Contribution of ammonia-oxidizing bacteria and archaea to nitrification in estuarine sediments along a eutrophic gradient Emily Stone Clark University Advisors: Julie Huber and Anne Giblin Abstract Eutrophication, the enrichment of ammonium and phosphate, is a major concern for coastal aquatic systems such as estuaries. Eutrophication may hinder the coupling of nitrification-denitrification in sediment, which is responsible for removing large amounts of nitrogen in aquatic ecosystems. The first and rate-limiting step in nitrification is catalyzed by the enzyme ammonia monooxygenase (amo) and is performed by two distinct groups of microorganisms: ammonia-oxidizing bacteria and ammonia-oxidizing archaea. Presence of ammonia-oxidizing bacteria and archaea was determined by amplifying the amoA gene. Sediment slurries were used to measure potential nitrification rates from estuarine sediments taken along a eutrophic gradient. Results showed lower potential nitrification rates at more eutrophic sites and higher nitrification rates at less eutrophic sites. Similarly, the relative abundance of ammonia oxidizing archaea was lowest at the most eutrophic site and highest at the least eutrophic site. The bacterial inhibitor, 1-octyne, which has been shown to inhibit ammonia oxidizing bacterial activity in soils, did not inhibit these bacteria in sediment. Although ammonia-oxidizing bacteria were detected in sediments, the relative abundance was not determined. However, results suggest that ammonia-oxidizing archaea are more abundant in estuarine sediments than ammonia-oxidizing bacteria. Trends indicate that the relative abundance of ammonia-oxidizing archaea increases as eutrophication decreases but no trends were detected for ammonia-oxidizing bacteria. Key words: eutrophication, nitrification rates, ammonia oxidizing bacteria, ammonia oxidizing archaea Introduction Anthropogenic inputs on ecosystems influence the nitrogen cycle and have resulted in many environmental problems including loss of biodiversity, global water acidification, increased levels of the potent greenhouse gas, nitrous oxide, and eutrophication (Gruber and Galloway 2008). Coastal regions receive much of the nitrogen-rich runoff from municipal and industrial wastewaters as well as urban and agricultural fertilizers. These regions are also susceptible to nutrient enrichment. Nutrient enrichment is mainly in the form of ammonium and leads to eutrophication in the water. Eutrophication, in particular, is a serious concern for estuaries. More than half of the estuaries in the US experience some type of effect from it (Meyer-Reil and Köster 2000). However, nitrogen levels in estuaries can typically be reduced through tightly coupled microbial processes in the nitrogen cycle. Nitrification converts ammonium to nitrite and then nitrate, which then can be converted to dinitrogen gas by denitrification or anammox. These microbially-mediated processes thus determine recycling and removal of dissolved inorganic nitrogen from aquatic systems. Denitrification in marine sediments is dependent on the supply of nitrate and is driven almost exclusively from sediment nitrification (Jensen et al. 1993). Coupling of nitrification-denitrification removes up to 50% of external dissolved inorganic nitrogen from ammonium inputs to estuaries (Seitzinger et al. 2006). Nitrate production requires oxygen, thus nitrification is restricted to aerobic environments (Ward et al. 2011). Because both ammonia-oxidizing bacteria and archaea require oxygen in order to nitrify, these microorganisms may be affected by the environmental changes brought about from nutrient loading by eutrophication such as reduced oxygen concentration in sediments. Thus, eutrophication may limit aerobic regions for nitrification, and thereby, may decrease nitrate production and removal of inorganic nitrogen in aquatic environments. The first and rate-limiting step in nitrification is ammonia oxidation catalyzed by the ammonia monooxygenase (amo) gene (Beman et al. 2007). Nitrification was long thought to be solely carried out by ammonia-oxidizing bacteria in soils and sediments (Bernhard et al. 2010). However, recent studies have shown that archaea from the phylum Thaumarchaeota can also oxidize ammonium and are present in various soils and sediments (Taylor, et al. 2013). Molecular studies have identified genes putatively encoding archaeal ammonia monooxygenase subunits (amoA, amoB, and amoC). The amoA subunit is homologous to the amoA gene found in ammonia-oxidizing bacteria (Mosier and Francis 2008). Thaumarchaeota carrying the archaeal amoA gene appear to be ubiquitous and have been detected in coastal and marine waters, cold sediments, subsurface of radioactive thermosprings, microbial mats, coral reefs, as