Phytoplankton Measurements

EOSC 473-573

What are Phytoplankton?

• Unicellular eukaryotes & prokaryotes• Photoautotrophs• Nano-Microplankton - 2 µm to 1 mm• All contain chl a as a photosynthetic

pigment

Key Taxa

• Cyanobacteria• Diatoms

• Dinoflagellates• Coccolithophores• Nanoflagellates

Diatoms

• 5,000 species• Opaline skeletal structure• Centric

– Chaetocerous spp.– Skeletonema costatum– Thalassiosira spp.

• Pennate– Pseudonitizschia spp.

• Often form large chains

Dinoflagellates

• 1,800 species• Theca• Motile- Flagellum• Mixotrophs• Red tides

Coccolithophores

• 100 species• Small (2-20 µm)• Produce Dimethylsulfide - DMS• Calcium Carbonate PlatesCa2+ + 2HCO3

- CaCO3 + H20 + CO2

Nanoflagellates

• Small (< 10 µm)• Flagella - motility• Mixotrophic

What can you estimate• Biomass• Diversity• Chla vs. accessory pigments, or phaepigments• Gross & net photosynthetic rates, &

respiration rates• Growth Rate vs. grazing rate by micrograzers• Rates of nutrient uptake (generate a

Michaelis Menten curve to determine Ks and Vmax)

• Photosynthetic efficiency (Fv/Fm) using DCMU• Sinking rates of phytoplankton

In red: things that you can do, but will not be covered in lecture. Talk to me, if interested

Research Questions• How does phytoplankton biomass

change over space/time ?• How does the abundance of specific

taxa change over space/time?• Are these changes related to specific

environmental factors?• What controls primary production?

Important Physical and Chemical Parameters

• Nutrients• Temperature• Light

– Measured in µE m-2 s-1

– High light ~ 2000 µE m-2 s-1

– Low light 100 µE m-2 s-1

– Important to determine compensation depth and depth of photic zone

Ih=Io*e-kHDepth (m)

Irradiance (µE m-2 s-1)

Ih = irradiance at depth H (µE m-2 s-1)Io = surface irradiance (µE m-2 s-1)K = light extinction coefficient (m-1)H = depth (m)

Beer-Lambert Law

Compensation Irradiance

Therefore, knowing the compensation irradiance (e.g. 7 µE m-2 s-1 for diatoms), surface irradiance and extinction coefficient, one can compute the compensation depth given surface.

Calculating k1. Measure irradiance depth profile - PAR sensor2. Measure turbidity depth profile - Turbidity sensor3. Secchi depth, d

k = 1.4/dk = 1.7/d (turbid waters)

Secchi disk

Field Collection

1. Surface sampling with underway pumping system

2. Bottles

Liters Flushing SentNansen 1.7 poor openNiskin 1.7-12 good openGo-flo 1.7-100 good closed

Field Collection

3. Rosette

4. Phytoplankton nets• Not as quantitative• Identify species• Rough estimate of

population biomass

Storage

Handling• Be gentle• Limit light exposure

Bottles• 50-100 ml• Glass (other material absorb preserving

solution)• Dark

Preservation

• Lugol’s solution (stains cells)– 1% final sample concentration– Acidic Lugol’s solution can deteriorate the

CaCO3 plates of coccolithophores - Lugol’s basic, acetate solution

• Formalin (does not stain cells)– must be buffered– 2 % final sample concentration– Disgusting smell

• Glutaraldehyde (does not stain cells)– 1 % final sample concentration

Biomass Estimation

Cell densities (cells/L, standing stock)• Light Microscopy

– Counting chamber with known volume– Know concentration factor

• Flow Cytometry• Coulter counter

Enumeration and Identification1. Pipette sample into an appropriate

counting chamber of known volume (ie. Sedgewick rafter).

2. Count a given number of light fields/or mm2 squares under a light microscope.

3. Average counts and scale up

Types of Counting Devices

Device Cell size (µm) Culture density (cells/ml)

Sedgwick Rafter 50-500 30-104

Palmer-Maloney 5-150 100-105

Speirs-Levy 5-75 104-106

Hemacytometer 2-30 104-107

Petroff-Hausser <1-5 106-108

Settling Chamber- Dilute samples must be concentrated

before counting by settling 100, 25, 10, 5 or 2 ml samples

- Need an inverted microscope

Has a bottom counting tray

Enumeration and IdentificationLight Microscopy

• < 200 X power• Count between 200-

400 cells to obtain accurate estimate

• Accuracy increases as the √N

N =cells counted• High taxonomic

resolution• Cheap• Time consuming

Enumeration and IdentificationFlow Cytometry• Fluorescence emission signals of

each cell are processed. Can be displayed on histograms

• Each phytoplankton has a specific fluorescence signature

• Rapid - 1000 cells per second• Precise• Can sort cells automatically• ExpensiveFlow Cam• Counting, measuring, and imaging

Enumeration and IdentificationCoulter Counter (only

appropriate for phytoplankton monocultures)

• Electronic particle counter• Non-conductive particle

(e.g. phytoplankton) cause a disruption of the electrical field as they pass through it

