Itaconic Acid Production Research Articles

Design of mutualistic microbial consortia for stable conversion of carbon monoxide to value-added chemicals

Carbon monoxide (CO) is a promising carbon source for producing value-added biochemicals via microbial fermentation. However, its microbial conversion has been challenging because of difficulties in genetic engineering of CO-utilizing microorganisms and, more importantly, maintaining CO consumption which is negatively affected by the toxicity of CO and accumulated byproducts. To overcome these issues, we devised mutualistic microbial consortia, co-culturing Eubacterium limosum and genetically engineered Escherichia coli for the production of 3-hydroxypropionic acid (3-HP) and itaconic acid (ITA). During the co-culture, E. limosum assimilated CO and produced acetate, a toxic by-product, while E. coli utilized acetate as a sole carbon source. We found that this mutualistic interaction dramatically stabilized and improved CO consumption of E. limosum compared to monoculture. Consequently, the improved CO consumption allowed successful production of 3-HP and ITA from CO. This study is the first demonstration of value-added biochemical production from CO using a microbial consortium. Moreover, it suggests that synthetic mutualistic microbial consortium can serve as a powerful platform for the valorization of CO.

Analysis of the Talaromyces flavus exometabolome reveals the complex responses of the fungus to minerals

Fungus-mineral interactions influence a variety of geochemical and biological processes on and near the Earth’s surface. The secondary metabolites produced by fungi during these interactions control mineral weathering and affect geochemical element cycling; however, the extent to which minerals exert their influence on fungal metabolism has not been quantitatively determined in detail. Here, we used mass spectroscopy to investigate the exometabolome of Talaromyces flavus, a serpentine soil-inhabiting fungus, during interactions with antigorite, chlorite, hornblende, lizardite, magnetite, and quartz at 28 °C under aerobic conditions. Our experimental results showed that all the minerals used promoted spore germination and hyphal growth. The glucose consumption rate during the initial stages of the fungus-mineral interactions indicated that the metabolic activity of T. flavus was enhanced by a factor of ∼7 by lizardite, antigorite, chlorite, hornblende, and magnetite and by a factor of ∼3 by quartz. A total of 1198 secondary metabolites were detected at 166 h, and 977 (81.55%) of them were common to the T. flavus interactions with antigorite, chlorite, hornblende, lizardite, and magnetite. Statistical and comparative analyses of the 28 most common organic acids revealed that the production of these secondary metabolites by T. flavus was highly mineral-specific and that their content varied significantly in response to the different minerals. Moreover, the component composition of the secondary metabolites of T. flavus interacting with lizardite was more similar to chlorite and magnetite than to antigorite, hornblende, and quartz. Strong correlations were found between the production of gluconic, oxalic, citric, and itaconic acid and the bioavailability of certain elements. Fumaric and orsellinic acid production were stimulated by magnetite and chlorite, respectively. These results demonstrate that fungi can significantly regulate their metabolic behavior during interactions with minerals and that their metabolic regulation is primarily related to the chemical composition, weatherability, and surface properties of the minerals. Such biotic and abiotic responses have important implications for the fungus-induced geochemical transformation of minerals and metals.

Antinociceptive and anti-inflammatory activity of α-d-mannan from Pseudozyma sp.

The online version contains supplementary material available at 10.1007/s13205-020-02635-1.

High level production of itaconic acid at low pH by Ustilago maydis with fed-batch fermentation

The metabolically engineered plant pathogen Ustilago maydis MB215 Δcyp3 Petefria1 has been cultivated to produce more than 80g/L itaconate in 16L scale pH and temperature controlled fermentation, in fed-batch mode with two successive feedings. The effect of pH as well as successive rounds of feeding has been quantified via elemental balances. Volumetric itaconic acid productivity gradually decreased with successive glucose feedings with increasing itaconic titers, with nearly constant product yield. Extracellular pH was decreased from 6 down to 3.5 and the fermentation was characterized in specific uptake, production, and growth rates. Notable is that the biomass composition changes significantly from growth phase to itaconic acid production phase, carbon content increases from 42% to around 62%. Despite the gradual decrease in itaconic acid levels with decreasing pH (nearly 50% decrease in itaconic acid at pH 3.5, compared to pH 6), significant itaconate production is still observed at pH 4 (around 63g/L). Biomass yield remained nearly constant until pH 4. Taken together, these results strongly illustrate the potential of engineered Ustilago maydis in itaconate production at commercial levels.

Bio-synthesis of itaconic acid as an anti-crease finish for cellulosic fiber fabric.

