Isolation and Identification of  PolyHydroxyButyrate (PHB) producing bacteria from Sewage sample

 

A. Ranganadha Reddy¹*, R. Bharath Kumar¹, K. Vidya Prabhakar²

¹Department of Biotechnology, Vignan University, Vadlamudi, Guntur 522213, Andhra Pradesh, India

²Department of Biotechnology, Vikrama Simhapuri University, Nellore, 524001  Andhra Pradesh, India.

 

ABSTRACT:

Plastics and synthetic polymers are synthesized from nonrenewable resources like petrochemicals and persist in the environment long after intended use, resulting into problems of solid waste management and global environmental pollution. Hence, an alternative source such as  Polyhydroxyalkanoates that are biodegradable, linear polyesters produced primarily by bacteria which   can be used as an effective thermoplastic, and has many characteristics similar to those of standard commercial plastics like polypropylene. Aliphatic polyester ,poly3-hydroxy butyrate was discovered and identified as a granular component in bacterial cells.PHB can grow in a wide variety of natural environments and is the reserve polymer found in many species of bacteria  found in nature, e.g. in soil, sea water, sewage waste or compost. In this present study high PHB producing strains were isolated from sewage sample. Five strains were showing PHB granules with Sudan Black B staining. The five strains were labeled as strain 2, 4, 5, 9 and 11. Further, they were morphologically and biochemically characterized. Growth profiles were studied for all these strains and were found that the PHB was produced maximum after 48 hrs at 37°C of incubation. Strain 2 showed high PHB production among the five strains isolated. The sugarcane molasses used in the medium for PHB production accounted for the least production cost.

 

 

 

1. INTRODUCTION:

The problem of environmental pollution caused by indiscriminate dumping of plastic waste has assumed global proportions. These conventional plastics that are synthetically derived from petroleum are not readily biodegradable and are harmful wastes^[1,2].  Petrochemical based plastics including polypropylene, polyethylene and polystyrene are almost exclusively made from the nonrenewable resource, viz., petroleum; which is also the main source of energy for today`s world^[3].

 

Plastic materials have become an integral part in our life as a basic need but they are causing serious environmental problems due to their non biodegradability ^[4].  Polyhydroxyalkanoate (PHA) is an important biopolymer of microbial origin. It is accumulated in the cells as intracellular granules in the presence of excess carbon source and limited nitrogen source^[5]. Poly-3-hydroxybutyrate (PHB), the most common representative of the polyhydroxyalkanoates (PHA), is widespread in different taxonomic groups of prokaryotes as an intracellular storage compound, acting as  a  carbon reserve  and reducing equivalent that facilitates cell survival during stressful conditions^[6,7]. Poly 3-hydroxybutyrate (PHB) is an important class of PHA which is water insoluble, resistant to ultraviolet radiation, impermeable to oxygen, biodegradable and biocompatible. These properties make PHB an attractive material which has several applications in medicine, food packaging, drug delivery, tissue engineering, etc^[8]. The bacterial flora in the presence of rich nutrients tends to accumulate certain storage materials like volutin granules, lipids and polyhydroxyalkanoates^[9]. These bacteria available abundantly in diverse ecosystem such as sewage sludge. However the potentials has not been adequately explored for bacteria accumulating polyhydroxyalkanoates (PHA) that are present in sewage. Hence we considered it to be a potential environment for screening of bacteria accumulating PHA. Both gram positive and gram negative bacteria were earlier reported to produce PHA^[10]. A number of bacteria producing PHAs are Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, Rhizobium sps, Bacillus sp, methylotrophs, pseudomonads, and recombinant Escherichia coli^[11,12,13].

 

The PHAs are classified according to the number of carbon atoms in their monomers. Polyhydroxybutyrate (PHB) and polyhydroxyvalerate carbon numbers of monomers are 3 to 5. Conversely, carbon numbers in medium chain-length PHA monomers range from 6 to 16. The PHB identified in Bacillus megaterium was characterized by its large accumulations of PHB ^[13]. PHAs are particularly important because of their good biodegradability, biocompatibility, thermoplastic nature that can also be extended for  drug delivery systems.PHB production is increased by excess of carbon source and limiting the nutrients such as nitrogen, phosphorus, sulfur, magnesium, iron, oxygen, and potassium^[14]. It is an intracellular polymer accumulated under stress conditions but with excess carbon source. It is produced by fermentation process of microorganisms such as Bacillus megaterium and Ralstonia eutropha. In the present study, several PHB accumulating bacteria from sewage sample were isolated, identified and characterized using morphological, biochemical and molecular techniques. They were identified as bacteria belonging to genus Bacillus and the isolates were characterized for the quantification of the PHB.

