Measurements of nitrogen fixation in the oligotrophic North Pacific Subtropical Gyre using a free-drifting submersible incubation device

Journal of Plankton Research, Jul 2015

One challenge in field-based marine microbial ecology is to achieve sufficient spatial resolution to obtain representative information about microbial distributions and biogeochemical processes. The challenges are exacerbated when conducting rate measurements of biological processes due to potential perturbations during sampling and incubation. Here we present the first application of a robotic microlaboratory, the 4 L-submersible incubation device (SID), for conducting in situ measurements of the rates of biological nitrogen (N2) fixation (BNF). The free-drifting autonomous instrument obtains samples from the water column that are incubated in situ after the addition of 15N2 tracer. After each of up to four consecutive incubation experiments, the 4-L sample is filtered and chemically preserved. Measured BNF rates from two deployments of the SID in the oligotrophic North Pacific ranged from 0.8 to 2.8 nmol N L−1 day−1, values comparable with simultaneous rate measurements obtained using traditional conductivity–temperature–depth (CTD)–rosette sampling followed by on-deck or in situ incubation. Future deployments of the SID will help to better resolve spatial variability of oceanic BNF, particularly in areas where recovery of seawater samples by CTD compromises their integrity, e.g. anoxic habitats.

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Measurements of nitrogen fixation in the oligotrophic North Pacific Subtropical Gyre using a free-drifting submersible incubation device

J. Plankton Res. ( Measurements of nitrogen fixation in the oligotrophic North Pacific Subtropical Gyre using a free-drifting submersible incubation device DENIZ BOMBAR 0 1 CRAIG D. TAYLOR 0 SAMUEL T. WILSON 0 3 JULIE C. ROBIDART 0 1 2 ARIEL RABINES KENDRA A. TURK-KUBO 0 1 JOHN N. KEMP DAVID M. KARL 0 3 JONATHAN P. ZEHR 0 1 0 CENTER FOR MICROBIAL OCEANOGRAPHY 1 OCEAN SCIENCES DEPARTMENT 2 NATIONAL OCEANOGRAPHY CENTRE 3 DEPARTMENT OF OCEANOGRAPHY One challenge in field-based marine microbial ecology is to achieve sufficient spatial resolution to obtain representative information about microbial distributions and biogeochemical processes. The challenges are exacerbated when conducting rate measurements of biological processes due to potential perturbations during sampling and incubation. Here we present the first application of a robotic microlaboratory, the 4 L-submersible incubation device (SID), for conducting in situ measurements of the rates of biological nitrogen (N2) fixation (BNF). The free-drifting autonomous instrument obtains samples from the water column that are incubated in situ after the addition of 15N2 tracer. After each of up to four consecutive incubation experiments, the 4-L sample is filtered and chemically preserved. Measured BNF rates from two deployments of the SID in the oligotrophic North Pacific ranged from 0.8 to 2.8 nmol N L21 day21, values comparable with simultaneous rate measurements obtained using traditional conductivity - temperature - depth (CTD) - rosette sampling followed by on-deck or in situ incubation. Future deployments of the SID will help to better resolve spatial variability of oceanic BNF, particularly in areas where recovery of seawater samples by CTD compromises their integrity, e.g. anoxic habitats. nitrogen fixation; in situ device; Pacific Ocean; diazotroph I N T RO D U C T I O N Biological nitrogen (N2) fixation (BNF), the conversion of N2 gas to ammonia (NH3), is performed by a select group of microorganisms, termed diazotrophs. BNF is a key component of the oceanic nitrogen cycle, with estimates of up to 200 Tg nitrogen (N) being fixed per year on a global scale (Karl et al., 2002; Capone et al., 2005; Gruber and Galloway, 2008). However, such estimates have large uncertainties, partly due to an incomplete understanding of the full diversity and ecology of marine diazotrophs (Goebel et al., 2010; Moisander et al., 2010; Farnelid et al., 2011). In the ocean, the major groups of diazotrophs include: (i) the filamentous, non-heterocystous cyanobacterium Trichodesmium (Mague et al., 1977; Capone et al., 1997; Capone et al., 2005; Laroche and Breitbarth, 2005), (ii) unicellular, free-living cyanobacteria such as Crocosphaera watsonii (UCYN-B) (Hewson et al., 2009; Webb et al., 2009; Bench et al., 2011; Foster et al., 2013) and (iii) cyanobacteria that form symbioses with eukaryotic algae, e.g. the heterocystous genera Richelia and Calothrix that are associated with diatoms (Villareal, 1990; Janson et al., 1995; Foster and Zehr, 2006) and unicellular Candidatus Atelocyanobacterium thalassa (UCYN-A) associated with prymnesiophytes (Zehr et al., 2001; Moisander et al., 2010; Thompson et al., 2012; Bombar et al., 2014). Although the major abiotic, nutrient and internal controls of BNF activity and its distribution appear to have been identified (Sohm et al., 2011; Voss et al., 