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Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10
Bioresource Technology 85 (2002) 69–71
Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10 C.S.K. Reddy *, R.P. Singh Department of Biosciences and Biotechnology, Indian Institute of Technology, Roorkee 247 667, India Received 5 December 2001; received in revised form 18 January 2002; accepted 5 February 2002
Abstract A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated from horticulture waste. Market refuse, apple and banana, were explored as novel substrates for itaconic acid production with yields of 20 2:0 and 20:0 1:0 g l1 , respectively. Itaconic acid yields of 28:5 2:2 and 31:0 1:7 g l1 were obtained with acid and a-amylase hydrolyzed corn starch. The efficiency of itaconic acid production by this wild type strain was improved by ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two mutants were capable of producing twice the yield of itaconic acid as the parent strain. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Itaconic acid; Aspergillus terreus; Organic acids; Corn starch; Fruit wastes
1. Introduction In the developing and developed countries, year after year there has been an increasing use of organic acids particularly itaconic, gluconic, lactic, fumaric and kojic acids. Itaconic acid (C5 H6 O4 ) is an unsaturated dicarboxylic acid, crystalline, relatively non-toxic with a melting point of 167–168 °C and density of 1.632 (Milsom and Meers, 1985). The property that makes itaconic acid a uniquely valuable compound is the conjunction of the two carboxyl groups and the methylene group. The methylene group is able to take part in addition polymerization giving rise to polymers with many free carboxyl groups that confer advantageous properties (Mattey, 1992). The widespread uses of itaconic acid in polymers and in synthesizing N-substituted pyrrolidones have resulted in its increased demand (Christiansen, 1980). Synthesis of itaconic acid has proven to be uneconomical because of high substrate costs and/or relatively low yields. Itaconic acid is known to be produced
* Corresponding author. Present address: Centre for Biochemical Technology (CSIR), Delhi University Campus, Mall Road, Delhi 110007, India. Fax: +91-11-7667471/7416439. E-mail address: [email protected] (C.S.K. Reddy).
on a commercial scale using only Aspergillus terreus and Aspergillus itaconicus (Milsom and Meers, 1985). In the present study, attempts were made to develop a potent strain for improved production of itaconic acid. Alternatively, with a view to reducing the substrate costs, cheap and abundantly available substrates such as fruit wastes, which had not been reported earlier, and corn starch were used.
2. Methods 2.1. Microorganism, growth conditions and inoculum development A. terreus SKR10 was isolated from horticulture waste in our laboratory (Identified by Indian Type Culture Collection ITCC, India; Accession No. ITCC 4826). A. terreus was grown and maintained on Czapek Dox agar medium at 34 °C for 6–8 days. A loopful of the culture was inoculated into 50 ml of the growth medium containing (g l1 ) glucose: 60, ammonium nitrate: 4.0, magnesium sulfate: 0.04, copper sulfate: 0.04 and potassium di-hydrogen-phosphate: 0.088 at pH 3.0. The growth medium was incubated in a 250 ml
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 7 5 - 5
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C.S.K. Reddy, R.P. Singh / Bioresource Technology 85 (2002) 69–71
