Ann Microbiol (2011) 61:375–381 DOI 10.1007/s13213-010-0137-x SHORT COMMUNICATION 16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a hypersaline pond in Sichuan, China Jie Tang & Ai-ping Zheng & Eden S. P. Bromfield & Jun Zhu & Shuang-cheng Li & Shi-quan Wang & Qi-ming Deng & Ping Li Received: 12 April 2010 /Accepted: 27 September 2010 /Published online: 22 October 2010 # Springer-Verlag and the University of Milan 2010 Abstract One hundred and twenty bacterial isolates were Keywords Bacteria . Salt tolerance . Hypersaline . obtained from a hypersaline pond (c. 22% salinity) in Sichuan, China . 16S rRNA genes China. Bacteria were isolated from hypersaline water, sediment and soil samples using three culture media and an incubation temperature of 37°C. Of these isolates, 47 were Introduction selected and examined by phylogenetic analysis of 16S rRNA gene sequences and by tests of salt tolerance. The phyloge- Hypersaline environments are defined as those in which salt netic analysis placed the 47 bacterial isolates either in the concentrations exceed that of seawater (Grant 2004). They phylum Firmicutes or in the class Gammaproteobacteria and include artificial and naturally occurring solar salterns in arid showed that they were affiliated with the genera Salimi- areas such as hypersaline lakes and the Dead Sea, crobium, Halalkalibacillus, Virgibacillus, Alkalibacillus, underground brines originating from marine evaporite Marinococcus, Halobacillus, Halomonas, Idiomarina, Chro- deposits, hypersaline soils, and brines in deep ocean zones. mohalobacter and Halovibrio. All tested isolates were either Because water availability is limited by high salt concentra- halophilic or halotolerant and several were capable of growth tion, hypersaline environments are considered to be extreme in the presence of 30% (w/v) NaCl. environments for microbial life (Oren 2008). Despite this fact, diverse taxa of halophilic (i.e requiring salt for growth) : : : : : and halotolerant bacteria have been recovered from a wide J. Tang A.-p.: Zheng J. Zhu S.-c. Li S.-q. Wang Q.-m. Deng P. Li variety of hypersaline environments (Caton et al. 2004; Rice Research Institute, Sichuan Agricultural University, Grant 2004; Jiang et al. 2006; Tsiamis et al. 2008;Xianget Wenjiang, Sichuan, China 611130 al. 2008). Moreover, bacteria such as Salinibacter ruber : : : : : have been found inhabiting solar crystallization ponds where J. Tang A.-p.: Zheng J. Zhu S.-c. Li S.-q. Wang Q.-m. Deng P. Li hypersaline brines approach saturation, conditions once Key Laboratory of Southwest Crop Gene Resource considered suitable only for the Archaea (Antón et al. & Genetic Improvement of Ministry of Education, 2000, 2002; Litchfield et al. 2009; Tsiamis et al. 2008). Sichuan Agricultural University, Ya’an, Sichuan, China 625014 The isolation and characterization of halotolerant and : halophilic bacteria from hypersaline environments is of J. Tang E. S. P. Bromfield practical importance because these bacteria have biotech- Agriculture and Agri-Food Canada, nological potential with regard to the production of useful 960 Carling Ave, Ottawa, Ontario, Canada K1A 0C6 biomolecules, such as osmolytes (compatible solutes), hydrolytic enzymes and exopolysaccharides (Margesin P. Li (*) and Schinner 2001). Rice Research Institute, Hypersaline subterranean aquifers in the Sichuan basin Dongbei Road No. 555, Liucheng Town, Wenjiang 611130 Sichuan, People’s Republic of China of China are located at depths of between 50 and 3,000 m e-mail: [email protected] and occur within sedimentary rocks ranging in age from the 376 Ann Microbiol (2011) 61:375–381 Sinian to the Cretaceous and especially the Triassic (Zhou Isolation of bacteria and tests of salt tolerance et al. 1997). These hypersaline brines are of marine origin (thalassohaline) and have been exploited by man for more Bacterial isolation was performed using SP medium (Caton et al. than 2,000 years by deep-well drilling and harvesting the 2004), Ma medium (Maturrano et al. 2006) and CM medium salt by boiling (Kuhn 2004). The hypersaline environment (Li et al. 2002), at four levels of NaCl (w/v): 10, 22, 30 and investigated in this work is located in the Sichuan Basin 35%. The final pH of each medium was adjusted to ∼7.2. and is a reservoir-storage pond of brines (c. 22% salinity) To isolate bacteria from hypersaline water, replicate used in the commercial extraction of salts and other aliquots of dilutions were surface plated (0.1 ml/plate) onto chemicals. The pond is maintained year round with warm the three media containing agar (15 g/l). Bacteria were (c. 40°C) geothermal brines pumped from a deep well isolated from sediment and soil samples by adding 1 g of (depth >1,000 m). each sample to 300 ml liquid SP, Ma and CM medium (each at The aim of this work was to isolate and characterize four