POLYHYDROXYALKANOATE-PRODUCTION BY A MODERATELY

HALOTOLERANT BACTERIAL STRAIN (Klebsiella pneumoniae) ISOLATED FROM

ACIDIC SOIL

URVIJA APPARAO13/PBYA/514

Guided by: Dr. K. Veena Gayathri

INTRODUCTION

• Petrochemical plastics from fossil fuels cause pollution and are toxic

to the environment.

• They are derived from non-renewable sources.

• Due to these reasons, researchers and industries are trying to

optimise the production process of bioplastics like polylactic acid

and polyhydroxyalkanoates.

• Polyhydroxyalkanoates (PHAs) are linear polyesters which are made

and degraded by some bacteria.

• They are accumulated intracellularly by bacteria under conditions of

nutrient stress as carbon reserve materials.

OBJECTIVES

1. To isolate PHA-producing bacterial consortium from soil and to check the

yield of PHA production under normal conditions prevalent in the lab.

2. To confirm the presence of PHA by UV Spectrophotometric and Fourier

Transform Infra-Red Spectrometric analysis.

3. To optimise PHA-production by the isolated bacterial strains by varying the

carbon and nitrogen sources.

4. To check the halotolerance of the isolated bacteria in different concentrations

of sodium chloride.

5. To biochemically characterise and molecularly identify the isolated PHA-

producing bacterial strains through 16s r-DNA sequencing and to construct a

phylogenetic tree.

MATERIALS AND METHODS

Collection of soil sample from rubber plantation area

Isolation and acclimatisation of PHA-producing bacterial consortium

Screening for PHA-production by the bacterial strain using Sudan Black B and Nile Blue A staining

Extraction of PHA by sodium hypochlorite digestion

Growth studies and PHA-production by the isolated

bacterial strain

Optimisation of PHA-production by varying the

carbon and nitrogen sources

Analysis of product by UV

Spectrophotometry and FTIR

Spectrometry

Screening of the PHA-producing bacterial isolates

for halotolerance

Biochemical characterisation of PHA-

producing isolate

Molecular identification of PHA-

producing isolate

Genomic DNA extraction Sequencing Phylogenetic tree

RESULTS

Enrichment and Isolation of PHA-Producing Bacterial ConsortiumIn Nitrogen-deficient medium, only one organism was observed (U1).

Bacterial isolate U1 on Nitrogen-deficient Agar

Screening of PHA-Production by Bacterial IsolateThe isolate was screened for PHA-production by staining methods.Sudan Black B Staining:

PHA granules stained by Sudan Black stain within the bacterial cell counter-

stained by Safranin

Nile Blue A Staining:

Intracellular PHA granules of isolate U1 stained by Nile Blue A visible as bright

orange spots under fluorescence microscope at 460 nm

Extraction of PHAA powdery white pellet was obtained after drying which was conserved in pre-weighed Eppendorf tubes and weighed. The weights obtained were recorded.

PHA obtained as a white powdery substance

DAY WEIGHT (mg/10 ml)

1 4

2 6

3 2

4 2

0 24 48 72 960

1

2

3

4

5

6

7

Weight of PHA

Weight (mg)

Time (hours)

Wei

ght o

f PH

A (m

g/10

ml)

0 24 48 72 960

5

10

15

20

25

30

35

Protein Content of U1

Protein Content

Time (hours)

Prot

ein

Con

tent

(µg/

2 m

l)

Analysis of PHA Produced by the Bacterial IsolateThe extract was confirmed for presence of PHA.UV Spectrophotometric Analysis: a)

b)

a) Crotonic acid, b) PHA converted to crotonic acid

Absorption maxima of crotonic acid and PHA converted to

crotonic acid

Fourier Transform Infra-Red Spectroscopy:O-H: 3408 cm-1; C-H: 2923 cm-1; C=O: 1745 cm-1.

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.00.0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100.0

cm-1

%T

U1

3408 2923

23042145

1745

1642

14171374

1243

11551029

714

575

Optimisation of Growth and PHA-Production of the Isolate U1

Optimisation by Varying the Carbon Source:Carbon sources used were Glucose, Glycerol, Sucrose and Sunflower Oil.

