Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice

Environmental Microbiology (2000) 2(4), 383±388

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice Adrienne L. Huston,1* Barbara B. Krieger-Brockett2 and Jody W. Deming1 1 School of Oceanography, Box 357940, University of Washington, Seattle, WA 98195, USA. 2 Department of Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195, USA. Summary Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 158C and maximally at 208C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucineaminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (158C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 208C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at 218C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation. Introduction By volume, 90% of the world ocean has a temperature of 58C or less (Baross and Morita, 1978), supporting communities of cold-adapted microorganisms, both psychrophilic and Received 16 June, 1999; revised 17 February, 2000; accepted 11 March, 2000. *For correspondence. E-mail [email protected]. edu; Tel. (11) 206 543 4558; Fax (11) 206 543 0275. Q 2000 Blackwell Science Ltd

psychrotolerant forms. Psychrophiles are defined as growing optimally at , 158C and to a maximal temperature of , 208C, whereas psychrotolerant microorganisms (previously known as psychrotrophs) are defined by their ability to grow at low temperatures, but with optimal and maximal growth temperatures above those of psychrophiles (Morita, 1975). In perennially cold habitats, microorganisms active at low temperatures play important roles in elemental cycles and the mineralization of organic matter (OM). The majority (. 95%) of OM in marine environments is composed of high molecular weight compounds largely unavailable for uptake by marine bacteria (Chrost, 1991). Before these polymeric compounds can be incorporated into microbial cells for catabolic and biosynthetic purposes, they must be degraded by a series of extracellular hydrolytic enzymes. These enzymes are located either on the bacterial cell surface or in the periplasmic space (cellattached extracellular enzymes) or are released from the bacterium into the environment (cell-free extracellular enzymes), where they may act in solution or be adsorbed to surfaces. Extracellular enzyme activity (EEA) has been recognized as the rate-limiting step in microbial degradation of high molecular weight OM in the marine environment (Hoppe, 1991; Meyer-Reil, 1991); it thus plays an important role in the concentration, speciation and cycling of OM. In cold environments, the degradation of surfaceadsorbed and polymeric OM depends on the capacity of extracellular enzymes to operate at ambient temperatures. In addition to their important roles in natural environments, microbial enzymes can be highly specific in their activity and thus are increasingly useful in industrial applications. Enzymes from aquatic bacteria adapted to moderate and high temperatures have already figured in a variety of applications, including polymerase chain reaction (PCR), laundry detergents, protein recovery and organic synthesis (e.g. Mullis and Faloona, 1987; Adams and Kelly, 1995). Although enzymes with optimal activity at cold temperatures have been understudied compared with their heat-stable counterparts, increasing attention is being paid to cold-active enzymes as potential applications are revealed (Margesin and Schinner, 1994; Brenchley, 1996; Hoshino et al., 1997; Morita et al., 1997). For example, bacteria that produce cold-active, heat-labile enzymes are attractive candidates for applications such as waste digestion in cold environments,

384 A. L. Huston, B. B. Krieger-Brockett and J. W. Deming food processing and preservation, and industrial processing that benefits from rapid inactivation of enzymatic reactions. This research addresses the fact that relatively little attention has been paid to the enzyme strategies of psychrophilic bacteria in polar marine environments or the biotechnological potential of their cold-active enzymes. Gerday et al. (1997) carefully considered psychrophily in reference to enzymatic activity as meaning a specific activity at low temperatures that is higher than mesophilic counterparts; they did not make distinctions based on temperature optima or maxima (as has been carried out with bacterial growth). They acknowledged, however, that high specific activity at low temperature may be the result of a relatively low activation energy which can give rise to limited enzyme stability, i.e. to lower temperature optima and maxima. In this sense, the temperature optima profiles of EEA reflect a degree of cold adaptation. In other work, where cold-active extracellular enzyme characteristics have been examined (whether from an environmental or biotechnological perspective), thermal optima are usually well above 208C, typically between 308C and 508C (Reichardt, 1987; 1988; Morita et al., 1997). Here we report the thermal characteristics of extracellular chitobiase and protease activity in Arctic sea ice and sediment samples, as well as cell-free leucineaminopeptidase activity measured in crude extract from an extremely psychrophilic bacterial isolate. The results provide evidence for the evolution of cold-adapted extracellular enzymes that display remarkably low temperature optima.

