STUDIES ON THE SUBMERGED FERMENTATION OF CITRIC ACID BY ASPERGILLUS NIGER IN STIRRED FERMENTOR

The present studies deal with the isolation, screening and selection of Aspergillus niger cultures for citric acid fermentation. Two hundred and fifty Aspergillus niger cultures were isolated from soil samples collected from different areas of Lahore District in sterile polythene bags. Pour plate method using malt extract agar medium was used for isolation. The agar plates were incubated at 30˚C for 2-3 days. The black conidia from individual colonies were picked up and transferred to potato dextrose agar (PDA) slants for culture maintenance. The cultures were incubated at 30˚C for 3-5 days until maximum sporulation and were then stored in a refrigerator at 4˚C for maintenance and further screening for citric acid fermentation. The selected culture was improved through ultraviolet (UV) radiations and chemical treatment by N-methyl N-nitroso N-nitroso guanidine (MNNG). The cultural conditions and nutritional requirements for citric acid production by the selected culture were optimized in 250 ml Erlenmeyer flasks by submerged mould culture technique prior to scale up studies in a stirred fermentor.\ud \ud The isolates were again propagated in the propagates on Czapek-Dox agar medium containing bromocresol green dye as an indicator. The fungal colonies that produced yellowish zones (0.5-1.5 cm dia) due to citric acid formation were picked up on the PDA slants. The fermentations using 200 ppm (w/v) ferrocyanide treated cane molasses medium (15.0 %, w/v sugar) were carried out in 250 ml cotton wool plugged conical flasks. Twenty-five milliliters of fermentation medium was used and thus 153 A. niger isolates were independently screened for citrate activity. The flasks were rotated in a shaking incubator (200 rpm) at 30˚C for 168 h. of all the isolates, A. niger GCI-79 which gave the highest citric acid production (25.20+0.66 g/l) was selected for mutation through UV irradiations. The conidial suspension of strain GCI-79 was exposed to the UV radiations (1.2x102 j/m2/S) for different time intervals (5-150min). mutants were picked up on the basis of yellow zones of citric acid from the plates having at least 90.0% death rate. One hundred and sixteen UV mutants were evaluated for citric acid biosynthesis. However, the mutant GCUV-47 gave maximum citric acid production i.e., 39.12+0.12g/l. this strain was selected after exposing the conidial suspension through UV radiations for 45 min.\ud \ud The mutant strain A. niger GCUV-47 was further improved through the chemical (MNNG) treatment of mycelial suspension (24 h old) in Vogel’s medium. Mycelial cells were exposed to the chemical mutagen (1.0-3.0mg/ml) for different time intervals (30-60min).Over 300 colonies from the plates having less than 10.0% survival rate were selected on the medium containing (w/v) oxgall 3.0% PDA 4.0%, agar 1.5 % and bromocresol green 1.0%. Out of the survivals, 91 MNNG treated mutants with larger yellow zones were selected for citric acid fermentation. Two mutant strains GCNG-7 and GCNG-41 produced relatively higher amount of citric acid i.e., 51.86+0.12 and 50.40+0.87 g/l, respectively. The best mutant strain A. niger GCNG-7 obtained after the 2.5 mg/ml MNNG treatment for 30min has about twice the value for y p/s (i.e., 0.55+0.14 g/g) than the original parental strain GCI-79. The improvement of GCNG-7 strain was further attempted by the alternate UV and MNNG treatment. Thirty-four A. niger mutants were picked up 45 min after the treatment. Of these, the mutant No. 3 gave maximum citric acid production (37.83+1.61 g/l), which is 1.37 fold lower than its parental strain. So, the mutant strain A. niger GCNG-7, a hyper citric acid producer was selected for its exploitation for the citric acid production in shake flask and stirred fermentor.