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Polyphasic Approach to Understand Bacterial Community Landscape in Soda Lake Environment

Vishal Dhundale, Vijayshree Hemke, Dhananjay Desai, Samruddhi Joshi, Pranali Shete*, Gurjinder Kaur

Smt. Chandibai Himathmal Mansukhani College, Ulhasnagar, Maharashtra

Abstract: Scientific advancement in exploration and exploitation of microbial community structure is fundamentally driven by discoveries and development of new technologies. This review summarizes the techno-scientific advancement based exploitation of the bacterial diversity of saline and soda lakes. Advancement in DNA based technologies have become a paradigm shift in microbiome community mapping of extreme environment. Further, we focused on alternative modern techniques that can be used as for the identification of bacteria and classification of bacterial diversity, as well as highlighted general description of hypersaline and soda lakes as microbial habitats based on molecular techniques. However molecular methods are detailed briefly as are the standard techniques for assessing genome content for taxonomic classification of bacterial diversity. This review provides advance microbial techniques insight for exploitation of microbial community landscape from extreme water bodies.

Keywords: polyphasic approach, extremophiles, soda lake, microorganism

Received July 02, 2021; Revised August 03, 2021; Accepted August 11, 2021

Citation: Vishal Dhundale, Vijayshree Hemke, Dhananjay Desai, Samruddhi Joshi, Pranali Shete, Gurjinder Kaur, Polyphasic Approach to Understand Bacterial Community Landscape in Soda Lake Environment. Applied Ecology and Environmental Sciences, 9 (8). 724-734. January 2021. doi:10.12691/aees-9-8-3

1. Ecological Importance of Soda Lake

Chemical composition of Soda Lake

Soda lakes are the most stable and productive naturally occurring alkaline environments in the world, with pH values generally higher than 10 and occasionally reaching 12. These alkaline environments are occurred by a combination of geological, geographical and climatic conditions. Soda Lake environments occur naturally in the world and these environments are described by their favorable with highly basic pH values ranging from 10-12. These environments exist due to the geological, geographical and climatic condition give to the saturation of sodium carbonate and sodium chloride which leads to generate soda lake environment [1]. Soda lakes are characterized by the presence with combination of a high concentration of sodium carbonate and Sodium chloride formed by evaporative processes, and are also associated with low concentration of both Mg²⁺ and Ca²⁺ ions [2]. The microbes present in such Soda Lake environments perform a crucial role in the remineralization of organic matter within the ecosystem. In the transformation of organic carbon, inorganic phosphorous, sulfur, nitrogenous compounds and metals with an important role in food webs and nutrient cycling contributed by microbiome.

Soda Lake environment often harbours remarkably high microbial cell densities and are biologically very productive ecosystems [3]. To evolve to such conditions, bacteria have exhibit several processes to exist in extreme soda lake environment. Exploring the bacterial diversity and understanding their process of adaptation into extreme environment and allow to development of investigation concerning the circumstances need for the origin and diversification of life on soda lake environment which are exist all over the world.

2. Concept of Microbial Community in Soda Lake

Importance of microbial ecology

Culture dependent approach of microorganisms is more important than the sequence data to understand the roles and importance of microbial communities in the extremophilic environment. Microbial isolates are important for understanding of bacterial genetics, physiology, and ecology [4,5]. The enrichment strategy is most important towards the study of bacterial diversity than the uncultured techniques. However, as this medium is rich in organic carbon, microorganisms which grow faster in liquid medium than slow growing on solid medium (Agar Medium). Previous studies of isolation of bacteria which were achieved by alkaline media described by Horikoshi (1999) [6]. The selection of culture media is very important for the isolation of the bacteria. The media selections are depend on the physicochemical parameter of water, sediment and soil samples [7]. The optimum isolation of bacteria is totally depending on the carbon, nitrogen, inorganic and organic content of the media (Table 1). However, the media contained ingredients dissolved in distilled water. Glucose, NaCl, Na₂CO₃ and trace element solutions required autoclaved separately and added to the organic components at 60°C before utilization of the medium to avoid the denaturation of medium content [1]. For the optimum isolation of bacteria, the enrichment of culture is most important. For these enrichment, media is prepared with the filtered soda lake water may be applicable for novel bacteria isolation for biotechnological potential.

