Lydia H. Zeglin MBL Microbial Diversity Final Project Report 29 July 2008 How Does Salinity Affect Aerobic Ammonia Oxidizer Abundance and Diversity?

Lydia H. Zeglin MBL Microbial Diversity Final Project Report 29 July 2008 How does salinity affect aerobic ammonia oxidizer abundance and diversity? Introduction Microorganisms are capable of making a living in diverse ways. For instance, chemolithoautotrophic microbes utilize inorganic electron donors and acceptors to supply cellular energy. Perhaps the most environmentally ubiquitous chemolithoautotrophic metabolic pathway is ammonia oxidation to nitrite, coupled with nitrite oxidation to nitrate, together + - - commonly referred to as nitrification (NH4 ⇒ NO2 ⇒ NO3 ). Ammonia oxidation to nitrite and nitrite oxidation to nitrate are separate steps performed by separate groups of organisms. + = + Ammonia oxidation (NH4 + 1.5 O2 ⇒ NO2 + H2O + 2H ) has been studied as an aerobic bacterial-mediated pathway for many years. Two monophyletic bacterial groups were thought to dominate this pathway: Nitrosomonas spp. (Betaproteobacteria: Nitrosomonadales: Nitrosomonadaceae) and Nitrosococcus spp. (Gammaproteobacteria: Chromatiales: Chromatiaceae). Recent insights have complicated this framework, as archaeal (Crenarchaeota, e.g. “Nitrosopumilis maritimus”, (Konneke et al., 2005)) microorganisms may perform a significant proportion of aerobic nitrification. A number of recent studies show a high abundance, diversity and activity of ammonia-oxidizing Crenarchaea in soils, marine waters and sediments (e.g. Francis et al. 2005, Leininger et al. 2006). There may also be a differential distribution of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) between habitats. Studies investigating AOA and AOB distribution along estuarine salinity gradients indicate that different functional assemblages are associated with different salinity levels and overall nitrification rates (Bernhard et al. 2007), with a stronger influence of salinity on AOB distribution than AOA (Sahan & Muyzer 2008, Santoro et al. 2008). While AOB display specialized metabolisms, Crenarchaea generally have more flexible metabolic capabilities, which may explain the higher sensitivity of AOB to changing salinity and the lower overall diversity of AOB in estuarine sediments. For my independent project, I attempted to measure the abundance and diversity of AOA and AOB along a natural gradient of salinity and in varying salinity lab enrichments from these environmental samples for ammonia oxidizing bacteria and archaea. Based upon the studies cited above, I expected AOA abundance and diversity to vary less with salinity, and that AOB would shift in dominance from β- to γ-Proteobacteria as salinity increased. Methods To evaluate these hypotheses, I first identified a site with a strong salinity gradient also corresponding with a shift from a freshwater to marine water source. Salt Pond is a shallow, stratified coastal pond with a freshwater cap and saltwater hypolimnion maintained by subsurface seawater intrusion. Water samples were collected from eight locations: seven through the Salt Pond water column plus one low salinity sample from the pond outflow. Concurrent with water sampling, lake water physiochemical parameters (including salinity and dissolved oxygen concentration) were recorded using a CTD meter. Also, inorganic nitrogen concentrations (ammonium, nitrite and nitrate) were measured in the lab using spectrophotometric methods and an AutoAnalyzer (J. Saenz). Environmental and lab enrichment AOA and AOB abundance and diversity were evaluated using three approaches. I enumerated cell abundances using mono-labelled and CARD-FISH oligonucleotide probes for the phylogenetic groups of interest, using protocols from the lab manual and published material (Table 1). 10-15 mL of water per sample were 1 Lydia H. Zeglin MBL Microbial Diversity Final Project Report 29 July 2008 filtered to provide a cell density appropriate for counting on the epifluorescent microscope. I attempted to amplify and construct clone libraries of the archaeal 16S rRNA gene and β- Proteobacterial, γ-Proteobacterial and Crenarchaeotal ammonia monoxygenase functional genes using protocols from the lab manual and published material (Table 2). Finally, the V6 regions of all 16S rRNA gene amplicons from the environmental samples were pyrosequenced using 454 technology (Josephine Paul Bay Center, MBL) to provide an exhaustive survey of bacterial diversity across the gradient (Sogin et al., 2006). Enrichments for AOA and AOB at three different salinity levels (25%, 50% and 75% salt water base) were assembled using a basic ammonia oxider selective medium (Watson, 1965; Koops and Moller, 1981) with several modifications. I added an antibiotic mixture to a final concentration of 50 ug/mL to one replicate of enrichments to encourage dominance