 45: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Reproducibility

Make up solvents by weight

Column thermostatted in a water bath (more stable than air oven)

Autoinjection

Tubing minimum length

HPLC Analysis

Page 46: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0 5 10 15 20 25 30

c2

pdn

ddx

dnx

dtx

apo8

a

BB

Graph showing reproducibility of retention times through the day for a series of dinoflagellate samples

RT

Sample #

Chl c2

peridinin

diaddinoxdiatoxInt Std

Chl aBB carot

Page 47: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Peak identification

Mixed standard every batch

RT table

Check column performance

Reference spectral library

HPLC Analysis

Page 48: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

The SCOR/UNESCO method • separated 52 pigments in 20 minutes C18 monomeric column Ternary gradient

Methanol + ammonium acetate Acetonitrile Ethyl acetate

Excellent resolution of marker carotenoids Inadequate resolution of polar chlorophylls and

divinyl chlorophylls

HPLC Method (1991): Wright et al., 1991

Page 49: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Waters Symmetry C8 (monomeric) column Binary gradient

A=Methanol:acetonitrile:pyridine (pH 5.0) (50:25:25 v/v/v) B= Methanol:acetonitrile:acetone (20:60:20 v/v/v) 25C

HPLC Method (2000): Zapata et al. (2000)

Main technique in our lab Good resolution of marker carotenoids, esp fucoxanthin derivs Excellent resolution of polar chlorophylls Divinyl chlorophylls resolved from chlorophylls Resolution order differs from Wright et al. (1991) Complementary techniques

Page 50: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Peak detection

Diode array detection436 nm470 nm665 nm400 – 480 nm (sum)

(summing the wavelengths on the diode array detector provides about 3 x improvement in signal : noise ratio

This greatly improves sensitivity)

Fluorescence detectionexcitation: Broad band blue (Turner fluorometer filter)emission: Long pass red (> 630nm)

Page 51: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Adjusting HPLC separation

HPLC separations will not look exactly like published methods:• different dead volume between pump and column• batch variations in columns

Resolution and retention time may require adjustment to suit particular samples• adjust gradient profile• adjust solvent composition

Page 52: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

• Samples integrated using Waters Millennium

software

• Peaks matched against library using Millennium

• Data exported to Excel (files concatenated in MS-

DOS)

Data tabulation and checking

Page 53: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

• Peaks tabulated and error checked in Excel

• Exported to Prepro files for CHEMTAX

• CHEMTAX output files tabulated using Excel macros

• Distributions plotted using Surfer

Data tabulation and checking

Page 54: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Things may be different in Bermuda

(You may need to modify these methods elsewhere)

Page 55: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Does CHEMTAX have problems?

Two examples where problems were encountered (from Henriksen et al.)• algae with unusual pigmentation• inconsistent pigment ratios (?)

Problems due to variable underwater light field follow:

Page 56: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

“Exceptional” blooms of Chattonella spp. (Raphidophyceae) and Gymnodinium chlorophorum (Dinophyceae) in Danish waters

• Peter Henriksen– National Environmental Research Institute, Denmark

• Helene Munk Sørensen– Århus County, Denmark

• Gert Hansen– IOC Science and Communication Centre on Harmful Algae,

Denmark

Page 57: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Chattonella spp. in Danish waters 1998

Page 58: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Sample dominated by Chattonella spp.(Danish west coast 12 May 1998)

Page 59: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Green dinoflagellate blooming in Danish waters 1999

Page 60: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Pigment profiles of strains GH-4 (Århus Bight 1999) and K-539 (Gymnodinium chlorophorum)

chl bneox

violax

lutein

chl a

Page 61: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Århus Bight 1999

0

500

1000

1500

2000

J F M A M J J A S O N D

Month

Bio

ma

ss

g C

/L)

Other groups

Cyanobacteria

Myrionecta

Diatoms

Dinoflagellates

Page 62: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

The two dominant organisms were:

• A raphidophyte, Chattonella sp., with pigments characteristic of pelagophytes and haptophytes

• A dinoflagellate with pigments characteristic of chlorophytes

Message: Must use a microscope

Page 63: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Species included in2 different pigment ratio-files

Dinoflagellates

Ratios_1

Scrippsiella sp.

