Folia Microbiol DOI 10.1007/s12223-013-0295-x
Isolation and characterization of cellulolytic bacteria from the Stain house Lake, Antarctica Itamar S. Melo & Tiago D. Zucchi & Rafael E. Silva & Elke S. D. Vilela & Mirian Lobo Sáber & Luiz H. Rosa & Vivian H. Pellizari
Received: 11 April 2013 / Accepted: 11 December 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014
Abstract The main aim was to evaluate the occurrence of cellulolytic bacteria from the Stain house Lake, located at Admiralty Bay, Antarctica. Thick cotton string served as a cellulose bait for the isolation of bacteria. A total of 52 bacterial isolates were recovered and tested for their cellulase activity, and two of them, isolates CMAA 1184 and CMAA 1185, showed significant cellulolytic activity on carboxymethylcellulose agar plates. Phylogenetic analysis placed the isolates into the Bacillus 16S ribosomal RNA gene subclade. Both isolates produced a cold-active cellulase which may play a crucial role in this extreme environment. Keywords Antarctic Peninsula . Cellulase . Bacillus Organisms capable to survive on extreme environments have often evolved unique biosynthetic pathways to overcome all the adversities (D’Amico et al. 2006). Thus, despite the harsh environmental conditions throughout the year, lakes of Antarctica can be colonized by microbial communities which survive even during winter (Henshaw and Laybourn-Parry
Electronic supplementary material The online version of this article (doi:10.1007/s12223-013-0295-x) contains supplementary material, which is available to authorized users. I. S. Melo (*) : T. D. Zucchi : R. E. Silva : E. S. D. Vilela : M. L. Sáber Embrapa Environment, CP 69, 13820-000 Jaguariúna, São Paulo, Brazil e-mail:
[email protected] L. H. Rosa Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil V. H. Pellizari Oceanographic Institute, University of São Paulo, São Paulo, São Paulo, Brazil
2002). In these environments, enzymes responsible for carbon cycling are of particular importance in the maintenance of communities and ecosystems. Microorganisms can attack amorphous cellulose to some degree, but relatively few taxa can completely degrade highly crystalline cellulose, i.e., cotton. Nevertheless, the contribution of different groups of microorganisms to cellulose degradation in aquatic ecosystems remains obscure. Also, the bacterial ability to utilize dissolved organic compounds at very low concentrations is important in oligotrophic lakes which have low concentration of available organic compounds (Kuznetsov et al. 1979; Ormerod 1983). Thus, the main aim was to access and evaluate cellulolytic bacteria from the Stain house Lake at Antarctica. Sterilized cellulose baits (thick cotton strings) were placed at different depths, and in equidistant points of the Stain house Lake, in Admiralty Bay, King George Island (62°04′221″S, 62°22′228″W) in midsummer (Dec. 2008–Jan. 2009). The baits provided matrices for colonizing biomass and source material for isolation of polysaccharide-degrading bacteria. The baits were recovered after 16 days and transferred into Falcon tubes containing freshwater of the same lake. Bacteria were isolated by vigorously vortexing the cotton baits in 0.85 % saline solution to remove the biofilm material. The suspension was serially diluted (up to 10−5), inoculated on tryptic soy agar plates (TSA; 100 μL from each dilution), and incubated at 18 °C for 7 days. Individual colonies were purified and stored in 20 % glycerol at −80 °C. Bacteria recovered from the cellulose baits were plated in TSA containing 1 % (w/v) carboxymethylcellulose (CMC) and incubated at 18 °C for 3–7 days. After this period, the agar medium was flooded with an aqueous solution of Congo red (0.1 %w/v) for 30 min, followed by washing with 1 mol/L NaCl for 30 min. Clear zones on a reddish background were scored as positive for cellulase production. This experiment was performed in triplicate.
