Biogeochemical and metagenomic analysis of nitrite accumulation in the Gulf of Mexico hypoxic zone

The Louisiana Shelf in the Gulf of Mexico experiences recurrent bottom water hypoxia under summertime eutrophic conditions. The onset, maintenance, and breakdown of hypoxia are associated with dynamic microbial biogeochemical cycles. However, the distribution of microbial taxa and metabolisms across Shelf oxygen gradients remains under-characterized. We combined biogeochemical analyses of nitrogen (N) distributions and metabolic rates with metagenomic and metatranscriptomic analysis of summertime Shelf waters. Samples from an east–west transect during July 2012 revealed an inverse relationship between nitrite and oxygen concentrations, with concentrations exceeding 1 lmol L at 50% oxygen saturation and reaching 4.6 lmol L at the most oxygen-depleted sites. Historical data confirms that nitrite accumulation occurs frequently in this region both above and below the hypoxic threshold. Experimental incubations demonstrated a strong decoupling between the two steps of nitrification, with ammonia oxidation proceeding up to 30 times faster than nitrite oxidation under low oxygen. 16S rRNA gene, metagenome, and metatranscriptome sequencing revealed a diverse microbial community, stratified over shallow (< 10 m) depth gradients, with an enrichment of ammonia-oxidizing Thaumarchaeota genes and transcripts in deeper more oxygendepleted layers, and a comparatively low representation of sequences related to nitrite oxidation. A range of factors, including temperature and substrate availability, which may be linked indirectly to bottom water oxygen content, potentially drives decoupling of ammonia and nitrite oxidation on the Louisiana Shelf. Nitrite accumulation in hypoxic zones remains understudied and may have important effects on microbial nitrogen flux, algal dynamics, and production. Oxygen concentration is a fundamental driver of microbial metabolism and biogeochemical cycling in aquatic ecosystems. In coastal ecosystems, such as the shallow waters of the Louisiana Shelf in the northern Gulf of Mexico (GoM), oxygen concentrations vary substantially over seasonal and spatial gradients (Turner et al. 2008). Shelf oxygen gradients are associated with variations and fluxes of key bioactive substrates, including organic carbon, and macronutrients and micronutrients, the dynamics of which have been well characterized as part of a comprehensive long-term monitoring program (Rabalais et al. 2002; Turner et al. 2007). In contrast, the microorganisms regulating material and energy cycling in the pelagic Shelf ecosystem remain undercharacterized. Oxygen is a primary determinant of the taxonomic composition of Shelf microbial communities (King et al. 2013; Tolar et al. 2013). However, it remains unclear how changes in microbial abundance or activity affect processes such as nitrification or denitrification, which involve interdependent metabolic transformations by different microbial guilds. Varied responses of microbial taxa to oxygen depletion may decouple important steps in elemental cycles, thereby influencing bulk fluxes, as well as standing stocks of bioavailable intermediates such as nitrite. The accumulation of nitrite may affect the pathways of microbial nitrogen cycling, as nitrite is a pivotal intermediate in microbial N transformations (Thamdrup et al. 2012). Accumulation may also influence phytoplankton dynamics, given that the kinetics of nitrite utilization vary greatly between different algal species (Collos 1998; Malerba et al. 2012). Additional Supporting Information may be found in the online version of this article. Conflict of Interest: The authors have no competing commercial interests in relation to this work. Laura A. Bristow and Neha Sarode contributed equally to this work. *Correspondence: [email protected] 1 LIMNOLOGY and OCEANOGRAPHY Limnol. Oceanogr. 00, 2015, 00–00 VC 2015 Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10130 Episodically occurring patches of low oxygen water are common features of Louisiana Shelf waters with important implications for ecosystem structuring (Diaz and Rosenberg 2008; Rabalais et al. 2010; Zhang et al. 2010). Hypoxia occurs where microbial respiration of organic biomass depletes oxygen concentrations to below 2 mg L ( 63 lmol kg). Louisiana Shelf hypoxia typically encompasses 20% to 50% of the total water column during summer (Rabalais et al. 2001) when nutrients from the Mississippi and Atchafalaya rivers fuel high levels of primary production. Although Shelf hypoxia can form naturally, enhanced riverine nutrient influx due to human activities has increased the intensity and extent of hypoxic layers in the northern GoM and throughout the world’s oceans over the last half-century (Diaz and Rosenberg 2008; Rabalais et al. 2010). In years of high nutrient runoff, the GoM hypoxic zone is one of the largest in the world (Rabalais et al. 2002, 2007; Bianchi et al. 2010), extending over an area of up to 22,000 km. In other years, including at the time of this study in 2012 when