Indian Journal of Geo-Marine Sciences Vol.44(10), October 2015, pp. 1646-1653

Studies on Haloalkaliphilic from hypersaline Sambhar Lake, Rajasthan, India

Makarand N. Cherekar & Anupama P. Pathak* School of Life Sciences (DST-FIST & UGC-SAP Sponsored), Swami Ramanand Teerth Marathwada University, Nanded-431606, India. *[E-mail: [email protected]]

Received 19 December 2013; revised 04 March 2014

The diversity of haloalkaliphilic gammaproteobacteria from hypersaline Sambhar soda lake, India was analyzed. Four different sampling points were selected within the lake. Nine moderate and extreme isolates were identified using morphological, biochemical and 16S rRNA sequencing method. These isolates were screened for salt, pH and temperature tolerance. In the gammaproteobacteria group, all the selected isolates belonged to one genus represented by four different species Halomonas pantelleriensis, Halomonas venusta, Halomonas alkaliphila, and Halomonas sp. All these isolates were capable to grow in 10 -20% NaCl concentrations, 8-12 pH and 20 to 50 oC temperature. These isolates were competent to produce industrially important extracellular hydrolytic enzymes. Most of them were shown to produce cellulase, pectinase and xylanase.

[Keywords: Gammaproteobacteria, Halomonas, haloalkaliphilic, Sambhar soda lake, 16S rRNA sequencing]

Introduction Sambhar Lake is the largest inland saline lake located Hypersaline soda lakes are considered as extreme in Thar Desert of Rajasthan, India (26o 52’- 27 o 2’ N, environments for microbial life. These lakes are 74 o 53’- 75 o 13’E). It is an elliptical and shallow characterized by high salinity and high alkalinity with lake, with the maximum length of 22.5 km. The width extremely productive environment for extreme of the lake ranges from 3.2 km to 11.2 km. The total organisms1,2. Hypersaline soda lakes have been catchments area of the lake is 7560 km2. The lake has studied by focusing on the isolation and occupied an area of about approximately 225 Sq. Km characterization of organisms with their industrial and average depth of water is about 1 m whereas the potential3,4. maximum depth is about 3m. Sambhar lake is In India Sambhar soda lake located in arid land of declared as Ramsar site in 1990 for receiving large Rajasthan is an example of such unique ecosystem. It number of migratory birds5-7 (Fig. 1). is an ancient soda lake and a rich source of salt in India. Extreme alkaline pH and high salt concentration made this site more suitable for growth of extreme haloalkaliphiles. In the present study, we reported culturable gammaproteobacterial diversity form Sambhar soda lake using biochemical and 16S rRNA gene sequencing methods. These belong to the genus Halomonas and had an obligate requirement for high pH and NaCl concentration for growth. The isolates were further characterized and screened for production of industrially important hydrolytic enzymes. Materials and Methods Fig.1—Sampling sites from Sambhar lake (Adopted from Sahay et.al 2012) CHEREKAR & PATHAK: HALOALKALIPHILIC GAMMAPROTEOBACTERIA FROM SAMBHAR LAKE, RAJASTHAN 1647

