Elevated Temperature Increases Carbon and Nitrogen Fluxes Between Phytoplankton and Heterotrophic Bacteria Through Physical Attachment
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The ISME Journal (2017) 11, 641–650 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE Elevated temperature increases carbon and nitrogen fluxes between phytoplankton and heterotrophic bacteria through physical attachment Nestor Arandia-Gorostidi1, Peter K Weber2, Laura Alonso-Sáez1,3, Xosé Anxelu G Morán1,4 and Xavier Mayali2,5 1Centro Oceanográfico de Gijón/Xixón, Instituto Español de Oceanografía, Centro Oceanográfico de Gijón/ Xixón, Gijón/Xixón, Asturias, Spain; 2Nuclear and Chemical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA, USA; 3AZTI, Sukarrieta, Bizkaia, Spain; 4Red Sea Research Center, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia and 5Oregon State University, Microbiology Department, Corvallis, OR, USA Quantifying the contribution of marine microorganisms to carbon and nitrogen cycles and their response to predicted ocean warming is one of the main challenges of microbial oceanography. Here we present a single-cell NanoSIMS isotope analysis to quantify C and N uptake by free-living and attached phytoplankton and heterotrophic bacteria, and their response to short-term experimental warming of 4 °C. Elevated temperature increased total C fixation by over 50%, a small but significant fraction of which was transferred to heterotrophs within 12 h. Cell-to-cell attachment doubled the secondary C uptake by heterotrophic bacteria and increased secondary N incorporation by autotrophs by 68%. Warming also increased the abundance of phytoplankton with attached heterotrophs by 80%, and promoted C transfer from phytoplankton to bacteria by 17% and N transfer from bacteria to phytoplankton by 50%. Our results indicate that phytoplankton-bacteria attachment provides an ecological advantage for nutrient incorporation, suggesting a mutualistic relationship that appears to be enhanced by temperature increases. The ISME Journal (2017) 11, 641–650; doi:10.1038/ismej.2016.156; published online 6 December 2016 Introduction a large part of which is ultimately phytoplank- ton derived (Buchan et al., 2014). At the same The oceans cover 70% of our planet and have a key time, bacteria sustain algal growth by providing role in the global C cycle through CO2 exchanges substrates such as remineralized nutrients or with the atmosphere and organic C transformations vitamins (Croft et al., 2005; Amin et al., 2012). within marine food webs. In the upper ocean, The transfer of C from phytoplankton to bacteria single-celled cyanobacteria and eukaryotic algae, may occur through two different strategies: as collectively known as phytoplankton, fix inorganic free-living cells that incorporate DOC previously C and partly release it as dissolved organic carbon released by autotrophs to the surrounding water, (DOC), feeding heterotrophic bacteria through the and through physical attachment (Grossart and microbial food web. A critical but often overlooked Simon, 2007a; Mével et al., 2008), where bacteria characteristic of these ecosystems is the interaction are in contact with their main sources of fresh between these two major components that dom- DOC. Because of the physical, direct interaction inate the biogeochemical cycles of C and N in the between bacteria and phytoplankton cells, the upper oceans (Azam and Malfatti, 2007). In surface waters, heterotrophic bacterial biomass production attachment strategy could provide both compo- (BP) depend on the availability of labile DOC, nents with an ecological advantage compared with their free-living counterparts (Rooney-Varga et al., 2005; Grossart and Simon, 2007a; Grossart Correspondence: N Arandia-Gorostidi, Centro Oceanográfico de et al., 2007b). Indeed, it has been recently observed Gijón/Xixón, Instituto Español de Oceanografía, Avenida Príncipe de Asturias 70 bis, Xixón, Asturias E-33212, Spain. that symbiotic relationships between cyanobacteria E-mail: [email protected] and microalgae through attachment are key or X Mayali, Nuclear and Chemical Sciences Division, Avenida strategies for C and N exchange between them Príncipe de Asturias 70 bis, Lawrence, Asturias Livermore (Thompson et al., 2012; Cornejo-Castillo et al., National Laboratory, Livermore, CA 94550, USA. E-mail: [email protected] 2016). Received 25 February 2016; revised 21 September 2016; accepted According to climate models, a sea surface 5 October 2016; published online 6 December 2016 temperature increase as high as 6.4 °C may occur Temperature-induced carbon–nitrogen flux N Arandia-Gorostidi et al 642 by the end of this century (Meehl et al., 2007), Materials and methods likely modifying oceanic C fluxes (Sarmiento et