Results and discussion Field results Rates of extracellular leucine-aminopeptidase (LAPase) and chitobiase activities in our Arctic environmental samples were measured and replicated over sufficiently resolved temperature increments to illustrate the temperature dependence of EEA (Figs 1 and 2). The most significant finding from these environmental assays was the detection of EEA temperature optima that are remarkably low relative to literature values. Sea ice in particular was identified as a potential source for unusually cold-optimized enzymes (Fig. 1). The less cold-optimized EEA measured in the sediment slurries (Fig. 2) and in one sea-ice sample (Fig. 1B) were more representative of previous studies indicating that psychrophilic and psychrotolerant bacteria release enzymes with activity optima between 25 and 458C, well above the upper growth limit of the EEA-producing organisms (McDonald et al., 1963; Helmke and Weyland, 1991; Schinner et al., 1992). Because environmental observations represent the combined activities of many enzymes, even those samples scored as less cold optimized may contain enzymes with very low temperature optima (as suggested by LAPase activity in sediment retaining 75% of maximal activity at 08C, as discussed below). Rates of EEA in ice-core samples at in situ temperatures compared favourably with those reported in a previous study (Helmke and Weyland, 1991). When compared with rates measured in our sea-water samples (data not shown), ice core rates were higher by two orders of

Fig. 1. EEA as a function of temperature in ice-core samples. A. Chitobiase activity in mid-chlorophyll band (ice core 157). B. LAPase activity in mid-chlorophyll band (ice core 157). C. LAPase activity in bottom chlorophyll band (ice core 171). Error bars indicate 95% confidence intervals. Weighted LLS results (not shown) did not differ significantly from LLS (see text). Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 383±388

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Table 1. Temperature characteristics of EEA.

EEA assayed

Samplea

Optimum temperature (8C)

LAPase

Ice core 157 Ice core 171 Boxcore 18 Strain 34H

35 15 ?b 20

12 42 75 15

Chitobiase

Ice core 157 Boxcore 18

15 25

5 2

EEA at 08C (% of maximum)

a. See Table 2 for sample information. b. Not clear (see Fig. 2b).

Fig. 2. EEA as a function of temperature in Arctic surficial sediment slurries from Boxcore 18. A. Chitobiase activity. B. LAPase activity. Error bars indicate 95% confidence intervals. Weighted LLS results (not shown) did not differ significantly from LLS (see text).

magnitude, suggesting an important role for extracellular enzymes in carbon cycling within sea-ice communities. Comparison of EEA at 08C with maximal observed EEA (Table 1) revealed that LAPases retained a greater percentage of activity (12±75%) at near in situ temperatures than did chitobiases (2±5%). The observation that warming increased proteolytic activity relatively less than chitobiolytic activity is consistent with previous findings and supports the hypothesis that extracellular proteases remain more closely associated with the cell membrane (Vetter and Deming, 1994), and thus the active bacterium (as suggested in Hoppe, 1983), than do other extracellular enzymes. Warming of the cell membrane may disrupt the molecular orientations of other proteins acting in conjunction with the protease to slow substrate hydrolysis more so than for proteins acting independently of the organism (Vetter and Deming, 1994). An alternative explanation is that proteases may be inherently more cold adapted than other types of hydrolytic degradative enzymes

Colwellia psychroerthryaea (A. Huston, K. Junge, S. Carpenter and J. Deming, unpublished results). Studies of LAPase activity in a cell-free extract from the strain revealed a pH optimum at neutrality (data not shown) and a temperature optimum of 208C, remarkably low for an extracellular enzyme. It is presently the lowest known optimum, by at least 58C (Fig. 3), for cell-free proteases derived from either psychrophilic or psychrotolerant bacteria. The apparent activation energy for the enzyme preparation (50.5 kJ mol21, calculated from the linear portion, 5±158C, of an Arhennius plot of the data) falls within the range of activation energies reported for other cold-active cell-free proteolytic extracts (Margesin et al., 1991). That the temperature optimum for the cell-free LAPase extract (208C) is still higher than that for growth of strain 34H (88C) is consistent with past conclusions that EEA is less well adapted to cold temperatures than is bacterial metabolism or growth (Reichardt, 1987). Although EEA may not be greatest at the temperature optimum for bacterial growth, it may be sufficient to support growth (Brenchley, 1996). Even at 08C, the LAPase extract from

