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Phytoplankton Biomarkers OCN 621-Biological Oceanography Bob Bidigare
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Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ......

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Page 1: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt

Phytoplankton Biomarkers

OCN 621-Biological Oceanography

Bob Bidigare

Page 2: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt

Outline• Introduction to “biomarkers”• Phytoplankton pigment biomarkers• Introduction to chromatography• HPLC analysis of pigments• Pigments and marine biotechnology

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Introduction to “Biomarkers”

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Page 5: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt
Page 6: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt

Lipid Biomarkers

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Page 8: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt
Page 9: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt
Page 10: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt
Page 11: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt
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Plastid

1-Deoxy-D-xylulose-5-P

DMAPP IPP

+IPP

GPP (C10)+IPP+IPP

GGPP (C20)

Plastoquinone-9 (C45)

+5 IPP

Phytol

2x

Pyruvate + GA-3P

Acetyl-CoA

C18 Fatty acids

Cytoplasm

DMAPP IPP

+IPP+IPP

FPP (C15)2x

Sterols

Mitochondrion

Ubiquinone-10

?FPP

IPP

Pyruvate + GA-3P

1-Deoxy-D-xylulose-5-P

Carotenoid Synthesis

Green Algae

Elongation & Desaturation

Endoplasmic Reticulum

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Carotenoid Biosynthesis

HO

OH

OO

violaxanthin

HO

OH

O

antheraxanthin

HO

OH

zeaxanthin

lycopene

β-carotene α-carotene

HO

OH

lutein

OHO

neoxanthinHO

OH

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Carotenoid Pigments

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Page 20: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt
Page 21: Phytoplankton Biomarkers - SOEST | School of Ocean and ... · PDF filePhytoplankton Biomarkers ... Pigment-based Chemotaxonomy Prasinoxanthin Prasinophytes ... OCN621-20060130-pigmentbiomarkers-bidigare.ppt

Pigment AnalysisSpectrophotometry• Relatively rapid & shipboard compatible method• Poor sensitivity (~10 L sample volume)• Trichromatic equations: Chl a, Chl b & Chl c• Interferences by accessory Chl degradation products• Inexpensive (~$10 K)

Fluorometry• Very rapid & shipboard compatible method• High sensitivity (~300 mL sample volume) • Provides estimates of Chl a and Phaeophytin a• Interferences caused by Chl b (spectrofluorometric correction)• Inexpensive (~$10 K)

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Thin-layer Chromatography (TLC)• Very time consuming and not shipboard compatible • Poor sensitivity (~10 L sample volume) • Spectrophotometric analysis after separation • Separates many pigments (Chls, phaeopigments & carotenoids) • Co-elution problems (fucoxanthin & its acyloxy derivatives)• Inexpensive (~$10 K)

HPLC• Relatively rapid & shipboard compatible method • Intermediate sensitivity (1-5 L sample volume) • Separates most pigments (Chls, phaeopigments & carotenoids) • Co-elution problems can be solved by monitoring 2 ls • Expensive (~$50 K)

Pigment Analysis

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ApplicationsPigments are useful as tracers for:• Characterizing marine organic matter (living & dead) • Determining rates of key oceanographic processes

Pigment analyses can be made in conjunction with:• Light measurements to estimate 1o production rates • 14C tracer experiments to determine algal growth rates • Dilution experiments to determine grazing rates• Sediment trap collections to identify the origins/sources of

sinking organic matter• Ultrastructural and molecular analyses (rDNA sequences)

to identify new groups of marine microbes

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Pigment-based Chemotaxonomy

PrasinophytesPrasinoxanthinCryptophytesAlloxanthin

Pelagophytes19’-but-fucoxanthinPrymnesiophytes19’-hex-fucoxanthinChlorophytesLutein

DinoflagellatesPeridininDiatomsFucoxanthinProchlorococcus spp.Divinyl Chl aPhytoplankton biomassTotal chlorophyll a

Taxonomic GroupPhytoplankton Pigment

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• Early applications were for the separation of plant photo-pigments (e.g., chlorophylls & carotenoids)

• Column chromatography was developed by a Russian botanist, Mikhail Tswett, in the early 1900s

• There have been twelve Nobel Prize awards for research relating to chromatography

• Today, it is unquestionably the most widely used of all analytical techniques encompassing all branches of scientific research

Introduction to Chromatography

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Introduction to Chromatography

Origin of Name: Chroma - Graphein (Color - Writing)

