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16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a hypersaline pond in Sichuan, China

16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a... 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;Xiang et 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, The isolation and characterization of halotolerant and Ya’an, Sichuan, China 625014 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, biomolecules, such as osmolytes (compatible solutes), Ottawa, Ontario, Canada K1A 0C6 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, of China are located at depths of between 50 and 3,000 m Wenjiang 611130 Sichuan, People’s Republic of China and occur within sedimentary rocks ranging in age from the e-mail: liping6575@163.com 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;Wuet al. 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 m . 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- MgCl , 10 mM Tris-HCl (pH 9.0), 10 μM of each dNTP, tralia) and anions determined by Ion Chromatography 0.1 μMofeachprimer, and1Uof EX TaqDNA (model: ICS 3000; USA). polymerase (Takara, Japan). PCR cycling conditions con- Ann Microbiol (2011) 61:375–381 377 − 2+ sisted of denaturation at 95°C for 2 min, 35 cycles of 95°C by trace amounts of Br (0.03±0.02) and Mn (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. The range of different colony according to the manufacturer’s protocol. PCR products morphologies of bacterial isolates recovered from each of were verified by agarose gel electrophoresis. the hypersaline water, sediment and soil samples were For 16S rRNA gene sequencing, purified PCR products generally similar. Although both Gram-positive and Gram- were ligated into vector pMD18 (Takara Inc., Japan), and negative bacterial isolates were obtained from all samples, transformed into Escherichia coli. Clones were screened by the majority (60%) were Gram-positive. PCR for inserts of the correct size (c. 1,500 bases) using The phylogenetic tree reconstructed from full length 16S universal primers (Edwards et al. 1989). Inserts were rRNA gene sequences of the 47 bacterial isolates recovered commercially sequenced (Invitrogen, China) to provide from hypersaline water, sediment and soil samples is shown full-length 16S rRNA gene sequences. in Fig. 1. Data for the source of isolation, NaCl tolerance and closest phylogenetic relative (type strains) of these Phylogenetic analysis bacterial isolates are given in Table 1. Analysis of 16S rRNA gene sequences divided the bacterial isolates into the 16S rRNA gene sequences of the bacterial reference strains, families, Bacillaceae (phylum Firmicutes), Halomonada- type strains and closest phylogenetic relatives were selected ceae and Idiomarinaceae (class Gammaproteobacteria). from GenBank by subjecting the nucleotide sequences of The Bacillaceae accounted for the majority of the isolates bacterial isolates to similarity searches using BLASTn (http:// (c. 72%) whereas the Halomonadaceae and Idiomarinaceae www.ncbi.nlm.nih.gov/blast) and SeqMatch (Release 10 accounted for c. 21% and c. 6% of the isolates, respectively. Update17, Ribosomal Database Project) (Cole et al. 2009). Among bacteria assigned to the Firmicutes, a cluster of Multiple alignments of sequences were done using ClustalW 15 isolates was closely related to the type strain of as implemented in MEGALIGN (DNASTAR Lasergene Salimicrobium luteum (98.5–99.0% similarity) whereas v.8.0; Madison, USA). one isolate (C8-1) grouped with, and was closely related A phylogenetic tree was reconstructed using MEGA 4 to, the type strain of Salimicrobium halophilum. Isolates (Tamura et al. 2007). The model of nucleotide substitution S25-1 and S15-3 grouped closely with the type strain of was selected on the basis of the Akaike information Halobacillus hunanensis and Halobacillus alkaliphilus, criterion implemented in Model Test 3.7 (Posada 2006). respectively, whereas the nearest neighbor of isolates S27- Evolutionary histories were inferred using the neighbor- 1, S30-2, S101-1 and S105-2 was the type strain of joining method (Saitou and Nei 1987) and bootstrap Halobacillus trueperi (98.7–99.8% similarity). These four consensus trees inferred from 1,000 permutations of the isolates were also closely related to the non-type strain, datasets (Felsenstein 1985). Evolutionary distances were Halobacillus sp., QW1018 (GenBank Accession no. computed using the Tamura-Nei method (Tamura and Nei EU124360) isolated from hypersaline well water in China 1993) as the number of base substitutions per site. The rate (98.8–99.9% similarity). variation among sites