Microbial Ecology

, Volume 55, Issue 2, pp 163–172

Cultivable Bacterial Diversity of Alkaline Lonar Lake, India

Authors

  • Amarja A. Joshi
    • Microbial Sciences DivisionAgharkar Research Institute
    • Microbial Sciences DivisionAgharkar Research Institute
  • Anita S. Kelkar
    • Microbial Sciences DivisionAgharkar Research Institute
  • Yogesh S. Shouche
    • National Centre for Cell Sciences
  • Aijaz A. Vani
    • National Centre for Cell Sciences
  • Suchitra B. Borgave
    • Microbial Sciences DivisionAgharkar Research Institute
  • Seema S. Sarnaik
    • Microbial Sciences DivisionAgharkar Research Institute
Original Article

DOI: 10.1007/s00248-007-9264-8

Cite this article as:
Joshi, A.A., Kanekar, P.P., Kelkar, A.S. et al. Microb Ecol (2008) 55: 163. doi:10.1007/s00248-007-9264-8

Abstract

Aerobic, alkaliphilic bacteria were isolated and characterized from water and sediment samples collected in the winter season, January 2002 from alkaline Lonar lake, India, having pH 10.5. The total number of microorganisms in the sediment and water samples was found to be 102–106 cfu g−1 and 102–104 cfu ml−1, respectively. One hundred and ninety-six strains were isolated using different enrichment media. To study the bacterial diversity of Lonar lake and to select the bacterial strains for further characterization, screening was done on the basis of pH and salt tolerance of the isolates. Sixty-four isolates were subjected to phenotypic, biochemical characterization and 16S rRNA sequencing. Out of 64, 31 bacterial isolates were selected on the basis of their enzyme profile and further subjected to phylogenetic analysis. Phylogenetic analysis indicated that most of the Lonar lake isolates were related to the phylum Firmicutes, containing Low G+C, Gram-positive bacteria, with different genera: Bacillus, Paenibacillus, Alkalibacillus, Exiguobacterium, Planococcus, Enterococcus and Vagococcus. Seven strains constituted a Gram-negative bacterial group, with different genera: Halomonas, Stenotrophomonas and Providencia affiliated to γ-Proteobacteria, Alcaligenes to β-Proteobacteria and Paracoccus to α-Proteobacteria. Only five isolates were High G+C, Gram-positive bacteria associated with phylum Actinobacteria, with various genera: Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus. Despite the alkaline pH of the Lonar lake, most of the strains were alkalitolerant and only two strains were obligate alkaliphilic. Most of the isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.

Introduction

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 [20]. These alkaline environments are caused by a combination of geological, geographical and climatic conditions. They are characterized by large amounts of sodium carbonate formed by evaporative concentration [12]. In the course of formation of alkalinity, other salts particularly (NaCl) also concentrate, giving rise to an alkaline saline environment.

The most extensively studied soda lakes are those of the East African rift valley [11, 20]. Phylogenetic diversity of alkaliphiles from East African soda lake has revealed presence of aerobic chemoorganotrophic bacteria and archaea [10], while microbial diversity with respect to the depth of Mono lake in California have revealed the sequences were related to five major lineages—α- and β-Proteobacteria, CFB, High G+C, Low G+C—plus Chloroplasts and Candidate divisions [18]. The diversity and activity of microorganisms were studied in hyperalkaline spring waters in Maquqrin, Jordan [34] and the diversity of aerobic heterotrophic populations from nonsaline alkaline environment was assessed [45]. Recently diversity of the Inner Mongolian Baer soda lake and Kenyan soda lake was studied by molecular methods [27, 37]. The microbes present in such alkaline saline environments play an important role in the reminerlization of organic matter within the ecosystem. They are the major contributors in the transformation of organic carbon, sulfur, nitrogenous compounds and metals with an important role in food webs and nutrient cycling. The ecology and diversity of an East African soda lake was studied and extensively reviewed for their biotechnological potential [11]. The microbial diversity of saline lakes has been studied primararily by focusing on the isolation and characterization of individual organisms with potential industrial application [17, 20]. Lipase-producing microorganisms from lake Bogoria were isolated and characterized [48], while starch-hydrolyzing microorganisms were isolated from a Kenyan alkaline soda lake as well as from Ethiopian soda lakes [14, 30]. As far as Indian soda lakes are concerned, a culture-dependent approach has not been yet applied to analyze bacterial diversity. We have applied this strategy to explore the diversity of aerobic bacteria of Lonar lake.

