PHA – A Novel Storage Compound

1. PHA – A Novel Storage Compound Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar Email – bhavesh1968@rediffmail.com

2. Typical Bacterial Cell

3. Inclusions - granules • Intracellular storage bodies • Vary in size, number & content • Bacterial cell can use them when environmental sources are depleted • Examples: glycogen, poly-b-hydroxybutyrate, gas vesicles for floating, sulfur and polyphosphate granules, Cyanophycin granules (Cyanobacteria – Nitrogen storage), Carboxysome (reduce CO2 to get Carbohydrate)

4. Plastic – A necessary evil • General -Plastic is a material made up of one or more polymer. -Main source of material needed to manufacture plastics are fossil fuels (petrochemicals). -Take very long time to biodegrade when disposed. • Consumption rate -India:2 Kg / Person / year -Europe:60 Kg / Person / year • Concerns -Rising cost of fossil fuel. -Environmental impact.

5. Biopolymers and Bioplastics • Polymers synthesized by biological system are Biopolymers. • Plastics manufactured by using biopolymers are Bioplastics. • Concept of bioplastic is not new, Henry ford developed a method of manufacturing plastic car parts from soybean in mid of 1900s. • Today bioplastics are gaining popularity once again as newer techniques developed through biotechnology.

6. Biopolymers • Biopolymers produced by living organisms - Cellulose, Soy protein, Starch, Polyesters • Polymerizable molecules - Lactic acid, Triglycerides

7. PHA – Novel compound • Polyhydroxyalkanoates (PHA) is a biological polyesters accumulated by wide range of bacteria. • It is an intracellular carbon and energy storage compound. • Produced under limiting nutritional condition of N,P,S,Mg etc. and in presence of excess of carbon. • Many companies have started producing PHA using pure culture of Ralstonia eutropha.

8. Detecting PHA • PHA producer can be isolated from various locations particularly from oil and molasses contaminated soil. • Bacteria accumulating PHAs can be easily identified on solid media, since they appear as more turbid colonies then non PHA producers. • Accumulation of PHA granules can be detected by electron or phase contrast microscope. • Staining with Sudan black B or Nile blue sulfate has been used for PHA. • For quantification gas chromatography is commonly used as it is more convenient, reliable and more accurate. • Recently Fourier Transformed Infrared Spectroscopy (FT-IR) is used which detect intracellular PHA within few seconds.

9. Getting acquainted - PHA • PHAs can be divided into three groups on the basis of chain length and monomer i.e. SCL, MCL, and Copolymer. • MCL PHA have advantage over SCL i.e. low crystallinity and high elasticity. • Copolymer produced by many bacteria (SCL-MCL) have superior mechanical properties similar to Low Density Poly Ethylene (LDPE), depending on the monomer composition.

10. PHA - Properties • Properties of PHAs depends on monomers, chain length and molecular weight. • About 120 monomer units with different R-group have been reported. • Piezoelectric, Nonlinear optical activity, Biocompatibility, Biodegradable. • Applications: Tissue engineering, drug delivery, smart materials, degradable packaging.

11. PHB – A most common PHA • Poly-3 Hydroxy Butyrate (PHB), the most commonly found member of PHA family, is a SCL-PHA with monomer containing 4-5 carbon atoms • PHB is thermoplastic or elastomeric material with melting point 40-180oC. • Properties of PHB are similar to polypropylene but its high melting point makes the processing difficult. • Copolymers of PHB are less stiff, tougher and has melting point about 100oC.

12. PHA - Producers • Ralstonia eutropha (earlier A.eutrophus) • Alcaligenes latus • Pseudomonas sp. • Recombinant E.coli Substrates: Glucose, sucrose, n-alkanes, n-alcohols, organic acids.

13. Fermentation v/s Field • Fermentation is a powerful way to produce different biopolymers using bacteria. • Production of bacterial polyesters and lactic acid by fermentation is already started. • Using GE gene responsible for plastic production can be transferred to plants to synthesize plastic. • Successful transformation of the above gene is already achieved Arabidopis thaliana.

14. Its not easy being green • Production cost -Synthetic Plastic ~ 1 € / Kg -Poly lactic acid ~ 3 - 4 € / Kg -Starch compound ~ 2 - 4 € / Kg -PHA ~ 3 - 5 € / Kg

15. Its not easy being green • Little hope Transgenic plants can produce large quantity of PHAs which lowers the cost of PHA to a level comparable to conventional plastic.

16. Lowering the production cost • Pure culture - Expensive raw materials - High investment and operational cost - High yield of PHA up to 80% of cell dry wt. • Mixed culture (activated sludge) - Cheap substrate - Low operational cost - Low yield of PHA up to 60% of cell dry wt.

17. Production strategies in majority of bacteria • Step 1 Two acetyl CoA are condensed to form aceto acetyl-CoA by β ketothiolase (pha A) • Step 2 Acetoacetyl-CoA is reduced to R-3-hydroxy butyryl CoA by NADPH linked reductase (pha B) • Step 3 PHA synthase (pha C) finally links R-3-hydroxy butyryl CoA to the growing chain of PHB.

18. Sustainable development & PHA • PHA is the main component in creating sustainable plastic industry. • PHA reduces our dependency on non renewable fossil fuels. • Biodegradable.

20. Thank You Q & A