2013), the relative strengths with which these different factors govern BNF under different environmental settings remain elusive. These variable controls are likely responsible for the unexplained large spatiotemporal variability in the abundances of diazotrophs in the surface ocean, recently highlighted by high-resolution sampling using a drifting robotic gene sensor (Robidart et al., 2014). It is currently unknown how these abundance fluctuations affect the variability in BNF. Thus, conducting corresponding rate measurements at similarly high spatiotemporal resolution is critical for better understanding the role of diazotrophs in oceanic N cycling. BNF field measurements are typically conducted using versions of the 15N2 tracer gas technique (Montoya et al., 1996). This method requires the collection of seawater using a conductivity – temperature – depth (CTD) – Niskin rosette sampling system, and subsequent on-deck or in situ incubations of tracer-amended seawater, lasting anywhere from a few hours to a few days. Seawater samples in traditional BNF studies experience changing pressures, light levels and temperatures upon recovery and seawater transfer, while characterization from cultured representatives and sorted cells suggest that many of these processes affect diazotroph populations (e.g. Thompson et al., 2012). Further, methodological improvements on the 15N2 tracer technique have demonstrated that BNF rates have been underestimated in most studies, with the addition of 15N2 tracer in a dissolved form more representative of the actual rates (Grosskopf et al., 2012). Automated sampling devices capable of conducting sampling and incubation in situ are a promising approach for resolving variability associated with biogeochemical cycling in the marine environment. Such devices have been developed and successfully employed to increase the spatial and temporal resolution of planktonic primary production rates (Dandonneau and Bouteiller, 1992; Taylor and Howes, 1994), as well as for comparing manipulations performed in situ and under “simulated” in situ conditions (Gundersen, 1973; Lohrenz et al., 1992). The submersible incubation device (SID) was originally designed for primary productivity measurements (Taylor and Doherty, 1990) and has since been adapted for a range of oceanographic measurements (Lohrenz et al., 1992; Taylor and Howes, 1994; Albert et al., 1995; Edgcomb et al., 2011, 2014; Pachiadaki et al., 2014). Integrating the BNF method protocol with a large capacity submersible in situ device (4L-submersible incubation device, 4L-SID) became more feasible with recent developments of the 15N2 assimilation technique, which requires dissolving the 15N2 gaseous tracer in sterile seawater prior to its addition to the samples (Mohr et al., 2010; Grosskopf et al., 2012). In the present study, a modified version of the 4L-SID was deployed, which is capable of autonomously executing the entire sampling, tracer amendment ( predissolved 15N2, 13C-bicarbonate), incubation and filtration processes associated with BNF and primary production measurements in situ. By conducting the entire sampling and incubation procedure directly in the water column, delays in the onset of the incubations and perturbations of the microbial community assemblages during sampling are minimized. Further, such devices have the potential to help overcome the major hurdle of achieving higher sampling resolution, which could reveal currently unknown heterogeneity in BNF rates and the key environmental factors that control them. M E T H O D Cruise overview The BioLINCS cruise (Biosensing Lagrangian Instrumentation and Nitrogen Cycling Systems) was conducted in the North Pacific Subtropical Gyre (NPSG) (24.39 –25.138N, 158.20 –157.298W) in September 2011, aboard the R/V Kilo Moana (Fig. 1). The overall goal of Fig. 1. Sampling stations north of Station ALOHA visited during the BioLINCS cruise (gray dots), locations of the SID on the mornings of all incubation starts (black crosses) and stations at which the in situ array was deployed (black circles). The gray contour lines (mesoscale altimetry) indicate the presence of two anticyclonic eddies that influenced the drift paths of the SID (described in the text). Color-coded near-surface chlorophyll concentrations are averages of satellite data from Archiving, Validation and Interpretation of Satellite Oceanographic data (AVISO) and Moderate Resolution Imaging Spectroradiometer (MODIS) Aqua, for 6–20 September 2011. the scientific cruise was to examine microbial biogeochemical cycling associated with the nitrogen cycle and was an ideal context for implementing the 4L-SID test. To characterize the hydrographic and biogeochemical conditions of the upper water column, vertical profiles were conducted daily using a CTD system coupled to a rosette consisting of 24 12 L Niskin bottles. Oxygen (O2) and fluorescence sensors were calibrated against discrete measurements of dissolved O2 (Carritt and Carpenter, 1966) and chlorophyll extracted and analyzed by fluorometry (Strickland and Parsons, 1972). Seawater for determination of nutrient concentrations was sampled and analyzed as documented in the online manual for “HOT Laboratory Protocols” ( .html). Regional ocean color and sea-level anomaly for the NPSG were analyzed using satellite-derived images from the Moderate Resolution Imaging Spectroradiometer (MODIS). Operation of the submersible incubation device Since the original description of the SID in the 1990’s (Taylor and Doherty, 1990), there have been several subsequent versions of the SID concept which have adapted the instrumentation (Lohrenz et al., 1992; Taylor and Howes, 1994; Albert et al., 1995; Edgcomb et al., 2014; Pachiadaki et al., 2014). This study is the first time that the 4L-SID has been used for conducting 15N2 rate measurements and therefore the entire instrument configuration relevant to quantifying N2 fixation is outlined here. The SID, as configured for this study, consists of a hydraulically driven, syringe-like 4-L incubation chamber, an 18-port fluidic distribution valve (FDV) for directing fluid flows, an array of 8 “version 1” Fixation Filter units (FF1s, Taylor et al., 2013) for collection and preservation of incubated particulate samples and a controlling electronics/battery pack (Fig. 2A). The incubation chamber consists of a precision bore borosilicate glass chamber [interior silane treated with SurfaSilTM siliconizing fluid (Thermo Scientific) for biological inertness]. Each end of the incubation chamber is capped with silicone O-ring-sealed polycarbonate end caps and it contains a silicone O-ring-sealed polycarbonate floating piston. The rotor/stator components of the FDV in contact with sample are made of PVC and Teflonw and interfacing tubing between the incubation chamber, FDV and FF1s are made of Teflonw. All interiors were acid-washed and rinsed with deionized water prior to deployment. Communication with the instrument prior to and after each deployment for programming and data retrieval was via a serial RS-232 link with a laptop PC. The 4L-SID was mounted to a free-drifting spar float system (Fig. 2B) for deployments at a fixed depth of 25 m. During the instrument operation in situ, the location of the spar float system was constantly monitored via two iridium GPS transponders. The FF1s are unique in-line filter units that each contain an appropriate chemical preservative that is delivered (with no moving parts) through the filter by density-driven laminar convection after completion of filtration (Supplementary data, Fig. S1). The SID was configured for deployment with the hydraulically driven floating piston flushed against the check valve-containing end cap (Fig. 3A). The space behind the floating piston was filled with deionized water. After deployments in the afternoon, the incubation sequence was programmed to automatically commence the next morning at 0530. To condition the interior of the incubation chamber with environmental sample, 500 mL of seawater from the depth of deployment was drawn into the chamber via the inlet check valve (ICV, red inset, Fig. 3B) and expelled back into the environment via the FDV “Waste” outlet (Fig. 3A). A total of two flushes were executed. The flushing operation was immediately followed by complete filling of the incubation chamber with sample via the ICV, advancement of the FDV rotor to the first FF1 filter unit and immediate filtration of the entire 4 L to obtain a natural abundance time zero (T0) particulate sample. The ICV has a large enough internal spacing that will not select against larger organisms (21 mm diameter annulus with a 1.63-mm spacing, through six 2.38-mm diameter holes, ultimately into the chamber via a 4.76-mm diameter orifice; see Fig. 3B inset). During filling, the ICV exerts low shear stress of 1.2 pascals (Pa); max. 1.7 Pa, at a flow rate of 200 mL/min (Taylor et al., 2015). The FDV advanced to the next valve port connected to the first bag of tracer and the chamber then re-filled as described above. The slight negative pressure that developed within the chamber during filling also quantitatively draws the entire 15N2 contents from the flexible tracer bag, which also quantitatively sweeps the 13C-bicarbonate contained within the in-line injector coil into the chamber as shown in Fig. 3B (tracer details described below). The gentle turbulence generated from the main bulk of the sample entering the chamber via the ICV completely mixes the tracer with the sample as it enters the chamber (confirmed by dye studies). The 4-L sample was then incubated for a preprogrammed 23.5 h, followed by direction of sample to the next FF1 to obtain the Tincub sample (Fig. 3C). Upon completion of the incubation the chamber was flushed 4 as described above to remove tracer. The taper of floating piston and front end cap were machined to the same angle, minimizing the dead volume remaining when the piston meets the front endcap. Assuming an interior dead volume of 4 mL when the chamber is empty, the 4 flushing cycle dilutes the tracer contents by 5.6 orders of