2. Introduction:
* Growth of the human population.
* The accumulation of huge amounts
of non degradable waste materials.
* Affecting the potential survival of
many species.
3. Introduction:
plastic based petroleum:
• Plastics are ubiquitous in everyday life.
• Plastic is one of the major toxic pollutants.
• over 60 to 100 million barrels of oil are used in the
manufacturing of plastic bags alone.
4. Introduction:
A- The advantages of plastics based petroleum:
• Light weight.
• Chemically resistant.
• Can easily be shaped.
• Durable.
• Easy to colour in the mass.
• Thermically insulating.
• Energy saving.
• Easily portable and impermeable to water.
5. B- The disadvantages of plastic based petroleum:
B-1- Environmental Plastic Pollution.
• Problems such as the greenhouse effect and global
warming.
6. B- The disadvantages of plastic based petroleum:
B-1- Environmental Plastic Pollution.
• Plastic bottles take almost
1000 years to decompose.
• An estimated 14 billion
pounds of plastic waste is
dumped in the ocean every
year.
• Plastic recycling is the only
way to make sure the safe
deposition of plastic but most
people don't even know that.
• Americans generate more
than 10.5 million tons of
plastic waste ,they recycle
only 2% of it.
7. How are Plastic Bags Harmful to the Environment?
• Plastic bags litter the landscape.
8. How are Plastic Bags Harmful to the Environment?
• Plastic bags kill animals.
9. How are Plastic Bags Harmful to the Environment?
• Plastic bags are non-biodegradable.
10. How are Plastic Bags Harmful to the Environment?
• Petroleum is required to produce plastic bags.
11. B- The disadvantages of plastic based petroleum:
B-2- Health Effects of Plastics:
• Many gases vaporize and pollute the air.
• Dioxin emissions from plastic burning
• Toxic chemicals (eg. ethylene
oxide, benzene, vinyl chloride and
xylenes) to air and water and cause
cancer in humans.
• Many of the toxic chemicals can cause
Endocrine disruption, cancer and birth
defects and damage the nervous
system, blood, kidneys and immune
systems.
• These chemicals can also cause serious
damage to ecosystems.
12. Bio-based plastics:
• The inclusion bodies observed in
1920s by Maurice Lemoigne
• Bio-based plastics developed
rapidly
• Rising petroleum prices
• Plastic pollution
• Biopolymers
• Bioplastics
(polyhydroxyalkanoates)
• More than 40
polyhydroxyalkanoates
(PHAs)biocompatibility (non- toxic)
13. Bio-based plastics:
• The cost of this (PHA) is still around ten times higher than that of
conventional plastics.
• Polyhydroxyalkanoates (PHA) synthesised completely by many types
of bacteria.
• PHA has been identified in more than 20 bacterial genera.
• The accumulation of PHA can be stimulated under unbalanced
growth conditions
• poly(3-hydroxybutyrate) [P(3HB)] or PHB. P(3HB) was the first type of
PHA to be discovered.
14. Types of Bacterial Plastics Synthesized by
Microorganisms:
• Polyhydroxyalkanoates
(PHA).
• poly(lactic acid) (PLA).
• poly(butylenes
succinate) (PBS).
• polyethylene (PE).
• poly(trimethylene
terephthalate) (PTT).
• poly(p-phenylene) (PPP)
• They are the best
studied polymers
containing at least one
monomer synthesized
via bacterial
transformation.
15. Monomers of Bacterial Plastics Synthesized by
Microorganisms.
• Six types of monomers produced by microbial fermentation are
the most common bio-based polymer building blocks.
17. PolyhydroxyaIkanoates (PHA) Types
• PHAs are classified into two groups based on the number of carbon atoms in
the monomers.
• This includes short chain length (scl) polymers consisting of 3–5 carbon
atoms containing monomers,
• including Cuprivadus necator and Alcaligenes latus.
• The other group is the medium chain length (mcl) polymers, consisting of 6–
14 carbon atom containing monomers,
• including Pseudomonas putida and Pseudomonas mendocina.
19. Properties of PHA:
Thermal Properties and Mechanical
Properties:
* Melting temperature (Tm), 60 and 177.
* Glass-transition temperature (Tg), 4−50 .
* Thermodegradation temperature (Td),
227 and 256°C .
* The mechanical properties include a very
flexible.
* An elongation at break ranging from 2
to 1,000%, a tensile strength of 17–104
MPa .
20. Properties of PHA:
Molecular Weights:
• PHA has the most diverse
structural varieties, resulting in
the most variable molecular
weights.
