A Survey of Extremophilic Bacteria in Lake Magadi, … - A Survey of Extremophilic...Reagent Amount...

Columbia International Publishing American Journal of Molecular and Cellular Biology (2013) Vol. 2 No. 1 pp. 14-26 doi:10.7726/ajmcb.2013.1002

Research Article

______________________________________________________________________________________________________________________________ *Corresponding e-mail: [email protected] 1* University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya 2 Bondo University College, P.O. Box 210-40601, Bondo, Kenya

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A Survey of Extremophilic Bacteria in Lake Magadi, Kenya

Bancy N. Muruga1* and Beatrice Anyango2 Received 7 October 2012; Published online 15 December 2012 © The author(s) 2012. Published with open access at www.uscip.org

Abstract Lake Magadi highly saline with a pH of around 11 harbors extremophilic eubacteria and archaeobacteria that range from haloalkaliphilic to thermophilic. Samples of water were taken primarily from hot springs along the shoreline. Bacteria isolated using media suitable for haloalkaliphiles were found to be mostly Gram negatives of varying morphology. They were found to hydrolyze casein and/- or starch, indicating their ability toproduce proteases and amylases, respectively. Optimal temperatures for growth ranged from 40℃ to 45℃, while optimal salt concentrations for growth ranged from 6% to 9% w/v. PCR products obtained from genomic DNA using 16S primers were sequenced and aligned to sequences in the NCBI database. Three of the isolates had 98% similarity to members of the gamma Proteobacteria while one had a separate lineage with only 97% similarity to its closest comparison. Phylogenetic analysis by means of a rooted tree showed clustering of the three isolates with species of gamma Proteobacteria. Keywords: Biochemical characterization; 16S rRNA; NCBI GenBank; Phylogeny

1. Introduction Lake Magadi, covering an area of 90 km2 is located about 2o S, 36o 20 E and has a salinity of up to 30% w/v (Grant, 1992). The lake has an almost solid deposit of sodium chloride and sodium carbonate, the latter existing as sodium sesquicarbonate or trona. The climatic zone receives erratic rainfall below 800 mm per year, with substantial annual variation. Most of the rain falls between December and May, followed by a long dry season with daily temperatures frequently above 40°C. There are no permanent rivers entering the lake basin and solutes are supplied mainly by a series of alkaline springs with temperatures as high as 86°C. The lake is known to harbor a diverse population of halophilic, alkaliphilic and alkalitolerant representatives of major bacterial and archealphyla.

