J. Phycol. 49, 502–516 (2013) © 2013 Phycological Society of America DOI: 10.1111/jpy.12057 MEASURING CARBON AND N2 FIXATION IN FIELD POPULATIONS OF COLONIAL AND FREE-LIVING UNICELLULAR CYANOBACTERIA USING NANOMETER-SCALE SECONDARY ION MASS SPECTROMETRY1 Rachel A. Foster,2 Saar Sztejrenszus,3 and Marcel M. M. Kuypers Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Celsiusstr 1, Bremen D-28359, Germany Unicellular cyanobacteria are now recognized as ocean, cyanobacteria are one of the dominant important to the marine N and C cycles in open N2-fixing (diazotroph) groups of organisms where ocean gyres, yet there are few direct in situ abundances of several lineages, including filamen- measurements of their activities. Using a high-resolution tous and unicellular types, often co-occur. The nanometer scale secondary ion mass spectrometer larger filamentous and symbiotic types (i.e., Trich- (nanoSIMS), single cell N2 and C fixation rates were odesmium spp. and Richelia intracellularis) have long estimated for unicellular cyanobacteria resembling been considered the primary N2 fixers in open Crocosphaera watsonii Crocosphaera watsonii N2 fixer . - ocean gyres (Mague et al. 1974, Capone et al. 1997, like cells were observed in the subtropical North Karl et al. 2002). However, more recently, unicellu- Pacific gyre (22°45′ N, 158°0′ W) as 2 different lar cyanobacteria are now thought of as an equally phenotypes: colonial and free-living. Colonies important new source of N due to their high abun- containing 3–242 cells per colony were observed and dances and broad distributions (Needoba et al. cell density in colonies increased with incubation 2007, Moisander et al. 2010). Further evidence for time. Estimated C fixation rates were similarly high an important role of unicellular diazotrophic cyano- in both phenotypes and unexpectedly for unicellular bacteria is also provided by high N2 fixation rates cyanobacteria 85% of the colonial cells incubated reported in the smaller size fractions (<10 lm) of 15 15 during midday were also enriched in N above bulk stable isotope ( N2) uptake experiments 15 natural abundance. Highest N enrichment and N2 (Dore et al. 2002, Montoya et al. 2004, Zehr et al. fixation rates were found in cells incubated 2007a, Grabowski et al. 2008, Bonnet et al. 2009, overnight where up to 64% of the total daily fixed N Kitajima et al. 2009, Sohm et al. 2011a), as well as in the upper surface waters was attributed to both results from recent modeling efforts (Goebel et al. phenotypes. The colonial cells retained newly fixed 2007, Monteiro et al. 2010). C in a sulfur-rich matrix surrounding the cells and Three groups of marine N2-fixing unicellular often cells of both phenotypes possessed areas cyanobacteria, A, B, and C, can be distinguished (<1 nm) of enriched 15N and 13C resembling storage based on their nifH gene sequence, which encodes granules. The nanoSIMS imaging of the colonial for the nitrogenase enzyme for N2 fixation (Zehr cells also showed evidence for a division of N2 and et al. 2001, Foster et al. 2007). More recently, the C fixation activity across the colony where few groups are referred to as UCYN-A, UCYN-B, and individual cells (<34%) in a given colony were UCYN-C. UCYN-B is the only group with a cultured enriched in both 15N and 13C above the colony representative, Crocosphaera watsonii WH8501, which average. Our results provide new insights into the was isolated from the western tropical South Atlan- ecophysiology of unicellular cyanobacteria. tic Ocean in the mid 1980s (Waterbury and Rippka Key index words: Crocosphaera 1989). Currently, there are up to 10 different strains ; diazocyte; N2 fixation; nanoSIMS; unicellular of C. watsonii isolated from the open ocean (Atlan- tic and Pacific Oceans; Webb et al. 2009) which pos- sess great phenotypic plasticity while maintaining high genetic conservation (Zehr et al. 2007b, Webb Nitrogen fixation, or the reduction of di-nitrogen et al. 2009, Bench et al. 2011). The isolated strains (N2) gas to bio-available ammonium is an important of Crocosphaera can be distinguished based on cell new source of nitrogen (N) in the World’s oceans diameter where the larger cell diameter (>4 lm) (Karl et al. 1997, Capone et al. 2005), and is only type strains produce substantial amounts of extracel- catalyzed by a small number but broad diversity of lular material (ECM) recently defined as an extra- microorganisms (Zehr et al. 1998). In the open cellular polysaccharide (EPS; Webb et al. 2009, Sohm et al. 2011b). The smaller cell diameter (<4.0 lm) strains also produce EPS, however, in les- 1Received 6 August 2012. Accepted 5 February 2013. ser amounts (Webb et al. 2009, Sohm et al. 2011b). 2Author for correspondence: e-mail [email protected].. 