The alkaline Lonar lake (Latitude 19°58′, Longitude 76°36′) is a unique basaltic rock meteorite impact crater, ranking third in the world. Lonar crater is filled with saline water. The uniqueness of the lake water is its salinity and high alkalinity. A review of literature revealed that its salinity was 40.78, 31.52 and 30.87 in 1910, 1958 and 1960, respectively. The salinity of lake is now lowered down to 7.9% [28]. The observed alkalinity is ascribed to an interaction between sodium chloride, calcium carbonate and water over a long period of time. Some geological and chemical reports are available on Lonar lake [19, 32]. However, there is meager data on the bacterial diversity of Lonar lake. Eutrophication and presence of blue green algae in Lonar lake have been described [3]. Some workers [6] have studied the alkaline metalloprotease from alkaline Streptomyces isolated from Lonar lake silt sample. Bioremediation of phenol-using alkaliphilic bacteria isolated from Lonar lake sediments was an interesting finding [22]. A preliminary account of bacterial diversity of the Lonar Lake ecosystem has been reported [21], which includes some of the biochemically identified isolates.

In the present study, culture dependent phenotypic characterization and 16S rDNA-based phylogenetic analyses were applied to study aerobic, cultivable bacterial populations present in the alkaline Lonar lake. The isolates were further studied for their biotechnological potential.

Materials and Methods

Sampling Sites and Sample Collection

Lonar lake has a periphery of 1.7 km and is situated in a hallow, 0.14 km below the ground level, with an amphitheater of practically vertical cliffs (Fig. 1). Six different samples, comprising of four sediment samples (35–40 cm) and two water samples, were collected (10–20 cm) in the winter season from different sites, as described in Table 1. The sediment samples were collected with the help of a scooper in sterile polyethylene bags, while water samples were directly into sterile bottles. They were labeled, transported on ice and stored at 4°C until analysis. The pH (with pH paper), temperature and depth of sampling were noted immediately at the site.
https://static-content.springer.com/image/art%3A10.1007%2Fs00248-007-9264-8/MediaObjects/248_2007_9264_Fig1_HTML.gif
Figure 1

Schematic representation of Lonar lake

Table 1

Chemical analysis of sediment and water samples from Lonar Lake

Sample type

Location

pH

Alkalinity, mg l−1

Salinity, NaCl mg l−1

SD1

1-Starting point 1

9.38

1,875

710.82

SD4

2-Rammagaya mandir

9.42

2,750

1,445.6

SD2

3-Kamalaja Devi temple

9.27

2,000

586.08

SD3

4-Near banana farm

8.84

1,750

507.9

WI

5-Point 2

9.82

2,750

4,532

WC

6-Dhara temple

9.48

2,275

3,887

The alkalinity of the samples was estimated by potentiometric titration in terms of CaCO3, while the salinity of the samples was estimated by the argentometric method both in terms of Cl and NaCl [13]. The pHs of the samples were noted using a pH meter (Systronics, India).

Enumeration and Isolation of Bacterial Strains

Determination of total viable count (TVC) of microorganisms present was performed using two representative samples designated as SD (mixture of four sediment samples) and W (mixture of two water samples). TVC was carried out using nutrient agar media [5] of various pH (7, 8, 9, 10, 11 and 12) by the spread plate technique [35]. The pH of the medium was adjusted using 1N NaOH solution and checked before and after autoclaving. The plates were incubated at RT (28 ± 2°C) and 37°C for 48 h. The TVC of both the samples were determined at the respective pH and temperatures.

