Introduction

Microbial isolates are important in expanding our understanding of bacterial physiology, genetics, and ecology (Palleroni 1997; Zinder and Salyers 2001). Most strains isolated from the Kenyan soda lakes belong to the class Gammaproteobacterium, including a large number of aerobic organotrophic strains of the halomonad lineage (Duckworth et al. 1996; Jones et al. 1994). For example, Alkalimonas delamerensis (Ma et al. 2004) and the cyanobacteria Arthrospira fusiformis and A. abijatae (Ballot et al. 2004) were isolated from Lake Elmenteita, while Anabaenopsis arnoldii was described in the phytoplankton communities of Lakes Nakuru and Elmenteita (Vareschi 1978; Melack 1988). Several alkaliphilic saccharolytic clostridia were reported to thrive in lakes Elmenteita, Bogoria and Magadi (Jones et al. 1998). Previous attempts at isolation have been based on the alkaline media described by Horikoshi (1991). However, as this medium is rich in organic carbon, heterotrophic microorganisms which grow faster most probably mask other slow growing organisms on a solid medium. The objective of this study was to enrich for and isolate novel groups from Lake Elmenteita for subsequent investigation of their biotechnological potential, using media prepared with lake water.

Materials and methods

Lake Elmenteita is situated at 0°27′S, 36°15′E on the floor of the Kenyan Rift Valley at 1776 m above sea level and has no direct outlet (Melack 1988). The region is characterised 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 (1999) the water temperature ranges between 30 and 40°C, the alkalinity of the water is high (1200 mg CaCO3/l) and the pH is above 9 with a high concentration of carbonates, chlorides and sulphates. The sampling sites for this study are described elsewhere (Mwirichia et al. 2009).

Enrichment and isolation of microorganisms

The media used for isolation were prepared with water collected from Lake Elmenteita. The water was filtered through a 0.45 μm and then through a 0.22 μm membrane filter (Whatman). The pH of the water from the lake was established to be 8.7. An enrichment strategy was established whereby 14 different liquid media were prepared using the filter-sterilised water collected from the lake. Five additional media were prepared targeting isolation of myxobacteria as described by Shimkets et al. (2006). The various media used in enrichment and isolation are shown in Table 1. Mud and water samples were collected from five different sampling points. Approximately 2 g of mud from each site was used to make a mastermix using filtered and sterilised 50 ml of water from the lake. An aliquot (100 μl) was used to inoculate 5 ml of each of the liquid media and grown for 24 h. These enriched samples were plated onto the respective solid media and allowed to grow for 24 h at 28°C. A second aliquot (100 μl) was plated directly onto solid media and allowed to grow for 48 h at 28°C. Strains obtained were purified on tryptic soy broth supplemented with 15 g Bacto agar (Difco), 3.5% NaCl and 1% NaCO3. The strains were finally stocked in tryptic soy broth supplemented with 3.5% NaCl, 1% NaCO3 and 20% (v/v) glycerol.

Table 1 The different media used in this study for enrichment and isolation of bacteria
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Screening for enzymes

Utilisation of various polymers which is an indication of the enzymes produced by an organism was assayed on a basal media containing per litre 1 g yeast extract (Difco), 1 g KH2PO4, 0.1 g MgSO4·7H2O, 0.05 g CaCl·2H2O, 4% NaCl, 1% NaCO3 and 15 g agar. The following substrates were added separately: starch 10 g (Merck), carboxymethylcellulose 10 g (Serva, Heidelberg), xanthan 5 g (Sigma) and xylan 2.5 g (Fluka). Esterase/lipase activity was tested using olive oil (domestic grade) at 3 ml/l. These tests were done for all the isolates recovered in this study. After 48 h of growth the plates containing xylan, xanthan and carboxymethylcellulose were flooded with Congo red solution (1 mg/ml) and allowed to stand for 10 min. The dye was then replaced with NaCl (1 M) and subsequently rinsed with distilled water. The plates were observed for halos around the colonies, as indication of positive polymer degradation. The starch-containing plates were flooded with iodine solution (Sigma–Aldrich) and observed for clearings around the colonies. Esterase/lipase activity was indicated by formation of precipitate around the colonies. Chitinase activity was tested by use of 4-methylumbelliferyl N-acetyl-β-d-glucosaminide as described by the manufacturer (Sigma–Aldrich).