Ratios_2

Scrippsiella sp.

Pelagophytes Literature values Literature values

Diatoms Ditylum brightwellii 5 Pseudo-nitzschia1 Ditylum

Haptophytes Chrysochromulinapolylepis

TYPE 1 (fuco):

8 Chrysochromulina,

1 Phaeocystis,

2 Prymnesium

TYPE 2 (hex):

7 Chrysochromulina

Cryptophytes Rhodomonas salina Rhodomonas salina

Prasinophytes TYPE 1 (+prasino):Pseudoscourfeldiamarina

TYPE 2 (- prasino):Pyramimonasdisomata

TYPE 1:

5 species/genera

TYPE 2:

7 Pyramimonas,

1 Tetraselmis,

1 Nephroselmis

Chlorophytes Brachiomonas sp. Brachiomonas sp.

Euglenophytes Eutreptiellagymnastica

Eutreptiellagymnastica

Cyanobacteria Synechococcus sp. Synechococcus sp.

In another expt they compared microscopically estimated C biomass (itself selective) with CHEMTAX attribute of chl a, starting from 2 different ratio files (above)

Page 64: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Århus Bight 1997-99Dinoflagellates

0

20

40

60

80

100

Co

ntr

ibu

tio

n t

o c

hlo

ro

ph

yll a

/ C

bio

mass (

%)

Rat_1

Rat_2

C biomasse

Gymnodinium chlorophorum

Page 65: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Århus Bight 1997-99Diatoms

0

20

40

60

80

100

Co

ntr

ibu

tio

n t

o c

hlo

rop

hy

ll a

/ C

bio

ma

ss

(%

)

Rat_1

Rat_2

C biomasse

Page 66: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Århus Bight 1997-99Haptophytes

0

10

20

30

40

50

Co

ntrib

utio

n t

o c

hlo

ro

ph

yll a

/ C

bio

mass (

%)

Rat_1

Rat_2

C biomasse

Page 67: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

I believe these correlations were so bad because a whole year’s data (summer and winter) was included in the analysis.

The pigment ratios would have changed between seasons, contravening CHEMTAX’s assumption that pigment ratios are constant through the data set.

Page 68: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Taxon specific subsurface chlorophyll Taxon specific subsurface chlorophyll maxima in the Southern Oceanmaxima in the Southern Ocean

south of Tasmania south of TasmaniaMarch 1998March 1998

S.W.Wright, R. L. van den Enden, F. B. Griffiths, A. C. Crossley

(in prep)

Page 69: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

S A Z T o t a l c h l a ( u g / L )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Subantarctic zone (SAZ)

Sub

trop

ical

fro

nt

Sub

anta

rctic

fro

nt

Pol

ar f

ront

The chlorophyll in the SAZ is relatively uniformly distributed vertically, but CHEMTAX found the populations to be stratified as follows:

Page 70: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.000.020.040.060.080.100.120.140.160.180.200.220.240.26

Diatom chl a

Page 71: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Haptophyte3 Chl a (ug/L)

Page 72: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.000.010.020.030.040.050.060.070.080.090.100.110.120.13

Haptophyte4 chl a (ug/L)

Page 73: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.000.010.020.030.040.050.060.070.080.090.100.110.12

Prasinophyte chl a (ug/L)

Page 74: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.000.010.020.030.040.050.060.070.080.090.100.11

Chlorophytes chl a (ug/L)

Page 75: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.0200.0220.024

Cryptophyte chl a (ug/L)

Page 76: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.000

0.004

0.008

0.012

0.016

0.020

0.024

0.028

Dinoflagellate chl a (ug/L)

Page 77: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.000.010.020.030.040.050.060.070.080.090.100.110.12

Cyanobacteria chl a (ug/L)

Page 78: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Was the observed stratification a result of splitting the data into depth strata and computing them independently?

