Int. J. Biosci.

2011

International Journal of Biosciences (IJB)

ISSN: 2220-6655 (Print) 2222-5234 (Online)
Vol. 1, No. 6, p. 27-32, 2011
http://www.innspub.net

RESEARCH PAPER OPEN ACCESS

Screening of PHA (poly hydroxyalkanoate) producing bacteria

from diverse sources
Nandini Phanse1*, Amruta Chincholikar1, Bhavesh Patel1, Pragya Rathore2, Priti Vyas1,
Mital Patel1
1
P.G. Dept. of Microbiology & Biotechnology, P.M.B. Gujarati Science College, Indore, India
2
P.G. Dept. of Biotechnology,Sanghvi Institute of Management and Science, Indore, India
Received: 11 July 2011
Revised: 04 August 2011
Accepted: 06 August 2011
Key words: Bioplastic, polyhydroxyalkanoates, screening.
Abstract

Synthetic plastics are non-degradable and cause waste disposal problems leading to environmental pollution.
Bioplastics (polyhydroxyalkanoates) are considered good substitutes for petroleum derived synthetic plastics because
of their similar physical and chemical properties. Main advantage of bioplastics is that they are of biological origin
and can get degraded completely to CO2 and water under natural environment by the enzymatic activities of
microorganisms. Poly-β-hydroxyalkanoates (PHA) are polyesters of various hydroxyalkanoates, synthesized by
numerous bacteria as an intracellular carbon and energy storage compound under limited nutrient conditions and
with excess carbon. Poly- β- hydroxy butyrate (PHB) is the best known polyhydroxyalkanoate. Considering the
industrial interest of PHA, this work has been undertaken for the screening of PHA producing bacteria from diverse
sources. In the present study, an attempt was made to isolate efficient PHB producing bacteria from diverse
environmental samples. Different industrial wastes and soil samples were screened for bacteria possessing the ability
to accumulate poly hydroxyalkanoate (PHA) granules. About 23 bacterial isolates were found to be promising PHA
accumulating bacteria. Screening for PHA producers was performed by using E 2 medium. Accumulation of PHB
granules in the organisms was analyzed by Sudan black method.

Corresponding Author: Nandini Phanse  [email protected]

27 Phanse et al.
 Int. J. Biosci. 2011

Introduction bacteria capable of PHA synthesis accumulate PHA

In response to problems associated with plastic waste during the stationary phase of growth and these PHA
and its effect on the environment, there has been granules facilitate cell survival during stressful
considerable interest in the development and conditions. Bioplastics have a wide range of
production of biodegradable plastics. Polyhydroxy agricultural, marine and medical applications (Arun et
alkanoates (PHA) are polyesters that accumulate as al.2006 and Kitamara et al. 2004).
inclusions in a wide variety of bacteria. Because of their
inherent biodegradability, PHA are considered to be This study includes screening of different bacterial
good candidates for biodegradable plastics, since they isolates producing PHA from sewage and soil samples.
possess material properties similar to those of
synthetic polymers currently in use and are completely Materials and methods
biodegradable after disposal. Collection of samples
Soil samples, waste water samples (including industrial
Poly-hydroxyalkanoates (PHAs) are inclusion bodies effluents, dairy waste, domestic sewage) and activated
accumulated by some bacterial genera as reserve sludge samples were collected from various sources
material, when culture medium is unbalanced due to and used for the isolation of bacteria.
limited oxygen, nitrogen, phosphorous, sulphur or
magnesium and an excess of carbon source (Kim et al., Enrichment of PHA producing microorganisms
1994; Lee, 1996). Lafferty et al. (1988) stated that the The samples were inoculated in E2 broth medium
accumulation of PHA by microorganisms can be (Lageveen et al., 1988) and incubated in a rotary
stimulated under unbalanced growth conditions, i.e., shaker (150 rpm) at 30°C for 24-48 hrs. The growth
when nutrients such as nitrogen, phosphorus or sulfate obtained was then inoculated on E2 agar plates and
become limiting, when oxygen concentration is low, or incubated at 30°C for 48 hrs.
when the C: N ratio of the feed substrate is higher.
PHB is accumulated by numerous microorganisms and Rapid screening of isolates for PHA production by
is the best characterized PHA (Madison and Huisman, Plate assay method
1999). A number of bacteria such as Azotobacter, All the isolates were qualitatively tested for PHA
Bacillus, Archaebacteria, Methylobacteria, production using Sudan Black B dye (Juan et al.,
Pseudomonas have been found to synthesize PHA to 1998). Ethanolic solution of (0.05%) Sudan Black B
varying levels. Ralstonia eutropha (formerly was spread over the colonies and the plates kept
Alcaligenes eitrophus) has been the subject of much undisturbed for 30 minutes. They are washed with
published research work because it can accumulate ethanol (96%) to remove the excess stain from the
PHAs upto 80 per cent dry weight (Lee, 1996). colonies. The dark blue coloured colonies were taken
as positive for PHA production.
Plastics produced from PHAs have been reported to be
truly biodegradable in both aerobic and anaerobic Screening for PHA by Sudan black staining
environment unlike many of the “so-called” After 48 hrs of incubation on E2 agar medium, the
biodegradable plastics made synthetically. PHAs are isolates were screened for PHA production by Sudan
composed mainly of poly-betahydroxybutyric acid black staining (Burdon, 1942a; Lee, 1996). The isolated
(PHB) and poly-beta hydroxyvaleric acid (PHV), bacterial colonies were subsequently analyzed for
although other forms are possible. β- hydroxy butyrate Gram character. Identification of isolates was carried
(PHB) is the best known polyhydroxyalkanoate. All out by performing biochemical tests (Holt, 1994).