was modeled with a gamma distribu- Several isolates clustered with the type strain of a species tion (shape parameter =4.44404). All positions containing belonging to the genus Virgibacillus (three isolates), gaps and missing data were eliminated from the dataset Halalkalibacillus (one isolate), Alkalibacillus (four isolates) (complete deletion option). Nucleotide sequences generated and the genus Marinococcus (four isolates). in this study were deposited in GenBank and their The isolates placed in the class Gammaproteobacteria accession numbers are shown in the phylogenetic tree. were subdivided into four genera. The nearest neighbor of isolates S35-1, S83-1 and S96-2 was the type strain of Halomonas janggokensis (99.7–99.9% similarity). These Results three isolates also grouped closely with the type strain of Halomonas subterraneum (GenBank Accession no. Analysis of the chemical composition of hypersaline water (g/l ± EF144148) from saline well water in China. Isolate S78-2 + - SE) indicated that Na and Cl were the most abundant ions was closely related to the type strains of Halomonas 2− (63.58±3.14 and 94.42±3.58, respectively), followed by SO aquamarina and Halomonas meridiana at the same level 2+ + 2+ (18.64±1.37), Mg (12.73±2.92), K (11.29±2.31), Ca of sequence similarity (99.1%). The nearest neighbors of 3− 2− isolate S74-1, isolates S15-2 and S24-1, and isolates S55-1 (6.78±1.54), HC0 (3.62±1.02), and CO (2.38±0.93) and 3 378 Ann Microbiol (2011) 61:375–381 Fig. 1 Phylogenetic relation- Salimicrobium flavidum ISL-25 (FJ357160) ships of halophilic and haloto- 15 Isolates 99 65 lerant bacteria isolated from a Salimicrobium luteum BY-5 (DQ227305) 100 Salimcrobium halophilum DSM 4771 (AJ243920) hypersaline pond in Sichuan, C8-1(EU868887) China. The phylogenetic tree, Salimicrobium album DSM 20748 (X90834) based on 16S rRNA gene S25-1 (EU868841) Halobacillus hunanensis JSM 071077 (FJ425898) sequences, was constructed by S15-3 (EU868841) the neighbor-joining method. Halobacillus alkaliphilus FP5 (AM295006) Numerals at nodes indicate T Halobacillus salinus HSL-3 (AF500003) bootstrap percentages derived Halobacillus sp. QW1018 (EU124360) S27-1 (EU868843) from 1,000 replications. Bar S105-2 (EU868845) represents 2% base substitu- Halobacillus locisalis MSS-155 (AY190534) tions. Strain designations and Halobacillus trueperi DSM 10404 (AJ310149) S101-1 (EU868844) accession numbers in bold are Halobacillus aidingensis AD-6 (AY351389) from this study. The cluster of S30-2 (EU868846) 15 isolates from this study des- Halobacillus dabanensis D-8 (AY351395) ignated with an asterisk are Halobacillus karajiensis DSM 14948 (AJ486874) Halobacillus litoralis SL-4 (X94558) C11-3, C13-2, M13-3, M13-5, Halobacillus yeomjeoni MSS-402 (AY881246) C14-2, C22-2, M22-4, M25-10, Halobacillus halophilus NCIMB 9251 (X62174) C15-8, M17-8, M22-5, M23-6, Virgibacillus olivae E308 (DQ139839) S79-2 (EU868856) M1-1, M14-4 and C15-4 with S57-2 (EU868858) Gen-Bank Accession numbers S57-1 (EU868857) EU868860 through EU868874, T Virgibacillus marismortui 123 (AJ009793) Virgibacillus dokdonensis DSW-10 (AY822043) respectively M24-1 (EU868883) 73 Halalkalibacillus halophilus BH2 (AB264529) M18-3 (EU868876) M18-4 (EU868877) M18-8 (EU868878) M18-1 (EU868875) 89 Alkalibacillus salilacus BH 163 (AY671976) Alkalibacillus silvisoli BM 2 (AB264528) Marinococcus luteus KCTC 13214 (FJ214659) 84 C17-6 (EU868881) C22-3 (EU868882) 92 M14-5 (EU868880) M22-6 (EU868879) Marinococcus halotolerans YIM 70157 (AY817493) Marinococcus halophilus DSM 20408 (X90835) Halomonas janggokensis M24 (AM229315) 91 S96-2 (EU868853) S35-1 (EU868847) S83-1 (EU868850) Halomonas subterranea ZG16 (EF144148) Halomonas arcis AJ282 (EF144147) Halomonas venusta DSM 4743 (AJ306894) Halomonas hydrothermalis Slthf2 (AF212218) S78-2 (EU868849) Halomonas aquamarina DSM 30161 (AJ306888) Halomonas meridiana DSM 5425 (AJ306891) 59 75 Halomonas axialensis ATCC BAA-800 (AF212206) S74-1 (EU868848) Halomonas gudaoensis SL014B-69 (DQ421808) Halomonas campisalis ATCC 700597 (AF054286) S15-2 (EU868851) 100 S24-1 (EU868852) 99 T Halomonas elongata 1H9 (X67023 ) Halomonas eurihalina ATCC 49336 (NR_026250) 99 T Halomonas almeriensis M8 (AY858696) Chromohalobacter marismortui ATCC 17056 (X87219) Chromohalobacter canadensis DSM 6769 (AF211861) S55-1 (EU868855) C3-1 (EU868854) Chromohalobacter salexigens DSM 3043 (CP000285) Chromohalobacter israelensis ATCC 43985 (AJ295144 C4-2 (EU868859) Halovibrio denitrificans HGD 3 (DQ072718) Idiomarina abyssalis KMM 227 (AF052740) S1-2 (EU868886) S87-1 (EU868885) S5-1 (EU868884) Idiomarina loihiensis L2-TR (AF288370) Idiomarina ramblicola R22 (AY526862) 