Hapto4 Pigment Ratios vs Depth

0

0.5

1

1.5

2

2.5

0 100 200

Depth (m)

Pig

men

t:C

hl

a R

atio

chlc3/chla

19butfu/chla

fucox/chla

19hexfu/chla

diadinox/chla

Remember this?

Only the Hapto3 data suggested not - the subsurface chl maximum deepened between layers toward the south of the transect, as follows.

Page 79: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 5 0 5 1 5 2 5 3 5 4 5 5

L a t i t u d e ( S )

-250

-200

-150

-100

-50

0

Dep

th (

m)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Haptophyte3 Chl a (ug/L)

Page 80: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

The microscopic data had neither the sampling density nor the statistical precision to determine whether these patterns were real (hence the need for CHEMTAX in the first place).

The only comparable data were flow cytometric counts of cyanobacteria for four stations in the SAZ.

Comparing cyanobacterial counts with CHEMTAX estimates of cyanobacterial chlorophyll showed that CHEMTAX consistently underestimated cyanobacterial abundance near the surface and overestimated at depth (data for 4 stations follows)

Page 81: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120 160 200 240

Depth (m )

0E+000

2E+007

4E+007

6E+007

8E+007

Cells / L

0

0.02

0.04

0.06

0.08

Chla (ug/L)

SAZ CTD 004

CyanobacteriaFlow cytom eter vs. CHEM TAX

Page 82: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120

Depth (m )

0E+000

1E+007

2E+007

3E+007

4E+007

5E+007

Cells / L

0

0.02

0.04

0.06

0.08

Chla (ug/L)

SAZ CTD 019

CyanobacteriaFlow cytom eter vs. CHEM TAX

Page 83: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 50 100 150 200 250

Depth (m )

0E+000

2E+007

4E+007

6E+007

8E+007

1E+008

Cells / L

0

0.02

0.04

0.06

0.08

0.1

Chla (ug/L)

SAZ CTD 029

CyanobacteriaFlow cytom eter vs. CHEM TAX

Page 84: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120

Depth (m )

0E+000

2E+006

4E+006

6E+006

8E+006

Cells / L

0

0.004

0.008

0.012

0.016

Chla (ug/L)

SAZ CTD 041

CyanobacteriaFlow cytom eter vs. CHEM TAX

Page 85: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

If you calculate the amount of cyanobacterial chlorophyll per cell vs depth (CHEMTAX cyano chl / flow cytometer counts), you find that it is relatively constant near the surface, then increases dramatically at depth.

Four stations follow with cyano chl per cell and cell counts vs depth.

NB. Ignore noisy data where cell counts approach zero at depth.

Page 86: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120 160 200 240Depth (m )

0E+000

2E+007

4E+007

6E+007

8E+007

Cells / L

0

2

4

6

8

10

Chla/cell (fg)

SAZ CTD 004

CyanobacteriaFlow cytom eter vs. Chl a / cell

Page 87: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120

Depth (m )

0E+000

1E+007

2E+007

3E+007

4E+007

5E+007

Cells / L

0

2

4

6

8

10

Chla/cell (fg)

SAZ CTD 019

CyanobacteriaFlow cytom eter vs. Chl a / cell

Page 88: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 50 100 150 200 250Depth (m )

0E+000

2E+007

4E+007

6E+007

8E+007

1E+008

Cells / L

0

4

8

12

16

Chla/cell (fg)

SAZ CTD 029

CyanobacteriaFlow cytom eter vs. Chl a / cell

Page 89: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120Depth (m )

0E+000

2E+006

4E+006

6E+006

8E+006

Cells / L

0

1

2

3

4

Chla/cell (fg)

SAZ CTD 041

CyanobacteriaFlow cytom eter vs. Chl a / cell

Page 90: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120

Depth (m )

0

2

4

6

8

10

Chla/cell (fg)

SAZ CTD 041

CyanobacteriaCHEM TAX Chl a / cell

vs. Depth

SAZ CTD 004SAZ CTD 019SAZ CTD 029

Summary

Page 91: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

How does cellular pigment content respond to irradiance?

