Abstract Citric acid production from cane molasses using free and immobilized cells of manganese resistant mutant of Aspergillus niger (Mn II) has been studied. The optimum conditions for citric acid production with free cells of A. niger (Mn II) using cane molasses were: sugar concentration, 10%; inoculum level, 10%; NH4NO3, 0.3%; K2HPO4, 0.1%; MgSO4.7H20, 0.03%; starch, 0.3%; pH, 2 at 28oC in 12 days which were comparable with that of standard medium. The Ca-alginate immobilized A. niger (Mn II) cells gave the optimum yield at 30oC with 14% inoculum level in 14 days. Submerged fermentation process (95 KgM-3) was found to give more yields as compared to surface fermentation (76 KgM-3). The used immobilized A. niger (Mn II) cells were reutilized for citric acid production using molasses as substrate under optimized conditions using submerged fermentation process and the substantial yield (47 KgM-3) obtained after reutilization was about 50% as that of first usage. The results obtained are very encouraging and thus, indicates the great potential for cost effective conversion of molasses, a bye product, into an organic acid of high market value.
Keywords- Aspergillus niger, citric acid, fermentation, immobilized cells, molasses
Citric acid is a well known industrially important organic acid being utilized immensely in medicine, dairy, pharmaceutical and biochemical industries. Various agrowastes [1, 2] have been used for of citric acid, Aspergillus niger being most commonly used as microbial source [3, 4]. Starchy and sugar substrates have long been used for citric acid production in the presence of A. niger due to its efficient enzymatic system [5, 6, 7,8]. Many strains of A. niger have been used for good yield of citric acid such as Aspergillus niger ATCC9142, the hyper productivity of citric acid is also studied by using mutant GCBT7 [9, 10, 11].
Commercially, citric acid is produced by fermentation method. For fermentation processes, substrates as molasses , orange peel with molasses , jackfruit with molasses , sucrose, whey, corn syrup, pumpkin , enzyme- treated starch and normal paraffin  have been used. The surface or submerged fermentation by mold (A. niger)  and submerged fermentation by yeast (Candida guilliermondii, C. lipolytica)  or by Candida oleophila  are also studied. The production of citric acid by using batch system with Yarrowia lipolytica strain also in consideration . Under static conditions as solid state fermentation by naturally occurring A. niger MAF3 is used for citric acid production from industrial solid potato waste . The commercial baker's yeast Saccharomyces cervisieae is also used for the production of citric acid by utilizing potato processing wastes . According to other studies the production of citric acid from apple pomace by using surface culture medium by using A. niger also been reported [23, 24].
For economical development, citric acid is produced by using cheap raw material because it is highly used in food and pharmaceutical industries. The main focus is to utilize molasses, a cheap byproduct of sugar industry, as potential sugar substrate along with optimization of various variables for citric acid production using A. niger. The major hindrance in accomplishing this objective is the presence of large amount of manganese in cane molasses which inhibits the production of citric acid , although there has been report of contrary results also .
The main objective of the present investigation is the comparison and optimization of various parameters for citric acid production from molasses by utilizing free and immobilized cells  of high manganese resistant mutant strain of A. niger (Mn II).
2. Materials and methods
2.1 Culture maintenance
A. niger strain (Mn II) which had been isolated, mutanized and maintained in our laboratory was used in this study. The culture has previously been developed by treatment of wild A. niger by ultraviolet irradiation. The culture of Mn II was maintained on sterilized potato dextrose agar medium (Diced potato 200 g/l, Dextrose 20 g/l and Agar 15 g/l) and stored at 4C in the refrigerator.
2.2 Molasses pre-treatment
Cane molasses was procured from Shamli sugar mill, U. P. Cane molasses contains water 20%, non-sugar contents 10%, sugar contents 62%, and inorganic salts (ash contents) 8%, making it a high viscosity and blackish homogenous liquid. Some ions as Mg, Mn, Al, Fe and Zn are present in ash content in variable ratio . Molasses was pre-treatment by diluting the sugar concentration to about 25% level. After adding 35 ml of 1N H2SO4 per litre, the molasses solution was boiled for half an hour, cooled, neutralized with lime-water (CaO) and was left to stand overnight to make clear solution . The clear supernatant liquid was finally diluted to 15% sugar level.
2.3 Optimization of parameters for citric acid production using molasses as medium
Molasses was used as fermentation medium for citric acid production and compared with the conditions using standard medium. The pH of the medium was adjusted to 2.0 with 2 M HCl and sterilized by autoclaving at 121oC, 15 psi for 15minutes. The components for the standard growth medium were: NH4NO3, 0.3%; K2HPO4, 0.1%; MgSO4.7H20, 0.03%; starch, 0.3%. Effects of concentration level of NH4NO3, K2HPO4, MgSO4.7H2O, starch, inoculum and physical parameters like pH, fermentation time and temperature were optimized for molasses and compared with the standard medium.
2.4 Fermentation methods for citric acid production
2.4.1 Submerged fermentation process: Pretreated molasses being used as medium was inoculated with Mn II cells and incubated at 30o C for 10-12 days in aerobic conditions on shaker. After completion of fermentation, the mycelium was separated from the broth after autoclaving, and the citric acid produced was quantified by standard assay.