The surface and Sediment water samples were For detection of amylase starch was used as collected in the winter (December) 2011 from four substrate and zone of clearance was observed by sampling stations located in main lake and salt pans flooding incubated plates with iodine. For detection of towards Sambhar Lake city. Samples were collected protease skim milk was used as substrate and zone of in presterilized polypropylene bottles these were clearance was observed. For detection of cellulase average of ten samples spanning the whole sampling carboxy methyl cellulose was used as substrate and point (Fig. 1). Temperature and pH were measured on zone of clearance was observed by flooding with field. Collected samples were transported on ice and congo red solution. For detection of pectinase pectin preserved at 4oC until analysis. Experiments were and for lipase olive oil was used as substrate and zone performed to determine selected physicochemical of clearance was observed. For detection of xylanase parameters like TS, DO, BOD, alkalinity, salinity, xylan was used as substrate and zone of clearance was sodium, magnesium, calcium etc8, 9. observed by flooding congo red solution12,15. Enrichment and isolation of bacteria were 16S rRNA analysis was performed by extracting performed by inoculating composite water sample DNA of isolates. DNA was extracted by phenol– into nine different broth media viz Alkaliphilic broth chloroform extraction. DNA was washed with 70% media pH- 10.0[A], Marine broth pH- 10.5 [MA], ethanol and dissolved in Tris–EDTA buffer (pH 8.0). Alkaline Nutrient broth pH- 10.5 [ANA] with 5-30 % Extracted DNA was analyzed by electrophoresis on a sodium chloride, Halophilic medium [H], modified 1% agarose gel and visualized by ethidium bromide Horikoshi II medium [H II], Synthetic Sea water staining16. The amplification of 16S rRNA fragments medium [S], Alkaline peptone water [AP], Alkaline were performed by using (PCR) thermocycler, Bacillus medium [AB] and Tindal’s medium [T]. (Eppendorf) with 530F These media were incubated at 30oC for 8 days on a (5′ GTGCCAGCAGCCGCGG 3′) and 1392R (5′ shaking incubator at 100 rpm speed. After enrichment ACGGGCGGTGTGTAC 3′) primer pair. PCR bacteria were isolated using respective agar media and products were run on a 1% agarose gel. PCR products pure cultures were obtained5,10-12. were purified by the PEG/NaCl method17 and directly All the isolates were screened for casein, gelatin, sequenced using Applied Biosystem model 3730 oxidase, catalase, starch hydrolysis, urease, nitrate DNA analyzer (Foster, California, USA). The 16S reduction, H2S production and utilization of sugars. rRNA sequences were initially analyzed using The Gram-staining was performed according to BLAST program Dussault13. Sensitivity of the strain to antibiotics was (www.ncbi.nlm.nih.gov/blast/blast.cgi). Multiple tested by using alkaline nutrient agar medium. sequence alignments of approximately 800 Chloramphenicol (30mcg), streptomycin (10mcg), sequences were performed using CLUSTALW 18 gentamycin (10mcg), tetracycline (30mcg), penicillin program . Phylogenetic and molecular evolutionary 19 G (10mcg), ampicillin (10mcg) and erythromycin analysis was conducted using MEGA version 5.2 . (15mcg) were used for antibiotics sensitivity test14. Bootstrap analysis was performed on 1000 random All the isolates were screened for tolerance level of samples taken from the multiple alignments. The 16S NaCl using alkaline nutrient agar medium with rRNA sequences from GenBank used in the different salt concentration ranging from 5 to 30% phylogenetic analysis are shown in Fig. 4. (5% intervals). The growth was measured at 600 nm The 16S rRNA sequences determined were with regular intervals. Similarly for screening of pH deposited in the GenBank and accession numbers and temperature tolerance the isolates were grown in KF288960- KF288961, KC434452- KC434453, alkaline nutrient agar medium with pH ranging from 7 KC434456, KC934936, KC934938- KC934940 were to 12 (Increment of 0.5) and the temperature ranging obtained. from 20 to 50oC (10 degrees intervals)5,10-12. All hydrolytic assays were performed by Results and Discussion inoculating isolates on alkaline nutrient agar medium Chemical composition of Sambhar lake water with respective substrate at 9 pH, 10% Salt significantly varies in monsoon and winter seasons. o o concentration, 40oC temperature and 7-15 days The maximum 31.7 C and minimum 22.3 C incubation period12,15. temperature of surface water was recorded in winter 1648 INDIAN J. MAR. SCI., VOL.44, NO.10 OCTOBER 2015

2011. The average pH values recorded for water 0.9 sample was 9.2 to 10.5. Surface algal blooms and 0.8 eutrophication of lake was also observed in peripheral 0.7 0.6 SSL8 locations. Lake water was considerable turbid and SSL11 0.5 SSL13 opaque. The Total Solids and Total dissolved Solids 0.4 SSL14 were recorded as 146293 mg/l and 88263 mg/l in 0.3 SSL15

ptical Density600nm at 0.2 monsoon and winter 2011 respectively. These values O 0.1 were higher as compared to the well studied African 0 soda lake, Kenyan soda lakes and Lonar soda lake, 7 8 8.5 9 9.5 10 10.5 11 12 pH India. Most abundant constituent present in lake water was sodium (9063mg/l). Next to sodium, (a) chloride (6633mg/l) was the most abundant constituent in lake water. Sulphate was also recorded 1.2 in considerable amount. The amounts of calcium and 1 11 0.8 magnesium ions were found in least amount . SSL3 SSL4 Considerable amount of various metal ions were 0.6 SSL5 recorded from this water samples. 0.4 SSL6 pticalDensity at 600nm