• Cannot distinguish between detritus and cells

• Precision depends on # of particles counted - 2000 units 3% error

• Rapid

Biomass Estimation

In-vivo Chlorophyll determination (µg chla/L)A fluorometer is lowered into the water column. Often

attached to CTD.Chlorophyll a naturally absorbs blue light and emits red

light

Transmits blue light (440-460 nm)

Fluorometer PhytoplanktonChl a

Detects red light (685 nm)fluoresced by chlorophyll

Chl a biomass over timeRivers Inlet 2007

March April JuneMay

Dep

th (m

)

0

10

20

In-vivo Chlorophyll Determination

• Interference with degraded plant material, DOM, Turbidity - anything that might contain Chl or its degradation products

• Temperature– Fluorescence 1.4% for every oC– Already compensated by instrument

• Calibrate in-vivo fluorescence with lab measurements of field samples

Chlorophyll DeterminationChlorophyll determination of field samples (µg chla/L)

1. A known volume of seawater is filtered onto filter of a given porosity (glass fiber : GFF (0.7 µm); or polycarbonate, many porosities) - 100 ml to 5 L depending on phytoplankton concentration

2. Pigments are extracted in 90% acetone3. [chl] determined spectrophotometrically or

fluorometrically (5-10 x more sensitive)

Diversity Estimation1. Cell identification2. Size fractionationUsing filters to characterize coarse

community diversity– 0.2-2 µm bacteria– 2-5 µm nanoflagellates, coccolithophores,

diatoms– 5-20 µm diatoms, dinoflagellates

Diversity Estimation

3. Signature Chloroplast Pigments (high performance/pressure liquid chromatography - HPLC)Each phytoplankton group has a specific accessory pigment

Filter 2-4 L of seawater onto a GFF filter and store in liquid nitrogen

Productivity

Productivity - the rate at which a quantity of organic material is produced (mg C/L/hr)

Primary Productivity -Measuring rate of photosynthesis

6CO2 + 6H20 C6H12O6 +12O2

Photosynthesis

Respiration

ProductivityGross Primary Productivity GPP

Rate of total amt of C fixed by photosynthesis

Net Primary Productivity NPPGPP - Phytorespiration

Net Community Productivity NCPGPP - Water column respiration

Productivity Estimation

Measure change in any photosynthetic product over time

• 14C method• 18O method• Winkler O2 titration method (only for

phytoplankton monocultures or bloom conditions)

• O2 electrode (Clark-type polarographic cell, only for phytoplankton monocultures or bloom conditions)

The 14C MethodMeasures the rate at which 14C inorganic C is

incorporated into organic C

1. Pour sample into two incubation bottles (500 ml polycarbonate bottles); one will be the control (dark)

2. Add H14CO3- to incubation bottles

3. Incubate bottles at depth4. At the end of incubation filter sample 5. Measure PO14C on filter

The 14C MethodPP= {(F/W)*1.05 }/t

F = 14C taken up (dpm on your filter, dpm)W (specific activity of C, dpm per mol)= H14CO3

-added(dpm/L) /

2000 µmol inorganic C/L 1.05 =factor accounting for the preferential uptake of lighter

carbon, 12Ct = incubation timeGPP - short incubations, t = 2-6 hrsNPP - long incubations, t = 12-24 hrs

2000 µM = ~ [inorganic C] in seawater

The 18O MethodMeasures the rate at which 18O2 is evolved

1. Take water sample in gas tight container2. Add H2

18O water and incubate in the light3. Measure production rate of 18O24. Approximate GPP - as respiration removes

more abundant 16O2

Winkler Titration & O2 electrode

Measure change in O2 before and after incubation

• O2 production in light = NPP• O2 consumption in dark = Respiration

**Only works in very productive environments measurement error = 0.1%

Growth RateA change in phytoplankton biomass over time, dP/dt

dP/dt (mg m-2 d-1) = [growth - losses (sinking, advection, grazing) rates] * P

= (r - l) P

r = growth rate (day-1)l = loss rate (day-1)

P= Standing stock (mg/m2)OR

Pt = Po e(k-g)t, where k = growth constant, g = grazing constant

Growth Rate

• If sinking and grazing are low compared to growth, the phytoplankton population grows exponentially. This is common during bloom conditions.

• One can calculate an average phytoplankton exponential rate of increase

Depth Integrated Chlorophyll (20 m) in Rivers Inlet in 2007

Measuring uptake ratesto generate a MM curve

• Collect seawater• Concentrate the phytoplankton using

polycarbonate filters of various porosities: collect the cells on filter & resuspend them in nutrient free water

• Split the phytoplankton concentrate into different flasks with different nutrient concentrations

• Measure nutrient drawdown

Measuring uptake kinetics to determine ability of phytoplankton to take up NO3at various concentrations

0.002 0.005 0.010 0.05 0.750.25 1.00

3.00 4.00 5.00 8.0 25.0 15.0 30.00

Additions of NO3 to seawater (µM)

Nut

rient

upt

ake

rate

(pm

ol N

cel

l-1h-

1 )

Able to calculate Ks and Vmax