magnitude, which is well below background concentrations. A given incubation cycle consumes three ports of the FDV and two FF1s (Fig. 3A). The 4L-SID, as configured, was thus able to conduct four in situ incubations. All filtrations were collected onto 47-mm diameter precombusted glass fiber filters (GFF) and chemically preserved in a pH 2 acid buffer inside the FF1s (Taylor and Doherty, 1990; Taylor and Howes, 1994; Supplementary data, Fig. S1), which terminates biological activity and preserves the sample for at least 1.5 month in warm water (confirmed by a Bermuda Test Bed mooring SID deployment where data agreed well with Bermuda Atlantic Time-Series measurements made at the same depth [C. D. Taylor, unpublished data]). Once the SID was recovered aboard ship, the filters were immediately recovered and dried for 48 h at 608C in a drying oven and then stored at room temperature until analyzed. In the laboratory, the filters were pelleted and sent for isotopic analysis at the University of California, Davis Stable Isotope Facility. BNF rate calculations followed the protocol of Montoya et al. (Montoya et al., 1996). To test for leakage of low molecular weight (LMW) metabolites into the acidic preservative, the remaining preservative contents of the FF1s were also recovered, evaporated onto GFF filters (soaking the GFFs with the preservative and putting them in a drying oven), and these filters were treated as described above for the particulate filters. We found only very low or even undetectable amounts of carbon and nitrogen on these filters, and more importantly, the d13C and d15N values were equal or even lower than those of the respective non-tracered T0 samples. Thus, the SID-derived rates were not underestimated due to loss of tracer to the LMW fraction. The two tracers added to the SID incubations were 15N2 gas to obtain estimates of BNF and 13C-bicarbonate for measurements of primary productivity. 15N2 gas (98 atom%; Sigma-Aldrich) was added to seawater samples as “15N2-enriched seawater” which was prepared on land prior to the cruise using sterile-filtered surface seawater from Station ALOHA (10 mL 15N2 per liter of seawater; Wilson et al., 2012). The 15N2 gas used in this study was from a batch manufactured from 2008 to 2009 by Sigma-Aldrich, and we identified it as not causing severe contamination with other bioavailable inorganic N species (Dabundo et al., 2014). After enrichment, the tracer water was stored in 200-mL gas-tight tri-layer aluminized polyethylene bags ( The bags were individually connected to the 18-port FDV via Luer locks and 1.6-mm I.D. Teflon tubing and coiled in-line tracer loops made of Teflonw tubing, as illustrated in Fig. 3A. A complete 200-mL bag was added to each 4-L incubation, providing a final atom enrichment of 5%. For the 13C additions, 400 mL of a 0.1-M solution of H13CO32 were stored within a coiled section of the Teflon tubing (see inset in Fig. 3A). To facilitate loading of the 13C-tracer into the coil using a syringe, small bubbles (volume 50 mL) were introduced at the beginning and end of the injection. The leading bubble isolates the tracer from the water contained within the tubing leading to the FDV, allowing it to be introduced as a “plug flow” instead of the spreading of tracer by the parabolic laminar flows that would otherwise occur. The trailing bubble provides isolation from the 15N2-enriched seawater in the bag. The surface tension of the small bubbles quite effectively confines the tracer within the loop and resists modest vibration. Complementary 15N2 measurements conducted during the cruise Measurements of BNF were also conducted during the cruise by sampling the water column using the CTD–rosette Fig. 3. Diagrammatic illustration of 4L-SID functions. (A) Deployed configuration. FDV: 18 port fluidic distribution valve. The inset shows an enlarged view of the 13C-bicarbonate stored in the tracer coil. (B) Procurement of sample and introduction of tracer. The inset illustrates the water flow through the inlet check valve (ICV). The poppet is normally closed by a light duty, Teflonw coated spring, except for when sample is drawn into the chamber. When the piston reaches the full extent of its travel in either direction, it is “lugged down” and the reduction in pump rounds per minute sensed by the electronics turns off the pump. (C) Delivery of incubated sample through an FF1 filter holder. The inset illustrates the seating of the Poppet O-ring against an annular “Knife Edge” (KE) to prevent loss of sample through the check valve during emptying of the chamber. and incubating the seawater samples either using an in situ array or on-deck incubators which simulated in situ conditions. The in situ incubations were used to obtain vertical profiles of BNF. Seawater was collected from depths of 5, 25, 45, 75, 100 and 125 m into replicate 4.3- L polycarbonate bottles, amended with 15N2-enriched water and attached to a free-floating in situ array at the appropriate depth for a 24-h period (Church et al., 2009). The on-deck incubations