28 Phanse et al.
 Int. J. Biosci. 2011

Results and discussion bacteria was observed in waste water samples

For the past two decades, there has been a growing (including industrial effluents, dairy waste, and
public and scientific interest in the development and domestic sewage) and activated sludge samples. Out of
use of biodegradable polymers as an ecologically useful the 23 PHA producing isolates obtained from various
alternative to plastics. Polyhydroxyalkanoates (PHA), samples, 14 were from sewage samples and 9 from soil
synthesized by different genera of microorganisms has samples (Table 1). PHA producers found in different
attracted attention as biodegradable plastics. They are soil samples were comparatively low. Similar results
accumulated intracellularly, as high as 90 per cent of were reported by Sujatha et al (2005). They obtained
cell dry weight under conditions of nutrient stress and higher PHA producers from tannery effluent and
act as a source of carbon and energy (Madison and sewage sludge samples compared to garden and field
Huisman, 1999). soil samples. While isolating PHA accumulating
bacteria from nature, it is necessary to screen rapidly
In this work, attempts were made to isolate PHA a wide collection of bacteria in a short time. Stains
accumulating bacteria from diverse sources and to specific to PHA are made use of in the detection of the
select the efficient strains. Various samples collected granules. Viable colony staining technique was
from diverse areas were used for isolating PHA performed as a method for rapid screening of PHA
producers. The relative occurrence of PHA accumulating bacteria. The PHA granule forming
accumulating bacteria from these of samples was ability of the isolates was further studied by Sudan
studied. A striking prevalence of PHA producing black staining method.

Table 1. Screening of PHA producing bacteria.

PHA Accumulation
Sample Source Designation of Gram Reaction Plate Assay Sudan Staining
isolate
Khan river sewage KS-1 Gram Positive bacilli ++++ ++++
Khan river sewage KS-2 Gram Positive bacilli ++++ ++++
Khan river sewage KS-3 Gram negative bacilli +++ ++++
Khan river sewage KS-4 Gram negative bacilli ++ ++
Khan river sewage KS-5 Gram Positive cocci ++ ++
Sewage treatment plant STP-1 Gram Positive bacilli ++++ ++++
Sewage treatment plant STP-2 Gram Positive bacilli ++++ ++++
Sewage treatment plant STP-3 Gram Positive cocci + +
Sewage treatment plant STP-4 Gram negative bacilli +++ +++
Industrial effluent IE-1 Gram negative bacilli +++ +++
Industrial effluent IE-2 Gram Positive bacilli ++++ ++++
Industrial effluent IE-4 Gram positive bacilli ++++ ++++
Industrial effluent IE-5 Gram negative bacilli ++ ++
Industrial effluent IE-6 Gram negative bacilli +++ +++
Industrial soil IS-1 Gram Positive bacilli +++ ++
Industrial soil IS-2 Gram negative bacilli +++ +++
Industrial soil IS-3 Gram Positive bacilli ++ ++
Industrial soil IS-4 Gram positive bacilli ++ +++
Agricultural soil AS-1 Gram negative bacilli +++ +++
Agricultural soil AS-2 Gram negative bacilli +++ +++
Compost C-1 Gram Positive bacilli ++++ ++++
Compost C-2 Gram positive bacilli ++++ ++++