Bancy N. Muruga and Beatrice Anyango

/ American Journal of Molecular and Cellular Biology (2013) 1: 14-26

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1.1 Significance of soda lake microbes Chemoorganotrophs produce hydrolytic enzymes such as proteinases, cellulases, lipases and amylases in order to utilize the products of primary production. Nitrogen cycling involving production of ammonia by fermentative bacteria such as Tindalia magadii (Kevbrin et al., 1998) which is used by methylotrophs and nitrifiers, producing nitrate that is used by the chemoorganotrophs. Sulfate-reducing bacteria provide sulfide as the electron donor for the sulfur-oxidizing bacteria, anaerobic Ectothiorhodospira sp.and anaerobic autotrophs and heterotrophs (Sorokin et al., 1996). Methanogenic bacteria are obligately methylotrophic but use methanol, methyl amine and dimethyl sulfides. Methane is not lost from the system as methane oxidizers such as Methylobacter may be present in soda lakes (Jones et al., 1998). Extremophilic microorganisms produce extracellular enzymes with potential applications in biotechnology; Bioplastic or polyhydroxyalkanoates (PHA) is a biodegradable heteropolymer that exhibits total resistance to water and is degraded in human tissue; hence it is biocompatible. It has pharmaceutical and clinical importance, including for use in delayed drug release, bone replacement and surgical sutures. PHA is produced by Haloferax mediterranei, a halobacterium that possesses high genomic stability a prerequisite for industrial purposes (Rodriguez-Valera and Lillio, 1992).Microbial exopolysaccharides are used as stabilizers, thickeners, gelling agents and emulsifiers in thepharmaceutical paint and oil recovery, paper, textile and food industries. Haloferax mediterranei produces a highly sulfated and acidic heteropolysaccharide (up to 3g/l) containing mannose as a major component (Dubey and Maheshwari, 2004).Cancer detection can be done using a protein from Halobacterium halobium as an antigen to detect antibodies against the human c-Myc oncogene product in the sera of cancer patients suffering from leukaemia (Kushner, 1985). Lipid from Halobacteria spp is used in liposome preparation which has great value in the cosmetic industry. Such liposomes would be more resistant to biodegradation, good shelf-life and resistance to other bacteria (Dubey and Maheshwari, 2004).Some Halobacterium spp. produce intracellular gas filled vacuoles which provide buoyancy. In the future, the genes for such properties could be engineered in other microorganisms to produce gas vacuoles to enable them to float in water (Grant et al., 1990). A sauce called ‘nampla’ is prepared in Thailand from fish fermented in concentrated brine containing Halobacteria. These are responsible for aroma production because they produce salt- stable extra cellular proteases. Large scale cultivation of the cyanobacterium Spirulina platensis in Israel uses brackish water unsuitable for agriculture and the Spirulina product is marketed as a food (Dubey and Maheshwari, 2004). Horikoshi (1971) reported the production of an extracellular protease by alkaliphilic Bacillus clausii 221. A haloalkaliphilic archaeon Natronococcus sp strain Ah-36 produces an extracellular maltotriose- forming amylase. The gene encoding this enzyme has been cloned and expressed in Haloferax volcanii (Kobayashi et al., 1992). Nakamura et al. (1975) discovered that alkaliphilic Bacillus halodurans produces an extracellular pullulanase in Horikoshi II medium. This enzyme has an optimal pH of 8.5-9.0 and is stable for 24 hr at pH 6.5-11.0 at 4℃ and can be used as dishwashing detergent additive. Taq polymerase used in molecular biology is found in Thermus aquaticus and is active at 80˚C and pH 8 (Chien, 1976). Previously published work on Lake Magadi includes the isolation of an alkaliphilic, obligately anaerobic, gram-positive bacterium from the soda deposits (Kevbrin et al., 1997). A new genus and species, Tindallia magadii, for this strain was proposed as a result of 16S rRNA gene analysis. Natronobacterium vacuolata sp. nov. of type strain NCIMB 13189, a novel haloalkaliphilic archaeon with cells containing large gas vacuoles in the stationary phase, was isolated by Mwatha and Grant (1993). Zhilina et al., (2001) isolated Halonatronum saccharophilum gen. nov. sp. nov. with the type



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strain Z-7986. The spore-forming, alkaliphilic, Gram negative rod was found to grow within 3–17% NaCl concentration range, pH range of 7.7–10.3 and a temperature of 36–55°C. Alkaliphilic spirochaetes such as the α-amylase-producing haloalkaliphilic archaeon Natronococcus amylolyticus have also been isolated (Haruhiko, 1995).

2. Materials and Methods 2.1 Water sampling methods Samples of water were collected from seven hot springs along the shoreline and one sample along the Western Causeway. Sampling was done in January, 2009. Three samples of water were collected from each site and placed in separate, sterile and labeled bottles. These were carried in a cool box and stored at about 4o C prior to analysis. 2.2 Determination of physicochemical conditions in the lake A water multi-parameter Meter (HANNAS HI 769828 model) was used to measure pH, total dissolved solutes (TDS) and salinity while the water temperature was measured by laser beam thermometer. 2.3 Bacterial isolation and characterization The microbial diversity was described in terms of the different colony characteristics, bacterial morphology and physiology. The following selective enrichment medium was used for isolation;

Medium for haloalkaliphiles(Designed by Prof Duboise, personal communication)

Reagent Amount (g/l)

Sodium carbonate (Na2CO3) 53.0 Sodium bicarbonate (NaHCO3) 42.0 Tryptic Soy Broth (TSB) 1.5 NaCl (Sea salt) 29.5 Agar 15.0

Salts were sterilized in an autoclave at 121 ℃ for 20 min separately then mixed with the rest of the sterilized ingredients at 60 ℃ j u s t before pouring onto plates.