Erlenmeyer flask under shake flask conditions for 24 h in a rotary shaker (120 rpm) at 34 °C. 5 ml was inoculated into 98 ml of production medium containing (g l1 ) glucose or fruit waste extract or hydrolyzed corn starch: 30–120 (as sugars), ammonium nitrate: 4.0, magnesium sulfate: 0.095, copper sulfate: 0.04 at pH 3.0 and incubated on a rotary shaker (120 rpm) at 34 °C for 6 days. 2.2. Pretreatment of fruit waste and corn starch The processing of market refuse fruits (apple and banana) was done following the procedures described by Ward (1985). Briefly, the market refuse apple and banana were individually washed and mashed into a coarse pulp and the juice was extracted in water at 90 °C, in a four to five step extraction procedure. The extracts were referred to as ‘banana waste extract’ and ‘apple waste extract’ respectively. The total sugars were estimated by routine methods and the extract was suitably diluted for use as substrate. Corn starch was hydrolyzed by acid and enzyme hydrolysis methods. Acid hydrolysis of corn starch was carried out at 140 °C using nitric acid at pH 2.0. The hydrolyzate was neutralized with calcium carbonate and filtered. The filtrate was analysed for total sugars (as glucose) by routine methods and suitably diluted for use in production media. Commercially available a-amylase of Bacillus sp. (Sreenath, 1992) was added directly to the medium for hydrolysis of corn starch (1 U/g of corn starch) and use as substrate simultaneously. 2.3. Mutagenesis Itaconic acid is produced by microorganisms in a secondary pathway and hence not secreted in high concentrations under natural conditions. Hence, strain improvement was done by mutagenic treatments to improve the yields of itaconic acid. Mutagenic treatment of A. terreus SKR10 was done by exposure to ultraviolet radiations (UV), N-methyl-N 0 -nitro-N-nitrosoguanidine (NTG), colchicine and sodium azide individually as well as mixed mutagenic treatments in all possible combinations of the agents. Spore suspensions (2 106 spores/ ml) were chosen from 6 to 8 day old cultures of A. terreus SKR10 and were made up in 0.85% (w/v) sodium lauryl sulfate. The spore suspension was exposed to UV from a 15 W, UV lamp from a distance of 70 cm for periods ranging from 10 to 60 min with 10 min intervals. Plates were wrapped with black paper and incubated at 34 °C for 5–7 days. NTG mutagenesis was done following the method of Thoma (1971). Spore suspensions were treated with NTG (100 lg/ml) solution for varying times (30–60 min with an interval of 5 min). The spores were plated on Czapek Dox agar medium and incubated
for 6–8 days. The surviving colonies were isolated and subcultured. Colchicine treatment was done as described by Claudimara and Glaucia (1989). The spore suspension was suspended in distilled water and treated with different concentrations of colchicine; 0.01, 0.02, 0.1, 0.2, 0.5 1 and 2 lg/ml for 24 h and plated in Czapek agar medium. Sodium azide treatment was done at concentrations 1, 2, 3, 4 and 5 lg/ml with the spore suspension for 24 h as described by Zohrer et al. (1996). The surviving colonies were maintained on Czapek Dox agar medium. The strains were assigned identity based on the treatment time and concentrations (UV10 to UV60, N35 to N60, C.01 to C.2 and S1 to S5). Potent mutants capable of producing high levels of itaconic acid, obtained after single treatment (UV or chemical mutagens) were chosen for further mutagenic treatments. The highest itaconic acid yielding mutants obtained after every single treatment were further exposed to all possible combinations of the rest of the mutagens used above. 2.4. Screening Screening for high itaconic acid producers was done by the gradient plating method as described by Yahiro et al. (1995). This provided a gradient plate with a gradual increase in itaconic acid concentration from 0 to 60 g l1 (left to right) along one horizontal axis. The plates were incubated for 7 days at 34 °C and the mutants capable of growing in the highest gradient were isolated and subcultured in Czapek Dox agar medium. The yield of itaconic acid produced was determined in submerged fermentations. 2.5. Analytical procedures Itaconic acid was estimated by the calorimetric method of Hartford (1962) and by the HPLC method of Kautola et al. (1989). The itaconic acid yield was calculated from itaconic acid formed (g l1 ). The biomass was filtered from the fermented broth by Whatman No. 1 paper, washed with distilled water and dried at 55 °C for 24 h. The weight of the biomass was referred to as dry mycelial weight. Calculation of Yields: The yields were determined as described by Yahiro et al. (1995).
