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