 

2. MATERIALS AND METHODS:

All chemicals used were of analytical grade procured from Strata gene, SRL and Sigma. Media components were purchased from Hi Media Laboratories.

 

2.1. Sample collection and isolation of pure cultures:

Sewage waste water sample was collected in sterile plastic bottle from dump yard at outskirts of Guntur; Andhra Pradesh. Bacterial isolates were obtained by serial dilution-pour plate technique. One ml of sewage sample was dispensed in 10ml of sterile distilled water. This was mixed vigorously and 1ml from this is taken and added to another tube with 9ml sterile distilled water to get a dilution of 10^-1. This serial dilution was repeated to get dilutions of 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8 and 10^-9. Luria Bertani (LB) agar plates were used.  Using different dilutions (10^-7, 10^-8) sewage sample from sewage treatment plant were pour plated onto LB agar plates. After incubation of 24 h at room temperature (30 C), around 5–9 isolates were randomly picked from each of these 2 plates (two dilutions) with sterile tooth picks and were transferred onto a fresh plate. Colonies with different characteristic features were maintained as pure cultures on nutrient agar slants and stored at 4°C.

 

2.2 Maintenance of bacterial cultures:

The bacteria were streaked on to nutrient agar slants, incubated at 30˚C overnight and then stored at 4˚C for further use.

 

2.3. Screening of PHB producing isolates by Sudan Black B staining:

The thin smear of bacterial culture was prepared on microscope slide and thoroughly air dried. Then the strain was stained with Sudan black B solution for 10-15 minutes. The slides were washed with distilled water and counter stained with safranin for 10 seconds. The slides were again washed with distilled water and dried on   tissue paper. The bacterial cultures positive for PHB production were selected by observing the granules under fluorescence microscope, (OLYMPUS Reflected Fluorescence System, (Olympus Corporation, Japan) using BXRFA fluorescence illuminator, fitted with Image Analyzer. Organism shows positive in blue-violet and shows negative in yellow-brown ^[15].

 

2.4. Characterization of PHB producing isolates:

The PHB-positive bacterial isolates were identified and characterized by morphological and biochemical characterization according to the Bergey’s Manual of Determinative Bacteriology ^{[16, 17]}. 

 

2.4.1 Morphological characterization:

Morphological features were identified by growing the cultures on nutrient agar media and gram staining was performed.

 

2.4.2 Biochemical characterization:

Different Biochemical tests were carried out includes IMVIC tests, catalase test, urease test and starch hydrolysis ^[18].

 

2.4.3 Cell dry weight:

After 48hrs incubation at 37°C, culture medium was collected and centrifuged at 10,000 rpm for 15min. Supernatant was discarded and the cell pellet was dried to estimate the dry cell weight (DCW) in units of      g/ml ^[19].

 

2.4.4 Extraction and Quantification of PHB

After 48hrs incubation at 37°C, culture was collected and centrifuged at 10,000 rpm for 15min and lyophilized. The lyophilized pellet was digested with 4% sodium hypochlorite solution at 37°C for 20min. Then pellet was collected by centrifugation at 10,000 rpm for 15min, washed with water, acetone, ethanol respectively for washing and extraction. Finally polymer was dissolved in chloroform and kept for complete evaporation^[20]. Dry weight of extracted PHB was estimated as g/L. Residual biomass was estimated as the difference between dry cell weight and dry weight of PHB^[21]. The percentage of intracellular PHB accumulation is estimated as the percentage composition of PHB present in the dry cell weight.

 

Residual biomass (g/ml) =

DCW (g/ml) – Dry weight o f extracted PHB (g/ml)

 

PHB accumulation (%) =

Dry weight of extracted PHB (g/ml) × 100 / DCW (g/ml)

 

3. RESULTS AND DISCUSSION

3.1. Isolation of microorganisms:

Microorganisms were isolated from sewage sample and independent colonies were obtained by serial dilution. A total of 15 bacterial colonies with different morphological features were selected and the numbers were given to each colony. These colonies were streaked on nutrient agar plates and preserved for further studies (Fig. 1).

 

Figure- 2: Strains 2, 4, 5, 9and 11 showing PHB granules with Sudan Black B staining

 

3.2. Screening of PHB Producing Bacteria:

Among 14 colonies, 5 colonies showed positive for Sudan Black B staining. These 5 colonies are 2, 4, 5, 9and 11. They were named as strain 2, strain 4, strain 5, strain 9 and strain 11. All the strains except 2 are showing high color intensity with Sudan black B. The Microscopic pictures were depicted in figure 2.