Enrichment and isolation The enrichment culture technique was employed to build up the population of the microbes by mixing each water sample with media for haloalkaliphiles at a ratio of 1: 1 and incubating at 37℃ for 12-48 hrs. The microbial suspension obtained was cultured by the pour plate method (Brown, 2005). Subculturing by streaking was done to obtain pure colonies. The pure colonies were distinguished by configuration, size, elevation, color, margin and opacity. Agar slants were used for preservation at 4℃. Gram - Stain reaction A thin film of each isolate was smeared over the surface of a slide and heated gently over a flame in order to fix the material. Gram-stain tests were then conducted on each isolate as outlined by Brown, 2005. Photographs of the isolates at 1000x magnification were taken.



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Motility test Motility medium, a semisolid medium that barely gels at room temperature (containing 7 g/l of agar) (Brown, 2005), was inoculated with the bacterial isolates. This was done using an inoculating needle, stabbing the media in a straight line and then incubating at 37 ℃ for 12-24 hr. Migration away from the original line of inoculation meant that the test organism was motile. Salt concentration range for optimal growth The medium for haloalkaliphiles was made with different NaCl concentrations of 0, 3, 6, 9 and 15 %w/v, inoculated and incubated for 24 hr at 37℃. Enhanced growth (when compared to growth in 3 % w/v) was recorded as positive and no enhancement as negative (Mwatha, 1997). Temperature range for optimal growth Ten ml stock broth culture for each isolate was prepared. Exactly 1 ml of each was used to inoculate exactly 6 ml of freshly prepared nutrient broth and incubated at 30°C for 24 hrs. The procedure was repeated at 35°C, 40°C, 45°C and 50°C.The optical density of each broth culture was measured using a spectrophotometer (Bausch and Lomb Spectronic 20 model) at a wavelength of 686 nm (Brown, 2005). pH range for optimal growth Using concentrated hydrochloric acid and universal paper strips to estimate pH, 200 ml each of haloalkaliphilic broth media of pH 11 was adjusted to pH 7 and 9. Six ml of each media with pH of 7, 9 and 11 were filter-sterilized with a 0.45 µm pore size filter, inoculated with each isolate and incubated at 37°C for 24 hr. The optical densities were measured at a wavelength of 686 nm (Brown, 2005). Utilization of different carbon sources The medium used (Mwatha, 1997) had the following composition; Yeast 1.0 g/l; KH2PO4 1.0 g/l; MgSO4.7H2O 0.2 g/l ; KNO3 1.0g/l; NaCl 40.0g/l; Na2CO3 18.0g/l; Agar 2.0g/l (Salts were sterilized separately then mixed with the rest of the sterilized ingredients at 60℃. Each carbohydrate solution (glucose, galactose and mannose) was filter- sterilized and added to the basal minimum medium to a final concentration of 0.5% w/v just before pouring. The plates were inoculated and incubated at 37℃ for 24hr.Growth was compared to growth on a plate containing basal minimum medium. Enhanced growth was recorded as positive and no enhancement as negative. Starch hydrolysis Starch agar plates were prepared by adding 100g/l starch to the media for haloalkliphiles inoculated and incubated at 37℃ for 24 hr. A few drops of Gram’s iodine were then added to the plate and the color c hange observed ( Brown, 2005). A b lac k /blue color i ndic ated l a c k of hydrolysis. Casein hydrolysis A solution of skim milk powder of 10% w/v concentration was sterilized by placing in the autoclave for 20min. After cooling to 55℃ (Brown, 2005) it was added to sterile medium for haloalkaliphiles whose salts were sterilized separately then mixed with the rest of the sterilized ingredients at 60℃. The white opaque milk salt agar plates were inoculated and incubated at 37 ℃

for 24 hr. Clear zones around the colonies indicated casein hydrolysis.