29 Phanse et al.
 Int. J. Biosci. 2011

The bacterial isolates were stained by Sudan black fatty material in bacteria by preparing microscopic
staining method and observed microscopically. A total slides of bacteria stained with alcoholic Sudan Black B
of 23 strains were found to accumulate PHA granules solution and counterstained with safranin.
after observing microscopically by Sudan black
staining method. All 23 isolates were also tested for The cultural characteristics of the PHA producing
PHA production following the viable colony screening bacterial colonies obtained on E2 were studied (Figure
method based on the intensity of staining. (Fig. 1) The 2). Gram staining showed that among 23 isolates, 12
black stained isolates were ranked in terms of + were Gram positive bacilli, 2 were Gram positive cocci
symbol. The poorly stained colonies were indicated and 9 were Gram negative bacilli. Biochemical analysis
with + symbol, medium stained colonies as ++ symbol, (Holt, 1994) revealed that the PHA producing strains
strongly stained colonies as +++ symbol while belonged to the genera- Bacillus (12 isolates),
excellently stained as ++++ symbol. Out of the 23 Pseudomonas (5 isolates), Azotobacter (4 isolates) and
isolates screened for PHA production 8 showed Staphylococcus (2 isolates).
excellent staining by Sudan Black in Plate assay
method.

Fig. 2. Colony morphology of PHA producing bacteria

on E2 medium.

Polyhydroxyalkanoic acids (PHA) represent a complex

Fig. 1. Rapid screening of isolates for production by
class of storage polyesters that are synthesized by a
Plate assay method.
wide range of different Gram-positive and Gram-
Juan et al (1998) employed viable colony screening negative bacteria. In our study, most isolates were
method for the rapid detection and isolation of PHA Bacillus species and showed large intracellular
producing Rhizobium meliloti strains by using 0.02% accumulation of PHA. Many workers have reported
alcoholic solution of Sudan Black B. Colonies unable production of PHA by various species of Bacillus.
to incorporate the Sudan Black B appeared white, Yuksekdag et al. (2004) have reported PHA production
while PHA producers appeared bluish black. Hartman by Bacillus subtilis and B. megaterium. Production
(1940) was the first to suggest the use of Sudan Black and characterization of PHA produced by Bacillus
B, as a bacterial fat stain. Subsequently, Burdon et al megaterium NCIM 2475 was also reported by Otari et
(1942a) confirmed the greater value of this dye and al (2009). Full et al (2006) have studied on production
modified the procedure for demonstrating intracellular of PHA by Bacillus species from industrial wastes.

30 Phanse et al.
 Int. J. Biosci. 2011

Yilmaz et al. (2005) have determined production of Burdon KL, Stokes JC, Kimbrough CE. 1942a.
PHA by Bacillus spp. Studies of the common aerobic spore-forming Bacilli
staining for fat with Sudan Black B- stain. Journal of
PHA production by Psuedomonas species has also Bacteriology, 43, 717-724.
been studied. Lageveen et al. (1988) have reported the
formation of Poly-(R)-3-Hydroxyalkanoates by Full TD, DO Jung, MT Madigan. 2006.
Pseudomonas oleovorans. Microbial production of Production of poly-β-hydroxyalkanoates from soy
polyhydroxy alkanotes (PHA) from Pseudomonas molasses oligosaccharides by new, rapidly growing
oleovorans using different carbon sources was Bacillus species. Letters of Applied Microbiology 43,
reported by Santhanam and Sasidharan (2010). Ayub 377-384.
et al. (2004) isolated Pseudomonas sp. 14-3, a strain
from Antarctic environments that accumulated large Hartman TL. 1940. The use of Sudan Black B as a
quantities of polyhydroxybutyrate (PHB) when grown bacterial fat stain. Staining Technology, 15, 23-28.
on octanoate.
Holt JG, Krig NR, Sneath Pefer HA, Staley JT,
Followed by genus Bacillus and Psuedomonas, Williams ST. 1994. Bergey’s Manual of
Azotobacter and Staphylococcus were the other Determinative Bacteriology 9th edition, Williams and
bacterial genera obtained in our work. Parshad et al. Wilkins, Baltimore.
(2001), Page (1992), have studied on PHA produced by
Azotobacter spp. Roy et al (2009) have reported poly Juan ML, Gonzalez LW, Walker GC. 1998. A
beta hydroxybutyric acid production by Novel Screening Method for Isolating
Staphylococcus species isolated from activated sludge. Exopolysaccharide deficient Mutants. Applied and
Environmental Microbiology 64, 4600-4602.
Conclusion
The search for promising strains of PHA producers is a Kim BS, Lee SC, Lee SY, Chang HN, Chang YK,
continuous process and development of efficient Woo SI. 1994. Production of Poly (3hydroxybutyric–
polyhydroxyalkanoate producing bacteria is the need co-hydroxyl valeric acid) by fed batch culture of
of the hour. On the basis of data obtained in the Alcaligens eutrophus with substrate control using on
present work it can be concluded that Bacillus species line glucose analyzer. Enzyme Microbial Technology
isolated can be employed in the production of PHA. 16, 556-561.

