Research Task
Polyhydroxyalkanoates (PHA)
Introduction
Polyhydroxyalkanoates (PHA) is a naturally occurring biopolymer which are produce by various fermentation of microorganisms. Polyhydroxyalkanoates are a class of natural polyesters that are derived from bacterial fermentation. The most common kind of PHA is Polyhydroxybutyrate (PHB), The poly(3-hydroxybutyrate) form of PHB is the most common type of PHB found in micro-organisms and plants alike, but many other isomers can be also available, such as poly(4-hydroxybutyrate) and can be anywhere between 1000 to 30000 monomers. This PHA can be produced through genetically modified plants, but industrially it is produce naturally in microorganism such as bacteria, when the organism experiences a manner of physiological stress, which starts to produce PHB as an energy storage molecule, to be metabolized when all the other energy sources are not available. Nowadays plastics and synthetic polymers are mainly produced using petrochemical materials that cannot be decomposed. Therefore, they contribute to environmental pollution and are a danger to many animals. The significance of PHA is that it is produced naturally by renewable agricultural resources, and most importantly, other natural bacteria can consume this polyester, so it is being promoted as fully biodegradable polymer.
Structure of poly(3-hydroxybutyrate)
History and development
The first PHA identified in nature for possible industrial use was PHB in 1925, Maurice lemoigne of the Pasteur institute in Paris found that certain bacteria grew PHB to store energy as in starch in plants and fat in mammals. PHB remained academic curiosity until W. R. Grace in the US produced small quantities for commercial evaluation in the late 1950s.
In the 1970s, the British chemical company IC began growing large quantities of Acaligenes Eutrophus bacterium to create PHB. 1987, Douglas Dennis succeeded in transferring three genes from Acaligenes Eutrophus that control PHB synthesis into the common bacterium Escherichia coli. In the late 1980s, ICI began worldwide commercialization of a family of (Poly(3HB- co-3HV ) copolymers with the tradename of Biopol. In 1996, Monsanto acquired the Biopol and emphasized on producing Biopol by improving their properties for different end-use applications. This continued until Monsanto stopped their research program and their business came to an end in 1998.
Genetic Engineering – In 1988, scientists found a way to make PHB is large quantities, economically and soon after a movement towards mass production of PHB began after 1988, pushes have been made by individuals and governments to see it looked into further as an environmentally friendly plastic alternative. And it is now mass produced at many fermenting factories around the world.
PHB is being researched to develop less expensive and more effective means of production. In 1992, a Michigan State University team genetically modified plants to enable them to produce PHB. They took genes from PHB-making bacteria and inserted them directly into two cress plants. Some of the offspring plants from this cross incorporated both the new genes and produced PHB in their leaves. They had managed to create a plant that could grow plastic. About 14% of the dry weight of the genetically modified leaves is PHB.
Currently, the method of producing the polymer PHB costs 5-7 times more than a similar petrochemical-based product. There have been attempts to grow polymer more economically and have utilized genetic engineering (biotechnology). The GMO bacteria include E. coli, can be used to produce PHB which would help in faster growth, better yield, easy recovery, less waste produced and can utilize cheaper biomass. But is more expensive to produce than petrochemical plastics, but they are biodegradable and made from renewable resources. This can be confronted by using GMO plants/organisms which will help to lower cost.
Researchers in industry are working on methods with which transgenic crops will be developed that express PHA synthesis routes from bacteria to produce PHA as energy storage in their tissues. There is an ongoing research on the growth of cynobacteria in olive mill wastewaters to produce PHAs as well as genetic modification to increase the yield in PHA
Enzyme used
There are variety of cultures of microorganism which can cater for the production of PHA. Over all, more than 300 different microorganisms are known that generate PHAs as natural energy reserves such as Cupriavidus necator, Bacillus subtilis and Alcaligenes Eutrophus. Essentially, the choice of microorganisms for industrial applications depends on the microorganism’s stability and biological safety and its PHA production rates.
This report will consider Alcaligenes Eutrophus for the production of PHA. Alcaligenes Eutrophus, now renamed to Cupriavidus metallidurans are non-spore-forming, Gram-negative Bacteria. PHB is produced in response to conditions of physiological stress and can be produced either by pure culture than mixed culture of bacteria the polymer is mainly product of carbon from glucose or starch which is employed by the microorganism as a form of storage energy molecules, is metabolized when other common energy sources are not available intentionally.
