INTRODUCTION^1-10

Polyhydroxyalkanoates (PHB) are polyesters of various hydroxyalkanoates, synthesized by many bacteria as inclusion bodies. Polyhydroxyalkanoates (PHBs) are accumulated as microbial intracellular carbon and energy reserves. These polymers correspond to a class of compounds with physical-chemical characteristics similar to petroleum-derived plastics such as polypropylene, polyethylene and polystyrene, but are environmentally compatible and totally biodegradable to carbon dioxide and water many bacteria store carbon as PHBs to protect themselves from nutrient limitation, including lack of nitrogen, phosphorous and magnesium ⁽¹⁾.

Received on 18.07.2015 Modified on 24.07.2015

Research J. Pharm. and Tech. 8(9): Sept, 2015; Page 1276-1280

DOI: 10.5958/0974-360X.2015.00231.0

PHBs are synthesised by most genera of bacteria and Archaea⁽²⁾. The most excellent characterized microorganisms that produce PHBs are Alcaligenes sp.⁽³⁾, Bacillus sp.⁽⁴⁾, Rhodopseudomonas sp.⁽⁵⁾,and Pseudomonas sp.⁽⁶⁾. Halotolerant bacteria were reported to produce high amounts of PHBs (40-60% DCW) were accumulated in halotolerant bacteria under the starvation condition ^(7-10).

Halotolerant photosynthetic bacteria have the advantage over the other microorganisms in their ability to adjust themselves to both presence and absence of light as well as able to live in saline condition which offers a multitude of actual or potential applications in various fields of biotechnology ^(11,
12). This report reveals the occurrence of PHB in halo tolerant bacteria isolated from Jeppiaar salt pane, Chennai, Tamilnadu, India.

MATERIALS AND METHODS:

Sample Collection:

Condenser water having a salinity of 155% was collected from the solar salt in Jeppiaar salt pan, Chennai, Tamilnadu, India. Samples were collected in sterile polythene bags, transported to the laboratory aseptically and stored at 4ºC for further use.

Isolation and Purification of Bacteria:

Water samples were serially diluted 10^-1 to 10^-6in sterile distilled water and 100ml of each dilution was spread onto sterile nutrient agar plates containing 5% NaCl (Composition of Nutrient Agar: 0.5% peptone, 0.3% beaf extract/ Yeast extract, 0.5% NaCl, pH of the medium was adjusted to 6.8 at 25 ºC. The plates were incubated at 37 ºC for 24h, after the incubation period morphologically different colonies selected and purified in nutrient agar and preserved in nutrient agar slants for further use.

Purification and preservation of isolates:

All the isolates were purified on nutrient agar medium with 5% of NaCl by quadrant streaking and preserved on nutrient agar slants containing 5% NaCl.

Screening for PHB producing bacteria:

Nile Blue staining by smear method:

Nile blue A staining is a more specific stain for Polyhydroxy butyrate (PHBs) and it is a more rapid and sensitive method for detecting PHBs. All the isolates were cultured for 2-3 days at 37^◦ C in Minimal Davis Media (Dipotassium phosphate 7g/l, Monopotassium phosphate 2g/l, Sodium citrate 0.5g/l, Magnesium sulphate 0.1g/l, Ammonium sulphate 1g/l, pH-7.0±0.2) supplemented with dextrose (10ml of 10% in 1l of Minimal Davis Media) as carbon source. From each, a loop full culture was taken on clean, sterile glass slides and heat fixed followed by staining with +Nile blue stain. The samples were allowed to get stained for 20 min at room temperature and then slides were washed with sterile water. Then the slides containing the samples were allowed to air dry for few minutes and observed under fluorescence microscope at wavelength 490 nm. PHB granule producing bacterial isolates flourish bright yellowish orange color ⁽¹³⁾.

Nile blue A dye viable colony method:

Positive isolates were checked for PHB production by a more rapid and sensitive, viable colony method by ⁽¹⁴⁾. The Nile blue A dye at concentrations of only 0.5𝜇g/mL was directly included in carbon rich nutrient agar medium (glucose 1%, beef extract 0.3%, peptone 0.5%, sodium chloride 0.8%, and agar 1.5%) and growth of the cells occurred in the presence of the dye. This allowed an estimation of the presence of PHBs in viable colonies at any time during the growth experiment and a powerful discrimination between PHB-negative and PHB-positive strains. The PHB accumulating colonies, after Nile blue A staining, showed bright orange fluorescence on irradiation with UV light and their fluorescence intensity increased with the increase in PHB content of the bacterial cells. The isolates which showed bright orange fluorescence on irradiation with UV light after Nile blue A staining were selected as PHB accumulators.

Extraction of PHB from the Potent Isolates:

The bacteria positive for PHB production were selected based on the screening results for further study of production of PHB. They were cultured in Minimal Davis Media supplemented with dextrose as carbon source for 3 days at 37º C at 150 rpm in a rotary shaker.

Dry weight estimation:

Dry weight was estimated from 100 ml of culture broth. The cell suspension was centrifuged at 10,000 rpm for 10 min at 20°C washed with warm distilled water several times, transferred to pre weighed vials and dried in an oven at 105°C till constant weight.