Laboratory results Strain 34H proved to be a motile, rod-shaped, Gramnegative, oxidase-positive bacterium with an optimal growth temperature of 88C (data not shown). Analysis by 16S rRNA sequencing has indicated that the strain belongs to the genus Colwellia and is most probably Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 383±388

Fig. 3. Cell-free extracellular LAPase activity as a function of temperature. Solid line, strain 34H (this study) representing a compilation of data from duplicate experiments (error bars indicate 95% confidence intervals for triplicate measurements taken at each temperature); dotted lines, literature data for other cultures (V Pseudomonas sp., strain PL-4,Hoshino et al., 1997; B Strain P105, Morita et al., 1997).

386 A. L. Huston, B. B. Krieger-Brockett and J. W. Deming thermo-stable counterparts (Davail et al., 1994; Feller et al., 1994). Current environmental models (see Vetter et al., 1998) predict that the nutritional benefit of releasing degradative enzymes will be a function of both enzyme activity and enzyme lifetime (lability). The discovery of extracellular enzymes in the environment with lower temperature optima (and thus potentially more labile structures) than previously expected provides new material for understanding the biochemical and evolutionary basis of cold adaptation in the bacterial world. Ongoing experiments investigating the activity, stability and lifetime of such cold-adapted enzymes produced by extremely psychrophilic bacteria may also reveal enzymes or production strategies useful for applied purposes in biotechnology or bioremediation in cold climates. Fig. 4. Zymogram SDS±PAGE gel of cell-free extracts of strain 34H: lanes 1 and 4, molecular weight markers; lane 2, cell-free extract of 34H grown at 218C; lane 3, cell-free extract of 34H grown at 88C. Zones of clearing depict protease activity; equal concentrations of total protein (1.9 mg) were loaded in lanes 2 and 3.

strain 34H retained 15% of maximal activity at 208C (Table 1). Zymogram SDS±PAGE gels revealed the presence of three proteolytic bands (37±45 kDa; Fig. 4) in the cellfree fractions of 34H cultures grown at both 21 and 88C. These bands may represent individual isozymes or active subunits of a larger proteolytic complex. The proteolytic bands in the extracts from both growth temperatures hydrolysed casein at 0, 4, 13, and 208C. Semi-quantitative observations suggested that the hydrolysis occurred maximally at 208C, in agreement with results of the MUF assays (Fig. 3). Although strain 34H synthesized the same group of proteolytic bands at each growth temperature tested, the activity of the enzymes differed: protease degradation was enhanced in the 218C extract, as seen by enhanced zones of clearing in lane 2 compared with lane 3 (Fig. 4). The presence of this effect on each of the gels incubated at 0, 4, 13 and 208C suggests that the enhanced activity was not due to expression of different thermal characteristics at the 218C and 88C growth conditions, but simply to the presence of a greater abundance of proteases in the 218C fraction. This observation agrees with the suggestion of Reichardt (1987; 1988) that enhanced enzyme production to compensate for reduced reaction rates at low temperatures may be the preferred bacterial strategy to producing fewer enzymes with activity optimized to lower temperatures. Neither molecular nor environmental constraints on cold-adapted bacterial enzymes are well understood. Current biochemical theory predicts that in order to retain sufficient activity at lower temperatures, a cold-adapted enzyme must possess a lower activation energy, due to a more flexible (and thus more labile) structure, than their

Experimental procedures Sample collection Samples for this study were obtained from several locations (Table 2) during two Arctic ice-breaking expeditions: an international expedition (called NEWP 93; Deming and the Newater Steering Committee and Principle Investigators, 1993) in the North-east Water Polynya, located on the continental shelf off the North-east coast of Greenland, during June±July of 1993; and a US Coast Guard cruise of opportunity (called AWS 96) in the permanent ice-cap and deep-sea environment of the Chukchi Sea during June of 1996. Sea-ice cores were obtained using an ice auger, as described by Tucker et al. (1999). Surficial sediments were recovered in sterile plastic subcores from deployments of a 0.25 m2 boxcore, as described by Rowe et al. (1997). Sea water was sampled at depths corresponding to the subsurface chlorophyll maximum (between 15 and 30 m), using 10 litre Niskin bottles. In all cases, samples were carefully guarded against temperature increases during recovery and processing.