were performed for 24 h using blue shaded incubators cooled with running surface seawater and additional neutral mesh shading to mimic the corresponding light irradiances for each depth. Both sets of BNF measurements were also amended with 400 mL of a 0.1-M solution of H13CO32 injected through the septum cap with a syringe. Upon termination of the incubations, the seawater samples were gently filtered through precombusted Whatman GFF filters (0.7-mm nominal pore size) and processed as described for the SID filters. Quantitative PCR Different diazotrophs present in the water column were quantified using quantitative PCR (qPCR) enumeration of specific nifH gene copies. Water column samples were collected from between 5 and 175 m depth. Once the CTD was recovered, the seawater was immediately drained from the Niskin bottles into acid-washed 4-L polycarbonate bottles. Using peristaltic pumps, 2 L from each depth was filtered in-line through 10 mm (Polyester, Sterlitech, Kent, WA, USA) and 0.2 mm (Supor; Pall Life Sciences, Ann Arbor, MI, USA) pore-size filters, held in 25-mm-diameter Swinnex filter holders (Millipore, Billerca, MA, USA). The filters were placed into sterile 1.5-mL cryotubes containing 0.1 g autoclaved glass beads, frozen in liquid nitrogen and stored at 2808C until processing in the laboratory. DNA extractions were carried out as described previously (Bombar et al., 2013). We used previously designed Taq-Manw primer-probe sets, including cyanobacterial phylotypes UCYN-A and UCYN-B (Moisander et al., 2010), Trichodesmium (Church et al., 2005) and two Diatom–Diazotroph Associations (DDAs) (Foster et al., 2007) termed het-1 (Rhizosolenia-Richelia) and het-2 (Hemiaulus-Richelia). Additionally, we quantified presumed heterotroph diazotroph phylotypes HM210397 (g-Proteobacteria) and KC013231 (Cluster 3), described in Bombar et al. (Bombar et al., 2013). qPCR optimizations and quantifications have been described in detail in Moisander et al. (Moisander et al., 2010), Halm et al. (Halm et al., 2012) (specifications for phylotype HM210397) and Bombar et al. (Bombar et al. 2013) (specifications for phylotype KC013231). R E S U LT S A N D D I S C U S S I O N The application of in situ devices for observing physical, chemical and biological parameters in the ocean is an important approach for better understanding the complex relationships between the physical and chemical environment and microbial distributions and activities (Taylor and Howes, 1994; Johnson et al., 2010; Ottesen et al., 2011; Robidart et al., 2014). This study reports the first successful deployment of an autonomous device capable of conducting sampling, incubation and filtration processes for BNF measurements in situ. We evaluate the operation of the SID as an in situ instrument for BNF measurement, compare the SID-derived BNF rates with commonly applied sampling and incubation methods and discuss the SID rates in the context of physical, chemical and microbial data obtained during the BioLINCS cruise. SID operation and measurements of BNF rates The SID was deployed twice during the 2011 BioLINCS cruise (Fig. 1). During the first deployment (4L-SID Deployment 1) from 9 to 14 September 2011, it followed a 47-km drift path in a north-easterly direction, and during the second deployment (4L-SID Deployment 2) from 16 to 20 September 2011, it drifted 28 km westwards (Fig. 1). During both deployments, the 4L-SID performed four autonomous tracer incubations over a 4-day period at a depth of 25 m in the water column. With respect to the proximity of each incubation event, the 4L-SID sampled approximately every 16 km during Deployment 1 and every 10 km during Deployment 2. The 4L-SID-derived BNF measurements ranged from 0.8 to 1.9 nmol N L21 day21 (average 1.4 + 0.5 nmol N L21 day21) during Deployment 1 and from 1.4 to 2.8 nmol N L21 day21 (average 2.0 + 0.6 nmol N L21 day21) during Deployment 2 (Table I). Thus, during each 4-day deployment, a 2-fold variation of BNF was recorded, and BNF rates were overall higher during Deployment 2. The simultaneous rate measurements of 13C primary production by the 4L-SID also revealed higher values for Deployment 2 (344 + 40 nmol C L21 day21) compared with Deployment 1 (207 + 48 nmol C L21 day21). The higher rates of both 13C primary production and BNF suggest that the difference in BNF rates between the two deployments was not due to methodological errors that were only specific to the BNF measurements (Table I, Fig. 4). Compared with 4L-SID-derived rates of 15N2 assimilation, BNF rates obtained from incubations in the on-deck incubator (with water from 25 m) were higher during Deployment 1 (3.3 + 0.2 nmol N L21 day21) and more variable (ranging from 1.4 + 0.1 to 6.6 + 1.9 nmol N Date (month/ day) in 2011 September 10–11 September 11–12 September 12–13 September 13–14 Primary N2 fixation production (nmol N L21 (nmol C L21 day21) day21) On-deck incubations September 10–11 September 16–17 September 18–19 September 8–9 September 10–11 Parallel to the first incubation of each