Enrichment of sediment samples (SD1, SD2, SD3, SD4) and water samples (WI and WC) was carried out in various enrichment media, such as Horikoshi I (A), Horikoshi II (B) [16], nutrient agar at pH 10 (C), nutrient agar at pH 10.0 with 30 g l−1 sodium chloride (D), Davis Mingiolis Synthetic Medium [8] at pH 10.0 with 5 g l−1 peptone (E) and Tindall’s medium (F) [46]. All the flasks were incubated at RT on arotary shaker (120 rpm) for 7 days. After enrichment, the organisms were isolated on respective media agar plates and incubated at RT for 7 days. Well isolated and differentiated colonies from these enrichment media were transferred on the respective medium slants and cultures were maintained as glycerol stocks.

Media Composition

The media contained ingredients given in the list below as g l−1 of distilled water. Glucose, NaCl, Na2CO3 and trace element solutions were autoclaved separately and added to the organic components at 60°C before pouring the agar media.

Medium A: glucose 10.0, peptone 5.0, yeast extract 5.0, KH2PO4 1.0, MgSO4.7H2O 0.2, Na2CO3 10.0, agar 20.0.

Medium B: soluble starch 10.0, peptone 5.0, yeast extract 5.0, KH2PO4 1.0, MgSO4.7H2O 0.2, Na2CO3 10.0, agar 20.0.

Medium C: peptic digest of animal tissue 5.0, yeast extract 1.5, beef extract 1.5, sodium chloride 5.0, agar 20.0. pH adjusted to 10.0 with 1N NaOH solution.

Medium D: peptic digest of animal tissue 5.0, yeast extract 1.5, beef extract 1.5, sodium chloride 35.0, agar 20.0. pH adjusted to 10.0 with 1N NaOH solution.

Medium E: K2HPO4 7.0, KH2PO4 3.0, MgSO4.7H2O 0.1, peptone 1.0, trace element solution (FeSO4.7H2O 0.5, ZnSO4.7H2O 0.5, MnSO4.3H2O 0.5, H2SO4 0.1N 10 ml, pH adjusted to 10.0 with 1N NaOH solution.

Medium F: Potassium chloride 1.0, sodium glutamate 1.0, NH4Cl 1.0, KH2PO4 1, yeast extract 5.0, casein hydrolysate 5.0, FeCl2.4H2O 0.036, MnCl2.4H2O 0.36, NaCl 20.0, Na2CO3 5.0, agar 20.0.

Morphological and Biochemical Characteristics of the Isolates

Bacterial strains were examined for their colony and cell morphology, motility, Gram and spore staining and standard biochemical tests (catalase, oxidase, oxidation and fermentation of D-glucose, production of acid from D-glucose, L-arabinose, D-xylose, D-mannitol, glycerol, D-ribose, mannose, sorbitol and sucrose; nitrate reduction and denitrification, methyl red reaction, acetoin production, citrate utilization by Simmon’s, urease activity, indole production, hydrolysis of casein, gelatin, starch, arginine, carboxy methyl cellulose, tributyrin and esculin, growth on nutrient agar with different NaCl concentrations 0.5–3.5 M and growth at various temperatures (from 5, 10, 30, 40, 50, 55 to 65°C) according to Bergey’s Manual of Systematic Bacteriology [23, 42]. The growth was measured as optical density at 600 nm for 1–5 days. To determine whether the isolates were alkaliphilic or alkalitolerant, cultures were inoculated in respective liquid enrichment media with pH ranging from 7 to 12 and incubated at 37°C. Cell growth was monitored as the optical density at 600 nm after 48 h. The effect of various concentrations of NaCl on the growth of isolates was studied by growing them in medium containing 0–5 M NaCl at their respective optimum pH for 48 h. The isolates were tested for the production of exopolysaccharides using nutrient agar (pH 10) with 50 g l−1 glucose medium and selected on the basis of colony morphology [29]. Presence of polyhydroxyalkanoate (PHA) was studied as described earlier [33, 36].

