Metabolic Diversity of Heterotrophic Bacterioplankton Over Winter and Spring in the Coastal Arctic Ocean

Environmental Microbiology (2008) 10(4), 942–949 doi:10.1111/j.1462-2920.2007.01513.x Metabolic diversity of heterotrophic bacterioplankton over winter and spring in the coastal Arctic Ocean Maria Montserrat Sala,1* Ramon Terrado,2 advection, and sedimentation events that contributed Connie Lovejoy,2 Fernando Unrein1,3 and organic matter that enhanced bacterial metabolism. Carlos Pedrós-Alió1 1Departament de Biologia Marina i Oceanografia, Institut Introduction de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain. Arctic Ocean ecosystems are particularly sensitive to 2Dépt de Biologie and Québec Océan, Université Laval, climate changes, for example small temperature differ- Québec City, Québec G1K 7P4, Canada. ences may have large effects on the thickness and extent 3Instituto de Investigaciones Biotecnológicas-Instituto of sea ice (Smetacek and Nicol, 2005). Snow and ice cover Tecnológico de Chascomús (IIB-INTECH), Camino de are biologically important, influencing light availabity and Circunvalación Laguna Km 6 (B 7130 IWA) Chascomús, the timing of phytoplankton production in the Arctic Buenos Aires, Argentina. (Carmack et al., 2004). Primary production in oceanic areas constitutes the main organic source for bacterioplankton growth (Bird and Kalff, 1984; Cole et al., 1988), Summary mainly owing to direct bacterial use of dissolved com- Metabolic diversity of heterotrophic bacterioplankton pounds released by growing phytoplankton cells (Baines was tracked from early winter through spring with and Pace, 1991). However, recent evidence suggests that Biolog Ecoplates under the seasonally ice covered bacteria do not respond immediately to phytoplankton arctic shelf in the Canadian Arctic (Franklin Bay, production in cold waters (Bird and Karl, 1999; Pomeroy Beaufort Sea). Samples were taken every 6 days from and Wiebe, 2001; Duarte et al., 2005; Morán et al., 2006). December 2003 to May 2004 at the surface, the halo- The Arctic coastal ocean is also influenced by the input of cline where a temperature inversion occurs, and at large rivers and riverine inputs may also contribute signifi- 200 m, close to the bottom. Despite the low nutrient cant amounts of terrigenous organic matter that bacteri- levels and low chlorophyll a, suggesting oligotrophy oplankton could use for substrate (Emmerton, C.A., in the winter surface waters, the number of substrates Lesack, L.F.W. and Vincent, W.F., in press). used (NSU) was greater than in spring, when chloro- The only seasonal study to date of Arctic bacterioplank- phyll a concentrations increased. Denaturing gradient ton under the ice was over the Central Arctic Ocean, gel electrophorisis analysis also indicated that the where bacterial biomass and production in the upper winter and spring bacterial communities were phylo- water column responded strongly to phytoplankton growth genetically distinct, with several new bands appear- in summer and activity was restricted to the upper 60 m ing in spring. In spring, the bacterial community (project SHEBA, Sherr and Sherr, 2003; Sherr et al., would have access to the freshly produced organic 2003). This Central Arctic Ocean study measured the bulk carbon from the early phytoplankton bloom and the production of bacterioplankton, but it did not analyse the growth of rapidly growing specialist phenotypes possible organic substrates. Moreover, it was conducted would be favoured. In contrast, in winter bacteri- from an ice camp that drifted with the pack ice, making it oplankton consumed more complex organic matter difficult to separate changes due to seasons from those originated during the previous year’s phytoplankton due to different water masses. Despite this pioneering production. At the other depths we tested the NSU effort, winter bacterial activity under the Arctic ice remains was similar to that for the winter surface, with no largely unknown, and a detailed view into the seasonal seasonal pattern. Instead, bacterioplankton metabo- changes in bacterial utilization of carbon sources under lism seemed to be influenced by resuspension, ice cover is lacking. The CCGS Amundsen was frozen into first-year pack ice in Franklin Bay (Beaufort Sea) from December 2003 to May 2004 thus providing an excellent platform for detailed sampling throughout the winter. This Received 7 June, 2007; accepted 29 October, 2007. *For correspondence. E-mail [email protected]; Tel. (+34) 93 2309500; cruise was part of the Canadian Arctic Shelf Exchange Fax (+34) 93 2309555. Study (CASES). © 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd Bacterial metabolism in the Arctic 943 Garland and Mills (1991) first used the Biolog plates to 16 POLYMERS CARBOHYDRATES CARBOXYLIC PHEN AMINO ACIDS evaluate the metabolic potential of a whole microbial com- ACIDS 14 munity, as they allow simultaneous detection of the utilization