Bacilli community of saline–alkaline soils from the Ararat Plain (Armenia) assessed by molecular and culture-based methods

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Abstract

The bacterial community composition in the A horizon of a natural saline–alkaline soil located in Ararat Plain (Armenia) was studied using molecular and culture-based methods The sequence analysis of a 16S rRNA gene clone library and denaturing gradient gel electrophoresis (DGGE) profiles indicated dominance of Firmicutes populations. The majority of the sequences of the bacterial 16S rRNA gene library were close relatives of representatives belonging to the genera Halobacillus (41.2%), Piscibacillus (23.5%), Bacillus (23.5%) and Virgibacillus (11.8%). Eight novel moderately halophilic bacilli isolates were successfully obtained from the enriched cultures of the saline–alkaline soil samples. 16S rRNA gene sequence analyses of isolates revealed their affiliation (97.7–99.7% similarity) to representatives of the genera Bacillus, Piscibacillus and Halobacillus. All isolates were able to tolerate high concentrations of NaCl and highly alkaline conditions. This is the first study combining cultivation-independent and -dependent approaches to reveal the bacterial diversity of the saline–alkaline soils of Ararat Plain and it suggested an important role of bacilli as key microbes in biogeochemical cycles of these environments.

Introduction

Halophiles are found in all three life domains and are distributed in habitats covering a wide range of salinities, such as saline lakes, saline and saline–alkaline soils, solar salterns and salt mines [11], [14], [19], [24], [28], [33]. Halophiles can survive and flourish in environments that limit the growth of most other organisms. Salinity is sometimes associated with alkalinity, and many saline or hypersaline environments have alkaline or extremely alkaline pH values [14]. Saline–alkaline soils differ from each other in terms of the salt concentrations caused by primary or secondary salinization processes, and their chemical compositions that determine the nature of inhabiting microorganisms [3], [33], [14]. Salinity has been found to influence the quantity and activity of microbes, which in turn play a key role in biogeochemical cycles [3], [31]. Despite previous studies on halophilic and haloalkaliphilic bacteria in hypersaline aquatic habitats, the current understanding of moderately halophilic bacteria and their activity and functional role in saline terrestrial environments, particularly saline–alkaline soils, is still limited [3], [14], [21].

Moderately halophilic bacteria are capable of growing at moderate salinities and have the ability to adjust rapidly to changes in the external salt concentration [35]. They constitute a very heterogeneous group of extremophiles that include both Gram-positive and Gram-negative (mainly) heterotrophic bacteria [14]. In contrast to hypersaline aquatic habitats, from which predominantly Gram-negative species have been obtained, saline or hypersaline soils have yielded many Gram-positive species, including species of the genera Bacillus, Halobacillus, Filobacillus, Tenuibacillus, Virgibacillus, Lentibacillus, Thalassobacillus, Marinococcus, Salinicoccus, Nesterenkonia and Tetragenococcus as dominant representatives [6], [14], [34].

The unique properties of many halophiles suggest that they have great potential for biotechnology, as they produce a large variety of stable unique biomolecules that may be useful for practical applications. Stable hydrolytic enzymes (such as DNases, lipases, amylases and proteases), pigments and compatible solutes (stress protectants) produced by halophilic microbes are widely used in the food, chemical, environmental, biofuel and medical sectors [4], [15], [20], [26], [27]. In this sense, the finding of novel enzymes and different compounds produced by halophiles is of great importance. Many enzymes, stabilizers and valuable compounds from halophiles may provide advantages for the development of novel and current biotechnological processes.

The rapid increase in understanding the microbial diversity of hypersaline ecosystems is due to the intensive use of molecular techniques. Using a combination of traditional microbiological methods and state-of-the-art molecular biology techniques has provided the basis for the current understanding of such microbial diversity in high-salt environments [3], [12]. Halophilic microbial communities of saline–alkaline soils have been studied recently in the USA, Spain, China, Egypt, Iraq, Iran, Pakistan and several other regions of the world [6], [34]. Hydromorphic saline–alkaline soils (occupying more than 29,000 ha) with up to 3% salinity and highly alkaline conditions (pH 9–11) are also found in the territory of Ararat Plain, Armenia [2]. The microbial diversity of saline–alkaline soils in Ararat Plain, as well as the microbial processes and key microbes involved in soil formation and biogeochemical cycles, are not yet well characterized [23].

In the present study, culture-dependent and -independent approaches were applied to describe the bacterial diversity in the A horizon of a natural saline–alkaline soil located in Ararat Plain (Armenia). The culture-independent studies involved denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes and the construction of clone libraries to reveal dominant members of halophilic microbial populations. Cultivation efforts included enrichment and isolation of aerobic organotrophic halophilic microorganisms.

Section snippets

Site description and sampling

Hydromorphic saline–alkaline soils occur in the areas of Ararat Plain, Armenia, where subsoil groundwater is mineralized and located 1–2 m below the surface. These soils are found at 850–900 m above sea level and have been formed on alluvial–proluvial stratified sediments from the River Araks. They are characterized by strong salinity (total soil content 1–3%), considerable carbonization, low humus content (<1.0%), high alkalinity (pH 9) and a high absorbed sodium content. The vegetation

Bacterial clone library construction and DGGE

The bacterial diversity in the saline–alkaline soils was examined by analyzing the PCR-amplified 16S rRNA gene library generated from total DNA extracts using universal bacterial oligonucleotide primer sets. A total of 20 clones were obtained, from which three cloned 16S rRNA sequences were found to be chimeric and were excluded from further analysis. Sequence analysis of the remaining 17 clones indicated that they originated from genera belonging to phylum Firmicutes. BLAST results of the

Discussion

Currently, it is widely accepted that the majority of microorganisms in the environment cannot be cultured by using traditional microbiological methods, hence the vast majority of the microbiota remains undiscovered [32]. This limitation has promoted the use of molecular approaches to detect both cultured and uncultured microbes for evaluation of the microbial diversity in ecosystems [13]. Therefore, in this present study a combination of cultivation-dependent and molecular techniques was used

Author contribution

Conceived and designed the experiments: HP, NKB. Performed the experiments: AH, HP. Performed and analyzed the sequencing data: HP, AH. Analyzed and interpreted the data: HP, NKB. Wrote the manuscript: HP. Critically revised the manuscript: NKB, AT.

Human and animal rights statements

This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgement

The financial support of the CPEA-2011/10081 grant from the Norwegian Cooperation Programme in Higher Education with Eurasia (Norway) for carrying out this project is greatly acknowledged.

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    Present address: Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany.

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