Diverse microbial groups inhabit multi-pond solar saltworks, in which a gradient of salinities ranges from seawater to NaCl precipitation. Along the salinity gradient, the majority of the microbial community changes, from moderately halophilic bacteria to extremely halophilic archaea, and biodiversity decreases as the environment becomes increasingly hypersaline(Oren and Rodríguez-Valera, 2001; Oren, 2002). In crystallizer ponds, the blooms of carotenoid-enriched microalgae Dunaliella, halobacteria, and archaea ensure increased heat absorption and the reduction of dissolved organics, which eventually results in enhanced evaporation and improved salt crystallization(Jones et al., 1981; Javor, 2002).
Aside from their ecological importance, the use of halophiles in biotechnology has attracted special attention recently. High salinity and long-term selection pressure have resulted in unique cell structures, physiological functions, and metabolic mechanisms of the halophiles(Zahran, 1997). The majority of these microorganisms produce a variety of special biologically active substances, which are directly related to their halophilic behavior(Litchfield, 2011). Halophilic bacteria and archaea are useful biological sources of poly-β-hydroxybutyrate (Quillaguamán et al., 2005; Tan et al., 2011), carotenoid pigments such as bacterioruberin(Fang et al., 2010) and canthaxanthin(Asker and Ohta, 1999, 2002). Moreover, the ease of cell lysis in the absence of salt and low contamination danger in highconcentration salt culture facilitate the industrial application of these halophilic microorganisms(Oren, 2002).
A number of studies have been conducted on the isolation and characterization of bacterial and archaeal strains and new species have been discovered in marine solar salterns. These include the extremely halophilic archaea Haloferax alex and rinus from Egyptian saltworks(Asker and Ohta, 2002), Haloferax volcanii, Haloarcula japonica, and Halobacterium salinarum from Puerto Rico and the Caribbean saltworks(MontalvoRodríguez et al., 1997), Halorubrum litoreum from Fuqing solar salterns, Fujian, China(Cui et al., 2007), and Halorubrum sp. from Indian saltworks(Pathak and Sardar, 2012), as well as halophilic bacteria such as Salinimicrobium flavidum from Korean saltworks(Yoon et al., 2009) and Halobacillus sp. from Ludaokou saltworks, Sh and ong, China(Chen et al., 2010). Carotenoid accumulation and anti-microbial activity in some of the above strains have also been studied(Asker and Ohta, 2002; Chen et al., 2010). However, little information is currently available on the distribution and diversity of microorganisms in Bohai Bay saltworks, China, the main salt production site. In this study, we attempted to isolate and identify the cultivable bacteria and archaea from solar saltern ponds of different salinities, aiming to elucidate the diversity of microbial resources in saltern ponds.2 MATERIAL AND METHOD 2.1 Sample collection
Sediment samples were collected from salinity 100, 150, 200, and 250 salt ponds in Hangu Saltworks Co. Ltd., Tianjin, China in March, 2011. The samples were kept in sterile plastic bags and stored in the refrigerator at 4°C.2.2 Colony isolation
The modified CM culture medium was prepared by dissolving yeast extract(10 g/L) and acid hydrolyzed casein(7.5 g/L)into the brine water taken from the original sampling salt ponds. pH was adjusted to 7.2– 7.4. One gram of sediment sample was added to an Erlenmeyer flask containing 50 mL culture medium. After incubation in an orbital shaker at 150 r/min at 37°C for 30 min, 2 mL supernatant was added to 98 mL culture medium. After 3-day incubation, 100 μL culture suspension was streaked on an agar plate. After incubation at 37°C for 5–14 days depending on the salinity, a single colony with different morphology was then picked up and inoculated into liquid medium. The above procedure was repeated twice for the purification.2.3 DNA extraction and PCR amplification of 16S rRNA gene
Genomic DNA was extracted from the isolated colonies using an Bacterial Genomic DNA Miniprep Kit(Axygen, USA). The 16S rRNA gene was amplified using both the bacterial universal forward primer F27: 5′-AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer R1492: 5′-GGTTACCTTGTTACGACTT-3′, and the archaeal forward primer F8: 5′-TTGATCCTGCCGGAGGCCATTG-3′ and the reverse primer R1462: 5′-ATCCAGCCGCAGATTCCCCTAC-3′(Lizama et al., 2001). The 50-μL PCR reaction mixture contained 1 μL primer, 4 μL dNTP, 5 μL 10× buffer, 0.5 μL Taq(TaKaRa, Japan), and 1 μL template DNA. The PCR conditions were 94°C for 5 min, following by 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 3 min, and a final 10-min extension at 72°C. Sequencing of the 16S rRNA gene was conducted by Beijing Genomics Institute, China.2.4 Phylogenetic analysis
Alignment of 16S rRNA gene sequences was carried out in ClustalX software. Sequences of the isolated strains were compared with sequences in the GenBank database, via BLAST searches(Chenna et al., 2003). A phylogenetic tree was constructed using the neighbor-joining method as implemented in MEGA5, and bootstrap consensus trees were inferred from 1 000 permutations of the datasets(Tamura et al., 2011).2.5 Morphological, physiological, and biochemical characterization of the isolated strains
Morphological characterizations of the isolated strains were determined by Gram-staining and electron microscopy(Hitachi, Japan). Salt tolerance for bacterial growth was tested by inoculating fresh culture into the modified CM medium(1:103 v/v)at pH 7.2–7.4(Mettler, Germany) and salinity 0, 50, 100, 150, 200, and 250 as measured by an refractometer (Atago, Japan). Growth at different pHs was evaluated using the modified CM medium at their original salinities and an initial pH of 5, 6, 7, 8, 9, and 10. Growth was determined at OD600 after incubating for 24 h at 37°C.