Table 1. Table describes different media composition used in this study for isolation and enrichment of bacteria

How and at what extent the soda lake ecology or microbial community is different from normal environment

To investigate the structural role of microbial diversity, sequence data are not enough, but culturable ability of microorganisms is very important for physiological and metabolic studies. Archaea and halo-alkalophiles are mostly found in soda lakes. High salinity and large amount of carbonates makes it unique ecosystem. Soda lakes are formed in the areas rich in CO2 and lacking Magnesium and Calcium. Sodium bicarbonate get formed due to increased evaporations, and the environment becomes anionic. Presence of these compounds makes the environment of soda lake suitable for halo-alkalophiles to grow there [8]. When compared with other environments, soda lakes are rich in prokaryotic communities. A few eukaryotes are present in these environments which may reduce with increasing salt concentrations [9]. Studies show that bacterial population and algal biomass in fresh water lakes are much more affected by grazers than those in soda lakes. Similarly in other lakes such as oligotrophic and mesotrophic lakes the response of bacterial populations to grazers is same as that of fresh water. This may happen due to the types of flagellates present in fresh water and oceans [10]. Soda lakes have been formed in archean times and many alkaliphilic species have evolved in these ecosystems. Most of the soda lakes are redish or greenish in colour because of microbial communities present in it [11]. The colour of soda lakes is a result of primary productivity of these lakes. Cynobacteria are mainly responsible for the primary productivity. At medium salinity cyanobacteria are dominant whereas green algae can survive at higher salt concentrations [8]. Archeal communities are also present in large amount at higher salt concentrations [11]. The usual red colour of soda lakes is due to haloalkaliphilic Archaea. Alkaliphiles are not restricted to soda lakes and are present in other environments also. But few microbial communities such as haloalkaliphiles are unique to soda lake ecosystems. As methanotrophs are unable to grow at high salinities, carbon and nitrogen cycles are partly inhibited in soda lakes. Still methanogenesis as well as nitrogen fixation takes place in soda lake environments. Nitrogen is fixed by haloalkaliphilic anaerobes. The conversion of ammonium formed during nitrogen fixation is oxidized to nitrate by nitrifying haloalkalophiles. Sulfur cycle is major process occurring in soda lakes. Sulfur reducing bacteria (SRB) are dominantly present in these environments and lead to sulfur cycling. These belong to the group Desufonatronum or Desulfonatronovibrio. The bacteria of these genera carry out sulfidogenesis. The formed sulfide then oxidized to sulfate by sulfur oxidizing bacteria (SOB) [8].

Dominated taxa / who have researched and found the community of microbes:

Halophiles and alkaliphiles are vigorously grown in soda Lake environments. Soda lakes are widely distributed; however, as a result of their inaccessibility, few such lakes have been explored for bacterial diversity. The bacterial diversity of these lakes, which is considerably phylogenetically diverse, includes Archaea, Cyanobacteria, Proteobacteria and Firmicute [11,12,13]. The well-studied soda lakes are those of the East African rift valley. Some alkaliphilic bacterial diversity of East African Soda Lake has studied for the aerobic archaea and chemoorganotrophic bacteria [14]. Bacterial diversity were studied from Mono lake in California with respect to depth distribution and the data obtained from these studies were belong to—α- and β- Proteobacteria, CFB, High G+C, Low G+C—plus Chloroplasts and Candidate divisions [15]. Hyperalkaline spring waters in Maquqrin (Table 2), Jordan were studied for diversity and activity of bacteria. Kenyan soda lake was studied by molecular methods with their biotechnological potential [16,17]. Lake Elmenteita harbours phylogenetically diverse groups of Cyanobacteria Firmicutes, Betaproteobacteria and other bacteria involved in complex metabolic interactions within the Lake’s ecosystem [18]. The Soda Lake has been adversely affected by climatic, geological, geographical changes and long periods of drought leads to change in physicochemical properties which affect on the bacterial diversity.

Importance of soda Lake ecology

Alkaliphilic bacterial diversity has attention toward scientific community due to ability to produce extracellular, intracellular metabolites and extremozymes which are stable at high pH [19]. Generally Haloalkaliphiles are extensively studied which focused on bacterial classification and genetic analysis, with limited work to discover their industrial application of these extremophiles. With increasing importance towards the environmental protection and decreasing pollution for these situations, the use of extremozymes and different metabolites gained considerable attention in the last few decades. The priorities of cultivability of microorganisms are most important for cellular studies and molecular studies. Extremophiles which produces a great extent of thermohaloalkali-stable enzyme and proteins are suitable and serve in commercial enterprises. Studies show that East African soda lakes consist of phylogenetically diverse group of prokaryotes. Amylase and Lipase producers are found in Kenyan Soda Lake. Similarly, in Lonar Lake amylase and chitinase producing microbes are found along with protease producers [20,21,22,23]. The bacterial diversity of Soda lake were well studied from all over the world with modern techniques but application of these diversity is not studied in detail so far.

