Introduction

The soda lakes environments are extremely productive because of high ambient temperatures, high light intensities and unlimited supplies of CO2 [1]. They are also regarded as naturally eutrophic reservoirs hence they feature considerable microbial diversity [2]. From culture-dependent studies, the soda lakes of the East African Rift Valley have been shown to support a dense and diverse population of aerobic, organotrophic, halophilic, alkaliphilic and alkalitolerant representatives of major bacterial and archaeal phyla [2–5]. A combination of both culture-dependent and culture-independent methods has been used previously to document the microbial diversity of Lake Magadi [6] and some other lakes [7]. Results from the culture-independent studies on Lake Elmenteita pointed towards an underestimation of the microbial diversity [7] when compared to other well-studied lakes such as the Wadi al Natrun in Egypt [8]. Like other environments, Lake Elmenteita has been adversely affected by climatic changes and long periods of drought have led to fluctuation in the water levels. In ecological terms Lake Elmenteita serves as an important refuge for vast flocks of flamingos during the dry seasons. The aim of this study was to assess the microbial diversity of Lake Elmenteita using a culture-independent approach.

Materials and Methods

Study Site

Lake Elmenteita is situated 0°27′S, 36°15′E on the floor of the Kenyan Rift Valley at 1,776 m above sea level and has no direct outlet [9]. The region is characterized by a hot, dry and semi-arid climate with a mean annual rainfall of about 700 mm. Due to the high temperatures; there are very high evaporation rates during the drier seasons leading to a reduction in the total surface area. The present size of Lake Elmenteita is roughly 20 km2 and the depths rarely exceed 1.0 m According to Mwaura [10] the water temperature ranges between 30 and 40°C, the alkalinity of the water is high (1,200 mg CaCO3/l) and the pH is above 9 with a high concentration of carbonates, chlorides and sulphates.

Sampling Site Description and Sample Collection

The sample site, sampling and DNA extraction are described elsewhere [11].

PCR Amplification

Almost full-length 16S rDNA bacterial genes were amplified using the primers bac8f (5′-AG(A/G)GTTTGATCCTGGCTCAG-3′) and bac1492r (5′-CGGCTACCTTGTTACGACTT-3′). These primers for PCR amplification of bacteria were based on the fD1 and rP1 primers described by Weisburg et al. [12] and modified by Sørensen et al. [13]. PCR cycling consisted of a 3 min initial pre-incubation step at 94°C followed by 28 cycles of a denaturation step at 93°C for 1 min, a 1 min annealing step at 58°C, and a 1 min elongation step at 72°C and a final extension step at 72°C for 5 min. The PCR mix consisted of 5 μl of 10× PCR buffer [100 mM Tris–HCl (pH 9), primers at a concentration of 0.5 mM, each deoxynucleoside triphosphate at a concentration of 200 mM, 2.0 mM MgCl2, 20 ng of bovine serum albumin (BSA), 0.5 μl of template DNA, and 2.5 U of Taq DNA polymerase (Roche). The volume was adjusted to a final volume of 50 μl with sterile MQ water. The presence of PCR products and their concentration were determined by analysing 5 μl of product on 2% agarose gels after staining with ethidium bromide. The products were compared with a molecular weight marker (Smartladder; Eurogentec). The ladder has a molecular weight ranging from 200 to 10,000 bp and is used for easy quantification as well as size determination of DNA.

Clone Library Construction and ARDRA Screening

These methods were done as previously described [11]. The presence of clone inserts with the expected size was determined by PCR using the primers M13F (5′-GTA AAACGACGGCCAG-3′) and M13R (5′-AGGAAACAGCTATGAC-3′). These primers flank the cloning site on the vector. The PCR products were checked on 1% agarose gel. Further screening of PCR fragments was done via ARDRA to determine unique pattern profiles, representatives clones of which were selected for subsequent sequence analysis. A double digestion was done using the restriction enzymes EcoNI and StuI (New England Biolabs [NEB], Beverly, Mass.). The fragments were then separated on 2% agarose gel at 37°C for 2 h and 100 V. Representative profiles were picked and the PCR amplicons purified with QIAquick® spin columns (Qiagen, Hilden, Germany).

