BIO SYNTHESIS OF DEGRADABLE POLYMER

FROM AZOTOBACTER VINELANDII USING

COCONUT WATER

SATHYA.K
SURYA.K.K
VIJAYALAKSHMI.J

ANNA BIO-RESEARCH FOUNDATION

DEPARTMENT OF BIO-TECHNOLOGY
ARUNAI ENGINEERING COLLEGE
TIRUVANNAMALAI
INTRODUCTION

Plastic materials are causing serious environmental problems due to

their non biodegradability.

The polymer(poly hydroxyalkanoic acid) [PHA] having properties

excellent biodegradability.

PHAs are natural storage compounds synthesized and

accumulated by numerous microorganisms

Alcaligenes eutrophus,
Alcaligenes latus,
Azotobacter vinelandii,
Psudomonas oleovorans, and
recombinant Escherichia coli.
Factors affecting P(3HB) formation

pH,
temperature,
composition of the media,
aeration, etc.
pH
Bacterial growth rates are usually maximum in the range of 6.5 – 7.5.

Azotobacter vinelandii was achieved using cheese whey at a pH of 7.

Temperature
The optimum temperatures forA.Vinelandii 37oc,
Media composition

Carbon,hydrogen,
Oxygen,Nitrogen,
Phosphorus, Calcium,
Sulfur, potassium and magnesium
 MATERIALS AND METHODS
Bacterial strain and maintenance
The strain Azotobacter vinelandii (MTCC 124*) was used for the
production of P(3HB).
Agar slant contains the Burk’s mineral salts supplemented with
0.05%(w/v) yeast extract and 2%(w/v) sucrose.
Inoculum preparation.
About 50ml of Burk’s salts medium containing 2%(w/v) carbon
source with pH of 7.2

A loop full of test organism is inoculated and incubated at 30oc.

Estimation of cellmass
The estimation of growth is carried out by spectrophotometric
method. The optical density of standard culture is measured using
Elico – SLV 164, Double beam UV – VIS spectrophotometer
at the peak wave length of 545 nm,
Estimation of P(3HB)

P(3HB) was assayed by the method of Law and Slepeeky.

OD was read at 235nm, with a Elico – SLV 164, Double beam
UV – VIS spectrophotometer.

RESULT AND DISCUSSION

Effect of different substrates on biosynthesis of P(3HB)

Substrates such as white sugar- 1.05% (w/v),

jaggery- 1.37% (w/v),
palm sugar-10% (w/v),
coconut water- 10% (w/v),
tender coconut-16.6% (v/v)
Initial pH: 7.2; Inoculum size: 8%(v/v); Fermentation time : 48(h)

Sugar sources Dry cell weight P(3HB)

(gl-1) (gl-1)

White sugar 1.400 0.938

Jaggery 0.600 0.402

Palm sugar 0.867 0.581

Coconut water 1.333 0.893

Tender coconut 0.767 0.514

Effect of buffer solution of biosynthesis of P(3HB)
Initial pH:7.2 ; temperature : 30oc ; inoculum size : 8% (v/v)

Buffer solution Dry cell weight P(3HB) weight

(gl-1) (gl-1)

Sodium hydroxide 0.778 0.521

Sodium carbonate 0.962 0.645

Disodium hydrogen 1.874 1.256

phosphate

Dipottasium 1.428 0.957

hydrogen phosphate
Effect of fermentation time on biosynthesis of P(3HB)
Initial pH:7.2; temperature : 30oc; inoculum size : 8% (v/v)

Fermentation time Concentration (gl-1) Final pH

(h) Dry cell weight P(3HB)

0 0.115 0 7.2

24 1.280 0.588 6.1

48 1.96 1.395 6

72 1.844 1.275 5.9

96 1.642 1.044 5.8

120 1.567 0.969 5.8

144 1.40 0.801 5.7

Effect of Coconut Water Concentration on Biosynthesis of P(3HB)

The effect of coconut water concentration on P(3HB) biosynthesis

using A. vinelandii (MTCC 124*) is studied by conducting
the experiments at different initial coconut water concentration

Sucrose inoculum of 8% (v/v) is used in the experiment.

As the coconut water concentration is increased from 10% (v/v) to

30%(v/v) the production of cellmass and P(3HB) increases and
there is no appropriate change in cellmass and P(3HB) when the
concentration increases from 30% (v/v) to 50% (v/v) .

The maximum cellmass (1.968gl-1) and P(3HB) (1.319gl-1) are

produced at 30% (v/v) of coconut water.

At higher substrate concentrations, the cellmass and P(3HB)

concentration remains constant, this may be due to the substrate
utilization rate of the strain reached maximum.
 Effect of different sources of medium ingredients on P(3HB)
biosynthesis using A.vinelandii MTCC 124 utilizing coconut water

Experiment no P(3HB)(gl-1)
Fermentation time (h)
24 48
1 1.03 1.79
2 1.32 1.68
3 0.92 1.57
4 1.15 1.81
5 1.22 1.61
6 1.18 1.58
7 0.79 1.21
8 0.47 1.28
9 0.78 1.41
10 0.62 1.18
11 0.82 1.48
12 0.83 1.67
13 1.22 1.76
14 0.68 1.14
15 1.31 1.64
16 0.72 1.13
17 0.68 1.32
18 0.79 1.29
CONCLUSION

In order to develop an economical process for the production of

different sources ingredients on Biosynthesis of P(3HB) coconut
water was used as a substrate.

A maximum concentration of different sources ingredients on

biosynthesis of P(3HB(1.319gl-1) was obtained with a coconut
water concentration of 30%(v/v) with a fermentation time of
48hours.

Further different sources ingredients on biosynthesis of P(3HB)

yield is improved by optimizing the culture medium using
placket – Burman optimization technique.

By placket - Burman optimization an increased different sources

ingredients on biosynthesis of P(3HB) concentration of (1.99gl-1)
was obtained which is 50% more than that of the yield obtained
from conventional optimization.
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