STUDIES ON PRODUCTION OF ITACONIC ACID BY ASPERGILLUS TERREUS

Abstract

The present study was focussed on isolating a potent microbial strain for producing itaconic acid and to explore the possibility of employing cheaper raw materials for economizing the fermentation process. Further, in order to achieve a higher itaconic acid yield under submerged conditions, the parental strain was subjected to mutagenesis. Whole cell immobilization was also undertaken to see the feasibility ofreusing the fungus in repeated batch fermentations and to cut down the fermentation time. A local wild type strain (SKR-10), isolated from decomposing apple waste was found to be a promising strain. It was identified to be Aspergillus terreus. This strain was capable ofutilizing a large variety ofsugars. The maximum itaconic acid was obtained with sucrose followed by glucose and D-xylose. The physicochemical and biological parameters were optimized for obtaining maximum product yields. The effects of various regulators were analyzed for assessing the sensitivity ofA terreus and to find their influence on itaconic acid formation. In order to economize the process of itaconic acid production, the synthetic substrates were substituted with cheap and high sugar containing rawmaterials such as sugarcane molasses, fruit wastes and corn starch. The crude sugarcane molasses was found to be unsuitable for itaconic acid production, due to higher heavy metal contents which were detrimental to the process. The excess of heavy metals was successfully removed by 0.1% (w/v) hexacyanoferrate (HCF) treatment. The fruit (i) wastes (apple and banana) were suitably processed and evaluated for use in itaconic acid fermentation. Corn starch in the native form was found unsuitable for itaconic acid production as Aspergillus terreus SKR-10 lacked the capacity of hydrolyzing corn starch. To achieve maximum hydrolysis both acid and enzyme hydrolysis was done. Acid hydrolysis was done using nitric acid at pH 2.0, while a-amylase was chosen for enzymatic hydrolysis. The physico-chemical and biological factors were evaluated and the conditions were defined for maximum itaconic acid production using these substrates. The itaconic acid yield obtained with sugarcane molasses (33 g/1), enzyme hydrolyzed corn starch (31 g/1) and fruit waste (19.5 - 20.0 g/1) were comparable to the yields obtained with sucrose (34 g/1), glucose (32.5 g/1) and D-xylose (20 g/1). In an attempt to further increase the itaconic acid production strain improvement was done by genetic manipulation of the wild type isolate, Aspergillus terreus SKR-10. This was done by single step and mixed mutagenic treatments involving ultraviolet radiation, nitrosoguanidine, colchicine and sodium azide treatments. The mutants were screened by the zone formation method and gradient plating method. Finally, two high itaconic acid producing mutants N45 and UNCS1 were obtained by single step and mixed mutagenic treatments, respectively. These two mutants were analyzed for itaconic acid production with all the substrates used above and yields obtained were compared with that of wild type strain. Both these strains were found promising, as they were capable ofproducing (ii) very significant levels of itaconic acid in comparison to the wild type strain under identical conditions. Whole cell immobilization was carried out with a view to further enhance the itaconic acid production. The mutant N45 was immobilized in three different matrices and the immobilized cells were used in repeated batch fermentation using 12% (w/v) sucrose (defined) medium for a total of 15 days (3 cycles of five days each). The maximum itaconic acid productivity (average from 3 cycles) was obtained from celite R-626 immobilized cells (0.41 g/l/h), followed by sodium alginate immobilized cells (0.40 g/l/h) and polyacrylamide immobilized cells (0.31 g/l/h). Product recovery was done from the fermented broth containing sucrose/molasses as substrate in which free cells/immobilized cells were used. About 90% recovery of the itaconic acid was achieved.