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Catalase test A small amount of each isolate was smeared onto a clean glass slide and a drop of 70 % hydrogen peroxide added. Visible bubbles of oxygen indicated catalase positivity (Brown, 2005). 2.4 Nucleic acid analysis (16S rRNA gene sequencing) Due to the highly conserved nature of the translational material of cells, the 16S rDNA that encodes for the small ribosomal subunits (the 16S rRNA), provides a nucleotide sequence highly useful as a molecular basis for phylogeny. The degree of similarity between the DNA sequences encoding the 16S rRNAs of two organisms was used as a measure of the relatedness between the two organisms (Dubey and Maheshwari, 2004).

DNA Isolation Bacterial cells were harvested in the early log phase of growth in liquid broth (about 24 hr after innoculation).Total DNA of the microbial isolates was isolated using a DNEasy Blood and Tissue kit, from Qiagen, according to the manufacturer’s instructions(http//www.qiagen.com). The genomic DNA obtained was stored at -25℃. Polymerase Chain Reaction (PCR) Base sequences of the primers used for amplification were: 16S F 5’- AGA GTT TGA TCH TGG CTY AG -3’ 16S R 5’- ACG GNT ACC TTGTTACGACTT – 3’ UA 751 F 5’ – CCG ACG GTG AGR GRY CAA – 3’ UA 1406 R 5’ – ACG GGC GGT GWG TRC AA – 3’

The reaction mixture contained the following components; 38.25µl PCR water; 4µl DNA template diluted 1: 10 in PCR water; 5µl thermopol – 10 x buffers; 1.25µl dNTP mix; 0.5µl each of forward and reverse Universal Archaea or 16S primer diluted 1: 10 with PCR water; 0.5µl Taq polymerase, totalling 50µl. The thermocycler pro Gram used was, 95 ℃ for 3 min, 95 ℃ for 30 sec, 55 ℃ for 30 sec, 72℃ for 1 min for extension of the PCR product, 30 cycles were performed with a final extension of 3 min at 72℃. The reaction was held at 4℃ and the product removed and stored at -25 ℃.To purify the PCR products a QIAquick Purification KIT from Qiagen was used according to instructions(http//www. qiagen.com).About 50µl of eluate was collected and stored at -25 ℃. Agarose gel electrophoresis To make 1 litre TAE, 242 g Tris –base, 57.1 ml glacial acetic acid, 18.6 g EDTA was mixed with 900 ml distilled water .To make 1% agarose gel, 0.5g agarose was dissolved in 50ml TAE. About 0.014% ethidium bromide was added. Six µl of each PCR product with about 1.5µl of loading dye was loaded using a DNA molecular marker of 0.12-23.1 Kbp in the outermost well. The electrophoresis was left to run for about 20 min at 5V/cm. The gel was observed in a transilluminator and band images taken using a digital camera and the Doc-It LS Image Analysis Software. Sequencing of the PCR Products The PCR product obtained was sequenced using the same primers used in the polymerase chain reaction. BioEdit pro Gram was used to remove ambiguity and comparisons done with the NCBI GenBank databases using the Basic Local Alignment Tool (BLAST). Alignment was done using the multialin program (http;//www-archbac.u-psud.fr/genomics/multialin.html).The differences

http://www.qiagen.com/



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i n the nucleotides were converted into distance matrices using the Jukes and Cantor (1969) neighbor joining m ethod .Construction o f a phylogenetic tree was done using Kimura two-parameter calculation model(Van de Peer and De Wachter,1994).

3. Results 3.1 Characterisation of bacterial isolates The temperature of the water in the sampled sites varied from as low as 30°C to as high as 47°C in one of the hot springs. The pH lay within a narrow range of 10 to 11 while salinity, as reflected by the electrical conductivity and total dissolved solutes, ranged from 7.9 to 25.6. Observation of colony characters revealed 26 bacterial isolates which had shapes varying from rods to cocci (Fig 1). However some colonies may have appeared different yet could have belonged to the same species.