Itaconic acid is an organic acid with a wide range of applications in the fields of polymer chemistry, pharmacy, agriculture, textile industry, etc. A bio-synthetic process of itaconic acid production in this study was carried out with Aspergillus terreus K17 having empty palm oil fruit bunches as a feedstock. Bio-synthesis of itaconic acid was compared with commercial maleic acid, itaconic acid and 1, 2, 3, 4-butanetetracarboxylic acid (BTCA) as anti-crease agents with sodium hypophosphate (SHP) as an esterification catalyst for cotton fabric finishing. The results showed that mechanical properties of cotton fabrics treated with bio-synthesized itaconic acid were better than those treated with the commercial ones whereas their whiteness index was lower. The best conditions for crease recovery were obtained from 8% w/v itaconic acid with 8% w/v SHP applied on cotton fabrics with a technique of 2-dip-2-nip, dried at 85 °C for 3 min and cured at 180 °C for 2 min. Even though the anti-crease properties of cotton fabrics treated with bio-synthesized itaconic acid were still lower than those treated with commercial maleic acid and BTCA, the finished cotton fibers retain the mechanical properties of cotton fabric. This study would be beneficial in producing itaconic acid as an eco-friendly anti-crease agent for cotton fabrics from waste empty palm oil fruit bunches by a bio-synthesis process.

An Optimized Ustilago maydis for Itaconic Acid Production at Maximal Theoretical Yield.

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillus terreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed.

Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis

BackgroundItaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation.ResultsThe efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation.ConclusionThis study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Aspegillus terreus: From Soil to Industry and Back.

Aspergillus terreus is an important saprophytic filamentous fungus that can be found in soils. Like many other soil microorganisms, A. terreus demonstrates multiple functions and offers various important metabolites, which can be used in different fields of human activity. The first application of A. terreus on an industrial level is the production of itaconic acid, which is now considered as one of the most important bioproducts in the Green Chemistry field. The general schemes for itaconic acid production have been studied, but in this mini-review some lines of future research are presented based on analysis of the published results. A. terreus is also intensively studied for its biocontrol activity and plant growth-promoting effect. However, this microorganism is also known to infect important crops such as, amongst others, rice, wheat, potato, sugar cane, maize, and soybean. It was suggested, however, that the balance between positive vs. negative effects is dependent on the soil-plant-inoculant dose system. A. terreus has frequently been described as an important human pathogen. Therefore, its safety manipulation in biotechnological processes for the production of itaconic acid and some drugs and its use in soil-plant systems should be carefully assessed. Some suggestions in this direction are discussed, particularly concerning the uses in crop production.

Economic Assessment of Itaconic Acid Production from Aspergillus Terreus using Superpro Designer

Itaconic acid is a metabolite produced from biotechnological pathway using the microorganism Aspergillus terreus. It is used in a wide number of sectors such as industry, agriculture and health. Based on its chemical properties, itaconic acid is a great interest for replacing polymers and resins derived from petroleum. However, its production is still expensive and this latter hinders large scale studies regarding itaconic acid production. Nowadays, simulation is a great alternative to overcome this problem. It allows researchers to make a more effective evaluation and self-assessment, reducing costs and avoiding losses at large scale operation. For that reason, the purpose of this research is performing an Itaconic acid production simulation on industrial scale using SuperPro Designer software. Costs reduction improvement is also analyzed. 90g/L of glycerol, 1g/L of NH4NO3 and 1.25 of KH2PO4 are set up on simulation as media composition reported from references. Results regarding itaconic acid crystals showed a productivity of 171 kg/h and an operating cost closed to 42.0 USD/kg. Interestingly, a 17 % productivity increase is reached by proposing a stream recycling based on itaconic acid recovering from downstream centrifugation processing. The latter based on a higher productivity estimated (200 kg/h). Also average costs are reduced at 12 % since 38.1 USD/kg is found using improvements mentioned. Results found here demonstrate the potential usage of simulators for estimating costs and production which allows predicting the bioprocess feasibility.

Next-generation itaconic acid production using novel Aspergillus japonicas from Citrullus lanatus rind through solid-state fermentation

Itaconic acid production was investigated by using isolated A. japonicas on pretreated Citrullus lanatus rind under solid-state fermentation. The isolated elite fungal strain A. japonicas showed maximum itaconic acid production was identified by using 18 S rRNA sequencing method. Citrullus lanatus rind was collected and pretreated using microwave radiation and its impact on itaconic acid production was investigated. Further, the effect of parameters like incubation period, inoculum percentage, moisture content, several nitrogen sources, nitrogen and KH2PO4 concentration were explored. Pretreated Citrullus lanatus rind exhibited maximum itaconic acid production of 17.68 ± 0.65 g of itaconic acid/kg of CLR. The optimum level of incubation period and inoculum percentage was found to be 192 h and 6% (v/w), respectively. Further, 60% (w/w) of moisture content, 2% (w/w) of peptone and 0.6% (w/w) of KH2PO4 resulted in 21.52 ± 1.87 g itaconic acid/kg of CLR was produced using novel A. japonicas.