levels of salt) in Erlenmeyer flasks, incubating for 3 days bacteria from the hypersaline pond ecosystem with a view on an orbital shaker, and then plating dilutions (0.1 ml) onto to screening for halotolerant and halophilic types. Bacterial the respective agar media. Plates were incubated for 10– isolates were obtained by plating on three agar media and 14 days and individual bacterial colonies selected on the basis by liquid enrichment. Characterization was achieved by of differences in morphology and Gram-staining reaction. analysis of 16S rRNA gene sequences. Single colonies were streaked on agar media and repicked. Incubation of plates and liquid media were done at 37°C, because this temperature is widely used for the isolation of Materials and methods diverse bacteria from hypersaline environments (e.g., Matur- rano et al. 2006; Wen et al. 2009;Wuetal.2006)and Site description and sample collection because warm (about 40°C) subterranean brines are fed to the hypersaline pond. Bacterial isolates were subjected to The hypersaline pond (30°36′04.56″N, 105°15′22.79″E, alti- Gram-staining using a Gram-staining kit (Fisher Diagnostics, tude 326 m) has a maximum depth of 2.5 m and a surface area USA), according to the manufacturer’s instructions. Purified of c. 1,200 m2. The pond is protected from rain by means of a cultures were maintained at −80°C in 50% (w/v) glycerol. plastic roof and is maintained year round by being supplied Salt tolerance for growth was tested by streaking fresh with geothermal brines from a subterranean aquifer. The bacterial cultures in duplicate on SP agar medium containing warm brines (temperature c. 40°C) derive from a deep well 0, 1, 5, 10, 15, 20, 25, 30 and 40% (w/v) NaCl. Growth optima and are fed into the hypersaline pond to maintain levels. The were assessed as described by Caton et al. (2004) and plates climate at this location is of the humid subtropical monsoon were incubated for 10 days at 37°C. type with average annual rainfall exceeding 900 mm. All water, soil and sediment sampling was carried out Genomic DNA extraction, PCR amplification of 16S rRNA during May 2008. Air temperature at the time of sampling genes and nucleotide sequencing varied between 25 and 35°C, while water temperature (0.5 m depth) varied between 25 and 30°C. Bacterial genomic DNA was extracted and purified using a Sixteen soil samples were randomly collected to 15 cm Takara MiniBEST Bacterial Genomic DNA Extraction kit depth from unvegetated areas within a 0.5-m border at the Ver.2.0 (China) according to the manufacturer’s instruc- edge of the pond. Water samples (500 ml) were collected at tions. Purified genomic DNAs were subjected to electro- 0.5, 1 and 1.5 m depth at each of 12 randomly selected phoresis on 1% agarose gels, followed by ethidium bromide locations, and 19 randomly selected samples of sediment were staining and visualization under UV light. 16S rRNA genes collected from the bottom of the pond. Each of the soil, were amplified from bacterial genomic DNA using bacte- sediment and water samples was pooled so as to provide rial universal primers (forward primer: 5′-AGAGTTT respective composite samples. Sampling was done with GATCCTGGCTCAG-3′, reverse primer: 5′-ACGG aseptic precautions. CAACCTTGTTACGACT-3′) (Edwards et al. 1989). The Samples were transported to the laboratory and analyses forward primer corresponds to positions 8–27 and the performed immediately or within 3 days. Portions of the reverse primer to positions 1,493–1,512 of the 16S rRNA composite water sample were sent to a commercial gene sequence of E. coli (Accession no. U00096). PCR was laboratory (Sichuan University, China) for chemical analy- performed using a thermal cycler (Thermo Scientific, sis. Cations were determined by Inductively Coupled Model Px2) with a 50-μl reaction containing 1.5 mM Plasma Mass Spectrometry (model: OptiMass 9500; Aus- MgCl2, 10 mM Tris-HCl (pH 9.0), 10 μM of each dNTP, tralia) and anions determined by Ion Chromatography 0.1 μMofeachprimer,and1UofEXTaqDNA (model: ICS 3000; USA). polymerase (Takara, Japan). PCR cycling conditions con- Ann Microbiol (2011) 61:375–381 377 sisted of denaturation at 95°C for 2 min, 35 cycles of 95°C by trace amounts of Br− (0.03±0.02) and Mn2+ (0.013±0.009); for 1 min, 53°C for 1 min, and 72°C for 1 min 30 s, and pH 7.5±0.3. final extension at 72°C for 10 min. A total of 120 isolates were obtained from hypersaline Amplified PCR products were separated by 1% agarose water, sediment and soil samples. Forty-seven of these gel electrophoresis. DNA fragments of the correct size (c. isolates were selected on the basis of differences in colony 1,500 bases) were excised from the gel and purified using a morphology and Gram-staining reaction for further analysis TIANgel Midi purification Kit (TIANGEN Inc., China) by 16S rRNA sequencing.