Award Dates: May 1, 2012 — June 30, 2014
Awardee: Luke J. McKay (University of North Carolina, Chapel Hill)
Advisor: Andreas P. Teske (University of North Carolina, Chapel Hill)
Abstract
Members of the Marinobacter genus play an important role in hydrocarbon degradation in the ocean – a topic of special significance in light of the recent Deepwater Horizon oil spill of 2010. The Marinobacter group has thus far lacked a genus level phylogenetic probe that would allow in situ identification of representative members. Here, two new 16S rRNA-targeted oligonucleotide probes (Mrb-0625-a and Mrb-0625-b) were developed to enumerate Marinobacter species by fluorescence in situ hybridization (FISH). In silico analysis of this probe set demonstrated 80% coverage of the Marinobacter genus. A competitor probe was developed to block hybridization by Mrb-0625-a to six Halomonas species with which it shared a one base pair mismatch. The probe set was optimized using pure cultures, and then used in an enrichment experiment with a deep sea oil plume water sample collected from the Deepwater Horizon oil spill. Marinobacter cells rapidly increased as a significant fraction of total microbial abundance in all incubations of original contaminated seawater as well as those amended with n-hexadecane, suggesting this group may be among the first microbial responders to oil pollution in the marine environment. The new probe set will provide a reliable tool for quantifying Marinobacter in the marine environment, particularly at contaminated sites where these organisms can play an important role in the biodegradation of oil pollutants. The next sections of this dissertation focus on the hydrothermally active sediments at Guaymas Basin, which show a wide range of shallow subsurface temperatures: from 3°C to 200°C in the first 45 cm depth. A combination of extreme thermal gradients and compressed geochemical and metabolic zones limits the depth range of microbial colonization in Guaymas sediments. Using stable carbon isotopic values for methane and dissolved inorganic carbon compared to associated temperatures the upper thermal limits for the anaerobic oxidation of methane and organic carbon remineralization in Guaymas sediments are suggested to be 80oC and 100oC, respectively. At higher temperatures the isotopic imprints of these microbially mediated processes cannot be detected. Additionally, 16S rRNA gene clone libraries demonstrate differential biogeographical zonation patterns for archaea versus bacteria, with archaeal community structure being more heavily influenced by hydrothermal regimes. Chloroflexi and Deltaproteobacteria dominated the bacterial clone libraries, and anaerobic methane-oxidizing (ANME) archaea represented nearly half of the total archaeal clone library. Thermal zonation of ANME archaeal subgroups is strong: ANME-2c is restricted to low temperature sediments (<25oC), ANME-1 is dominant at warmer temperatures, and the ANME-1 Guaymas archaea appear to have access to the deepest and hottest sediment horizons up to approximately 80oC. In the last chapter of this dissertation, microbial life at extreme temperatures was investigated further by RNA-based methodologies. Using push core samples collected by the Alvin submarine at four high temperature sites with 40-cmsbf thermal maxima ranging from 100°C to 185°C, the composition of the active microbial community and its possible influence on carbon and sulfur cycling was investigated. Here, evidence is presented indicating that hydrothermal fluctuations are frequent enough to restrict hyperthermophilic life to sediments with average in situ temperatures between 70°C and 95°C, where temperatures may vary by 25°C in as little as a day. Strong microbially mediated sulfate reduction is implicated by sharp decreases in porewater sulfate within the upper 15 cm of all four high temperature cores, while stable isotopic evidence of methane oxidation is only expressed in a single core. Archaeal sequence recovery was greater than bacterial sequence recovery in six out of eight samples from the four cores, but bacterial sequence recovery was particularly strong for a single core, yielding 35% of the total archaeal and bacterial recovery from all samples. Although putative anaerobic methane oxidizing (ANME) archaea were very common, distinct cores hosted diverse and distinct sequence assemblages, including ANME-1 Guaymas, ANME-2c, and ANME-2d/GoM Arc-1/Methanoperedenaceae. Dominant bacterial groups fell within the Thermodesulfobacteriaceae family in the Thermodesulfobacteria phylum, the Helicobacteriaceae family in the subphylum Epsilonproteobacteria, or were close relatives of Desulfocapsa exigens in the subphylum Deltaproteobacteria. The most probable thermo- or hyperthermophilic groups were investigated by co-occurrence of OTUs across the four hottest samples within the sediment cores and appear to be ANME-1 Guaymas and an uncultured representative of the Miscellaneous Crenarchaeotal Group (MCG)-15 for archaea, and members of the Thermodesulfobacteriaceae family for bacteria.