3. Results and discussion A local strain capable of producing itaconic acid was isolated from horticulture waste. This fungus could utilize a wide variety of pure sugar substrates as well as market refuse fruits and hydrolyzed corn starch. It was
C.S.K. Reddy, R.P. Singh / Bioresource Technology 85 (2002) 69–71
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Table 1 Effect of substrates on the culture parameters of A. terreus SKR10 and its mutants UNCS1 and N45 Substrate supplied
Strain 1
Acid hydrolyzed corn starch (120 g l )
Enzyme hydrolyzed corn starch (120 g l1 )
Market refuse apple (90 g l1 )
Market refuse banana (90 g l1 )
A. terreus SKR10 Mutant UNCS1 Mutant N45 A. terreus SKR10 Mutant UNCS1 Mutant N45 A. terreus SKR10 Mutant UNCS1 Mutant N45 A. terreus SKR10 Mutant UNCS1 Mutant N45
Yield Y ðp=sÞa
Y ðx=sÞa
Y ðp=xÞa
0.25 0.38 0.40 0.27 0.41 0.42 0.23 0.36 0.37 0.22 0.35 0.35
0.125 0.116 0.108 0.117 0.110 0.101 0.117 0.110 0.104 0.107 0.103 0.101
2.0 3.3 3.7 2.3 3.7 4.2 2.1 3.3 3.4 2.2 3.3 3.4
a Based on sugar consumed, Y ðp=sÞ ¼ Itaconic acid from substrate, Y ðx=sÞ ¼ Biomass yield from substrate, Y ðp=xÞ ¼ Itaconic acid from cells (s ¼ substrate, p ¼ product, x ¼ dry mycelial mass).
identified as A. terreus and designated as A. terreus SKR10. The fruit wastes and corn starch were suitably processed and the physico-chemical and biological parameters were optimized for the production of itaconic acid. A. terreus SKR10 produced 28.5 and 31.0 g l1 of itaconic acid from acid hydrolyzed and a-amylase hydrolyzed corn starch, espectively. The use of market refuse apple and banana extract as substrates gave yields of 20 g l1 of itaconic acid each. Mutagenesis resulted in two potent itaconic acid producing mutants, N45 and UNCS1. The mutant N45, was the result of a single mutagenic treatment i.e. NTG (100 lg/ml) for 45 minutes, which gave a yield of 46:0 3:0 and 50:0 3:0 g l1 of itaconic acid with acid and amylase hydrolyzed corn starch (120 g l1 ), respectively. The other mutant, UNCS1 was obtained as a result of treatment with NTG, colchicine and sodium azide at concentrations of 100, 0.02 and 2 lg/ml sequentially. With UNCS1 mutant, the yield of itaconic acid with acid and enzyme hydrolyzed corn starch (120 g l1 ) was 43:6 3:0 and 48:0 3:0 g l1 of itaconic acid, respectively. On the other hand, the two mutant strains N45 and UNCS1 used banana and market refuse apple extract (90 g l1 ), as substrates to yield and 29:0 1:2 to 30:0 2:0 g l1 and 31:0 1:7 to 32:0 2:0 g l1 of itaconic acid, respectively. In the present study, the evaluation of the effect of substrates on the culture parameters (Table 1) revealed that the strain (N45) having a highest Y ðp=sÞ, Y ðp=xÞ value and a low Y ðx=sÞ was the ideal high itaconic acid yielding strain. Kirimura et al. (1997) succeeded in doing interspecies protoplast fusion between A. usamii and A. terreus for simultaneous starch hydrolysis and itaconic acid production with a yield of 35.9 g l1 . Petrucciolli et al. (1999) used a variety of starchy materials, and obtained 18.4 g l1 of itaconic acid from corn starch. Hence, the present study signifies the use of corn starch and fruit wastes as potential substrates for itaconic acid fermentation.
Acknowledgements The authors are very much thankful to the Council of Scientific and Industrial Research (CSIR), India for the financial support.