Fig 1. Images of pure bacterial isolates (rods and cocci)cultured in the media for haloalkaliphiles, Gram - stained and observed under high power (x1000). Scale bar = 5µm

Characterisation data (Table 1) showed that the isolates had growth pH optima ranging from 9 to 11 and an optimum salt (NaCl) concentration ranging from 3% to 9 % w/v. Most of them were

5µm

5µm



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slight or moderate thermophiles with growth temperature optima ranging from 40 ℃ to 45 ℃. Some of the isolates were able to utilize glucose, galactose or mannose as carbon sources and hence are chemoorganotrophs. Out of 26 isolates 17 were catalase positive. The presence of catalase can be used as a way of differentiating strict anaerobes and aerotolerant from aerobic bacteria.About 50% of isolates hydrolyzed starch indicating production of amylases. About 30%of the isolates hydrolyzed casein, i.e., they produce proteases. A few of the Gram– negative rods were motile. Table 1 Summary of morphological and biochemical characteristics of isolates from Lake Magadi cultured in media for haloalkaliphiles and incubated at 37°C for 24 hr

Key G.S.-Gram-stain; M-Motility; S.O.- Salt Concentration optima; T.O.-Temperature optima; P.O.- pH optima; S.Hy-Starch hydrolysis; C.H.-Casein hydrolysis; I.P.-Indole production; C.P.-Catalase production; H- Halotolerant; S.H. - Slightly halophilic; M.H.-Moderately halophilic; S.T.-Slightly thermophilic; M.T.–Moderately thermophilic

Isolate shape G.S. M S.O. T.O. P.O. S.Hy. C.H I.P. C.P.

M1-1 rod - - H ST 11 - + - +

M1-2 Rod - - SH ST 9 - - - +

M1-3 spirilla + - MH ST 9 - + - -

M2-1 Rod - - SH ST 11 - + - +

M2-2 Rod - - H ST 11 - + - +

M2-3 rod - + SH MT 11 - - - +

M3-1 Coccus - - SH ST 9 + - - +

M3-3 Coccus - - MH ST 11 + + - -

M4-1 Rod - - MH MT 9 + - - +

M4-2 Rod + - SH ST 9 + - - +

M4-3 Rod - - MH MT 11 + - - +

M4-4 Rod - - H MT 9 + - - -

M5-1 Coccobacilli - - MH ST 11 + - - +

M5-2 Spirillum + - MH MT 9 - + - +

M5-3 Rod - + MH ST 9 - - - +

M6-2 Coccus - - MH ST 11 - - - +

M6-3 Rod - + MH ST 11 - - - -

M6-4 Rod - - H MT 11 + - - +

M6-5 Rod + - H MT 11 + - - +

M7-1 Rod - + H ST 11 - - - +

M7-3 Coccobacilli - - H MT 9 + + - +

MC-1 Rod - - MH ST 9 - - - +

MC-2 Coccus - - MH ST 11 - + - -

MC-3 Coccus - + MH MT 11 + - - +

MC-4 Rod - - MH ST 9 - + - +

MC-5 Rod - - MH ST 9 + - - +



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3.2 Nucleic acid analysis

Fig 2. Image of Ethidium bromide-stained electrophoresis gel of PCR product on 1% agarose. Lanes labeled1-14 correspond to PCR product generated from genomic DNA of the following bacterial isolates; lane1, M7-1; lane2, M7- 3; lane3, M6-2; lane 4, M1-3;lane 5, M2-3;lane 6 , M3-3; lane 7,M4-3; lane 8,M6-1; lane 9,MC-3;lane10,M5-1;lane11, M4-1; lane 12,MC-1; lane 13, DNA molecular marker of 0.12 -23.1 kbp Table 2 A comparison of 16S rRNA gene sequences of the isolates with sequences of close relatives from NCBI GenBank

Isolates G+C Mol% Accession no Names of comparative isolates Maximum identity %

M4-1 55.50 EU870505.1 EF527873.1

Halomonas sp. Sua-BAC009 Halomonas salifodinae strain BC7

99 98

M7-1 53.29 AY730234 EU723884.1 FJ170028.1

Gamma Proteobacterium M6-24A Gamma Proteobacterium CF12-14 Idiomarina sp. CF12-14

