Polar Biol (2009) 32:1539���1547 DOI 10.1007/s00300-009-0654-x 123 ORIGINAL PAPER Extracellular enzymes of cold-adapted bacteria from Arctic sea ice, Canada Basin Yong Yu �� Huirong Li �� Yinxin Zeng �� Bo Chen Received: 27 November 2008 / Revised: 12 May 2009 / Accepted: 29 May 2009 / Published online: 16 June 2009 �� Springer-Verlag 2009 Abstract A total of 338 aerobic heterotrophic bacterial strains were isolated from Arctic sea ice, Canada Basin (77��30 N���80��12 N). The capability of the isolates to pro- duce protease, lipase, amylase, chitinase, -galactosidase, cellulase and/or agarase was investigated. Isolates that were able to degrade tributyrin, skim milk, starch, lactose and chitin accounted for 71.6, 65.7, 38.5, 31.6 and 16.9% of sea ice strains, respectively. Lipase producers and/or protease producers were phylogenetically widespread among the isolated strains. Starch and/or lactose hydrolytic strains were mainly distributed among Colwellia, Marinomonas, Pseudoalteromonas, Pseudomonas and Shewanella iso- lates. Pseudoalteromonas tetraodonis, Pseudoalteromonas elyakovii, Bacillus Wrmus and Janibacter melonis isolates all have the ability to degrade chitin. Only some strains belonging to Pseudoalteromonas genus scored positive for agarase (6) and cellulose (9). The temperature dependences for lipase activities were determined for Wve psychrophilic and six psychrotolerant bacteria. At low temperatures, the psychrophilic bacterial lipase activity was not signiWcantly higher than psychrotolerant bacterial lipase, though all lipases showed remarkably high activity with 10���36% residual activity at 0��C. Keywords Extracellular enzymes �� Psychrophilic �� Psychrotolerant �� Cold-active lipase �� Sea ice �� Arctic Introduction Sea ice with temperatures ranging from 0 to ��35��C (Haas et al. 1997) provides one of the coldest habitats on the earth for marine life. In contrast to ice formed from freshwater, that from seawater is a semisolid matrix being permeated with a labyrinth of brine-Wlled channels and pores. Such a semi-enclosed, or even completely enclosed, matrix is a microhabitat characterized by highly changeable salinity, acidity, dissolved gas and light signatures (Eicken et al. 2000 Thomas and Dieckmann 2002) in which very dense and diverse microbial populations develop annually (Horner et al. 1992). Heterotrophic bacteria represent a major group within sea ice microbial assemblages (Sullivan and Palmisano 1984 Mock and Thomas 2005), and about 20���30% of the ice-bound primary production was found to cycle through them (Palmisano and Garrison 1993). In recent years, progress in the understanding of the phyloge- netic composition of bacterial communities associated with sea ice has been made. The Arctic sea ice bacteria fall into eight phylogenetic groups: subclasses , , and of Proteo- bacteria, the Cytophaga/Flexibacter/Bacteroides (CFB) phylum group, the high- and low-G + C Gram positives, and the orders Verrucomicrobiales and Chlamydiales (Brown and Bowman 2001 Junge et al. 2002 Brinkmeyer et al. 2003 Groudieva et al. 2004). However, the functional roles of bacteria in regards to the mineralization of organic matter in sea ice remains poorly understood. The high concentration of dissolved organic matter that has been reported from sea ice in the Arctic, Antarctic and Baltic Sea, exceeds surface water concentration by factors Electronic supplementary material The online version of this article (doi:10.1007/s00300-009-0654-x) contains supplementary material, which is available to authorized users. Y. Yu (&) �� H. Li �� Y. Zeng �� B. Chen SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, People���s Republic of China e-mail: yuyong@pric.gov.cn

1540 Polar Biol (2009) 32:1539���1547 123 of up to 500 (Mock and Thomas 2005). The majority (95%) of organic matter in marine environments is com- posed of high molecular weight compounds that are too large for direct uptake by marine bacteria (Chrost 1991). Before these polymeric compounds can be incorporated into microbial cells, they must Wrst be degraded by a series of extracellular hydrolytic enzymes. Extracellular enzyme activity has been recognized as the rate-limiting step in microbial degradation of high molecular weight organic matter in the marine environment (Hoppe 1991). The deg- radation of organic matter in sea ice depends on extracellu- lar enzymes functioning at low temperature. Environmental assays at in situ temperatures revealed that high chitobiase, aminopeptidase, phosphatases, -glucosidases and -gluco- sidases activities were present in sea ice (Helmke and Wey- land 1995 Huston et al. 2000). Extracellular enzyme activity is relative to bacterial concentration, which sup- ports the observation that sea ice with high bacterial con- centrations exhibits high maximal hydrolysis rates (Helmke and Weyland 1995). In addition to their important roles in permanently cold habitats, microbial extracellular enzymes with optimal activity at low temperature provide opportunities to study the adaptation of life in cold habitats and the potential for biotechnological exploitation (Aguilar 1996). Applications of these roles are being considered in all areas of industry and products. Examples of technology aVected by these dis- coveries includes: cleaning agents, leather processing, deg- radation of xenobiotic compounds in cold environments, food processing (cheese manufacture, bakery, meat tenderi- zation, dairy, sea food, fruit and vegetable products), phar- maceuticals (organic synthesis of enantiomerically pure drugs and pharmaceutical intermediates), biofuels (low- energy ethanol production process), molecular biology (heterologous gene expression), and enzyme nanobiotech- nology (Huston 2008). Besides low temperature, dehydra- tion caused by the high-brine salinities is the other major stressor for sea ice bacteria, which may experience salini- ties three times that of seawater (Thomas and Dieckmann 2002). Most of the work that has been conducted on sea ice bacteria has indicated highly cold-adapted and salt-tolerant proteases, -galactosidases, phosphatase, and amylase (Thomas and Dieckmann 2002). These enzymes may be particularly adept at catalyzing organic synthesis reactions in aqueous/organic and nonaqueous media, as salt tolerant enzymes are well adapted to low water activity conditions (van den Burg 2003). In the present study, the extracellular enzymes produced from culturable heterotrophic bacteria associated with Arc- tic sea ice were investigated. Isolation of 338 Arctic sea ice bacteria, followed by molecular identiWcation and the detection of their extracellular enzymes was performed in order to expand our knowledge of the roles that cultivable bacteria play in biopolymer decomposition in sea ice. In addition, some experimental eVorts focused on screening for cold-active lipase. Materials and methods Sample collection A total of Wve sea ice samples 150���340 cm core length with 9 cm diameter were collected using a MARK II ice auger (Kovacs Enterprises, INC., USA) during the Second Chi- nese National Arctic Research Expedition cruise of the Chi- nese icebreaker Xue Long into the Canada Basin in August 2003 (Fig. 1). During sampling and processing, careful attention was put to maintaining sterile conditions. No visi- ble algal assemblages were observed in all Wve ice cores. The ice cores were cut into 10���20 cm sample sections using a sterile saw. Each ice section was melted at 4��C in equal volume unamended, pre-Wltered (0.2- m pore size) and autoclaved natural seawater (NSW, from 5 m below the ice). Isolation of bacterial strains To maximize the isolation of a diverse group of heterotro- phic bacteria, three media were used for the cultivation of samples. These included marine R2A (Suzuki et al. 1997), marine 2,216 (Difco) and NSW. All media contained 15 g of agar per liter. Ice-melt samples were diluted in NSW and 100 l of each dilution plated on a media. The plates were incubated in the dark at 4��C for up to 8 weeks. Colonies Fig. 1 The location of the Wve sampling sites