"REPORT ONPRODUCTION OF POLY-3-HYDROXYBUTYRATE USINGWASTE WATER FROM BIOREFINERY PLANT INTRODUCTIONToday, plastics are a part of humanities everyday life and are indispensable for a varietyof consumer goods and applications (Chanprateep, 2010). An area of increasing research is onmaterials that can be produced from microorganisms. Bio plastics are an example, to be used asan alternative to petro-derived plastics which accumulate in the environment. Most Plastics aremade overwhelmingly of non-biodegradable organic polymers with high molecular weight. As aresult, they are, and accumulate in the environment as waste and landfill. In comparison,bioplastics are commonly derived from biological materials, and are capable of degradation bybacteria and can be classified into two cases. The first kind of bioplastics are made up ofcomponents derived from renewable raw material and the other derived from petrochemicalswith additives that are biodegradable and which will enhance biodegradation. A family of thefirst kind of said biodegradable plastics are known as Polyhydroxyalkanoates (PHAs). ? Poly-3-hydroxybutyrate (PHB)? Polyhydroxyvalerate (PHV)? Polyhydroxyhexanoate (PHH)All the polymers above are naturally synthesized from many micro-organisms such asRalstoniaeutropha, Escherichia coli, etc. during their life cycle using renewable source ofsubstrate. PHB is the polymeric ester of D (-) 3 hydroxybutyric acid and the most common type ofbiopolymer because of its excellent material properties such as its physical and mechanicalsimilarities to synthetic plastics (example: polypropylene). It is stiff and brittle and the degree ofbrittleness depends upon the degree of crystallinity and glass temperature (Marandet al., 2000). Fig1. Structure Poly-3-hydroxybutyrate Unlike synthetic polymers, PHB does not contain any residues of catalysts, allowing the plasticto flow easily in the process. The biocompatibility of PHB makes it one of the major sources forvarious applications in the field of medicine. It is said to be compatible with the blood andtissues of mammals. The monomer of PHB is metabolic in blood and as the body reabsorbs itmight be used in surgical implants as seam threads for healing wounds and blood vessels(Williams & Miller, 1987). In the field of pharmacology, it is used in microcapsules in therapyand tablet packing. In the food industry, laminated foils, bottles, potted flower, one way cups andfibers in textiles can be made with PHB.The metabolic pathway of microorganism is involved in the regulation and synthesis ofPHB in microbial cells. The organisms that produce PHB in its cell accumulate them as storagecompounds in response to substrate limiting conditions. PHB was produced by a variety ofmicroorganism under theenvironmental stresses such as nutrient limitation i.e. nitrogen,phosphorus or oxygenlimitation (Steinbüchel and Füchtenbusch, 1998; Thakor, 2003).The large scale production of PHB from micro-organisms is dependent on thedevelopment of process with low cost and also which produces biodegradable plastics superior topetrochemical plastics (Apostoliset al., 2006). Several methods have been studied in the pastusing cheap substrates such as methanol (Suzuki et al., 1986), ethanol (Alderetet al., 1993), beetmolasses, starch and whey (Ghaly., 2003), cane molasses (Mona et al., 2001), wheat hydrolysateand fungal extract (Apostoliset al., 2006) and also soy cake (Fabianeet al., 2007) as sole carbonsource. It has been reported that, PHB production is enhanced under limiting conditions ofnitrogen and phosphorus/ sulfur (Verlindenet al., 2007).The optimization of nitrogen source hasalso been studied using casein hydrolysate, yeast extract, tryptone, corn steep liquor and collagenhydrolysate in nitrogen rich media for PHB bioconversions using either Cupriavidusnecator orrecombinant E.coli strains (Lee and Chang., 1995, Bormann et al., 1998, Khanna andSrivastava., 2005). In this report, it has been decided to produce PHB from wastewater of starchproductionfrom cassavas.The justification for doing so lies mainly in the idea of convertingwaste into a useful product, along with comparable yields to rival processes. Cassava is also called a tapioca root or a woody shrub of the euphorbiaceae family and is extensively