• Ranging from 10×104 to
10×106 .
• PLA has Mw ranging from
5×104 to 50×104
21. Biodegradability:
1- lntracellular degradation (mobilisation) of
PolyhydroxyaIkanoates (PHA), ((dehydrogenase reaction))
2- Extracellular Degradation of Polyhydroxyalkanoates (PHA), ((PHA
depolymerases))
23. Degradation of PHB by Microorganisms
• Many Aerobic and anaerobic PHA-degrading
microorganisms ( bacteria, cyanobacteria) isolated
from various ecosystems
• Fungi
27. Microbiology of Polyhydroxyalkanoate (PHA) Synthesis:
A- Natural PHA Producers
• Prokaryotic
microorganisms, including
bacteria and archaea
• Bacillus
• Alcaligenes
• Pseudomonas
• Azotobacter
• Ralstonia
• Vibrio
• Enterobacter
• Cupriavidus . Necator Bacteria (red) produce PHB, a
• Methylobacterium polymer similar to solid plastic,
which they stockpile as food
28. Microbiology of Polyhydroxyalkanoate (PHA) Synthesis
B- Engineering of Non-PHA Producers
• Fast growing population.
• Able to utilize cheap carbon and having a high production rate
• Escherichia coli
• Poly (3-hydroxybutyrate) PHB biosynthesis genes including phbA
(encoding 3-ketothiolase).
• phbB (encoding acetoacetyl-CoA reductase).
• phbC (encoding PHB synthase).
29. Screening of PHB Producing Bacteria:
• Optical microscope operated in
phase-contrast mode
• Dye Nile blue A results in a
bright orange fluorescence of
PHA granules if observed under
an ultraviolet (UV) light
microscope
• Observation of cells without
PHA granules (a), with PHA
granules (b) and cells with PHA
granules and spores; and (c)
under phase-contrast light
Microscopy.
30. Screening of PHB Producing Bacteria:
Sudan black B staining method
31. Screening of PHB Producing Bacteria:
• Fluorescence
Staining Method
(Acridine
Orange) then
Smear prepared
on a clean
microscopic slide
observed under
the fluorescence
Microscope
32. Screening of PHB Producing Bacteria:
• using a freeze-fracture replica technique coupled
with Transmission electron microscopy (TEM)
33. Screening of PHB Producing Bacteria:
• For qualitative
determination of PHB gas
chromatography (GC) was
used.
• The Gas chromatography /
mass spectrometry (GC /
MS) instrument separates
chemical mixture (The GC
component) and identifies
the components at a
molecular level.
• Also, PHB was qualitatively
and quantitatively analyzed
by high performance liquid
chromatography (HPLC).
34. Extraction and Purification of Polyhydroxyalkanoates
(PHA) from Microbial Cells
• Chloroform extraction results in a high
level of polymer purity without polymer
degradation
• Sodium hypochlorite is a well-known
cell solubiliser which has been used for
extraction of poly(3-hydroxybutyrate)
• The PHA granules separated by
centrifugation, cause severe
degradation of P(3HB).
• A modified method of recovery using a
dispersion solution of sodium
hypochlorite and chloroform
• Among these chemicals, SDS, NaOH and
KOH were more efficient in recovering
P(3HB) from recombinant Escherichiu
coli
35. Substrates and growth conditions for PHB production:
Cheaper substrates for PHB production :
A- Available Waste Streams in Different Global Regions
• Plant oils.
• Molasses.
• Starch.
• Whey.
• Industrial wastes.
37. Examples of the most important companies
that produce PHB over the worled
• PHB Produced by Chemie Linz, Austria, Using
Alcaligenes latus
• PHB Produced by PHB Industrial Usina da Pedra-
Acucare Alcool Brazil Using Bhurkolderia sp.
• PHB Produced by Tianjin Northern Food and Lantian
Group China Using Ralstonia eutropha and
Recombinant Escherichia coli, Respectively.
• Industrial Production of PHBV
Biopol products did not succeed and the PHBV
patents were sold to Monsanto and further to
Metabolix. NingBo TianAn, China
38.
39. References;
• Isolation, Cloning and Sequencing of Poly
(3-Hydroxybutyrate) Synthesis Genes From
a Producing Bacterium. Seminar submitted
to the University of King Abdulazez, Jeddah in
partial fulfillment of the requirements for the
of Degree. PhD Of Science in Microbial
Biotechnology. By Mona Al Bureikan. 16 May
2012.