3Department of Microbial Ecophysiology, University of Bremen, Field populations of Crocosphaera-like cells have also Bremen D-28359, Germany been observed forming loose groupings or colonies Editorial Responsibility: J. Raven (Associate Editor) presumably held together by the EPS (Biegala and 502 NANOSIMS IMAGING OF C AND N2 FIXATION 503 Raimbault 2008, Webb et al. 2009, Sohm et al. throughout the year (Leteliar and Karl 1996, Karl 2011b). The function of the EPS in Crocosphaera et al. 1997, Dore et al. 2002, Falcon et al. 2004, spp. is not known; however, other phytoplankton Fong et al. 2008, Grabowski et al. 2008, Church produce EPS as a carbon (C) reserve during nutri- et al. 2008, Sohm et al. 2011a). ent limitation (Corzo et al. 2000), while in the uni- In this study, we report single cell N2 and C fixa- cellular diazotroph, Cyanothece spp., EPS has an tion rates for two phenotypic field populations of acidic character, which may be beneficial for bind- C. watsonii-like cells: free-living and colonial. Both ing metals and the thick layering of EPS could phenotypes were present at the field site, and were potentially aid in oxygen protection (Reddy et al. amongst the more abundant diazotrophic cyanobac- 1996). teria observed by microscopy. We initially hypothe- Since the nitrogenase enzyme is irreversibly inacti- sized that the unicellular cyanobacterial cells living vated by the presence of oxygen (Postgate 1998), colonially could have an advantage similar to Trich- N2-fixing cyanobacteria have developed strategies odesmium spp. where N2 fixation and photosynthesis to protect their nitrogenase enzyme from oxygen could occur contemporaneously during the daytime. evolved during photosynthesis (Gallon and Chaplin Although nanoSIMS imaging of both phenotypes of 1988, Burris 1991, Fay 1992). In unicellular diazo- the C. watsonii-like cells showed an enrichment of 15 trophic cyanobacteria, N2 fixation is separated tem- N after incubations during the day, our results are porally to the night while photosynthesis occurs still speculative since other N2 fixers were present during the day (Griffiths et al. 1987, Fay 1992). and could contribute to a labeled and available dis- Hence, the highest nifH transcription and N2 fixa- solved inorganic nitrogen (DIN) pool. However, the tion of C. watsonii and UCYN-B field populations nanoSIMS imaging did allow us to visualize a spatial are observed during the night (Tuit et al. 2004, heterogeneity of 15N and 13C across the colonies Church et al. 2005a,b, Zehr et al. 2007a, Webb et al. that suggested a division of metabolic activity 2009, Mohr et al. 2010a, Pennebaker et al. 2010, Shi amongst the cells. The results presented here pro- et al. 2010, Saito et al. 2011). In heterocystous vide new insights into the ecophysiology of two phe- cyanobacteria, N2 fixation is separated spatially into notypes of unicellular cyanobacteria common in the specialized thick-walled cells called heterocysts while oligotrophic waters of the World’s oceans. photosynthesis occurs in vegetative cells (Wolk et al. 1976). A combination of both spatial and temporal separation can be found in the nonheterocystous MATERIALS AND METHODS colonial filamentous marine cyanobacterium, Trich- Water collection and incubation experiments. Whole seawater odesmium spp., where N2 fixation occurs during the was collected during the HOT series cruise on 23–27 July day in only some of the cells along a filament (Berg- 2009 (HOT-213) to station ALOHA (A Long-Term Oligo- man et al. 1997, Berman-Frank et al. 2001, Lund- trophic Habitat Assessment; 22°45′ N, 158°0′ W) in the North Pacific subtropical gyre. Seawater samples were col- gren et al. 2001). The localized N2-fixing cells are referred to as diazocytes. Others have shown in field lected from Niskin bottles on 24 July 2009 from 15 m using a Conductivity Temperature Depth (CTD) rosette. Whole and culture populations of Trichodesmium that in seawater was transferred directly into 12 acid rinsed 4.4 L addition to a spatial separation, segregation of C light transparent polycarbonate bottles (Nalgene, Fisher and N2 fixation can be influenced by temporal fac- Scientific, Pittsburgh, PA, USA). The bottles were filled with- tors (Berman-Frank et al. 2001, Finzi-Hart et al. out headspace and capped without air bubbles, and each 13 bottle was amended with 1 mL of 0.2 M NaH CO3 and 2009). The colony formation in Trichodesmium and 15 other cyanobacteria could therefore be strategic 4mLof99% N2 (Cambridge Isotopes, Andover, MA, USA) through the septa cap (Thermo Fisher Scientific, since physiological activities, e.g., photosynthesis Waltham, MA, USA) using gas tight syringes (Hamilton, and N2 fixation, can occur simultaneously by parti- Reno, NY, USA). The bottles were manually inverted for tioning the activity in time and space within the several minutes (5 min) to mix in the isotopes and placed cells of a colony. on their sides in an on deck incubator provided by the ship Recent methodological advancements in single (R/V Kilo Moana). cell technologies have made it possible to directly The bottles were incubated for ~0, 3.8, 12.3 and 18.3 h at measure metabolic activities and variability of meta- ambient surface water temperatures (25.9°C) by continuously flowing surface seawater and shading to 50 % incident sur- bolic rates in individual cells (reviewed in Wagner face irradiance in the on deck incubator.