BIOCONVERSION OF AGRO-FOOD BY-PRODUCTS TO GLUCONIC ACID BY ASPERGILLUS NIGER

Abstract

In this investigation attempt has been made to obtain a microorganism that has potential for gluconic acid production on a commercial scale. The strategy involved was as follows: Fungi were isolated from the soil at the sites where sugarcane wastes were decomposing. Substantial amounts of gluconic acid were produced by a fungal strain identified to be Aspergillus niger. This strain was subjected to both physical and chemical mutagenesis. UV mutagenesis in two stages resulted m a ORS-4.410 which was a high yielding strain of A. niger. Chemical mutagenesis by nitrous acid and N-methyl-n'-nitro-N-nitrosoguanidine led no improvement in gluconic acid production. ORS-4.410 was dirty white in colour and had a morphology that was characteristically distinct from the yellow colouredparental strain of A. niger. Mutant cells had hyphae that were thick and short with rough cell surface while the parental strain possessed ones that were thin flat thread like and highly branched. Once it was established based on acid unitage values that ORS-4.410 was highly productive strain, the performance of the fungus was put to test under standard fermentation conditions with glucose as substrate. It was observed that the optimal conditions for the parental and mutant strains were identical with respect to the type and concentration of sugars, pH, temperature and several other requirements. However, the production of gluconic acid and the generation of biomass was substantially higher in the mutant ORS- 4.410 compared to that of the parental strain. In order to improve further the yield of gluconic acid, vegetable oils, starch and H202 (ii) were evaluated for their role as regulators. H202 was found to be the best stimulator of gluconic acid production by A. niger strain. Pure sugars in fermentation mediumwas substituted with locally available natural carbohydrate sources with the intention of economizing the production of gluconic acid by ORS-4.410. Agro-food by-products e.g. grape must, banana must and sugarcane molasses were tried as alternate substrate in batch fermentations. Initial experiments showed that crude fruit must and sugarcane molasses cannot be used with success. Fermentations carried out with enzymatically clarified fruit must and hexacyanoferrate (HCF) treated sugarcane molasses proved to be promising. Fermentation using rectified grape must was found to be superior to banana must and sugarcane molasses. However, because of a change in viscosity a higher aeration was required in fermentations carried out with natural carbohydrate sources. The efficiency of batch fermentation was then evaluated under submerged, surface and solid state surface fermentation condition. Surface fermentation required aeration but the amount of gluconic acid produced was found to be far greater than submerged cultures with shaking. The yield of gluconic acid was found to be further enhanced when solid state surface fermentation was carried out with bagasse as support. In order to develop a system for the semicontinuous production of gluconic acid attempts were made to recycle the fungus using immobilization technology. Two types of immobilization were tried in fermentation broth containing agro-food by-products. Submerged conditions were simulated by calcium alginate immobilization while surface fermentation condition was generated by

immobilization on polyurethane sponge (PUS). The size of matrix, level of inoculum, concentrations of spores, rate of aeration and duration of fermentation were quite different in the two cases. However, in terms of cost and effort the PU sponge based of immobilization yielded better results; since the fungi could be reused in more cycles thereby increasing the overall yield of gluconic acid.