Is this real?

We compared calculated pigment per cell with data obtained in our lab over the last 2 years measuring :

Page 92: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Variation of pigment content in response to irradiance

Several species cultured under a range of irradiances • 10 – 888 uE m-2 s-1

• ‘marine blue’ filtered light• log phase cultures used

Pigments analysed using Zapata et al. (2000)

Cells counted by flow cytometry

Page 93: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Species employed

• Phaeodactylum tricornutum - Diatom• Pavlova gyrans - Haptophyte• Emiliania huxleyi - Haptophyte• Dunaliella tertiolecta - Chlorophyte• Pelagococcus subviridis - Pelagophyte• Synechococcus sp. -

Cyanobacterium• Amphidinium carterae - Dinoflagellate

• Phaeocystis antarctica x 2 - Haptophyte• Polarella glacialis - Dinoflagellate

• Homo sapiens• Lana Pirrone - University of Tasmania• Suzanne Roy - Université de Québec, Canada• Peter Henriksen - Danish Environmental Research Inst.

Page 94: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Inlet water flow

Insulation tank

Air flow

Air flowAir flow

Fan

Fan

Fan

Light regime

Powermeter

DC

DC

DC

Light sourceLight source

AC

Outlet water flow

AC

Powermeter

AC

AC

Insulation box

Powersource

Thermo-regulator

Powersource

Light gradient apparatus

Page 95: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division
Page 96: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 200 400 600 800 1000

Irradiance (uE)

0

0.2

0.4

0.6

0.8

fg /

ce

ll

0

0.1

0.2

0.3

0.4

0.5

0.6

fg/c

ell

Pigment content per cellvs Irradiance

Pavlova gyrans

Chlorophyll a Fucoxanthin

Page 97: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 200 400 600 800 1000

Irradiance (uE)

0

0.04

0.08

0.12

0.16

fg /

ce

ll

0

0.04

0.08

0.12

0.16

fg/c

ell

Pigment content per cellvs Irradiance

Pavlova gyrans

Chl c1Chl c2

Page 98: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 200 400 600 800 1000

Irradiance (uE)

0

0.1

0.2

0.3

fg /

ce

ll

0

0.01

0.02

0.03

0.04

fg/c

ell

Pigment content per cellvs Irradiance

Pavlova gyrans

Diadinoxanthin Diatoxanthin

Page 99: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 200 400 600 800 1000

Irradiance (uE)

0

0.2

0.4

0.6

0.8P

igm

en

t / c

hl a

ra

tio

0 . 1

0 . 2

0 . 3

0 . 4

Pig

me

nt

/ Ch

l a r

ati

o

Pigment / Chl a ratio vs Irradiance

Pavlova gyransDiadinox / Chl a Diatox / Chl a

Page 100: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 200 400 600 800 1000

Irradiance (uE)

0

0.04

0.08

0.12

0.16

0.2

Pig

me

nt

/ ch

l a r

ati

o

0 .05

0.1

0.15

0.2

0.25

Pig

me

nt

/ Ch

l a r

ati

o

Pigment / Chl a ratio vs Irradiance

Pavlova gyransChl c1 / Chl a Chl c2 / Chl a

Page 101: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 200 400 600 800 1000

Irradiance (uE)

0

0.2

0.4

0.6

0.8P

igm

en

t / c

hl a

ra

tio

0 .04

0.08

0.12

0.16

Pig

me

nt

/ Ch

l a r

ati

o

Pigment / Chl a ratio vs Irradiance

Pavlova gyransFuco / Chl a B,B-car / Chl a

Page 102: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

We obtained equations for these lines so that we could model the pigment /cell vs irradiance, then put those equations into an underwater light field to model pigment /cell vs depth.

