Temperature Regulation of Nitrate-Respiring Microbial Communities in Permanently Cold Arctic Sediments

Nitrogen (N) is often a limiting nutrient for primary productivity in the ocean and plays a critical role in the global carbon cycle. Fixation of N2 into reactive N (Nr) by photosynthetic prokaryotes in surface waters is a primary source of N to the marine nitrogen cycle. Removal of reactive nitrogen from the marine environment is accomplished primarily through two microbial processes, denitrification and anammox, that produce gaseous N as terminal metabolic byproducts. Denitrification is the respiratory reduction of nitrate (NO3-) to gaseous end products (N2, N2O), is mediated by a phylogenetically diverse group of microbes, and may be heterotrophic or autotrophic. Anammox involves the anaerobic oxidation of ammonium (NH4+) with nitrite (NO2-) and is an autotrophic process. Only one genus, Scalindua, within the Planctomycetes has been identified as an anammox bacterium in the marine environment. Historically, all gaseous N production in the oceans was attributed to heterotrophic denitrification, but it is now clear that a more diverse group of microbes mediate Nr removal. An estimated 50 to 70% of the global removal of marine Nr occurs in sediments, and Nr removal from continental shelves comprises approximately half of the total sediment contribution. It follows that Arctic continental shelves, which contribute 18% of the global continental shelf area, may contribute significantly to global Nr removal. High export production in the subarctic Bering Sea and in the arctic Chukchi and Barents Sea fuels locally high rates of N2 production that largely overlap with those of temperate shelf sediments. It appears that the permanently cold (< 4 °C) conditions do not limit Nr removal rates from sediments in these regions of the Arctic. This lack of inherent temperature limitation has been observed for other microbial respiratory processes in permanently cold sediments and has been attributed to the activity of psychrophilic ("cold-loving") sediment microbial communities. The main objective of this dissertation was to further elucidate the temperature adaptation of naturally occurring sediment microbial communities that mediate denitrification and anammox in polar sediments. The Arctic Ocean faces, among other things, increases in water temperature and shifts in export production as a result of climate change. Therefore, an understanding of the physiology of denitrifying and anammox communities is necessary to understand the potential sensitivity of these communities to climate change related effects. Adaptation of sediment microbial communities to permanently cold temperatures was examined in both sediment samples and bacterial isolates from fjords of the west coast of the island of Spitsbergen in the Svalbard archipelago (79 °N). A comparative study of subtropical (St. George Island, Florida), temperate (Sylt, Germany), and polar (Svalbard) sites was undertaken to further understand the role of temperature adaptation on latitudinal spatial scales. Psychrophilic adaptation of naturally occurring sediment microbial communities that mediate denitrification and anammox was examined in Arctic fjord sediments from western Svalbard (Chapter 2). Rates of denitrification and anammox in sediments of three western fjords of Svalbard with contrasting total organic carbon (TOC) and C:N ratios were quantified using the isotope pairing technique (IPT). Rates of sediment N2 production ranged between 7.8 - 333 µmol N m-2 d-1 and varied directly with sediment organic carbon content and the solid phase C:N ratio. The temperature dependency of denitrification and anammox rates was investigated using anaerobic slurry incubations in a temperature gradient block incubator for temperatures ranging from -1 °C to 40 °C. The optimal temperatures (Topt€) for denitrification and anammox were 25 to 27 °C and 12 to 17 °C, respectively. One site (Station J), exhibited a broad optimum for denitrification between 9 °C to 30 °C that may be influenced by the high concentration of labile marine organic matter observed at this site. The Topt values provided evidence for predominantly psychrophilic adaptation in sediment communities and were consistent with a previous study of N2 production in Arctic sediments. Apparent activation energy (Ea), calculated from the Arrhenius function, varied with site from 24 - 64 kJ mol-1. When compared to previous temperature response data for other respiration processes in sediments, a considerable amount of variability in Topt and activation energy (Ea) was observed and was hypothesized to be caused by substrate availability. Long term (weeks) warming experiments indicated that increases in temperature of 5 to 10 °C above in situ temperatures had little effect on the temperature response of denitrification and anammox, but increases of 25 °C shifted