References Christiansen, A., 1980. Surface active amides and imidazolines. Br Pat 1 574 916 (Chem. Abstr. 94, 109 083w). Claudimara, F.P.L., Glaucia, M.M.S., 1989. Comparative analysis of colchicine induced micronuclei in different cell types in vitro. Mut Res 349, 77–83. Hartford, C.G., 1962. Rapid spectrophotometric method for the determination of itaconic acid. Anal Chem 34 (3), 426–428. Kautola, H., Vassilev, N., Linko, Y.Y., 1989. Itaconic acid production by immobilized Aspergillus terreus on sucrose medium. Biotechnol Lett 11, 313–318. Kirimura, K., Sato, T., Nakashima, N., Terada, M., Usami, S., 1997. Breeding of starch utilizing itaconic acid producing molds by interspecific protoplast fusion between Aspergillus terrreus and Aspergillus usamii. Appl Microbiol Biotechnol 47, 127–131. Mattey, M., 1992. The production of organic acids. Crit Rev Biotechnol 12 (1/2), 87–132. Milsom, P.E., Meers, J.L., 1985. Gluconic and itaconic acids. In: MooYoung, M. (Ed.), Comprehensive Biotechnology, vol. 3. Pergamon Press, Oxford, pp. 672–700. Petrucciolli, M.P., Pulci, V., Federici, F., 1999. Itaconic acid production by Aspergillus terrreus on raw starchy materials. Lett Appl Microbiol 28 (4), 309–312. Sreenath, H.K., 1992. Studies on starch granules digestion by alphaamylase. Starch 44 (2), 61–83. Thoma, R.W., 1971. Use of mutagens in the improvement of production of microorganism. Folia Microbiol 16, 197–204. Ward, O.P., 1985. Hydrolytic enzymes. In: Industrial Biochemicals, Biochemicals and Fuels, vol. I. Pergamon Press, Oxford, pp. 819– 824. Yahiro, K., Takahama, T., Park, Y.S., Jai, S., Okabe, M., 1995. Breeding of Aspergillus terreus mutant TN-484 for itaconic acid production with high yield. J Ferment Bioengg 5, 506–508. Zohrer, E., Albertini, S., Gocke, E., Knasmuller, S., 1996. Mutation induction and mutation spectra of Salmonella typhimurium TA100 after exposure to isohistidines. Mut Res 356, 155–161.
Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10 C.S.K. Reddy *, R.P. Singh Department of Biosciences and Biotechnology, Indian Institute of Technology, Roorkee 247 667, India Received 5 December 2001; received in revised form 18 January 2002; accepted 5 February 2002
Abstract A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated from horticulture waste. Market refuse, apple and banana, were explored as novel substrates for itaconic acid production with yields of 20 2:0 and 20:0 1:0 g l1 , respectively. Itaconic acid yields of 28:5 2:2 and 31:0 1:7 g l1 were obtained with acid and a-amylase hydrolyzed corn starch. The efficiency of itaconic acid production by this wild type strain was improved by ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two mutants were capable of producing twice the yield of itaconic acid as the parent strain. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Itaconic acid; Aspergillus terreus; Organic acids; Corn starch; Fruit wastes
1. Introduction In the developing and developed countries, year after year there has been an increasing use of organic acids particularly itaconic, gluconic, lactic, fumaric and kojic acids. Itaconic acid (C5 H6 O4 ) is an unsaturated dicarboxylic acid, crystalline, relatively non-toxic with a melting point of 167–168 °C and density of 1.632 (Milsom and Meers, 1985). The property that makes itaconic acid a uniquely valuable compound is the conjunction of the two carboxyl groups and the methylene group. The methylene group is able to take part in addition polymerization giving rise to polymers with many free carboxyl groups that confer advantageous properties (Mattey, 1992). The widespread uses of itaconic acid in polymers and in synthesizing N-substituted pyrrolidones have resulted in its increased demand (Christiansen, 1980). Synthesis of itaconic acid has proven to be uneconomical because of high substrate costs and/or relatively low yields. Itaconic acid is known to be produced
* Corresponding author. Present address: Centre for Biochemical Technology (CSIR), Delhi University Campus, Mall Road, Delhi 110007, India. Fax: +91-11-7667471/7416439. E-mail address: [email protected] (C.S.K. Reddy).
on a commercial scale using only Aspergillus terreus and Aspergillus itaconicus (Milsom and Meers, 1985). In the present study, attempts were made to develop a potent strain for improved production of itaconic acid. Alternatively, with a view to reducing the substrate costs, cheap and abundantly available substrates such as fruit wastes, which had not been reported earlier, and corn starch were used.