99 98 98

M5-1 55.98 DQ077911.1 DQ077910.1 DQ077909.1 DQ077908.1

Halomonascampisalis strain LL6 Halomonascampisalis strain LL5 Halomonascampisalis strain LL4 Halomonascampisalis strain LL3

97 96 96 96

MC-1 53.35 FJ170017.1 EU723884.1 EF554894.1 AY914068.1

Idiomarina sp. CF11-10 G. Proteobacterium CF12-14 Idiomarina sp. JK16 Gamma Proteobacterium A-7B

99 98 98 98

1 2 3 4 5 6 7 8 9 10 11 12 13



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Isolate M4-1 is 98% similar to Halomonassalifodinae, M7-1 and MC-1 are 98% similar to Idiomarinasp and M5-1 is 97% similar to Halomonascampisalis

Fig 3. Rooted phylogenetic tree to show relationship between 16S rRNA sequences of isolates and

their comparisons from NCBI GenBank Genomic DNA was isolated for all the 26 isolates but only 12 PCR products were successfully obtained (Fig 2). Sequencing was successfully done for six of the PCR products. The PCR products obtained with Universal Archaea primers had sequences of less than 400 bp and were too small to be used for comparison. The longest sequences were obtained with 16S primers with about 1400

FJ764791.1| Gamma proteobacterium E-410 FJ764791.Gamma proteobacterium E-410 M7-1 1

X92129.1| Bacterial sp. M C-1

FJ170017.1 Idiomarina sp. CF11-10 AY914068.1 Gamma proteobacterium A-7B... FJ170028.1 Idiomarina sp. CF12-14 CCA... EU723884.1| Gamma proteobacterium CF1... EU723884.1 Gamma proteobacterium CF12...

AY730234| Gamma proteobacterium M6-24A EF554894.1 Idiomarina sp. JK16 TGAACG... EF554894.1 Idiomarina sp. JK16

DQ077911.1 H. campisalis TCCAGAGTTTGAT... DQ077908.1 H. campisalis strain LL3 DQ077910 H. campisalis DQ077909.1 H. campisalis

M5- 1

AJ515365 H. campaniensis EF527873.1 H. salifodinae strain BC7

M 4-1

EU870505.1| Halomonas sp. Sua-BAC009 DQ289061 H. campisalis DQ289060 H. campisalis iso. HKS 3

AB006899.1| P. carotinifaciens

66 90

96 98

91

100

78

100

100 83

97

75 98

94

100

48 57

100

58 60

0.02



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bp. These were sequences of isolates labeled as M4-1, M5-1, M7-1, and MC-1, which were later compared to sequences of closely related organisms in the NCBI GenBank (Table 2), and a rooted phylogenetic tree generated (Fig 3). The tree was generated by the neighbor joining method. Distance matrices were calculated by Jukes and Cantor (1969). Bootstrap values at the nodes of the branches are based on 1,000 replications. Accession numbers of the comparable isolates are provided in the text. The scale bar represents 2 nucleotide substitutions per 100 nucleotides.

