Monterey Bay Aquarium Research Institute
Celebrating our 20th Anniversary

Undersea genetics lab detects marine microbes and toxins

Diagram of the Environmental Sample Processor
Model of the second-generation ESP mechanism, with the electrical and most fluidic components omitted for clarity. Overall dimensions are approximately 41 centimeters in diameter by 76 centimeters high.

Celebrating 20 yearsDetecting toxic organisms that are invisible to the naked eye has long presented a daunting challenge to public officials charged with ensuring the health of our oceans and waterways. By the time a water sample is collected, transported to a laboratory, and observed under controlled conditions to determine whether it poses a public health risk, it may be too late. In the time that it takes to obtain a result, for example with the standard culturing technique used by the Environmental Protection Agency (EPA), hospital emergency rooms and emergency medical workers are already aware that there’s a problem in the ocean or waterway, or the problem itself may be long gone.

For this reason, the 2004 report of the U.S. Commission on Ocean Policy highlighted the importance of developing a device to rapidly and remotely detect microorganisms such as bacteria or harmful algae based on unique attributes, such as their genetic codes. Now, just a few years after the U.S. Commission report was published, the technical barriers to what must seem like a futuristic capability are being overcome. MBARI’s Environmental Sample Processor (ESP), a “portable laboratory,” has proven its ability to detect harmful algae and other microorganisms in real time. Originally envisioned as a research tool for in situ exploration of the microbial ocean, the ESP has successfully completed a number of high-profile field trials. The ESP team of scientists and engineers is working with coastal managers to create versions of this instrument that can be used not only for research, but for public health and resource management missions as well.

In ocean research, application of genetic analytical techniques is pervasive, yet to date the vast majority of work typically occurs in shore-based laboratories on samples collected from the ocean. Therefore, although the analyses are state-of-the-art, there is still a large gap in time between when the water is sampled and when the results are obtained.

Launching the ESP
Members of the ESP team deploy the second-generation ESP off the R/V Zephyr in Monterey Bay in March 2006.
Furthermore, this mode of collection and analysis does not adapt itself to the new paradigm of ocean observatories in which scientists seek to have data, not samples, returned to shore by fielding instruments that perform their analyses in situ. Conducting the requisite genetic analyses outside of a laboratory, let alone under the water, poses many technological challenges.

The original ESP underwent field trials from 2001 through 2005 during which mechanical functions and genetic tests for several different species were successfully implemented for deployments lasting up to 20 days. These field trials emphasized engineering design iterations and refining assay chemistry and methods for validating results obtained using the instrument. Based on that work, a “second generation” (2G) ESP was developed with a grant from the National Ocean Partnership Program/National Science Foundation, and is now being refined with support from several federal agencies and private foundations, including the David and Lucile Packard Foundation and the Gordon and Betty Moore Foundation.

The 2G ESP is basically a molecular biology lab packed inside a canister 41 centimeters across and 91 centimeters tall. It can acquire water samples, concentrate small organisms by filtration, and apply molecular probe technology remotely. The instrument is used for handling small to moderate sized samples (milliliters to several liters) at depths to 50 meters. The core instrument offers a means of detecting numerous microorganisms, and the substances they produce, using DNA probe and protein array technology. The ESP can also be used to collect and store samples for later use on shore after the instrument is recovered.

Organisms detected by the ESP
Example of a DNA probe array developed using a natural seawater sample and the first-generation ESP. Printed for demonstration purposes are chemistry controls (top center), and (clockwise, from top right) probes for “all eukaryotes,” “pennate diatoms,” specific groups of marine bacteria, mussels, and barnacles. Spots on the arrays glow when targeted organisms are present in a sample. This array illustrates the detection of mussel larvae, two different groups of marine bacteria, and pennate diatoms. Barnacle larvae were not present. Actual size of the array is approximately 20 millimeters in diameter, about the size of a U.S. quarter.

