MATERIAL AND METHODS

REVIEW OF LITERATURE

Han and Woodams (2002) investigated Corn husks could serve as a potential substrate for the production of citric acid by Aspergillus niger NRRL 2001. Combined treatments of corn husks with dilute NaOH and Rapidase Pomaliq (a commercial apple juice processing enzyme preparation) significantly enhanced the yield of citric acid. Under favorable conditions (pretreated with 0.5 mol/L NaOH, followed by 120 h of fermentation at 30°C in the presence of Rapidase Pomaliq), the yield of citric acid was 259±10 g per kg of dry matter of corn husks.

Rodriguez and Sanroman (2004) invested that Solid-sate fermentation (SSF) has received new interest not only from researchers but also from industry. This technique has become a very attractive and alternative to submerged (SmF) for specific application due to the recent improvements in reactor designs. .

Kim et al (2006) investigated that citric acid is an effective remediation agent for soils contaminated by heavy metals. The large-scale field use of this organic acid requires the development of efficient production techniques using low cost substrates such as sugar rich wastes. To develop such techniques, the objective was to simultaneously optimize the initial glucose, nitrogen (N), phosphorus (P) and NaCl levels of a nutrient solution used to wet peat moss (PM) simulating a sugar rich waste on which Aspergillus Niger NRRL 567 was grown to produce citric acid. Seventeen different combinations of nutrients were tested to grow A. Niger at 30 °C for 48 and 72 h, and to measure the resulting citric acid production. With the central composite design method (CCD), the results were used to produce a second order equation defining citric acid production as a function of initial glucose, N, P and NaCl levels. Initial glucose and N levels were found to have a significant positive and negative effect on citric acid production, while P and NaCl were found to have a limited positive and an insignificant effect, respectively. A peak citric acid production of 82 g kg−1 dry peat moss (DPM) was reached after 72 h with the following optimized nutrient solution, in terms of g kg−1 DPM: 967.9 glucose, 15.4 (NH4)2SO4, 43.9 KH2PO4 and 4.0 NaCl. This represented a 50% increase in production, over a shorter fermentation period, compared to a basal nutrient solution optimize solely for initial glucose level.

MATERIAL AND METHODS

Microorganism

Aspergillus niger carrier will be grown on potato-dextrose-agar 

Medium. The slant will be incubated at 28oC for seven days and preserved at 4oC for two months.

Substrate

The corncobs will be obtained from the University of the Agriculture Faisalabad, will be dried in oven; cut into small pieces and will be sieved to obtain fractions between 0.8-2.0 mm particle size.

Inoculum preparations

. Spores of A. niger was produced by growing the fungus in inoculumfor 10 days. The inoculum media will be containing (g/l)ZnSo4.7H2O 0.2and FeCl3.H2O 0.014 was sterilized at 121oC for 15 min. After cooling methanol will be added .Solid substrate were inoculated with suspension, mix with nutrient medium. The medium will be adjusted at pH 4.5 and autoclave at (121oC) for fifteen minutes. A loopful culture of A. niger will be transferred to sterilized medium to get maximum growth.

Experimental Procedure

Experiment will be performed using 250 ml Erlenmeyer flasks in a temperature-controlled incubator. Flasks will be prepared in Duplicate. Each flask will be sterilized and autoclave at (121oC) for fifteen minutes and will be in kept at 30 oC for different time periods. Sample will be harvested after 24, 48, 72hours

Optimization of conditions

Effect of tempratur

The duplicate flask will be run at different temprature viz 20,25,30,35oC. The medium with maximum citric acid production will be choosen for further study.

Effect of pH

The duplicate flask will be run at different pH 3,3.5,4.0,4.5,5.0. The medium with maximum citric acid production will be choosen for further study.

Effect of nitrogen sources

Different nitrogen sources such as ammonium sulphate, yeast extract, peptone, corn steep liquor and urea will be used under preoptimized conditions for high citric acid production.