0.02 Bacillaceae Idiomarinaceae Halomomadaceae Firmicutes Gammaproteobacteria Ann Microbiol (2011) 61:375–381 379 Table 1 The isolation source, NaCl tolerance and closest phylogenetic relatives of bacterial isolates from a hypersaline pond in Sichuan, China Isolate No. Isolation NaCl Closest phylogenetic Sequence Isolation source Reference - b c source tolerance relative (type strain) similarity (%) Accession No. Bacillaceae, Bacilliales, Firmicutes M1-1 Water 0–30 (15) Salimicrobium luteum BY-5 98.5–99.0 Saltern sediments DQ227305 C15-8; C11-3; C13-2 ; C14-2; Sediments 0–30 (15) (Korea) C15-4; M13-3; M13-5; M14-4 M22-4; C22-2; M17-8; M22-5; Soil 0–30 (10) M23-6; M25-10 C8-1 Water 1–30 (25) Salimicrobium halophilum 99.1 Solar saltern (Korea) AJ243920 DSM 4771 S25-1 Soil 1–25 (10) Halobacillus hunanensis JSM071077 99.5 Brine, salt mine (China) FJ425898 S15-3 Soil 0–25 (10) Halobacillus alkaliphilus FP5 99.3 Solar saltern (Spain) AM295006 S105-2; Water 0–25 (5) Halobacillus trueperi DSM 10404 98.7–99.8 Sediments (Great Salt AJ310149 S30-2; S27-1; S101-1 Water 0–20 (5) Lake, USA) S57-1; S57-2; S79-2 Water 1–15 (5) Virgibacillus marismortui 123 99.7–99.9 Water (Dead Sea) AJ009793 M24-1 Soil 5–25 (10) Halalkalibacillus halophilus BH2 99.8 Non-saline soil (Japan) AB264529 M18-1; M18-3; M18-4; M18-8 Soil 5–25 (15) Alkalibacillus salilacus BH163 99.6 Sediments, salt lake AY671976 (China) M14-5 Sediments 0–30 (15) Marinococcus luteus KCTC 13214 99.8–99.9 Saline soil FJ214659 C22-3; C17-6; M22-6 Soil 0–30 (10) (Barkol Lake, China) Halomonadaceae, Oceanospirillales, Gammaproteobacteria, Proteobacteria S35-1; S83-1; S96-2 Water 0–15 (5) Halomonas janggokensis M24 99.7–99.9 Solar saltern (Korea) AM229315 S78-2 Water 0–20 (10) Halomonas aquamarina DSM 30161 99.1 Sea water (Hawaii) AJ306888 and Halomonas meridiana Saline lake (Antarctica) AJ306891 DSM 5425 S74-1 Water 0–20 (10) Halomonas gudaoenis SL014B-69 99.4 Contaminated saline DQ421808 soil (China) S15-2 Sediments 0–20 (10) Halomonas elongata IH9 99.0–99.2 Solar saltern (Bonaire, Antilles) X67023 S24-1 Soil 0–20 (10) C3-1; S55-1 Water 0–30 (20) Chromohalobacter salexigens 99.7–99.9 Solar saltern (Bonaire, CP000285 DSM 3043 Antilles) C4-2 Water 10–30 (15) Halovibrio denitrificans HGD 3 98.5 Sediments, salt lake DQ072718 (Mongolia) Idiomarinaceae, Alteromonadales, Gammaproteobacteria, Proteobacteria S1-2; S5-1; S87-1 Water 1–25 (10) Idiomarina loihiensis L2-TR 99.4–99.5 Deep sea hydrothermal AF288370 vent (Hawaii) Isolates tested for NaCl tolerance are shown in bold Range of NaCl concentrations (w/v%) at which bacterial growth was recorded; values in parentheses represent salt concentrations for optimal growth Type strains of validly published species and C3-1, were the type strains of Halomonas gudaoensis, Isolates representing five genera of the Firmicutes and Halomonas elongata and Chromohalobacter salexigens, two genera of the Gammaproteobacteria were halophilic respectively. Isolate C4-2 clustered with the type strain of and required between 1 and 10% (w/v) NaCl for growth. Halovibrio denitrificans (98.5% similarity) whereas isolates Optimal growth of the 22 halotolerant and halophilic S1-2, S5-1 and S87-1 grouped with the type strain of isolates was between 5 and 25% (w/v) NaCl. Idiomarina loihiensis isolated from a deep sea hydrother- mal vent in Hawaii. Data for the salt tolerance of 22 representative isolates Discussion (Table 1) indicate that all were either halotolerant or halophilic and were capable of growth on agar media In this work, a culture-dependant approach was used to containing between 15 and 30% (w/v) NaCl. isolate diverse halophilic and halotolerant bacterial The halotolerant isolates belonged to three genera of the representatives of the genera Halalkalibacillus, Virgiba- Firmicutes and to two genera of the Gammaproteobacteria. cillus, Marinococcus, Salimicrobium, Halobacillus and Of the halotolerant bacteria, only isolate S57-1 (genus Alkalibacillus (phylum Firmicutes)and Halomonas, Idio- Virgibacillus) and isolate S35-1(genus Halomonas) did not marina, Chromohalobacter and Halovibrio (class Gam- grow at salt concentrations above 15% w/v NaCl. The maproteobacteria) from a hypersaline environment in remaining isolates grew at salt levels exceeding 15% (w/v). China. 380 Ann Microbiol (2011) 61:375–381 A comparison of these findings with other cultivation and Halovibrio showed 98.7% or less sequence similarity dependant studies suggests that the Firmicutes and Gammap- to the type strain of the closest relative. Further work is roteobacteria are indeed predominant among members of required to ascertain the species identity of these isolates. the cultivable bacterial community in a wide variety of The majority of bacterial isolates in this study grew on hypersaline habitats worldwide (Baati et al. 2010;Hediet al. media containing NaCl at concentrations of between 15 and 2009; Xiang et al. 2008;Yeonetal. 