Indian Journal of Natural Products and Resources Vol. 3(4), December 2012 pp. 483-488
Isolation and characterization of carotenoid producing Haloarchaea from solar saltern of Mulund, Mumbai, India Anupama P Pathak* and Aparna G Sardar School of Life Sciences, S R T M University, Nanded, Maharashtra, India Received 26 March 2012; Accepted 4 July 2012 Seven efficient carotenoid producing Halophiles were isolated and characterized from Solar saltern of Mulund, Mumbai, Maharashtra. Of these, a Gram negative aerobic rod shaped organisms SS-12 was selected for carotenoid production. It was identified as Halorubrum sp. using cultural, microscopic and biochemical characterization. Isolate SS-12 showed luxuriant growth at 20% NaCl concentration, 35°C temperature and at 7 pH. Spectophotometric analysis of pigment revealed that the pigment is bacterioruberin type. The antioxidant activity exhibited by extracted pigments was equivalent to the antioxidant activity of lutein and astaxanthin at 4 mM concentration. Keywords: Antioxidant, Carotenoids, Halorubrum sp., Solar saltern, Mumbai. IPC code; Int. cl. (2011.01)—A61K 36/00.
Introduction Carotenoids are naturally occurring yellow to orange red pigments synthesized as hydrocarbons (carotene; eg. lycopene, α-carotene and β-carotene) or their oxygenated derivatives (xanthophylls, eg. lutein, α-cryptoxanthin and β-cryptoxanthin, zeaxanthin, canthaxanthin and astaxanthin) by plants, bacteria, algae and fungi1-3. These pigments represent the largest and most diverse class of natural products known to mankind. It is the ability of carotenoids to confer color therefore, commercial interest has been developed in these molecules predominantly as natural colorants. In addition to their utilization as colorants many carotenoid have proved beneficial in prevention and treatment of certain cancers such as prostate cancer and in prevention of heart diseases by quenching free radicles4. Carotenoids are also widely used as colorant in various food products, to color yolk, meat and fish products. Amongst many natural resources of carotenoids; red, salt resistant archaea have gained more importance due to possible recovery of carotenoid at low ionic strength solution and under non aseptic condition5. In present investigation, for recovery and isolation of carotenoid producing haloarchaea we have spanned multi pond —————— *Correspondent author: E-mail:
[email protected]; Phone +91 9404732162, 09420718679; Fax: +91 (2462) 229245
solar saltern located in coastal region of Arabian Sea at Mulund (E) near Mumbai, India. The higher biodiversity of these nutrient deficient solar salterns also promoted us to search for carotenoid producers. Besides food coloring properties the anticarcinogenic, anti-inflammatory, radical scavenging properties of carotenoid are attracting researchers to develop new therapeutic products. Materials and Methods Sample collection from interconnected ponds
Brine Samples from seven shallow interconnected ponds were collected aseptically from traditional seasonal salt pans of Mulund (E) (19°10’12’’N, 72°57’18’’E), Mumbai, Maharashtra, India in February 2010. The samples from each pond were the average of 10 samples spanning the whole pond. The samples were filtered and stored in a refrigerator during investigation. The temperature was measured at the time of sampling in order to study abiotic characters of brine samples. We have determined following physicochemical parameters for seven samples individually, viz. total soilds, total dissolved solids and total suspended solids6. Salinity was measured with the Refractometer (Erma Tokyo). Electrical conductivity (EC) and pH was measured with a conductivitimeter (VSI-01C, VSI Electronics Pvt. Limited) and pH meter, respectively. Dissolved oxygen was measured by an oxygenometer (YSI 57).
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Ca++ and Mg++ were determined by EDTA titrimetric method. The K+ and Na+ were determined with flame photometry (CL 361 ELICO)7. Isolation of carotenoids producing Halophiles
Composite brine samples were spreaded on nutrient agar (Hi-media) with 20% salt and halophiles agar8 and plates were incubated at 35°C for 14 days. After incubation plates were observed and red pigmented colonies were selected for further analysis. Identification of selected isolates
Selected colonies were subcultured on nutrient agar plates with 20% salt and incubated at 35°C. Microscopic and macroscopic features of grown isolates were recorded. Amongst many pigmented colonies seven intense red colored fast growing colonies were selected for further analysis. Gram’s staining was performed by Dussalt’s modified method9. Catalase and oxidase activities, indole production, citrate utilization, lipase H2S productions, hydrolysis of gelatin, casein, starch, cellulose and urea were observed, Methyl red, Vogues–Proskauer tests and carbohydrate fermentation tests were performed using standard procedures10,11. Appropriate positive and negative controls were used in all these tests. Antibiotic susceptibility was tested by disc diffusion method using disc containing Bacitracin (10 units/disc), Polymyxin (300µg/disc), Ciprofloxacin (5µg/disc), Gentamycin (10 µg/disc) and Tetracycline (30 µg/disc) (Hi media Mumbai)12. Effect of pH, temperature and incubation time on growth of selected pigment producer
Effect of pH, temperature and NaCl on growth of pigment producers was determined by incubating inoculated cultures at various pH (4-10), temperature (20-60oC) and NaCl concentration (5-30%). pH, temperature and salt concentration showing highest growth was selected and used for further analysis. Optimum incubation time at predetermined pH and temperature was calculated by incubating culture and recording optical density at 540 nm. Out of seven isolates, isolate showing remarkable pigment production and fast growth was selected and used for pigment production.