References Kitamara S, Doi Y. 2004. Staining method of poly

Arun A, Murrugappan RM, David Ravindran, (3- alkanoic acid) producing bacteria by Nile blue.
Veeramanikandan V, Balaji S. 2006. Utilization Biotechnological Techniques 8, 345-350.
of various industrial wastes for the production of poly-
hydroxy butyrate (PHB) by Alcaligenes eutrophus. Lafferty RM, Korsatko B, Korsatko W. 1988.
African Journal of Biotechnology 5, 1524-1527. Microbial production of poly- -hydroxybutyric acid.
Biotechnology Special microbial Processes, 136-176.
Ayub ND, Pettinari MJ, Ruiz JA, Lopez NJ.
2004. A polyhydrobutyrate –producing Pseudomonas Lageveen RG, GW Huisman, H Preusting, P
sp. isolated from Antarctic environments with high Ketelaar, G Eggink, B Witholt. 1988. Formation
stress resistance. Current Microbiology, 49, 170-174. of Polyesters by Pseudomonas oleovorans: Effect of

31 Phanse et al.
 Int. J. Biosci. 2011

Substrates on formation and composition of Poly-(R)- Roy B, Banerjee R, Chatterjee S. 2009. Isolation
3-Hydroxyalkanoates and Poly-(R)-3- and identification of poly beta hydroxybutyric acid
Hydroxyalkenoates. Applied Environmental accumulating bacteria of Staphylococcal sp. and
Microbiology 54, 2924-2932. characterization of biodegradable polyester. Indian
Journal of Experimental Biology, 47: 250-256.
Lee SY. 1996. Bacterial Polyhydroxyalkanoates.
Biotechnology and Bioengineering, 49: 1-14. Santhan, A. and Sasidharan. S. 2010. Microbial
Microbiology Review, 103, 131-140. production of polyhydroxy alkanotes (PHA) from
Alcaligens spp. and Pseudomonas oleovorans using
Madison LL, Huisman GW. 1999. Metabolic different carbon sources. African Journal of
engineering of poly(3 hydroxyalkanoates): from DNA Biotechnology, 9 (21), 3144-3150.
to plastic. Microbiology Molecular Biology Reviews.
63, 21–53. Sujatha K, Mahalakshmi A, Snehbagarathai.
2005. A study on accumulation of PHB in native
Otari SV, and Ghosh JS. 2009. Production and Pseudomonas isolates LDC-5 and LDC-25. Indian
Characterization of the Polymer Polyhydroxy Butyrate- Journal of Biotechnology, 4, 216-221.
copolyhydroxy Valerate by Bacillus Megaterium NCIM
2475. Yilmaz M, Soran HB, Yavuz. 2005. Determination
Current Research Journal of Biological Sciences 1(2), of poly-β-hydroxybutyrate (PHB) production by some
23-26. Bacillus spp. World Journal of Microbiology and
Biotechnology, 21 (4), 565-566.
Page WJ. 1992. Production of
Polyhydroxyalkanoates by Azotobacter vinelandii Yuksedag ZN, Aslim B, Beyadliti X, Mercan N.
Strain UWD in Beet Molasses Culture. FEMS 2004. Effect of carbon and nitrogen sources and
Microbiological Reviews, 103, 149-158. incubation time on poly-β-hydroxybutyrate (PHB)
synthesis by Bacillus subtilis 25 and Bacillus
Parshad J, Suneja S, Kukeja K, megaterium 12. African Journal of Biotechnology, 3
Lakshminarayana K. 2001. Poly hydroxybutyrate (1): 63-66.
production by Azotobacter chroococcum. Foliar
Microbiology, 46 (4), 315-320.

32 Phanse et al.