Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2013, 3 (4):13-21
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ISSN : 2231 –3168

CODEN (USA) : JMBRB4

Evaluation of biodegradtion of polyurethane by enzyme assay and GC-MS

J. Roshnee and V. Mahalakshmi

Department of Microbiology, Madras Christian College, Chennai, India

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ABSTRACT

Polyurethanes are present in many aspects of modern life. They represent a class of polymers that have found a
widespread use in the medical, automotive and industrial fields. The Polyurethane films subjected for study were
buried in compost soil and the organisms involved in the degrading the films were isolated and characterized. The
strains Micrococcus sp., Corynebacterium sp., Pseudomonas putida and Pseudomonas mediteranean were
inoculated in minimal salt agar medium containing PU as the sole source of carbon. The enzymes responsible for
the degradation were extracted by ammonium sulphate precipitation and dialysis. The molecular mass of the enzyme
was found to be approximately 62kDa. Also the monomers released as a result of degradation of PU film were
analysed by GC-MS and were detected as phthalic acid, 1.2- benzenedicarboxylic acid, dibutyl phthalate, eicosane
and hexacosane. The enzyme activity was confirmed by clear zone method and degradation of the PU film.
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INTRODUCTION

POLYURETHANE
Polyurethanes, abbreviated commonly as PU, were invented back in the 1930s by Professor Dr. Otto Bayer (1902-
1982) [6].

Polyurethanes are present in many aspects of modern life. They represent a class of polymers that have found a
widespread use in the medical, automotive and industrial fields. Polyurethanes can be found in products such as
furniture, coatings, adhesives, constructional materials, fibres, paddings, paints, elastomers and synthetic skins. They
have been used as biomaterial application for the manufacture of medical devices such as artificial heart diaphragms,
valves, vascular grafts, catheters, neurological lead insulation and connecting modules for cardiac pacemakers [8].

Advantages of polyurethanes are that they have increased tensile strength and melting points making them more
durable. Their resistance to degradation by water, oils, and solvents make them excellent for the replacement of
plastics. As coatings, they exhibit excellent adhesion to many substances, abrasion resistance, electrical properties
and weather resistance for industrial purpose. Depending on the chemical structures of the polyisocyanates and
polyols, PU can adopt various forms ranging from flexible to rigid and from low density to solid elastomer. The
chemical composition of PU precludes them from being classified as pure plastics but rather as a mixed polymer.
The urethane group, which is the basis of this class of plastics, represents a small part of the macromolecule and
some PU products do not contain a urethane group. Despite the lack of this base unit, all PU are based on the
composition of polyisocyanates. The polyisocyanate polyaddition is distinct from polymerization and
polycondensation for the production of synthetic polymers and this feature explains their versatility.

BIODEGRADTION OF POLYURETHANE:
Polyurethanes are classified into two types: polyester PUs and polyether PUs.

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Polyester-polyurethanes
polyurethanes having different chemical structure were synthesized and their biodegradability was
investigated. Average molecular weight of the synthesized polyurethanes ranged from 79 106 to 161 715(Mw) by
gel permeation chromatography. Degradation
Degradation experiments were conducted by: hydrolytic degradation in NaOH
solution; enzymatic degradation by lipase; and composting. Hydrolytic and enzymatic degradation decreased with
the increase of the diol carbon chains in polyol, and increased by substitutin
substitutingg aromatic diisocyanate with aliphatic
diisocyante. It is considered that hydrophobicity and hard segment formation seem to resist the hydrolytic and
enzymatic degradation of polyurethanes. Synthesized polyurethanes were biodegradable under composting cond condition
to a certain extent depending on their chemical structures.