2005). However, a 30% and can be considered to be extremely halotolerant number of other studies have also reported the isolation of (Margesin and Schinner 2001). The remaining isolates were members of the Actinobacteria (Jiang et al. 2006; Tsiamis et halophilic and required salt for growth. Most of these al. 2008;Wuet al. 2006), Bacteroidetes (Benlloch et al. halotolerant and halophilic isolates are related to bacterial 2002; Caton et al. 2004;Oren 2008) and the Alphaproteo- genera that have the ability to colonize and survive in bacteria (Benlloch et al. 2002) from different hypersaline diverse habitats. For example, several bacterial isolates are ecosystems. affiliated with different Halomonas species that have been Because many of the bacteria inhabiting saline environ- isolated from contrasting saline environments including soda ments are intractable to cultivation, it is perhaps not surprising lakes, solar salterns, mineral pools, marine habitats, animals, that culture-independent approaches, such as oligonucleotide mural paintings and from sewage treatments (Xu et al. microarrays and sequencing 16S rRNA genes from denatur- 2007). Isolate M24-1 was isolated from hypersaline soil ing gradient gel electrophoresis (DGGE) and clone libraries, whereas the closest phylogenetic relative (Halalkalibacillus have identified far greater bacterial diversity than has been halophilus,BH2 ) was originally isolated from non-saline achieved using cultivation-based methods (Benlloch et al. soil in Japan (Echigo et al. 2007). The recovery of isolates 2002; Jiang et al. 2006; Lefebvre et al. 2006; Perreault et al. related to the genera Salimicrobium and Halomonas from 2007; Tsiamis et al. 2008). However, culture-independent hypersaline water, sediment and from soil samples further approaches have the disadvantage that bacterial isolates are emphasizes the ability of these bacteria to adapt to differing not obtained for further investigation. There is an urgent saline environments. need for new media and approaches for culturing halophilic Several bacterial isolates identified in this work may and halotolerant bacteria from hypersaline environments. have strong biotechnological potential. For example, Xiang et al (2008) reported the isolation of bacteria members of the genera Halomonas and Marinococcus have related to the genera Halomonas (class Gammaproteobac- been reported to have the ability to degrade phenol and oil teria), Planococcus, Halobacillus, Oceanobacillus and pollutants (Nicholson and Fathepure 2004), whereas bacte- Virgibacillus (phylum Firmicutes) from subterranean hy- rial representatives of the genus Idiomarina have been persaline well water (20–25% salinity) in Zigong, Sichuan reported to produce phytases that have potential applica- Province, China. In contrast, we isolated bacteria affiliated tions in food processing and the improvement of crop plant with four genera of the Gammaproteobacteria and with six nutrition in agriculture (Jorquera et al. 2008). genera of the Firmicutes from a hypersaline ecosystem that Research is underway to assess the biotechnological is also supplied with brines from a subterranean aquifer in potential of the halophilic and halotolerant bacterial isolates Sichuan Province. Of these bacteria, only representatives of obtained in this work. the genera Halobacillus, Halomonas and Virgibacillus were Acknowledgment This study was supported by a grant from the common to both studies. A notable difference between the National High Technology Research and Development Program of studies was our isolation and identification of bacterial China (Program 863; No. 2006AA02Z189). representatives of the genera Salimicrobium, Halalkaliba- cillus and Halovibrio. To our knowledge, this is the first report of the isolation of bacteria related to these three References genera from a hypersaline environment in China. Interestingly, Halobacillus sp. strain QW1018 that was Antón J, Rosselló-Mora R, Rodríguez-Valera F, Amann R (2000) isolated from hypersaline well water in Sichuan (Xiang et Extremely halophilic bacteria in crystallizer ponds from solar salterns. Appl Environ Microbiol 66:3052–3057 al. 2008), is closely related to isolates S27-1, S101-1, S105- Antón J, Oren A, Benlloch S, Rodríguez-Valera F, Amann R, 2 and S30-2 in the present study. Moreover, the type strain Rosselló-Mora R (2002) Salinibacter rubber gen. nov., sp. nov., of Halomonas subterranea (ZG16 ), also isolated from a novel, extremely halophilic member of the bacteria from saltern saline well water in Sichuan (Xu et al. 2007), is closely crystallizer ponds. 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16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a hypersaline pond in Sichuan, China

