Flow Cytometric Analyses and RTCM of Water Quality around Hawaii Island

Jason E. Adolf and Judy K. WalkerUHH Marine Science

Study sites

Pauoa Bay(W. Hawaii)

Hilo Bay(E. Hawaii)

1 km100 m

Leeward sideLow rainfallGround water plume Windward side

High rainfallGroundwater plume(s)

SGD

SGD

Rivers

Pauoa Bay

Hilo Bay

Hawai‘i Island

EPSCoR MARINE sites, including Pauoa BayMarine ecosystem response to environmental variation, such as climate change (atmospheric-oceanic) and submarine groundwater discharge

Kīholo Plot

Kaloko Plot

Hawai‘i Island

Kīholo Bay

Pauoa Bay

Why examine phytoplankton responses to coastal hydrology?

• A complex, fast-responding microbial assemblage– many types, different needs,

different fates– A good ‘indicator’

• Biomass serves as a nutrient vector (down); productivity; HABs

• Size structure responds to environmental forcing

• Size structure can determine the ‘fate’ of phytoplankton

-sinking rate-grazing losses

Environment

Phytoplankton

Ecosystem

Flow Cytometry

What can a flow cytometer do?

1. Count cells (up to 10,000 per second)2. Optically classify each cell, one at a time, 10,000 per second

• Can distinguish phytoplankton from other cells3. Quantify optical properties of cells and generate statistics

• Side and forward scatter – indicator of cell size and shape• Fluorescence (green, orange, red) – indicator of pigmentation

Flow Cytometry is an important tool for our research

PMT

PMT

PMT

PMT

DichroicFilters

BandpassFilters

Flow Cytometry Optics

Laser

1

2

3

4

Flow cell

J. Paul Robinson, Purdue University

Light scatterdetectors

Calibration with size standards(latex beads)

FSC-H and FSC-A poorly resolved 1 & 2 micron beadsSSC-H and SSC-A didn’t have this problem

The machine needs to be calibrated against phytoplankton for absolute sizing

Flow Cytometry: a monitoring tool for Hawaiian waters

• Exploring two parameters from each sample– Total phytoplankton

(cells / mL)• Sum events within

regions– ‘Avg ESD’

• Weighted average of cell diameter

Avg ESD = (F1*ESD1)+ (F2*ESD2)+ (F3*ESD3)+ (F4*ESD4)

F = fractional count for the regionESD = geo. mean ESD for that region

Red

Flu

ores

cenc

e

Cell Size

Bacteria are analyzed similarly in each sample by applying a fluorescent stain

Phytoplankton in SGD plumes ofWest Hawaii Island

• Counting and optical characterization– Abundance, size and pigmentation

Size

Chl

orop

hyll

fluor

esce

nce

Typical off-shore(surface)

Kaloko: near-shore(surface)

Kiholo: near-shore(surface)

Groundwater-influenced waters

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

33 34 35 36 37

Salinity (ppt)

Dep

th (

m)

Depth profile of salinity and phytoplankton (determined by flow cytometry) from Station 14 on July 27, 2010.

Surface

Bottom

FSC-A = forward scatter area (size)

FL3A = levels of red fluorescence

Middle

Larger phytoplankton are constrained to surface plume (Kaloko Bay)

Euglena-likecells

Synechococcus

Euglenoid blooms of West Hawaii (green / black water)

~70 mm

1. A Euglenoid causes the dark water-Not toxic

2. It’s a large cell with potential to sediment, but the blooms are localized

3. Reduced circulation set favorable conditions for the bloom to occur

We now have a tool for monitoring this Euglenoid to learn better how it fits in to the ecosystem

Phytoplankton Characteristics atDifferent Sites of West Hawaii

Euglenoid bloom

Kīholo Plot

Kaloko Plot

Pauoa Bay

Conclusion• Research – Do phytoplankton ‘respond’ to

nutrients in SGD?

• Approach: Flow cytometry– A tool to address this question (numbers and

size of phytoplankton)– Developing a broadly applicable monitoring

tool

Real-time Continuous Water Quality Buoy Deployed in Hilo Bay

Western portion ofHilo Harbor

Moored with >100 lbs steel

Want to capture storm / base flow conditions

April 7, 2010Still there!

Tides

FWSGD

Buoy for Kiholo Bay is ready to go!

Solar powered

Data logger / cell phone modemUSCG beacon

bumper

floatwaterline

probes

ballast /battery

Mooringattachment

~6 feet

YSI EMM 68

Hilo BayWQ Buoy

Hilo Bay Buoy Data:Salinity and Wailuku Flow

Apx one day lag betweenbuoy and stream gauge

Peak dischargeMinimum salinity

Storm Surges: Turbidity in Hilo Bay

Turbidity subsides before salinity ‘recovers’

Nov 7, 2010 – Hilo Bay From Rt 19 overlook

Hamakua coast

Hilo Bay Chl a and Salinity

Storms flush Chl a from Hilo Bay

Recovery of Chl a?

Vertical migrations?

Conclusions• Many tools in place at UH Hilo to examine

hydrologic influences on coastal phytoplankton / microbes– Developing indicator tools

• Understanding the influence of groundwater and surface water is a key research drive

• Linkages to terrestrial biomes is key

Acknowledgements

• NSF EPSCoR III (UH System)– Kiholo / Kaloko

• Fairmont Orchid Green Committee– Pauoa Bay

• NSF EPSCoR II (UH System)– Hilo Bay

Tracy Wiegner (UHH Marine Science)

Students: Judy Walker, Erick Johnson, Rebecca Most, Gillian Wysock, Ambyr Mokiao-Lee, Javez Mooteb, John Burns (Corals)

Conclusions• East Hawaii – Ground water nutrients likely fuel

productivity; Storm flows flush Hilo Bay, reduce productivity.– How has the biology adapted to these

conditions?

• West Hawaii – Phytoplankton differ between SGD / non-SGD plume areas

• Physical conditions limit use of nutrients