1. Introduction This survey is about fermentation technology.

Over the past ten years we have seen considerable growth in biotechnology. The use of filamentous fungi for the production of commercially important metabolites has increased rapidly over the past half century and the production of enzymes in submerged fermentation has long been established. Aspergillus niger is capable of producing much kind of acid like citric acid and gluconic acid. Through the fermentation process, Aspergillus niger can produced citric acid in both surface and submerged culture. Since this project is subject to fermentation in shake flask, submerged culture is used. Production in submerged culture is now the favoured process. In submerged fermentation, agitation is important for adequate mixing, mass transfer and heat transfer. The effects of mixing and mass transfer can be important in order to observe the morphological of fungi and to determine the viscosity. Inoculum morphology has been studied and correlated with process yields for a number of important submerged fermentation processes. The influence of agitation on growth rate is complicated by its other effects on mixing and oxygen transfer. The isolated study of shear on growth rate and biomass production is difficult because of effects like increased oxygen transfer and better mixing also brought about by agitation. The accumulation of citric acid is strongly influenced by the composition of the fermentation medium, particularly in the submerged process.

2. Objectives Two objectives of this project are: 1 To study the influence of agitation speed on Aspergillus niger fermentation broth in shake flask 2 To observe the viscosity and cell morphological of Aspergillus niger at different agitation speed.

3. Literature Review 3.1 History of citric acid Citric acid derives its name from the Latin citrus, the citron tree, the fruit of which resembles a lemon. The acid was first isolated from lemon juice in 1784 by Carl Scheele, a Swedish chemist (1742-1786), who made a number of discoveries important to advance of chemistry. Citric acid was thus one amongst many natural organic acids. Citric acid was produced commercially from Italian lemons from about 1826 in England by John and Edmund Sturge, but with the increasing importance of citric acid as an item of commerce, production was started in Italy by lemon growers, who establish a virtual monopoly during the rest of the nineteenth century. Lemon juice remained the commercial source of citric acid until 1919 when the first industrial process using Aspergillus niger began in Belgium (Kristiansen et al, 1999). 3.2 Application of Citric Acid Citric acid is used in food, confectionery and beverages, in pharmaceuticals and in industrial fields. Its uses depend on three properties: acidity, flavour, and salt formation. It also is being used for metal complexion, buffering, and antioxidant and sequestering. 3.3 Aspergillus niger Aspergillus niger is capable of producing very high levels of citric acid (about 70% of the theoretical yield from carbohydrate source) because this organism is very efficient in metabolizing carbon (A. Bodie et al, 1994). Although the production of citric acid by fermentation using Aspergillus niger has been a commercial process since 1923 (Prescott & Dunn, 1959), there still remains many gaps in our knowledge and understanding of the fermentation. (Berry et al, 1977).

3.4 Fermentation Broth Mycelia broths are used in the pharmaceutical industry in the production of penicillin, citric acid, amylase, modified steroids, and other products. Broth of filamentous mycelia organisms are known to exhibit highly viscous shear-thinning behavior as the biomass concentration increases (Shivla, et al, 1995). 3.5 Viscosity Filamentous suspensions of Aspergillus niger exhibited apparent viscosities three times greater than pelletal suspensions for similar biomass concentrations and that broth of filamentous organisms with short, thin filaments exhibited lower yield stresses than did broths of mycelia organisms with longer hyphae. High viscosity tends to promote bubble coalescence, reducing the dispersion of gas (Shivla et al, 1995) 3.6 Morphology Ujcova, et al (1980) reported that oxygen limitation occurred at low speeds, but there was also a drop in productivity at higher speeds, although growth remains rapid. Large morphological changes were observed at higher speeds, resulting in thicker, twisted and densely branched hypae. A maximum citric acid production level is reached in a diffusely growing mycelium: the production level being dependent on the agitation speed. 3.7 Agitation speed The changes in the specific rate of citric acid formation observed by Chmiel (1977) resulted from inhibition of its synthesis caused by a decrease in the level of dissolved oxygen at high mycelia densities. Kwapisz (1976) maintained the dissolved oxygen level at 60% of saturation by increasing the air flow rate and the agitation speed. 4. Material and Method

4.1 Preparation of Maintenance Media for Spore Development 9.76 g Potato Dextrose Agar (PDA) was dissolved in 250 ml of distilled water. It then was boiled to dissolve completely. The agar then was poured into test tube with 5 ml each. The test tube was covered with cotton wool and aluminum foil and gum with masking tape. Then it was sterilized by autoclaving. The test tubes were taking out before agar become a solid and allowed it to sit on the table in a slanted way.

4.2 Microorganism Aspergillus niger strains that obtained from Biochemical Engineering Laboratory, Faculty of Engineering, University of Putra Malaysia was used through out this work. Stock culture was maintained on PDA slants. This stock was kept in oven incubator for five days with temperature at 30oC in order to grow the fungi. 4.3 Preparation of fermentation media Shake flask experiments were performed using 150ml of sucrose medium contained in 250ml conical flasks. The fermentation media described in table below: Component Sucrose KH2PO4 MgSO4.7H2O Fe2+a Zn2+b Cu2+c pH
a b

Composition (g/L) 140 1.00 0.25 1.510-4 1.010-4 1.010-5 2.6

Fe2(SO4)2.7H2O ZnSO4.7H2O

CuSO4.5H2O

4.4 Inoculum To prepare for an experiment, a fresh well-grown culture was selected. Spores were washed from agar surface with10 ml sterilized distilled water. The spores then were transferred into sterilized bottle and were kept in a freezer at 4oC. 4.5 Cultivation The suspension was then poured into a 250ml flask containing sterile growth media. The flasks containing approximately 7.2106 of spores were incubated at 30oC on a mechanical incubator shaker at an operating speed at different speed (150rpm, 180rpm, and 210rpm). The fermentation was carried out for 12 days. 5ml samples were drawn at different time intervals from first day of fermentation until day 12. These samples were analyzed for citric acid concentration, viscosity and cell morphology. 4.6 Analytical Methods 4.6.1 Determination of viscosity Viscosity was measured afterwards with a portable viscometer model Helipath with T-Bar Spindles at 30 C which was the fermentation temperature. 4.6.2 Morphology Analysis Morphology was characterized by using a Leica/Q-Win, Q500 1W image analyzer which produced the digitized image from a microscope, on which was mounted with Sony CCD video camera, under magnification of 5. References

Bjrn Kristiansen, Michael Mattey, Joan Linden, 1999.A Brief Introduction to Citric Acid Biotechnology. Citric Acid Biotechnology.1 Elizabeth A. Bodie, Ben Bower, Randy M. Berka, Nigel S. Dunn-Coleman, 1994. Economically Important Organic Acid and Enzyme Products. Aspergillus:50 years on.563 Prescott, S.D. & Dunn, C.G, 1959. Industrial Microbiology D.R. Berry, A. Chmiel and Z. Al Obaidi, 1977. Citric Acid Production by Aspergillus niger. Genetic and Physiology of Aspergillus.405 C. Kurt Shivla, Sundeep N. Dronawat and Thomas R. Hanley, 1995. Steady State Shear Characteristics of Aspergillus niger Broths. Applied Biochemistry and Biotechnology. 51/52, 355-366. E. Ujcova, Z Fenci, M. Musilkova and L. Seichert, 1980. Biotechnology and Bioengineering. 22, 237. Chmiel, A, 1977. Kinetics of Citric Acid Production by Pre-cultivated mycelium of Aspergillus niger. Transactions of the British Mycological Society. Kwapisz, E, 1976. Dr. Thesis, Technical University of Lodz, Poland.