of a large number of carbon sources. Biolog plates 12 have found application for the assessment of microbial 10 metabolic diversity in freshwater (Grover and Chrza- 8 nowski, 2000; Sinsabaugh and Foreman, 2001), estua- Surface 6 rine (Schultz and Ducklow, 2000) and, to a lesser extent, Number ofpositive results marine bacterioplankton (Hollibaugh, 1994; Jellet et al., 4 1996). The Biolog-Ecoplate contains 31 carbon sources in 2 triplicate (Insam, 1997) and has provided information, not 0 available using other techniques, on the functional diver- 16 sity of bacterioplankton in marine environments such as 14 western Antarctica (Sala et al., 2005a) and the Mediterra- nean Sea (Sala et al., 2005b; 2006b) and can also be 12 used to estimate bacterial use of dissolved organic nitro- 10 gen compounds in seawater (Sala et al., 2006a). TI 8 We used the Biolog plates as a way to estimate the 6 metabolic diversity of the heterotrophic bacterioplankton Number of positive results positive Number of through winter and at the onset of the productive spring 4 season, when phytoplankton were likely to become the 2 main source of dissolved organic matter. Our main aim 0 was to determine changes in the carbon sources poten- 16 tially used by bacterioplankton during winter and spring at 14 three different layers within the ice-covered water column of the Arctic shelf. Additionally, we also followed the com- 12 position of the bacterial community in the surface with 10 denaturing gradient gel electrophoresis (DGGE) in order 8 to assess if eventual changes in metabolic diversity were Bottom 6 related to changes in phylogenetic diversity. Number of positiveresults 4 2 Results 0 Out of the 31 substrates tested, 21 were used by communities in the Arctic water samples. Ten substrates did not -Cyclodext -Hydroxybut show any positive result: the two amines (putrescine and Glycogen α Tween 80 Tween 40 D-Mannitol D-Cellobiob N-Acetyl-D- D-Xylose Glucose-1-P Pyruvic Acid D-Galacturon γ Itaconic Acid 4-Hydroxy B 2-Hydroxy B Glycyl-L-Glu L-Threonine L-Phenylala L-Asparagine L-Serine L-Arginine phenylethylamine); three carboxylic acids (a-ketobutyric, Fig. 1. Number of positive results for each of the 31 substrates in D-malic and D-glucosaminic acid), five carbohydrates the Biolog plate along the period of the study. Substrates are (b-methyl-D-glucoside, D,L-a-glycerol phosphate, a-D- grouped into categories and results are shown for the three different layers: surface (n = 26), temperature inversion depth lactose, D-galactonic acid g-lactone and i-erythritol). Gen- (n = 24) and bottom (n = 25). erally, polymers and carbohydrates were the categories with higher number of positive results (Fig. 1). The most often used substrates (more than 25 positives out of 75 Tween 80 and N-acetyl-D-glucosamine showed more samples) were Tween 80 (a trademark for polysorbate positive results at the bottom than at other layers (not 80), glycogen, D-cellobiose, D-mannitol, N-acetyl-D- significantly different). glucosamine, and glycyl-L-glutamic acid. Chlorophyll a concentrations at the surface varied Although the differences were not significant, surface between 0.02 and 0.11 mgl-1 from December to 15 March. bacteria utilzed a greater number of substrates (number Then, as the ice started thinning, chlorophyll a increased of substrates used, NSU) than bacteria at deeper depths. rapidly, reaching values from 0.20 to 0.36 from 17 April to Likewise, most of the substrates had a higher number of the end of the study, 21 May (Fig. 2). Bacterial abundance positive results at the surface, but this was only significant did not show a clear seasonal pattern and varied between for three of them (one-way ANOVA): pyruvic acid methyl 1.5 ¥ 105 and 5.2 ¥ 105 bacteria ml-1 over the period of ester (F = 5.26, P = 0.01), D-mannitol (F = 4.12, P = 0.02) study. There was a distinct seasonal pattern in chlorophyll and D-cellobiose (F = 3.17, P = 0.03). Two substrates, concentrations. Initially, from December to the end of © 2008 The Authors Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 942–949 944 M. M. Sala et al. (A) At the deepest layer just above the bottom, chlorophyll Winter Spring a concentrations were even lower than at the TI depth 6 10 0.4 (0.01–0.05 mgL-1) and there was no clear seasonal Bacteria Chlorophyll pattern (Fig. 4). As was the case at the TI depth, bacterial 0.3 Chlorophyll abundances had no seasonal signal. The highest values -1 over the study period and among all three layers were 5 105 0.2 from this deep region, with up to 5.6 ¥ 10 in January. a ( μ Bacteria ml Bacteria There was no pattern in the NSU, but the greatest values g l -1 were found during a short period between February and 0.1 ) March, similar to that of the TI layer. The eight substrates with greatest number of positive 104 0 results over the period of study were chosen for a detailed (B) analysis (Fig.

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