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Springer Journals
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Copyright © 2010 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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10.1007/s13213-010-0137-x
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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;Xiang et 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, The isolation and characterization of halotolerant and Ya’an, Sichuan, China 625014 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, biomolecules, such as osmolytes (compatible solutes), Ottawa, Ontario, Canada K1A 0C6 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, of China are located at depths of between 50 and 3,000 m Wenjiang 611130 Sichuan, People’s Republic of China and occur within sedimentary rocks ranging in age from the e-mail: liping6575@163.com 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;Wuet al. 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 m . 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- MgCl , 10 mM Tris-HCl (pH 9.0), 10 μM of each dNTP, tralia) and anions determined by Ion Chromatography 0.1 μMofeachprimer, and1Uof EX TaqDNA (model: ICS 3000; USA). polymerase (Takara, Japan). PCR cycling conditions con- Ann Microbiol (2011) 61:375–381 377 − 2+ sisted of denaturation at 95°C for 2 min, 35 cycles of 95°C by trace amounts of Br (0.03±0.02) and Mn (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. The range of different colony according to the manufacturer’s protocol. PCR products morphologies of bacterial isolates recovered from each of were verified by agarose gel electrophoresis. the hypersaline water, sediment and soil samples were For 16S rRNA gene sequencing, purified PCR products generally similar. Although both Gram-positive and Gram- were ligated into vector pMD18 (Takara Inc., Japan), and negative bacterial isolates were obtained from all samples, transformed into Escherichia coli. Clones were screened by the majority (60%) were Gram-positive. PCR for inserts of the correct size (c. 1,500 bases) using The phylogenetic tree reconstructed from full length 16S universal primers (Edwards et al. 1989). Inserts were rRNA gene sequences of the 47 bacterial isolates recovered commercially sequenced (Invitrogen, China) to provide from hypersaline water, sediment and soil samples is shown full-length 16S rRNA gene sequences. in Fig. 1. Data for the source of isolation, NaCl tolerance and closest phylogenetic relative (type strains) of these Phylogenetic analysis bacterial isolates are given in Table 1. Analysis of 16S rRNA gene sequences divided the bacterial isolates into the 16S rRNA gene sequences of the bacterial reference strains, families, Bacillaceae (phylum Firmicutes), Halomonada- type strains and closest phylogenetic relatives were selected ceae and Idiomarinaceae (class Gammaproteobacteria). from GenBank by subjecting the nucleotide sequences of The Bacillaceae accounted for the majority of the isolates bacterial isolates to similarity searches using BLASTn (http:// (c. 72%) whereas the Halomonadaceae and Idiomarinaceae www.ncbi.nlm.nih.gov/blast) and SeqMatch (Release 10 accounted for c. 21% and c. 6% of the isolates, respectively. Update17, Ribosomal Database Project) (Cole et al. 2009). Among bacteria assigned to the Firmicutes, a cluster of Multiple alignments of sequences were done using ClustalW 15 isolates was closely related to the type strain of as implemented in MEGALIGN (DNASTAR Lasergene Salimicrobium luteum (98.5–99.0% similarity) whereas v.8.0; Madison, USA). one isolate (C8-1) grouped with, and was closely related A phylogenetic tree was reconstructed using MEGA 4 to, the type strain of Salimicrobium halophilum. Isolates (Tamura et al. 2007). The model of nucleotide substitution S25-1 and S15-3 grouped closely with the type strain of was selected on the basis of the Akaike information Halobacillus hunanensis and Halobacillus alkaliphilus, criterion implemented in Model Test 3.7 (Posada 2006). respectively, whereas the nearest neighbor of isolates S27- Evolutionary histories were inferred using the neighbor- 1, S30-2, S101-1 and S105-2 was the type strain of joining method (Saitou and Nei 1987) and bootstrap Halobacillus