Among nine broth media used alkaliphilc broth O 0.2 medium [A], alkaline nutrient broth [ANA], modified 0 Horikoshi II broth medium [H II], Marine broth [MA] 7 8 8.5 9 9.5 10 10.5 11 12 pH have supported highest growth during enrichment. Agar media of same type supported diversity were (b) used in further investigation. Isolates showing distinct Fig. 2 (a and b)—Effect of pH on growth colony characters were selected from alkaliphilc medium [A], alkaline nutrient agar [ANA], modified 0.4 Horikoshi II medium [H II] and Marine agar [MA] 0.35 0.3 SSL3 plates. Small colonies were appeared after incubation 0.25 SSL4 of 10 days, further incubation of 20-30 days have 0.2 SSL5 SSL6 yielded large colonies. The Alkaline peptone water 0.15 SSL8 0.1 [AP], Alkaline Bacillus medium [AB] and Tindal’s ptical Density at600nm O medium [T] supported least population and diversity. 0.05 0 Out of sixty four, 9 extreme haloalkaliphiles were 5 10 15 20 25 30 selected for further characterization and designated as NaCl Concentration(w/v)% SSL3 to SSL6, SSL8, SSL11 and SSL13 to SSL154,11,12. (c) All the isolates were aerobic, Gram negative, rods 0.7 and oxidase positive. Out of nine, 4 isolates (SSL4, 0.6

SSL6 SSL8 and SSL15) were motile and 5 isolates 0.5 SSL11 (SSL3, SSL5, SSL11, SSL13 and SSL14) were non 0.4 SSL13 motile. The isolates SSL13 was positive for gelatinase 0.3 SSL14 activity, remaining all were negative for Starch. They SSL15 0.2 ptical Density600nm at

did not produce acid from lactose and none of the O isolate could produce gas either from glucose, lactose, 0.1 0 mannose, and arabinose. Further arabinose, fructose, 5 10 15 20 25 30 glucose, galactose, mannose, ribose and lactose were NaCl Concentration (w /v)% not utilized when provided as the sole carbon source. All isolates were susceptible to the antibiotic (d) penicillin G and chloramphenicol20,21 (Table 1). Fig.3 (c and d)—Effect of Salinity on growth

CHEREKAR & PATHAK: HALOALKALIPHILIC GAMMAPROTEOBACTERIA FROM SAMBHAR LAKE, RAJASTHAN 1649

Table 1—Morphological and Biochemical characteristics of Sambhar lake isolates

SSL3 SSL4 SSL5 SSL6 SSL8 SSL11 SSL13 SSL14 SSL15

Morphology Rod Rod Rod Rod Rod Rod Rod Rod Rod Gram nature ------Size (mm) (1-2) 0.5-2 (1-2) (1-2) (0.5-2) (1-0) 0.5-2 (1-2) (0.5-2) Colony pigmentation cream cream cream Ye llow cream white cream cream cream non non non non Motility motile motile non motile motile motile motile motile motile motile

Oxidase + + + + + + + + + Catalase - + - + + - - - + pH range 8.0-11 8.0-11 8.0-11 8 -10.5 8.0-11 8.0-11 8.0-11 8.0-11 7.0-11 Optimum pH 9.5 8.5 9.5 9.5 8.5 9 9 9.5 8.5 Salt range % 7-15 5-15 5-20 5-20 5-15 8-25 8-25 5-25 5-15 Optimum Salt % 10 10 10 15 10 20 20 10 10 Temperature oC 20-40 20-40 20-40 20-45 20-40 20-40 20-40 20-40 20-40 Urease - + - - + + - - + Nitrate reduction - + - + + - - - + H2S production - + - - + - - - + Hydrolysis of:

Casein - - - + - + - - -

Gelatin ------+ - -

Starch ------

Utilization of: Arabinose ------ND - - Fructose - - - + - - - - - D-Glucose - - - + - - - - - D-Galactose ------Mannose ------ND ND -

Ribose ------Lactose ------Growth on streptomycin + - - - - + + - - ampicillin ------+ - - gentamycin - + - - + - + - + penicillin G + ------

chloramphenicol ------

tetracycline - + - - + - + - +

erythromycin + - + - - + - - - + Positive, - Negative, ND Not Detected

All isolates were capable to growing in NaCl (SSL6) was able to tolerate 15 % of NaCl and 6 concentration of 10-20%. Isolates (SSL11, SSL13) isolates (SSL3, SSL4, SSL5, SSL8, SSL14 and could tolerate 20% of NaCl concentration, isolate SSL15) could tolerate 10% of NaCl concentration. 1650 INDIAN J. MAR. SCI., VOL.44, NO.10 OCTOBER 2015

SSL6(KC434453) SSL13(KC934938) Halomonas sp.(JF340230) Halomonas alkaliphila(JQ680444) Halomonas hydrothermalis(NR 027220) Halomonas hydrothermalis(GU938192) Halomonas stevensii(AM941388) SSL11(KC934936) Halomonas meridiana(KF500395) Halomonas aquamarina(NR 042063) Halomonas sp.(JF499685) SSL5(KC434452) Halomonas alimentaria(EF517969) Halomonas sp.(EU870505) SSL3(KF288960) SSL14(KC934939)

