PHB production by bacteria and its applications

polyhydroxybutyrate production

1. I SEMINAR PHB production by Bacteria a nd its application SHANTHANU, K. GOWDA Sr. M.Sc ( Agri ) PALB-4144

2. Sequence of the seminar

3. Introduction Polyhydroxybutyrates (PHBs) are members from family of polyesters known as Polyhydroxyalkanoates (PHAs). Accumulated in intracellular granules by Gram-positive and Gram-negative microorganisms. PHB are produced when there is excess carbon source with the limitation of one of the essential nutrients. Also known as Biopolymers as they are produced from microorganisms. They are thermoplastic polymers and are totally biodegradable .

4. Cupriavidus necator Bacillus megaterium Delftia acidovorans Bacillus megaterium

5. Many different types of PHAs are available and PHB is the most common one Empirical formula - [C 4 H 6 O 2 ] n Structural formula for the linear chain of PHB

6. History PHB was discovered in 1925 by French scientist Maurice Lemoigne. Found that PHB as the intracellular inclusions in many bacteria. In 1982, the Imperial Chemical Industry in England announced product development program of this biopolymer. A pilot production of 2 tonnes of PHB was made in 1991. Maurice Lemoigne

7. Properties

8. Chen and Wu, 2005 Samples Melting temp. (◦C) Glass transition temp. (◦C) Tensile strength ( Mpa ) Elongation at break ( % ) PHB 177 4 43 5 P(HB-co-10% HV) 150 — 25 20 P(HB-co-20% HV) 135 — 20 100 P(HB-co-10% HHx ) 127 -1 21 400 P(HB-co-17% HHx ) 120 -2 20 850 Polypropylene 170 — 34 400 Polystyrene 110 — 50 — Physical properties of various PHA in comparison with conventional plastics

9. Important PHB producing bacteria Ralstonia Bacillus Pseudomonas Alcaligenes Azotobacter Hydrogenomonas Chromatium Methylobacterium Recombinant Escherichia coli and many others.

10. Chee et al., 2010

11. Carbon Cycle

12. PHB Biosynthesis It consists of three enzymes β - ketoacyl-CoA thiolase ( phb A) NADPH dependent Acetoacetyl-CoA dehydrogenase ( phb B) P(3HB) polymerase ( phb C) Huisman et al ., 1989

13. Maurice Lemoigne (1926) PHB biosynthesis

14. Why PHB are produced ? Polyhydroxybutyrates (PHBs) are polymers that bacteria produce under conditions of low concentrations of important nutrients (typically nitrogen, but sometimes oxygen) and high concentrations of carbon sources. This process occurs because the excess carbon leads to bacteria creating carbon reserves (PHAs) to save for a time with more plentiful nutrients in which they need energy to carry out regular functions. Bacteria store PHBs in granules for later use . These polymers are accumulated intracellularly under conditions of nutrient stress and act as a carbon and energy reserve .

15. Poly-β- hydroxybutyrate (PHB) is synthesized as an intracellular storage material and accumulates as distinct white granules during unbalanced growth in the cell, these are clearly visible in the cytoplasm of the cell. Many bacteria including those in the soil, are capable of PHB production and breakdown .

16. Production of PHB

17. Extraction of PHB from Bacteria Heinrich et al., 2012

18. Organic solvent to release PHB from cells Commercial production of PHB from Bacteria Centrifugation B

19. Example for bioplastic produced from microorganisms ICI, 1982 : BIOPAL - Alcaligenes eutrophus

20. APPLICATIONS OF PHB

21. Agriculture

22. Medicine In medicine , used as a surgical implant, seam threads, screws, plates.

23. Pharmaceuticals

24. Automobile industry

25. AGRICULTURE

26. FOOD Service & Product Packaging EcoBags Food industry

27. BIOFUEL

28. Body of Sony Walkman Other Applications

29. Bioenvelop – Canada – BioP – food containers EarthShell – USA - utensils EverCorn . Inc. – Japan – EverCorn – resin for coating National Starch Company – UK - packaging Novamont – Italy – Mater-Bi – films and moulded products VTT Chemical Technology – Finland – COHPOL Plastobag Industries – India Companies involved in production of PHB

30. Research Findings

31. Research Findings - 1

32. Effect of different carbon sources on PHB yield PHB production (g/100ml)

33. Effect of different N sources on PHB yield PHB production (g/100ml)

34. Effect of different C : N ratios on PHB yield PHB production (g/100ml)

35. Effect of different pH levels on PHB yield PHB production (g/100ml)

36. Research Findings– 2

37. Media: Treated date molasses dissolved in nutrient broth, supplemented with glucose. PHB production (g/50ml) 2 days 6 days 4 days 8 days Eschericia coli Bacillus subtilis Lactobacillus acidophilus Bacillus thuringiensis Staphylococcus aureus

38. Media: Treated date molasses dissolved in nutrient broth. 2 days 6 days 4 days 8 days PHB production (g/50ml) Bacillus subtilis Lactobacillus acidophilus Bacillus thuringiensis Staphylococcus aureus Eschericia coli

39. Media: Whey supplemented with peptone, yeast extract and glucose. 2 days 6 days 4 days 8 days PHB production (g/50ml) Bacillus subtilis Lactobacillus acidophilus Bacillus thuringiensis Staphylococcus aureus Eschericia coli

40. Media: Whey supplemented with peptone, yeast extract and sucrose. 2 days 6 days 4 days 8 days PHB production (g/50ml) Bacillus subtilis Lactobacillus acidophilus Bacillus thuringiensis Staphylococcus aureus Eschericia coli

41. Media: whey supplemented with peptone and yeast extract 2 days 6 days 4 days 8 days Bacillus subtilis Lactobacillus acidophilus Bacillus thuringiensis Staphylococcus aureus PHB production (g/50ml) Eschericia coli

42. Research Findings - 3

43. Cell dry weight (g/l) PHB (g/l) PHB yield (%) Pre-mutation (Control) 0.58 0.091 15.68 UV mutation 0.81 0.192 23.70 Acridine Orange mutation 0.65 0.121 18.61 PHB production by wild and mutant strain [Agriculture isolate (M1)]

44. Cell dry weight (g/l) PHB (g/l) PHB yield (%) Pre-mutation (Control) 0.51 0.060 11.76 UV mutation 0.36 0.090 25 Acridine Orange mutation 0.53 0.080 15.09 PHB production by wild and mutant strain [MTCC453]

45. Conclusion PHB derived plastics can serve as a better replacement for conventional plastics Eco-friendly High cost Approaches required to reduce the cost Strain development is the needed