Bio-plastic From Bacteria. Presented by MONA AL BUREIKAN
Introduction:* Growth of the human population. * The accumulation of huge amounts of non degradable waste materials.* Affecting the potential survival ofmany species.
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.
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.
B- The disadvantages of plastic based petroleum: B-1- Environmental Plastic Pollution.• Problems such as the greenhouse effect and global warming.
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 dont even know that.• Americans generate more than 10.5 million tons of plastic waste ,they recycle only 2% of it.
How are Plastic Bags Harmful to the Environment?• Plastic bags litter the landscape.
How are Plastic Bags Harmful to the Environment?• Plastic bags kill animals.
How are Plastic Bags Harmful to the Environment?• Plastic bags are non-biodegradable.
How are Plastic Bags Harmful to the Environment?• Petroleum is required to produce plastic bags.
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.
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)
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.
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.
Monomers of Bacterial Plastics Synthesized by Microorganisms.• Six types of monomers produced by microbial fermentation are the most common bio-based polymer building blocks.
PolyhydroxyaIkanoates (PHA) Types• There are many types of monomer constituents of PHA• Natural and unnatural.
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.
Biosynthesis of PHA:Biosynthesis of Poly -hydroxybutyrate (PHB):
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 .
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
Biodegradability:1- lntracellular degradation (mobilisation) of PolyhydroxyaIkanoates (PHA), ((dehydrogenase reaction))2- Extracellular Degradation of Polyhydroxyalkanoates (PHA), ((PHA depolymerases))
Biodegradability:3- Blending of PHAs with Other Polymers• Totally Biodegradable Blends.• Non-Totally Biodegradable Blends.
Degradation of PHB by Microorganisms• Many Aerobic and anaerobic PHA-degrading microorganisms ( bacteria, cyanobacteria) isolated from various ecosystems• Fungi
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
Microbiology of Polyhydroxyalkanoate (PHA) SynthesisB- 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).
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.
Screening of PHB Producing Bacteria:Sudan black B staining method
Screening of PHB Producing Bacteria:• Fluorescence Staining Method (Acridine Orange) then Smear prepared on a clean microscopic slide observed under the fluorescence Microscope
Screening of PHB Producing Bacteria:• using a freeze-fracture replica technique coupled with Transmission electron microscopy (TEM)
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).
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
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.
Industrial Production of Bioplastic.Production of Polyhydroxyalkanoates (PHA).
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
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.