Directional Breeding of High Itaconic Acid Yielding Strain of Aspergillus terreus with a New Plate Technique

Itaconic acid is commercially produced by the cultivation of Aspergillus terreus using starch hydrolysate as carbon source. The degree of hydrolysis had a great influence on itaconic acid production which was suitable when corn starch was saccharified at 35 DE. The α-amylase was sufficient to drive the starch hydrolysis to the degree. The agar plate assay with LiCl treatment provided a rapid, simple and unequivocal method for screening large numbers of colonies for itaconic acid producing strains. It was learned by experience that the strains on the plates with thick hyphae and light-colored spores often accompanied high itaconic acid production. A strain, designated Ast165, producing itaconic acid with a high yield, was successfully obtained by directional breeding of metabolic end products resistant strains. The itaconic acid concentration produced by Ast165 was 53.8 g/l from 100 g/l of starch hydrolysate in shake flasks. The conversion rate was 61.3%, which was the highest value found in tests.


Introduction
With a new interest in sustainable development, the chemical industry is making many attempts to replace petrochemical-based monomers with natural ones.Itaconic acid (IA), or methylene succinic acid, is an unsaturated acid with conjugated double bonds and two carboxyl groups (Figure 1).IA and its ester are used for the synthesis of fiber, resin, plastic, rubber, paints, surfactant and lubricant [1][2][3].IA was discovered by Baup as a thermal decomposition product of citric acid at 175˚C [4].This was the most primitive preparation method of IA.But chemical synthesis processes can not really compete with fermentation by fungi because the cost remains high [5].The biosynthesis by fungi from carbohydrates was first reported by Kinoshita [6], who isolated IA from the growth medium of an osmophilic fungi, Aspergillus itaconicus.Later, other fungal strains, mainly of the species Aspergillus terreus, were found to be more suitable [7].New biotechnological methodologies involving fermentation processes and technologies that use alternative cheap substrates as the carbon source are currently under investigation and development [8].The favorite substrates for IA production by the A. terreus are hydrolysates of starchy materials [9][10][11].Corn starch is one of the best carbon sources, since it is very pure, inexpensive, and stable in a mass supply [12].However, corn starch is not a popular fermentation raw material because it is very difficult to sterilize, due to gelatinization upon heating.The problem of gelatinization of corn starch upon heat sterilization was solved by hydrolyzing the starch using acid or enzymes.Acid hydrolysis of starch required the use of corrosion resistant materials as vessel, gave rise to high colour and salt ash content (after neutralisation), needed more energy for heating and was relatively difficult to control.Therefore, only the starch hydrolysate prepared with enzymes was used as carbon source for IA production in experiments.Strain improvement is another method to reduce the cost of IA production.To date very little research has been directed at the improvement of itaconic acid production.The screening of an excellent strain will be a promising choice to get the successful results [13].an 4 , 0.2; LiCl, 10; Bromocresol green, 0.1 and pH 3.0. of pH, total sugar, reducing sugar and IA concentrations.p, China.Corn starch was hydrolyzed using enzymes.

rolysis of Starch for
sed as carbon sources for IA production by A. terreus.

Screening of Strains with High Itaconic
vealed using scanning electron microscopic techni f total sugar, reducing sugar and IA concentrations.

2.6.
Metabolic End e indicator medium agar plates for screening of strains.total sugar content-residual sugar content) × 100%.