Optimisation by Varying the Nitrogen Source:Nitrogen sources used were Ammonium Nitrate, Ammonium Chloride, Sodium Nitrite and Peptone.

Optimisation by Varying the Carbon Source:

0 24 48 72 960

1

2

3

4

5

6

7

8

9

10

Weight of PHA

GlucoseGlycerolSucroseSunflower Oil

Time (hours)

Wei

ght o

f PH

A (m

g/10

ml)

0 24 48 72 960

50

100

150

200

250Protein Content of U1 in Various Carbon Sources

GlucoseGlycerolSucroseSunflower Oil

Time (hours)

Prot

ein

Con

tent

(µg/

2 m

l)

Optimisation by Varying the Nitrogen Source:

0 24 48 72 960

50

100

150

200

250Protein Content of U1 in Various Nitrogen Sources

Ammonium NitrateAmmonium ChlorideSodium NitritePeptone

Time (hours)

Prot

ein

Con

tent

(µg/

2 m

l)

0 24 48 72 960

2

4

6

8

10

12

Weight of PHA

Ammonium NitrateAmmonium ChlorideSodium NitritePeptone

Time (hours)

Wei

ght o

f PH

A(m

g/10

ml)

Screening for HalotoleranceScreening for halotolerance of the isolate was done in 0%, 5%, 7% and 10% sodium chloride.

0 24 48 72 960

5

10

15

20

25

30

35

Protein Content of U1 in Various NaCl Concentrations

0%5%7%10%

Time (hours)

Prot

ein

Con

tent

(µg/

2 m

l)

0 24 48 72 960

2

4

6

8

10

12 Weight of PHA

0%5%7%10%

Time (hours)

Wei

ght (

mg/

10

ml)

0% 5% 7% 10%0

1

2

3

4

5

6Average Weight of PHA

Average Weight of PHA (mg/10 ml)

NaCl Concentrations %

Ave

rage

Wei

ght o

f PH

A (m

g/10

ml)

Biochemical CharacterisationCHARACTERISTICS U1

Gram Staining +

Cell Shape Rod

Motility -

Oxidase -

Catalase +

Urease +

Indole -

Methyl Red +

Voges-Proskauer -

Citrate Utilisation +

Triple Sugar-Iron Agar Acid butt, Acid slant

Isolate Klebsiella pneumoniae

Genomic DNA Isolation 1 2 3 4 5 6

Lane 2 – Genomic DNA of Isolate U1

Phylogenetic Analysis Super kingdom : Bacteria

Phylum : ProteobacteriaClass : GammaproteobacteriaOrder : EnterobacterialesFamily : EnterobacteriaceaeGenus : Klebsiella

Klebsiella pneumoniae strain DSM 30104 16S ribosomal RNA gene partial sequence.

Klebsiella oxytoca KCTC 1686 strain KCTC 1686 16S ribosomal RNA complete sequence.

Klebsiella pneumoniae subsp. ozaenae strain ATCC 11296 16S ribosomal RNA gene partial sequence (U1)

Klebsiella variicola At-22 strain At-22 16S ribosomal RNA complete sequence.

Klebsiella pneumoniae strain NBRC 14940 16S ribosomal RNA gene partial sequence.

Klebsiella pneumoniae strain JCM 1662 16S ribosomal RNA gene partial sequence.

Klebsiella pneumoniae subsp. pneumoniae MGH 78578 strain ATCC 700721 MGH 78578 16S ribosomal RNA complete sequence. (U1)

Klebsiella variicola strain F2R9 16S ribosomal RNA gene partial sequence.

Klebsiella pneumoniae subsp. ozaenae strain ATCC 11296 16S ribosomal RNA gene partial sequence.

Klebsiella pneumoniae subsp. rhinoscleromatis strain ATCC 13884 16S ribosomal RNA gene partial sequence.94

9529

27

12

15

12

1

(U1)

SUMMARY

• A bacterial strain was isolated from the acidic soil of rubber plantation which could survive in a medium of nitrogen-deficiency.

• The bacterial isolate could produce PHA at a maximum concentration of 0.8 g/L with glucose as carbon source and ammonium nitrate as nitrogen source after 48 hours.

• The product extracted from the bacterial isolate U1 was checked for presence of PHA by UV Spectrophotometric analysis using crotonic acid as a standard, and PHA was found to be present.