Sequencing

Partial sequences were generated using the primer 518r which targets one of the conserved regions. The reads were manually edited and the sequence data were BLAST (www.ncbi.nlm.nih.gov/BLAST/) analyzed against the GenBank 16S rRNA database. The sequences were then aligned using the CLUSTAL W program against the nearest neighbours [14], and checked for chimeric structures by using the Mallard program [15]. Phylogenetic relationship of the partial sequences was determined using neighbour-joining [16] and maximum-likelihood analyses [17]. These analyses were conducted in MEGA 4 [18]. The evolutionary distances were computed using the Maximum Composite Likelihood method [19]. The resultant tree topologies were evaluated in bootstrap analyses of the Neighbour-joining method based on 1,000 re-samplings [20]. Only representative partial sequences are indicated in dendrograms.

Nucleotide Sequence Accession Numbers

A total of 560 clone sequences that could be properly aligned to the closest neighbours without distorting the phylogenetic trees were deposited in the GenBank under accession numbers FJ764199–FJ764759.

Results

A total of 1,663 clones were picked (sediment samples: 694 clones; water: 644 clones; dry mud: 325 clones). All the clones from sampling sites 1 and 2 were sequenced in order to obtain high coverage of the bacterial diversity. In addition, ARDRA was done for the samples from sites 1 and 2 and these patterns were used as a reference in selecting representative ARDRA profiles from most of the other sites for subsequent clone sequence analyses. In total 875 clones were selected. Supplementary Table S1 gives a summary of the number of clones picked per site, selected ARDRA profiles that were sequenced per site and the number of non-chimeric sequences per site. Supplementary Fig. S1 shows an example of the range of ARDRA pattern diversity for clones from sample site 2 (ES2-065 to ES2-080), resulting in the formation of 11 unique pattern.

A total of 655 clones gave non-chimeric partial sequences and the affiliation of these clones to different orders of the Domain Bacteria is shown on Table 1. Analysis of the sequences via BLAST showed that 525 (80.15%) sequences were related to as yet uncultured members as recorded in the BLAST database. About 75% of the 655 clones had similarity values between 80 and 97% to both cultured and as yet uncultured microorganisms. Eighty-five clones (13%) had a similarity value of 98%, whereas only 45 clones (6.9%) had similarity values between 99 and 100% to cultured and uncultured microorganisms, indicating that they may be affiliated to described species. The four dominant phyla determined by in the clone library analyses were the Cyanobacteria (24.4% of sequences), Firmicutes (18.4%), Proteobacteria (16.8%) and Bacteroidetes (17.4%).

Table 1 Summary of phylotypes represented in the 16S rRNA gene clone library
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A few unique groups were site-specific: clones only retrieved from the hot spring sampling site 2 include members of the orders Methylophilaceae (13 clones), Polyangium (1 clone), Thermosynechococcus (14 clones), Chloroflexi (1 clone) and from the phylum Acidobacteria (7 clones). From water sample W2, only clones related to Limnothrix (5 clones) were retrieved. All the 13 clones related to the phylum Actinobacteria were from water of sampling site 1. All the other clones were retrieved from two or more samples.

Predominance of Different Groups

The Phylum Cyanobacteria

As judged from the 160 sequences affiliated to Cyanobacteria, these aerobic phototrophs comprise the most abundant group of microorganisms within the soda lake environment. Four orders of Cyanobacteria were represented with clones originating from members of the genus Synechococcus being the most common (Table 2). Cyanobacteria groups reported so far from Lake Elmenteita include Arthrospira spp., A. fusiformis, A. arnoldii, Synechococcus spp., Synechocystis spp., Spirulina subtilissima, Spirulina subsalsa and Pseudanabaena spp. ([4, 5, 9, 21–24], respectively). Except for the latter taxon members of these genera were also retrieved in this study which increased the range of diversity significantly by reporting the presence of additional 11 genera. Figure 1 shows the phylogenetic affiliation of representative clones to other groups in the phylum.

