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International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.9, No.11 pp 224-228, 2016
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Isolation and Identification of Polyhydroxybutyrate(PHB) Producing Bacillus cereus BB613-A Novel Isolate.
B. Bharathi, D. Gowdhaman* and V. Ponnusami
Bioprocess Intensification Lab, School of chemical & Biotechnology, SASTRA University, Thanjavur, India
Abstract : Poly-3-hydroxy butyrate (PHB) is a copolymer of polyhydroxyalkonate family. It accumulates in the intracellular granules of bacteria under nitrogen limiting environment. The PHB producing bacteria was isolated from farm soil by serial dilution method. The Sudan Black B staining and Nile Blue A staining were carried out to confirm that the isolated strain was capable of producing PHB. Further, it was subjected to morphological, biochemical and molecular characterization. The 16S rRNA sequencing and phylogenetic analysis confirmed the organism was Bacillus cereus BB613 with accession number LN613102. The growth parameter and usage of low cost substrate for production was optimized. The maximum PHB accumulation was found at 30°C and pH 7.0 after 48h incubation. For the production of PHB, 1% sucrose was used as carbon source. Sucrose was then substituted by low cost substrates such as mosambi peel, orange peel, banana peel and molasses which gave yields 35.2%, 26.7%, 32.1% and 33.9% respectively.
Key words: Poly-3-hydroxybutyrate, 16Sr RNA sequencing, Phylogenetic analysis, Mosambi peel.
1. Introduction
Synthetic thermoplastics is a type of solid waste pollutant which affects the environment and cannot be degraded [1]. To overcome this problem is to producing biodegradable polymer. Poly-3-hydroxy butyrate (PHB) is a copolymer of polyhydroxyalkonate family. It accumulates in the intracellular granules of bacteria in the excess of carbon and less nitrogen. Poly-3-hydroxy butyrate is ecological polymer, reusable, insoluble in water and non-toxic [2]. The nature of the material cause suitable for the application of packaging [3]. Many of the bacterial species has the ability to produce PHB, they are Bacillus megaterium, Bacillus thuringiensis, Halomonas campisalis, Brevibacterium casei, Pseudomonas sp, etc[4-10] . It has a wide application in various fields like drug delivery vehicles in medicine [11-13], plant germination and plant growth in agriculture [14], Nano fibrous scaffolds are used for cell proliferation, cell signaling in tissue engineering [15-18]. Though there are many reports to minimize the production cost of PHB by agro industrial residues [19-25] like palm oil, molasses, rice bran, wheat bran, and soya bean etc.,. The main aim of the work was to produce PHB from isolated bacteria and also to improve the production of PHB by using economically low cost carbon sources like mosambi peel, orange peel, banana peel, molasses.
2. Materials and Method
2.1 Isolation of Bacteria from Soil
The bacteria were isolated from farm soil, because of the presence of partially degraded plant waste and usage of fertilizers there will be rich in carbon and less nitrogen source. The bacteria was isolated by serial dilution method and inoculated onto the nutrient agar plate. The Bacterial colony was preserved in a slant at 4°C for further characterization.
2.2 Screening of PHB Producer
The bacteria which produce PHB granules were screened by Sudan black B and Nile blue A staining. 0.3% of Sudan black B in 70% of ethanol was prepared. Heat fixed smear was stained with Sudan black B solution and incubated for 10minutes. Then rinsed with tap water, counter stained with safranin for 5minutes, visualized under phase contrast microscope at 1000X magnification. For Nile blue staining, 0.1% of Nile blue A solution was added into the heat fixed smear followed by incubation at 55°C for 10 minutes. After incubation rinsed with tap water to remove excess stain and observed under fluorescent microscope.
2.3 Biochemical and Molecular Characterization
The isolated bacterial strain was described by Biochemical Test as per Bergey’s Manual of Determinative Bacteriology and 16s rRNA sequencing, in an ABI 3730xl sequencer (Applied Bio systems). Phylogenetic studies was done for the conformation of the bacteria using Phylogene.fr. The sequence was submitted in webin to obtain the accession number.
2.4 Production of PHB
Bacillus cereus BB613 (1ml) was inoculated in the media containing (Yeast extract 2.0g/l), Peptone (2.0g/l), K2HPO4 (2.0g/l), (NH4)2SO4(2.0g/l), Mgso4(0.3g/l), Sucrose (10g/l) for the production of PHB and it was incubated at 37°C for 48 hrs at 150rpm. Every 12 h, samples were with drawn and it was subjected to centrifugation at 10,000rpm for 10 min and the cell pellet was collected.
2.5 Optimization of Process Parameters for PHB Production
The optimization of various process parameters like temperature (20, 30, 40, 50°C), pH (5.0, 6.0, 7.0, 8.0and 9.0), time duration (12, 24, 36, 48h), carbon source (1%) like orange peel, banana peel, mosambi peel, molasses were used. 1ml of inoculum was added into the medium and PHB production was determined.