trueperi (98.7–99.8% similarity). These four consensus trees inferred from 1,000 permutations of the isolates were also closely related to the non-type strain, datasets (Felsenstein 1985). Evolutionary distances were Halobacillus sp., QW1018 (GenBank Accession no. computed using the Tamura-Nei method (Tamura and Nei EU124360) isolated from hypersaline well water in China 1993) as the number of base substitutions per site. The rate (98.8–99.9% similarity). variation among sites was modeled with a gamma distribu- Several isolates clustered with the type strain of a species tion (shape parameter =4.44404). All positions containing belonging to the genus Virgibacillus (three isolates), gaps and missing data were eliminated from the dataset Halalkalibacillus (one isolate), Alkalibacillus (four isolates) (complete deletion option). Nucleotide sequences generated and the genus Marinococcus (four isolates). in this study were deposited in GenBank and their The isolates placed in the class Gammaproteobacteria accession numbers are shown in the phylogenetic tree. were subdivided into four genera. The nearest neighbor of isolates S35-1, S83-1 and S96-2 was the type strain of Halomonas janggokensis (99.7–99.9% similarity). These Results three isolates also grouped closely with the type strain of Halomonas subterraneum (GenBank Accession no. Analysis of the chemical composition of hypersaline water (g/l ± EF144148) from saline well water in China. Isolate S78-2 + - SE) indicated that Na and Cl were the most abundant ions was closely related to the type strains of Halomonas 2− (63.58±3.14 and 94.42±3.58, respectively), followed by SO aquamarina and Halomonas meridiana at the same level 2+ + 2+ (18.64±1.37), Mg (12.73±2.92), K (11.29±2.31), Ca of sequence similarity (99.1%). The nearest neighbors of 3− 2− isolate S74-1, isolates S15-2 and S24-1, and isolates S55-1 (6.78±1.54), HC0 (3.62±1.02), and CO (2.38±0.93) and 3 378 Ann Microbiol (2011) 61:375–381 Fig. 1 Phylogenetic relation- Salimicrobium flavidum ISL-25 (FJ357160) ships of halophilic and haloto- 15 Isolates 99 65 lerant bacteria isolated from a Salimicrobium luteum BY-5 (DQ227305) 100 Salimcrobium halophilum DSM 4771 (AJ243920) hypersaline pond in Sichuan, C8-1(EU868887) China. The phylogenetic tree, Salimicrobium album DSM 20748 (X90834) based on 16S rRNA gene S25-1 (EU868841) Halobacillus hunanensis JSM 071077 (FJ425898) sequences, was constructed by S15-3 (EU868841) the neighbor-joining method. Halobacillus alkaliphilus FP5 (AM295006) Numerals at nodes indicate T Halobacillus salinus HSL-3 (AF500003) bootstrap percentages derived Halobacillus sp. QW1018 (EU124360) S27-1 (EU868843) from 1,000 replications. Bar S105-2 (EU868845) represents 2% base substitu- Halobacillus locisalis MSS-155 (AY190534) tions. Strain designations and Halobacillus trueperi DSM 10404 (AJ310149) S101-1 (EU868844) accession numbers in bold are Halobacillus aidingensis AD-6 (AY351389) from this study. The cluster of S30-2 (EU868846) 15 isolates from this study des- Halobacillus dabanensis D-8 (AY351395) ignated with an asterisk are Halobacillus karajiensis DSM 14948 (AJ486874) Halobacillus litoralis SL-4 (X94558) C11-3, C13-2, M13-3, M13-5, Halobacillus yeomjeoni MSS-402 (AY881246) C14-2, C22-2, M22-4, M25-10, Halobacillus halophilus NCIMB 9251 (X62174) C15-8, M17-8, M22-5, M23-6, Virgibacillus olivae E308 (DQ139839) S79-2 (EU868856) M1-1, M14-4 and C15-4 with S57-2 (EU868858) Gen-Bank Accession numbers S57-1 (EU868857) EU868860 through EU868874, T Virgibacillus marismortui 123 (AJ009793) Virgibacillus dokdonensis DSW-10 (AY822043) respectively M24-1 (EU868883) 73 Halalkalibacillus halophilus BH2 (AB264529) M18-3 (EU868876) M18-4 (EU868877) M18-8 (EU868878) M18-1 (EU868875) 89 Alkalibacillus salilacus BH 163 (AY671976) Alkalibacillus silvisoli BM 2 (AB264528) Marinococcus luteus KCTC 13214 (FJ214659) 84 C17-6 (EU868881) C22-3 (EU868882) 92 M14-5 (EU868880) M22-6 (EU868879) Marinococcus halotolerans YIM 70157 (AY817493) Marinococcus halophilus DSM 20408 (X90835) Halomonas janggokensis M24 (AM229315) 91 S96-2 (EU868853) S35-1 (EU868847) S83-1 (EU868850) Halomonas subterranea ZG16 (EF144148) Halomonas arcis AJ282 (EF144147) Halomonas venusta DSM 4743 (AJ306894) Halomonas hydrothermalis Slthf2 (AF212218) S78-2 (EU868849) Halomonas aquamarina DSM 30161 (AJ306888) Halomonas meridiana DSM 5425 (AJ306891) 59 75 Halomonas axialensis ATCC BAA-800 (AF212206) S74-1 (EU868848) Halomonas gudaoensis SL014B-69 (DQ421808) Halomonas campisalis ATCC 700597 (AF054286) S15-2 (EU868851) 100 S24-1 (EU868852) 99 T Halomonas elongata 1H9 (X67023 ) Halomonas eurihalina ATCC 49336 (NR_026250) 99 T