Photo-pigments

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Sample Collection & Storage

• Seawater is collected using Niskin bottles, Go-Flo samplers or in situ pumps

• Seawater is dispensed into opaque bottles

• Samples are concentrated using Whatman GF/F glass fiber filters (25 mm dia and porosity of 0.7 μm) via vacuum or positive-pressure filtration

• Filters are placed in Al foil packets & stored in LN2(preferable) or at –80oC for later analysis

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Sample Extraction• Filters are extracted in 90% acetone (v:v)

• An internal standard (canthaxanthin) is added in order to determine the final extraction volume

• Cell disruption can be aided by grinding or sonication at 0oC

• Samples are allowed to extract for 24 hours in darkness at 0oC

• Extracts are clarified via centrifugation or filtration (HPLC syringe cartridge filters with PTFE membranes)

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HPLC Analysis

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Implementation of a HPLC Method

• Method of Wright et al. (1991) as modified by Bidigare et al. (2005)

• Solvent systems (HPLC-grade solvents)Solvent A: 80:20 (v:v) methanol: 0.5 M ammonium acetate aqueous (pH=7.2) & 0.01% BHT (w:v)Solvent B: 87.5:12.5 (v:v) acetonitrile:water & 0.01% BHT (w:v)Solvent C: Ethyl acetate

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Solvent Gradient

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Implementation of a HPLC Method

• Flow rate: 1 mL min-1

• Injection volume: 200 μL

• Column temperature: 27oC (tR stability)

• Guard column: 50 x 4.6 mm (same stationary phase as analytical column)

• Analytical column: Reverse-phase column with endcapping (250 x 4.6 mm, 5 μm particle size, ODS-2 Spherisorb C18 stationary phase)

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Structure of a RP-C18 Column

Pigment analyte

C18 moiety

Si(OH)4with endcapping

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Pigment Detection & Identification• Fixed λ absorbance detectors (chlorophylls & carotenoids)• Fluorescence detectors (chlorophylls only)• Diode-array spectrophotometry (DAS, 200 – 800 nm)

BChl a

Chl b

Chl aPEβ-Car

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RP-HPLC Chromatogram(λ = 436 nm)

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Peak Identities

β,β-carotene19

Chlorophyll a'18

Chlorophyll a17

Chlorophyll b 16

Canthaxanthin (IS)15

Zeaxanthin14

Lutein13

Diatoxanthin12

Alloxanthin11

Diadinoxanthin10

Violaxanthin9

Prasinoxanthin8

19΄-hex-fucoxanthin7

Neoxanthin6

Fucoxanthin5

19΄-but-fucoxanthin4

Peridinin3

Chlorophyll c1+22

Chlorophyll c31

Peak IdentityPeak Number

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Retention Time (tR)

where, tM = elution time of un-retained component (dead time) & tR1 = elution time of retained component 1

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Column Resolution

Rs = 1.0 gives rise to 3% peak overlapRs = 1.5 gives rise to 0.2% peak overlap

(cross-contamination)

( )R2 R1s

B1 B2

2 t tR

w w−

=+

W2

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Calculation of the Minimum Detection Limit (MDL)

( ) c= 6,0.99MDL t S

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LC/MS/MS

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LC/MS/MS

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Carotenoids & Marine Biotechnology

• Photo-protective pigments in microalgae

• High-value bioproducts:- Fish/poultry feed additives (color enhancers)- Nutraceuticals (strong anti-oxidant activities)- Pharmaceuticals (e.g., anti-cancer activity)

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Carotenoids & Marine Biotechnology

• Target compounds include:

Primary target:- Zeaxanthin (green algae & cyanobacteria)

Secondary targets:- Canthaxanthin (cyanobacteria)- Lutein (green algae)- Astaxanthin (green algae)

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Green Algae & Carotenoid Production• Dunaliella salina: A rich source of lutein,

zeaxanthin & β,β-carotene

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Induction of High-Value Xanthophylls in Dunaliella salina

XanthophyllContent

(106 mol/cell)

ControlCells

InducedCells

Difference(%)

Lutein 80 370 +460

Zeaxanthin 4 440 +11,000

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Reference

Bidigare, R. R., L. Van Heukelem and C. C. Trees. 2005. Analysisof algal pigments by high-performance liquid chromatography. In: Culturing Methods & Growth Measurements (R. A. Andersen, Ed.), Academic Press, New York, pp. 327-345.

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