Table 2. Globaly distribution of soda lakes and soda desert

3. Recent Overview of Soda Lake Microbial Ecology Research

Techniques for identiﬁcation of the bacteria:

The traditional method for performing this classification task is dependent on the comparison of standard morphologic and phenotypic description of species with the accurate morphologic and phenotypic description of the isolate. Phenotype includes morphological, physiological and biochemical characters of the bacteria. Traditional phenotypic tests used in classical microbiological laboratories for this kind of analysis include characteristics of the organism on different growth on carbon and nitrogen sources, growth of bacteria on different conditions of pH, temperature and NaCl. The disadvantages with phenotypic methods are the gene regulation wherein the identical bacteria show different phenotypic characters in variable environmental conditions. However, the conventional bacterial classification methods based on the morphology, physiology and biochemical test were time consuming and not fully reliable. But at the same time it is necessary for the exact identification of the bacterial strain in combine with molecular techniques [7]. Dendrogram for the cluster analysis should be performed using different measure of similarity and distance and different clustering algorithms.

4. Identiﬁcation of the Bacteria on the Basis of Genetic Marker

Microbial taxonomy or microbial systematics deals with the classiﬁcation, identification, nomenclature of microorganisms and taxonomy is the theory and practice of classifying organisms. Systematic refers to the study of diversity of organisms and all relationships among them including their evolutionary relatedness (phylogeny) and all possible biological interactions based on genetic marker. From the last few decades a new standard techniques are developed for bacterial identification based on 16S rRNA this marker was generally used for phylogenetic relationships, identification and taxonomic purpose of bacteria (Figure 1) [24]. This is an area in which 16S rRNA gene sequence identiﬁcation might have strong effect on soda lake bacterial diversity. However, identiﬁcation of these organisms which survive in Soda Lake by phenotypic methods is difficult and subject to error. The 16S rRNA gene sequence analysis provides accurate identiﬁcation at the species level. Tambekar and Dhundale (2012) [19] compared a variety of identiﬁcation systems including phenotypic, sugar fermentation, and conventional biochemical identiﬁcation with the 16S rRNA gene sequence to evaluate aerobic gram- positive bacilli isolated from Lonar soda Crater Lake. They found that 16S rRNA gene sequence provided more rapid, correct identiﬁcation of the difficult to bacterial isolates than did conventional methods and found that only a minority of the soda lake isolates of aerobic gram-positive bacillus could be correctly identiﬁed by phenotypic methods whereas rRNA gene sequencing is an excellent method for identifying these organisms, which are difficult to identify by traditional methods. Thus, Proteobacteria and Firmicute were an important group of organisms, in early important studies establishing the usefulness of 16S rRNA gene sequencing for soda lake bacterial diversity. In all of these studies, the accuracy of 16S rRNA gene sequencing in the identiﬁcation to the strain level was better than to phenotypic methods and traditional methods. Overall, by providing for the accurate identiﬁcation of species in the database and the taxonomic placement cannot complete identiﬁcation of novel species, 16S rRNA gene sequence analysis of organism seems to be the most precise method available. The phylogenetic tree revealed the genus to which the strain belongs and its closest neighbors i.e. those sharing the clade >97% 16SrRNA gene sequence similarity are obtained from various culture collections to execute advanced genotypic analysis. The 16S rDNA sequence of some isolates from Lonar Lake showed the 93% and 96% with Planococcus maritimus and Bacillus cohnii revealed that less than 97% similarity with the previously known culture. This could be the novel bacterial species require and confirmed by the chemotaxonomic and DNA DNA Hybridization technique etc [25].

DNA DNA Hybridization (DDH):

DNA–DNA hybridization (DDH) values have been used by bacterial taxonomists since from the few decades to investigate the relatedness between species, strain and yet most important technique and criteria in bacterial classification [26]. The regulatory, structural, functional and taxonomic information about a bacterium is incorporated in the nucleic acid of its genome sequence [27]. DDH technique has been used to investigate relatedness between species and this technique was universally accepted for genome-wide comparisons between bacteria. The recommended value of DDH for classification of species is 70% [28]. But this value is not a standard one; it may differ. But all prokaryotes cannot be identified using DDH. Using this technique only closely related species can be differentiated. As it is expensive and complicated technique, not used for routine identification.