Table 2 Cyanobacterial groups represented in the 16S rRNA gene clone library for Lake Elmenteita
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Fig. 1
figure 1

16S rRNA gene sequence relationships of taxa in the phylum Cyanobacteria. Clones from Lake Elmenteita are indicated in bold font. ES wet sediment, EW water, ED dry mud, U uncultured

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The Phyla Firmicutes, Spirochetes, Actinobacteria and Planctomyces

Of the 121 clones affiliated to the phylum Firmicutes, the majority of 85 clones were affiliated to the Clostridiales. Also present were clones from the orders Lactobacillales (15 clones), Dethiobacter (8 clones), Bacillales (7 clones), Thermoactinomycete (3 clones) and Thermoanaerobacter (3 clones). Clones belonging to the order Clostridiales were found to be related to the genera Alkaliphilus, Anoxynatronum, Clostridium and Sporacetigenium; the closest affiliates of clones were in most cases clones from diverse environments (Fig. 2b). Four clones related to the genus Bacillus (similarity values 88–98%) and were from hotspring sediment samples and one clone (ED3-020) was from a dry mud sample. Low G+C isolates from soda lake environments are represented especially by Bacillus alcalophilus [25] and Bacillus clarkia group, [3]. Clones related to Alkalibacteria were similar to Alkalibacterium olivapovliticus and clone ED4-015 was related to Isolate WN16 from the geographically neighbouring Lake Nakuru (Fig. 3)

Fig. 2
figure 2

16S rRNA gene sequence relationships of taxa in the phyla Firmicutes and Actinobacteria. Clones from Lake Elmenteita are indicated in bold font. Abbreviationsas in legend to Fig. 1

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Fig. 3
figure 3

16S rRNA gene sequence relationships of taxa in the phylum Planctomyces. Clones from Lake Elmenteita are indicated in bold font. Abbreviations as in legend to Fig. 1

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Only two clones belonging to the phylum Spirochetes were detected and they were related to a uncultured spirochete clone WN-FWB from the Wadi An Natrun, Egypt [8]. Spirochaeta halophila, a facultative anaerobe, was also previously detected in clone libraries of Wadi Al Natrun, Egypt [8]. The species was fist isolated from lake Eilat in Israel. This indicates that members of this species are present in the soda lakes and probably participate in the sulphur cycle through anaerobic sulphate reduction [26].

Thirteen clones were affiliated to uncultured members of the family Microbacteriaceae in the Actinobacteria. All these clones were affiliated to as-yet uncultured bacteria. Within the Planctomyces, 18 clones were retrieved. Five of the clones aligned to the Pirellula group, eight to the Blastopirellula and one clone formed a deep branch within the Rhodopirellula. Clone EW-053 was affiliated to Planctomycete str. 449 [27]. Two clones (ES1-102 and EW2-020) were not affiliated to any cultured planctomycete. Clone ES2-165 was related to a clone retrieved from the Yellowstone National Park.

The Class Alphaproteobacteria

A total of 44 clones were affiliated to members of the class Alphaproteobacteria. The most common representatives of this group are related to members of the genera Rhodobaca and Rhodobacter. 17 clones were related to Rhodobaca bogoriensis. Ten clones were related to Rhodobacter, six of them aligning to uncultured members of this genus. Other genera represented include Mesorhizobium (clone ES1-022), Rhodoblastus (clone ES1-09), Methylobacterium (clone ED5-027), Erythrobacter (clones EW4-036 and EW2-036). The genera Sphingomonas, Rubrimonas and Sandracinobacter were represented by one clone each. Four clones affiliated to the Methylococcales were retrieved from the wet sediments. Several clones in this group especially those from the hotspring (sampling site 2) show phylogenetic affiliation to phylotypes retrieved from similar environments around the world (Fig. 4).