2. Methods 2.1. Microorganism, growth conditions and inoculum development A. terreus SKR10 was isolated from horticulture waste in our laboratory (Identified by Indian Type Culture Collection ITCC, India; Accession No. ITCC 4826). A. terreus was grown and maintained on Czapek Dox agar medium at 34 °C for 6–8 days. A loopful of the culture was inoculated into 50 ml of the growth medium containing (g l1 ) glucose: 60, ammonium nitrate: 4.0, magnesium sulfate: 0.04, copper sulfate: 0.04 and potassium di-hydrogen-phosphate: 0.088 at pH 3.0. The growth medium was incubated in a 250 ml
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 7 5 - 5
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C.S.K. Reddy, R.P. Singh / Bioresource Technology 85 (2002) 69–71
Erlenmeyer flask under shake flask conditions for 24 h in a rotary shaker (120 rpm) at 34 °C. 5 ml was inoculated into 98 ml of production medium containing (g l1 ) glucose or fruit waste extract or hydrolyzed corn starch: 30–120 (as sugars), ammonium nitrate: 4.0, magnesium sulfate: 0.095, copper sulfate: 0.04 at pH 3.0 and incubated on a rotary shaker (120 rpm) at 34 °C for 6 days. 2.2. Pretreatment of fruit waste and corn starch The processing of market refuse fruits (apple and banana) was done following the procedures described by Ward (1985). Briefly, the market refuse apple and banana were individually washed and mashed into a coarse pulp and the juice was extracted in water at 90 °C, in a four to five step extraction procedure. The extracts were referred to as ‘banana waste extract’ and ‘apple waste extract’ respectively. The total sugars were estimated by routine methods and the extract was suitably diluted for use as substrate. Corn starch was hydrolyzed by acid and enzyme hydrolysis methods. Acid hydrolysis of corn starch was carried out at 140 °C using nitric acid at pH 2.0. The hydrolyzate was neutralized with calcium carbonate and filtered. The filtrate was analysed for total sugars (as glucose) by routine methods and suitably diluted for use in production media. Commercially available a-amylase of Bacillus sp. (Sreenath, 1992) was added directly to the medium for hydrolysis of corn starch (1 U/g of corn starch) and use as substrate simultaneously. 2.3. Mutagenesis Itaconic acid is produced by microorganisms in a secondary pathway and hence not secreted in high concentrations under natural conditions. Hence, strain improvement was done by mutagenic treatments to improve the yields of itaconic acid. Mutagenic treatment of A. terreus SKR10 was done by exposure to ultraviolet radiations (UV), N-methyl-N 0 -nitro-N-nitrosoguanidine (NTG), colchicine and sodium azide individually as well as mixed mutagenic treatments in all possible combinations of the agents. Spore suspensions (2 106 spores/ ml) were chosen from 6 to 8 day old cultures of A. terreus SKR10 and were made up in 0.85% (w/v) sodium lauryl sulfate. The spore suspension was exposed to UV from a 15 W, UV lamp from a distance of 70 cm for periods ranging from 10 to 60 min with 10 min intervals. Plates were wrapped with black paper and incubated at 34 °C for 5–7 days. NTG mutagenesis was done following the method of Thoma (1971). Spore suspensions were treated with NTG (100 lg/ml) solution for varying times (30–60 min with an interval of 5 min). The spores were plated on Czapek Dox agar medium and incubated
