Brent Roman

Underwater application of quantitative PCR on an ocean mooring.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

The Environmental Sample Processor (ESP) - An Autonomous Robotic Device for Detecting Microorganisms Remotely using Molecular Probe Technology

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

Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor

Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The D-ESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.

Controlling a Robotic Marine Environmental Sampler with the Ruby Scripting Language

The Environmental Sample Processor (ESP) is an autonomous robotic instrument developed at the Monterey Bay Research Aquarium Institute (MBARI) that operates below the oceans 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.

Simultaneous monitoring of faecal indicators and harmful algae using an in-situ autonomous sensor.

Faecal indicator bacteria (FIB) and harmful algal blooms (HABs) threaten the health and the economy of coastal communities worldwide. Emerging automated sampling technologies combined with molecular analytical techniques could enable rapid detection of micro‐organisms in‐situ, thereby improving resource management and public health decision‐making. We evaluated this concept using a robotic device, the Environmental Sample Processor (ESP). The ESP automates in‐situ sample collection, nucleic acid extraction and molecular analyses. Here, the ESP measured and reported concentrations of FIB (Enterococcus spp.), a microbial source‐tracking marker (human‐specific Bacteriodales) and a HAB species (Psuedo‐nitzschia spp.) over a 45‐day deployment on the Santa Cruz Municipal Wharf (Santa Cruz, CA, USA). Both FIB and HABs were enumerated from single in‐situ collected water samples. The in‐situ qPCR efficiencies ranged from 86% to 105%, while the limit of quantifications during the deployment was 10 copies reaction−1. No differences were observed in the concentrations of enterococci, the human‐specific marker in Bacteroidales spp., and P. australis between in‐situ collected sample and traditional hand sampling methods (P > 0·05). Analytical results were Internet‐accessible within hours of sample collection, demonstrating the feasibility of same‐day public notification of current water quality conditions.

Co-registered Geochemistry and Metatranscriptomics Reveal Unexpected Distributions of Microbial Activity within a Hydrothermal Vent Field

Despite years of research into microbial activity at diffuse flow hydrothermal vents, the extent of microbial niche diversity in these settings is not known. To better understand the relationship between microbial activity and the associated physical and geochemical conditions, we obtained co-registered metatranscriptomic and geochemical data from a variety of different fluid regimes within the ASHES vent field on the Juan de Fuca Ridge. Microbial activity in the majority of the cool and warm fluids sampled was dominated by a population of Gammaproteobacteria (likely sulfur oxidizers) that appear to thrive in a variety of chemically distinct fluids. Only the warmest, most hydrothermally-influenced flows were dominated by active populations of canonically vent-endemic Epsilonproteobacteria. These data suggest that the Gammaproteobacteria collected during this study may be generalists, capable of thriving over a broader range of geochemical conditions than the Epsilonproteobacteria. Notably, the apparent metabolic activity of the Gammaproteobacteria—particularly carbon fixation—in the seawater found between discrete fluid flows (the intra-field water) suggests that this area within the Axial caldera is a highly productive, and previously overlooked, habitat. By extension, our findings suggest that analogous, diffuse flow fields may be similarly productive and thus constitute a very important and underappreciated aspect of deep-sea biogeochemical cycling that is occurring at the global scale.

The environmental sample processor (ESP) software design: software for detection and quantification of microorganisms

The Environmental Sample Processor (ESP) instrument has been designed by the Monterey Bay Aquarium Research Institute (MBARI) for ocean sampling and monitoring. The ESP is an in situ sampling and processing device that enables near real-time detection of specific microorganisms through the application of molecular probes. The intended use of ESP is a 1 to 3 month deployment in 50 meters maximum depth for detection of harmful algal blooms. The authors present an overview of the software architecture deployed on the ESP instrument. The ESP software design is applied on two prototype instruments with similar mechanical design, but different control electronics. Presented in this work is the software architectural framework used that allows for controlled start up, shutdown, task and event handling in a concurrent software environment. They discuss how object-oriented design patterns such as the Adapter pattern are used to solve design problems and how testing improved reliability. A description and examples are given of the flexible ESP macro language that allows scientists to automate chemical processing steps. And finally, an algorithm for DNA probe array image registration and data extraction involving low-pass filtering, connected components, rotational translation, and component recognition and interpretation is presented.