Fig. 4
figure 4

16S rRNA gene sequence relationships of taxa in the class Alphaproteobacteria. Clones from Lake Elmenteita are indicated in bold font. Abbreviations as in legend to Fig. 1

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The Class Betaproteobacteria

A total of 38 clones were affiliated to members of the class Betaproteobacteria (Fig. 5). All the clones in this group were from the water samples. BLAST analysis showed that two clones from the sampling site 2 were similar to uncultured members of the genus Macromonas [28]. Seventeen clones were affiliated to uncultured members of Hydrogenophaga. Clone EW2-021 was found to be affiliated to Aquabacterium citratiphilum [29]. Others clones were affiliated to the genera Alcaligenes and Lautropia and the family Methylophilaceae was represented by six clones all affiliated to uncultured members of the genus.

Fig. 5
figure 5

16S rRNA gene sequence relationships of taxa in the class Betaproteobacteria. Clones from Lake Elmenteita are indicated in bold font. Abbreviations as in legend to Fig. 1

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The Class Gammaproteobacteria

A total of 20 clones representing various groups were retrieved (Fig. 6). The order Xanthomonadales was represented by three clones affiliated to as yet uncultured bacteria. Other genera detected were Nitrosococcus (7 clones) and Thioalkalivibrio (1 clone). Four clones affiliated to the family Ectothiorhodospiraceae were identified. Clone ED5-037 was closely related to Ectothiorhodospira shaposhnikovii while clone EW4-003 and EW2-059 were related to Ectothiorhodosinus mongolicum isolated from a Mongolian soda lake. Three clones from the hotspring samples were related to Thiothrix nivea [30]. This genus comprises filamentous sulphide oxidizing bacteria.

Fig. 6
figure 6

16S rRNA gene sequence relationships of taxa in the class Gammaproteobacteria and Deltaproteobacteria. Clones from Lake Elmenteita are indicated in bold font. Abbreviations as in legend to Fig. 1

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The Class Deltaproteobacteria

Eight clones were affiliated to members of the class Deltaproteobacteria (Fig. 6). Clone S2-056 from the hotspring sample was closely affiliated to Polyangium. Other taxa include Bacteriovorax (EW4-075), Desulfobulbus (ED5-036) and Desulfosarcina (ES1-119). Clone ES1-134 was closely affiliated to Desulfonatronum cooperativum (50) and D. thiodismutans [31], the latter being a sulphate reducing bacteria isolated from Mono Lake California. Three clones were affiliated to unclassified Deltaproteobacteria.

Phylum Bacteroidetes, Chloroflexi and Chlorobi

Hundred and fourteen clones belonging to the phyla Bacteroidetes, Chloroflexi and Chlorobi were retrieved (Fig. 7). The Chitinophaga-Flexibacter clone sequences were from sediment sample, site 1. All clones in the family Saprospiraceae aligned to members of Haliscomenobacter and all of them were from the hot spring sediment sample. Three clones from the hot spring were affiliated to Sphingobacteriaceae. Eight clones from the Saprospiraceae, four from Lewinella and seven from the phylum Chlorobi formed distinct clades which were only distantly related to other members of their respective groups. Phylogenetic analysis reveals similarity in the habitat from which the clones were retrieved. Three clones were affiliated to Chloroflexi group. “Candidatus Chlorothrix halophilus” is an obligately anaerobic sulphide-dependent phototrophic green nonsulfur bacterium that was cultured from a hypersaline microbial mat [32].

