Polar Biol (2012) 35:543-554 DOI 10.1007/s00300-011-1100-4 ORIGINAL PAPER Heterotrophic bacterial diversity in aquatic microbial mat communities from Antarctica Karolien Peeters • Elie Verleyen • Dominic A. Hodgson • Peter Convey • Damien Ertz • Wim Vy ver man • Anne Willems Received: 16 June 2011/Revised: 25 August 2011/Accepted: 26 August 2011/Published online: 11 September 2011 © Springer-Verlag 2011 Abstract Heterotrophic bacteria isolated from five aquatic order to explore the variance in the diversity of the samples at microbial mat samples from different locations in conti­ genus level. Habitat type (terrestrial vs. aquatic) and specific nental Antarctica and the Antarctic Peninsula were com­ conductivity in the lacustrine systems significantly pared to assess their biodiversity. A total of 2,225 isolates explained the variation in bacterial community structure. obtained on different media and at different temperatures Comparison of the phylotypes with sequences from public were included. After an initial grouping by whole-genome databases showed that a considerable proportion (36.9%) is fingerprinting, partial 16S rRNA gene sequence analysis was currently known only from Antarctica. This suggests that in used for further identification. These results were compared Antarctica, both cosmopolitan taxa and taxa with limited with previously published data obtained with the same dispersal and a history of long-term isolated evolution occur. methodology from terrestrial and aquatic microbial mat samples from two additional Antarctic regions. The phylo- Keywords Microbial diversity • Cultivation • types recovered in all these samples belonged to five 16S rRNA gene sequencing • ASPA • PCA major phyla.Actinobacteria, Bacteroidetes, Proteobacteria, Firmicutes and Deinococcus-Thermus, and included several potentially new taxa. Ordination analyses were performed in Introduction Microbial mats and surface crusts that may develop in wet Electronic supplementary materialThe online version of this article (doi:10.1007/s00300-011-1100-4) contains supplementary Antarctic habitats (Vincent 2000; Layboum-Parry and Pearce material, which is available to authorized users. 2007) are dense communities of vertically stratified micro­ organisms and are believed to be responsible for much of the K. Peeters • A. Willems (H ) primary production under the extreme polar conditions. The Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Science, Ghent University, mats and crusts typically consist of mucilage, in which cya­ K. L. Ledeganckstraat 35, 9000 Ghent, Belgium nobacteria and other algal cells are embedded, together with e-mail: [email protected] other heterotrophic and chemoautotrophic microorganisms, sand grains and other inorganic materials (Femández- E. Verleyen • W. Vyverman Protistology and Aquatic Ecology, Department of Biology, Valiente et al. 2007). Particularly, the lacustrine ecosystems, Faculty of Science, Ghent University, which range from relatively deep freshwater and hypersaline Krijgslaan 281, S8, 9000 Ghent, Belgium lakes, to small ponds and seepage areas (Verleyen et al. 2011) act as true biodiversity and primary production hotspots in a D. A. Hodgson • P. Convey British Antarctic Survey, Natural Environment matrix of polar desert and ice. Research Council, High Cross, Madingley Road, In recent years, Antarctic microbial mats have attracted Cambridge CB3 0ET, UK a lot of scientific interest, with the photoautotrophic taxa such as cyanobacteria (Taton et al. 2006), green algae D. Ertz Department Bryophytes-Thallophytes, National Botanic (De Wever et al. 2009) and diatoms (Sabbe et al. 2003) Garden of Belgium, 1860 Meise, Belgium probably being the best-studied groups. Water depth (and Ô Springer 544 Polar Biol (2012) 35:543-554 hence light climate), liquid water availability and conduc­ features expected of the organisms obtained based on their tivity or related parameters are the most important vari­ phylogenetic position. ables in structuring these communities (Hodgson et al. The aims of this study were (1) to contribute to a better 2004; Verleyen et al. 2010). Surprisingly, only a small understanding of the diversity of heterotrophic bacteria in number of studies have focussed on the heterotrophic microbial mat communities from a range of terrestrial and bacterial diversity in these microbial mats (Brambilla et al. aquatic habitats in coastal and inland ice-free regions in 2001; Van Trappen et al. 2002). Other land-based habitats Continental and Maritime Antarctica and (2) to explore the in Antarctica that have been studied for their heterotrophic relationship between the bacterial communities and a set of bacterial diversity include soils in dry valleys (Aislabie environmental parameters. We applied a cultivation-based et al. 2006b) and maritime Antarctica (Chong et al. 2010), approach using several media and growth conditions to the plankton in freshwater lakes (Pearce. 