• The extract was further confirmed for presence of PHA by detection of functional groups using FTIR Spectroscopy.

• Optimisation of PHA-production by varying the carbon and nitrogen sources was carried out and glucose was found to give the maximum yield of 1.1 g/L with either ammonium chloride or peptone as nitrogen sources. This was closely followed by glucose and sodium nitrite at 1 g/L and glycerol and ammonium nitrate at 0.9 g/L. Maximum yield was achieved after 48 hours of growth.

• Halotolerance of the isolated organism was checked by inoculating in medium containing sodium chloride in increasing concentrations of 0%, 5%, 7% and 10%. Although growth was found in all four concentrations of sodium chloride, PHA production on average was found to be optimal in concentrations of 0% and 5%.

• The isolated bacterial strain was identified through 16S rDNA sequencing as Klebsiella pneumoniae subsp. pneumoniae MGH 78578 strain.

Future Work• Application of the strain Klebsiella

pneumoniae in production of PHA from effluents or industrial wastes.

• Optimisation of production process by cultivating in anaerobic conditions in different temperatures and pH.

• Characterisation of the polymer by GC-MS and thermal studies by Thermo-Gravimetric analysis.

CONCLUSION

• Cost-efficient production of PHAs from bacteria will ensure the reduction in production of petrochemical-based plastics.

• PHAs are a better option than other bioplastics as the production does not involve large areas of agricultural land.

• PHAs can be produced from wastes and effluents.• Klebsiella pneumoniae has the ability to produce

PHAs under aerobic conditions in acidic medium, and is moderately halotolerant.

REFERENCES

1. Anderson, Alistair J. and Dawes, Edwin A. (1990). Occurrence, Metabolism, Metabolic Role, and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiological Reviews. 54(4) p. 450 – 472.

2. Kannahi, M. and Rajalakshmi, M. (2012). Production and Optimisation of PHB by Bacillus megaterium and Azospirillum spp. International Journal of Chemical and Pharmaceutical Sciences. 3(3) p. 15 – 18.

3. Van-Thuoc, Doan, Huu-Phong, Tran, Thi-Binh, Nguyen, Thi-Tho, Nguyen, Minh-Lam, Duong, and Quillaguaman, Jorge (2012). Polyester Production by Halophilic and Halotolerant Bacterial Strains Obtained from Mangrove Soil Samples Located in Northern Vietnam. Microbiologyopen. 1(4) p. 395 – 406.

4. Tekin, Ebru, Ates, Mustafa and Kahraman, Ozge (2012). Poly-3-hydroxybutyrate-Producing Extreme Halophilic Archaeon: Haloferax sp. MA10 Isolated from Camalti Saltern, Izmir. Turk J Biol. 36(2012) p. 303 – 312.

5. Chaijamrus, S. and Udpuay, N. (2008). Production and Characterisation of Polyhydroxybutyrate from Molasses and Corn Steep Liquor produced by Bacillus megaterium ATCC 6748. Agricultural Engineering International: the CIGR Ejournal. X(2008) p. 1 – 12.

6. Lopez-Cortes, Alejandro, Lanz-Landazuri, Alberto and Garcia-Maldonado, Jose Q. (2008). Screening and Isolation of PHB-Producing Bacteria in a Polluted Marine Microbial Mat. Microb Ecol. 56 p. 112 – 120.

7. Prasanna, T., Babu, Ajay P., Lakshmi, Dhanavara P., Chakrapani, R., and Rao, Ramachandra CSV. (2011). Production of Poly(3-hydroxybutyrates) by Bacillus species Isolated from Soil. Journal of Pharma Research and Reviews ISSN: 2249-8214 p. 15 – 18.

ACKNOWLEDGMENT

I would like to thank a few people who have supported me throughout the duration of this project:• The Principal, Dr. Sr. Jasintha Quadras, for the opportunity to

be part of Stella Maris College. • Dr. Annie Cherian, the Head of the Department of

Biotechnology, for providing us constant support.• The faculty of the Department of Biotechnology, who

encouraged us and cleared our doubts with patience.• My guide, Dr. K. Veena Gayathri, for taking the time and

efforts to be a wonderful teacher and guide.• My friends who were a source of comfort during the entire

project.THANK YOU.