1. Jason E. Adolf and Judy K. Walker UHH Marine Science
Flow Cytometric Analyses and RTCM of Water Quality around Hawaii Island
Jason E. Adolf and Judy K. Walker
UHH Marine Science

2. Study sites Hilo Bay Pauoa Bay Hilo Bay (E. Hawaii) Pauoa Bay
Hawai‘i Island
Pauoa Bay
Hilo Bay
(E. Hawaii)
Rivers
Pauoa Bay
(W. Hawaii)
SGD
SGD
1 km
100 m
Leeward side
Low rainfall
Ground water plume
Windward side
High rainfall
Groundwater plume(s)

3. EPSCoR MARINE sites, including Pauoa Bay
Marine ecosystem response to environmental variation, such as climate change (atmospheric-oceanic) and submarine groundwater discharge
Pauoa Bay
Hawai‘i Island
Kīholo Plot
The Marine Agenda is developing research capacity to investigate ecosystem responses to primarily environmental factors, such as climate change, ocean warming, and groundwater discharge. The research activities will be conducted in the two marine plots established on the Kona coast of the Island of Hawaii in the watersheds where terrestrial plots were established.
Kīholo Bay
Kaloko Plot 3

4. 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

5. Flow Cytometry What can a flow cytometer do?
Flow Cytometry is an important tool for our research
What can a flow cytometer do?
Count cells (up to 10,000 per second)
Optically classify each cell, one at a time, 10,000 per second
Can distinguish phytoplankton from other cells
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

6. Flow Cytometry Optics Laser Dichroic Filters Flow cell Bandpass
PMT 4
Dichroic
PMT
Light scatter
detectors
Filters 3
Flow cell
PMT 2
Bandpass
Filters
PMT 1
Laser
J. Paul Robinson, Purdue University

7. Calibration with size standards (latex beads)
FSC-H and FSC-A poorly resolved 1 & 2 micron beads
SSC-H and SSC-A didn’t have this problem
The machine needs to be calibrated against phytoplankton for absolute sizing

8. 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
F = fractional count for the region
ESD = geo. mean ESD for that region
Avg ESD = (F1*ESD1)+ (F2*ESD2)+ (F3*ESD3)+ (F4*ESD4)
Red Fluorescence
Bacteria are analyzed similarly in each sample by applying a fluorescent stain
Cell Size

9. Phytoplankton in SGD plumes of West Hawaii Island
Counting and optical characterization
Abundance, size and pigmentation
Groundwater-influenced waters
Typical off-shore
(surface)
Kaloko: near-shore
(surface)
Kiholo: near-shore
(surface)
Chlorophyll fluorescence
Size

10. Larger phytoplankton are constrained to surface plume (Kaloko Bay)
Depth profile of salinity and phytoplankton (determined by flow cytometry) from Station 14 on July 27, 2010.
Middle
FSC-A = forward scatter area (size)
FL3A = levels of red fluorescence
Bottom

11. Euglenoid blooms of West Hawaii (green / black water)
A Euglenoid causes the dark water
-Not toxic
It’s a large cell with potential to sediment, but the blooms are localized
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
Euglena-like
cells
~70 mm
Synechococcus

12. Phytoplankton Characteristics at Different Sites of West Hawaii
Euglenoid bloom
Kīholo Plot
Kaloko Plot
Pauoa Bay

13. 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

14. Real-time Continuous Water Quality Buoy Deployed in Hilo Bay
Western portion of
Hilo Harbor
Moored with >100 lbs steel
Want to capture storm / base flow conditions
April 7, 2010
Still there!
Tides FW
SGD
Buoy for Kiholo Bay is ready to go!

15. Hilo Bay WQ Buoy USCG beacon Data logger / cell phone modem bumper
Solar powered
~6 feet
float
waterline
probes
ballast /
battery
Mooring
attachment
YSI EMM 68

16. Hilo Bay Buoy Data: Salinity and Wailuku Flow

17. Apx one day lag between buoy and stream gauge
Peak discharge
Minimum salinity

18. Storm Surges: Turbidity in Hilo Bay

19. Turbidity subsides before salinity ‘recovers’
Nov 7, 2010 – Hilo Bay From Rt 19 overlook
Turbidity subsides before salinity ‘recovers’
Hamakua coast

20. Hilo Bay Chl a and Salinity

21. Storms flush Chl a from Hilo Bay
Recovery of Chl a?
Vertical migrations?

22. 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

23. Acknowledgements NSF EPSCoR III (UH System)
Tracy Wiegner (UHH Marine Science)
Students: Judy Walker, Erick Johnson, Rebecca Most, Gillian Wysock, Ambyr Mokiao-Lee, Javez Mooteb, John Burns (Corals)
NSF EPSCoR III (UH System)
Kiholo / Kaloko
Fairmont Orchid Green Committee
Pauoa Bay
NSF EPSCoR II (UH System)
Hilo Bay

24. 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