2005) and anoxic access heterotrophic bacteria. A large number of isolates waters in meromictic lakes (Franzmann et al. 1991). The was obtained and identified through genotypic character­ few studies focussing on the heterotrophic bacterial ization using rep-PCR fingerprinting and phylogenetic diversity in aquatic microbial mats comprised samples analysis of the 16S rRNA gene sequences. Comparison of from lakes in the McMurdo Dry Valleys, the Vestfold Hills the sequences with those available in public databases and the Larsemann Hills and included culture-dependent as allowed identification of the bacteria and an assessment of well as independent approaches. They reported a large their geographical distribution. diversity with an important number of previously unknown taxa (Brambilla et al. 2001; Van Trappen et al. 2002). As a result, several new species have been described in the Experimental procedures phyla Bacteroidetes, Proteobacteria, Actinobacteria and Firmicutes (Reddy et al. 2002a, b, 2003a, b; Van Trappen Source of samples et al. 2003, 2004a, b, c, d; Shivaji et al. 2005). The rela­ tionship between the bacterial diversity of microbial mats Five samples (PQl, LA3, SK5, WO10 and S06) from and environmental parameters has not yet been studied lacustrine habitats in different locations in Continental although Brambilla et al. (2001) suggested some general Antarctica and the Antarctic Peninsula (Fig. 1) were S06, Schirmacher Oasis BB11S, 498 isolates LA3, Utsteinen 79 phylotypes Syowa Oasis 331 isolates 253 isolates 37 phylotypes 39 phylotypes BB50, WO 10, Utsteinen Syowa Oasis 46555 isolates 1 498 isolates 63 phylotypes 48 phylotypes SK5, Syowa Oasis Pourquoi-Pas Island 550 isolates 426 isolates 59 phylotypes 89 phylotypes ■ Actinobacteria ■ Alphaproteobacteria TM4, ■ Betaproteobacteria Shackleton Range m Gammaproteobacteria 421 isolates 34 phylotypes ■ Bacteroidetes Pensacola Mountains ■ Deinococcus-Thermus 364 isolates 56 phylotypes u Firmicutes Fig. 1 Division of the phylotypes over the different phylogenetic groups. The number of obtained isolates and phylotypes are mentioned for the different samples. Information for samples BB50, BB115, TM2 and TM4 was based on Peeters et al. 2011a, b Ô Springer Polar Biol (2012) 35:543-554 545 analysed (Table 1). All samples were kept frozen contin­ All plates were incubated for several weeks dining which uously after collection (in January 2003 [PQ1] and January the number of colony forming units (CFUs) was counted. 2007 [LA3, SK5, WO10 and S06]) until processing in the When the number of CFUs had stabilized, the total number of laboratory. Specific conductivity and pH were measured in CFU/g for each combination of culture conditions was cal­ the field using a YSI 600 m. Details regarding the analysis culated for the plates showing between 20 and 400 colonies. of the concentration of the major ions and nutrients have At the end of the incubation period, three colonies (or less in been described by Hodgson et al. (2010) and Verleyen case of insufficient growth) of each morphological type et al. (2011). (colony parameters used include colour, margin, elevation, Data for the new samples were also compared with shape, diameter, surface appearance) were isolated and puri­ information on four further samples previously studied fied. Pure cultures were cryopreserved at —80°C using broth using the same methods, including two terrestrial mat medium plus 15% glycerol or the MicroBank™ system (Pro- samples from Utsteinen (Sor Rondane Mountains, East Lab Diagnostics, Ontario, Canada). Antarctica) (Peeters et al. 2011a) and two microbial mat samples from lakes in the Pensacola Mountains and the Genotypic fingerprinting Shackleton Range (Peeters et al. 2011b). To reduce the large number of isolates, duplicates were Enumeration and isolation of heterotrophic bacteria eliminated using a whole-genome fingerprinting technique, repetitive element palindromic (rep)-PCR, resulting in a One gram of sample was aseptically weighed and smaller number of clusters and unique isolates. DNA prepa­ homogenized in 9 ml sterile cold (4°C) physiological sal­ ration was carried out as described by Baele et al. (2003). Rep- ine (0.86% NaCl) using a vortex. Tenfold dilution series PCR fingerprinting using the GTG5 primer (5'-GTG GTG (kept at 4°C) were plated on four different media (Marine GTG GTG GTG-3') was performed according to Gevers et al. agar 2216 (MA) (BD Difeo™), R2A (BD Difco™), ten (2001). Resulting fingerprints were processed using the Bio- times diluted R2A (R2A/10), and PYGV (peptone-yeast-