Understanding the distribution of deep life in the subsurface biosphere is a major challenge to advancing our understanding of the evolution life and ecosystems on Earth. To date, all methods developed to detect microbial life in the deep biosphere involve ex-situ analysis of recovered materials from boreholes. Materials are analyzed for microbial biomass by extracting cells or cellular components, or by application of dyes to samples and performing cell counts. This latter methodology being the most standard in the industry, and represents a very time and labor intensive hands-on by eye counting of fluorescent cells under a microscope. Current methods are highly laborious and inefficient processes that involve both cell loss and the loss of information about the mineralogical context that may have influence on the microbial ecology.
To meet the challenges associated with detecting and quantifying microbial life within a natural matrix, The PI requests RAPID funding to develop a novel in-situ downhole logging tool for detecting microbial life in subseafloor boreholes. "Deep UV" (DUV) is an optical method that enables detection and imaging of single bacterial cells on natural and opaque surfaces, including assessment of bacterial density and distribution of single cells to biofilms over spatial scales ranging from centimeters to microns. DUV induces and detects native fluorescence (DUV) of organic components intrinsic to the cell or spore while avoiding autofluorescence interference from the substrate, enabling detection of bacteria at spatial scales ranging from tens of centimeters to micrometers - i.e., both communities of microbes and single cells.
Broader Impacts
This project Leverages $350K from outside sources. It is anticipated that this technology could become widely used as a tool for detection and mapping of subsurface remote life everywhere it may occur, becoming the "satellite imager" applicable to distal environments such as represented by the entire deep sea. A JPL/Caltech post-doc with expertise in microbial physiology and fluorescence spectroscopy will be supported.
The ocean crust remains one of the last unexplored frontiers on our planet. In many cases, we know more about the surface of the moon than we know about the ocean crust. Over the last decade, scientists have discovered the existence of life deep in sediments on the seafloor, raising the possibility that life might extend deep into the oceanic crust. This life exists in the form of microorganisms, most of which are bacteria and archaea. However, to date we have very little direct evidence of the presence of life in the ocean crust, and no direct evidence that these organisms are active. Until now, most researchers exploring this area have relied on traditional techniques used in microbiology research like DNA extraction and fluorescence microscopy. However, that requires that scientists bring up cores from the ocean floor via drilling, and that the organisms be removed from the recovered rocks. Recovering rocks from beneath the seafloor is a very complex process and leads to very low rates of recovery. Additionally, ensuring that the recovered samples do not get contaminated during extraction from the crust, transport up to the surface and then during handling by scientists, is also extremely difficult. Finally, extraction of DNA from microbes bound to a rock matrix is difficult (there is currently no universally established technique for this process) and using fluorescence microscopy to look for cells on the rocks is complicated by the fact that many minerals auto-fluoresce when interrogated with the light used in fluorescence microscopes. In response to these issues, the University of Southern California, Photon Systems, Inc., Caltech-JPL and The Lamont-Doherty Earth Observatory collaborated to develop the Deep Exploration Biosphere Investigative tool (DEBI-t). DEBI-t is a biosensor that uses what is known as deep Ultraviolet laser to excite organic molecules that are on the surface of a rock. The excited organics emit at wavelengths that are shorter than those used in typical fluorescence microscopy, thus, there is no interference from the mineral matrix. Microbes like bacteria and spores emit light at specific wavelengths and in specific patterns that make them distinguishable from organic molecules. DEBI-t was designed to be deployed as a wireline logging instrument. These instruments are common in the oil and gas industry and are used to explore the deep subsurface. In order to explore the crust, a borehole is drilled that allows instrumentation to be lowered down into it. The logging tools are then lowered through the drill pipe and into the borehole to collect information about the crust, such as density, conductivity mineralogy and porosity, as they travel up and down. In DEBI-t's case, the laser shines on the surface and causes the organic molecules to fluoresce; that fluorescence is then recorded by detectors onboard DEBI-t. The instrument has a small sapphire window that allows the laser pulse to exit the instrument and the fluorescence signal to pass back into the instrument. DEBI-t also contains a small pinhole camera to record where in the borehole the instrument is so that we have a visual context for what the instrument is detecting. That enables the scientists to know how dirty the water was, or what kind of rocks the instrument might have been scanning, etc. The camera uses the same sapphire window to look outside. Because we know where in the borehole DEBI-T is, we know where the different fluorescence signals come from. DEBI-t was successfully deployed into 3 borehole during expedition 336 (Fig. 1). The instrument was deployed as part of an instrument suite known as the microbiology combination tool string (Fig. 2). The microbiology combo tool obtains a number of parameters in addition to the fluorescence collected by DEBI-t; namely, 3-axis downhole acceleration, 3-axis magnetic field, temperature and gamma ray measurements. This allows for the signals collected by DEBI-t to be interpreted in a geophysical context. Results indicated that Hole 395A, a legacy borehole that had been sealed for over 10 years, harbored a large amount of material within it, some of which appears to be spectrally similar to spores. Holes U1382A and U1383C, two new holes drilled during the expedition, had different fluorescence signatures, which are still being interpreted. The successful deployment of DEBI-t is a positive prospect for further development of novel technologies that can be used to explore the deep ocean subsurface. This in turn will provide us with a better understanding of how bit the subsurface biosphere is, and the importance of this environment to global nutrient cycling.
