Ann Microbiol (2017) 67:203–214 DOI 10.1007/s13213-016-1252-0 ORIGINAL ARTICLE A glimpse of the diversity of complex polysaccharide-degrading culturable bacteria from Kongsfjorden, Arctic Ocean Anand Jain1 & Kottekkatu Padinchati Krishnan1 Received: 8 September 2016 /Accepted: 22 December 2016 /Published online: 12 January 2017 # Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract Cold-adapted, complex polysaccharide-degrading within the bacterial genera indicates a role in regulating carbon/ marine bacteria have important implications in biogeochemical carbohydrate turnover in the Kongsfjorden, especially by reduc- processes and biotechnological applications. Bacteria capable of ing recalcitrance. degrading complex polysaccharide substrates, mainly starch, have been isolated from various cold environments, such as sea Keywords Bacteria . Diversity . Culturable . ice, glaciers, subglacial lakes, and marine sediments. However, Polysaccharides . Cold active enzymes . Kongsfjorden the total diversity of polysaccharide-degrading culturable bacteria in Kongsfjorden, Arctic Ocean, remains unexplored. In the study reported here, we tested 215 cold-adapted heterotrophic bacterial Introduction cultures (incubated at 4 and 20 °C, respectively) isolated from Kongsfjorden, for the production of cold-active extracellular Kongsfjorden is an open glacial fjord located on the west coast polysaccharide-degrading enzymes, including amylase, of Spitsbergen, an island in the Svalbard archipelago, situated pectinase, alginase, xylanase, and carboxymethyl (CM)-cellu- in the Arctic Ocean between mainland Norway and the North lase. Our results show that 52 and 41% of the bacterial isolates Pole (79°N, 12°E). It has been identified as a key European tested positive for extracellular enzyme activities at 4 and 20 °C, site for Arctic biodiversity monitoring (Hop et al. 2002)andis respectively. A large fraction of the bacterial isolates (37% of the also suitable for exploring the impacts of possible climate positive isolates) showed multiple extracellular enzyme activi- changes (Włodarska-Kowalczuk and Węsławski 2001;Hop ties. Alginase and pectinase were the most predominantly active et al. 2002). Earlier studies in this fjord have focused on the enzymes, followed by amylase, xylanase, and CM-cellulase. All physical characteristics of the environment (Svendsen et al. isolates which tested positive for extracellular enzyme activities 2002), bacterial abundance and biomass (Jankowska et al. were affiliated to microbial class Gammaproteobacteria.The 2005), phytoplankton and zooplankton diversity (Hop et al. four genera with the highest number of isolates were 2002), benthic microbial community (Srinivas et al. 2009), Pseudomonas, followed by Psychrobacter, and marcobenthos (Wlodarska-Kowalczuk and Pearson Pseudoalteromonas,andShewanella. The prevalence of com- 2004). However, in the recent past there have been reports plex polysaccharide-degrading enzymes among the isolates indi- on culturable (Srinivas et al. 2009;Choidashetal.2012; cates the availability of complex polysaccharide substrates in the Prasad et al. 2014) and non-culturable (Zeng et al. 2009; Kongsfjorden, likely as a result of glacial melting and/or Piquet et al. 2010) bacterial diversity of the Kongsfjorden. macroalgal load. In addition, the observed high functional/ These studies revealed that exceptionally diverse bacterial phenotypic diversity in terms of extracellular enzyme activities communities thrive in the perennially cold (<5 °C) water col- umn of the Kongsfjorden. To date, cultivation-based studies have focused primarily on the bio-prospecting potential of the * Anand Jain Kongsfjorden bacterial community with respect to the produc- [email protected] tion of various cold-active enzymes, mostly amylases, lipases, and proteases (Srinivas et al. 2009; Chattopadhyay et al. 2013; 1 Cryobiology Laboratory, National Centre for Antarctic and Ocean Prasad et al. 2014). Despite these findings, however, little is Research, Headland Sada, Vasco da Gama, Goa 403804, India known of the ability of the culturable Kongsfjorden bacterial 204 Ann Microbiol (2017) 67:203–214 community to produce extracellular cold-active polysaccha- addition, low temperature decreases the rate of chemical reac- ride-degrading enzymes other than amylases. tions and increases protein compactness, thereby affecting en- Kongsfjorden receives 5–10% of its total organic carbon of zyme catalysis (Rasmussen et al. 1992). To overcome these terrestrial origin from glacier melting, and about 90–95% of challenges, cold-active enzymes have evolved high specific the organic carbon in the Kongsfjorden is contributed by pri- activity and a lower optimum temperature for activity (Feller mary production (Kuliński et al. 