Fig. 7
figure 7

16S rRNA gene sequence relationships of Taxa in the phylum Bacteroidetes, Chlorobi and Chloroflexi. Clones from Lake Elmenteita are indicated in bold font. Abbreviations as in legend to Fig. 1

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Discussion and Conclusion

The results from this study suggest that the bacterial diversity within Lake Elmenteita is significantly higher than reported before [7]. This is confirmed by the finding that of the 655 clones sequences selected for analysis, 525 (80.2%) were more closely related to uncultured members of the Domain Bacteria than to described species. Interesting is the occurrence of clones representing members of the class Betaproteobacteria which have not been previously reported from the East African soda lakes [7] or the other soda lakes such as Wadi al-Natrun in Egypt [8]. The functional role of members of this phylum in the soda lake environment needs to be further investigated preferably using a culture-dependent approach. The dominance of Cyanobacteria concurs with earlier reports that the less alkaline lakes such as Lake Elmenteita are usually dominated by dense blooms of Cyanobacteria while the hypersaline lakes occasionally support blooms of both cyanobacteria and alkaliphilic anoxygenic phototrophs belonging to the genera Halorhodospira and Ectothiorhodospira [4, 5, 21, 33, 34]. Members of the latter genus have also been identified, though in small numbers only.

Primary productivity is driven by several groups within the soda lake environment. The Cyanobacteria form the dominant group as shown by their diversity. Organic matter is also produced by anoxygenic phototrophic purple bacteria of the family Ectothiorhodospiraceae [2] as well as the order Chromatiales [35]. Eight clones affiliated to the order Chromatiales were retrieved from the water, dry mud and wet sediment samples. The alkaliphilic Rhodobaca bogoriensis, originally isolated from Lake Bogoria in Kenya, is capable of both phototrophic and chemotrophic growth and is a representative of purple nonsulfur bacteria described from soda lake environments [36]. The filamentous green nonsulfur bacteria are among the most abundant organisms in both oxic and anoxic layers of microbial mats from a variety of environments [37–40]. The occurrence of clones from both Chlorobi and Chloroflexi shows that these groups are present and contribute to primary production in the Lake.

A number of aerobic chemoorganotrophic, alkaliphilic isolates obtained from several soda lakes in East Africa have been studied in detail [3, 41]. Organotrophic bacteria of the Actinobacteria phylum, i.e., Bogoriella caseilytica [42] and Cellulomonas bogoriensis [43] have also been described from Lake Bogoria in Kenya. Others are members of the genus Dietzia natronolimnaea [16], the genera Arthrobacter and Terrabacter [3] from Lake Oloiden, Kenya. The phylum Planctomyces probably contribute to the mineralization of organic material created by phototrophic primary producers. Saccharolytic spirochetes utilize sugars and a limited range of polysaccharides to produce acetate, lactate, ethanol and hydrogen under anaerobic conditions [2]. Two haloalkaliphilic strains Spirochaeta alkalica and S. africana have been isolated from Lake Magadi, and an alkaliphilic species S. asiatica from Lake Khatyn, Central Asia [44]. The Bacilli are aerobic or facultatively spore forming heterotrophs whereas clostridia are anaerobic fermentors and some groups are sulphate reducers. Other heterotrophic groups detected in the clone libraries were from the phylum Bacteroidetes. The orders Sphingobacteriales and Flavobacteriales consist mainly of aerobic heterotrophic organisms while strains of order Bacteroidales are all anaerobic [45, 46].

The occurrence of clones affiliated to the orders Desulfobacterales, Desulfovibrionales and Desulfomonadales and the genus Desulfitibacter confirms the fact that the microbial sulphur cycle seems to be one of the most active in the soda lakes with anaerobic phototrophic purple sulphur bacteria and sulphate reducing alkaliphiles as the main actors [47]. It has been proposed that sulphate reduction is responsible for generating alkaline conditions as a result of transformation of sulphate to sulphide [2]. The genus Methylobacterium contains strictly aerobic, facultatively methylotrophic bacteria that are able to grow on C1 compounds more reduced than carbon dioxide as the sole carbon and energy sources [48]. In spite of the limitations of the molecular approach such as differential DNA extraction, primer selectivity, and variable rRNA gene copies [49], the approach remains as an effective method to identify the most prominent microorganisms in a natural environment. From the data obtained in this study it can be concluded that the microbial communities within the soda lakes are complex and major trophic groups are represented. Novel approaches for enrichment and isolation will further deepen our understanding of the roles played by the different groups within the ecosystem.