Phytoplankton Biomarkers OCN 621-Biological Oceanography Bob Bidigare
Phytoplankton Biomarkers
OCN 621-Biological Oceanography
Bob Bidigare
Outline• Introduction to “biomarkers”• Phytoplankton pigment biomarkers• Introduction to chromatography• HPLC analysis of pigments• Pigments and marine biotechnology
Introduction to “Biomarkers”
Lipid Biomarkers
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
Carotenoid Biosynthesis
HO
OH
OO
violaxanthin
HO
OH
O
antheraxanthin
HO
OH
zeaxanthin
lycopene
β-carotene α-carotene
HO
OH
lutein
OHO
neoxanthinHO
OH
Carotenoid Pigments
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)
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
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
Pigment-based Chemotaxonomy
PrasinophytesPrasinoxanthinCryptophytesAlloxanthin
Pelagophytes19’-but-fucoxanthinPrymnesiophytes19’-hex-fucoxanthinChlorophytesLutein
DinoflagellatesPeridininDiatomsFucoxanthinProchlorococcus spp.Divinyl Chl aPhytoplankton biomassTotal chlorophyll a
Taxonomic GroupPhytoplankton Pigment
• 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
Introduction to Chromatography
Origin of Name: Chroma - Graphein (Color - Writing)
Photo-pigments
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
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)
HPLC Analysis
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
Solvent Gradient
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)
Structure of a RP-C18 Column
Pigment analyte
C18 moiety
Si(OH)4with endcapping
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
RP-HPLC Chromatogram(λ = 436 nm)
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
Retention Time (tR)
where, tM = elution time of un-retained component (dead time) & tR1 = elution time of retained component 1
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
Calculation of the Minimum Detection Limit (MDL)
( ) c= 6,0.99MDL t S
LC/MS/MS
LC/MS/MS
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)
Carotenoids & Marine Biotechnology
• Target compounds include:
Primary target:- Zeaxanthin (green algae & cyanobacteria)
Secondary targets:- Canthaxanthin (cyanobacteria)- Lutein (green algae)- Astaxanthin (green algae)
Green Algae & Carotenoid Production• Dunaliella salina: A rich source of lutein,
zeaxanthin & β,β-carotene
Induction of High-Value Xanthophylls in Dunaliella salina
XanthophyllContent
(106 mol/cell)
ControlCells
InducedCells
Difference(%)
Lutein 80 370 +460
Zeaxanthin 4 440 +11,000
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.
Questions???