I SEMINAR
PHB production by Bacteriaand its application
SHANTHANU, K. GOWDASr. M.Sc(Agri)
PALB-4144
Sequence of the seminar
Introduction
Properties of PHB
Biosynthesis of PHB
Commercial Production of PHB
Applications
Research Findings
Conclusion
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.
Cupriavidus necator
Bacillus megateriumDelftia acidovorans
Bacillus megaterium
• 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
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
Properties
Thermoplastic
Water insoluble (Hydrophobic)
Good oxygen permeability
Good ultra-violet resistance
Poor resistance to acids and bases
Soluble in chloroform and other chlorinated hydrocarbons
Biocompatible
Tensile strength is 40MPa
Sinks in water
Brittle to elastic
Non toxic
Piezoelectrical
Can have functional groups
Biodegradable
Chen and Wu, 2005
SamplesMelting
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
Important PHB producing bacteria
Ralstonia
Bacillus
Pseudomonas
Alcaligenes
Azotobacter
Hydrogenomonas
Chromatium
Methylobacterium
Recombinant Escherichia coli and many others.
Chee et al., 2010
Carbon Cycle
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
Maurice Lemoigne (1926)
PHB biosynthesis
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.
• 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.
Production of PHB
Extraction of PHB from Bacteria
Heinrich et al., 2012
Organic solvent
to release PHB from
cells
Commercial production of PHB from Bacteria
Centrifugation
B
Example for bioplastic produced from microorganisms
ICI, 1982 : BIOPAL - Alcaligenes eutrophus
APPLICATIONS OF PHB
Agricultu
re
Medicine• In medicine, used as a surgical implant, seam threads,
screws, plates.
Pharmaceuticals
Automobile industry
AGRICULTURE
FOOD Service & Product Packaging
EcoBags
Food industry
BIOFUEL
Body of Sony Walkman
Other Applications
• 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
Research Findings
Research Findings - 1
Effect of different carbon sources on PHB yield
PHB
pro
duct
ion
(g/1
00m
l)
Effect of different N sources on PHB yield
PHB
pro
duct
ion
(g/1
00m
l)
Effect of different C : N ratios on PHB yield
PHB
pro
duct
ion
(g/1
00m
l)
Effect of different pH levels on PHB yield
PHB
pro
duct
ion
(g/1
00m
l)
Research Findings– 2
Media: Treated date molasses dissolved in nutrient broth, supplemented with glucose.
PHB
pro
duct
ion
(g/5
0ml)
2 days6 days
4 days8 days
Eschericiacoli
Bacillussubtilis
Lactobacillusacidophilus
Bacillusthuringiensis
Staphylococcusaureus
Media: Treated date molasses dissolved in nutrient broth.
2 days6 days
4 days8 days
PHB
pro
duct
ion
(g/5
0ml)
Bacillussubtilis
Lactobacillusacidophilus
Bacillusthuringiensis
Staphylococcusaureus
Eschericiacoli
Media: Whey supplemented with peptone, yeast extract and glucose.
2 days6 days
4 days8 days
PHB
pro
duct
ion
(g/5
0ml)
Bacillussubtilis
Lactobacillusacidophilus
Bacillusthuringiensis
Staphylococcusaureus
Eschericiacoli
Media: Whey supplemented with peptone, yeast extract and sucrose.
2 days6 days
4 days8 days
PHB
pro
duct
ion
(g/5
0ml)
Bacillussubtilis
Lactobacillusacidophilus
Bacillusthuringiensis
Staphylococcusaureus
Eschericiacoli
Media: whey supplemented with peptone and yeast extract
2 days6 days
4 days8 days
Bacillussubtilis
Lactobacillusacidophilus
Bacillusthuringiensis
Staphylococcusaureus
PHB
pro
duct
ion
(g/5
0ml)
Eschericiacoli
Research Findings - 3
Cell dry weight (g/l)
PHB (g/l) PHB yield (%)
Pre-mutation (Control) 0.58 0.091 15.68
UV mutation0.81 0.192 23.70
Acridine Orange mutation 0.65 0.121 18.61
PHB production by wild and mutant strain [Agriculture isolate (M1)]
Cell dry weight (g/l)
PHB (g/l) PHB yield (%)
Pre-mutation (Control) 0.51 0.060 11.76
UV mutation0.36 0.090 25
Acridine Orange mutation 0.53 0.080 15.09
PHB production by wild and mutant strain [MTCC453]
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