Scientific discovery and commercial interest of many new and useful polysaccharides were obtained from microbial fermentations. The polysaccharides or gums are in great demand in food processing and a large number of industrial operations. The production is unlimited throughout the year. Three distinct types of carbohydrate polymers which are extracellular polysaccharides, structural polysaccharides and intracellular storage of the cell wall can be produced by microorganisms such as bacteria. Extracellular polysaccharide can be classified into two major groups based on their monosaccharide composition which are homo-polysaccharides (composed of a single structural unit) and hetero-polysaccharides (constructed from two or more monomers). However, the extracellular polysaccharides such as dextrans, pullulans and xanthan gum, have a great commercial significance and marketed extensively. Therefore, the interest of this research was to study the production of xanthan gum.
Xanthan gum is a high molecular weight extracellular hetero-polysaccharide and currently produced by a bioprocess using a plant-pathogenic bacterium, Xanthomonas campestris (X.campestris). This bacteria strain is strictly aerobic. The important properties of the xathan gum is the ability to form high viscosity solution at low shear forces, highly pseudoplastic and may also display a viscosity yield value. The gum is also stable over a wide range of temperatures (up to 90oC), in both alkaline and acidic conditions with wide range of pH (2-11), shear, enzyme degradation and excellent compatibility with salts at salt concentration up to 150g/L NaCl over a wide pH range. According to the study done by Ashtaputre et al. (1995), the viscosity of 4 g/L of xanthan gum was stable in the pH range 4 to 8 and only 26% of the original viscosity was retained at 90oC. The viscosity of xanthan gum was stable up to 90oC in the presence of 50 g/L NaCl. The superior properties of xathan gum have enabled it to compete with most of natural gums, and also become the preferred product due chemical reproducibility and relatively easy supply.
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Xanthan gum is widely used in abroad range of industries, such as foods, toiletries, cosmetics, as water-based paints, etc., due to its superior rheological properties and is used as rheological control agent in aqueous systems and as stabilizer for emulsion and suspension. In the agriculture industry, xanthan has been used to improve the flow-ability in fungicides, herbacides, and insecticides formulations by uniformly suspending the solid component. As it is non-toxic, United States Food and Drug Administration (FDA) approved xanthan for food use in 1969 without any specific quantity limitations (Becker et al., 1998). According to Sharma et al. (2006), xanthan is listed as item E- 415 in Annex-I of the European Economic Community as the emulsifier or stabilizer. The acceptable daily intake (ADI) for xanthan was issued by the Joint Expert Committee of the Food and Agriculture Organization (FAO) and World Health Organization (WHO) of the United Nations. In addition, many other countries have approved xanthan for various food uses.
In petroleum industry, xanthan is commonly used in drilling fluids and in enhancing oil recovery processes due to its high viscosity of solutions and water solubility of the polymer (Garcia-Ochoa et al., 2000). Its excellent compatibility with salt and resistance to thermal degradation make it useful as an additive in drilling fluids. The pseudoplastic behavior of its solutions provides low viscosity at the drill bit where the shear rate is high and high viscosity in the annulus where shear is low. Therefore, xanthan serve as a dual purpose by allowing faster penetration at the bit and suspending cuttings in the annulus.
According to Rosalam et al. (2006), approximately two of it remains in the ground for every barrel of oil produced. Therefore, enhanced oil recovery by using xanthan gum in the next decades will be important. The basic principle applied is to improve the separation of water and oil thereby would increase oil recovery. Xanthan gum is also used in micellar-polymer flooding as tertiary oil recovery operation in which the polymer-thickened brine is used to drive the slug of the surfactant through porous reservoir rock to immobilize residual oil. This polymer prevents bypassing of the drive water through the surfactant band and ensures good area sweeping. The polymers reduced the mobility of injected water by increasing its viscosity in both applications.
USA, Europe, China and Japan are the world xanthan gum producers. According to an article written in the website of AP-Food Technology (2006), currently, there is three factories globally produce xanthan gum which is in San Diego, California, a plant in Okmulgee, Oklahoma and one in Knowsley, UK. In China, one of a leading manufacturer, Fufeng Group Limited (2009), reported an outstanding annual results on the sales of xanthan gum of the company for the year ended 31 December 2008. The sales of xanthan gum increased significantly from approximately 6,200 tonnes in 2007 to approximately 20,000 tonnes and completely absorbed the 12,000 tonnes additional production capacity created in 2008. It shows that the sales volume increased significantly by more than 200% as compared with 2007, which makes the company emergence as one of the top three leading xanthan gum manufacturers in the world.
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Malaysia is one of the importers of xanthan gum for the food and non-food industries. Thus, the research would pave way for the development of a bioprocess for the production of xanthan gum in Malaysia. Therefore, this research will help to develop an economical process in the production of xanthan gum. The results of this research could be useful in food and non-food industries such as petroleum extraction in Malaysia. The knowledge and experience obtained in this research could be shared with various industries intending to produce xanthan gum for their needs by using palm oil, our local resource.