Effect of inoculum size

To check which inoculum is best for high production of citric acid, follow range of inoculum size 2ml-6ml will be used under preoptimized conditions for high citric acid production.

Analytical Methods

After completion of fermentation, the myclium will be removed by filtration and the filterate obtained will be treated with milk of Lime Ca(OH)2 at 80-90oC. Calcium citrate will be precipitated and precipitate were separated by filtration. The residue was washed with boiling distilled water. The precipitates from filter paper were transferred to beaker and treated with transferred to beaker and treated with equal amount of H2SO4. The Ca(SO4)2 formed was separated by filtration filterate obtained was concentrated by water bath heating.

Crystals of citric acid will be collected and percentage of citric acid formed was determined.

Statistical Analysis

The data collected was subjected to analysis of variance and treatment means will be compared by Duncan’s Multiple Range Test

RESULTS AND DISCUSSION

Aspergillus Niger was cultured on corn cobs for the production of citric acid. Effect of different parameters like fermentation period, substrate water ratio, ph, nitrogen sources viz, urea, (NH4)2 SO4, peptone, corn steep liquor and yeast extract and Agitation of citric acid production was studies on laboratory scale.The results obtained during the investigations are discussed here under.

Five different growth media adjusted at different levels of PH of i.e. 3, 4, 5, 6 and 7 were used in the experimental study. For each treatment, duplicate flasks of growth media containing 1.0% corn cobs were used. All the samples were harvested after 72 hours incubation and subjected to analysis for citric acid content.

Citric acid was estimated gravimetrically, using pyridine–acetic anhydride method as reported by Marrier, J.R. and Boulet, M., 1958. Direct determination of citric acid in milk with an improved, pyridine acetic anhydride method. J. Dairy Sci. 41, p. 1683.Marrier and Boulet .One ml of the diluted culture filtrate along with 1.30 ml of pyridine was added in the test tube and swirled briskly. Then 5.70 ml of acetic anhydride was added in the test tube. The test tube was placed in a water bath at 32 ± 0.25 °C for 30 min. The optical density was measured on a spectrophotometer (405 nm) and citric acid contents of the sample were estimated with reference (run parallel, replacing 1.0 ml of the culture filtrate with distilled water) to the standard. The % of citric acid was determined on the basis of sugar used

It was found that maximum citric acid production was observed at Ph 6 while Ph 3 showed comparatively less citric acid production . Graph was plotted between ph of media and citric acid (mg/ml) production, which showed lower citric acid production at ph 3 and 7 than all other levels .

Analysis of variance of the date showed significant (P> 0.05) difference in the production of citric acid with different ph levels tested . Comparison of treatment means by DMR-test showed significant difference between all treatments. Control and ph 6 differed non-significantly. The media with ph 3 and ph 7 also showed non-significant difference between their treatment means.

Among the factors that determine morphology and the general course of fungal fermentations, the type and size of inoculum is of prime importance. Five different growth media adjusted at different inoculum size of of i.e. 3, 4, 5, 6 and 7ml were used in the experimental study at pH 6. For each treatment, duplicate flasks of growth media containing 1.0% corn cobs were used. All the samples were harvested after 72 hours incubation and subjected to analysis for citric acid content.

The temperature of fermentation medium is one of the critical factors that have a profound effect on the production of citric acid. Five different growth media adjusted at different incubation temprature of i.e. 25, 30, 35, 40 and 45ºC were used in the experimental study at pH 6 and 5ml of inoculum size.

Different nitrogen sources (0.2%) were added into the growth medium for maximum production of citric acid. These included urea, (NH4)2 SO4, peptone, corn steep liquor and yeast extract. Duplicate flasks of growth media containing corn cobs adjusted at ph 6,incubation temprature 35ºC and 5ml of inoculum size were fermented for 72 hours. The control without any additional nitrogen source was also proceeded under similar conditions. Maximum production of citric acid was observed in the medium containing 0.4% corn steep liquore as nitrogen source but urea also has significant effect. The other nitrogen sources having same concentration gave less production of citric acid.