Page 103: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Pavlova Pigment vs Irradiance

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 200 400 600 800 1000 1200

Irradiance (uE)

Pig

men

t /

cell

(p

g)

Fuc

Ddx

chl a

c2

c1

Page 104: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Dunaliella Pigment vs Irradiance

0

0.5

1

1.5

2

2.5

3

3.5

0 200 400 600 800 1000 1200

Irradiance (uE/m-2/s)

Pig

men

t /

cell

(p

g)

Zea

Lutein

Chlb

Chla

Page 105: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Synechococcus Pigment vs Irradiance

0

2

4

6

8

10

12

14

16

18

20

0 200 400 600 800 1000 1200

Irradiance (uE /m-2/ s)

Pig

men

t /

cell

(fg

)

Zea

Chl a

Page 106: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Irradiance vs Depth

0

200

400

600

800

1000

1200

0 50 100 150 200

Depth (m)

Irra

dia

nc

e (

uE

/ m

-2 /

s)

Light

Vertical Atten. Coeff. = 0.046

Page 107: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Combining these models showed that pigment / cell varied with depth and produced a subsurface chl maximum in all species.

Page 108: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Dunaliella Pigment vs Depth

0

0.5

1

1.5

2

2.5

3

3.5

0 50 100 150 200

Depth (m)

Pig

men

t /

cell

(p

g)

Zea

Lutein

Chlb

Chla

Page 109: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Pavlova Pigment vs Depth

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150

Depth (m)

Pig

men

t (f

g/c

ell)

Fuc

Ddx

chl a

c2

c1

Page 110: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Modelled Synechococcus Pigment vs Depth

0

2

4

6

8

10

12

14

16

18

20

0 50 100 150 200

Depth (m)

Pig

men

t /

cell

(fg

)

Zea

Chl a

Page 111: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Compare the last graph with the only real data we have:

Page 112: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

0 40 80 120

Depth (m )

0

2

4

6

8

10

Chla/cell (fg)

SAZ CTD 041

CyanobacteriaCHEM TAX Chl a / cell

vs. Depth

SAZ CTD 004SAZ CTD 019SAZ CTD 029

Page 113: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Changes in pigment ratios with depth

It seems that CHEMTAX accurately calculated the pigment response of cyanobacteria and hence presumably the other categories.

Page 114: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

More data required on pigment content vs light fields

Need to model algal responses to depth

Methods to convert chlorophyll estimates to cells / L or total carbon?

What about variable light climates?

Message

Page 115: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

• Lack of data on pigment composition of

phytoplankton

• Few species done

• Ideally should know characteristics of major species

• But usually don’t know what proportion the species are

in, so can’t calculate average

• Refine the limits range

• Normally have to let CHEMTAX calculate average ratio

• Nutrient effects?

How do we model pigment content?

Page 116: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Thought:

Maybe we can use cells such as cyanobacteria and cryptophytes (each readily distinguishable in both flow cytometry and CHEMTAX) to determine their pigment / cell and use them as proxies for the underwater light field.

Page 117: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Current directions

New pigmentsChl c derivsNon polar chl csGyroxanthin diester4 keto-acyl fucoxanthinsUn421

Tracking particular species e.g toxic dinos

Page 118: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Differentiate between nanoplankton and microplankton

• ecologically meaningful • remove contribution of large diatoms from pools

of fucoxanthin etc.• Simplifies CHEMTAX interpretation

Use of size fractionation

Page 119: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Current workup software (Excel, Surfer macros)

Translate CHEMTAX to Excel?

Software should incorporate changing pigment ratios with depth or (preferably) light field.

