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Over the last several years, scientists and engineers at the Monterey Bay Aquarium Research Institute (MBARI) have successfully developed and deployed two generations of deep ocean laser Raman spectrometers. There are many advantages to this type of spectroscopy: it is rapid, non-destructive, and can be used to analyze solids, liquids, and gases. Unfortunately, one of the disadvantages of using Raman spectroscopy is that the return signal measured is very weak. Thus, it is difficult to detect dilute chemical compounds in solution, such as bicarbonate and carbonate ions at natural concentrations. This paper discusses the efforts we have made to increase the sensitivity of our second generation Deep Ocean Raman In Situ Spectrometer (DORISS2). In developing this second generation instrument, we incorporated new components which improved system performance. These components include a new ruggedized U-shaped spectrometer and a back-illuminated CCD camera which is much more sensitive than our original front-illuminated CCD camera. The element which has had the most impact on system sensitivity is a set of new custom-made fiber optic cables. We had built several sets of custom fiber optic cables, but despite our efforts, their performance degraded substantially over time. Recently, we developed a new oil-filled pressure-compensated fiber optic cable which performs far better in pressure testing and is much more robust. At 6000 psig, this cable shows losses of only 2-3 dB versus our previous cables where losses of 25-30 dB were common. This new fiber optic cable was field tested in May and showed markedly improved performance.

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.