Halomonas almeriensis M8 (AY858696) Chromohalobacter marismortui ATCC 17056 (X87219) Chromohalobacter canadensis DSM 6769 (AF211861) S55-1 (EU868855) C3-1 (EU868854) Chromohalobacter salexigens DSM 3043 (CP000285) Chromohalobacter israelensis ATCC 43985 (AJ295144 C4-2 (EU868859) Halovibrio denitrificans HGD 3 (DQ072718) Idiomarina abyssalis KMM 227 (AF052740) S1-2 (EU868886) S87-1 (EU868885) S5-1 (EU868884) Idiomarina loihiensis L2-TR (AF288370) Idiomarina ramblicola R22 (AY526862) 0.02 Bacillaceae Idiomarinaceae Halomomadaceae Firmicutes Gammaproteobacteria Ann Microbiol (2011) 61:375–381 379 Table 1 The isolation source, NaCl tolerance and closest phylogenetic relatives of bacterial isolates from a hypersaline pond in Sichuan, China Isolate No. Isolation NaCl Closest phylogenetic Sequence Isolation source Reference - b c source tolerance relative (type strain) similarity (%) Accession No. Bacillaceae, Bacilliales, Firmicutes M1-1 Water 0–30 (15) Salimicrobium luteum BY-5 98.5–99.0 Saltern sediments DQ227305 C15-8; C11-3; C13-2 ; C14-2; Sediments 0–30 (15) (Korea) C15-4; M13-3; M13-5; M14-4 M22-4; C22-2; M17-8; M22-5; Soil 0–30 (10) M23-6; M25-10 C8-1 Water 1–30 (25) Salimicrobium halophilum 99.1 Solar saltern (Korea) AJ243920 DSM 4771 S25-1 Soil 1–25 (10) Halobacillus hunanensis JSM071077 99.5 Brine, salt mine (China) FJ425898 S15-3 Soil 0–25 (10) Halobacillus alkaliphilus FP5 99.3 Solar saltern (Spain) AM295006 S105-2; Water 0–25 (5) Halobacillus trueperi DSM 10404 98.7–99.8 Sediments (Great Salt AJ310149 S30-2; S27-1; S101-1 Water 0–20 (5) Lake, USA) S57-1; S57-2; S79-2 Water 1–15 (5) Virgibacillus marismortui 123 99.7–99.9 Water (Dead Sea) AJ009793 M24-1 Soil 5–25 (10) Halalkalibacillus halophilus BH2 99.8 Non-saline soil (Japan) AB264529 M18-1; M18-3; M18-4; M18-8 Soil 5–25 (15) Alkalibacillus salilacus BH163 99.6 Sediments, salt lake AY671976 (China) M14-5 Sediments 0–30 (15) Marinococcus luteus KCTC 13214 99.8–99.9 Saline soil FJ214659 C22-3; C17-6; M22-6 Soil 0–30 (10) (Barkol Lake, China) Halomonadaceae, Oceanospirillales, Gammaproteobacteria, Proteobacteria S35-1; S83-1; S96-2 Water 0–15 (5) Halomonas janggokensis M24 99.7–99.9 Solar saltern (Korea) AM229315 S78-2 Water 0–20 (10) Halomonas aquamarina DSM 30161 99.1 Sea water (Hawaii) AJ306888 and Halomonas meridiana Saline lake (Antarctica) AJ306891 DSM 5425 S74-1 Water 0–20 (10) Halomonas gudaoenis SL014B-69 99.4 Contaminated saline DQ421808 soil (China) S15-2 Sediments 0–20 (10) Halomonas elongata IH9 99.0–99.2 Solar saltern (Bonaire, Antilles) X67023 S24-1 Soil 0–20 (10) C3-1; S55-1 Water 0–30 (20) Chromohalobacter salexigens 99.7–99.9 Solar saltern (Bonaire, CP000285 DSM 3043 Antilles) C4-2 Water 10–30 (15) Halovibrio denitrificans HGD 3 98.5 Sediments, salt lake DQ072718 (Mongolia) Idiomarinaceae, Alteromonadales, Gammaproteobacteria, Proteobacteria S1-2; S5-1; S87-1 Water 1–25 (10) Idiomarina loihiensis L2-TR 99.4–99.5 Deep sea hydrothermal AF288370 vent (Hawaii) Isolates tested for NaCl tolerance are shown in bold Range of NaCl concentrations (w/v%) at which bacterial growth was recorded; values in parentheses represent salt concentrations for optimal growth Type strains of validly published species and C3-1, were the type strains of Halomonas gudaoensis, Isolates representing five genera of the Firmicutes and Halomonas elongata and Chromohalobacter salexigens, two genera of the Gammaproteobacteria were halophilic respectively. Isolate C4-2 clustered with the type strain of and required between 1 and 10% (w/v) NaCl for growth. Halovibrio denitrificans (98.5% similarity) whereas isolates Optimal growth of the 22 halotolerant and halophilic S1-2, S5-1 and S87-1 grouped with the type strain of isolates was between 5 and 25% (w/v) NaCl. Idiomarina loihiensis isolated from a deep sea hydrother- mal vent in Hawaii. Data for the salt tolerance of 22 representative isolates Discussion (Table 1) indicate that all were either halotolerant or halophilic and were capable of growth on agar media In this work, a culture-dependant approach was used to containing between 15 and 30% (w/v) NaCl. isolate diverse halophilic and halotolerant bacterial The halotolerant isolates belonged to three genera of the representatives of the genera Halalkalibacillus, Virgiba- Firmicutes and to two genera of the Gammaproteobacteria. cillus, Marinococcus, Salimicrobium, Halobacillus and Of the halotolerant bacteria, only isolate S57-1 (genus Alkalibacillus (phylum Firmicutes)and Halomonas, Idio- Virgibacillus) and isolate S35-1(genus Halomonas) did not marina, Chromohalobacter and Halovibrio (class Gam- grow at salt concentrations above 15% w/v NaCl. The maproteobacteria) from a hypersaline environment in remaining isolates grew at salt levels exceeding 15% (w/v). China. 