"PROCESS FLOW CHART: PHB PRODUCTIONFed-batch fermentation has been the efficient process and it has been used in almost most of theresearch on PHB production from microorganism. Fed batch fermentation has been used wherePHB is produced from agro industrial waste products (Hamiehet al., 2013) also studies onoptimization of PHB production from Ralstoniaeutropha have been done Patanik, 2008. Similarresearch has also been performed on optimal adaptive control of PHB in fed batch fermentationby Wu et al., 2013.Acidogenic MediaCassava roots fermentationpreparationFermentedMediaStarchcassava wasteManufacturing formulationwasterWaste water Sterilizationfrom StarchplantMICROORGANISM Fermentation Downstream ProcessingFreezeCentrifuga dryingSeparator Wash tanktionCentrifuga PHB tionEvaporator Fig4. Simple process flow diagram for PHB production. CULTURE MEDIUM & CULTURE CONDITIONS The effect of substrate concentration and nutrients was first investigated with respect tonitrogen and phosphorus concentrations by means of chemical oxygen demand (COD). Thechemical oxygen demand I used to indirectly measure the amount of organic compounds presentin water. The application of COD determines the amount of organic pollutants present in wastewater making it a useful measure of water quality. In this case, NH Cl and KH PO are used as4 2 4 nitrogen and phosphorus source along with FCSW.The ratio of soluble COD: N: P was studied and it was found from the previous literaturesthat 100:0.5:11 was the most optimum ratio (Sangyokaet al., 2012) that can be used forfermentation in order to obtain high amount PHB in the process.The parameters for thefermented cassava waste water are shown in the following table. PARAMETER (mg/L) FCSWpH 5.30tCOD 17,200sCOD 12,400Total nitrogen 71Total phosphorus 81Acetic acid 31Butyric acid 16Propionic acid 16DOWNSTREAM PROCESSING The most common method of poly-3-hydroxy butyrate recovery is the method of solventrecovery (Ramsay et al., 1994) and high purity of 95-98% can be achieved through this method(Zinnet al., 2003). The solvent modifies the cell membrane and dissolves PHB and finally theseparation is important which involves the polymer to be separated from the solvent. In the simple schematic flow diagram depicted above, the steps involved in thedownstream processing are shown. The cells, along with the fermentation medium are first transferred in to the centrifuge where the bacterial cells are separated from the culture broththrough continuous centrifugation. After centrifugation the cells are freeze dried in a lyophilizer.The lyophilized cells are then transferred to a separator containing sodium hypochlorite. TheSodium hypochlorite 13% (v/v) is used for digesting the non poly-3-hydroxybutyrate cell matterand the separation of polymers is done with internal cooling and sedimentation. Aftersedimentation, the non digested biomass remains at the top leaving the polymer to be extractedfrom the bottom. The polymer is then washed with distilled water and isopropanol and the cellsare recovered through centrifugation. The solvent modifies the cell membrane and dissolvesPHB which is separated by drying the solvent isopropanol in an evaporator. The extractedpolymer appeared as white powder with high purity. It is also to be noted that the sodium hypochlorite used in the process is non-volatile orcombustible and therefore it requires less safety measures than other solvents used (Berger et al.,1989). DISCUSSION AND EVALUATIONThe quality of a non-experimental paper such as this can only be as high as theinformation available to it. While there are many papers concerned with microbial production ofPHB, very few appear to carry out studies with an industrial process in mind (e.g. mediumoptimization, pilot scale experiments etc.). It may be that experimental studies of industrial scalemicrobial PHB production are sponsored by companies in industry planning to use the results fora proprietary process, thereby keeping the relevant studies a secret.Lack of optimized data is an issue, especially with regards to PHB production ingenetically modified (GM) microbes. Park et al.,1995demonstrates production of PHB usingEscherichia coli containing genes transferred from Ralstoniaeutropha, and compares the PHBproduction capabilities the GM microbe and the naturally occurring R. eutropha microbe. It isshown that the GM (transgenic) microbe is superior in terms of PHB production kinetics. Despitethese findings, made almost 20 years ago, available studies that attempt to optimize productionof PHB from transgenic E. coli were found to be severely lacking (Centeno-Leijaet al.,2013)optimizes chemical levels within the transgenic E. coli to improve PHB accumulation, andwas cited in the investigation of PHB production from sugarcane bagasse in this report. No papers were found that study optimization of the medium in which microbial growth and/or PHBaccumulation occurs.In the case of PHB production from cassava wastewater, the bio-reactor medium hasactually been optimized by (Sangyokaet al., 2012) to maximize PHB accumulation, which, whilenot quite ideal, is a step in the right direction. Ideally, research would result in formulation of anon-linear programming problem to be optimized to maximize annual profit (£/yr) for a givenmicrobe and carbon source, with the independent variables:? Cultivation time/Bio-reactor residence time? Starting specific biomass? C-source concentration (along with later addition of C-source in the case of a fed batchreactor)? Concentration of other nutrients (N-source etc.)? Desired yield or annual feedstockOf course, this is a far cry from the actual situation.Location of the process is significant; it affects the market price of the products alongwith land price, raw materials cost, crop yields, environmental and ethical responsibilities andother costs such as labor and electricity. This has not been addressed in this report.Much of the decision making in this report has been made qualitatively. While there isnothing inherently wrong with this, it is preferable to make business decisions with the certaintyprovided by a quantitative economic analysis of the situation. Lifecycle analysis is also a usefulindicator of environmental harm or benefit (In fact, in the case of PHB production from CSW,lifecycle analysis is possibly more important than economic analysis). Ideally, an economicevaluation and cradle-to-gate lifecycle analysis would have been carried out for each potentialprocess, before continuing with the report, and then a final evaluation can be carried out whenthe process is understood in more detail. (Side note: in terms of life cycle analysis, cradle-to-gateis chosen as opposed to cradle-to-grave as it is not something that can be controlled within thescope of the investigation.). "