Culture Conditions
The experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml of medium.A spore suspension of 10 8 /ml was incubated at 35˚C for 48 h on a rotary shaker at 200 rpm.Then 10% of the seed medium was transferred into a 250 ml Erlenmeyer flask containing 45 ml of production medium.The strain cultivation was performed at 35˚C for 120 h on a rotary shaker at 220 rpm.Samples were taken for analysis

Fermentation Substrate
Corn starch used was a commercial grade manufactured by Global Bio-chem Technology Grou

Enzymatic Hyd Fermentation
Enzymatic hydrolysis was carried out by the two-step hydrolysis of corn starch: liquefaction and saccharification.The corn starch and distilled water were mixed to the concentration of 200 g/l with stirrer.The slurry was blended with 0.5% (v/w dry starch) of α-amylase.The liquefaction was performed at 90˚C, pH 6 in a stirred reactor.The termination of liquefaction was determined by the colour of reaction of resultant in enzymatic hydrolysis of starch with iodine chemical.The downstream process was undertaken by following the general processes of saccharification.According to the catalyzing characteristics of glucoamylase, saccharification was carried out at 60˚C, pH 4.3.Dextrose equivalent (DE) was determinated in process of starch hydrolysis.Corn starch hydrolysate at different dextrose equivalent (DE) were u

Acid Yield
Primary screening: The obtained rearrangement strains with good mycelia growth and spore-production abilities were successively transferred and cultured.Then their spore suspension were transferred onto the selective medium agar plate cultured at 30˚C for 4 d.Single colonies growing fast on the plates with a larger color change zone diameter were selected as the high-output strains through primary screening.Other colonies were discarded.The surface structures of A. terreus on the plates were re que.
Secondary screening: The strains obtained by primary screening were activated in agar slope culture according to the seed medium at 30˚C for 4 d, then transferred into 50 ml of the production liquid medium in a 250 ml flask and cultured at 35˚C for 120 h.At the end of fermentation, the supernatant fluid was used for determinations o The Directional Breeding of Products Resistant Strains 2 g/l itaconic acid was added to the production liquid medium.The spore suspension was transferred into 50 ml of the mixed medium in a 250 ml flask and cultured at 35˚C for 48 h.The fermented liquor was spread on th

Analytical Methods
The pH value of culture broth was measured by a pH meter (Model UB-10, DENVER).For the total sugar, the phenol-sulfuric acid method was used [14].The DNS method was used for the reducing sugar measurement [15].IA concentration was measured by the bromine absorption method [16]."Dextrose equivalent" refers to the industry standard for measuring the concentration of total reducing sugars, calculated as glucose on a dry weight basis [17].Conversion rate is defined as: conversion rate = IA production/(initial medium

FTIR Spectroscopic Measurement
The fermented liquor was centrifuged.The supernatant was decolorized with actived carbon, and filtered.The filtrate was passed through ion exchange resin beds, then concentrated and dried to produce white powder.The powder was examined by Fourier transform infrared (FTIR) spectroscopy, on Nicolet Avatar 370 using a KBr pellet.

3.1.
Based on the Enzyme te as carbon urce in later fermentation medium was 35.

IA Production Hydrolysate
Enzyme hydrolyzed corn starch was used as carbon source for IA production by fermentation.Scanning electron micrographs of A. terreus showed some characteristics of the spores (Figure 4) and the hyphae (Figure 5).The scanning electron microscope magnified the object 2000 times.A. terreus on the selective medium agar plates had smaller conidia heads, less spores and thicker hyphae than A. terreus on the indicator medium plates.The observation revealed that LiCl treatment retarded spore germination and yet the hyphae grew strong.A positive correlation existed between LiCl resistance and IA production.It was concluded that A. terreus with thicker hyphae had a higher apparent metabolizability and the spores germination rate signified the speed of growth and reproduction of A. terreus.The above conclusion has subsequently been verified by numerous experiments.The efficiency of strain selection is Copyright © 2013 SciRes.AiM greatly raised, and the intensity of labor is decreased.

