Search

The Environmental Sample Processor (ESP) is an autonomous robotic instrument developed at the Monterey Bay Research Aquarium Institute (MBARI) that operates below the ocean's surface, sampling raw seawater and executing a variety of sample manipulation and analytical protocols, in situ. It uses DNA and antibody probes to identify marine planktonic organisms and substances they produce. Initial prototypes of the ESP were hosted on an Intel i486 CPU running a commercial real-time operating system (OS). The application, coded in C++, included a custom ‘macro’ language interpreter to direct biochemical analyses. To achieve greater flexibility and minimize the development effort for the 2nd generation of the ESP (2G ESP), MBARI replaced its ‘macro’ language with a general purpose, open-source scripting language, selecting Ruby for its unique combination of a succinct, English-like syntax with a seamless underlying object-oriented paradigm. The 2G ESP application, aside from custom servo control firmware, is coded entirely in Ruby, hosted on a low-power ARM9 CPU running Linux. Servo control was distributed onto a network of dedicated microcontrollers to cope with the nondeterministic delays inherent in the Linux operating system and Ruby interpreter.

In this contribution, we assess the application of methodology used in a novel in situ remote sampling platform, the environmental sample processor (ESP), for the identification and enumeration of a harmful algal species, the domoic acid-producing diatom Pseudo-nitzschia australis, using 2 molecular assays: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). Both the SHA and FISH assays were initially designed as laboratory-based methods that are now emulated in the ESP. Response of the SHA to a range of concentrations of P. australis using the laboratory (96-well plate) and ESP (DNA probe array) formats showed that the two were highly correlated. Enumeration of cells filtered and archived for FISH using a manifold designed for laboratory applications agreed well with counts of cells filtered and archived in the ESP at ≥2.5 × 104 cells L−1. At lower concentrations, it becomes statistically unlikely to derive a reliable abundance estimate, suggesting that FISH is better suited for qualitative analyses unless the target organism is very abundant. We also assessed the suitability of an oligonucleotide as synthetic target sequence to act as a SHA reagent quality control and internal standard for the plate assay. This was successful, but the probe and/or associated reagents were stable for only ~60 days. Our results show that the ESP is capable of detecting P. australis in near real-time and also supports whole-cell archival samples, making it a potentially useful tool for future research and monitoring initiatives.

A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10-1.98 and 4.43- 12.54 fmole ml(-1) homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.

The Simple Cabled Instrument for Measuring Properties In Situ (SCIMPI) is a proposed new long-term seafloor observatory system for sub-seafloor studies. The SCIMPI sensors, initially proposed, include temperature sensors, resistivity sensors, and pore pressure transducers. The design easily accommodates integration of other sensors (e.g., geophones, X-ray fluorescence, spectrophotometry). SCIMPI can be deployed autonomously with a battery and data module for 1-2 year deployments or connected to a seafloor fiber optic cable for real time data streaming. Applications include the study of sub-seafloor hydrological and deep biosphere studies; pore pressure measurements for hazards assessments; long-term temperature measurements; and earthquake monitoring. Potential near-term SCIMPI sites include Monterey Bay, Cascadia Margin and the Gulf of Mexico. In Monterey Bay, SCIMPI is proposed to be part of the Monterey Accelerated Research System (MARS) cabled observatory. On Cascadia Margin, SCIMPIs are proposed to be part of a cabled observatory, and are specifically targeted for a location called Hydrate Ridge. Hydrate Ridge studies are focused on understanding the mechanisms, rates, and extent of methane hydrate formation in this area. SCIMPIs at Hydrate Ridge will measure temperature, pore pressure, electrical resistivity, and micro-seismicity. The proposed plan is to install SCIMPIs by the IODP and then link the SCIMPI observatories into the Regional Cabled Observatory in the ORION program. Potential Gulf of Mexico sites are in the deep water environments where overpressures have been observed in shallowly buried, loose sand deposits.

We are developing an instrument to conduct molecular biological analyses below the ocean surface, autonomously. The device is known as the Environmental Sample Processor, or ESP. The system is based on a modular design consisting of a core sample processor (the ESP), analytical modules and sampling modules. The core ESP provides the primary interface between the environment and a set of DNA and antibody-based tests that are carried out onboard the instrument in real-time. In addition, the ESP can be used to archive samples for a variety of analyses after the instrument is returned to a laboratory. Sampling modules are devices external to the core ESP that can be added to meet specialized needs, such as operating in the deep-sea (etc). Analytical modules are conceived of as stand-alone devices that can be added to the core ESP to impart different suites of analytical functions downstream of common sample processing operations. At the time of this writing we have worked most extensively on the core ESP and detection chemistries that involve DNA probe and protein arrays. The ESP has been deployed successfully in coastal ocean surface waters. We are also developing a sample collection module and pressure housing suitable for deploying the ESP at depths to 1000m. This version of the instrument is known as the deep-sea ESP, or D-ESP. The long-term objective of the D-ESP program is to provide a molecular analytical capability at deep-sea hot vents and cold seeps. The D-ESP will be deployed using an ROV and later transitioned to benthic moorings and a cabled observatory. Finally, we are just starting work to incorporate a microfluidic analytical module to support assays that require DNA purification and amplification

Many of the diagnostic protocols for detecting molecular signatures share an overlapping set of requirements, such as collecting, concentrating, preserving and disrupting cells, applying a series of reagents in a timed sequence, removing particulates, and applying solid phase extraction chemistries, among others. Operationally, these methods can be divided into those that depend on intermolecular reactions, such as antibody/antigen binding, nucleic acid hybridization and enzyme mediated processes, and those that rely on separation and identification of target molecules based on their inherent physical properties. Optical and electrochemical transducers are used commonly to detect and quantify numerous molecular signatures. By developing a relatively low-power, compact instrument that meets this core set of functional requirements, it would be possible to conduct many commonly applied molecular biological analyses remotely, in situ. Toward that goal, a group of scientists and engineers at the Monterey Bay Aquarium research Institute (MBARI) undertook development of the Environmental Sample Processor. The ESP is an electromechanical/fluidic instrument system designed to collect discrete water samples from the ocean subsurface, concentrate microorganisms (particulates), and automate application of molecular probes to identify microorganisms based on ribosomal RNA (rRNA) signature sequences. In addition, the ESP archives discrete samples for a variety of nucleic acid analyses, microscopy and other types of analytical procedures after the instrument is recovered. To date, the ESP team has focused on detecting a suite of marine planktonic organisms ranging from heterotrophic and photosynthetic bacteria, archaea and eucarya to small invertebrates found in the upper ocean. "First generation" (or "1G") prototypes of the ESP have been deployed in Monterey Bay and the Gulf of Maine