8 days at 35°C. Pigment production was carried out using a medium composed of 5 g yeast extract, 5 g cas-amino acids, 1g Na-glutamate, 2g KCl, 3 g Na3-citrate, 20 g MgSO4.7H2O, 200 g NaCl, 36 mg FeCl2.4H2O, 36 mg MnCl2.4 H2O, 1000 mL distilled water, pH was adjusted to 7.0-7.2. Effect of NaCl concentration on pigment production was determined by varying it in the range of 0-30% and optimum concentration was used for production. Effect of temperature on pigment production was determined by varying production temperature in the range of 25-55oC and optimum temperature was recorded. Effect of pH was determined by varying it in the range of 4-10. Carotenoid extraction and analysis
The extraction of carotenoid was performed as described by Asker & Ohta5,13. Five mL of culture broth was centrifuged at 10,000 rpm for 10 min in cooling centrifuge. The harvested cells were re-suspended in distilled water. Spontaneous cell lysis occurred and then the pigments were extracted with methanol and transferred to hexane. The carotenoid extract was scanned in the wavelength region 400-800 nm using a UV-vis spectrophotometer (Shimadzu). Further carotenoid production and spectrophotometric characterization of crude pigment was carried out. Antioxidant activity of carotenoid DPPH radical scavenging assay
The DPPH assay was carried out according to the standard method. An aliquout of carotenoid (100 µL) and equal amount of acetone and MeOH were added to 950 µL of 100 µM DPPH methanol solution. The mixture was shaken vigorously and then left to stand at room temperature for 30 min in the dark. The absorbance was measured spectrophotometrically at 580 nm against an acetone/MeOH (1/1, v/v) blank. Percent DPPH radical scavenging activity was determined using the formula14,15 Control absorbance – sample absorbance × 100 Control absorbance Results and Discussion Physicochemical analysis of water sample
Optimization of carotenoid production
Optimum conditions for pigment production were determined by inoculating the selected strain in 100 mL nutrient broth in a 500 mL Erlenmeyer flask and incubating it in shaking incubator at 120 rpm for
The traditional seasonal salt pan of Mulund, Mumbai, was sampled in February 2010. The physicochemical characteristics of water samples collected are presented in Table 1. Water samples were pale yellow in color and viscous (Plates 1-3).
PATHAK & SARDAR: CAROTENOID PRODUCING HALOARCHAEA
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Table 1—Physicochemical characteristics of the seven ponds studied Initial pond
2nd Pond
3rd pond
4th pond
5th pond
6th pond
Crystallizer pond
Salinity (%)
10
12
18
22
23
32
34
pH
7
7.12
7.13
7.12
7.0
7.1
7.0
Physicochemical parameter
Temperature EC
35
40
40
42
42
55
58
16.11
16.15
16.2
16.31
16.42
16.42
16.56
Dissolved O2 (mg/l)
7.1
6.4
5.3
4.3
2.1
2.11
1.06
TS (g/L)
96
112
188
258
268
428
474
TDS (g/L)
82.8
96.0
170
239
260
420
426
TSS (g/L)
13.2
16.0
18.0
19.2
7.9
8.0
4.8
Ca++ (g/L)
1.06
1.33
1.29
1.78
1.20
2.4
1.96
Mg++ (g/L)
3.41
3.9
6.7
6.7
7.1
12.8
12.2
K+ (g/L)
5.7
6.2
6.6
7.3
7.5
7.8
7.2
69.2
68.5
81.7
89.0
108.1
96.3
94.2
+
Na (g/L)
Plate 1—Solar saltern at Mulund, Mumbai, India
Plate 3—Workers at solar saltern Isolation and screening of carotenoid producing halophilic bacteria
Plate 2—Crystallizer pond of saltern
Total 53 halophilic organisms were isolated and designated as SS-1 to SS-53. Amongst the 53 isolates, 21 were pigmented of which 7 were intense coloured colonies and they were selected and characterized. Colonies of all selected isolates were circular, red to orange in colour, small in size having raised elevation and entire margin. Out of seven four isolates have developed red colonies. Two developed orange colonies while one isolate has developed pink colony. All seven isolates were Gram negative aerobic rods and showed luxuriant growth at 20% salt concentration. Out of seven isolates only SS-1 has used glucose as carbon source. Four isolates SS-36, SS-37, SS-6 and SS-32 have used xylose and SS-36 has used maltose. However, none of the isolate has
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Table 2—Biochemical characteristics of isolated carotenoid producers Characteristics Morphology Gram nature Cell size (µm) Motility Colony pigmentation NaCl Range for growth (%) Temp. optimum °C PH Optimum Utilization of Glucose Sucrose Arabinaose Lactose Maltose Fructose Mannitol Xylose Sorbitol Cellobiose Galactose Indole production Enzyme Profile Catalase Amylase Gelatinase Urease Protease Cellulase Oxidase Antibiotics suspetibility Bacitracin (10 unit/disc) Polymyxin (300 mcg/disc) Ciprofloxacin (5 mcg/disc) Gentamycin (10 mcg/disc) Tetracyclin (30 mcg/disc) Identified as genus