\ud \ud All optimizations of the process parameters were carried out in 250 ml shake flasks using blackstrap sugarcane molasses, and easily and abundantly available carbon source by-product of sugar industries. To reduce the trace metal contents in molasses, 200 ppm (w/v) ferrocyanide ions using K4Fe(CN)6 as a source were added into the medium under hot conditions before inoculation. This ferrocyanide pre-treated molasses medium resulted in 2.38 folds higher citric acid production the control, which is a highly significant value (LSD=0.314+0.02). The fermentations were carried out at 30˚C following growth of A. niger GCNG-7 in a rotary incubator shaker (200 rpm). In the present investigation, different cultural conditions such as sugar concentration (150 g/l), rate of citric acid biosynthesis (168 h), initial pH (6.0), volume of fermentation medium (10.0% and conidial inoculum (4.0% v/v, 1.2x106 Conidia/ml) were optimized. After 168 h of fermentation, the mutant GCNG-7 showed the highest value for qp i.e.,0.0537+0.03 g/g/h which is 2.17 folds improved over the original parental strain. To stimulate citric acid production, 1.0% (v/v) methanol was added into the fermentation medium 24 h after inoculation. The addition of 0.25 % N (w/v) with NH4 NO3 0.15% P(w/v) with K2 HPO4 and 0.020% Mg(w/v) with MgSo4 7H2O were found optimal for the enhanced citric acid production. The use of organic nitrogen sources, however, gave significantly less citric acid production than the control at all levels. So, their addition was not continued in the subsequent experiments.\ud \ud In the present study, 0.02% Ca (w/v) using CaCl2 and 0.035% (w/v) vermiculite were added during medium preparation to achieve the desirable mycelial morphology and subsequent citric acid production, In all the product formation kinetic parameters such as Yp/s,Qp and qp showed higher values for the mutant strain A. niger GCNG-7 compared to the parental as well as other mutant strains at all rates. The re-use of fungal mycelium was also undertaken with no encouraging results except the results of 1st batch (74.10+1.93 f/l citric acid) when 20.0 g/l mycelia were added into the medium. Citric acid production was however, markedly decreased in the re-use of fungal mycelium for 2nd and 3rd batches i.e., 65.45+3.05 and 33.80+ 1.56 g/l, respectively. It may be due to the increased mycelial age and decreased efficiency of metabolic pathways. The maximum amount of citric acid produced during the shake flask studies was recorded to be 75.52+1.02 g/l (88.08% yield) by the mutant GCNG-7. The consumption of sugar was 86.33+1.17 g/l with small round pellets of mycelium. However, the dry cell mass was 13.73+1.55g/l.\ud \ud The development of a microbial process for the formation of citric acid is aimed at maximizing three things; the yield of product per gram of substrate, the concentration of product and the rate of product formation. To compare cane molasses with sucrose salt medium in terms of citric acid productivity, 84 best stock cultures of A. niger (isolates and mutants) taken randomly from each category were screened in 250 ml shake flasks using sucrose salt medium. The mutant strain A. niger GCNG-31 was found to be the best citric acid producer (14.83+0.48 g/l). on the basis of kinetic relations, it was found that the mutant GCNG-31 has greater ability to consume the substrate ad faster growth rate, which finally increased the actual citric acid productivity rate. The cultural conditions such as initial sugar concentration (150 g/l), rate of fermentation (192 h), initial pH (3.0) and NH4 NO3 (3.0% N, w/v), K2HPO4 (2.0% P,w/v) and MgSO4 7H2O (0.20 % Mg, w/v) were optimized by the mutant GCNG-31. The highest citric acid production (42.88+1.22 g/l) was obtained when the ratio between K4Fe(CN)6 and EDTA was maintained at 100:150 ppm (w/v). The % citric acid yield was improved by 1.27 fold over the control (without the addition of metal complexing agents). Sugar consumption and dry cell mass were 93.93+1.90 and 15.73+0.64 g/l, respectively. The mycelial morphology was in the form of intermediate size round pellets.