Denaturation Gradient Gel electrophoresis:

Denaturation Gradient Gel electrophoresis (DGGE) is a promising technique has been used by many researchers to estimate bacterial diversity scenario of Soda Lake [17,29]. It gives information about the microbial communities and need not cultivation of bacteria [30]. In this technique, DNA can be recovered from gels which will then be used for sequencing. When it is used in combination with PCR, complex microbial communities can be easily identified. For this, V2 – V3 regions of 16S rDNA is amplified and then separate bands obtained on DGGE. Studies show that using these technique microbes can be identified up to strain level also (Figure 2). DGGE is not helpful for lower number of microorganisms [31]. Here polyacrylamide gel is used which contains denaturating agents. Partially melted double stranded DNA samples are separated on it by decreasing their electrophoretic mobility. These DNA molecules migrate up to certain distance and then stop depending on their melting behavior. Visualization of these bands can be done by staining with ethidium bromide. But melting temperature of the DNA fragments should be checked before DGGE [32]. Sulfate reducing bacteria present in various soda lakes of Russia were identified by DGGE using 16S rRNA genes. This is then followed by phylogenetic analysis. According to DGGE analysis, two types of sulfur reducing bacteria: alphaproteobacteria and Brevundimonas are present in that lake. Members of Delta and gamma proteobacteria are found at higher salinities [33]. In Kenyan Rift valley soda lakes, different DNA showed the same bands which clearly indicated that the microbial community is more complex than showed by DGGE analysis. However, Flavobater and bacteroides are group of microbes found in those soda lakes which were analyzed by DGGE [17]. Though this technique is useful, there are some disadvantages too. It may show amplification errors, contamination in isolated DNA samples as well as in PCR. Another major disadvantage of this technique is only small fragments of DNA (less than 500 bp) can be separated. These problems can be overcome by third as well as fourth generation sequencing techniques.

Figure 1. It describes about different techniques used for identification of microbial community and their classification in various taxonomic ranks

Figure 2. It describes how the diverse group of culturable bacteria are analysed and identified using different molecular techniques

G+C content

Density gradient centrifugation and thermal denaturation techniques were used previously for GC analysis. High performance liquid chromatography is a technique which uses deoxynucleotides to determine G+C content of DNA [34,35]. GC content is the characteristic feature of bacterial genome. Coding DNA occupies major part of the genome. Studies show that GC content is about 5% more in coding region as compared with non coding region of DNA. GC content is useful for taxonomical identification of Bacteria [36]. Thermal denaturation and subsequent renaturation technique is used to determine G+C content of DNA. Genomic size, exposure to oxygen and habitat of microbes are dependent on G+C content [37]. AT rich bacterial genomes are smaller in size while genomes with higher GC content are larger. Availability of nitrogen affects the GC content; increase in nitrogen increases the GC content in DNA. High coding density is found in bacterial genomes but when compared to eukaryotes, variation is less [37,38,39,40]. According to Genbank database, the GC DNA content varies from 16.6 mol% in Carsonella species to 74.9 mol% in Anaeromyxobacter species [37]. Soda lakes act as suitable habitat for a variety of microorganisms belonging to gyamma and delta subdivisions of Proteobacteria [41]. These are mainly Gram positive bacteria and show high and low G+C content [42]. Studies show that methylophaga species differ from other methylobacteria by low G+C content and found to be 48.3 mol% [43]. In Lake Magadi, a halobacterium species has been isolated which differs from other halobacterium in their GC content. It is analyzed by using buoyant density and melting point techniques and found to be 59 mol% which is very low as compared with other halobacterium species [44]. Similarly, in another study a novel alkalophilic, fermentative bacteria named as Tindalliamagadii from same lake was analyzed by GC content (37.6 mol%) and it was belonging to low GC subphylum of Gram positive bacteria [45]. Methylomicrobium buryatense is a novel bacteria isolated from a southeastern soda lake of Trasbaikal region and identified from its percent GC DNA content which is reported as 49.6 – 51.5 mol% [46]. The GC content of the bacterium Bacillus aurantiacus is determined by deoxyguanosine to thymine ratio and was about 42.9mol%. Through this overview of this study it can be concluded that GC content analysis is not enough for identification of prokaryotes, it should be followed by 16S rRNA sequencing [47]. However, G+C DNA content of different species can show similar bands which give false results [48]. Hence more advanced techniques such as NGS should be used for identification of microbes.