16S rDNA Sequencing and Phylogenetic Analysis

DNA was extracted from selected cultures using standard phenol chloroform extraction procedures [39]. The 16S rRNA genes were amplified from the extracted DNA as described here by PCR with two bacterial primers: 16F 27 (5′-CCAGAGATTGATCMTGGCTCAG-3′) and 16R 1525 (5′-TTCTGCAGTCTAGAAGGAGGTGWTCCAGCC-3′), corresponding to E. coli 16S rRNA numbering [25]. The reaction mixture contained 1.5 mM MgCl2, 0.2 M each dNTP, 25 pM each primer, 100 ng template and 2.5 U Taq DNA polymerase (Bangalore Genei, India) with a reaction buffer supplied by the manufacturer in a total volume of 100 μl. PCR was performed with an initial denaturation at 98°C for 2 min, 35 cycles of 45 s at 95°C, 45 s at 55°C and 90 s at 72°C, followed by a final 12 min extension at 72°C. The PCR products were purified by the PEG/NaCl method as described previously [2] and directly sequenced on the Applied Biosystems model 3730 DNA sequencer (Foster City, Calif., USA).

All the sequences were compared with 16S rRNA gene sequences available in the GenBank databases by BLASTn search [1]. Multiple sequence alignments of approximately 900-bp sequences were performed using CLUSTAL W, version 1.8 [44]. A phylogenetic tree was constructed from evolutionary distances using the neighbor-joining method [38]. Tree topologies were evaluated by performing bootstrap analysis of 1,000 data sets by use of the MEGA 3.1 package [24]. Trees generated were analyzed with the TREEVIEW program [47]. The 16S rDNA sequences from this study have been submitted to NCBI GenBank Database under accession numbers as DQ333285 to DQ333302, DQ354090 to DQ354108, DQ157467 to DQ157468, DQ166854 to DQ166855, DQ077906 to DQ077911, DQ409330 to DQ409331 and AY935689.

Results and Discussion

The data on the chemical analysis of the four sediment and two water samples (shown in Table 1) indicated that the Lonar lake water and sediment samples were alkaline with the pH ranging from 8.84 to 9.82. Sediment samples had alkalinity in terms of CaCO3 ranging from 1,750 to 2,750 mg l−1, while for water samples 2,275–2,750 mg l−1. The salinity in terms of NaCl for sediment samples was found in the range of 586–1,445 mg l−1 and for water samples 3,887–4,532 mg l−1.

The TVC of the aerobic bacteria from sediment samples was found to range from 102 to 105 cfu g−1 and comparatively more than that of the TVC of the water sample, 102–104 cfu ml−1. It is seen from Table 2 that the maximum TVC of bacteria was present in the alkaline pH range (8–10) than neutral pH. As regards microbial population density in soda lakes, TVCs of aerobic organotrophic bacteria of diluted lakes indicate 105–106 cfu ml−1 [11], whilst from the hyper-alkaline spring waters of Maqarin in Jordan 103–105 cfu ml−1 [34]. In our studies, the TVC of the Lonar lake sediment and water samples was somewhat lower than other soda lakes, but comparable with hyper-alkaline water.
Table 2

TVC of bacteria

Sample

Temperature (°C)