well as, terrestrial forest soils and grasslands (Erguder, Boon et al. 2009). Several studies have found that ammonia-oxidizing archaeal amoA gene copy numbers are more abundant than the bacterial amoA gene in soil, estuaries, and open waters (Mosier and Francis 2008). Beman et al. (2008) determined that ammonia-oxidizing archaea dominated ammonia-oxidizing bacteria by a factor of 37-217 in surface waters from the Gulf of California while Caffrey et al. (2007) found an abundance ratio of archaeal to bacterial amoA gene around 80 in estuarine sediments. Although the archaeal amoA gene has been found in higher abundance than the bacterial amoA gene, the relative contributions of ammonia-oxidizing bacteria and archaea to nitrification are still not fully understood. Recent studies suggest that selectively blocking ammonium oxidation by ammonia oxidizing bacteria may help estimate ammonia-oxidizing archaea nitrification rates. This, in return, may aid in determining the relative contribution of both ammonia-oxidizing bacteria and archaea to nitrification in soils and sediments. For example, Taylor et al. (2013) found that 1- octyne inhibited a range of ammonia-oxidizing bacterial activity in soils by inactivating the amo enzyme. It had no effect on ammonia-oxidizing archaeal activity in the soil. They also found that 1-octyne inhibited the bacterial activity in less than 48 hours. With its relative efficiency, this inhibitor may also help differentiate potential nitrification rates attributable to either ammonia- oxidizing bacteria or archaea in estuarine sediments. Using a combination of potential nitrification rate measurements and PCR-based methods, this paper investigates i) the effects of eutrophication in estuarine sediments around Falmouth, Massachusetts on nitrification rates and ii) the relative contribution of ammonia- oxidizing bacteria and archaea to nitrification in eutrophic sediments. Methods Sampling Sites Four inch diameter sediment cores were taken in duplicates from four estuaries located around Falmouth, Massachusetts in early November 2015. Sediment taken from Little Pond and Eel pond did not show a clear separation of aerobic and anaerobic regions. Sediment cores collected from these locations were near black and viscous indicating more anaerobic conditions and higher eutrophication. However, sediment collected from Quissett Harbor and Waquoit Bay was less viscous and had more distinction between the aerobic and anaerobic regions indicating less eutrophic sediments. Additionally, amphipod tubes were present in sediment taken from Waquoit Bay, which indicates well-oxygenated sediment. To determine level of eutrophication in sediment, subsamples of sediment from cores from each of the four sites were burned at 500ºC in a muffle furnace (Thermo Scientific™ Thermolyne™ Industrial Benchtop Muffle Furnaces). Percent organic matter within sediments was calculated by subtracting original weight of sediment from post-burned sediment. The four sites were then organized along a eutrophic gradient based on percent organic matter in sediment subsamples with higher percent organic matter reflected more eutrophic sediment and lower percent organic matter reflected less eutrophic sediment. Potential Nitrification Rates Potential rates of nitrification in sediments were measured using a shaken sediment-slurry method with added nutrient solution (Bernhard et al. 2007). Two treatments were analyzed to look at total nitrification and archaeal nitrification: (i) environmental control and (ii) with addition of the bacterial inhibitor, 1-octyne (10 uM). Octyne was used as in inhibitor to block ammonia-oxidizing bacteria activity while allowing ammonia-oxidizing archaea activity to continue (Taylor et al. 2013). Cores were sectioned off to isolate the aerobic region of the sediment by removing the top 2 centimeters of sediment. Approximately one gram of wet weight of sediment from each site was added to 50 mL falcon tubes for destructive sampling at time points 12 hours, 24 hours, 48 hours, and 72 hours, and several blanks in duplicates. Archaeal nitrification from Little Pond and Waquoit Bay had additional time points at 120 hours and 168 hours for a total 7 day incubation period to determine if the archaeal population would rapidly increase without bacterial ammonia oxidizers. Subsamples from cores of each site were weighed, dried at 60ºC for two days and re-weighed to obtain wet/dry conversion factors. Seawater was collected from each site and filtered through GF/F filters and re-filtered again with 0.20 nanometer pore sized membrane filters to remove microorganisms. A nutrient solution (300 µM 3 NH4, 60 µM PO4 ) was created with filtered seawater from each location and 30 mL was added to each tube to create a