BIOCONVERSION OF MOLASSES TO CITRIC ACID

Abstract

India produces a huge quantity of sugarcane molasses every year as a by-product of the sugar industry, which can be used as a potential source ofsubstrate for the production of citric acid through fermentation using Aspergillus niger . However, to accomplish this, there are two major requirements that must be fulfilled. Firstly, the sugarcane molasses contains some inhibitory substances, including heavy metal ions (Fe , Cu , Zn and Mn2 +)which should be removed prior to using itas substrate for citric acid fermentation. Secondly, availability of a high citric acid yielding strain ofA niger which would be less susceptible to heavy metal toxicity. The first requirement was met by treating the diluted molasses, containing 12% sugars, with 0.1% hexacyanoferrate (HCF) at pH 4.5 and 70-90°C for 15min. After this pretreatment, molasses wasfound to be a better substrate for citric acid fermentation. For the second requirement, the wild typeA niger cells were subjected to a two-stage UV mutagenesis resulting in a high citric acid-yielding mutant (KCU520) ofA niger which was isolated and characterized. The mutant cells were found to be morphologically distinct. These were white in colour, short and thick with branches compared to yellow colour, thin and smooth structures of the wild type. The mutant cells were easily maintained in culture without any loss of efficiency in citric acid production. Optimum conditions for citric acid production byA niger KCU520 were as follows: pH, 4.5; temperature, 28°C; sugar concentration, 12% and fermentation period, 10 days. Under these conditions 60%, 75% and 80-85% sugars were converted to citric acid in submerged, surface and solid state surface fermentation (SSF) systems, respectively. Various regulators, such as 2% groundnut oil, 0.1% H2O2 or 0.3% starch in the fermentation medium were also found to enhance the citric acid yield by 5-10%. In the case of SSF, the microbial bed grown on the solid surface could be reused, at least in three subsequent fermentation cycles with fresh medium, with equal efficiency. In fact, in the 2nd and 3rd cycle, maximum yield was achieved in 5days. The SSF process was scaled-up to 10 liters without loss of efficiency. Similar results were obtained when Ca- alginate immobilized cells were used for fermentation. It was found that A niger KCU520 cells requie a 6day acclimatization period after which these can be used repeatedly in 4 fermentation cycles, each giving maximum yield in 6-7 days. The polyacrylamide gel immobilization ofA niger KCU520 cells was also tried in atwo-stage bioreactor. After a 6 day acclimatization period under optimum conditions of fermentation, these immobilized cells were found to convert about 20% sugars into citric acid in 24 hand the cells remained viable for 3to 4weeks without any significant loss of activity. Hence, by using immobilized cells in atwo-stage bioreactor it was possible to obtain acontinuous production of citric acid from sugarcane molasses at the rate of 20 g/lit/day. Scaling-up of this procedure is in progress.