Production of Bioplastic from Bacteria Asst. Prof. Dr. Wala’a Shawkat Ali (Microbial Biotechnology)
Department of Biology College of Science University of Baghdad E-mail:
[email protected]
Bioplastics are a special type of biomaterial. They are polyesters, produced by a range of microbes, cultured under different nutrient and environmental conditions. These polymers, which are usually lipid in nature, are accumulated as storage materials, allowing microbial survival under stress conditions. The number and size of the granules, the monomer composition, macromolecular structure and physico-chemical properties vary, depending on the producer organism
Bioplastics are made also from organic biomass sources, such as vegetable fats, oils, corn starch and as well as from agricultural by-products. Bioplastic (poly(3-hydroxybutyrate) (PHB)) was discovered for the first time by French microbiologist Maurice Lemoigne at 1925 in Gram-positive bacterium Bacillus megaterium.
Polyhydroxyalkanoates (PHAs): The polymers with properties similar to conventional plastics Naturally occurring, lipid-like water-insoluble polyester molecule PHA acts as an intracellular, granular energy storage reserve; its synthesis and accumulation is triggered by unfavourable growth conditions
Molecular masses ranging from 50,000 to 1,000,000 Da PHAs vary in their physical and chemical characteristics. Factors affecting the monomer content include: 1. Type of microorganisms (e.g. Gram-negative or Grampositive) 2. Media ingredients 3. Fermentation conditions 4. Modes of fermentation (batch, fed-batch, continuous) 5. Recovery
PHAs are biodegradable (The hydrolytic cleavage action and complete degradation process of the PHA depolymerases are quite short, taking about 3-9 months) The major advantage of PHAs is that both the physical properties and the rate of degradation of PHAs can be altered by changing the bacterial source of the polymer Immunologically inert To date, more than 160 different polyesters with plastic properties have been described and this number is growing exponentially by means of genetic and metabolic engineering techniques.
There are three genes required for PHA production: phaA, phaB, and phaC
General structure of polyhydroxyalkanoates (PHA)
Classification of microbial bioplastics according to different criteria Biosynthetic origin
Natural bioplastics: those produced by microorganisms from general metabolites (i.e. PHBs and aliphatic PHAs).
Semisynthetic bioplastics: those that require the addition to the culture broth of some precursors that cannot be synthesized by the microbe (i.e. PHAs containing aromatic monomers)
• Synthetic bioplastics: those polyesters that resemble the natural ones but that can only be obtained by chemical synthesis (i.e. synthetic thermoplastic polymers)
Chemical nature of the monomers Bioplastic containing aliphatic fatty acid derivatives: saturated or unsaturated (with double or triple bonds) monomers; linear or branched monomers; substituted or not (with functional groups in the monomers). Bioplastics containing aromatic fatty acid derivatives Bioplastics containing both aliphatic and aromatic fatty acid derivatives • Bioplastics containing other different compounds (e.g. poly-g-glutamic acid, poly-e-L-lysine, poly-b-Lmalic acid, polyglycolic acid, cianophicin)
Monomer size Bioplastics containing a short-chain length (sclPHB and derivatives sclPHAs; C3–C5 monomers) Bioplastics containing a mediumchain length (mclPHAs; C6–C14) • Bioplastics containing a long-chain length (lclPHAs; >C14)
Number of monomers in the polyesters
Homopolymeric bioplastic: a single monomer is present in the bioplastic • Heteropolymeric bioplastic (copolymer): more than one monomer is present in the bioplastic
Type of polyesters accumulated by the microbe Unique (a single bioplastic) • More than one (mixed bioplastics)
PHAs Bacterial Production requirements: Microbes have been reported to be the potent producers of PHA due to their high adaptability in various extreme environmental conditions. Over 300 species from over 90 genera of Grampositive and Gram-negative bacteria have been reported to accumulate PHA. Bacillus spp., Pseudomonas spp. and Vibrio spp. are found to be more efficient for PHA production due to their higher stability and reproducibility under environmental stress.
Depending on the culture conditions that favor PHA accumulation, bacteria that are used for the production of PHA can be classified into two groups: The first group of bacteria requires limitation of essential nutrients such as nitrogen, oxygen and presence of excess carbon source for the efficient synthesis of PHA. The representative bacteria belonging to this group include C. necator, Protomonas extorquens and Protomonas oleovorans. The second group of bacteria does not require nutrient limitation for PHA synthesis and can accumulate PHA during exponential growth phase. Some of the bacteria included in this group are Alcaligenes latus, a mutant strain of Azotobacter vinelandii and recombinant E. coli harboring the PHA biosynthetic operon of C. necator.
Media (cheap and available; molasses, corn steep liquor, whey, wheat and rice bran, starch and starchy wastewaters, effluents from olive mill and palm oil mill, activated sludge and swine waste) Fermentation conditions depend on the demands of the microbe (temperature, pH, etc…)
Cultivation techniques used in the large scale production of PHA: 1.Batch fermentation 2.Fed-batch fermentation 3.Continuous fermentation 4.A two-stage cultivation method
Recovery & purification of PHAs from biomass: Solvent Extraction: most commonly used (very simple and effective). Among various solvents, chloroform is the most preferred solvent. Sodium hypochlorite method: simple and effective method. Surfactant-hypochlorite method ; a modified method by to obtain PHA with higher degree of purity and Mw Chloroform-hypochlorite method Enzymatic digestion: is a gentle but a selective separation method. Enzymes such as proteases (trypsin, chymotrypsin, rennin, papain and bromelain), cellulases and lysozyme, are commonly used.
Characterization of PHA • Granule analysis
• Molecular weight • Monomer compositions • Other physical properties, such as crystal structure, melting temperature, and mechanical properties
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