Extraction by acetone-alcohol:

Cell mass (g/L) obtained after 48 h growth in Minimal Davis media was separated by centrifugation at 10,000 rpm for 10 min and lysed by sodium hypochlorite at 37^oC for 1 h. Contents were re centrifuged at (10,000 rpm for 10 min) and the lysed cell mass was sequentially washed with distilled water, acetone: ethanol (1:1) followed by precipitation in boiling chloroform (10ml). The precipitate was allowed to evaporate at room temperature to drive PHB in powder form ⁽¹⁵⁾. Percent production of PHB was calculated by using the formula

% of PHB = Total weight of PHB / Total weight of Pellet X 100.

RESULTS:

Isolation of bacteria:

A salt pane condensed water sample was collected from Jeppiaar salt pane, Chennai, INDIA. A total of twenty morphologically different bacteria was isolated from the sea shore sediment sample by serial dilution method. The morphology of the all twenty isolates were recorded and were purified and preserved on Nutrient agar slants with 5% of NaCl for further usage.

Screening of bacteria for PHB Production:

All the 20 isolates were initially screened for the PHB production in Minimal Davis Medium and the ability to synthesize PHB granules was confirmed using Nile blue dye. About 55% of the isolates were positive to Nile blue staining. A total of 11 isolates showed different intensity of reddish orange coloration when stained with Nile blue A. Figure.4.1.Though 11 isolates showed reddish orange coloration internally the isolate AJ 8, AJ 10 and AJ 11 were emits high intensity of the reddish orange colour of granules.

AJ8

AJ11

AJ10

Figure: 4.1 Fluorescence of PHB granules using Nile blue staining

All the positive isolates of Nile blue A smear method were grown on nutrient rich medium with the dye concentrations of only 0.5 𝜇g/ml. Among the11 positive isolates the isolate AJ8, AJ10 and AJ11 were emitted pink florescence under UV light Figure.4.2. This viable colony method confirming the isolates AJ8, AJ10 and AJ11 are must be the potential producers of PHB.

Figure :4.2 (a)Pink fluorescence under UV light by PHB producers isolate (a) AJ8 (b)AJ10 (c) AJ11 fluorescence under UV light by PHB producer with Nile blue A staining by viable colony method

Extraction of PHB:

The screening results of Nile Blue A smear method and Nile blue dye viable colony methods shows the isolates AJ8, AJ10, and AJ11 are the prominent PHB producers Figure:4.3. These positive isolates were selected for PHB production by cultivating in Minimal Davis media (100ml) with Dextrose as carbon source.

Figure: 4.3 Selected Potential PHB producers isolate AJ11, AJ10 and isolate AJ8

After 3 days of incubation in Minimal Davis Medium the presence of PHB was checked in 5 ml of cultured broth initially by following sodium hypochlorite-chloroform method. All the three isolates were given positive for PHB production by forming a white precipitate at the end of the extraction procedure. Then the whole 100ml broth was subjected to PHB extraction and the dry weight of producing PHB was recorded. The isolate AJ11, AJ10, AJ8 produced 0.225, 0.210, 0.125g/100ml of PHB respectively Table:4.1 and Figure:4.4.

Table 4.1 Percentage of PHB produced by the selected isolates

Isolates code	Dry weight (g/l)	PHB (g/l)	Yield of PHB %
AJ8	2.86	1.250	43.7
AJ10	2.9	2.10	72.4
AJ11	2.75	2.25	81.8

Figure: 4.4 The extracted PHB from the the isolates AJ11 and AJ 10 using Sodium hypochlorite - Chloroform method

DISCUSSION:

In this course of isolation and screening of poly hydroxy butyrate (PHB) producing bacteria from Jeppiaar salt pane, Chennai, twenty isolates were obtained from condensed salt water. Nile blue A, a preliminary screening agent for lipophilic compounds ⁽¹⁶⁾ used to screen the potential PHB producing bacteria from the soil. Nile blue stains the PHB granules in the intracellular environment of the isolated bacteria. Based on the intensity of the fluorescence observed by the stained PHB granules, the potential PHB producers were identified⁽¹⁷⁾The granules were observed as reddish orange fluorescence at an emission wavelength of 580nm.

For the rapid detection and isolation of PHB producing bacteria, Nile blue A staining viable colony method was used. The growth of the cells occurred in the presence of the dye, the PHB accumulating colonies, after the growth showed pink fluorescence on irradiation with UV-light and their fluorescence intensity increased with increase in PHB content of the bacterial cells. Ramachandran and Abdullah⁽¹⁸⁾ also observed the colonies formed on nutrient rich medium under ultraviolet light (UV) to screen for the pink fluorescence which indicated the presence of PHB producers. The percentage of PHB production was high for the isolate AJ11 (81.8%), AJ10 (72.4%) and minimum production was observed for AJ8 was (43.7%) (Table 4.1). These PHB will be characterized for its morphological, chemical and functional group analysis. In a similar study Kalaivani R and Sukumaran⁽¹⁹⁾ produced PHB by using the simplified glucose peptone medium may be attributed to the presence of complex organic nitrogen source, peptone favoring the growth as well as PHB accumulation. They produced PHB yields of 0.601 0.052 0.431 0.346 0.531g/100 ml, respectively.

ACKNOWLEDGEMENTS:

The authors are grateful to the management of Sathyabama University. We also thank Biotechnology department, Sathyabama University, for providing facilities required for the successful completion of the research work.

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