Next Generation Sequencing (NGS)

NGS is a technique which mainly includes pyrosequencing and ion torrent. Pyrosequencing is one of the advanced technique which gives detailed information about diversity of such environments. As compared with Sanger sequencing, bar code tag primer based pyrosequencing is cost effective and can give the classification of microbial community up to genus level. It is a bioluminescence technique in which inorganic pyrophosphate is released producing colour [49]. In pyrosequencing, dNTPs are added only one at a time. If the dNTP added is complementary to the base in template DNA pyrophosphate is released. This released pyrophosphate can be converted to ATP by enzyme ATP sulfurylase. This ATP along with luciferin and oxygen is further utilized by enzyme luciferase which results in light formation. Light emission is detected by flowgram. And if the dNTP is not utilized, the pyrophosphate is removed by apyrase enzyme. Pyrosequencing allows sequencing of different template in single run [50]. Pyrosequencing is used for characterization of bacterial and archaeal communities present in alkaline environment. Ikaite columns are very similar to soda lakes. The microbes found closely related groups of alkalophiles which are present in soda lakes. In ikaite columns, cyanobacteria are mainly found at surface while heterotrophs and chemolithotrophs are found at the bottom [51]. In Mexican Isabel soda lake sequence analysis by pyrosequencing show that Halotolerant as well as Halophilic bacteria are dominant [29]. During DNA polymerization, hydrogen ions get released and are detected by a chip using Ion Torrent sequence technique. Ion Torrent instrument can sequence megabases of raw sequence reads on a single chip [52]. During the process, pyrophosphates are released and simultaneously hydrogen ions get removed from 3’ end and are detected by the sensor. Natural nucleotides are used in this technique [53]. 16S rRNA gene sequencing using Ion torrent system explained a diverse and distinct group of bacterial fauna in both sediments and water samples with dominance of the phyla Gammaproteobacteria, Alphaproteobacteria, Actinobacteria and Firmicutes [54]. Sequencing of a haloalkaliphilic methanotroph isolated from Khilganta Soda Lake was done using ion torrent technique [55]. In case of pyrosequencing, the analysis system may misinterpret the signal density or its relation with nucleotides added. As the process requires sample preparation which includes PCR, it is time consuming [56]. While in Illumina technique, the structure of nucleotide get damaged which indirectly decreases the efficiency of reaction [57].

Fatty acid methyl ester (FAME):

FAME is widely used technique for bacterial identification. Fatty extraction of fatty acid from cells, methylation of fatty acids, extraction of FAMEs and analysis of these FAMEs through Gas Chromatography are the four steps involved in FAME analysis [58]. Methylation includes trans-esterification of acylglycerols and esterification of free fatty acids converting them into FAME. Capillary column makes the separation and identification easy. FAME analysis helps in providing accurate quantification of fatty acids [59]. Esterification reagents are of two different types: Acid catalysed and base catalysed reagents. However, the base catalysed reagents are not useful as they cannot convert free fatty acids to FAMEs. This technique is very useful as it can identify living as well as dead cells [60]. The quantification of FAME is mainly affected by changes in composition of fatty acids during esterification and incomplete conversion of lipids into FAMEs [61]. This technique is very sensitive to changes in community structures and shows specificity in identifying microorganisms. To identify the microbial community of the mierlei lake, ester linked fatty acid methyl esters (EL-FAME) is used and the results show that most of the isolated bacteria are Gram negative in nature. The results of EL-FAME analysis show that the actinomycetes are present in this lake but none of the actinomycetes are isolated from the lake [62]. Similarly, for the Soap saline lake microbes, the structure of amphiphilic siderophores produced by Halomonas and presence of fatty acid chain is analysed by FAME technique [63]. One newly identified strain of Clostridium form Owens lake California, is differentiated from two already known bacterial strains: Clostridium formicaceticum and Clostridium aceticum using fatty acid profiles. The major fatty acids found in the new strain were C14:0, iso-C15:0, C16:1ω 9c and C16:0 [64]. In Lonar Lake, one novel bacteria Indibacter alkaliphilus was found and identified by FAME analysis. It consists of 29 different fatty acids and the dominant fatty acids are C15:0 and C17:0 [65]. Besides all the advantages, it has few disadvantages too.