MICROBIAL ACTION:
Despite its xenobiotic origins PU has been found to be susceptible to biodegradation by naturally occurring
microorganisms. Several reports have appeared in the literature
literature on the susceptibility of PUs to microbial and fungal
attack [6].. Both bacteria and fungi are reported to have the potential of polyurethane degradation. Fungal isolates
from soil like Fusarium solani, Curvularia sengalensis, Aureobasidiumpullulans and Cladosporium sp. sp have been
found to have polyurethanolytic potepotential. Bacterial isolates like Pseudomonas fluorescens, Bacillus subtilis,
Pseudomonas chlororaphis and Comamonas acidovorans are reported to have the ability to degrade polyurethane
[1].

The present study was designed to isolate bacteria capable of degrading polyurethane from compost soil and to study
the involvement of bacterial esterases in the breakdown of polyurethane.

AIM AND OBJECTIVE:

To isolate the bacteria capable of degrading polyurethane
polyu from compost soil.
To extract the extracellular esterase produced by the degrading bacterium involved in the polyurethane
degradation.
To partially purify the polyurethane hydrolyzing esterase by SDS-PAGE.
SDS
To study the end products present in the m
medium
edium containing polyurethane as a sole carbon source by GC-MS.
GC

MATERIALS AND METHODS

Polyurethane (PU) used for study was Polyurethane resins which were procured from Bharat enterprises, New
Delhi (shown in fig.)

A B

Figure. A. Polyurethane beads. B. Dissolved Polyurethane.

Figure.1:

The PU beads were made into films by dissolving in polar solvents such as chloroform, toluene and acetone (1g of
PU in 20ml of the solvent). They were then poured on slides or thick glass plates to a thickness
thicknes of approximately
1mm and were allowed to dry, giving PU films.
Nutrient Agar (HIMEDIA).
Nutrient broth (HIMEDIA).
Minimal salt agar medium, devoid of any carbon source.

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SAMPLE COLLECTION:
Compost soil was collected from CIPET, Guindy. This soil mixture was used for the isolation of Polyurethane
degrading micro-organisms.

ISOLATION OF PLASTIC DEGRADING MICROORGAISMS:

The polyurethane degrading microorganisms were isolated by different techniques:

Soil burial experiment:

Prepared Polyurethane films were washed and buried in compost soil at room temperature for a period of 2 months.
The plates were sprinkled with water at intervals of 2 weeks to maintain the moisture. After the incubation period,
the films were removed from the soil and added into 100ml saline. The microorganisms responsible for degradation
were isolated by spread plating onto nutrient agar plates.

Identification of the selected isolates:

The identification of bacterial isolates with the ability to degrade PU was performed on the basis of macroscopic and
microscopic examination and biochemical tests according to Bergey’s manual of Determinative Bacteriology (16).
The bacterial isolates were identified macroscopically by examining colony morphology; pigment production,
shape, size, margin, surface on nutrient agar plates and microscopic examination by gram’s staining to study the
staining behaviour, shape and cell arrangement and granulation.

Biochemical tests:
For the biochemical characterization of the bacterial isolates following tests were performed:

Indole test, MR, VP, Citrate tests; Carbohydrate fermentation tests (glucose, fructose, sucrose, lactose, maltose,
mannose); urease; nitrate reduction, TSI, oxidase and catalase.

BIODEGRADATION STUDIES:
Plate assay:
Minimal salt agar medium was prepared with PU as the sole carbon source (0.5%w/v). The agar plates were
individually inoculated with the bacterial cultures isolated during burial of PU film in compost soil and incubated at
37˚c for 5-7 days.

Clear zone method:

Polyurethane plates were prepared with LB medium so as to give 3.0 g/l and 15g/l as a final concentration of PU and
agar respectively. When assaying for enzyme activity, wells of 1.0 cm diameter were cut and loaded with 100µl of
the crude enzyme preparation. The plates were incubated at either 30˚c or 37˚c for 18-20 hours. After an appropriate
incubation period, agar plates were loaded with 0.1% (w/v) Coomassie blue R-149 solution in 40% (v/v) methanol
and 10% (v/v) acetic acid for 20 min. The Coomassie blue solution was then poured off and the plates were flooded
with 40% (v/v) methanol and 10% (v/v) acetic acid for 20 min. Visualized clear zones of degradation were observed
in a blue background [3].

