Research J. Pharm. and Tech.

10(4): April 2017

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Isolation and Identification of PolyHydroxyButyrate (PHB) producing

bacteria from Sewage sample
A. Ranganadha Reddy1*, R. Bharath Kumar1, K. Vidya Prabhakar2
1
Department of Biotechnology, Vignan University, Vadlamudi, Guntur 522213, Andhra Pradesh, India
2
Department of Biotechnology, Vikrama Simhapuri University, Nellore, 524001 Andhra Pradesh, India.
*Corresponding Author E-mail: rangaaluri@gmail.com

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.

KEYWORDS: Polyhydroxy Butyrate, Biopolymer, Thermoplastic, Municipal sewage.

1. INTRODUCTION: Plastic materials have become an integral part in our life

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].
Received on 28.01.2017 Modified on 16.02.2017
Accepted on 20.03.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2017; 10(4): 1065-1069.
DOI: 10.5958/0974-360X.2017.00193.7
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

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biocompatible. These properties make PHB an attractive to get a dilution of 10-1. This serial dilution was repeated
material which has several applications in medicine, to get dilutions of 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8 and
food packaging, drug delivery, tissue engineering, etc[8]. 10-9. Luria Bertani (LB) agar plates were used. Using
The bacterial flora in the presence of rich nutrients tends different dilutions (10-7, 10-8) sewage sample from
to accumulate certain storage materials like volutin sewage treatment plant were pour plated onto LB agar
granules, lipids and polyhydroxyalkanoates[9]. These plates. After incubation of 24 h at room temperature (30
bacteria available abundantly in diverse ecosystem such C), around 5–9 isolates were randomly picked from each
as sewage sludge. However the potentials has not been of these 2 plates (two dilutions) with sterile tooth picks
adequately explored for bacteria accumulating and were transferred onto a fresh plate. Colonies with
polyhydroxyalkanoates (PHA) that are present in different characteristic features were maintained as pure
sewage. Hence we considered it to be a potential cultures on nutrient agar slants and stored at 4°C.
environment for screening of bacteria accumulating
PHA. Both gram positive and gram negative bacteria 2.2 Maintenance of bacterial cultures:
were earlier reported to produce PHA[10]. A number of The bacteria were streaked on to nutrient agar slants,
bacteria producing PHAs are Alcaligenes eutrophus, incubated at 30˚C overnight and then stored at 4˚C for
Alcaligenes latus, Azotobacter vinelandii, Rhizobium further use.
sps, Bacillus sp, methylotrophs, pseudomonads, and
recombinant Escherichia coli[11,12,13]. 2.3. Screening of PHB producing isolates by Sudan
Black B staining:
The PHAs are classified according to the number of The thin smear of bacterial culture was prepared on
carbon atoms in their monomers. Polyhydroxybutyrate microscope slide and thoroughly air dried. Then the
(PHB) and polyhydroxyvalerate carbon numbers of strain was stained with Sudan black B solution for 10-15
monomers are 3 to 5. Conversely, carbon numbers in minutes. The slides were washed with distilled water and
medium chain-length PHA monomers range from 6 to counter stained with safranin for 10 seconds. The slides
16. The PHB identified in Bacillus megaterium was were again washed with distilled water and dried on
characterized by its large accumulations of PHB [13]. tissue paper. The bacterial cultures positive for PHB
PHAs are particularly important because of their good production were selected by observing the granules
biodegradability, biocompatibility, thermoplastic nature under fluorescence microscope, (OLYMPUS Reflected
that can also be extended for drug delivery systems.PHB Fluorescence System, (Olympus Corporation, Japan)
production is increased by excess of carbon source and using BXRFA fluorescence illuminator, fitted with
limiting the nutrients such as nitrogen, phosphorus, Image Analyzer. Organism shows positive in blue-violet
sulfur, magnesium, iron, oxygen, and potassium[14]. It is and shows negative in yellow-brown [15].
an intracellular polymer accumulated under stress
conditions but with excess carbon source. It is produced 2.4. Characterization of PHB producing isolates:
by fermentation process of microorganisms such as The PHB-positive bacterial isolates were identified and
Bacillus megaterium and Ralstonia eutropha. In the characterized by morphological and biochemical
present study, several PHB accumulating bacteria from characterization according to the Bergey’s Manual of
sewage sample were isolated, identified and Determinative Bacteriology [16, 17].
characterized using morphological, biochemical and
molecular techniques. They were identified as bacteria 2.4.1 Morphological characterization:
belonging to genus Bacillus and the isolates were Morphological features were identified by growing the
characterized for the quantification of the PHB. cultures on nutrient agar media and gram staining was
performed.
2. MATERIALS AND METHODS:
All chemicals used were of analytical grade procured 2.4.2 Biochemical characterization:
from Strata gene, SRL and Sigma. Media components Different Biochemical tests were carried out includes
were purchased from Hi Media Laboratories. IMVIC tests, catalase test, urease test and starch
hydrolysis [18].
2.1. Sample collection and isolation of pure cultures:
Sewage waste water sample was collected in sterile 2.4.3 Cell dry weight:
plastic bottle from dump yard at outskirts of Guntur; After 48hrs incubation at 37°C, culture medium was
Andhra Pradesh. Bacterial isolates were obtained by collected and centrifuged at 10,000 rpm for 15min.
serial dilution-pour plate technique. One ml of sewage Supernatant was discarded and the cell pellet was dried
sample was dispensed in 10ml of sterile distilled water. to estimate the dry cell weight (DCW) in units of
[19]
This was mixed vigorously and 1ml from this is taken g/ml .
and added to another tube with 9ml sterile distilled water
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2.4.4 Extraction and Quantification of PHB Residual biomass (g/ml) =

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.
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- 1: Isolated colonies were streaked on nutrient agar plates

Strain 2 Strain 4 Strain 5 Strain 9 Strain 11

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.

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

A) Indole test B) Methyl red test

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.
H2SO4 and heated for 10 minutes to convert PHB into
3.3.2. Quantification of PHB
crotonic acid, which gave 200 mg/ml of crotonic acid.
The spectrometric chemical assay for the determination
From the above stock, working standard solutions were
of PHB from the sample was estimated by using Law
prepared by diluting it to obtain different concentrations
and Slepecky, 1961method [22]. Pure PHB (Sigma, USA)
ranging between 10 mg/ml to 150 mg/ml. Absorbance of
was used to prepare the standard curve of PHB. Two
all the dilutions was read at 235nm against a
gram of PHB was dissolved in 10 ml of concentrated
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concentrated H2SO4 blank on UV-VIS polyhydroxyalkanoates, a family of Biodegradable plastics and

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