pH

7

8

9

10

11

12

SD2 cfu g−1

28 ± 2

6 × 103

7 × 103

9 × 103

1 × 105

3 × 103

1 × 102

37

1 × 103

1 × 106

8 × 105

1 × 105

6 × 103

1 × 103

WI cfu ml−1

28 ± 2

1 × 103

4 × 103

6 × 103

7 × 103

1 × 104

6 × 103

37

4 × 103

1 × 104

2 × 103

2 × 103

1 × 104

5 × 103

Morphological characteristics, optimum pH and salt tolerance of all the strains were studied and 64 isolates were selected on the basis of their pH and salt tolerance. The isolates were identified on the basis of biochemical characteristics as described earlier and further confirmed by 16S rDNA sequencing. Out of 64 isolates, 31 were selected on the basis of their enzyme profile as described in Table 3. Our attempts to isolate different morphotypes led to diversity either in cell morphology or in colony morphology. All of the strains were catalase positive; most of them were oxidase positive, except for five. Majority of isolates were Gram positive. Among the selected strains, 14 isolates grew optimally at pH 9, eight isolates at pH 10, four at pH 8, two isolates (ARI 345 and ARI 347) at pH 11, while only two (ARI 332 and ARI 358) were able to grow optimally at pH 12. One isolate (ARI 85) had seven as optimum pH for growth. ARI T1 and ARI T2 could grow only at pH above 8. We tested salt tolerance of all strains. Fourteen strains could grow optimally in 0.86 M salt. Only two isolates (ARI T1 and ARI T2) required 2.5 M and 0.86 M salt for growth, respectively. Among all the isolates, 24 were able to produce lipase, 16 isolates could produce amylase, and ten isolates could produce caseinase, while six isolates produced the enzyme cellulase at alkaline pH.
Table 3

Characteristics of representative strains of Lonar lake and their phylogenetic affiliations (+ positive, negative, NR not required)

Isolate no.

Sample type

Isolation medium

Colony color

Cell morphology

Gram stain

Catalase

Oxidase

PH tolerance

Optimum pH

Salt tolerance, M

Optimum salt, M

Production of extracellular enzymes

Division

Nearest phylogenetic neighbour and (%) similarity to nearest neighbour

Amylase

Lipase

Caseinase

Cellulase

ARI 12

SD2

C

White

Rod

+

+

7–11

9

0–3.4

0.86

+

+

+

Firmicute

Bacillus cereus (99%)

ARI 15

SD2

C

White

Rod

+

+

7–12

9

0–5.1

NR

+

+

+

+

β-Proteobacteria

Alcaligenes sp. A 72 (99%)

ARI 22

SD1

C

Colourless

Rod

+

+

7–10

10

0–3.4

0.86

+

γ-Proteobacteria

Providencia rustigianii (99%)

ARI 72

SD3

C

Colourless

Rod

+

+

+

7–10

10

0–1.7

0.86

+

+

+

Firmicute

Arthrobacter mysorens (98%)

ARI 77

SD2

C

Colourless

Rod

+

7–12

8

0–5.1

NR

+

+

γ-Proteobacteria

Stenotrophomonas sp. AHL 1 (99%)

ARI 83

SD4

C

Yellow

Cocci

+

+

+

7–12

8

0–2.5

NR

+

+

+

Firmicute

Micrococcus luteus (99%)

ARI 85

SD1

C

Orange

Cocci

+

+

+

7–12

7

0–2.5

NR

+

+

Firmicute

Planococcus maritimus (93%)

ARI 195

SD2

C

White

Cocci

+

+

7–12

10

0–2.5

NR

+

α-Proteobacteria

Paracoccus sp. (99%)

ARI 317

SD3

A

Colourless

Rod

+

+

7–11

9

0–2.5

NR

+

 

Firmicute

Bacillus cereus (100%)

ARI 318

SD3

A

White

Rod

+

+

+

7–10

9

0–1.7

NR

+

+

Firmicute

Paenibacillus sp. L 55 (96%)

ARI 319

SD3

A

White

Rod

+

+

+

7–10

9

0–2.5

NR

+

+

Firmicute

Bacillus benzoevorans (99%)

ARI 324

WI

A

Colourless

Rod

+

+

+

7–12

8

0–2.5

NR

+

+

Firmicute

Bacillus flexus (99%)

ARI 329

SD2

B

Colourless

Rod

+

+

+

7–10

9

0–2.5

NR

+

Firmicute

Bacillus cohnii (96%)

ARI 332

SD2

B

White

Rod

+

+

+

8–12

12

0–2.5

NR

+

+

+

+

Firmicute

Bacillus sp. 199 (100%)

ARI 336

WC

B

White

Cocci

+

+

7–11

9

0–2.5

NR

+

Firmicute

Enterococcus caseliflavus (100%)

ARI 338

SD2

C

White

Rod

+

+

+

7–10

10

0–2.5

NR

+

+

Firmicute

Bacillus firmus (99%)