POLYURETHANE DEGRADING ENZYME STUDIES:

Enzyme assay and purification:
The bacterial culture was inoculated into minimal broth medium containing PU films as the sole carbon source and
incubated at 37˚c for 3 months. The enzyme was extracted by first centrifuging the culture broth and removing the
cells. The supernatant was transferred to sterile beakers and Ammonium sulphate was added to the crude enzyme
solution to 20% saturation, and the mixture was centrifuged at 12000 rpm for 20 min. The precipitate was added
with potassium phosphate buffer. All the purification procedures were performed at 4°C. Ammonium sulphate was
added to the crude extract with gentle stirring until the solution reached 70% saturation. The solution was stirred for
a further 30 min and centrifuged at 7000 rpm for 10min. The precipitate was dissolved in potassium phosphate
buffer (pH 7). Then the sample was loaded into the dialysis membrane and kept for overnight. The purified
enzyme was stored in freezer until use [13].

Electrophoresis of Extracellular Proteins:

Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at room
temperature with 12.5% resolving polyacrylamide gel and Tris-glycine buffer (pH 8.5) at 100 V for 90 min. After

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electrophoresis, the gel was fixed and stained using coomasie blue staining to visualize the protein bands. The
molecular mass of the extracellular proteins were evaluated using standard protein marker (molecular weight
ranging from 18-72kDa).

Table 1 Identification of polyurethane degrading bacterial strains

Characteristics Isolate 1 Isolate 2 Isolate 3

Colony characteristics:
Shape Round Round Round
Size Large Small Large
Colour White/Opaque Yellow Pale
Surface Dull, Granular, Wrinkled Shinny Convex
Margin Irregular Entire Undulate

MORPHOLOGY:
Gram Stain Straight Rod Cocci Straight rod
Cell arrangement + + -
Short Chains, Single Irregular clusters Short chains, single
Spore + - +
Motility + - +

CARBOHYDRATE FERMENTATION:
Glucose Acid/No Gas No acid/no gas No acid/no gas
Fructose Acid/No Gas No acid/no gas No acid/no gas
Lactose Acid/No Gas No acid/no gas No acid/no gas
Mannose Acid/No Gas No acid/no gas No acid/no gas
Sucrose Acid/No Gas No acid/no gas No acid/no gas
Citrate + + +
TSI Yellow/Yellow Red/red Y/Y
Oxidase + - +
Catalase + + +
Nitrate reduction + + +
Identified organism: Bacillus sp. Micrococcus sp. Pseudomonas sp
Characteristics Isolate 4 Isolate 5 Isolate 6
Colony characteristics:
Shape Round Round Round
Size Large Pin point Small
Colour Off white Off white Yellow
Surface Raised Convex Shinny
Margin Undulate Entire Entire
MORPHOLOGY: Straight rod Straight rod Cocci
Gram Stain + + +
Cell arrangement Long, club shaped,single Irregular clusters Short chains, single
Spore - - -
Motility - + -

CARBOHYDRATE FERMENTATION:
Glucose Acid/no gas No acid/no gas No acid/no gas
Fructose No Acid/no gas No acid/no gas No acid/no gas
Lactose Acid/no gas No acid/no gas No acid/no gas
Mannose Acid/no gas No acid/no gas No acid/no gas
Sucrose No Acid/no gas No acid/no gas No acid/no gas
Citrate - - +
TSI Yellow/Yellow Yellow/Yellow Red/red
Oxidase + + +
Catalase - - +
Nitrate reduction - - +
Identified organism: Corynebactreium sp. Arthrobacter sp Pseudomonas putida

Determination of enzyme activity:

Degradation of PU film (20µm thick) by the enzyme was observed by wetting the film with 30µl purified enzyme
solution (10-80µg/ml in 10mm Tris-HCl buffer, pH8.0). After incubation at 37°c for 12 hours, the filter was
removed & the film was washed with water. Degradation of the film surface was estimated visually.