380 Ann Microbiol (2011) 61:375–381 A comparison of these findings with other cultivation and Halovibrio showed 98.7% or less sequence similarity dependant studies suggests that the Firmicutes and Gammap- to the type strain of the closest relative. Further work is roteobacteria are indeed predominant among members of required to ascertain the species identity of these isolates. the cultivable bacterial community in a wide variety of The majority of bacterial isolates in this study grew on hypersaline habitats worldwide (Baati et al. 2010;Hediet al. media containing NaCl at concentrations of between 15 and 2009; Xiang et al. 2008;Yeonetal. 2005). However, a 30% and can be considered to be extremely halotolerant number of other studies have also reported the isolation of (Margesin and Schinner 2001). The remaining isolates were members of the Actinobacteria (Jiang et al. 2006; Tsiamis et halophilic and required salt for growth. Most of these al. 2008;Wuet al. 2006), Bacteroidetes (Benlloch et al. halotolerant and halophilic isolates are related to bacterial 2002; Caton et al. 2004;Oren 2008) and the Alphaproteo- genera that have the ability to colonize and survive in bacteria (Benlloch et al. 2002) from different hypersaline diverse habitats. For example, several bacterial isolates are ecosystems. affiliated with different Halomonas species that have been Because many of the bacteria inhabiting saline environ- isolated from contrasting saline environments including soda ments are intractable to cultivation, it is perhaps not surprising lakes, solar salterns, mineral pools, marine habitats, animals, that culture-independent approaches, such as oligonucleotide mural paintings and from sewage treatments (Xu et al. microarrays and sequencing 16S rRNA genes from denatur- 2007). Isolate M24-1 was isolated from hypersaline soil ing gradient gel electrophoresis (DGGE) and clone libraries, whereas the closest phylogenetic relative (Halalkalibacillus have identified far greater bacterial diversity than has been halophilus,BH2 ) was originally isolated from non-saline achieved using cultivation-based methods (Benlloch et al. soil in Japan (Echigo et al. 2007). The recovery of isolates 2002; Jiang et al. 2006; Lefebvre et al. 2006; Perreault et al. related to the genera Salimicrobium and Halomonas from 2007; Tsiamis et al. 2008). However, culture-independent hypersaline water, sediment and from soil samples further approaches have the disadvantage that bacterial isolates are emphasizes the ability of these bacteria to adapt to differing not obtained for further investigation. There is an urgent saline environments. need for new media and approaches for culturing halophilic Several bacterial isolates identified in this work may and halotolerant bacteria from hypersaline environments. have strong biotechnological potential. For example, Xiang et al (2008) reported the isolation of bacteria members of the genera Halomonas and Marinococcus have related to the genera Halomonas (class Gammaproteobac- been reported to have the ability to degrade phenol and oil teria), Planococcus, Halobacillus, Oceanobacillus and pollutants (Nicholson and Fathepure 2004), whereas bacte- Virgibacillus (phylum Firmicutes) from subterranean hy- rial representatives of the genus Idiomarina have been persaline well water (20–25% salinity) in Zigong, Sichuan reported to produce phytases that have potential applica- Province, China. In contrast, we isolated bacteria affiliated tions in food processing and the improvement of crop plant with four genera of the Gammaproteobacteria and with six nutrition in agriculture (Jorquera et al. 2008). genera of the Firmicutes from a hypersaline ecosystem that Research is underway to assess the biotechnological is also supplied with brines from a subterranean aquifer in potential of the halophilic and halotolerant bacterial isolates Sichuan Province. Of these bacteria, only representatives of obtained in this work. the genera Halobacillus, Halomonas and Virgibacillus were Acknowledgment This study was supported by a grant from the common to both studies. A notable difference between the National High Technology Research and Development Program of studies was our isolation and identification of bacterial China (Program 863; No. 2006AA02Z189). representatives of the genera Salimicrobium, Halalkaliba- cillus and Halovibrio. 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Annals of Microbiology – Springer Journals

Published: Oct 22, 2010

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