deployment, a comparison incubation in the shipboard incubator was carried out, using water sampled from 25 m at stations close to the SID location. These comparisons, even though they are not perfect control measurements taken in closest proximity to the SID, suggest that the SID-derived rates are of realistic magnitude. Fig. 4. (A) BNF and (B) primary production rates obtained from both SID deployments in the context of vertical rate profiles obtained from in situ array deployments (n ¼ 2). Incubations at Station 11 are shown as well, but were done in the shipboard incubator due to the loss of the in situ array. No primary production was measured on Station 11. L21 day21) during Deployment 2 (Table I). There are several factors which might contribute to the difference in 4L-SID and on-deck measurements of 15N2 assimilation. The first is the potential for sampling different populations of diazotrophs by the SID and the shipboard CTD – rosette. The distance between the ship and the SID when the comparative samples were taken was 5.4 and 3.9 km for 4L-SID Deployment 1 and Deployment 2, respectively (Fig. 1). Another potential source of variability is the method of incubation itself. The on-deck incubators are a best-effort to mimic temperature and light levels equivalent to a depth of 25 m in the water column, but have a few limitations. For example, nearsurface seawater intake is used for cooling which has a slightly higher temperature than 25 m water, and the existence of variability in light intensity due to shading among incubation bottles or from ship structures. Potential perturbations of the natural abiotic conditions during sampling and incubations are a wellknown problem (Feike et al., 2012), and highlight the necessity for using in situ devices especially when longer incubations are required. This theory seems to be supported by the BNF measurements obtained using an in situ array which performed incubations at 25 m, the results of which are better aligned with the 4L-SID Deployment 1 values (Table I, 0.9–2.9 nmol N L21 day21). Unfortunately, corresponding in situ array incubations to match Deployment 2 were not obtained due to the loss of the array at sea. In contrast, the available rates of 13C primary production (Table I) do not mirror the high similarity of BNF observed between the SID and the in situ array measurements, with higher rates obtained by the in situ array compared with the SID. However, overall both the 4L-SID-derived BNF and primary production rates compare favorably with values obtained using traditional methods during the same oceanographic expedition and during time-series measurements conducted at nearby Station ALOHA. BNF and 14C primary production (in situ array incubations) rate measurements are conducted on a nearly monthly basis at Station ALOHA, situated 100 km to the south of the BioLINCS expedition region [Hawaii Ocean Time-series (HOT) and other funding programs (Grabowski et al., 2008; Church et al., 2009)]. BNF rates at 25 m depth ranged from 1 to 5 nmol N L21 day21 during the summer months (July – September) between 2004 and 2007 (Church et al., 2009, 15N2 bubble addition protocol). During a more recent cruise in June 2014, BNF rates were measured daily for 7 days and produced values of 5.9 + 1.1 nmol N L21 day21 (range 3.7 – 7.2 nmol N L21 day21) (S. T. Wilson, unpublished data, 15N2 added as predissolved in sterilefiltered seawater). The average rate of 14C primary production for the month of September during the years 1989 – 2011 is 463 + 126 nmol C L21 day21, which is quite similar to values obtained using different incubation techniques during our cruise (Table I). We can only speculate on the reasons for the comparably high primary production from in situ array 2 (11 September 2011, 594 + 68 nmol C L21 day21) or the relatively low values from SID deployment 1 (207 + 48 nmol C L21 day21). Interestingly, CTD chlorophyll fluorescence measurements were relatively high at 25 m during sampling for in situ array 2 (0.20 + 0.00 mg L21) compared with any measurements at 25 m at four stations in proximity to 4L-SID samplings during deployment 1 (0.13 + 0.04 mg L21). Given the high spatiotemporal variability in prokaryotic distributions and abiotic conditions for our particular expedition (Fig. 5, also see Robidart et al., 2014), the observed range in primary production appears realistic. Without more specific comparison experiments, at this point it is difficult to claim that one or the other method delivers more trustworthy rate measurements. Most importantly, the overall similar range of BNF and primary production values obtained using the different methods supports the efficacy of the SID for in situ rate measurements. Hydrographic and biogeochemical background 4L-SID Deployment 1 was on the northern edge of an anticyclonic eddy (Fig. 1). During the second deployment, the SID drifted west between the primary eddy and a smaller adjoined anticyclonic eddy (Fig. 1). The drift paths for both 4L-SID deployments followed the clockwise circulation