Fermentation in Shake Flasks by Directional Breeding
Single colonies on the indicator plates with a larger color change zone diameter, thick hyphae, light-colored and many spores were selected for shake flask fermentation.7 strains with high IA production were obtained from 600 single colonies.Table 1 showed the results of fermentation in shake flasks after directional breeding procedures.The IA concentration in the fermented liquor of parental strain on starch hydrolysate was 35.2 g/l.When the strains were picked up in the screening, IA production after fermentation had been greatly improved.The fermentation medium containing IA could reduce some IA-intolerant strains.The high producing mutant was obtained with strong aconitase activity and anti-feedback inhibition.The strain Ast352 gave the highest IA concentration of 54.5 g/l after fermentation.The highest conversion rate of 61.3% from starch hydrolysate to IA was obtained by strain Ast165 with IA concentration of 53.8 g/l, respectively.Strain Ast352 and strain Ast165 were serially transferred through agar slant culture.  of transfer of culture on IA production.A downward trend in the IA production of strain Ast352 had accelerated from the 4th transfer.However, the IA production of strain Ast165 was stable relatively.Simultaneous observation of plate culture showed that the spores of strain Ast352 enhanced the yellow pigment productin and the hyphae were shortened.It demonstrated that strain Ast352 was in degradation.Relative to the other strains, the IA production of strain Ast165 was high and stable; meanwhile, the conversion rate from starch hydrolysate to IA was the highest.

Characterization of IA by FTIR
The purified sample was characterized by the functional groups using FTIR technique.The IR spectrum was presented in Figure 6.The sample O-H stretch appears as a very broad band in the region 3100 -2900 cm −1 , centered at about 3000 cm −1 .An intense band at 1702 cm −1 is associated with the carbonyl C = O stretch of a carboxylic acid.It indicates that the sample is conjugated unsaturated carboxylic acid.The carboxylic acid C-O stretch appears at 1215 cm −1 , and the O -H bend is at 1440 cm −1 and 910 cm −1 .The band at approximately 1635 cm −1 is the C = C stretch.The results show that structure of sample is a carboxylic acid with conjugated double bonds, which is consistent with the structure of IA.

Conclusions
The results of the present study had indicated the possibility of utilizing the low DE starch hydrolysate as a raw material for IA production. A. terreus had the ability to produce IA by using partially hydrolyzed corn starch as carbon sources.Enzymatically hydrolyzed corn starch was used as substrates for IA production by A. terreus T730.The degree of hydrolysis had a great influence on IA production which was suitable when corn starch was saccharified at 35 DE.The α-amylase was sufficient to drive the starch hydrolysis to 35 DE.The agar plate assay with LiCl treatment provided a rapid, simple and unequivocal method for screening large numbers of colonies for IA producing strains.It was learned by experience that the strains on the plates with thick hyphae and light-colored spores often accompanied high IA production.From the results observed, the directional breeding method developed in the present study can be advocated as more rapid, sensitive, easily visible and reproducible than existing methods, and hence it will certainly be helpful in the rapid screening and isolation of IA producing strain.A strain, designated Ast165, producing itaconic acid with a high yield was successfully obtained by the screening with directional breeding of metabolic end products resistant strains.The production medium consisted of (g/l): starch hydrolysate, 100; NH 4 NO 3 , 3; MgSO 4 •7H 2 O, 4; corn steep liquor, 2; KH 2 PO 4 , 0.2 and pH 3.0.The DE value of starch hydrolysate in medium was 35.The fermentation was performed at 35˚C for 120 h on a rotary shaker at 220 rpm.The IA concentration produced by Ast165 was 53.8 g/l using starch hydrolysate in shake flasks, which was 52.8% higher than that of the parental strain.The conversion rate was 61.3%, which was the highest value found in tests.

Figure 2 Figure 2 . 3 . 2 .
Figure 2. Effect of degree of starch enzyme hydrolysis on itaconic acid production.Results were mean values of thre

Figure 3 .Figure 4 .
Figure 3. Colony shape on agar plate.(a) A. terreus on the indicator medium plate; (b) A. terreus on the selective medium plate.

Figure 5. Scanning electron micrographs of A. terreus hy- phae (×2000). (e) A. terreus hyphae on the indicator me- dium plate; (f) A. terreus hyphae on the selective medium plate.Table 1 . Results of fermentation in shake flasks by direc- tional breeding.
*Data were the means of triplicate experiments with standard deviations.