Autonomous in-situ qPCR in the Deep Sea

We are developing an instrument to autonomously detect microbial genes that mediate biogeochemical transformations in the deep sea as a step towards developing the scientific and technical capability for searching for life on other planets. This device, known as the Deep-sea Environmental Sample Processor (D-ESP), allows for autonomous collection of discrete 10L water samples at depths up to 4,000 m followed by application of DNA probe arrays as well as qPCR in support of microbial community ribotype analyses and detection of specific genes, respectively. The D-ESP can also be used to preserve particulate material for return to laboratory to validate information gleaned from in-situ sample processing as well as to support expanded studies of genomic diversity and gene expression. To provide a contextual framework for evaluating factors controlling microbial populations, the D-ESP is deployed with a custom suite of chemical and physical sensors including an in-situ mass spectrometer (ISMS), In Situ Underwater Spectrophotometer (ISUS) and CTD/optical sensor package. The D-ESP was deployed for 5 days during July 2010 on the crest of a methane-rich authigenic carbonate mound in Santa Monica Basin (~800 m depth) and two off-mound sites (70 m and 263 m due east of the mound). Bacterial mats mantle the mound, and streams of methane bubbles rise out of long linear cracks in the authigenic carbonate mound. Previous molecular investigations of the benthic water column over and surrounding the mound have demonstrated presence of a diverse assemblage of 16S rRNA and particulate methane monoxygenase subunit A (pMMO-A) genes belonging to putative aerobic methanotrophs making it a useful site for testing the D-ESP. For qPCR analysis, the D-ESP was thus configured to target the pMMO-A and 16S rRNA genes of two putative methanotrophic groups OPU1 and OPU3, the most widespread and abundant groups found in Santa Monica Basin. The pMMO-A and 16S rRNA genes of OPU1 and OPU3 were detected by D-ESP at the on- and off-mound sites as was expected based on previous work. The pMMO-A gene of OPU1 was more abundant than OPU3. In contrast, the 16S rRNA gene of OPU3 group was more abundant than OPU1. Shipboard measurements of on-mound methane show a high degree of meter-scale spatial and temporal heterogeneity (2.9 to 55,000 nM). At the two off-mound sites, methane was more uniform and substantially lower (70-m site = 8.4±0.3 nM; 263-m site = 2.2±0.1 nM). Water for background comparison collected by CTD rosette from 620 m depth at a site near Point Conception was processed by the D-ESP on the deck of the R/V Western Flyer. OPU1 and OPU3 were less abundant than at the off-mound sites, and although detected they were unquantifiable (<10 copies/mL seawater). Methane concentration was an order of magnitude lower than the off-mound sites (0.4±0.07 nM). Although the qPCR data indicate the increased presence of these microbes in a region of methane-enriched water relative to background, methane itself is not a good predictor of the abundance of genes involved with metabolizing that substrate highlighting the need to place biogeochemical analyses in a regional rather than site-specific context.