al., 2004). Raised temperature impacts on microbial Sample collection and water incubations food webs may include increases in bacterial Seawater samples were taken on April 2013 off the west coast of Gijón/Xixón, northern Spain (Southern abundances (Morán et al., 2015) and changes in ′ ″ DOC use (Sarmento et al., 2010; Duarte et al., 2013) Bay of Biscay; latitude 43° 40 12 /43.67°N longitude 5° 34′ 48″/5.58°W). Seawater was pre-filtered by ultimately resulting in increased bacterial produc- μ tion and respiration (López-Urrutia and Morán, 200 m to remove mesozooplankton. Samples were 2007; Vázquez-Domínguez et al., 2007). It has been collected at noon and transported to the laboratory in suggested that warming may also induce a stronger 20 l polycarbonate bottles (Nalgene, Rochester, NY, coupling between bacteria and phytoplankton (von USA) within 6 h. Six transparent 4 l polycarbonate Scheibner et al., 2014). Important questions bottles (Nalgene) were filled with 2 l of the sampled include whether warming will promote higher water and placed in a controlled temperature incuba- bacterial incorporation of phytoplankton-derived tor, 3 at 12.5 °C (ambient) and 3 at 16.5 °C (+4 °C). All DOC, and whether physical interactions through bottles were incubated under saturating irradiance μ − 2 − 1 attachment may impact the magnitude of this (~150 mol photons m s ) (Morán and Scharek, coupling. As productivity of attached bacteria 2015) with a 13:11 light:dark period. After the samples may be an order of magnitude higher than free- were placed in the incubators, the water was accli- living cells (Azam and Long, 2001), distinguishing mated for 12 h until the next morning. After the between these two ecological strategies is necessary acclimation period, 200 ml of each bottle were trans- ferred to Pyrex glass bottles, where we added to accurately quantify bacterial C fluxes. However, − 1 13 13 traditional methodologies such as bulk productiv- 0.1 mmol l of C-bicarbonate (99 atom% C, Cam- ity measurements are problematic for resolving C bridge Isotopes Laboratories Inc., Cambridge, MA, USA, equivalent to 5% 13C after dilution by ambient incorporation by attached cells, and in general the − 115 study of attached bacteria in aquatic ecosystems CO2) and 50 nmol l N-leucine (Cambridge Isotopes remains a challenge (Simon et al., 2002). The rise of Laboratories Inc.). The bottles were incubated under single-cell approaches (Wagner, 2009) now allows constant light conditions at the same temperatures. the study of microscale processes such as those that After 12 h, 5 ml of each sample were fixed with 4% occur in the phycosphere, that is, the surface of formaldehyde for 3 h at room temperature and filtered algal cells and the thin layer that surrounds it (Bell through polycarbonate filters (type GTTP; pore size, 0.2 μm; diameter, 47 mm; Millipore, Eschborn, Ger- and Mitchell, 1972), where compounds can be À transported by diffusion allowing direct exchange many). The collected samples were stored at 80 °C. between cells (Lazier and Mann, 1989). Although Samples for chlorophyll a and nutrients were thus far bacteria–phytoplankton interactions collected once per day during 3 days (Supplementary through attachment have been described mostly Figure S1), even after the stable isotope incubation, between heterotrophic bacteria and microphyto- in order to characterize the state of the auto- plankton cells such as diatoms or dinoflagellates trophic community. Both parameters were analyzed (Amin et al., 2012), the magnitude of bacterial following Calvo-Díaz and Morán (2006). attachment to smaller nano- and picophytoplank- ton, the most abundant autotrophs in the ocean (Stockner, 1988; Magazzù and Decembrini, 1995; Autotrophic cell abundance Marañón et al., 2001), remains unclear. Even The abundance of small autotrophic cells (Prochlor- though picophytoplankton may be too small to be ococcus, Synechococcus and picoeucaryotes) was detectable by membrane chemosensory mechanism determined by flow cytometry based on their orange by heterotrophic bacteria (Jackson, 1987), some (FL2, 585 nm) and red (FL3, 650 nm) fluorescence work has shown that pico and nanophytoplankton and light scatter at 90° (SSC, side scatter) signals are also susceptible to colonization by hetero- following standard procedures (Gasol and Del trophic bacteria (Salomon et al., 2003; Eiler et al., Giorgio, 2008) with a BD FACSCalibur (Becton 2006). Dickinson, Franklin Lakes, NJ, USA) flow cytometer, Here we investigated the effect of warming (4 °C equipped with an argon 488 nm laser. To estimate over ambient) in a temperate ecosystem on the cell- Synechococcus and eukaryotes abundance at the end specific activities of marine heterotrophic and of the 12 h incubation, a linear interpolation