Field trials of the 2G ESP began in March of 2006. In a single deployment, it successfully automated application of DNA probe arrays that targeted invertebrate larvae, harmful algae, and bacteria.

The basic instrument supports a generic, yet limited, set of sample collection and processing operations. The second generation version is designed to allow for the addition of specialized analytical modules: stand-alone devices that can be added to the existing core ESP to impart new and varied functions. This offers new possibilities for remote assessment of the genomic content and activities of microbial communities.

The 2G ESP may also be useful in the area of resource management. Researchers at the Marine Biotoxins Laboratory in Charleston, South Carolina, part of the National Ocean Service at NOAA, came to MBARI to work with the lab group on application of the ESP to detect algal toxins. They succeeded in developing a test for domoic acid, a neurotoxin produced by certain phytoplankton found in Monterey Bay and elsewhere. Domoic acid is linked to illness and mortality of humans and wildlife, with substantial economic impacts. In 2006 the ESP project reached a major milestone—the first record of sensing in situ both a harmful algal bloom species and the toxin it produces.

ESP group
Members of the MBARI ESP team with collaborators from the National Oceanic and Atmospheric Administration’s (NOAA) Marine Biotoxins Laboratory. Gathered around the instrument, from left to right are Greg Doucette (NOAA), Chris Scholin, Christina Preston, Scott Jensen, Tina Mikulski (NOAA), Roman Marin III, Dianne Greenfield, and Joe Jones.

In March 2007, researchers reached another milestone, with the successful deployment of the ESP down to a depth of 1,000 meters in Monterey Bay. In order to adapt this instrument for use in the deep sea, the researchers had to develop a "deep-water sampling module," which allows the ESP to collect samples of seawater at depths where the water pressure can be several hundreds of times that at the surface. Eventually this device may be used at deep-sea hydrothermal vents or hooked up to the MARS ocean observatory in Monterey Bay.

Nevertheless, solving the technical problems associated with performing in situ molecular analyses under water is not quite sufficient in terms of gaining acceptance of this new methodology in regulatory circles. The genetic approach to detecting toxics and harmful microbes may indeed be faster and more precise, but translating those measurements to new EPA standards is not a simple matter. Extensive comparisons are required to determine if the new methods provide results equivalent to or better than the EPA standard, which has its ultimate base in probabilistic models for people getting sick. And because there are large economic consequences with either complying, or not complying, with the EPA standards, until such comparisons can be completed, there will be issues surrounding how the 2G ESP can be used in water-quality monitoring. Fortunately, making those comparisons is a high priority for researchers and resource managers because it will ultimately improve their ability to protect public health. Figure 6.

The team is experiencing an increasing demand for the ESP. Three copies of the core 2G ESP are being built at MBARI in response to requests from outside groups for their own instrument. The initial units are aimed at toxic algae found along the coasts of Florida, New England, the Pacific Northwest, and Central California. Each area represents a unique ocean regime and poses different problems for studying and mitigating the impacts of harmful blooms. The MBARI ESP group will be working to validate the utility of these systems with researchers and managers in a coordinated, multi-region fashion, understanding that they will face a learning curve for operating the ESP in these different environments as well. The dream of the U.S. Commission on Ocean Policy—a device that resource managers can use to rapidly and remotely detect harmful organisms based on their unique genetic code—is indeed becoming a reality.

MBARI contributors to the Environmental Sample Processor: Nilo Alvarado, Mark Brown, Danelle Cline, Ed DeLong, Jon Erickson, Jason Feldman, Dianne Greenfield, Shana Goffredi, Scott Jensen, Joe Jones, Gene Massion, Roman Marin III, Peter Miller, Doug Pargett, Christina Preston, Brent Roman, Jim Scholfield, Chris Scholin, Rich Schramm, Alana Sherman, Mary Silver, John Tyrrell, Robert Vrijenhoek, Kevin Wheeler, Dave Wright

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