patterns of the two anticyclonic eddies revealed by sea surface altimetry (Fig. 1) and shipboard acoustic doppler current profiler measurements (Robidart et al., 2014). These mesoscale eddies introduced small-scale physical and chemical heterogeneity in the area, which clearly affected microbial distributions, especially of diazotroph cyanobacteria (Robidart et al., 2014). Several hydrographic and biogeochemical conditions may have influenced the 4L-SID measurements. Low average wind speeds of 4.8 m s21 for Deployment 1 and 6.9 m s21 for Deployment 2 contributed to a shallow mixed layer depth (MLD) during both deployments, but with an average of 17 m (range 10 – 27 m) during Deployment 1 and 30 m (range 14 – 57 m) for Deployment 2 (based on 0.03 density offset from 10 m criterion) (De Boyer Monte´gut et al., 2004). Accordingly, along the 25-m depth horizon seawater temperatures averaged 25.98C (range 25.7 – 26.18C) during Deployment 1 and 26.28C (range 26.1 – 26.28C) during Deployment 2 (Fig. 5). Therefore, possibly the majority of sampling conducted by the 4L-SID during Deployment 1 was beneath the mixed layer, and thus below the main accumulation of diazotrophs, which could partly explain why lower rates were found compared with Deployment 2. However, with the available qPCR data (samples from 5, 25, 45 m etc.) we cannot resolve whether the MLD had an influence on the vertical distribution of diazotrophs that would explain the variations in BNF. Nutrient concentrations in the upper 100 m were mostly low, which is common in oligotrophic oceanic gyres (Fig. 5). However, some of the NO22 þ NO32 (low-level nitrogen, LLN) and phosphate concentrations near the surface equaled concentrations much deeper in the water column at around 125 m (Fig. 5D, near the “apex”), which is atypical for NPSG waters (Robidart et al., 2014). Along the 25-m-depth horizon where the 4L-SID was situated, LLN concentrations ranged from 2 – 6 nmol L21 and phosphorus concentrations ranged from 20 to 137 nmol L21 for the complete 12-day oceanographic expedition (Fig. 5). During Deployment 1, the 4L-SID encountered remarkably steep gradients in salinity and phosphate concentrations (Fig. 5B and D). In turn, waters sampled during Deployment 2 were relatively rich in chlorophyll (Fig. 1). Overall, these data suggest that different water types were sampled during 4L-SID Deployments 1 and 2. The highest BNF and primary production rates were measured nearest to the “apex” of the cruise transit (Fig. 5), where nutrient concentrations were elevated. Possibly, diazotrophs in this region were stimulated by the nutrients and were able to respond with higher BNF, although it is not clear whether nutrient concentrations were elevated due to influx from depth, atmospheric deposition (Kim et al., 2014), or whether there was low demand in the microbial community which led to its accumulation within the surface layer. The 4L-SID drifts in a Lagrangian manner and can provide unbiased samples describing the variability in BNF within a complex setting of small spatiotemporal fluctuations in abiotic parameters. In order to pinpoint specific abiotic/biotic parameters responsible for observed variations in BNF rates, future versions of the SID need to include additional oceanographic sensors, like a CTD package including O2, nitrate and optical sensors (see next section). The qPCR quantifications of nifH gene copies suggest that unicellular cyanobacterial diazotrophs (Candidatus Atelocyanobacterium thalassa, “UCYN-A,” and Crocosphaera watsonii, “UCYN-B”) were the most abundant diazotrophs present in the water column throughout the sampled area (Fig. 6A). In the region near the apex, nifH genes of these organisms attained concentrations of 2.0 108 copies m22, which was 96% of all quantified nifH gene copies. Trichodesmium sp. was the next most abundant diazotroph (up to 3.0 107 nifH gene copies m22); heterocystous symbionts of diatoms (het-1 and het-2) as well as heterotrophic bacteria were present at much lower abundances (Fig. 6B). While nifH gene copy inventories appeared to co-vary for unicellular cyanobacterial diazotrophs (UCYN-A and UCYN-B, Fig. 6A), a different pattern was observed for the remaining five phylotypes, i.e. Trichodesmium, heterocystous cyanobacterial symbionts het-1 and het-2, and the two heterotroph diazotrophs (Fig. 6B). The nifH gene abundances in this latter group also co-varied, but appeared to be relatively more abundant at stations parallel to 4L-SID deployment 1 (Fig. 6B). This group includes the diazotrophs typically assigned to the .10-mm size fraction (Trichodesmium and heterocystous cyanobacteria), and was up to 42% of nifH inventories in the “Transit 1” area (Fig. 6C). These data suggest that the eddy-induced advection and mixing in the area had clear effects on the distribution of different diazotrophs. While such data cannot be used to infer which diazotrophs were responsible for the measured BNF rates, it is noteworthy that the overall higher rates obtained during 4L-SID deployment 2 coincided