SS-1
SS-36
SS-37
SS-6
SS-11
SS-12
SS-32
Rods Negative 2×1 Motile Pink 10-24 35°C 7
Rods Positive 3×1 Motile Orange 10-24 35°C 7
Short rods Positive 2×1 Motile Red 10-26 35°C 7
Rods Negative 3×1 Motile Orange 10-18 35°C 7
Rods Negative 2×1 Motile Red 10-26 35°C 7
Rods Negative 3×1 Motile Red 10-26 35°C 7
Rods Negative 3×1 Motile Red 10-22 35°C 7
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ND ND ND ND ND S ND ND S S ND ND S ND ND S S ND ND S ND ND S S ND ND R ND ND ND ND ND ND S ND Chromohalobacter Haloferex Halobacterium Haloferex Halorubrum Halorubrum Halobacterium
used sucrose, arabinose and lactose, fructose, mannitol sorbitol, galactose and cellobiose as carbon source. Remarkable diversity was observed in enzyme profile of isolates. In this out of 7, SS-1 and SS-36 have secreted extracellular amylase. SS-1, SS-6, SS-11 and SS-12 were recorded as urease producer, SS-36 has secreted gelatinase and all isolates showed positive oxidase test. All the 7 isolates were tested for antibiotic susceptibility isolate SS-36 and SS-37 were sensitive to polymyxin B, ciprofloxacin and gentamycin where as SS-12 showed sensitivity towards bacitracin, polymyxin B, ciprofloxacin and tetracycline. SS-12 showed resistance towards gentamycin. Pattern of
sugar utilization, enzyme profile and antibiotic sensitivity of isolates is given in Table 2. All the seven isolates showed remarkable growth at pH 7 and temperature 35°C. However, variation was recorded in resistance of isolates to salt concentration. Salt tolerance recorded in isolates SS-11, SS-12 and SS-37 was at 26% salt concentration followed by SS-1 and SS-36 which showed optimum growth at 24% of salt. While isolate SS-32 tolerated 22 % of salt, SS-6 achieved growth with 18% of salt concentration. Identification of isolate was carried by comparing results with standard strain characteristics given in Bergey’s Manual of Systematic Bacteriology16. SS-11 and SS-12 were identified as Halorubrum sp.
PATHAK & SARDAR: CAROTENOID PRODUCING HALOARCHAEA
SS-37 and SS-32 were identified as Halobacterium sp. SS-36 and SS-6 were identified as Haloferex sp. and SS-1 was identified as Chromohalobacter sp. Out of seven isolates, Halorubrum (SS-12) showing remarkable pigment production and fast growth was selected and used for pigment production (Plates 4, 5). Remarkable carotenoid production by SS-12 was recorded at pH 7, temperature 35°C and NaCl concentration of 20% (Figures 1-3). Initiation of carotenoid production was observed after 4 days of incubation and maximum carotenoid production was recorded after 7 days of incubation (Figure 4). Spectrophotometric analysis of pigment revealed that the type of pigment produced is of bacterioruberin group as it showed highest absorption at 490 and 511 nm17 (Figure 5). The antioxidant activity of crude carotenoid extracted from Halorubrum sp. was tested and recorded activity was equivalent to antioxidant activity of standard lutine and astaxanthin at their 4mM concentration18.
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Figure 1—Effect of pH on growth and carotenoid production
Figure 2—Effect of temperature on growth and carotenoid production
Plate 4 —Grown culture of Halorubrum sp.
Plate 5—Microscopic image of Halorubrum sp.
Figure 3—Effect of NaCl on growth and carotenoid production
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References 1
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Figure 4—Effect of Incubation time on growth and carotenoid production
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8 9 10
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13 Figure 5—UV-Vis absorption spectrum of carotenoid extract of Halorubrum sp. 14
Conclusion We have highlighted the presence of carotenoid producing Halorubrum sp. in solar saltern of Mulund, Mumbai, India. The carotenoid production, extraction and characterization was also carried out. The results obtained revealed that the isolate Halorubrum can be used as source of carotenoid. Acknowledgments Honourable Vice Chancellor Dr. S. B. Nimse of S.R.T.M. University is thanked for providing infrastructure facility and University Research fellowship to Miss Aparna G Sardar.
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17
18
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