4. Discussion The East African soda lakes, among the most productive aquatic environments in the world, have water with shades of red and green reflecting theirprimary productivity.This is due to their virtually unlimited supply of carbon dioxide, high ambient temperatures and high daily light intensities (Melack and Kilham, 1974). The bacterial population in the Kenyan soda lakes is limited by the total phosphate and nitrogen ions and conductivity levels. A study of organotrophic bacteria in the saline lake showed that eubacterial haloalkalophiles dominated at low conductivity and archeobacteria haloalkalophiles dominated at high conductivity (Mwatha,1997). The major primary producers in the lake are the anoxigenic, phototrophic, halophilic Ectothiorhodospira, a genus of bacteria in the family Chromatiaceae (Grant and Tindall, 1986). Cyanobacteria of the genus Cyanospira also contribute to primary production.However cyanobacterial blooms occur only occasionally after extensive rainfall causing dilution of the brine (Florenzano et al., 1998).Normally the trona crusts are colored red by the haloalkaliphilic Archaea which are classified into two genera Natronococcusand Natronobacterium (Kamekura et al.,1997). It is not unusual to find common types of alkaliphilic organisms inhabiting soda lakes in various widely dispersed locations throughout the world such as in the East African Rift Valley, in the western U.S.,Tibet, China and Hungary. An example is Natronobacteria which has been isolated and identified in soda lakes located in China (Wang and Tang, 1989), western U.S. (Morth and Tindall, 1985) and in India (Upasani and Desai, 1990). Such comparative studies are possible by analyzing ribosomal RNA (rRNA) which represents about 80% of total RNA of the cell. Isolate M4-1 was sampled from a hot spring, site 4, whose water had a temperature of 47°C, pH of 10.5 and salinity of 7.89. The isolate was a Gram-negative, aerobic, rod growing optimally at a temperature of 45°C, 6-9 % w/v NaCl and a pH range of 9-11. If the G+C content of the 16S rRNA gene were to be used as an estimation of the G+C content of the genomic DNA, then isolate M4-1 had an estimated G+C content of 55.55 mol% (Table 2). Comparisons of 16S rRNA gene sequences with sequences in the NCBI GenBank database showed that the isolate was 99% similar to Halomonas sp. Sua-BAC009 with accession no EU870505.1and 98% similar to Halomonas salifodinae strain BC7 with accession no EF527873.1. Phylogenetic comparisons indicated that the isolate clustered closely with Halomonas campisalis HKS 3. These characteristics match those of Halomonas species belonging to the Halomonadaceae family that are heterotrophic, Gram-negative, rod shaped, slight or moderate halophiles and aerobic.



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Isolate M5-1 which was isolated from a hot spring, site 5, with a temperature of 44°C, salinity of 20.4 and a pH of 10.2, was a gram negative aerobic coccobacillus, growing best at a temperature optimum of 40°C, NaCl concentration of 3-6%w/v and a pH of 11. The isolate had an estimated DNA G+C content of 55.98 mol%. It showed highest similarity of 97% with Halomonas campisalis strain LL6 with accession no DQ077911.1; 96% with Halomonas campisalis strain LL5 with accession no DQ077910.1 ; 96% with Halomonas campisalis strain LL4 with accession no DQ077909.1; and 96% with Halomonas campisalis strain LL3 with accession no DQ077908.1.The isolate formed a distinct out group in the phylogenetic tree but showed some clustering with Halomonas campisalis. This was likely to be a novel species and it would be necessary to do further analysis in order to verify this. Isolate M7-1 was isolated from a hot spring, site 7, where the water temperature was 43°C, pH of10.0 and salinity of 18.19. It was a motile Gram-negative aerobic rod, which grew optimally at a temperature of 40°C, 0%-6%w/v NaCl and a pH of 11. The estimated G+C content for isolate M7-1 was 53.29 mol%. It showed a 99 % similarity to AY730234.1GammaProteobacteriumM6-24A;98%similaritytoEU723884.1Gamma Proteobacterium CF12-14; 98% similarity to FJ170028.1 Idiomarina sp.CF12-14; the isolate clustered closely with a Gamma Proteobacterium E-410 strain that has not beencharacterized. Isolate MC-1, sampled from the lake water along the Western causeway, was a Gram-negative aerobic large rod (Fig 1),that grew best at a temperature range of 35°C-40°C, 6-9%w/v NaCl and at a pH of 9. The isolate had an estimated G+C content of 53.35 mol%. Close similarity of sequence was 99 % with FJ170017.1Idiomarina sp.CF11-10; 98% with EU723884.1 Gamma proteobacterium CF12-14; 98% with EF554894.1 Idiomarina sp. JK16; 98% with AY914068.1 Gamma proteobacterium A-7B. Highest clustering was with isolate M7-1 which clustered with Gamma proteobacteria. The gram negative isolates such as M4-1 and M5-1 are members of the gamma subdivision of the Proteobacteria that are capable of producing hydrolytic enzymes. The potential to produce different kinds of enzymes under specific culture conditions is observed in certain alkaliphilic bacterial strains. A good example is the facultatively alkaliphilic Bacillus halodurans C-125 which produces at least five kinds of enzymes of industrial interest (Takami and Horikoshi, 1999). The amount of each enzyme produced varies depending on the culture conditions particularly with different combinations of nitrogen and carbon sources. Species similar to the ones identified in this study have been found elsewhere in the world. The haloalkaliphile Halomonas campisalis, isolated near Soap Lake, Washington, grows under both aerobic and denitrifying conditions with optimal growth occurring at 2 to 3% w/v NaCl (Aston and Peyton,2007). Halomonas salifodinae, a Gram-negative rod that is aerobic, motile and halophilic, designated strain BC7, was isolated from a salt mine in China with optimum growth at 3 % (w/v) NaCl, pH 7.0 and 30 °C (Wang et al., 2008). Two novel bacterial strains, F23 and R22, were isolated from hypersaline habitats in Málaga and Murcia (Spain).They were Gram-negative rods, chemoorganotrophic, strictly aerobic and motile by a single polar flagellum. The DNA G+C composition being 46·0 mol% in strain F23 and 48·7 mol % in strain R22. It was proposed that they