\ud \ud For scale up studies, a 15.0 L locally designed stainless steel stirred fermentor (working volume 9.0 L) was used for all microbial cultivations. Sterilized silicone oil (10.0 %, v/v) was used to control the foaming problems during fermentation. Out of the 9 different A. niger stock cultures, the mutant strain A. niger GCNG-7 was found to be the best citric acid producer (68.42 g/l in fermentor. Sugar consumption and dry cell mass were 98.5 and 14.0 g/l, respectively. It is the same strain (GCNG-7), which gave the highest citric acid production in shake flask technique, using molasses medium. However, citric acid production in stirred fermentor was 24.44 % (w/v) improved over the shake flasks due to a better agitation and aeration rate. On the basis of kinetic parameters, it was found that the mutant GCNG-7 showed many folds improved values for Qp,Yp/x, Yp/s and qp over other strains. The comparison of specific growth rate for citric acid fermentation shows that the mutant strain A. niger GCNG-7 using molasses medium as a basal substrate has higher values than the corresponding cultures with sucrose salt medium as a basal substrate has higher values than the corresponding cultures with sucrose salt medium. This reveals the ability of raw molasses sugars to support mycelial growth, which is a primary step for higher citric acid yields. Also the values for Yx/s (0.123 g cells/g) and Qx (0.090 g cells/l/h) are higher at 200 ppm (w/v) K4 Fe (CN)6 than the control. In the present study, maximum value for specific citric acid productivity is more encouraging than many of the previous workers.\ud \ud The different cultural conditions such as volume of fermentation medium (60.0%), incubation period (144 h), temperature (30˚C), initial pH (6.0), aeration (1.0 l/l min) and agitation (160 rpm)were employed to enhance citric acid yields. Twenty-four hours old vegetative mycelial inoculum was used at a level of 4.0%(v/v). As a stimulating agent 1.0% (v/v) methanol was added 24 h after inoculation. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from mycelial cells. Among the mineral nutrients tested (w/v), NH4NO3(0.20 % N),KH2PO4 (0.10% P) and MgSO4 7H2O (0.02 % Mg) were optimized as nitrogen, phosphorus and magnesium sources, respectively. Calcium chloride (0.04 % Ca w/v)and copper sulphate (0.02 % Cu, w/v) were found to affect the mould morphology and thus have a profound effect on citric acid production. In comparison o product formation kinetic parameters such as Qp, Yps,qp, the results obtained at 0.02% Cu (w/v) were much improved over the control on all Cu levels. The addition of copper into fermentation medium is proved to be highly encouraging. In this regard the most notable finding is the reduction in incubation period from 144 to 120 h at 0.02 % Cu (w/v) using CuSO4 in fermentor. The maximum citric acid yield was 97.63 % (108.86 g/l). Sugar consumption and dry cell mass were 111.5 and 14.5 g/l, respectively. Mixed mycelia were observed in the culture broth. The final ferrocyanide concentration was determined to be 72.5 ppm (w/v)and final pH was reduced to 1.4.\ud \ud The fed-batch culture study was also carried out and found to give consistent citric acid yields although net productivity was lower than the control. In the present investigation, the maximum amount of citric acid (94.02 g/l) was obtained 24 h after incubation when the sampling vs. fed-batch level was maintained at 2.0 L (v/v). the mycelia were small round pellets having dry weight 14.0 g/l. the amount of sugar consumed was 115 g/l while % citric acid on the basis of sugar consumed was 81.76 %. The final pH and K4 Fe(CN)6 concentration were 1.9 and 30.0 ppm (w/v), respectively. From the results, it is clear that in every trial of fed-batch culture (v/v), citric acid production remained all the way less than the control. However, the citric acid volumetric productivity rate (qp=1.306 g/l/h) was about 1.74 fold improved over the control, which shows that fed- batch culturing may be useful for improved citric acid yields in a shorter time period