ARI 339

SD2

C

Orange

Rod

+

+

+

7–10

8

0–1.7

0.86

+

Firmicute

Dietzia natronolimnaea (99%)

ARI 341

SD3

C

Orange

Cocci

+

+

+

7–11

10

0–2.5

NR

+

+

Firmicute

Vagococcus carniphilus (98%)

ARI 343

WI

C

Cream

Rod

+

+

7–10

9

0–3.4

0.86

+

Firmicute

Uncultured bacterium clone (99%)

ARI 345

WI

C

White

Rod

+

+

+

7–11

11

0–0.86

0.86

+

+

Firmicute

Bacillus fusiformis (100%)

ARI 347

WC

C

Colourless

Rod

+

+

+

7–12

11

0–3.4

0.86

+

+

Firmicute

Exiguobacterium aurantiacum (99%)

ARI 351

SD1

D

Colourless

Rod

+

+

7–12

9

0–3.7

2.0

+

+

γ-Proteobacteria

Halomonas campisalis (100%)

ARI 357

SD3

D

Yellow

Rod

+

+

7–12

9

0–1.7

0.86

+

+

+

Firmicute

Bacillus horikoshii (99%)

ARI 358

SD4

D

Brown

Rod

+

+

7–12

12

0–2.5

0.86

+

γ-Proteobacteria

Lake Bogoria isolate 25 B1 (99%)

ARI 359

SD4

D

Orange

Cocci

+

+

7–10

10

0–3.4

0.86

Firmicute

Planococcus maritimus (99%)

ARI 360

SD4

D

White

Cocco-bacilli

+

+

7–11

9

0–3.4

0.86

+

γ-Proteobacteria

Lake Bogoria isolate 25 B1 (99%)

ARI 361

WI

D

Yellow

Rod

+

+

+

7–11

9

0–2.5

0.86

+

Firmicute

Glacial ice bacteriumSB12K-2-1 [99%]

ARI 371

SD3

E

White

Rod

+

+

+

7–12

10

0–1.7

NR

+

+

Firmicute

Cellulosimicrobium cellulans [99%]

ARI 375

SD1

E

White

Rod

+

+

+

8–11

10

0–0.86

0.86

+

Firmicute

Bacillus fusiformis [98%]

ARI T1

WI

G

Colourless

Rod

+

+

+

8–11

9

0.86–4.3

2.5

+

Firmicute

Alkalibacillus haloalkaliphilus [99%]

ARI T2

WI

G

Colourless

Rod

+

+

+

9–11

9

0.86–1.7

0.86

+

+

Firmicute

Bacillus sp (99%)

Primary goal of the present investigation was to determine bacterial diversity of Lonar lake using different culture media with various substrates as peptone, yeast extract, glucose and starch. The media employed in the present study were suitable for the growth of different bacteria associated mainly with Firmicutes and Proteobacteria. Media A and B favored the growth of several moderate halophilic alkalitolerant Bacillus species. Growth of Gram-positive bacteria of the genera Bacillus, Arthrobacter, Micrococcus, Planococcus, Dietzia, Vagococcus, Exigobacterium, and some of the Gram-negative isolates related to genera Alcaligenes, Providencia, Stenotrophomonas and Paracoccus was supported by medium C. Medium D was favorable for the growth of several Gram-negative bacteria related to Halomonas and Gram-positive isolates of Bacillus, while medium E for the growth of Gram-positive isolates of the genera Bacillus and Cellulosimicrobium. Obligately haloalkaliphilic bacteria related to genus Alkalibacillus could be grown only on medium F.

Different media with different substrates, like glucose, starch, peptone and olive oil etc., have been used earlier to study phylogenetic diversity of soda lake bacteria [10]. Their results indicated presence of Gram-negative bacteria related to the γ3 subdivision of Proteobacteria, Gram-positive isolates of High % G+C and Low % G+C divisions, with the majority of bacteria belonging to the Bacillus group, others to Arthrobacter and an anaerobic thermophilic isolate related to Thermotogales from samples collected from lake Bogoria, which has a temperature of 66–96°C.

