Douglas Pargett

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.

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.

Engineering Development of the Free Ocean CO 2 Enrichment (FOCE) Experiment

Globally, the burning of fossil fuels for energy production produces over 25 gigatons of CO2 per year and this material is released directly into the atmosphere. While approximately half of the CO2 has remained in the atmosphere long-term, most of the rest has been absorbed by the surface ocean. This has resulted in a lowering of the surface ocean pH by about 0.1 units since the beginning of the industrial revolution and if society is able to stabilize atmospheric CO2 levels at twice their pre-industrial concentrations will result in a lowering of surface ocean pH by 0.25 units. While many are asking the question of whether we should pursue direct ocean CO2 sequestration, the FOCE experiment is asking what will be the impact of the pH change on the ocean. In order to address this question, MBARI science and engineering have designed a small-scale in situ CO2 enrichment experiment to assess the chemical and biological impacts in a manner analogous to the land-based Free Air CO2 Enrichment (FACE) experiments. This prototype design is testing the ability to control pH within a fixed but freely exchanging volume of sea water. The technology concept for the experiment is based on a small ring structure using a central valve to direct the flow of pH altering fluid. The initial phase of the project uses concentrated HCl mixed with sea water and includes directional and volume control to achieve a desired pH offset. Control feedback is obtained by using pH sensors in the center of the control volume. Other aspects of the design that address the inherent time delays and noise of the associated pH signal are also discussed. Test results will show the capability of the system to maintain close loop control of pH in a given volume. Sea trials then demonstrate the ability of this initial system at a selected site to control pH to specified average level over a given amount of time. Further discussion includes systems in-situ results analysis, corrective actions, upgrades, and the anticipated next phase for FOCE including the use of CO2 addition to change the local chemistry

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.

Development of a mobile ecogenomic sensor

Modern ocean microbial research utilizes advanced molecular analytical techniques, such as polymerase chain reaction (PCR), DNA and protein probe arrays, and nucleic acid sequencing (etc.). Applying or at least initiating these techniques at the point and time of sample collection can enhance their effectiveness. To that end, in-situ sample processing and real-time molecular detection schemes have been implemented using deployable autonomous systems that can be operated in diverse ocean environments from shallow coastal waters to the deep sea. Such devices have been termed “ecogenomic sensors.” The size of these instruments currently requires that they be moored in a fixed location or passively mobile, drifting at fixed depth and observing microbial communities in a moving frame of reference with ocean currents. With the highly dynamic motion of open water and microbial life, the next frontier of ocean microbial research requires the improved capability of an actively mobile asset. A mobile ecogenomic sensor encompasses a fully maneuverable vehicle with weeks of persistence, environmental data analysis, detection of physical and biological features, autonomous sampling and in situ analysis, and near-real-time data reporting. This system is now being developed by integrating three components: a compact molecular analytical instrument (the 3rd generation Environmental Sample Processor), a long-range autonomous underwater vehicle, and software algorithms for AUV-based feature detection and sampling. A summary of the system and its initial application is presented.

Tracking and sampling of a phytoplankton patch by an autonomous underwater vehicle in drifting mode

Phytoplankton patches in the coastal ocean have important impacts on the patterns of primary productivity, the survival and growth of zooplankton and fish larvae, and the development of harmful algal blooms (HABs). We desire to observe microscopic life in a phytoplankton patch in its natural frame of reference (which is moving with the ocean current), thereby permitting resolution of time-dependent evolution of the population. To achieve this goal, we have developed a method for a Tethys-class long range autonomous underwater vehicle (AUV) (which has a propeller and a buoyancy engine) to detect, track, and sample a phytoplankton patch in buoyancy-controlled drifting mode. In this mode, the vehicle shuts off its propeller and actively controls its buoyancy to autonomously find the peakchlorophyll layer, stay in it, and trigger water sampling in the layer. In an experiment in Monterey Bay, CA in July 2015, the Makai AUV, which was equipped with a prototype 3rd-generation Environmental Sample Processor (3G-ESP), ran the algorithm to autonomously detect the peak-chlorophyll layer, and drifted and triggered ESP samplings in the layer.