Research Article
Selection of Fermentation for Citric Acid in Bioreactor
Government College University, Lahore, Pakistan
Sikander Ali
Government College University, Lahore, Pakistan
Ikram-ul -Haq
Government College University, Lahore, Pakistan
Citric acid (2-hydroxy propane-1, 2, 3-tricarboxylic acid) is a white solid at room temperature melts at 153°C (307oF) and decomposes at high temperature into other products (Srivasta and Kamal, 1979). It is the most important organic acid produced in tonnage and is extensively used in food and pharmaceutical industry. Because of its commercial and academic importance, the biosynthesis of citric acid by moulds has been a subject of numerous investigations (Mattey, 1992). Most of the citric acid produced commercially comes from Aspergillus niger (Fiedurek et al., 1996; Pera and Callieri, 1997; Maddox and Brooks, 1998). Carbon source is one of the factors that have been shown to exert an influence on citric acid fermentation (Papagianni et al., 1999). Various carbohydrate materials such as cane or beet molasses and crude infiltered starch hydrolysate may be used in citric acid production by Aspergillus niger in submerged fermentation (Haq et al., 1998). Workers have also investigated on the citric acid fermentation using sucrose salt media (Sanchez et al., 1970; Singh et al., 1998). However, the utilization of pure sugars as carbon source in fermentation media for large-scale is uneconomical (Islam et al., 1986). A number of reports have been published on the production of citric acid by submerged mould cultures using molasses and starch-based media (Vandenberghe et al., 1999; Kubicek and Roehr, 1977; Obaidi and Berry, 1979). Stainless steel fermentor of different working capacities can be employed for submerged citric acid fermentation (Sanjay and Sharma, 1994; Vergano et al., 1996; Rajoka et al., 1998). The present study is concerned with the selection of fermentation medium for citric acid fermentation a stirred tank reactor.
The experiments were conducted in Biotechnology Research Laboratory, Government College University Lahore, Pakistan during the year 2001. The cultures of Aspergillus niger were isolated from different soil samples of Lahore by serial dilution method (Clark et al., 1958). Twenty-five isolates were evaluated for citric acid bio-production (GCB-1 to GCB-25) and the best isolate (GCB-14) that produced 16.04 g L-1 was selected for further investigations. Cane molasses obtained from Kamalia Sugar Mills were clarified according to the method of Panda et al. (1984).
Fermentation conditions
Hundred ml of fermentation medium (clarified cane molasses; sugar 15%, pH=6.0) containing glass beads, in 250 ml cotton wool plugged Erlenmeyer flask was sterilized at 121°C for 15 min. One ml of conidial suspension (prepared in 10 ml of sterilized distilled water) was added. The flask was incubated at 30°C in a rotary incubator shaker at 200 rpm for 24 h. Stainless steel stirred fermentor of 15 l capacity (GLSC-AF-199-10, Pak made) with working volume of 9 l was used for fermentation. The fermentation medium and working vessel of the fermentor were sterilized at 121°C (15 lb inch-2) for 35 min using autoclave (GLSC-194-100). The vegetative inoculum was transferred at a level of 4% (v/v). K4Fe(CN)6 solution (200 ppm) was added during the time of inoculation. Agitation speed of the stirrer was kept at 200 rpm and aeration rate was maintained at 1.0 vvm. Sterilized silicone oil was used to control the foaming.
Assay methods
Sugar was estimated gravimetrically by DNS method (Tasun et al., 1970). Photoelectric colorimeter (Model: AE-11M Erma, Japan) was used for measuring colour intensity. Dry cell mass was determined by filtering the culture medium through weighed Whatmann filter paper No. 44. Mycelia were thoroughly washed with tap water and dried at 105°C for 2 h. Anhydrous citric acid was estimated colorimetrically, using pyridine-acetic anhydride method, as reported by Marrier and Boulet (1958).
In this study, cultures of Aspergillus niger isolated from various soil samples produced citric acid ranging from 0.24-16.04 g L-1 (Table 1). Cane molasses and sucrose salt media were evaluated for the basal fermentation media. The maximum amount of citric acid i.e. 55.43 g L-1 was produced using cane-molasses after 144 h of incubation (Fig. 1). The strain produced intermediate pellets when cane-molasses medium was used and it gave fine pellets when sucrose salt medium was utilized after 144 h of incubation (maximum citric acid production).
Table 1: | Screening of Aspergillus niger strains using molasses for citric acid production in 250 ml shake flask |
Initial sugar concentration 150 g L-1, incubation period 148 h, temperature 30°C, initial pH 6.0, ferrocyanide concentration 200 ppm, * On the basis of sugar used |
Table 2: | Comparison of the mycelial morphology of Aspergillus niger during citric acid fermentation using cane-molasses and sucrose salt media |
Fig. 1: | Comparison of citric acid production using cane-molasses and sucrose salt media Initial sugar concentration 150 g L-1, temperature 30°C, ferrocyanide concentration 200 ppm |
Fig. 2: | Comparison of sugar consumption using cane-molasses and sucrose salt media Initial sugar concentration 150 g L-1, temperature 30°C, ferrocyanide concentration 200 ppm |
Fig. 3: | Comparison of dry cell mass using cane-molasses and sucrose salt media Initial sugar concentration 150 g L-1, temperature 30°C, ferrocyanide concentration 200 ppm |
Fig. 2 shows the comparison of sugar consumption using cane molasses and sucrose salt media. Sugar consumption in case of cane molasses medium increases gradually with the incubation period. In the comparison of dry cell mass using the two media (Fig. 3) it increases continuously in both the cases but dry cell mass in case of molasses is more on the increase than that when sucrose salt medium was utilized as the fermentation medium. Ali et al. (2001) and Singh et al. (1998) used sucrose salt medium for citric acid fermentation and got good results. However, in this investigation, clarified cane molasses was optimized as the basal fermentation medium for subsequent studies. It was concluded that molasses medium as the best fermentation medium for enhanced and consistent yields of citric acid (Khan et al., 1970; Clark, 1962; Haq et al., 2001).