Continuous Underway Microbial Enzyme Activity Measurements in Seawater: The First Step Towards Autom

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Continuous Underway Microbial Enzyme Activity
Measurements in Seawater The First Step Towards
Automated In Situ Measurements (Or how to make
microbial rate measurements when everyone else is
doing genomics!)
Supported by NSF Biocomplexity (IDEA) DBI
0216154, Development of an Instrument for In
Situ Measurement of Microbial Enzyme Activities
in Aquatic Ecosystems Ammerman and Chant
(Rutgers), and Klinkhammer (Oregon St.), PIs
Institute of Marine Coastal Science 71 Dudley
Rd, Rutgers University New Brunswick, NJ 08901
USA [phone] ammmerman_at_imcs.rutgers.edu
James W. Ammerman and Brian M. Gaas
ABSTRACT The goal of this Biocomplexity (IDEA)
Project is to develop an instrument for automated
measurements of in situ microbial enzyme
activities at ocean observatories. As a first
step, we have automated shipboard underway ecto-
(cell-surface) enzyme activities using a flow
injection system. Three cruises to the eutrophic
Louisiana Shelf, supported by another project,
provided an opportunity for automated
measurements of alkaline phosphatase (AP)
activity during March, May, and July of 2004.
This system, which is capable of continuous
automated sampling, ran concurrently with manual
AP activity assays, conducted with a fluorescence
microplate reader, during all three cruises. In
July, both manual and automated systems
systematically mapped Louisiana Shelf AP activity
from Head-of-Passes (Mississippi River) to the
mouth of the Atchafalaya River. Contour maps
from both systems showed very similar features of
high activity near the mouth of the Mississippi
River, decreasing at higher salinities. The
automated system consistently yielded somewhat
higher AP activity than manual assays despite
identical solutions and water samples, probably
because of shorter incubation times and better
mixing. Sampling rates of the automated
instrument were approximately four times faster
than the manual assays, resulting in higher
density spatial coverage. Short incubation
times, limited substrate usage, greatly reduced
operator involvement, and potentially more
accurate results are the advantages of this novel
method for mapping microbial rate measurements at
nearly the same frequency as temperature and
salinity.
Raw and calculated data from the FIAlab
Comparisons with microplate assays
Basic schematic of flow injection system
Detector
Mixing Coil
Seawater
Sample
5-30 min incubation
Substrate (10uM difMUP, final concentration)
Contour plot of AP Activity (nmol l-1 h-1) as
measured by manual method with fluorescence
microplate reader, July 2004
Sample and substrate are mixed together and then
are incubated in the detector for specified time,
during which the machine measures fluorescence.
This is the STOPPED FLOW mode of the flow
injection system. Diagram from Jarda Ruzicka,
Flow Injection Analysis.
Raw fluorescence units vs. time
Flow injection system (FIAlab) in action

Contour plot of automated AP Activity (nmol l-1
h-1), July 2004
AP activity (nmol l-1 h-1) vs. time for 5.5 hours
(left) and the entire cruise (right)
Acknowledgements Thanks to Garth Klein at
FIAlab Instruments, the crew of the R/V Pelican,
the NOAA Coastal Ocean Program for shiptime, and
Jason Sylvan for poster preparation. Thanks also
to Gary Klinkhammer, Stephanie Jaeger, and
colleagues at Oregon State, and Bob Chant and
Eric Simms at Rutgers, our collaborators on this
Biocomplexity project. Prior support for
instrument purchase and assay development was
provided by NSF OCE 0196175.
The Substrate 6,8-difluoro-4-methylumbelliferyl-
phosphate (difMUP, for alkaline phosphatase),
from Molecular Probes, Inc. Excitation ? 360
nm Emission ? 440 nm No buffer required, low
background
Comparison of automated vs. manual AP Activity
from July 2004 cruise
Brian aboard the R/V Pelican
The Assay
CONCLUSION We now have the ability to
continuously measure any microbial ecto-
(cell-surface) enzyme which can cleave a
fluorescent substrate. Automated assays can
increase frequency and accuracy, as well as
reducing operator effort and reagent consumption.
Similar technology will next be incorporated
into an in situ version of the current
instrument that will measure multiple enzyme
activities at ocean observatories.
Product (fluorescent)
Current location of Brian and the FIAlab aboard
the R/V Oceanus near Bermuda!
Substrate (non-fluorescent)
Fluorescence assay for measuring AP activity
(this same basic assay is used for manual enzyme
measurements as well as automated ones)
Photo courtesy of Chris Linder on R/V Oceanus
website