Phylogenetic analysis of representative isolates (Fig. 2) indicated that most of the isolates were related to the phylum Firmicutes, which included Low G+C Gram-positive bacteria related to different families of Bacillaceae, with the four genera Paenibacillus, Bacillus, Alkalibacillus and Exiguobacterium, Planococcaceae with genus Planococcus and Enterococcaceae with genera Enterococcus and Vagococcus. Phylogenetic affiliations of Lonar lake isolates with nearest phylogenetic neighbor in the GenBank database is shown in Table 3.
https://static-content.springer.com/image/art%3A10.1007%2Fs00248-007-9264-8/MediaObjects/248_2007_9264_Fig2_HTML.gif
Figure 2

Unrooted phylogenetic tree based on a comparison of the 16S ribosomal DNA sequences of Lonar lake isolates and some of their closest phylogenetic relatives. The tree was created by the neighbor-joining method. The numbers on the tree indicates the percentages of bootstrap sampling derived from 1,000 replications. Isolates characterized in the present study are indicated in bold. Bar inferred nucleotide substitutions per nucleotides

In the present study, Gram-positive isolates obtained were found to be more diverse and predominant. Within the Gram-positive bacteria, Low G+C bacteria were associated with the members of the diverse Bacillus spectrum. Similar results were observed from studies on alkaliphilic diversity of the Hailaer soda lakes in Inner Mongolia [52] and the highly alkaline lake Van in Turkey [26]. This study indicated the presence of Bacillus, Exiguobacterium, Paenibacillus and Enterococcus, which were reported from various soda lakes as the highly alkaline lake, Van in Turkey, Inner Mongolian Baer Soda Lake, and a nonsaline alkaline lake and also from a Kenyan Soda Lake [7, 26, 27, 45, 48]. One of our isolates, Bacillus cohnii (ARI 332) has been found to have an optimum at pH 12, tolerate salt up to 2.5 M and produce all the four enzymes, thus exhibiting to be biotechnologically very important. The main industrial application of alkaliphilic enzymes is in the detergent industry. The most commonly used enzymes in detergent industries are proteinases, lipases and amylases. Cellulases have been introduced as laundry additives. Previously reported B. cohnii was isolated from alkaline soil, horse meadows and old horse feaces but not from soda lakes [43]. Vagococcus carniphilus described previously was isolated from ground beef [41] but the isolate (ARI 341) from Lonar lake was found to produce enzymes amylase and lipase at alkaline pH. It has an interesting property of secreting exoploysaccharide (EPS) using glucose as a carbon source at pH 10. This is probably the first report of EPS production by Vagococcus isolated from Lonar lake and becomes a new finding. There was a report on the production of EPS by a marine bacterium Pseudomonas sp. strain S9 due to stress [49]. Planococcus maritimus was reported from the Yellow Sea in Korea [50]. It could grow optimally at pH 6–8 the in presence of 0.34 M NaCl, while our isolate (ARI 359) could grow optimally at pH 10 with 0.86 M NaCl concentration.

In our studies, fewer bacteria were found in the High G+C group. The isolates (ARI 72, ARI 83, ARI 339, ARI 361 and ARI 371) were associated with the phylum Actinobacteria of various genera—Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus—belonging to the different families, Promicromonosporaceae, Dietziaceae and Micrococcaceae, respectively. Among them, Glacial ice bacterium was reported earlier from the highly alkaline lake Van, Turkey [26]. Micrococcus, Arthrobacter and Dietzia were isolated earlier from east African soda lakes [10]. One organism, Cellulosimicrobium cellulans described earlier [40] is not yet reported from any of the soda lakes. Thus, presence of three organisms—namely, Vagococcus carniphilus (ARI 341), Planococcus maritimus (ARI 359) and Cellulosimicrobium cellulans (ARI 371)—from the Lonar lake is a new finding, which extends our knowledge of the diversity of the soda lakes.