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be classified as novel species of the genus Idiomarina, with the names Idiomarina fontislapidosi sp. nov.and Idiomarina ramblicola sp. nov.(Martínez-Cánovaset al., 2004).

5. Conclusion The work presented here has produced some insight into the diversity of microbes in Lake Magadi but much more needs to be explored. The soda lakes are not entirely closed ecosystems. This is due to disturbance by wildlife including migratory birds such as flamingoes that use the Rift Valley as a migratory route and contribute to the spread of haloalkaliphiles. Constant sampling raises the chances of redeeming novel species which may be of commercial importance. Culturing the microorganisms is a major hurdle to the complete understanding of the microbial diversity. There is evidence that if experiments are conducted in situ then a different spectrum of organisms can be isolated (Mwatha, 1997). Such studies on the springs of Lake Magadi have hinted at uncultured thermoalkaliphiles. One cloned gene showed only 7 % similarity to all known Archeal sequences thus representing a new distinct Archeal line (Grant et al., 1998). There remains therefore the challenge of obtaining more of the microbes in culture in order to study their physiology and to classify them to the species and strain level. Such classification would require analyses of the polar lipids, fatty acid profiles and isoprenoid quinones, which are characteristic components of the plasma membrane, as well as the G+ C content of their DNA. Phylogenetic analyses based on 16S rRNA gene sequence comparisons are of utmost importance as they have helped to reorganize species into different genera. A good example is the family Halomonadaceae within the Gamma Proteobacteria whose species were previously assigned to other genera such as Deleya, now extinct, Alcaligenes, Pseudomonas, Halovibrio and Volcaniella (David and Ventosa, 1998). It is also important to determine all the information on the genome of the bacterial strains in order to control the extracellular enzyme production (Takami and Horikoshi, 2000).The genes encoding these alkalitolerant enzymes may be isolated, cloned and expressed in compatible expression hosts. This would provide a source of larger volumes of enzyme products especially if the wild-type strain has failed to produce sufficient amounts of the desired enzyme. Halophiles, having the largest plasmids so far known among all the known bacteria, (Dubey and Maheshwari, 2004) could be used for genetic manipulation. The isolates characterized in this study could be put to commercial use by optimizing production of metabolites for biotechnological applications.

Acknowledgements My acknowledgements go to the University of Nairobi fraternity for granting me a study scholarship and offering laboratory space at the Chiromo campus

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http://dx.doi.org/10.4319/lo.1974.19.5.0743

http://dx.doi.org/10.1016/S0723-2020(85)80026-6

A Survey of Extremophilic Bacteria in Lake Magadi, … - A Survey of Extremophilic...Reagent Amount...

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