Transcript Flow Cytometric Analyses and RTCM of Water Quality around Hawaii Island Jason E.

Flow Cytometric Analyses and RTCM
of Water Quality around Hawaii Island
Jason E. Adolf and Judy K. Walker
UHH Marine Science
Study sites
Hilo 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)
EPSCoR MARINE sites, including Pauoa Bay
Marine ecosystem response to environmental variation, such as climate
change (atmospheric-oceanic) and submarine groundwater discharge
Pauoa Bay
Kīholo Plot
Kīholo Bay
Kaloko Plot
Hawai‘i Island
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
Flow Cytometry is an important
tool for our research
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 cells
3. 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 Optics
PMT
4
Flow cell
Light
scatter
detectors
PMT
Dichroic
Filters
3
PMT
2
Bandpass
Filters
PMT
1
Laser
J. Paul Robinson, Purdue University
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
Flow Cytometry: a monitoring tool
for Hawaiian waters
• Exploring two
parameters from each
sample
Avg ESD = (F1*ESD1)+ (F2*ESD2)+ (F3*ESD3)+ (F4*ESD4)
• Sum events within
regions
– ‘Avg ESD’
• Weighted average of
cell diameter
Bacteria are analyzed similarly in each sample
by applying a fluorescent stain
Red Fluorescence
– Total phytoplankton
(cells / mL)
F = fractional count for the region
ESD = geo. mean ESD for that region
Cell Size
Phytoplankton in SGD plumes of
West Hawaii Island
• Counting and optical characterization
– Abundance, size and pigmentation
Chlorophyll fluorescence
Groundwater-influenced waters
Typical off-shore
(surface)
Kaloko: near-shore
(surface)
Size
Kiholo: near-shore
(surface)
Larger phytoplankton are constrained to
surface plume (Kaloko Bay)
Surface
Depth profile of salinity and phytoplankton (determined by flow
cytometry) from Station 14 on July 27, 2010.
Salinity (ppt)
33
34
35
36
37
0.0
0.5
1.0
1.5
Middle
FSC-A = forward scatter
area (size)
FL3A = levels of red
fluorescence
Depth (m)
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Bottom
Euglenoid blooms of West Hawaii (green / black water)
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
Euglena-like
cells
~70 mm
Synechococcus
Phytoplankton Characteristics at
Different 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 of
Hilo Harbor
Tides
Moored with >100 lbs steel
Want to capture storm /
base flow conditions
April 7, 2010
Still there!
Buoy for Kiholo Bay is ready to go!
FW
SGD
USCG beacon
Data logger / cell phone modem
bumper
Solar powered
~6 feet
waterline
float
probes
ballast /
battery
YSI EMM 68
Mooring
attachment
Hilo Bay
WQ Buoy
Hilo Bay Buoy Data:
Salinity and Wailuku Flow
Apx one day lag between
buoy and stream gauge
Peak discharge
Minimum salinity
Storm Surges: Turbidity in Hilo Bay
Nov 7, 2010 – Hilo Bay From Rt 19 overlook
Turbidity subsides before salinity ‘recovers’
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
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
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 / nonSGD plume areas
• Physical conditions
limit use of nutrients