Chemotaxonomy& Numerical Taxonomy

Bacterial taxonomy started with phenotypic characterization. Later on numerical taxonomy was used for classification. Now for more detailed classification, chemotaxonomy and genotypic analysis are widely used techniques. The present classification system is based on polyphasic approach which includes phenotypic, genotypic, phylogenetic and chemotaxonomic analysis. Chemotaxonomy is a technique used for classification based on similarities and differences in chemical composition of microbes. It gives detailed information about the presence and arrangement of various biochemical compounds in taxonomic ranks of microbes. These compounds act as biomarkers which include cell wall amino acids, proteins, polysaccharides lipids, fatty acids and polymeric compounds. It specifically gives information about differences between species and strains where phylogenetic analysis fails to explain. It is the most important method for polyphasic studies. However, the specificity of chemotaxonomic studies is low as compared with genetic characterization. This is because of the differences in the chemical structures of bacteria [66]. To study alkalophiles as well as halophiles present in Soda Water Lake, chemotaxonomic classification has been studied. In some cases phylogenetic analysis of halophiles is more useful than chemotaxonomic analysis as it showed similarities in taxonomic ranks [14]. Identification of cell wall components such as DAP in peptidoglycan was carried out by TLC. A novel species Streptomyces alkaliphilus was identified and confirmed by chemotaxomonic analysis. Study showed that it consists of LL-Diaminopimelic acid (LL-DAP) instead of meso-DAP which is rare [67]. In another case peptidoglycan of newly identified microbe composed of lysine and glycine. This type of peptidoglycan previously reported in few Corynebacteium taxa and some Bifidobacteium. It is then classified into genus Exoguobacterium[68]. Several types of isoprenoids are found in bacteria, in which most commonly found is Squalene and these isoprenoidsare analyzed by HPLC [69]. Polar lipids present in alkalophiles& halophiles are also identified for chemotaxonomic classification [70]. The technique used for its extraction and separation is 2D TLC [67]. Fatty acid profiles have been well introduced for chemotaxonomy of bacteria [68]. GC is the most useful technique for bacterial classification. Fatty acid methyl ester composition is determined by GC or GCMS [71]. The fatty acid composition can differentiate between facultative and obligate strains. It is found that obligate alkalophiles consists of high amount of branched chain, unsaturated fatty acids as compared to facultative alkalophiles [69]. This technique helps to differentiate any strain from its closely related strains [72]. Many distinct lineages of genus Bacillus have been determined by chemotaxonomic analysis and spore formers kept in separate genera [73]. Studies show that bacteria which primarily classified and kept in genus Microtetraspora and Thermomonospora were then identified to be the members of genus Nonomuraea using chemotaxonomic techniques [74]. However, the chemical structure of microorganisms likely to be affected by culture conditions, pH and other factors. Hence before proceeding to chemotaxonomic analysis it should be confirmed that the optimization of culture conditions has been done [75]. It is technologically challenging as well as demanding technique. However chemotaxonomy has several disadvantages that only culturable microbes can be identified by chemotaxonomic techniques. All the species of bacteria cannot be detected using this technique [58,60]. Using numerical taxonomy, it is possible to quantify the degree of similarity. Numerical taxonomy chooses various characters and uses to evaluate similarities in microorganisms which is then calculated by algorithm [76]. Ultimately, these similarities are used to differentiate the microbes. On the basis of numerical taxonomy, the microbial community from different lakes such as Bogoria, Elementieta, Nakuru and Sonachi were identified [14]. It is more efffecient technique than other traditional methods.

5. Prospectives

In the naturally occurring alkaline environments, Soda Lake are the most stable with pH values generally higher than 10 and occasionally reaching at 12. At the time of alkalinity formation, other salts also accumulate, giving rise to haloalkaliphilic environment in which the native microflora is subjected to a number of extreme ecological pressure. By Traditionally technique, the studies of bacterial communities were limited to a cultured isolates but recent techniques are applicable for whole microbial community from Soda Lake. Very few bacterial isolates are obtained from the soda lake but culture independent techniques have covered diverse microbial community. In future more advanced data will be able to use for classification and novel bacteria will be discovered with advanced tools and database. The improvement in evaluation of genomic level which leads to perfect and reliable classification of bacteria isolated from Soda Lake. The conventional bacterial classification methods based on the morphology, physiology and biochemical test were time consuming and not fully reliable. But at the same time it is necessary for the exact identification of the bacterial strain in combine with molecular techniques. So the identification based on traditional biochemical test cannot be replaced. Soda lake harbors wealth of diverse microorganism and through the culture independent technique, most of the bacteria will be identified but culture base technique is more important due to cellular and molecular studies of bacteria. The systematic studies of bacterial classification which required intellectual and ﬁnancial support for determine the roll of bacteria in ecosystem, diversity, biotechnological potential and environmental science.

Competing Interests

Nil

Ethical Approval

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