Biosynthesis Of PHA By Fusarium moniliforme

Biosynthesis Of PHA By Fusarium moniliforme

Parul Jindal1, D. P. Tiwari2

1 Research scholar in department of Chemical Engineering, DCRUST, Murthal, Sonipat (India)-131001

2 Den of department of Chemical Engineering, DCRUST, Murthal, Sonipat (India)-131001

* Corresponding author: Parul Jindal,

Abstract:

Fusarium moniliforme isolated from infected roots of rice can accumulate polyhydroxyalkanoates (PHA) upto 45% of its cell biomass.This microorganism can cause bakane disease in rice which results in premature growth of plant ultimately yield of rice become decreases. Production of PHA commercially and at comparable cost has been the main focus in this area.

Introduction:

Demand of plastic goes on increasing day by day. But the dependence on conventional plastic resulted in waste accumulation and green house gas emissions. Due to this reason recent technologies are directed towards the development of biogreen material that has negligible effect on environment. Now days, major interest is in the development of polyhydroxyalkanoates (PHA) by using some bioproduct. As it possess similar properties to conventional plastic and its biodegradability. PHA is produced by a number of microorganisms in the form of PHB (Polyhydroxybutyrate) .But it is not very useful because of its brittle nature. This limitation can be overcome by converting PHB into its copolymer with other PHA by using a varying combination of carbon substrates for growth.

Fusarium moniliforme accumulate upto45% PHA of the cell biomass and the copolymers are synthesized by using propionate or valerate along with sucrose as carbon sources. In the present work we describe the synthesis of PHA by Fusarium moniliforme in sucrose medium.

Fig.1-Fusarium moniliforme

Materials and methods:

Extraction of microorganism and its growth:

Fusarium moniliforme a fungus found in infected roots of rice plant. This causes bakane disease in plants resulting in reduced yield.

Fig:2- The disease: BAKANAE and FOOT ROT

For its culture infected portion of the root is cut and inoculate in the petridish in the form of strands with the help of loop having potato dextrose media (PDA). Labelled the petri dish and placed in BOD at 250C temperature for 8 days. After 8 days colonies come.

Fig.3- Cultured Fusarium moniliforme

Preparation of potato dextrose media (PDA):

Boiled 300g of potatoes in 1L of water for extraction of starch and after that add 2g glucose as carbon source and 0.2g agar-agar for solidification of media.

PHA production in Fusiform moniliforme:

PHA production was carried out in 500ml Erlenmeyer flask having 100ml liquid media contained different C:N ratios such as (NH4)2SO4FeSO41g/l with different amount of sucrose like 10g, 20g, 30g or 20g/l sucrose with 2g, 4g, & 6g of (NH4)2SO4FeSO4. Incubations were

carried out for 72 hours at 250rpm and 300C

temperature.

Biomass estimation:

After centrifugation of culture media cell biomass was collected and washing of pellet was carried out with distilled water. It was dried in airflow drier at temperature 700C.

Polysaccharides estimation:

To precipitate out polysaccharides isopropanol was used. Polysaccharides were dried at 700C upto a constant weight.

PHA estimation:

For the extraction of PHA from biomass solvent extraction was used. The solvent employed for this was chloroform. As chloroform extraction is a very simple and effective method to separate PHA granules from biomass. PHA obtained by this method was highly purified and without any degradation of PHA molecule.

PHA production with varying concentration of carbon and nitrogen:

PHAs are mainly synthesized in the presence of excess of carbon with limiting amount of other nutrients like N, O, S, P etc. Production of PHA increases with increasing concentration of substrate that provide carbon and decreases with increasing concentration of substrate that provide nitrogen. Results are shown in the following table:

Table1- Production of PHA with varying concentration of carbon and nitrogen

Sucrose (g/l)	Ammonium ferrous sulphate (g/l)	Biomass (g dry wt/l)	PHA (g/l)
10	1	3	0.85
20	1	4	1
30	1	4.5	1.5
20	2	4	1
20	4	2.5	0.8
20	6	1	0.5

Result and discussion:

PHA synthesized by Fusarium moniliforme is mainly polyhydroxybutyrate(PHB). As its NMR spectrum has given the following signals:

1.1ppm (doublet for methyl group)

2.5ppm ( doublet of quadruplet for methylene group)

5.28ppm (multiplet for methyne group)

PHB synthesized by Fusarium moniliforme in its homopolymer form is relatively stiff and brittle. PHB is 100% stereospecific with asymmetric carbon atoms having D(-) configuration, thats why highly crystalline. Its melting point is 1750C which is slightly lower than its degrading temperaturei.e.1850C.Yet PHB has several useful properties such as moisture resistance, water insolubility and optical purity. PHB also shows good oxygen impermeability. The various properties of PHB thus formed is shown in following table:

Property	PHB
Melting point (0C)	175
Glass-transition temperature (0C)	15
Crytalline (%)	80
Youngs modulus	3.5

Property	PHB
Melting point (0C)	175
Glass-transition temperature (0C)	15
Crytalline (%)	80
Youngs modulus	3.5

Table 2 shows the physical properties of PHB

Tensile strength (MPa)	40
Elongation to break(%)	6
Impact strength (v/m)	50

Biodegradability of PHA synthesized:

PHB synthesized by F.moniliforme degrades in microbially active environment.Microorganisms colonize on the polymer surface and secrete enzyme that convert PHB into hydroxybutyrate (HB). These monomer units are then utilized by cell as carbon source.

Degradability rate dependa on various factors like surface area, pH, temperature, pressure of other nutrients and moisture etc. In aerobic environment carbon dioxide and water are the end products of PHB degradation.

Future outlook:

PHAs production has drawn major attention due to its biocompatibility and biodegradability with conventional plastic. But the production of PHAs through this method is very costly which a major drawback is. Consequently, researches are going on to produce PHA at lower cost by developing some mutant strains and using some alternative carbon sources.

Acknowledgement:

The author acknowledges sincere thanks to DCRUST Murthal, Sonipat, and Haryana, India for carrying out the research wor successfully and for providing the assistance for doing the laboratory work.

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