Determination of environmental EEA Rates of EEA were estimated using fluorescently tagged substrate analogues as described in protocols from Hoppe (1983) for sea water and melted sea-ice samples, and from Mayer (1989) and Vetter and Deming (1994) for sediment samples. The sea-ice samples were taken from chlorophyll bands in the ice cores, using sterile cutting tools and collection containers. The samples were melted shipboard at 48C to a known dilution (usually 2.5) in prechilled sterile (0.2 mm filtered) sea water. For sediment samples, sections from the upper centimeter of the boxcore were slurried with nine parts chilled (08C) sterile artificial sea water. Sea-water samples were used for preliminary experiments at in situ temperatures (218C) to establish methodologies shipboard for two classes of EEA: chitobiase activity, using N-acetyl-b-D-glucosaminide (MUF-G) as a substrate analogue; and leucine-aminopeptidase (LAPase) activity, using L-leucine 7-amido-4-methylcoumarin (MCA-L; both from Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 383±388

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Table 2. Sample information. Sample

Station location

Sample origin

In situ temperature (8C)

Ice core 157a

72818 0 N 162809 0 W 76805 0 N 167807 0 W 76805 0 N 167807 0 W 79843 0 N 16814 0 W

1.1±1.2 m below ice±air interface (mid-ice chlorophyll band) 0.0±0.1 m above ice±water interface (bottom-ice chlorophyll band) Top 1 cm of boxcore (water depth of 612 m) Top 1 cm of boxcore (water depth of 305 m)

20.8

Ice Core 171a a

Boxcore 18

Strain 34Hb

21.5 0.2 0.7

a. From the AWS 96 expedition. b. From the NEWP 93 expedition.

Sigma). Sea-ice and sediment samples were split into homogeneous subsamples and incubated at five temperatures ranging from 21 to 358C. The fluorescently tagged substrates were added at saturating concentrations (100± 200 mM), as determined by substrate saturation experiments and incubated in the dark for a total period of 12±24 h. Over the course of incubation, EEA was measured in duplicate at each of the four time points chosen sequentially based on activity at the prior time points. At each time point, independently incubated subsamples were sacrificed, boiled for 60 s to stop EEA and buffered (Borax buffer, pH 10). Fluorescence was determined immediately using a PerkinElmer LS-5B spectrofluorometer with quartz cuvettes. Optimal fluorescence was observed at excitation and emission wavelengths of 355 and 440 nm respectively.

Isolation of EE-producing bacterial strains Yeast extract (0.01%) enrichments of environmental samples (kept at 218C in the dark) from both expeditions were transported back to the laboratory and stored (again at 218C in the dark) until subcultured. Twenty-one isolates were purified subsequently using an organic-rich liquid medium (Marine Broth 2216 from Difco laboratories; Zobell, 1946) at 218C via the dilution-extinction method. Isolates were screened for production of cold-active extracellular enzymes using the MUF substrate-based methods described above on unfractionated log-phase cultures grown at 218C. Strains producing significant amounts of EEA were further characterized for Gram stain, motility and oxidase reaction. The strain with the highest EEA was designated strain 34H.

cell-free extracts was confirmed by epifluorescence microscopy, using methods described in Porter and Feig (1980). At all times during the processing of samples, care was taken not to exceed a temperature of 48C. Thermal characteristics of EEA in the cell-free fractions were first estimated from enzyme assays at five temperatures between 21 to 378C; they were further resolved by assays at 15 temperatures within this range. Substrate analogues were added to the filtrate at a final saturating concentration of 100 mM and samples were incubated in the dark. For each temperature, independently incubated triplicates were sacrificed at each of the four time points over the course of the incubation, which never exceeded 8 h. Fluorescence was measured as described above. Effects of pH were determined at the optimal temperature by the addition of NaOH and HCl to achieve six incremental pH values between 5 and 10.