2.6 Extraction of PHB
After 48h of incubation, samples were with drawn and it was then subjected to centrifugation at 10000 rpm for 10 minutes. The cell pellet was dried at 60°C in a hot air oven and cell dry weight was measured. Then 3ml of 6% sodium hypochlorite was added and incubated at 50°C for 20minutes and 3ml of hot chloroform was added into the pellet. From the three phases, chloroform containing PHB was carefully collected and precipitated by 5ml methanol or hexane. The PHB content was determined by spectrophotometric assay as described by Law and Slepecky (1960) .
Figure 1: A) Micrograph of strain Bacillus Cereus BB613 with Sudan black B showing PHB accumulation when counter stained with safranin using Phase contrast microscope B) Nile blue A stain fluorescent micrograph of Bacillus Cereus BB613 showing green fluorescence due to PHB granules.
3.2 Strain Identification
B
The PHB producing positive isolate was identified as Bacillus cereus BB613, by the biochemical and molecular characterization analysis. Table 1 shows the biochemical characterization of isolated bacteria. The nucleotide sequence was subjected to Blast and Bacillus cereus BB613 was found to have 99% homology with Bacillus cereus.
Table 1: Morphology and biochemical characterization of Bacillus cereus BB613
Test |
Observation |
Gram’s stain |
Gram positive |
Spore staining |
Positive |
Cell shape |
Rod |
Colony character |
White, raised, irregular |
Motility |
Positive |
Starch hydrolysis |
Positive |
Catalase |
Positive |
Indole |
Negative |
Methyl Red |
Negative |
Voges-Proskauer |
Negative |
Citrate Utilization |
Positive |
Oxidase |
Positive |
Urea |
Positive |
TSI |
Acid slant/Acid butt, no gas, no H2S |
Glycerol |
Positive |
Arginine |
Positive |
Mannitol |
Positive |
Glucose |
Positive |
Sucrose |
Positive |
Lactose |
Positive |
The unrooted phylogram was constructed using phylogene.fr. (Fig.2) The bacterial sequence was submitted at ENA Webin and the accession number was obtained as LN613102.
Figure 2: Unrooted phylogram obtained by neighbor-joining of 16SrRNA sequence of the isolated Bacillus Cereus BB613 strain.
3.3 Optimization of Process Parameter for PHB Production
The effect of process parameters on the production of PHB by Bacillus cereus BB613 was assessed. Table 2 shows the maximum yield of the PHB produced by the Bacillus cereus BB613 was optimized and found to be maximum of 0.534 (g/l) PHB production occurred at 48 h time interval which is the result of consumption
of PHB by cells as carbon reserve when depletion occurs. The optimum growth and production of PHB was found to be 30°C yielding 0.383g/l. The increase in growth cause decrease in production due to disruption of cell wall and also due to alternation of metabolic activity. Increase in the temperature causes decrease in the production this is due to the disruption of cytoplasmic membrane. Therefore the cytoplasm content diffuses out and PHB cannot exist from the cell (Tripathi et al., 2012).The maximum PHB production was obtained at pH value of 7.0 with yield of 1.765 (g/l). Substrate sucrose was replaced by different carbon source and maximum yield of the PHB production was found on Mosambi peel yielding 0.352 (g/l).
Table: 2 Yield of PHB (g/l) was produced by Bacillus cereus BB613 at different time interval, pH, temperature and carbon source 1% (w/v)
Parameters |
Biomass (g/100ml) |
PHB (g/100ml) |
Yield % |
Time(h) |
|||
24 |
0.2113 |
0.409 |
40.96 |
48 |
0.3306 |
0.534 |
53.43 |
72 |
0.2603 |
0.203 |
20.30 |
96 |
0.1097 |
0.158 |
15.81 |
p H |
|||
5 |
0.1214 |
0.379 |
37.91 |
6 |
0.2312 |
0.603 |
60.14 |
7 |
0.3688 |
0.621 |
62.58 |
8 |
0.1312 |
0.236 |
23.63 |
9 |
0.0267 |
0.153 |
15.39 |
Temperature (°C) |
|||
20 |
0.0128 |
0.088 |
8.8 |
30 |
0.3332 |
0.383 |
38.8 |
40 |
0.1623. |
0.248 |
24.8 |
50 |
0.0161 |
0.096 |
9.61 |
Carbon source used |
|||
Banana peel extract |
0.8214 |
0.312 |
32.1 |
Orange peel extract |
0.8844 |
0.267 |
26.7 |
Mosambi peel extract |
0.9787 |
0.352 |
35.2 |
Molasses |
0.8486 |
0.339 |
33.9 |
Isolated novel bacterial stain was capable of producing PHB was confirmed by characteristics staining techniques. Based on the morphological, biochemical and molecular characterization of the bacteria was identified as Bacillus cereus BB613 and accession number was obtained as LN613102.The optimum growth and maximum PHB accumulation was found to be at temperature 37°C, pH 7.0, incubation time 48h. The maximum yield of the PHB 35.2% from 1% mosambi peel extract. From the above studies, it was concluded that the Bacillus cereus BB613 has a capability of producing PHB, from various low cost substrates.
5. References
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