Seven representative strains of the isolated populations belonged to the Gram-negative bacterial group. Five isolates (ARI 358, ARI 360, ARI 351, ARI 77 and ARI 22) were γ-Proteobacteria related to the families Halomonadaceae, Xanthomonadaceae and Enterobacteriaceae with the genera Halomonas, Stenotrophomonas and Providencia, respectively. One isolate (ARI 15) belonged to β-Proteobacteria with the family Alcaligeneceae and genus Alcaligenes, while the isolate (ARI 195) was found to be α-Proteobacteria belonging to the family Rhodobacteraceae of the genus Paracoccus.

There are many reports on the isolation and characterization of Gram-negative bacteria from different soda lakes. Studies on the Inner Mongolian soda lake [27] indicated dominance of Gram-negative bacteria of the α, β, γ and δ groups, with a low percentage of Gram-positive bacteria. Similar results were also observed in the case of the hyper-alkaline spring waters in Jordan [34]. Halomonas,Stenotrophomonas,Alcaligenes and Paracoccus were already reported from different soda lakes [9, 10, 27, 51]. Halomonas campisalis (ARI 351), isolated from a sediment sample of Lonar lake, is found to be different than a previously described one [31]. It was able to produce enzymes like amylase and lipase at alkaline pH. It showed the formation of polyhydroxyalkanoate (PHA) within 6 h of incubation, with a very good yield of 45%. Production of PHA by Halomonas boliviensis was described earlier [36]. This is the first report of the production of PHA by H. campasalis and thus a very important finding. Extremophilic bacteria are said to follow a different metabolic pathway and produce novel substances under stress. PHA is also produced under unbalanced nutritional conditions during growth. Our isolate was also able to produce this polymer under alkaline conditions. Thus, the alkaline saline environment of the lake has harbored a haloalkalitolerant organism producing PHA, which is produced under stress conditions. Providencia rustigianii was isolated from different clinical samples [15]. The presence of this organism (ARI 22) in Lonar lake may be due to possible contamination from the nearby soils or by human and animal activities.

In the present studies, Firmicutes belonging to the LowG+C group are more diverse and abundant than Gram-negative Proteobacteria. This is probably because of inability of culturing Gram-negative bacteria from the lake. Thus, nutrient media specially designed for the isolation of Gram-negative bacteria must be used. Similar results were obtained in the case of the Inner Mongolian Soda Lake using culture-dependent and culture-independent approaches.

The DNA sequences of the three isolates, ARI 85 (93% similarity with Planococcus maritimus, ARI 329 (96% similarity with Bacillus cohnii) and ARI 318 (96% similarity with Paenibacillus sp. L 55), showed less than 97% similarity with the previously known sequences in the GenBank database. Thus, these could be novel organisms, which needs to be further confirmed by fatty acid analyses, DNA-DNA hybridization etc.

The present study not only describes bacterial diversity of the Lonar lake ecosystem but also indicates many biotechnologically important cultures. Most of the cultures isolated from Lonar lake are able to produce lipase, amylase, cellulase and caseinase at alkaline pH. The Lonar lake is surrounded by forest and many trees grow in the peripheral water body. The litter is accumulated at the periphery of the lake. Green algae as well grow luxuriously in the peripheral lake boundaries. Thus, the lake water receives rich organic matter. It is no wonder that the organisms isolated from Lonar lake exhibit ability to produce hydrolytic enzymes like lipase, amylase, cellulase and caseinase. Many of the Lonar lake isolates produced all these enzymes at alkaline pH (Table 3). The stability of these enzymes at alkaline pH is attributed to their habitat (alkaline lake) and growth profile in a wide range of pHs.

To understand the roles and structures of microbial communities, sequence data only are not enough, but cultivability of microorganisms is very important [4]. Lonar lake harbors a wealth of diverse microorganisms with useful commercial properties. Thus, the culture-dependent approach used in the present study contributes to our understanding of Lonar lake diversity and provides useful information on many fascinating cultures in this extreme environment.

Acknowledgements

The work was carried out under All India Co-ordinated Project on Taxonomy - Centre for Research on Bacteria and Archaea (AICOPTAX) funded by Ministry of Environment and Forests, Government of India.

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© Springer Science+Business Media, LLC 2007