for 6–8 days. The surviving colonies were isolated and subcultured. Colchicine treatment was done as described by Claudimara and Glaucia (1989). The spore suspension was suspended in distilled water and treated with different concentrations of colchicine; 0.01, 0.02, 0.1, 0.2, 0.5 1 and 2 lg/ml for 24 h and plated in Czapek agar medium. Sodium azide treatment was done at concentrations 1, 2, 3, 4 and 5 lg/ml with the spore suspension for 24 h as described by Zohrer et al. (1996). The surviving colonies were maintained on Czapek Dox agar medium. The strains were assigned identity based on the treatment time and concentrations (UV10 to UV60, N35 to N60, C.01 to C.2 and S1 to S5). Potent mutants capable of producing high levels of itaconic acid, obtained after single treatment (UV or chemical mutagens) were chosen for further mutagenic treatments. The highest itaconic acid yielding mutants obtained after every single treatment were further exposed to all possible combinations of the rest of the mutagens used above. 2.4. Screening Screening for high itaconic acid producers was done by the gradient plating method as described by Yahiro et al. (1995). This provided a gradient plate with a gradual increase in itaconic acid concentration from 0 to 60 g l1 (left to right) along one horizontal axis. The plates were incubated for 7 days at 34 °C and the mutants capable of growing in the highest gradient were isolated and subcultured in Czapek Dox agar medium. The yield of itaconic acid produced was determined in submerged fermentations. 2.5. Analytical procedures Itaconic acid was estimated by the calorimetric method of Hartford (1962) and by the HPLC method of Kautola et al. (1989). The itaconic acid yield was calculated from itaconic acid formed (g l1 ). The biomass was filtered from the fermented broth by Whatman No. 1 paper, washed with distilled water and dried at 55 °C for 24 h. The weight of the biomass was referred to as dry mycelial weight. Calculation of Yields: The yields were determined as described by Yahiro et al. (1995).
3. Results and discussion A local strain capable of producing itaconic acid was isolated from horticulture waste. This fungus could utilize a wide variety of pure sugar substrates as well as market refuse fruits and hydrolyzed corn starch. It was
C.S.K. Reddy, R.P. Singh / Bioresource Technology 85 (2002) 69–71
71
Table 1 Effect of substrates on the culture parameters of A. terreus SKR10 and its mutants UNCS1 and N45 Substrate supplied
Strain 1
Acid hydrolyzed corn starch (120 g l )
Enzyme hydrolyzed corn starch (120 g l1 )
Market refuse apple (90 g l1 )
Market refuse banana (90 g l1 )
A. terreus SKR10 Mutant UNCS1 Mutant N45 A. terreus SKR10 Mutant UNCS1 Mutant N45 A. terreus SKR10 Mutant UNCS1 Mutant N45 A. terreus SKR10 Mutant UNCS1 Mutant N45
Yield Y ðp=sÞa
Y ðx=sÞa
Y ðp=xÞa
0.25 0.38 0.40 0.27 0.41 0.42 0.23 0.36 0.37 0.22 0.35 0.35
0.125 0.116 0.108 0.117 0.110 0.101 0.117 0.110 0.104 0.107 0.103 0.101
2.0 3.3 3.7 2.3 3.7 4.2 2.1 3.3 3.4 2.2 3.3 3.4
a Based on sugar consumed, Y ðp=sÞ ¼ Itaconic acid from substrate, Y ðx=sÞ ¼ Biomass yield from substrate, Y ðp=xÞ ¼ Itaconic acid from cells (s ¼ substrate, p ¼ product, x ¼ dry mycelial mass).