drought in the upper U.S.A led to decreased river outflow, the spatial extent of hypoxia is far less. However, dynamic oxygen and nutrient gradients occur on the Shelf in all years, as documented by LUMCON (Louisiana Universities Marine Consortium) monitoring over the past 30 yr (Turner et al. 2006, 2012). The internal turnover of nutrients and its potential linkage to Shelf hypoxia have been studied only rarely (Dagg et al. 2007). Nitrification plays a key role in aquatic systems, linking the most oxidized and most reduced species of the N cycle. The process involves a two-step conversion of ammonium to nitrate (via nitrite), carried out by phylogenetically distinct groups of organisms, namely ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitriteoxidizing bacteria (NOB). Nitrification is, thus, a key step in the regeneration of nitrate, which supports phytoplankton growth in the sunlit ocean, as well as N loss through denitrification under functionally anoxic conditions. The first step of nitrification (ammonia oxidation to nitrite) is mediated by both AOB and AOA (Francis et al. 2005; Mosier and Francis 2008). Under low oxygen conditions, marine ammonia oxidation appears to be conducted primarily by AOA in the phylum Thaumarchaeota (Lam et al. 2009; Beman et al. 2012; Stewart et al. 2012). The second step of nitrification (nitrite oxidation to nitrate) is carried out by NOB, notably the marine genera Nitrospina and Nitrospira. These clades are adapted for microaerophilic metabolism (L€ ucker et al. 2010, 2013), remaining efficient at nitrite consumption even at the nanomolar oxygen concentrations typical of OMZ boundaries (F€ ussel et al. 2012; Beman et al. 2013). In many aquatic environments, including sediments (Meyer et al. 2005), wastewater treatment systems (Schramm et al. 1999), and most marine waters (Ward 2008), the two steps of nitrification are tightly coupled such that nitrite does not accumulate to any appreciable extent. However, nitrite accumulation is observed consistently in some environments. The most extensive accumulation of nitrite occurs in the functionally anoxic core of oceanic oxygen minimum zones (OMZs) where oxygen concentrations fall below 0.1 lmol kg and dissimilatory nitrate reduction generates 1–10 lmol L nitrite (Thamdrup et al. 2012; De Brabandere et al. 2014), while nitrite oxidation is limited by oxygen availability (Cline and Richards 1972). Nitrite accumulation may also occur in environments where oxygen is not limiting, presumably due to a decoupling of ammonia and nitrite oxidation. Periodic accumulations of nitrite associated with Thaumarchaeota ammonia-oxidizer blooms have been observed across diverse coastal ecosystems (Pitcher et al. 2011; Hollibaugh et al. 2014). In estuaries, a nitrite maximum can be found at intermediate salinities, where high rates of ammonia oxidation may generate>10 lmol L nitrite while growth and activity of NOB are apparently limited by changing salinity combined with short hydraulic retention times (Billen 1975; McCarthy et al. 1984). Likewise, uncoupling of ammonia and nitrite oxidation represents one potential explanation for the formation of the “primary nitrite maximum” of 0.1–0.5 lmol L observed at the base of the photic zone in some oceanic settings (Brandhorst 1958; Lomas and Lipschultz 2006; Beman et al. 2013; Santoro et al. 2013). However, the potential drivers of uncoupling are best understood for artificial systems, for example in biological wastewater treatment where inhibition of nitrite oxidation is achieved in part through temperature (> 258C) limitation of NOB, or by a combination of high, inhibitory ammonia concentrations and low oxygen (< 10 lmol kg) (Schmidt et al. 2003; Van Hulle et al. 2010). In natural systems, an effect of temperature has not been observed (Ward 2008), ammonia typically does not reach inhibitory levels (Van Hulle et al. 2010), and other potential controls on nitrification steps (e.g., photoinhibition) (Olson 1981) remain poorly constrained (Bouskill et al. 2011). To date only a single profile of nitrification rates has been described for the hypoxic GoM, with observed rates up to 3.5 lmol L d (Carini et al. 2010). In that study, ammonia and nitrite oxidation rates were not determined independently, so the extent of coupling could not be resolved. Hence, the spatial distribution and drivers of this environmentally important two-step process, notably in relationship to the strong physiochemical gradients observed in the hypoxic GoM, remain poorly characterized. Only a handful of studies have characterized the microbial taxonomic composition of northern GoM waters (King et al. 2013; Tolar et al. 2013). King et al. (2013) quantified bacterioplankton 16S rRNA gene diversity within both the Mississippi River water outflow plume and onand off-shelf sites in the northern GoM. This work concluded that prior to the development of hypoxia, community composition varied substantially with depth but diversity indices showed no considerable variation over biological and Bristow et al. Nitrite accumulation in hypoxic zones