polyhydroxybutyrate production

2. SHANTHANU, K. GOWDA
Sr. M.Sc(Agri)
PALB-4144

3. Introduction
Properties of PHB
Biosynthesis of PHB
Commercial Production of PHB
Applications
Research Findings
Conclusion

4.  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.

5. Cupriavidus necator
Bacillus megateriumDelftia acidovorans
Bacillus megaterium

6. • Many different types of PHAs are available and PHB is the
most common one
• Empirical formula - [C4H6O2]n
• Structural formula for the linear chain of PHB

7. • 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

8. Thermoplastic
Water insoluble (Hydrophobic)
Good oxygen permeability
Good ultra-violet resistance
Poor resistance to acids and bases
Soluble in chloroform and other chlorinated
hydrocarbons

9. Biocompatible
Tensile strength is 40MPa
Sinks in water
Brittle to elastic
Non toxic
Piezoelectrical
Can have functional groups
Biodegradable

11. 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

12.  Ralstonia
 Bacillus
 Pseudomonas
 Alcaligenes
 Azotobacter
 Hydrogenomonas
 Chromatium
 Methylobacterium
 Recombinant Escherichia coli and many others.

14. Chee et al., 2010

16. 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

17. Maurice Lemoigne (1926)

20.  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.

21. • 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.

22. Purification of PHB
PHB recovery from bacterial extract
PHB synthesis
Bacteria
Physical stress ( pH, Temperature)
Production of PHB

23. Heinrich et al., 2012

24. B

25. ICI, 1982 : BIOPAL - Alcaligenes eutrophus

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

29. Pharmaceuticals

32. FOOD Service & Product Packaging
EcoBags

33. BIOFUEL

34. Body of Sony Walkman

35. • 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

37. Research Findings

38. Effect of different carbon sources on PHB yield
PHBproduction
(g/100ml)

39. Effect of different N sources on PHB yield
PHBproduction
(g/100ml)

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

41. Effect of different pH levels on PHB yield
PHBproduction
(g/100ml)

42. Research Findings– 2

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

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

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

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

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

48. Research Findings - 3

49. 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)]

50. 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]

51. 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