Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.     

Production of itaconic acid from pentose sugars by Aspergillus terreus

Itaconic acid (IA), an unsaturated 5‐carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low‐cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose‐derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, six strains (NRRL strains 1960, 1961, 1962, 1972, 66125, and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8 ± 0.3, 38.9 ± 0.8, 34.8 ± 0.9, and 33.2 ± 2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose, and arabinose by A. terreus. The results presented in the article will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1059–1067, 2017     

Production of itaconic acid by Aspergillus terreus immobilized in polyacrylamide gels

Summary Living Aspergillus terreus cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column reactors to produce itaconic acid from glucose.With the replacement batch reactor, maximum itaconic acid productivity was observed under the following conditions: pH 2.50, temperature at 35°C, addition of NH₄H₂PO₄ and MgSO₄·7H₂O. Using the continuous reactor, the maximum itaconic acid yield was 60 mg/h/40 g of gel. The biocatalyst activity or half-life was about 10 days.     

Efficient itaconic acid production by Aspergillus terreus: Overcoming the strong inhibitory effect of manganese

Itaconic acid (IA), a building block platform chemical, is produced industrially by Aspergillus terreus utilizing glucose. Lignocellulosic biomass can serve as a low cost source of sugars for IA production. However, the fungus could not produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate even at 100-fold dilution. Furfural, hydroxymethyl furfural and acetic acid were inhibitory, as is typical, but Mn²⁺ was particularly problematic for IA production. It was present in the hydrolyzate at a level that was 230 times over the inhibitory limit (50 ppb). Recently, it was found that PO₄³⁻ limitation decreased the inhibitory effect of Mn²⁺ on IA production. In the present study, a novel medium was developed for production of IA by varying PO₄³⁻, Fe³⁺ and Cu²⁺ concentrations using response surface methodology, which alleviated the strong inhibitory effect of Mn²⁺. The new medium contained 0.08 g KH₂PO₄, 3 g NH₄NO₃, 1 g MgSO₄·7H₂O, 5 g CaCl₂·2 H₂O, 0.83 mg FeCl₃·6H₂O, 8 mg ZnSO₄·7H₂O, and 45 mg CuSO₄·5H₂O per liter. The fungus was able to produce IA very well in the presence of Mn²⁺ up to 100 ppm in the medium. This medium will be extremely useful for IA production in the presence of Mn²⁺. This is the first report on the development of Mn²⁺ tolerant medium for IA production by A. terreus.     

Biosynthesis of itaconic acid by aspergillus terreus

Summary The formation of itaconic, aconitic and other acids from glucose in the presence of some enzyme inhibitors or organic acids by Aspergillus terreus was studied. Moreover, the metabolic activities of the preformed mats when floated on solutions of some organic acids were traced.When the resulting information were collected together a presumed condensation reaction between acetate and succinate could be formulated. The reaction product, presumably 1, 2, 3 propane tricarboxylic acid, would undergo a dehydrogenation reaction to yield aconitic acid and subsequently itaconic acid. It has also been suggested that aconityl CoA may be the metabolic form which suits the reactions leading to the formation of itaconic acid. The presumed aconityl CoA may be formed either through a condensation reaction between acetyl CoA and succinate or acetate with succinyl CoA.     

Oxygen requirement and energy relations of itaconic acid fermentation by Aspergillus terreus NRRL 1960

The effect of interrupting aeration on itaconic acid fermentation by A. terreus NRRL 1960 has been studied. Under the conditions used, stopping aeration for 5 min led to a complete cessation of itaconic acid production, which was only slowly restored after 24 h when aeration was resumed. After a 5-min break in aeration and in the presence of 0.1 mM cycloheximide no itaconic acid was formed even after 3 days. It seems that, upon oxygen shortage, a rapid destruction of the itaconic-acid-producing mechanism takes place, which is restored only aerobically in a slow process involving protein synthesis. Itaconic acid fermentation is also effectively stopped by metabolic inhibitors of ATP formation, pointing to the need for biochemical energy in maintaining the fermentation. ATP is possibly needed to maintain a proper physiological (i.e. near neutral) pH inside the cells, counteracting the acid produced in the fermentation process and the low external pH (below 2.0). Inhibitors of plasma membrane ATPase have no effect on itaconic acid fermentation. This indicates that the plasma membrane might be impermeable to H⁺ and that ATP might rather be involved in the transport of itaconic acid out of the cell. It is suggested that insufficient aeration may leads to insufficient production of ATP which, in turn, leads to damage of the metabolic machinery by acid produced in the fermentation process.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Comparison of air-lift and stirred tank reactors for itaconic acid production by Aspergillus terreus

In a 2-l stirred tank reactor (STR), maximum production rate ofitaconic acid was 0.48g/l.h , for an agitation rate of 400 rpm andan aeration rate of 0.5 vvm. In an air-lift reactor (ALR) themaximum production rate was 0.64 g/l.h at an O supply rate of0.41 l O /l. min. Power input per unit volume which gave themaximum production rates for STR and ALR were 1180 and 542 W/m 3,respectively. If O -enriched air was used in place of air for ALR,the corre-sponding power input per unit volume was decreased to 34W/m 3 . ALR requires less power input per unit volume in comparisonwith that of STR whether therefore air or O -enriched air is used.ALR would be a suitable bioreactor for a large production of itaconicacid.     