of archaeal growth. I also made a batch of 75% Saltwater Base enrichments anaerobically with sodium sulfide (to maintain reducing conditions) and sodium nitrate as a potential electron acceptor for ammonia oxidation. All recipes are listed in Table 3. All batch tubes were burned at 300°C for 2 h in a muffle furnace before inoculation to remove all organic material. The inoculation volume was 1 mL Salt Pond water into 10 mL liquid medium. Oxic enrichments were incubated on a shaker table at 30°C. I assessed cell growth in enrichments after one and two weeks using the light microscope. After two weeks, I selected the ten enrichments with the most dense cell growth to filter for CARD-FISH scans for Nitrosomonas spp., Nitrosococcus spp. and Crenarchaeota (a EUB I-III and NON probe were hybridized also as positive and negative controls). Results Lake water column conditions at the time of sampling are shown in Tables 1 and 2. Salinity ranged from 15 ppt in the upper water column to 25 ppt in the lower water column, and the transition between the two salinity conditions was sharp and closely corresponded with the oxycline. Salinity in the outflow water sample was approximately 5 ppt. Inorganic nitrogen species distribution also varied along the same spatial scale, with both oxidized and reduced nitrogen concentrations relatively high in the epilimnion, low through the chemocline, and ammonium concentrations increasing again to a high of 100 µM in the anoxic hypolimnion. Archaeal 16S rRNA and bacterial and archaeal ammonia monoxygenase genes could only be amplified from a subset of Salt Pond water column samples (Table 4). Cloning of the three strong amplifications of archaeal 16S rRNA was unsuccessful. Amplification of γ- Proteobacterial amoA was unsuccessful. Though a ~450 bp PCR product (the correct length) could be easily amplified from four of the water samples, cloning and sequencing of these products revealed the product to resemble a glycosyl transferase family protein from the Prosthecochloris vibrioformis DSM 265 complete genome (BLASTx). This organism is a member of the Chlorobi phylum, a taxonomic group that proved to be dominant in the 454 bacterial survey of lower water column samples. Though the archaeal amoA gene amplified in two of the lower water column samples, insufficient time was available to optimize the PCR reaction and attempt clone library construction for that gene. A number of enrichments for ammonia oxidizing bacteria and archaea at differing salinity levels displayed significant cell growth over the two week project period (Table 5, Figure 3). CARD-FISH screens for Nitrosomonas spp., Nitrosococcus spp., and Crenarchaeota from filters of these enrichments, however, revealed that only one enrichment was dominated by archaea (oxic high salinity inoculum in 75% SWB medium with antibiotics). The other enrichments 2 Lydia H. Zeglin MBL Microbial Diversity Final Project Report 29 July 2008 were comprised of bacterial cells that did not hybridize with oligonucleotide probes for the ammonia-oxidizing phyla. Overall cell counts through the Salt Pond water column ranged from 4.5 x 106 in surface waters to a high 1.97 x 107 at depth (Figure 4). CARD-FISH counts of Bacteria ranged from 88 – 100% of total cells; of Archaea from 1.1 – 9.3% of total cells; of β-Proteobacteria from 3.4 – 53% of total cells; and of γ-Proteobacteria from 1.6 – 11% of total cells (Figure 5). Archaea and β-Proteobacteria were most abundant in lower depths (> 3 m), with β-Proteobacteria comprising a large (53% of total cells) population within the 3.4 m sample. Cells from the Nitrosomonas and Nitrosococcus genera were undetectable. Time constraints prevented the full count of Crenarchaeota. Surveys of the bacterial 16S rRNA V6 region showed a shift in dominant Phyla from upper to lower depths in the water column (Figure 5). At shallow depths Proteobacteria, Actinobacteria and Cyanobacteria were dominant while at deeper depths, Chlorobi were dominant and Proteobacteria were also common. The distribution of V6 sequnces of Proteobacterial subphyla showed a decrease in γ-Proteobacteria with depth, with a similar magnitude of relative abundance as was tabulated from CARD-FISH counts (1.7 – 8.7% of total sequences). β-Proteobacteria were an extremely minor portion of the V6 survey, however, comprising <0.1% of total sequences and showing no depth distribution patterns. Seven sequences from the Nitrosomonas genus were detected in the entire 454 library (> 20,000 sequences): two at 1.8 m depth, four at 2.2 m depth and one at 3.1 m depth. Discussion Environmental conditions were appropriate to support the activity of ammonia oxidizing microorganisms in the oxygenated, ammonium rich eplilmnion. The lower concentrations of ammonium in deeper depths may limit the activity of these

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