with a generally lower abundance of diazotrophs in the .10-mm size fraction. Recommendations for future SID deployments In its current configuration, the SID was successfully deployed and recovered on two occasions, providing daily BNF and primary production measurements in the surface waters of the oligotrophic open ocean. The SID concept can contribute more environmentally relevant rates of BNF to inform global flux calculations, and this technology has been validated in this study and others (Taylor and Howes, 1994; Pachiadaki et al., 2014; Edgcomb et al., 2014) for expeditions in various marine provinces. The SID could be especially helpful for studies in “delicate” habitats (e.g. anoxic habitats), where the seawater samples are severely compromised when they are brought onboard the research vessel for on-deck incubations (Feike et al., 2012; Edgcomb et al., 2014). Fig. 6. Depth-integrated inventories of nifH gene copies of diazotroph microorganisms (quantified by qPCR) on ship stations in close proximity to the SID drift paths. Section distance on the x-axis is the total distance covered by shipboard stations along the northeast and following westward transit. (A) nifH inventories of UCYN-A (Candidatus Atelocyanobacterium thalassa) and UCYN-B (Crocosphaera watsonii). (B) nifH inventories of Trichodesmium sp., het-1 and het-2 [Diatom – Diazotroph associations between the diazotroph cyanobacteria Richelia intracellularis and two different diatom species, Rhizosolenia-Richelia (RR) and Hemiaulus-Richelia (HR)] and heterotroph phylotypes HM210397 (Halm et al., 2012) and KC013231 (Bombar et al., 2013). (C) Percentage of the total nifH gene copies m22 of diazotrophs that are usually found in the .10 mm size fraction (Trichodesmium, het-1, het-2). The triangles at the top axis indicate where the SID took samples along the section distance during its two consecutive deployments (four samplings per deployment). The current SID technology would be improved by conducting simultaneous replicate measurements. Furthermore, an increased number of FF1s would permit more samples to be processed and longer deployment periods. The ability to also collect replicate samples for metagenomic and metatranscriptomic analysis will enable investigators to link biological function with the identity and activity of the prokaryotic key players present in the water column at the exact time of sampling (Robidart et al., 2014). To this end, a new SID-implemented fixation filter (FF3, Taylor et al., 2015) capable of chemically preserving particulate microbial samples in a manner compatible with subsequent metagenomic and metatranscriptomic study (Edgcomb et al., 2014) has just been developed. Finally, the addition of further oceanographic sensors, like an ISUS for NO32 measurements (Johnson and Coletti, 2002), and the ability for adaptive sampling in response to thresholds in environmental parameters, would allow the SID to sample along environmental gradients. A relatively new version microbial sampling-SID (MS-SID, Edgcomb et al., 2014; Pachiadaki et al., 2014; with a host of sensors [CTD, turbidity sensors, oxygen optode]), that was not available during this study, is now in hand and possesses the ability of collecting/in situ chemically preserving up to 48 incubated samples and/or larger volume microbial samples as well as 24 samples in gastight bags. At present, a dual incubation chamber SID is in advanced development (Vent-SID, laboratories of C. Taylor and S. Sievert, WHOI). C O N C L U S I O N S The development of a device for conducting the entire sampling, incubation and filtration processes of 15N2 rate measurements in situ could help in the future to obtain higher resolution coverage of direct estimates of oceanic BNF. The two 4-day deployments conducted during the September 2011 BioLINCS cruise were successful with respect to instrument operation and obtaining BNF rates comparable with those achieved by traditional CTD – rosette sampling and incubation. Overall, the SID offers increased sampling resolution of BNF measurements and a platform for conducting in situ sampling of oceanic water columns where on-deck incubations are not feasible. This device will help in identifying driving factors of BNF in situ, and could be used to test important hypotheses about the regulation of BNF within the oceanic nitrogen cycle. S U P P L E M E N TA RY D ATA Supplementary data can be found online at http://plankt. AC K N O W L E D G E M E N T S We thank M. Hogan and T. Cote for help in cruise organization, the personnel onboard R/V Kilo Moana for technical assistance and John Ryan for help with Fig. 1. 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Deniz Bombar, Craig D. Taylor, Samuel T. Wilson, Julie C. Robidart, Ariel Rabines, Kendra A. Turk-Kubo, John N. Kemp, David M. Karl, Jonathan P. Zehr. Measurements of nitrogen fixation in the oligotrophic North Pacific Subtropical Gyre using a free-drifting submersible incubation device, Journal of Plankton Research, 2015, 727-739, DOI: 10.1093/plankt/fbv049