Xylanase and itaconic acid production by Aspergillus terreus NRRL 1960 within a biorefinery concept

Production of two industrially important products, xylanase and itaconic acid (IA), by Aspergillus terreus NRRL 1960 from agricultural residues was investigated within a biorefinery concept. Biological pretreatment was applied to lignocellulosic materials by using A. terreus, which produced xylanase while growing on agricultural residues. For IA production, already grown cells were transferred into a new medium. The first step provided not only the pretreatment of lignocellulosic material in order to be used as feedstock but also production of xylanase. For this purpose, cotton stalk, sunflower stalk and corn cob were used as carbon sources as lignocellulosic material. Among them, the highest xylanase production was obtained on corn cob. By application of two-step fermentation, about 70 IU/mL xylanase and 18 g/L IA production levels were achieved. This study shows the stepwise usage potential of the microorganism as a tool in a biorefinery concept.     

Effect of dissolved oxygen concentration and impeller tip speed on itaconic acid production by Aspergillus terreus

Summary Effect of aeration rate and impeller tip speed on mycelium growth and itaconic acid production was investigated in a batch culture of Aspergillus terreus IFO-6365. When impeller tip speed was 94.2 cm/sec at a fixed aeration rate of 0.5 vvm, itaconic acid concentration was 3.6 and 1.6 times higher than those in the impeller tip speed of 62.8 and 125.7 cm/sec, respectively. When an oxygen-enriched air was supplied at a fixed impeller tip speed of 94.2 cm/sec and dissolved oxygen concentration was maintained in the 20–60 % range, both itaconic acid concentration and mycelium growth were not affected by the dissolved oxygen concentration.     

Catabolism of itaconic acid by preformed mats of aspergillus terreus

Summary The breakdown of itaconic acid by preformed mats of a local strain of Aspergillus terreus was studied using the zone-strip technique. Low p_H values restrained the uptake of itaconic acid and the accumulation of various other acids while higher p_H values exerted an opposite effect. The results obtained when using various enzyme inhibitors were those anticipated on basis of the direct transformation of itaconic to aconitic acid through the fixation of CO₂ and of the existence of the T.A.C. (tricarboxylic acid cycle) as a main metabolic channel operating in this organism.     

Flocculation of Aspergillus terreus with polyelectrolyte complex and production of itaconic acid with the flocculated mycelia

Mycelia from Aspergillus terreus K 26 were flocculated with a polyelectrolyte complex consisting of potassium poly9vinyl alcohol) sulfate (KPVS) and poly(diallyldimethyl-ammonium chloride) (PDDA) by three different methods: (a) PDDA was added into the broth obtained from precultivation of the hyphal inoculum in the presence of KPVS; (b) use of KPVS and PDDA was reversed from that in method a; (c) after the precultivation in the absence of the polymer, the mycelia were harvested, dispersed in 0.1 M phosphate buffer containing PDDA, then flocculated by addition of KPVS. The three types of the flocculated mycelia were investigated concerning growth and itaconic acid production in shake flask cultures. Viscosity and sedimentation were further examined to characterize the flocculated mycelial broths. A slight inhibition caused by flocculation on growth and acid production was observed at the beginning of repeated cultivation, but this was eliminated when cultivation in the fresh medium was repeated. There was no marked difference in the specific rates of acid production between fre and flocculated cells. Viscosity of the flocculated mycelial system was close to that of the medium, even while maintaining a cell concentration of 2 g/dl. The poor sedimentation of mycelia was favorably imporved with these flocculation methods, especially with methods b and c.     

ImmobilizedAspergillus terreus in itaconic acid production from glucose

Summary The production of itaconic acid by immobilizedAspergillus terreus TKK 200-5-1 was studied both in shake flask cultures, and in continuous column bioreactors. The effect of glucose and ammonium nitrate concentrations, and of pH were examined using a statistical experimental plan. The highest itaconic acid product concentration could be reached at the highest investigated glucose concentration of 150 g/l and the highest initial pH of 3.75, in the absence of ammonium nitrate. In a continuous packed bed column system operated fro 4.5 months itaconic acid was obtained at a productivity of 328 mg/d per gram of polyurethane foam carrier.