Analysis of EEA rates For both environmental samples and culture filtrates, rate of EEA was calculated at each temperature by determining the linear least-squares (LLS) best fit of the replicated fluorescence measurements versus time. Because homoscedasticity (homogeneous variability of Y with X) of the data cannot be assumed, weighted LLS best fit was also determined for the increase in fluorescence with time. Observed fluorescence was converted to enzyme activity (nM substrate analogue degraded min21) using externally prepared standards.

Determination of cell-free EEA from laboratory isolates

Thermal characterization of cell-free protease extract

Cell-free enzyme fractions from strain 34H were prepared from late log-phase batch cultures (grown in 2 litres of Marine Broth 2216 at 218C) by centrifugation at 3000 g for 20 min and subsequent gentle filtration (using a 0.2 mm cellulose filter, in which filtration pressure never exceeded 30 cm Hg). The filtration step was introduced after microscopic observation showed the presence of up to 105 cells ml21 supernate after centrifugation. Potential contamination by intracellular enzymes due to cell lysis was determined by assaying for glucose-6-phosphate-dehydrogenase mediated conversion of MTT (a tetrazolium salt) to Formazan in the cell-free extracts (Sigma no. M5655). The absence of bacteria in

Cell-free protease extracts from strain 34H were examined using SDS±PAGE prestained zymogram gels (Novex no. EC6415). To determine whether growth temperature affected synthesis of extracellular proteases, cell-free extracts were obtained from batch cultures (as described above) grown at the near in situ environmental temperature of 218C and at the strain's optimal growth temperature of 88C (data not shown). Total protein was quantified using the Bio-Rad Protein Assay and bovine gamma globulin as a standard. The extracts were run on zymogram gels at 48C, with protease activity visualized as zones of clearing in the gel after incubation (24±96 h). The effects of temperature on protease activity

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388 A. L. Huston, B. B. Krieger-Brockett and J. W. Deming were also examined by incubating gels, containing extracts from both growth temperatures, at 21, 4, 13 and 208C.

Acknowledgements This research was supported by the NOAA-WA State SeaGrant Program and NSF grant no. OPP9113960 to J.W.D. with additional support from a NSF Graduate Student Fellowship awarded to A.L.H. We thank T. Tucker and D. Meese for access to ice-core samples, L. Clough for boxcore samples, and T. Yager for sea-water samples during AWS 96. We also thank S. Carpenter for growth characterization of strain 34H and Y.-A. Vetter for enlightening discussion. The expertise and help of US Coast Guard officers and crew of the `Polar Sea' during NEWP 93 and AWS 96 were essential to our success.

References Adams, M.W.W., and Kelly, R.M. (1995) Enzymes from microorganisms in extreme environments. Chem Eng News 18: 32± 42. Baross, J.A., and Morita, R.Y. (1978) Microbial life at low temperatures: Ecological aspects. In Microbial Life in Extreme Environments. Kushner, D.J. (ed.). London: Academic Press, pp. 9±71. Brenchley, J.E. (1996) Psychrophilic microorganisms and their cold-active enzymes. J Ind Microbiol 17: 432±437. Chrost, R.J. (1991) Environmental control of the synthesis and activity of aquatic microbial ectoenzymes. In Microbial Enzymes in Aquatic Environments. Chrost, R.J. (ed.). New York: Springer-Verlag, pp. 84±95. Davail, S., Feller, G., Narinx, E., and Gerday, C. (1994) Cold adaptation of proteins: purification, characterization, and sequence of the heat-labile subtilisin from the Antarctic psychrophile Bacillus TA41. J Biol Chem 269: 17448±17453. Deming, J., and the Newater Steering Committee and Principle Investigators. (1993) Northeast water polynya: Polar Sea cruise results. Eos Trans AGU 74: 185, 195±196. Feller, G., Payan, F., Theys, F., Qian, M., Haser, R., and Gerday, C. (1994) Stability and structural analysis of alpha-amylase from the Antarctic psychrophile Altermonas haloplanctis A23. Eur J Biochem 222: 441±447. Gerday, C., Aittaleb, M., Arpigny, J.L., Baise, E., Chessa, J.-P., Garsoux, G., et al. (1997) Psychrophilic enzymes: a thermodynamic challenge. Biochim Biophys Acta 1342: 119±131. Helmke, E., and Weyland, H. (1991) Effect of temperature on extracellular enzymes occurring in permanently cold marine environments. Kieler Meeresforsch Sonderh 8: 198±204. Hoppe, H.-G. (1983) Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl-substrates. Mar Ecol Prog Ser 11: 299±308. Hoppe, H.-G. (1991) Microbial extracellular enzyme activity: a new key parameter in aquatic ecology. In Microbial Enzymes in Aquatic Environments. Chrost, R.J. (ed.). New York: SpringerVerlag, pp. 60±83. Hoshino, T., Ishizaki, K., Sakamoto, T., Kumeta, H., Yumoto, I.,