cultivatedas an annual crop in tropical and sub tropical regions for its edible starchy tuberous root, a majorsource of carbohydrates. The wastewater obtained from the cassava starch manufacturingindustry can be used as one of the potential carbon source for PHB production along withlimiting concentrations of nitrogen and phosphorous source. Details have been provided on thedesign considerations and also the amount of PHB that can be produced annually from the biorefinery plant. LITREATURE REVIEWThe most popular method of PHB synthesis is through a fermentation process using bacteriaand various raw materials. It is required that the raw material provides a carbon source, alongwith nitrogen and phosphorous. Each raw material has its own merits and drawbacks. Thefollowing raw materials have been investigated and will be discussed:? Sugarcane bagasse? Palm oil? Glycerol? Cassava wastewaterPHB FROM SUGARCANESugarcane as a carbon source was investigated on the basis of extracting glucose from sugarcaneby hydrolysis, and using the glucose as a carbon source for growth of PHB. The reason forfocusing on sugarcane as a source of glucose was its high glucose content.The useful part of sugarcane is the sugar content, from which glucose can be extracted byeither acidic or enzymatic hydrolysis. (Rabelo, et al., 2008)provides a method by which glucoseis extracted from sugarcane bagasse enzymatically with lime pretreatment. A maximum yield ing/g is provided.The average worldwide yield of sugarcane per hectare is 70.24 tons per hectare. Peruvianfarms manage a very high national average yield (the highest worldwide, according to the Foodand Agriculture Organization of the United Nations) of 127.8 tons per hectare (FAO, 2012). Onspeculation, one might suggest that this is using energy intensive farming techniques, to account for low agricultural area caused by a lack of flat land – Peru is commonly known to be amountainous region. From experience, bio-processes are more economical favourable when non- energy intensive agriculture is used (Rosenberger et al., 2001). In the literature search, two papers were found that utilize glucose as a carbon source forPHB production from bacteria (Centeno-Leijaet al., 2013) details production of PHB usinggenetically modified Escherichia coli to achieve a high PHB yield with a minimal, glucose basedmedium. (Wang & Han-Quing, 2006) provide data on production of PHB usingRalstoniaeutropha with glucose as a carbon source which gives lower yield than the E. coliexample, but otherwise exhibits faster kinetics and also a higher end PHB concentration, whichwould both give lower production costs compared to E. coli. In practice there will be a trade-offbetween these methods, between profit from yield and expenditure due to bio-reactor residencetime and energy costs required for a higher yield. PHB FROM PALM OILIn the industrial production of poly-3-hydroxybutyrate,the carbon source has been identifiedas one of the major cost factors.It is important to find cheap and renewable carbon sources toreplace the sugar-based raw material. Comparing with sugar,the palm oil has been investigated asan economical and efficient feedstock in both laboratory and commercial scale since it offerscompetitive price and high yield of PHB per gram of carbon substrate(Kumaret al., 2011).Thepalm oil mainly consists of triacylglycerols(TAGs),which are formed by combining glycerol withthree molecules of fatty acid.The bacteria secretes lipases in order to utilize the plant oil.The fattyacids are catalyzed to release from the TAGs by some particular lipases(Jaeger et al., 1999).Aftertransportation into the cell;the fatty acids are catabolized by the ß-oxidation cycle (DiRussoet al.,1999).Palm oil creates a heterogeneous two-phase medium with water,in which the palm with lowdensity suspends on the top of a vessel due to its low density.The separation of the heterogeneousmedium makes the material unavailable to the bacteria and causes some errors in themeasurements of palm oil consumption and product yield.In addition, it is also impossible to takeearly samples in the palm oil medium since there is no way to determine the depth of the sampleport.In order to overcome this problem,a new method was developed by (Charles et al., 2011)in "