Molecular identification of the isolated microorganisms

DNA extraction was carried out using the SEQLAB bacteria-DNA-Kit and PCR amplification of the 16S rRNA genes was carried at SeqLab (Göttingen, Germany). Partial gene sequences were obtained using the primer 530 reverse (5′-GKATTACCGCGGCKGCTG-3′). The readings were manually edited and the sequence data was BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) analysed (Altschul et al. 1990) against the GenBank 16S rRNA database. Complete sequences were generated for all those 34 isolates that showed similarity values below 98% in the initial BLAST analysis. Partial and the complete sequences were assembled with the Sequencer software, version 4.1 for Macintosh (Genes Codes, Ann Arbor, MI). Phylogenetic relationship of all the sequences was determined using neighbour-joining (Felsenstein 1985) and maximum-likelihood analyses (Olsen and Woese 1993). These analyses were conducted in MEGA 4 (Tamura et al. 2007). The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al. 2004). The resultant tree topologies were evaluated in bootstrap analyses of the neighbour joining method based on 1000 re-samplings (Felsenstein 1985). Only representative sequences are indicated in the trees.

Nucleotide sequence accession numbers

The partial sequences of all the isolates that were generated using the primer 530r are deposited in the GenBank under accession numbers between FJ763850 and FJ764198. The sequence reads were between 400 and 593 base pairs. The almost complete sequences of the isolates listed on Table 2 are deposited under the accession numbers FJ764760–FJ764793 and the sequence length is between 1340 and 1606 base pairs.

Table 2 A summary of the isolates recovered using the different media
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Results

From a total of 367 isolates obtained in the isolation exercise, 221 isolates were selected for partial sequencing on the basis of colony morphology and for observation of cellular morphology under the microscope. This was done to avoid sequencing several identical isolates from the same media; 181 isolates gave readable sequences of excellent quality representing members of 51 genera in the domain bacteria. Figure 1a, b shows the phylogenetic relationship of representative sequences to the closest neighbours as per BLAST analysis. Due to the large number of closely related isolates in each taxon, only 51 sequences are shown in Fig. 1a, b. However, these represent all the species that were detected in this study.

Fig. 1
figure 1figure 1

a Phylogenetic affiliation of the isolates from this study to other members of 2 phyla; firmicutes and Actinobacteria. Isolates from this study are indicated in bold font. Only bootstrap values above 50 are shown. b Phylogenetic affiliation of the isolates from this study to other members of the class Gammaproteobacterium. Isolates from this study are indicated in bold font. Only bootstrap values above 50 are shown

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Though not more than between two and seven different isolates (representing different species) were recovered from any medium, the medium composition seems to have an effect on the taxa recovered. The distribution of genotypes on the different media is shown in Table 2. Members of the genus Bacillus were mostly encountered on media 3 and 4 and on media 10–14. Medium 12 only supported growth of bacilli. Halomonads were mostly from media 1 to 10 and 15 to 20. Media 15 to 20 were prepared targeting isolation of myxobacteria. Initial results showed the swarming morphology but after sequencing it was found out that these isolates were related to the genus Marinospirillum.

Analysis of the 181 partial sequences showed that 31 isolates had similarities between 97 and 98%, 15 isolates scored between 96 and 97%, while 8 isolates had lower than 96% similarities to their nearest neighbours. The rest of the isolates had similarity values between 99 and 100% to their nearest neighbours. All the 34 isolates that had similarity values of 97% and below were selected for full sequencing. This was to ascertain whether they represented novel phylotypes or not. Results from the 34 isolates that were fully sequenced indicate that some of them may represent novel taxa (Table 3). BLAST analysis of the full sequences indicated that in 26 isolates there was increase in the similarity values, in 6 isolates the values remained the same whereas in 2 isolates (M2-C23 and S-C28) there was a decrease in the similarity values from 100 to 99 and 99 to 98%, respectively. The full sequences confirmed the genera to which the various isolates belonged. It is only in the isolate M16-C8 where there was a change from Brevibacterium (partial sequence) to Vibrio metschnikovii (full sequence).

Table 3 BLAST results of the 34 isolates that were fully sequenced
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BLAST analysis of the partial sequences shows that 109 isolates (60%) belonged to the class Gammaproteobacteria. Among these, 68 isolates were affiliated to the genus Halomonas, 20 isolates were affiliated to microorganisms belonging to the genus Marinospirillum while 15 isolates were related to Idiomarina species. Several isolates from each taxon were full sequences to ascertain their affiliation (Table 3). The isolate (M7-C9) was affiliated to the genus Alkalimonas and the almost complete sequence shows that the closest neighbour is Alkalimonas delamerensis (98%), the type strain of which was previously isolated from Lake Elmenteita. Interestingly, five isolates from the genus Vibrio were closely related to the marine Vibrio metschnikovii.