Production process of the biopolymer
PHA is renewable source, thus the process begins with sunlight. Through photosynthesis carbon dioxide and water is converted to carbohydrates and oxygen. These carbohydrates are the raw material for the manufacture of PHA. PHA can be produced from glucose as a raw material. The sugar splits up the metabolism to C2 building blocks, which are converted, over several steps, to C4 monomers and Finally, PHB is polymerised.
Industrially PHA is produce through Fermentation process and selected Bacteria are used for prefermentation. This report will focus on the Alcaligenes Eutrophus as the preferred bacteria to produce PHB
Alcaligenes Eutrophus produces PHB when basic growth supporting substances are depleted while there is still an oversupply from a carbon source available. This is process is known as ‘Discontinuous synthesis’. Industrial production of PHB consists of 3 stages. Fermentation, extraction and compounding.
Fermentation
The process begins through bacterial fermentation, where bacteria required for the process multiply and grow. In industrial production, Eutrophus is grown in an environment favorable to its growth to create a very large population of bacteria. They are allowed to grow in steamy, aqueous mediums with temperatures from 20-30°C and is enriched with a balanced nutrition supply (C, N, P, S, O, Mg, Fe) achieving optimum condition for their production. They reproduce rapidly to form large population, one the population is big enough. Nitrogen is restricted, preventing further reproduction. Under these conditions, the organism is no longer able to increase its population but instead begins to make the desired polymer from absorbing carbon, which it stores for later use as an energy source. There are three enzymes that are responsible of this: 3-ketohiolase, acetoacetil-CoA and PHB synthase. At the end of this process PHB makes up around 80% of the bacterial dry mass, which subsequently 100 kg of PHB can be produced per m³ from the of fermentation process.
Extraction
After the fermentation process, there are two isolation process
1. The Extraction method
the bacteria are rounded up and cleaned. To isolate and purify, the cells are concentrated, dried and extracted, then the polymer is solved in chloroform or another solvent like methyl chloride, 1,2-dichloroethane, pyridine and propylene carbonate. The residual cell debris is removed from the solvent containing PHB by a Solid-liquid process such as centrifugation or filtration. The PHA is then precipitated by addition of a non-solvent and recovered by centrifugation and filtration. PHA is washed to enhance the quality and dried under vacuum and moderate temperatures. The product is fine, white powder with purity of over 98%.
2. The Enzymatic method
Digestive enzymes are used to for PHB recovery by destroying membrane wall (or cell wall in Plant). the process starts with thermal pretreatment prior to enzymatic digestion. Panreatin is used as enzymes and is most efficient ttemperature range between 50 °C and 70 °C. The recovery is reached 93.5% purity of PHB.
Compounding
Following Isolation, the PHAs are usually further purified and dried in vacuum processes and subsequently is in powered form. This PHA powder is then extrusion-granulated as made into pellets or small chunks using machinery, at the same time additives are added for improvement and achieving the desired properties. These pellets are then made into larger pieces by compaction.
Alternate process
PHB can be extracted from plants. By genetically modifying plants, there would be no need for a fermentation process, or microorganisms to grow PHB. This process is alternate of extracting PHB. This is process needs further research to become economically and industrially viable. The production process for plant production of PHB differ to the industrial production.
Properties of the Biopolymer
PHAs polymers are thermoplastic and they differ in their properties depending on their chemical composition. Some grades of additives are added to PHB to achieve similarities in their material properties to polypropylene (PP), and offer good resistance to moisture and aroma barrier properties. HB is a highly crystalline (60–70% crystallinity) and linear polymer and the crystallization speed is fast between 80 and 100°C but slow below 60°C or above 130°C so that the material then remains amorphous and sticky for hours. This the why sharp transition of solid to liquid can be used to achieve very fast processing speeds. Biodegradation is dependent on a number of factors such as microbial activity, moisture, temperature, pH of the environment, and the exposed surface area, molecular weight polymer composition and nature of monomer unit. PHB monomer units have been found to be degraded more rapidly and is estimated tthat 0.5%-9.6% of PHB-degrading microorganisms can be found in the environment
This is important because PHB is relatively brittle with elongation break below 15% and is generally stiff as PHB is a fragile material, which is why additives are added to achieve desire properties. These properties may include;
o Water insoluble
o Relatively resistant to hydrolytic degradation
o Biocompatible and hence suitable for medical application
o Good ultra-violet resistance
o Non-toxic
o Highly crystalline (60-70%)
o Very brittle
o biodegradable
o malleable
o durable solid at room temperature
o melting point of 180
o transparent and shiny
o denser than water
o high tensile strength
Use or Potential Use of the Polymer
because of these properties, PHA is useful in many applications, such as;
In medicine, PHB is