Development of Aspergillus NIGER Strain for Citric ACID Fermentation of Molasses

Abstract

Two hundred and fifty isolates of Aspergillus Niger were obtained from soil samples collected from different areas by pour – plate – method by using malt extract agar medium. The agar plates were incubated at 30 °C for 2 – 3 days. The black spores from individual colonies were picked up and transferred to potato dextrose agar slants for cultural maintenance. The cultures were incubated at 30 °C for 3-4 days for maximum sporulation and were stored in the refrigerator at 10°C for maintenance and screening for citric acid fermentation. The isolates were again propagated in the petriplates on Czapek Dox agar medium containing bromocresol green dye as an indicator. The fungal colonies that produce yellowish zone (0.5 – 1.5 cm dia.) due to citric acid production were further picked up on potato dextrose agar slants for citric acid fermentation in shake flasks. Citric acid fermentation in molasses medium (15%, w/v sugar) containing ferrocyanide (200 mg/L) was carried out in 1L cotton wool plugged conical flasks containing 100 ml fermentation medium at °C for seven days, using ninety-one isolates of A. niger independently in shake flasks. The flasks were shaken on a rotary shaker at 200 rpm. Of all the isolates, only two were found to be highly producers of citric acid i.e., 28.0-31.0 g/L. These were designated as GCB-13 and GCB-57. Aspergillus niger GCB-57 was further improved by treating the spore suspension by the UV irradiation for different time periods (15-180 min.) Fifty-eight isolates were picked up and examined for citric acid biosynthesis. Among the isolates examined, GCM-45 was found to be best producer of citric acid (49.5 g/l). Aspergillus niger GCM-45 strain was further improved y chemical treatment (NTG) of mycelial suspension (24 hours old) in Vogel medium contained in 1 liter shake flask. The mycelium was exposed to the chemical mutagen for different time periods (5-130 min.). Fifty isolates were picked up by dye method and screened for citric acid production. GCMC-7 and GCMC-14 strains produced maximum citric acid i.e., 80g/L and 73.5 g/L respectively, after seven days of ferrocyanide pretreated molasses medium (200 mg/L). The improvement of Aspergillus niger GCMC-7 strain was further attempted by giving alternate treatment of UV and chemical as above. Forty isolates were again picked up for citric acid production. The maximum citric acid production was obtained by strain No. 14 (58.6 g/l). It was lesser than that produced by the parent strain. The A. niger GCMC-7 strain, the best producer, has been preserved for exploitation for citric acid production in shake flask and stirred fermenter.