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Genomic DNA was extracted from the positive cellulolytic isolates using PureLink™ Genomic DNA kit (Invitrogen) following the manufacturer’s protocol. PCR amplification and 16S ribosomal RNA (rRNA) gene sequencing were achieved following the methods of Zucchi et al. (2011). The almost complete 16S rRNA gene sequences (1,417–1,422 nucleotides [nt]) were aligned manually using MEGA version 5 software (Tamura et al. 2011) against corresponding sequences of closely related type strains of Bacillus species retrieved from the GenBank database using the EzTaxon-e server (Kim et al. 2011). Phylogenetic trees and bootstrap analysis were inferred following the procedures described by Zucchi et al. (2012). The root position of the neighbor-joining
tree was inferred by using Salirhabdus euzebyi CVS14T (GenBank accession no. AM292417). The taxonomic position from the positive cellulolytic bacteria was further evaluated by fatty acid methyl ester (FAME) analysis using the extraction method described by Miller and Berger (1985). FAME was analyzed using an Agilent GC equipped with a 7683 Series injector, flame ionization detector, and HP-5 capillary column (30 m×0.32 mm). Peaks were identified with the Sherlock Microbial Identification System (MIDI Inc., Newark, DE, USA) and the TSB version 6 database (Sasser 1990). Cellulase production was stimulated by inoculating the selected isolates in conical flasks containing 50 mL tryptic
Fig. 1 Neighbor-joining tree based on nearly complete 16S rRNA gene sequences (~1,200 bp) showing relationships between isolates CMAA 1184 and CMAA 1185, and between them and the type strains of phylogenetically close Bacillus species. White circles indicate branches of the tree that were also recovered with the maximum likelihood and maximum parsimony tree-making algorithms; black and white diamonds
stand for branches which were recovered by the maximum parsimony or by the maximum likelihood tree-making algorithms, respectively. Numbers at the nodes are percentage bootstrap values based on a neighbor-joining analysis of 1,000 resampled datasets; only values above 50 % are given. Bar, 0.005 substitutions per nucleotide position
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soy broth amended with wheat bran (1 %w/v). The cultures were incubated at 20 °C for 24 h. Each flask was considered as one replicate and the experiment was conducted in triplicate. The enzyme produced was sampled by taking 5 mL from the culture flask for each replicate. The supernatant containing the cellulase was separated from the cellular fraction after centrifugation (15,000g, 15 min, 4 °C) and filtration (0.2 μm) and later used to test for cellulase activity. Total cellulase was measured following the procedures in accordance to Ruegger and Tauk-Tornisielo (2004), using a paper filter (6 cm2) as substrate in 1 mL sodium acetate buffer (pH 5.0) and 500 μL enzyme extract and incubated at 20 °C for 30 min. The reaction was interrupted with the addition of 3,5dinitrosalicylic acid (DNS) and heated at 100 °C for 5 min prior to quantification by spectrophotometry at 540 nm. The activity was calculated using a standard curve of glucose as a reference. One unity of enzyme activity (U) was defined as 1 μmol/min glucose per milliliter. The pH of the cellulases produced by isolates CMAA 1184 and CMAA 1185 was further evaluated in 50 mM sodium acetate buffer (pH 3–10) using the same conditions previously described. Cellulase activity was also assayed using dextrin, microcrystalline cellulose, and xylan as substrates. From 52 bacterial isolates recovered from the cellulose baits and screened for cellulase activity, only two strains, CMAA 1184 and CMAA 1185 (GenBank accession numbers KF425536 and KF425537, respectively), produced clear zones (>2.5-cm diameter) of hydrolysis on CMC. The classification of isolates CMAA 1184 and CMAA 1185 in the genus Bacillus was confirmed by their assignment to the 16S rRNA Bacillus gene tree (Fig. 1). Isolate CMAA 1184 (GenBank accession number KF425536) formed a distinct phyletic line that is associated with the Bacillus subtilis subsp. subtilis 16S rRNA subclade in the neighbor-joining analysis and supported by a bootstrap value of 86 %; its closest neighbor is the type strain of B. subtilis subsp. inaquosorum. These two strains shared a 16S rRNA gene similarity of 99.4 %, a value that corresponded to 7-nt differences at 1,161 sites. The relationship of isolate CMAA 1185 (GenBank accession number KF425537) and representatives of the Bacillus pumilus 16S rRNA gene subclade was also recovered by maximum parsimony analysis and supported by a high bootstrap value (98 %). The isolate is most closely related to the type strain of Bacillus safensis; these organisms shared a 16S rRNA similarity of 98.9 %, a value equivalent to 15-nt differences at 1,417 locations. Fatty acid profiles (“supplementary material”) were also in line with the taxonomic placement of the isolates into the Bacillus genus (Roberts et al. 1994). The existence of Bacillus species in Antarctica has been reported by various authors, and although several genera of cellulolytic bacteria were also isolated from Antarctic soils and ice core, the isolation methods applied so far have been
unable to access the culturable community of cellulolytic Bacillus (Antony et al. 2012; Soares et al. 2012). It also indicates the need for other isolation methods, for instance, the use of cotton baits. The few cellulolytic isolates obtained from the Stain house Lake greatly underrepresent the abundance and diversity of cold-adapted bacteria involved in cellulose degradation. Since some studies have isolated bacteria using similar procedures (cotton baits) in cold lake conditions (de Menezes et al. 2008), it should be considered that the incubation period of 16 days was too short to permit a massive bacterial colonization. The two putative cellulase producer isolates were assayed for production of cellulase in liquid medium. Both isolates yield cellulases active in low temperatures (below 20 °C) and at 30 °C; their activities decrease to only ~30 % of those observed at 20 °C (Supplementary Figure 1). The cellulases produced by isolates CMAA 1184 and CMAA 1185 presented a lower optimum temperature than Fibrobacter succinogenes (24 °C; Iyo and Forsberg 1999). Furthermore, isolate CMAA 1184 produced alkaline cellulase (optimum pH 10) which was inactive on the other tested substrate whereas cellulase produced by CMAA 1185 demonstrated a higher activity at pH 3 and activity on all tested substrate (Supplementary Figure 2). Finally, the relatively high levels of cellulase produced (>5 U) along with their psychrotolerant and pH features justify further studies to evaluate the extent of cellulose breakdown by microorganisms in Antarctic lakes and also their potential role as degraders of organic matter in extreme freshwater environment. Acknowledgments This study was made possible with the financial and logistic support of PROANTAR and Marine of Brazil. This is part of the IPY activity 403 “MIDIAPI Microbial Diversity of Terrestrial and Maritime Ecosystems in Antarctic Peninsula.” TDZ is also grateful to FAPESP for providing the funding for developing this research (grants 11/50243-1 and 11/14333-6).
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