 

3.3. Characterization of PHB producing isolates

3.3.1 Morphological and Biochemical characteristics

Morphological features were observed for PHB producing strains. Strains 2, 4, 5, 9 and   11 has bacillus in shape. Strain 2 is yellow in color and the remaining strains 4,5,9,11,16 were Creamish white in color. Strain 4 is Gram positive and the remaining strains 2, 5,9,11 and 16 were Gram negative to gram staining. Morphological features were represented in Table-1.

Different Biochemical tests have been performed for PHB producing strains. Strain 9 and 16 are positive to indole and the remaining are negative to indole test. Strain 5 is negative to MR and positive to VP test. Strains 2, 4, 9, 11 and 16 are positive to MR and negative to VP test. Only strain 2 is negative to citrate utilization test and the remaining strains are positive to citrate utilization test. Strain 2and 4 are negative to starch hydrolysis test and the remaining strains are positive to starch hydrolysis test. Biochemical tests were depicted in Table-1.

 

Table 1: Morphological and Biochemical characteristics of PHB isolates

	Strain 2	Strain4	Strain5	Strain 9	Strain 11
Morphological characteristics
Shape	Bacillus	Bacillus	Bacillus	Bacillus	Bacillus
Color	Creamish	Creamish	Creamish	Creamish	Creamish
	white	white	White	white	white
Gram staining	Gram +ve	Gram +ve	Gram +ve	Gram +ve	Gram +ve
Biochemical tests
Indole	Negative	Negative	Negative	Positive	Negative
production
MR	Positive	Positive	Negative	Positive	Positive
VP	Negative	Negative	Positive	Negative	Negative
Catalase	Positive	Negative	Negative	Negative	Negative
Starch	Positive	Positive	Positive	Negative	Positive
hydrolysis

 

C) Catalase test                                                                                 D) Starch hydrolysis test

Figure-3: Images of results of various biochemical tests: A) Indole test: Strains 2,4,5,9 and 11 positive for Indole test. B) Methyl red:  Strains 2,4,5,9 and 11 positive for Methyl Red test. C) Catalase test: Strain 2 is positive to catalase test. D) Starch hydrolysis test: Strains2, 4, 5 and 11 showing positive to starch hydrolysis.

 

3.3.2. Quantification of PHB

The spectrometric chemical assay for the determination of PHB from the sample was estimated by using Law and Slepecky, 1961method ^[22]. Pure PHB (Sigma, USA) was used to prepare the standard curve of PHB. Two

gram of PHB was dissolved in 10 ml of concentrated H₂SO₄ and heated for 10 minutes to convert PHB into crotonic acid, which gave 200 mg/ml of crotonic acid. From the above stock, working standard solutions were prepared by diluting it to obtain different concentrations ranging between 10 mg/ml to 150 mg/ml. Absorbance of all the dilutions was read at 235nm against a concentrated H₂SO₄ blank on UV-VIS spectrophotometer, and the standard graph was made by plotting the various concentrations on the x-axis and the respective optical densities on the y-axis. The standard curve was used for estimation of PHB yield of the bacterial isolates.

 

Table 2: PHB accumulation for PHB isolates

Name of the Organism	Cell  dry weight (g/l)	Dry Weight of PHB (g/l)	Residual biomass (g/l)	% PHB Accumulation
Strain 2	1.06	0.88	0.18	83.01
Strain 4	1.08	0.80	0.28	74.07
Strain 5	0.93	0.73	0.20	78.49
Strain 9	1.04	0.70	0.34	67.30
Strain 11	0.90	0.42	0.48	46.67

 

Figure4: Plot showing relation between CDW,PHB (g/l) and PHB(%) of 2,4,5,9 and 11 strains

 

The percentage of intracellular PHB accumulation was estimated as the percentage composition of PHB present in the dry cell weight. Residual biomass was estimated as difference between the dry cell weight and dry extract of PHB. The results were depicted in Table 2. From these results strain 2 showing high PHB production whereas the strain 11 showing less PHB production compared with other strains. Similar results have been showed by with glucose as a carbon source in the medium ^[23].

 

4. CONCLUSION:

Poly Hydroxy Butyrate producing bacteria were isolated from sewage sample and five isolates showed the positive response for polyhydroxybutyrate production. The maximum concentration of PHB by isolate 2 was found to be 0.88g/l at 48 hrs of incubation.

 

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