Halomonas ventosae(GQ903444)

Halomonas pacifica(EU373088)

Halomonas salina(AY505524)

Halophilic bacterium (KC142101)

Halomonas sp.(AM110974)

Halomonas campaniensis(NR 042157)

Halomonas ilicicola(NR 044436) Halomonas phoceae(AY922995) Halomonas xinjiangensis(NR 044350) SSL8(KC434456) Halomonas pantelleriensis(NR 026298) SSL4(KF288961) SSL15(KC934940)

0.005

Fig. 4—Phylogenetic analysis of haloalkaliphilic gammaproteobacteria isolated from Sambhar Lake. The 16S rRNA gene sequence based dendrogram shows Sambhar lake isolates were constructed using neighbour-joining method.

but optimum pH was 9.5. Isolates (SSL4, SSL8 and The species of genus Halomonas can tolerate or SSL15) were able to grow in an optimum pH 8.5 and required a high salt concentration for growth22 (Table the optimum pH for isolates (SSL11, SSL13) was 9. 2) (Fig. 2 and Fig. 3). Similarly pH tolerance was also All isolates were grown at temperature ranging from o o o observed; some of isolates (SSL3, SSL5, SSL6 and 20 C to 50 C with an optimum at 40 to 45 C. SSL14) were able to grow in a pH range 8 to 12

CHEREKAR & PATHAK: HALOALKALIPHILIC GAMMAPROTEOBACTERIA FROM SAMBHAR LAKE, RAJASTHAN 1651

KC934939 accession numbers respectively. Isolate Table 2—Characterization of haloalkaliphilic isolates from SambharSSL4, lake and SSL8 their and identification SSL15 deposited based on nearest in gene phylogenetic neighbour

solate Code solate I Accession Number Optimum NaCl percentage Optimum pH Amylase Protease Cellulase Lipase Pectinase Xylanase Nearest phylogenetic neighbour similarity% Phylum/ Subclass Gamma SSL3 KF288960 10 9.5 - - + - + + Halomonas sp. 99 Halomonas Gamma SSL4 KF288961 10 8.5 - - - - - + pantelleriensis 99 proteobacteria Gamma SSL5 KC434452 10 9.5 - - + - + - Halomonas sp. 99 proteobacteria Gamma SSL6 KC434453 15 9.5 ------Halomonas venusta 100 proteobacteria

Halomonas Gamma

SSL8 KC434456 10 8.5 - - - - + + pantellerinsis 97 proteobacteria

Gamma SSL11 KC934936 20 9 - - + - - + Halomonas sp. 99 proteobacteria

Halomonas Gamma SSL13 KC934938 20 9 ------alkaliphila 99 proteobacteria Gamma SSL14 KC934939 10 9.5 - - + - - + Halomonas sp. 99 proteobacteria

Halomonas Gamma SSL15 KC934940 10 8.5 - - - - - + pantelleriensis 99 proteobacteria

Amongst nine selected isolate SSL3 showed bank with KF288961, KC434456, and KC934940 remarkable production of xylanase, cellulase and accession numbers respectively, showed 97-99% pectinase enzyme. Isolate SSL3, SSL4, SSL8, identity with Halomonas pantellerinsis. The isolate SSL11, SSL14 and SSL15 showed production of SSL6 and SSL13 showed 100% and 99% similarity xylanase. Xylanase enzyme producers were found with Halomonas venusta and Halomonas alkaliphila highest as compared to cellulase and pectinase and deposited in gene bank with KC434453 and producing strains. All isolates were found to negative KC934938 accession numbers. All nine isolates SSL3 for production of amylase, protease and lipase to SSL6, SSL8, SSL11 and SSL13 to SSL15 are enzymes (Table 2). Based on 16S rRNA gene deposited in PG research laboratory number 5 School sequencing and phylogenetic tree the bacterial isolates of Life Sciences SRTM University Nanded and 11-12,23 were identified and studied (Fig. 4). All selected accessible to all . isolates were belonging to gammaproteobacteria group and related to one genus Halomonas, Conclusion represented with four different species. The isolates Biotechnologically important haloalkaliphilic SSL3, SSL5, SSL11 and SSL14 showed 99% gammaproteobacteria isolation and characterization is similarity with Halomonas sp. and deposited in gene carried out from Sambhar soda lake, Rajasthan, India. bank with KF288960, KC434452, KC934936, and All the bacterial isolates showed optimum growth in 1652 INDIAN J. MAR. SCI., VOL.44, NO.10 OCTOBER 2015

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