Matsuyama, H., et al. (1997) Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature. Lett Appl Microbiol 1997: 70±72. Margesin, R., and Schinner, F. (1994) Properties of cold-adapted microorganisms and their potential role in biotechnology. J Biotechnol 33: 1±14. Margesin, R., Palma, N., Knauseder, F., and Schinner, F. (1991) Proteases of psychrotrophic bacteria isolated from glaciers. J Basic Microbiol 31: 377±383. Mayer, L.M. (1989) Extracellular proteolytic activity in sediments of an intertidal mudflat. Limnol Oceanogr 34: 973±981. McDonald, I.J., Quadling, C., and Chambers, A.K. (1963) Proteolytic activity of some cold-tolerant bacteria from Arctic sediments. Can J Microbiol 9: 303±315. Meyer-Reil, L.A. (1991) Ecological aspects of enzyme activity in marine sediments. In Microbial Enzymes in Aquatic Environments. Chrost, R.J. (ed.). New York: Springer-Verlag, pp. 84± 95. Morita, R.Y. (1975) Psychrophilic bacteria. Bacteriol Rev 39: 144±167. Morita, Y., Nakamura, T., Hasan, Q., Murakami, Y., Yokoyama, K., and Tamiya, E. (1997) Cold-active enzymes from coldadapted bacteria. J Am Oil Chem Soc 74: 441±444. Mullis, K.B., and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzes chain reaction. Methods Enzymol 155: 335±350. Porter, K.G., and Feig, Y.S. (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr 23: 943±948. Reichardt, W. (1987) Differential temperature effects on the efficiency of carbon pathways in Antarctic marine benthos. Mar Ecol Prog Ser 40: 127±135. Reichardt, W. (1988) Impact of Antarctic benthic fauna on the enrichment of biopolymer degrading psychrophilic bacteria. Microb Ecol 15: 311±321. Rowe, G.T., Boland, G.S., Briones, E.G.E., Cruz-Kaegi, M.E., Newton, A., Piepenburg, D., et al. (1997) Sediment community biomass and respiration in the Northeast Water Polynya, Greenland: a numerical simulation of benthic lander and spade core data. J Mar Systems 10 (1): 497±515. Schinner, F., Margesin, R., and Pumpel, T. (1992) Extracellular protease-producing psychrotrophic bacteria from high alpine habitats. Arct Alp Res 24 (1): 88±92. Tucker, W.B., III, Gow, A.J., Meese, D.A., Bosworth, H.W., and Reimnitz, R. (1999) Physical characteristics of summer sea ice across the Arctic Ocean. J Geophys Res 104 (C1): 1489± 1504. Vetter, Y.-A., and Deming, J.W. (1994) Extracellular enzyme activity in the Arctic Northeast Water Polynya. Mar Ecol Prog Ser 114: 23±34. Vetter, Y.-A., Deming, J.W., Jumars, P.A., and Krieger-Brockett, B.B. (1998) A predictive model of bacterial foraging by means of freely released extracellular enzymes. Microb Ecol 36: 75± 92. Zobell, C.E. (1946) Marine Microbiology. Waltham, MA: Chronica Botanica.

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