identified as A. terreus and designated as A. terreus SKR10. The fruit wastes and corn starch were suitably processed and the physico-chemical and biological parameters were optimized for the production of itaconic acid. A. terreus SKR10 produced 28.5 and 31.0 g l1 of itaconic acid from acid hydrolyzed and a-amylase hydrolyzed corn starch, espectively. The use of market refuse apple and banana extract as substrates gave yields of 20 g l1 of itaconic acid each. Mutagenesis resulted in two potent itaconic acid producing mutants, N45 and UNCS1. The mutant N45, was the result of a single mutagenic treatment i.e. NTG (100 lg/ml) for 45 minutes, which gave a yield of 46:0 3:0 and 50:0 3:0 g l1 of itaconic acid with acid and amylase hydrolyzed corn starch (120 g l1 ), respectively. The other mutant, UNCS1 was obtained as a result of treatment with NTG, colchicine and sodium azide at concentrations of 100, 0.02 and 2 lg/ml sequentially. With UNCS1 mutant, the yield of itaconic acid with acid and enzyme hydrolyzed corn starch (120 g l1 ) was 43:6 3:0 and 48:0 3:0 g l1 of itaconic acid, respectively. On the other hand, the two mutant strains N45 and UNCS1 used banana and market refuse apple extract (90 g l1 ), as substrates to yield and 29:0 1:2 to 30:0 2:0 g l1 and 31:0 1:7 to 32:0 2:0 g l1 of itaconic acid, respectively. In the present study, the evaluation of the effect of substrates on the culture parameters (Table 1) revealed that the strain (N45) having a highest Y ðp=sÞ, Y ðp=xÞ value and a low Y ðx=sÞ was the ideal high itaconic acid yielding strain. Kirimura et al. (1997) succeeded in doing interspecies protoplast fusion between A. usamii and A. terreus for simultaneous starch hydrolysis and itaconic acid production with a yield of 35.9 g l1 . Petrucciolli et al. (1999) used a variety of starchy materials, and obtained 18.4 g l1 of itaconic acid from corn starch. Hence, the present study signifies the use of corn starch and fruit wastes as potential substrates for itaconic acid fermentation.
Acknowledgements The authors are very much thankful to the Council of Scientific and Industrial Research (CSIR), India for the financial support.
References Christiansen, A., 1980. Surface active amides and imidazolines. Br Pat 1 574 916 (Chem. Abstr. 94, 109 083w). Claudimara, F.P.L., Glaucia, M.M.S., 1989. Comparative analysis of colchicine induced micronuclei in different cell types in vitro. Mut Res 349, 77–83. Hartford, C.G., 1962. Rapid spectrophotometric method for the determination of itaconic acid. Anal Chem 34 (3), 426–428. Kautola, H., Vassilev, N., Linko, Y.Y., 1989. Itaconic acid production by immobilized Aspergillus terreus on sucrose medium. Biotechnol Lett 11, 313–318. Kirimura, K., Sato, T., Nakashima, N., Terada, M., Usami, S., 1997. Breeding of starch utilizing itaconic acid producing molds by interspecific protoplast fusion between Aspergillus terrreus and Aspergillus usamii. Appl Microbiol Biotechnol 47, 127–131. Mattey, M., 1992. The production of organic acids. Crit Rev Biotechnol 12 (1/2), 87–132. Milsom, P.E., Meers, J.L., 1985. Gluconic and itaconic acids. In: MooYoung, M. (Ed.), Comprehensive Biotechnology, vol. 3. Pergamon Press, Oxford, pp. 672–700. Petrucciolli, M.P., Pulci, V., Federici, F., 1999. Itaconic acid production by Aspergillus terrreus on raw starchy materials. Lett Appl Microbiol 28 (4), 309–312. Sreenath, H.K., 1992. Studies on starch granules digestion by alphaamylase. Starch 44 (2), 61–83. Thoma, R.W., 1971. Use of mutagens in the improvement of production of microorganism. Folia Microbiol 16, 197–204. Ward, O.P., 1985. Hydrolytic enzymes. In: Industrial Biochemicals, Biochemicals and Fuels, vol. I. Pergamon Press, Oxford, pp. 819– 824. Yahiro, K., Takahama, T., Park, Y.S., Jai, S., Okabe, M., 1995. Breeding of Aspergillus terreus mutant TN-484 for itaconic acid production with high yield. J Ferment Bioengg 5, 506–508. Zohrer, E., Albertini, S., Gocke, E., Knasmuller, S., 1996. Mutation induction and mutation spectra of Salmonella typhimurium TA100 after exposure to isohistidines. Mut Res 356, 155–161.