Only two actinobacterial isolates were recovered and fully sequenced. The first one (M9-C5) was related to Nocardiopsis exhalans while the second (M4-C21) was from the genus Streptomyces and it had 96% similarity to Streptomyces caelestis. The strain had an optimum growth temperature of 39°C and an optimum pH of 8.4.

From the phylum firmicutes, two isolates affiliated to the genus Enterococcus were recovered. The genus Enterococcus contains bacterial species associated with animals and plants. A total of 58 isolates from the genus Bacillus was recovered and this makes it the second most common group after the halomonads. In this study seven isolates were recovered which closely related to Alkalibacterium olivoapovliticus strain WW2-SN4a (AF143511), A. olivoapovliticus strain WW2-SN4c (AF143512), A. psychrotolerans (AB125938), Alkalibacterium sp. G-He1 (EF554904) and Alkalibacterium sp. NP13 (EU196343). One isolate (M7-C13) from the genus Amphibacillus was recovered and it had a 97% similarity to Amphibacillus sediminis. A single isolate (M14-C7) with 94% BLAST similarity to Marinilactibacillus sp. was recovered. The genus Marinilactibacillus (Ishikawa et al. 2003) has only two described species and both are from the ocean.

Enzyme assay

Of the 181 isolates screened, 67 were able to utilise between 1 and 5 of the substrates screened. The phylogenetic affiliation of these isolates was as follows: Bacillus (39), Halomonas (12), Vibrio (5), Alkalibacterium (4), Enterococcus (2), Idiomarina (2) and Alkalimonas, Halolactibacillus and Streptomyces had 1 isolate each. Only Bacillus species were able to utilise carboxymethylcellulose. Among the Bacillus, isolates M13-C22, M14-C13, M14-C3, M10-C9 AND M13-C14 were able to utilise xanthan, xylan, starch, olive oil and carboxymethylcellulose. The highest chitinase activity was recorded among the Vibrio species.

Discussion

The use of different media prepared using water from the lake could have helped retrieve novel taxa not detected in the soda lake before. Of the sequenced isolates, 77 isolates had similarity values ranging 94–98% to cultured members of the domain bacteria. The media composition seemed to have an effect on the recovery of different groups. Most of the Bacillus (40 isolates) species were from media 10 to 14. This could have been due to the various carbon sources in these media which supported the proliferation of heterotrophic groups. Only halomonads were recovered on media 6 and 20. The halomonads were recovered in 14 of the 19 media, mostly media that were low in organic carbon. Swarming morphology characteristic of the myxobacteria was observed in several media. However, partial sequencing indicated that the isolates were related to members of the genus Marinospirillum notably M. Alkaliphilum.

In terms of diversity, the majority of the isolates were from the class Gammaproteobacteria and the Bacillus group within the firmicutes. This concurs with earlier reports that the majority of Gram-negative isolates and culture-independent bacterial clones retrieved from soda lakes belong to the class Gammaproteobacteria (Jones et al. 1994, 1998; Ma et al. 2004), including strains related closely to typical aquatic bacteria such as Aeromonas and Pseudomonas (Duckworth et al. 1996), moderate halophiles from the Halomonas/Deleya group, and marine bacteria, e.g. Marinobacter (Rees et al. 2004). Species belonging to the family Halomonadaceae are ubiquitous and have been isolated from seawater, estuarine water, hypersaline soils, and bodies of hypersaline water, including Antarctic lakes, the Dead Sea, and several soda lakes of the Rift Valley, Kenya. They are aerobic, and some strains have the capacity for facultative anaerobic growth in the presence of nitrate. Members of Halomonadaceae have been shown to be of biotechnological importance in the production of compatible solutes as well as extracellular compounds such as exopolysaccharides and enzymes, and their use in environmental bioremediation processes (Nakayama et al. 2000; Garcia et al. 2004; Llamas et al. 2006; Wu et al. 2008).

The helical Gram-negative bacteria isolates retrieved here differed from the genus Marinospirillum in the physiological, biochemical, DNA G + C content and phylogenetically. These could also represent a new genus whose members differ from those of the genus Marinospirillum. Within the genus Idiomarina (Ivanova et al. 2000), 16 species are now validly published after the re-classification of the members of the genus Pseudidiomarina (Jean et al. 2009; Taborda et al. 2009). Although some isolates in this study were affiliated to the genus Idiomarina according to BLAST results, phylogenetic analysis of the full sequences indicates that they form a distinct cluster and hence could belong to a novel genus (data not shown).