Software should identify changes of oceanic region and/or gross species composition within a sample set.(following)

Software improvements

Page 120: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

Sample Depth (m) CTD NO. Lat Long chl c3 peri but fuc hex pras violax ddx allox lutein zea98MAR151 15.8 041 48.22445 141.4017 0.031392 0.027687 0.013293 0.072922 0.064616 0.00591 0.004732 0.035452 0.00051 0.003593 0.00314898MAR152 31.6 041 48.22445 141.4017 0.035344 0.027601 0.013483 0.071824 0.065545 0.006021 0.005369 0.037461 0.000958 0.003663 0.00327298MAR153 44.4 041 48.22445 141.4017 0.02762 0.027792 0.012789 0.070482 0.06295 0.005781 0.003962 0.035773 0.001032 0.003611 0.00288698MAR154 60.7 041 48.22445 141.4017 0.034245 0.028429 0.013522 0.073983 0.066931 0.006996 0.004527 0.037545 0.000895 0.003833 0.00340998MAR155 87.5 041 48.22445 141.4017 0.031448 0.026573 0.013022 0.070428 0.062184 0.005939 0.004872 0.033211 0.000988 0.003731 0.00319298MAR156 119.4 041 48.22445 141.4017 0.00206 0.001961 0.000882 0.005445 0.002631 0.000595 0.000181 0.000839 0.000285 0 0.00012398MAR157 8.3 043 49.5136 141.7885 0.032184 0.031934 0.0139 0.064827 0.073772 0.006293 0.005072 0.032724 0.000727 0.00307 0.00294498MAR158 17.6 043 49.5136 141.7885 0.02864 0.02996 0.01272 0.056662 0.065716 0.005188 0.003753 0.030954 0.001069 0.002544 0.00225498MAR159 31.7 043 49.5136 141.7885 0.035289 0.032554 0.013662 0.062159 0.071614 0.006577 0.00505 0.032701 0.001156 0.002741 0.00260398MAR160 49.2 043 49.5136 141.7885 0.034737 0.03299 0.013943 0.063439 0.072411 0.006559 0.00429 0.033522 0.000982 0.00305 0.00241398MAR161 62.3 043 49.5136 141.7885 0.027936 0.033016 0.013617 0.063596 0.072187 0.006351 0.005184 0.032443 0.000334 0.002721 0.00253698MAR162 74.4 043 49.5136 141.7885 0.029441 0.035429 0.014008 0.064851 0.073278 0.006214 0.00395 0.033054 0.000719 0.002975 0.00264698MAR163 89.8 043 49.5136 141.7885 0.037024 0.032282 0.013194 0.061935 0.068627 0.005887 0.004741 0.030555 0.001009 0.002916 0.00202298MAR164 104.4 043 49.5136 141.7885 0.008651 0.003389 0.004103 0.029902 0.008678 0.002161 0.000701 0.003551 0 0 0.00049798MAR165 120.6 043 49.5136 141.7885 0.001619 0.002375 0.001465 0.007307 0.003128 0.000336 0.000449 0.001023 0.000225 0 0.00012398MAR166 152.4 043 49.5136 141.7885 0.001276 0.002911 0.000611 0.003189 0.001495 0.000518 0 0.000829 0 0.000311 0.00012798MAR167 205.8 043 49.5136 141.7885 0.000909 0.001968 0.000797 0.006559 0.00127 0 0 0.001219 0.000147 0 0.00032998MAR168 250.3 043 49.5136 141.7885 0 0 0.000406 0.006389 0.001237 0 0 0 0 0 098MAR169 5.8 045 49.60053 141.8961 0.032677 0.040781 0.014809 0.064245 0.07578 0.007188 0.003831 0.047372 0.001394 0.003732 0.00263898MAR170 18 045 49.60053 141.8961 0.029472 0.034098 0.01317 0.051162 0.071568 0.006875 0.0044 0.040767 0.001243 0.003521 0.00277198MAR171 30.1 045 49.60053 141.8961 0.035932 0.037349 0.013961 0.062603 0.071785 0.006484 0.005567 0.04741 0.001365 0.003752 0.00309298MAR172 45.8 045 49.60053 141.8961 0.035648 0.039172 0.014342 0.064738 0.073931 0.005836 0.004241 0.042785 0.000873 0.00382 0.00288398MAR173 61.9 045 49.60053 141.8961 0.034915 0.038766 0.014196 0.056765 0.076588 0.006384 0.005122 0.033499 0.001295 0.003789 0.00300598MAR174 76.1 045 49.60053 141.8961 0.038247 0.048277 0.016592 0.068778 0.089713 0.007747 0.006305 0.03503 0.001201 0.003451 0.00271298MAR175 92.5 045 49.60053 141.8961 0.043461 0.04537 0.016037 0.065489 0.087526 0.0079 0.005406 0.033986 0.001375 0.003765 0.00304898MAR176 102.2 045 49.60053 141.8961 0.044594 0.047667 0.017678 0.069405 0.089403 0.007358 0.005895 0.033466 0.001255 0.004339 0.00385298MAR177 104.8 045 49.60053 141.8961 0.039747 0.039694 0.014335 0.057668 0.076858 0.006633 0.003966 0.027412 0.001107 0.00313 0.00253398MAR178 119 045 49.60053 141.8961 0.006998 0.004086 0.003308 0.022623 0.009092 0.001623 0.00065 0.003255 0.000228 0.000163 0.00038998MAR179 148.8 045 49.60053 141.8961 0.000827 0.001529 0.00052 0.004228 0.001453 0.000763 0.000283 0.000788 0 0 9.45E-0598MAR180 6.5 046 50.5319 141.7861 0.036056 0.022301 0.012625 0.11543 0.057005 0.005664 0.003268 0.038251 0.001917 0.001998 0.00121798MAR181 29.4 046 50.5319 141.7861 0.035386 0.021518 0.012865 0.110655 0.056887 0.007413 0.004481 0.039939 0.001911 0.003303 0.00202998MAR182 44.7 046 50.5319 141.7861 0.033412 0.020327 0.012383 0.107556 0.055265 0.005418 0.002989 0.039279 0.002011 0.002753 0.00190498MAR183 60.7 046 50.5319 141.7861 0.030896 0.018553 0.011819 0.105353 0.054373 0.005287 0.003228 0.032453 0.001792 0.002113 0.00142898MAR184 93 046 50.5319 141.7861 0.028317 0.020547 0.011769 0.081506 0.05888 0.007084 0.003525 0.025059 0.000992 0.002663 0.002216