2.4.2 Surface fermentation process: In this process, the flasks containing inoculated media were kept undisturbed at 30oC in an incubator. Air pump was used to circulate air over the mycelial mat formed on the surface of medium. After the desired time period, the citric acid produced was estimated after autoclaving and filtration of fermentation broth.
2.5 Calcium alginate entrapment of Mn II cells
The subcultured Mn II cells were incubated at 30oC for 4-5 days till the completion of sporulation.The spores were then aseptically harvested in sterile distilled water (107-108 spores/ml). Further sterilized sodium alginate solution (8%) was added to the spore suspension. The contents were mixed thoroughly and the mixture was poured, drop by drop, into 5% sterilized solution of CaCl2 solution under aseptic conditions. The beads thus formed, washed thoroughly and stored in 0.95% NaCl in refrigerator.
2.6 Reuse of Ca-alginate immobilized cells of Mn II for citric acid production
The Ca-alginate beads with entrapped Mn II cells were reutilized for citric acid production.
2.7 Citric acid Estimation
Citric acid was estimated by Marrier and Boulet (1958)  method.
2.8 Product recovery
The classical calcium hydroxide precipitation method  was used to recover citric acid.
3. RESULTS AND DISCUSSION
3.1 Comparative status of optimum conditions for the production of citric acid using molasses by free cells Mn II and standard medium. (Table 1.)
TABLE 1. Parameters for citric acid production by Mn II using standard medium and molasses
PARAMETERS STANDARD MEDIUM MOLASSES MEDIUM
Inoculum level 10% (v/v) 10% (v/v)
Sugar concentration 10% (w/v) 10% (w/v)
Fermentation time 12-14 days 10-12 days
Starch concentration 0.20% (w/v) 0.20% (w/v)
NH4NO3 0.30% (w/v) 0.30% (w/v)
K2HPO4 0.10% (w/v) 0.10% (w/v)
MgSO4.7H2O 0.03% (w/v) 0.03% (w/v)
pH 2.0 2.0
Temperature 28oC 28oC
3.2 Effect on fermentation time on citric acid production
The results (Fig. 1) show that in the case of Ca-alginate immobilized Mn II cells, maximum yield of citric acid was obtained in 14 days after a lag phase 5 days in contrary to shorter span (2 days) in free cells indicating the time requirement for immobilized cells for proper maturation and acclimatization. In free state Mn II cells took nearly 10-12 days for the completion of fermentation. The immobilized cells may be used for the continuous production of citric acid by adjusting the dilution rate and the optimum immobilized cell mass. Sigmoid shaped graph was obtained with a lag phase of 3-4 days followed by a log phase of 5-8 days, and then a stationary phase upto 18 days. The standard medium gave slightly better yield.
3.3 Effect of temperature on citric acid production
A temperature of 30oC was found to be optimum in case of immobilized Mn II cells which was slightly higher than the optimum temperature (28oC) obtained with free cells (Fig. 2).
3.4 Effect of inoculum level of immobilized and free cells of Mn II on citric acid production
The results (Fig. 3) indicate 14% inoculum level to be optimum for the acid production in case of immobilized Mn II cells, whereas the corresponding value for free cells was 10%.
3.5 Citric acid production by reuse of Ca-alginate immobilized Mn II cells
The Ca-alginate immobilized Mn II cells were reutilized for citric acid production using molasses as substrate under the same optimized conditions in submerged fermentation. The yield (47 KgM-3) obtained was about 50% as that of first usage, which is quite substantial.
3.6 Citric acid production in submerged and surface fermentation systems
Fig. 4 clearly indicates that the submerged fermentation (95 KgM-3) process gave better citric acid yield as compared to surface fermentation (76 KgM-3) process.
A manganese resistant mutant  of A. niger (Mn II) both, in free and immobilized state has been used for the production of citric acid from molasses in the present investigation. It was observed that the citric acid production was affected by various factors viz; substrate (sugar) concentration, nitrogen source, phosphorus source and trace elements along with some physical factors such as pH, temperature, and fermentation time. According to the results optimum conditions for citric acid production were: sugar concentration, 10%; inoculum level, 10%; NH4NO3, 0.3%; K2HPO4, 0.1%; MgSO4.7H20, 0.03%; starch, 0.3%; pH, 2 at 28oC in 12 days which were compared with that of standard medium.
The Ca-alginate immobilized cells gave the optimum yield at slightly higher temperature (30oC) with 14% inoculum level. Immobilization of Aspergillus niger cells enhanced the production of citric acid . Submerged fermentation process (95 KgM-3) was found to give better yield as compared to surface fermentation (76 KgM-3). The used immobilized Mn II cells were reutilized under optimized conditions and gave yield upto 47 KgM-3 which was about 50% as that of first usage. The results obtained are promising, as a byproduct (molasses) of sugar cane industry has been converted into high cost organic (citric) acid with high yielding manganese resistant mutant A. niger (Mn II), thus, making the process cost effective with good economical feasibility at industrial scale.
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Fig. 1. Effect of fermentation time on citric acid production.
Fig. 2. Effect of temperature on citric acid production.
Fig. 3. Effect of inoculum level of immobilized and free cells of Mn II on citric acid production.
Fig. 4. Citric acid production in submerged and surface fermentation systems.
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