The two isolates of the genus Nocardia recovered in this study were identical phylogenetically though the morphology was different. Therefore they could represent a new species from the soda lake environment. The closest neighbour in BLAST analysis was Nocardiopsis exhalans (Peltola et al. 2001). High G + C Gram-positive bacteria isolated from the Kenyan soda lakes so far belong to the genus Dietzia, Arthrobacter and Terrabacter (Duckworth et al. 1996, 1998; all the three being from Lake Oloiden). Bogoriella caseilytica (Groth et al. 1997) and Cellulomonas bogoriensis (Jones et al. 2005) have also been described from Lake Bogoria in Kenya. So far no isolate from the genus Streptomyces has been described from the East African soda lake environment. The single isolate recovered in this study could represent a novel genus. Members of the genus Streptomyces are involved in the biodegradation of various polymers abundant in soil owing to their ability to produce extracellular enzymes.

Bacillus species are among the most commonly found aerobic, eubacterial alkaliphiles both in soda lakes and in less selective environments (Horikoshi and Akiba 1982; Krulwich and Guffanti 1983; Guffanti et al. 1980, 1986; Takami et al. 1999). The Bacilli were the second group after the Halomonas in terms of diversity. The Bacilli are grouped into two clusters of alkaliphiles and alkaline-tolerant isolates based on physiological and biochemical characteristics as well as DNA base composition, hybridisation, and 16S rDNA analyses (Fritze et al. 1990; Nielsen et al. 1994, 1995).

The genus Alkalimonas was first proposed by Ma et al. (2004) to describe two novel alkaliphilic microorganisms described from Kenyan and Mongolian soda lakes, respectively. The single isolate from the genus Alkalimonas isolated was closely related to Alkalimonas delamerensis isolated from the same lake (Ma et al. 2004). These strains can be readily distinguished from close phylogenetic relatives by being alkaliphilic and halophilic meaning they are well suited for survival in soda lake conditions (Ma et al. 2004).

Members of the genus Alkalibacterium are found in the soda lake and in this study seven isolates were recovered. An isolate designated as WN16 from Lake Nakuru was isolated in a previous study by Duckworth et al. (1996). The genus Amphibacillus was first proposed by Niimura et al. (1990) and the genus currently comprises four recognised species, Amphibacillus xylanus (Niimura et al. 1990), Amphibacillus fermentum, Amphibacillus tropicus (Zhilina et al. 2001) and Amphibacillus sediminis (Sun-Young et al. 2007). The single isolate recovered in this study could therefore represent a new species within the same genus.

The genus Marinilactibacillus has only two described species and both are from the ocean. Isolation and taxonomic studies of lactic acid bacteria from marine environments to date are few and have generally been confined to those from cultured fish (Ringø and Gatesoupe 1998; Gatesoupe 1999). The single isolate recovered was 94% similar to Marinilactibacillus piezzotolerans. The low similarity value indicates that the isolate could represent a novel genus of lactic acid bacteria from the soda lake.

The name Vibrionaceae was formally proposed (Véron 1965) as a convenient grouping for fermentative bacteria that have polar flagella and a positive oxidase reaction. The isolates recovered in this study were affiliated to Vibrio metschnikovii, isolated from the ocean. Many factors probably govern their distribution, but four of the most important are: the particular animal or plant hosts, temperature, salinity, and depth below the surface for the species that are found in the ocean (Simidu and Tsukamoto 1985).

Microbial communities in natural alkaline environments such as soda lakes have attracted attention as a possible source of novel enzymes and metabolites for use in biotechnology. Microbes are a preferred source of enzymes since they are cheaper to produce and their enzyme content is more predictable and controllable (Adams and Kelly 1995; Plummer and Tarentino 1991). Phylogenetic analysis showed that some of the isolates retrieved belong to novel groups not reported in the soda lakes before.

Conclusion

The generic affiliation of cultured microbes reflects the sampling methods and culture conditions used in the isolation process and this has a major effect on the range of types encountered in the laboratory. Those methods that match most closely the physiology of members of a microbial community will be enriched and subsequently isolated. The use of different media helped retrieve novel groups not reported before from the soda lake environments. Some of the media used also targeted detection of useful extracellular enzymes produced by the alkaliphiles.