denitrification towards a predominately mesophilic community and eliminated anammox activity. Both denitrification and anammox exhibited temperature response characteristics consistent with a predominately psychrophilic community, but microbes with the anammox pathway appeared to be more adapted to permanently-cold sediments. Results also indicate that the effects of low temperature are modulated by other environmental factors, including carbon substrate availability, to control rates of N-removal in these Arctic coastal sediments. In order to identify the taxa potentially responsible for denitrification in polar sediments, denitrifying bacteria from Svalbard fjord sediments were isolated and physiologically characterized (Chapter 3). Identification of the active denitrifying bacteria in ecosystems using nucleic acid based techniques is hindered by a lack of PCR primer coverage and widespread horizontal gene transfer of denitrification functional genes. Thus, the verification of denitrification activity in cultivated bacteria is a crucial aspect of improving cultivation-independent approaches. A combined approach of MPN enumeration, cultivation, and physiological characterization was used to describe psychrophilic denitrifying bacterial communities in sediments of three Arctic fjords in Svalbard (Norway). A most probable number (MPN) assay showed the presence of 103 -106 cells of psychrophilic nitrate-respiring bacteria g-1 of sediment. Fifteen strains within the Proteobacteria were isolated using a systematic denitrifying enrichment approach with organic acids as electron donors and nitrate as an electron acceptor at 4 °C. Isolates belonged to five genera, including Shewanella, Pseudomonas, Psychromonas (Gammaproteobacteria), Arcobacter (Epsilonproteobacteria), and Herminiimonas (Betaproteobacteria). Members of the genera Shewanella, Pseudomonas, and Arcobacter were putatively detected in sediment samples by terminal restriction fragment polymorphism (TRFLP) profiling. All isolates were denitrifiers, except Shewanella, which exhibited the capacity for dissimilatory nitrate reduction to ammonium (DNRA). Growth from 0 to 40 C demonstrated that all genera except Shewanella were psychrophiles with optimal growth below 15 °C, and adaptation to low temperature was demonstrated as a shift from primarily C16:0 saturated fatty acids to C16:1 monounsaturated fatty acids at lower temperatures. This study provides the first targeted enrichment and characterization of psychrophilic denitrifying bacteria from polar sediments, and two genera, Arcobacter and Herminiimonas, are isolated for the first time from permanently cold marine sediments. A comparison of temperature adaptation of denitrification and anammox between polar, temperate, and subtropical permeable sediments was the objective of Chapter 4. Recent studies support the emerging paradigm that permeable sediments serve as a substantial sink for land-derived nitrogen along the land-to-sea continuum. Nitrogen removal rates and the temperature regulation of microbial communities that mediate nitrogen removal were investigated in nearshore permeable sediments over a 50° latitudinal gradient at subtropical (Gulf of Mexico), temperate (Wadden Sea), and polar (Svalbard archipelago) sites. Total N2 production rates determined with the isotope pairing technique (IPT) in intact core incubations ranged from 2 to 359 µmol N m-2 d-1 according to environmental conditions. While denitrification predominated at all sites, anammox comprised 6 - 14 % of nitrogen removal at temperate and polar sites. Pore-water advection had a substantial impact on N2 production at all sites, with rates up to 7 times higher under conditions with simulated advective transport. Optimal temperatures (Topt) of potential denitrification rates were highest at subtropical sites (35 - 36 °), varied seasonally at the temperate site (26 - 34 °C), and were lowest at the arctic site (21 °C). The apparent activation energy (Ea) of denitrification was two-fold higher in subtropical (102 - 124 kJ mol-1) versus temperate and polar sediments (52 - 65 kJ mol-1), further indicating dominance of mesophilic microbial communities at the subtropical sites. The Topt values for potential anammox rates were 9 °C and 26 °C at the polar and temperate sites, respectively, and anammox was not detected at subtropical sites. Values of apparent activation energy (Ea) for anammox were lower than denitrification (28 - 37 kJ mol-1), and along with lower Topt values as compared to denitrification, suggest that marine anammox bacteria may be more specialized for cold environments. This study reveals adaptation of denitrification and anammox processes to in situ temperatures in permeable marine sediments across a wide range of in situ temperatures (7 - 32 °C), and the first evidence for substantial nitrogen removal from permeable sediments in a polar environment is provided.