An API 20E Bacterial Identification Kit (Biomérieux, France)was used to determine the biochemical characteristics of the isolated strains. The kit contained 20 biochemical indicators, including seven enzymes, such as β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase and gelatinase, use of citrate, production of H2S, indole and acetoin, acid production from D-glucose, D-mannite, myoinositol, D-sorbierite, L-rhamnose, D-sucrose, D-melibiose, laetrile, and L-arabinose.3 RESULT 3.1 Phylogenetic analysis
A total of 26 isolates were obtained from different salinities. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates were from the bacterial genera Halomonas, Salinicoccus, Oceanobacillus, Gracibacillus, and Salimicrobium, and the archaea genus Halorubrum. The sequence similarity ranged from 99%–100%, except for strain SM.200-5, which had 95% similarity with Salimicrobium flavidum sp. ISL-25(Yoon et al., 2009) (Fig. 1). Halomonas sp. was the most dominant with eight strains distributed in salinities ranging from 100–200, followed by Halorubrum with six strains found exclusively in salinity 250. Based on the genus and their original habitat salinity, 10 isolates, namely H.100-16-2, H.150-7, H.200-1, SN.100-2-1, O.150-1, G.150-9, SM.150-6, HB.250-1, and HB.250-7, were selected for further morphological, physiological, and biochemical characterization.3.2 Phenotypic characterization
More than half of the isolated strains were white or yellowish in color, whereas strain SN.100-2-1 and HB.250-7 formed red colonies and strain HB.250-1 formed orange colonies on the agar plates(Table 1).The majority of the isolates were Gram-negative, while strains SN.100-2-1 and HB.250-1 were Gram-positive. The cells of the most frequently isolated strains were rod-shaped, while those of SN.100-2-1 and HB.250-1 were coccoid and HB.250-7 was oval(Fig. 2). There was a slight difference among the three Halomonas strains obtained from different salinities; they measured 0.5–0.8 μm×2.0–3.0 μm for H.100-16-2, 0.3–0.4 μm× 0.6–0.8 μm for H.150-7, and 0.3–0.4 μm×3.5–4.5 μm for H.200-1. A size difference was also observed between strains SM.150-6(0.3–0.4 μm×0.8–1.0 μm) and SM.200-5(0.3–0.4 μm×1.8–3.0 μm).3.3 Physiological and biochemical characterization
Optimal growth performance of the isolated strains was tested in a range of salinities(0, 50, 100, 150, 200, and 250) and pHs(5, 6, 7, 8, 9, and 10)(Table 2). The optimal salinity for growth for most isolates was 50, while it was 100 for strains SM.150-6 and SM.200-5, and 200 for strains HB.250-1 and HB.250- 7. Seven was the optimal pH for growth for most strains, while it was 9 for strain O.150-1 and 8 for strain HB.250-7.
None of the isolates produced enzymes, i.e. β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease and gelatinase, and did not use citrate or produce indole. Six isolates, e.g., strains O.150-1, SM.150-6, H.150- 7, G.150-9, H.200-1, and HB.250-1, produced tryptophan deaminase. Five isolates including strains SN.100-2-1, H.100-16-2, H.150-7, G.150-9, and H. 200-1 produced acetoin. Only one strain, SM.200-5, produced H2S.