After CHEMTAX has finished optimising ratios, it should look at how each sample responds to a change in ratio - e.g. increasing diatom fuco/chl_a ratio may increase total chl_a in some samples (pink) and decrease others (yellow). By looking at how individual samples respond to ratio changes, CHEMTAX may decide the data set can be spilt and optimized separately.

Page 121: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

What are the overall conclusions?

Page 122: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types

Power of pigment analysis

Page 123: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

Power of pigment analysis

Page 124: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT

Power of pigment analysis

Page 125: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios

Power of pigment analysis

Page 126: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios more algal cultures must be analysed by the

best methods

Power of pigment analysis

Page 127: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios more algal cultures must be analysed by the

best methods pigment:chl a ratios needed to validate

CHEMTAX in response to:

Power of pigment analysis

Page 128: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios more algal cultures must be analysed by the

best methods pigment:chl a ratios needed to validate

CHEMTAX in response to: light

Power of pigment analysis

Page 129: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios more algal cultures must be analysed by the

best methods pigment:chl a ratios needed to validate

CHEMTAX in response to: lightnutrient regimes

Power of pigment analysis

Page 130: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios more algal cultures must be analysed by the

best methods pigment:chl a ratios needed to validate

CHEMTAX in response to: lightnutrient regimes

need pigment / cell w.r.t. to environment

Power of pigment analysis

Page 131: Application of pigment analysis and CHEMTAX to field studies of phytoplankton communities Simon Wright Australian Antarctic Division

first step in discriminating algal types allows hundreds of samples to be analysed

BUT lack of pigment ratios more algal cultures must be analysed by the

best methods pigment:chl a ratios needed to validate

CHEMTAX in response to: lightnutrient regimes

need pigment / cell w.r.t. to environment improved computational methods needed

Power of pigment analysis