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ANALYSIS OF DEGRADED PRODUCTS BY GAS CHROMATOGRAPHY:
After 2 months of incubation period, the bacterial pellet (in case of bacterial culture) was removed by filtration, and
the filtrates were extracted with distilled ether. The degraded products of PU were determined by Gas
chromatography-mass spectrometer (Sargam laboratories, Guindy, Chennai.) using HP5 column, helium gas, was
programmed to raise the oven temperature from 70°C to 300°C (maximum temperature - 250°C at 15°C/min,
Injection liquid 1 micro litre). Mass spectrometer consists of tungsten filament as electron source which works with
70eV, a double focusing analyzer and photo multiplier tube as detector with resolution of maximum 5000. Using
PerFluoro Kerosene (PFK) as standard, mass spectrometer was calibrated [15].

RESULTS

ISOLATION AND IDENTIFICATION OF POLYURETHANE DEGRADING MICROORGANISM:

The compost soil collected was used as the source for isolating microorganisms having the ability to degrade
polyurethane.

After 3 months of incubation in the compost soil, the polyurethane film was removed and added to 100 ml saline.
The bacterial strains thus isolated by plating were tabulated in the given table 1.

The strains Micrococcus sp., Corynebacterium sp., Pseudomonas putida and Pseudomonas mediteranean were
selected for further studies.

BIODEGRADATION STUDIES
PLATE ASSAY
Polyurethane containing minimal salt agar plates were inoculated with the bacterial isolates isolated from soil. The
plates were incubated at 37˚c for 7 days. Growth of isolates following incubation confirmed the ability to use PU as
a carbon source.

(A) (B)

Figure 2: Polyurethane film after soil burial, minimal agar plates showing growth indicating the utilization of
PU as the sole carbon source

Clear zone method:

Polyurethane plates were prepared with LB medium containing. When assaying for enzyme activity, wells of 1.0 cm
diameter were cut and loaded with 100µl of the crude enzyme preparation. After incubation the plates were stained
with Coomasie blue solution and destained. The zones of clearance were observed clearly confirming enzyme
activity.

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Figure 3:: LB agar plates containing PU showing zones of clearance after staining with
wi Coomasie blue
solution.

EXTRACTION OF ENZYME BY AMMONIUM SULPHATE PRECIPITATION AND PARTIAL P

PURIFICATION BY DIALYSIS:
The extracellular esterase enzyme was extracted by 70% ammonium sulphate precipitation of the 100 ml culture
supernatant with the strains P. putida,
putida Corynebacterium sp.,., Pseudomonas mediterrenea and Micrococcus sp.
followed by partial purification of the eenzyme
nzyme by dialysis in potassium phosphate buffer for 2 days. The molecular
weight of the enzyme was determined by Sodium dodecyl sulphate
sulphate- polyacrylamide electrophoresis. Its molecular
mass was approximately 62 kDa when compared to the standard marker. The enzyme activity was confirmed by
degradation of the film surface due to the action of the enzyme.

Figure 4: SDS-PAGE
PAGE band: 1. Standard protein marker 2 & 3. Enzyme bands. 4. Film degradation by
extracted enzyme.

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DETECTION OF PU DEGRADED PRODUCTS BY GC-MS:
Monomers released as a result of degradation of PU film by Pseudomonas putida,, were analysed by GC-MS.
GC Eight
new peaks were detected in the samples at retention times 17.698, 18.177, 21.337, 22.980, 23.225, 23.756 and
24.505, after 3 months incubation,
on, corresponds to the compounds given in the table, when compared
compare to the standard
chromatograms.