Strains SN.100-2-1, H.150-7, and H.200-1 used D-glucose, D-mannite, myoinositol, D-sorbierite, L-rhamnose, D-sucrose, D-melibiose, laetrile, and L-arabinose as carbon substrates. Only H.100-16-2 used D-glucose, D-sucrose, D-melibiose, and Larabinose. O.150-1 used D-glucose, D-mannite, D-rhamnose, D-sucrose, and L-arabinose. G.150-9 could not use myoinositol or D-sorbierite. SM.200-5 could ferment all carbon sources listed in Table 2, but only weak use of D-mannite, myoinositol, D-sorbierite, D-sucrose, and D-melibiose was observed in this strain. SM.150-6 could not ferment D-sucrose or laetrile. Strain HB.250-1 could use all given carbon sources, but its use of D-glucose, D-melibiose laetrile, and L-arabinose was better than others’. HB.250-7 could only use L-arabinose and weakly use D-melibiose and laetrile.4 DISCUSSION
It had been commonly assumed that the microbial biodiversity in moderate environments is mostly contributed by halophilic bacteria, while archaea predominantly exist in crystallizer ponds(Olsen, 1994). Nevertheless, recently published molecular data have indicated the presence of archaea in moderate environments, while bacteria seem to be as widespread as archaea(Antόn et al., 2000; Oren, 2002). In this study, although both archaeal and bacterial primers were used for the 16S rDNA amplification of all isolates, only five bacterial genera were identified in salinities below 200 and one archaeal genus was observed in a crystallizer pond of 250. This could be explained by the limitation of the culture-dependent methods applied in this study, even though the culture medium prepared with brine from the investigated salt ponds should enable a greatly improved recovery of genera and related organisms from saltern brines(Wais, 1988).
Halomonas sp. was the most dominant and widespread genus among the isolates. It has been reported that some Halomonas species, i.e. H. elongata, can live in a wide variety of salt concentrations of 0.05–3.4 mol/L(salinity of 30–200) through increasing cell wall structure integrity and the amount of negatively charged lipids(Vreel and et al., 1980). In our study, eight Halomonas sp. strains were obtained at a salinity range of 100–200, accounting for 30% of the total isolates. The high recovery rate of this species can be explained by the easy cultivation and high numbers of this species in a wide range of salinities. Moreover, six colonies recovered from 250 brine were all identified as Halorubrum sp. This is in agreement with Pašić et al.(2005), who considered Halorubrum a fast-growing and widely distributed halophilic archaea in crystallizers. It should also be mentioned that the bacterial and archaeal strains isolated by traditional cultivation approaches cannot provide actual information on the structure of microbial communities in the salterns. Cultureindependent molecular approaches are required to investigate the actual biodiversity of salt ponds(Yeon et al., 2005).
In our study, most isolated bacterial strains could grow in a salinity range of 50–200 and the optimal salinity for growth was usually lower than that of their original habitat. Therefore, they should be considered moderately halophilic bacteria(RodríguezValera et al., 1985; Yeon et al., 2005). Furthermore, the hypersaline environments in solar saltworks are formed through seawater evaporation, thus the ionic composition of brine is similar to that of seawater, with dominating ions of sodium and chloride and a slightly alkaline pH. Compared with salt lakes, the microbial community in coastal solar salterns may be more similar to communities present in seawater (Yeon et al., 2005). Microorganisms originating in seawater gradually adapted to the hypersaline conditions during the process of seawater evaporation and became broadly salt tolerant.
The strain SM.200-5 was 95% similar to Salimicrobium flavidum sp. ISL-25 in terms of its 16S rRNA gene sequence(Yoon et al., 2009). It could grow optimally at salinity 100 and pH 7, which is consistent with Salimicrobium flavidum sp. ISL-25. However, the assays with the API 20E system indicated that this strain had different characteristics compared with Salimicrobium sp. ISL-2, in terms of producing H2S and acid production through fermenting rhamnose and arabinose. Therefore, strain SM.200-5 could be a new c and idate Salimicrobium species. Further characterization to gain sufficient morphological, physiological, and genomic information is necessary.5 CONCLUSION
In this study, 26 isolates were obtained from the salt ponds ranging from the intermediate to crystallizer ponds. The isolates belonged to the bacterial genera Halomonas, Salinicoccus, Oceanobacillus, Gracibacillus, and Salimicrobium and the archaea genera Halorubrum. Halomonas was predominant in salinity range of 100–200, followed by Halorubrum in salinity 250. The physiological and biochemical characterization of the 10 representative strains revealed that the bacterial strains were moderately halophilic with optimal growth at either salinity 50 or 100, while the archaeal strains were extremely halophilic with optimal growth at salinity 200. A microbial biodiversity study is required for community structure of the salt ponds using culture-independent approaches, such as DGGE analysis.
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