Solutions of xanthan are highly viscous and pseudoplastic. The broth viscosities will increase dramatically as the product accumulates. This broth rheology presents serious problems to mixing, heat transfer and oxygen supply in a bioreactor, which limits the process efficiency. Various approaches have been adopted to improve the xanthan productivity which includes new impeller designs, different reactor configurations and modified media. As regards impeller and reactor design, Intermig and Ecomix impellers have been proved to be very efficient for mixing highly viscous non-Newtonian fluids (Galindo et al., 1995). Therefore, in the bioreactor studies, the Intermig and Rushton impeller were used and the effect between these impellers was studied.
Another important approach is to enrich the growth media with locally available source. It has been found that the use of oil in the fermentation of xanthan gum lowers the viscosity of the solutions in which the aqueous fermentation phase with its microbial growth and metabolic processes takes place in a finely dispersed homogenous oil phase (Engelskirchen et al., 1986). This results in lower energy requirements for the process and also enhanced yields of the xanthan. Recently, vegetable oil is used as the oil phase for overcoming the problems related with high broth viscosity in xanthan fermentations (Kuttuva et al., 2004). In addition to decreasing the viscosity of the reaction mixture as the concentration of xanthan gum therein increases, 0.2 to 0.8 v/v of oil used in the fermentation significantly increased oxygen transfer efficiency leading to an increased rate of reaction (Maury et al., 1982). The oil used has to be in liquid fraction at the temperature of fermentation. RBD palm-olein was in a liquid form at room temperature. This makes it suitable as the organic medium in the fermentation. On this basis, RBD palm-olein was selected to be used in this research to solve the problem of high viscosity of xanthan gum which brings serious problems to mixing, heat transfer, and oxygen supply.
Oils are primarily having antifoaming properties as well as nutritional particularities. It also considered essential components of many fermentation media and serve as a supplemental nutrient source for growth which is important for the maintenance of the microbial cells (Sena et al., 2006). Lipases are activated only when adsorbed to an oilââ‚¬"water interface. It does not hydrolyze dissolved substrates in the bulk fluid. It will split emulsified esters of glycerine and long-chain fatty acids such as triolein and tripalmitin. Lipases also display little activity in aqueous solutions containing soluble substrates. Bacteria, fungi, yeasts, and actinomyces are lipase-producing microorganisms (Sharma et al., 2001). Lipase enzyme released by cells will hydrolyze fats and oils to their basic components, fatty acids and glycerol. Cells might utilize these components as secondary carbon source. Therefore, in order to identify the potential of X. campestris to consume oil as secondary carbon source, a study on the lipase activity in the production of xanthan gum with addition of oil was determined.
Objectives of Research
The objectives of the present work are as follows:
To study the effect of fresh and recycled palm-oil in formulating growth medium on the xanthan production and growth of Xanthomonas campestris NRRL B-1459 (ATCC 13951).
To study the effect of palm-oil on the viscosity and rheological behavior of xanthan gum produced.
To identify the utilization of palm-oil as a secondary carbon source by the cells based on the substrate consumption and lipase activities in the fermentation.
To study the effect of different impellers design on xanthan gum production with palm-oil based growth medium.
1.4 Scope of Research
The research was conducted within the following range of operating conditions:
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As per literature review, the optimum temperature and pH for the production of xanthan gum was found to be 28oC and 7, respectively. The focus of this study was not on optimizing the operating condition, therefore, the optimum temperature and pH was chosen to run the fermentations.
In water-in-oil technology, more than 0.5 v/v oil was used. It was proved that this technology enhanced the production of xanthan gum and could overcome the problems related with high broth viscosity in xanthan production. However, there was no study basically on the oil-in-water technology in enhancing xanthan production. Therefore, this research approached oil-in-water technology by using RBD palm-olein (CP10) as the organic phase at oil fractions of 5, 15, 25, 35 and 50% (v/v).
InterMIG impeller is suitable in mixing the highly viscous fluid such as xanthan gum and Rushton impeller is common impeller use in the fermentation. On this basis, these two types of impeller were installed in order to study their effect on xanthan production when different fractions of oil were added in the fermentation broth. The agitation speed was set to 400 rpm and the aeration rate was 0.5vvm to compare the efficiency between these two impellers. The effect of increasing the agitation and aeration to a certain rate was also studied.
1.5 Thesis Overview
The characteristic of xanthan gum and palm oil were further discussed in the literature review which is in Chapter 2. The literature also included on the study of lipase enzymes and the relevant research which used oil as one of the component in the media. The following chapters of this thesis elaborate on the research methodology. In Chapter 3, all the method applied in the research including the bacteria cultivation was thoroughly explained. The bacteria were subcultured every 2 weeks. The inoculum development for bioreactor studies was made from this culture and incubated at 28oC for 48 hours. Then, the effects of different fraction of palm oil in the fermentation range from 0.05 to 0.5 v/v oil were investigated. The lipase activity in the xanthan fermentation in the shake flask and bioreactor was also studied.
In Chapter 4, the effects of palm oil the cells growth, glucose consumption and xanthan production in the shake flask and bioreactor studies were discussed. The potential of palm oil as carbon source was also proven from the results obtained. The lipase activity in the fermentation also managed to present in this chapter. The rheological behavior of xanthan gum obtained in the fermentation also presented in this chapter. In Chapter 5, all the result discussed in the previous chapter was concluded and recommendations were made based on the results obtained. These recommendations could be beneficial if a study on the similar area is planning to be done in the future.