DISCUSSION

Polyurethanes have found widespread acceptance for a variety of applications. Recycling of waste plastics has
become very important because of shortages in raw materials for their synthesis. Complete recycling is difficult and
limited to special cases due to the incompatibility of most polymers and the enormous difficulties encountered in
segregating polymeric articles of different chemical composition found in plastic waste. Therefore, hydrolytic
processes are being developed for the utilization of plastics
plastics scrap and waste as either an energy source or as raw
material for the production of valuable chemicals as described by Schnabel, 1981 [10].

In the present study, microorganisms capable of degrading polyurethane were isolated from compost soil, and the
major
ajor isolates obtained were identified as Pseudomonas sp., Bacillus sp., Comomonas acidovorans, Micrococcus
sp., Corynrbacterium sp., and Arthrobacter sp. The isolated colonies were grown in minimal salt agar medium
containing PU as sole carbon source. The growth was observed within 5-77 days indicating that the biodegrading
capability of the isolated organisms. This is a rapid confirmation method as described by Amaer Ali Shah et al, 2008
[1].

The enzyme was extracted and purified by ammonium sulphate precipitation

precipitation and dialysis in potassium phosphate
buffer (pH- 7) and molecular mass of the enzyme was approximately 62 kDa when compared to the standard protein
marker. The involvement of the purified enzyme in the degradation of PU was confirmed by degradation
degradatio of film
surface. Howard et al. purified a thermally labile membrane bound esterase having molecular weight of 62kDa [3].
Carmen Ruiz et al, purified two polyurethane degrading enzymes from P. chlororaphis,, utilizing PU as sole source
of carbon. He determinedined that both the enzymes were extracellular, soluble proteins with molecular weight
approximately 63 kDa and 31kDa. Curvalaria sengalensis was also found to secret an extracellular enyme of 28kDa
having PU degrading activity [2]. Yukie Shigeno-
Shigeno Akutsu et al. purified two types of polyesterase enzyme from
PUR degrading bacterium Comamonas acidovorans,
acidovorans, i.e. cell bound esterase (PUR esterase) and other secreted in the
culture broth (CBS esterase) with molecular weight approximately 62kDa [13]. [13] Studies by Lori
Lor Rowe et al (2002)
revealed an extracellular protein produced by Bacillus subtilis of molecular weight 28kDa. The involvement of
esterase in the degradation of polyurethane was confirmed by the detection of ester hydrolysis products [7].

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Figure 5: GC-MS Chromatogram of PU samples treated with Pseudomonas putida culture.

GC-MS chromatograms of diethyl ether extract of the strain Pseudomonas putida, using PU as the sole carbon
source revealed phthalic acid, 1.2- benzenedicarboxylic acid, dibutyl phthalate, eicosane and hexacosane as
degraded products. Nakajima-Kambe et al. investigated PU degradation metabolites, when exposed to C.
acidovorans strain TB35 by GC-MS and reported the detection of diethylene glycol, trimethylolpropane, adipic acid
and 2, 4- diaminotoluene [13]. These compounds were derived from both the polyester and polyisocyanate portions
of the polyurethane. G.T. Howard et al. investigated the presence of PU degradation metabolites, 1.4- butanediol and
adipic acid, which were not present in biotic and abiotic controls [3].

CONCLUSION

The Polyurethane films subjected for study were buried in compost soil and the organisms involved in the degrading
the films were isolated and characterized. The strains Micrococcus sp., Corynebacterium sp., Pseudomonas putida
and Pseudomonas mediteranean were inoculated in minimal salt agar medium containing PU as the sole source of
carbon. The enzymes responsible for the degradation were extracted by ammonium sulphate precipitation and
dialysis. The molecular mass of the enzyme was found to be approximately 62kDa. Also the monomers released as a
result of degradation of PU film were analysed by GC-MS and were detected as phthalic acid, 1.2-
benzenedicarboxylic acid, dibutyl phthalate, eicosane and hexacosane. The enzyme activity was confirmed by clear
zone method and degradation of the PU film.

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