2014). However, Buchholz et al. 1996). Thus, the detection of cold-adapted bacteria with and Wiencke (2016) reported that macroalgae, in particular cold-active polysaccharide-degrading enzymes is important kelps, also contribute significantly to the pool of organic mat- for elucidating the mechanism of organic matter transforma- ter in the Kongsfjorden. It is important to note that high- tion in cold marine environments as well as to fulfill the needs molecular-weight carbohydrates or polysaccharides constitute of various industries (Georlette et al. 2004; Kumar et al. 2011). a large proportion of the particulate organic matter (POM) and The aim of the study reported here was, therefore, to explore dissolved organic matter (DOM) in the ocean (Benner et al. both the phylogenetic and functional/phenotypic (in terms of 1992; lee et al. 2004). Marine microbial communities possess enzyme activity) diversity among polysaccharide-degrading extracellular enzymes for hydrolyzing these high-molecular- cold-adapted culturable bacteria isolated from the weight substrates to yield sufficiently smaller substrates Kongsfjorden in order to decipher their possible role in organ- (<600 Da) for microbial uptake and to make them available ic matter transformation. for higher trophic levels (Azam et al. 1994;Arnosti2011). Therefore, there is a strong possibility that complex polysaccharide-degrading cold-adapted bacteria will be pres- Material and methods ent in the waters of Kongsfjorden. The ability to produce cold-active enzymes is important for Study area and sampling the growth and survival of microorganisms living in the per- manently cold marine environment. Bacterial activity at low Six different stations located in the Kongsfjorden were sam- temperature requires higher levels of DOM compared to pled, starting at the mouth of Kongsbreen and Kronebreen warmer environments (Pomeroy and Wiebe 2001). In glaciers and continuing to the open ocean waters (Fig. 1). A Fig. 1 Map of Kongsfjorden showing locations of the sampling stations (red stars 1–6) Ann Microbiol (2017) 67:203–214 205 Conductivity Temperature Depth (CTD) instrument was de- inoculation of the bacterial isolates, the plates were incubated at ployed at each station to determine depthwise variations in 4 °C and 20 °C, respectively, for 7 days. The zone of clearance temperature, salinity, and fluorescence. Depthwise distribu- around bacterial colonies due to extracellular polysaccharide- tion of bacteria in Kongsfjorden is affected by the hydrologi- degrading enzyme activities was observed by flooding the plates cal stratification and/or phytoplankton distribution. Moreover, with appropriate reagents. Xylan- and CMC-containing plates bacterioplankton and phytoplankton generally tend to accu- were flooded with 1% Congo Red for 30 min followed by rinsing mulate in the layers around the pycnocline in the with 1.5 M NaCl (Khandeparker et al. 2011). Gram’s iodine Kongsfjorden (Jankowska et al. 2005). Therefore, in order to solution was used for detecting bacterial isolates which could increase the probability of encountering diverse hydrolyze starch, pectin (Sunnotel and Nigam 2002), and algi- polysaccharide-degrading bacteria we collected 5-l water sam- nate (Sawant et al. 2015). ples from the deep chlorophyll maxima (DCM) depths using Niskin bottles in June and July 2015. Immediately after col- Genomic DNA extraction and PCR assay lection, the water samples were filtered through a 0.22-μm pore-size polycarbonate membrane (Merck Millipore, Genomic DNA from bacterial cultures which tested positive for Billerica, MA). enzyme activity was extracted using the Purelink Genomic DNA Extraction kit (Invitrogen, Carlsbad, CA) following the manufac- Isolation of heterotrophic bacteria turer’s protocol. The extracted DNA was stored in Purelink elu- tion buffer at −20 °C until analyzed. The 16S rRNA gene from The bacterial population retained on the 0.22-μm pore-size the genomic DNA was amplified using universal bacterial 16S polycarbonate membrane was cultivated. Specifically, imme- primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′)and diately after filtration of a water sample, the filter paper was 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Lane 1991). immersed in a flask containing 25 ml of Zobell Marine Broth PCR assays were carried out in 50-μl reaction mixtures contain- (ZMB; Hi-Media, Mumbai, India) and incubated at 4 °C until ing 20 pmol of each primer, 1.5 mM MgCl2,200μMdNTPs, visible bacterial growth, identified as increased turbidity, was and 2.5 U Taq DNA polymerase. Amplifications were carried recorded. At the end of the incubation period, 500 μlofthe out in a thermocycler (model CFX 96; BioRad, Hercules, CA) medium was preserved and transported back to the