Investigation of Itaconic Acid Separation by Operating a Commercialized Electrodialysis Unit with Bipolar Membranes

Nowadays, the merging of membrane and fermentation technologies is receiving significant attention such as in the case of itaconic acid (IA) production, which is considered as a value-added chemical. Its biotechnological production is already industrially established; however, the improvements of its fermentative and recovery steps remain topics of significant interest due to sustainable development trends. With an adequate downstream process, the total price of IA production can be reduced. For the task of IA recovery, a contemporary electro-membrane separation processes, electrodialysis with bipolar membranes (EDBM), was proposed and employed in this work. In the experiments, the laboratory-scale, commercialized EDBM unit (P EDR-Z/4x) was operated to separate IA from various model solutions compromised of IA (5–33 g/L), glucose (varied in 15–33 g/L as a residual substrate during IA fermentation) and malic acid (varied in 0–1 g/L as a realistic by-product of IA fermentation) under different initial pH (2–5) and applied potential conditions (10–30 V). Unambiguously negative effects related to the glucose and malic acid as impurities were found neither on the IA recovery ratio nor on the current efficiency, falling into the ranges of 90–97% and 74.3–98.5%, respectively. The highest IA recovery ratios of 97% and 98.5% of current efficiency were obtained with the model fermentation solution containing 33 g/L IA, 33 g/L glucose at 20 V and an initial pH of 5. However, the selective separation of IA needs further investigations with a real fermentation broth, and the findings of this research may contribute to further studies in this field.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Valorizing a hydrothermal liquefaction aqueous phase through co-production of chemicals and lipids using the oleaginous yeast Yarrowia lipolytica

Hydrothermal liquefaction is a promising technology to upgrade wet organic waste into a biocrude oil for diesel or jet fuel; however, this process generates an acid-rich aqueous phase which poses disposal issues. This hydrothermal liquefaction aqueous phase (HTL-AP) contains organic acids, phenol, and other toxins. This work demonstrates that Y. lipolytica as a unique host to valorize HTL-AP into a variety of co-products. Specifically, strains of Y. lipolytica can tolerate HTL-AP at 10% in defined media and 25% in rich media. The addition of HTL-AP enhances production of the polymer precursor itaconic acid by 3-fold and the polyketide triacetic acid lactone at least 2-fold. Additional co-products (lipids and citric acid) were produced in these fermentations. Finally, bioreactor cultivation enabled 21.6 g/L triacetic acid lactone from 20% HTL-AP in mixed sugar hydrolysate. These results demonstrate the first use of Y. lipolytica in HTL-AP valorization toward production of a portfolio of value-added compounds.

Production of Itaconic Acid from Carbohydrate-Rich Material by using Fungi

Production of Itaconic Acid from Carbohydrate-Rich Material by using Fungi

Second-generation itaconic acid: An alternative product for biorefineries?

The ability to produce second-generation itaconic acid by Aspergillus terreus, and the inhibitory effects of hydrolysis by-products on the fermentation were evaluated by cultivation in a synthetic medium containing components usually present in a real hydrolysate broth from lignocellulosic biomasses. The results showed that A. terreus NRRL 1960 can produce itaconic acid and consume xylose completely, but the conversion is less than the fermentation using only glucose. In addition, compared to fermentation of glucose, or even xylose, the mix of both sugars resulted in a lower itaconic acid yield. In the inhibitory test, the final itaconic acid titer was reduced by acetic acid, furfural, and 5-hydroxymethylfurfural concentrations of, respectively, 188, 175, and 700 mg L−1. However, the presence of any amount of acetic acid proved to be detrimental to itaconic acid production. This research sheds some light on doubts about the biorefinery implementation of itaconic acid production.

Production of Itaconic Acid from Cellulose Pulp: Feedstock Feasibility and Process Strategies for an Efficient Microbial Performance

This study assessed the feasibility of using bleached cellulose pulp from Eucalyptus wood as a feedstock for the production of itaconic acid by fermentation. Additionally, different process strategies were tested with the aim of selecting suitable conditions for an efficient production of itaconic acid by the fungus Aspergillus terreus. The feasibility of using cellulose pulp was demonstrated through assays that revealed the preference of the strain in using glucose as carbon source instead of xylose, mannose, sucrose or glycerol. Additionally, the cellulose pulp was easily digested by enzymes without requiring a previous step of pretreatment, producing a glucose-rich hydrolysate with a very low level of inhibitor compounds, suitable for use as a fermentation medium. Fermentation assays revealed that the technique used for sterilization of the hydrolysate (membrane filtration or autoclaving) had an important effect in its composition, especially on the nitrogen content, consequently affecting the fermentation performance. The carbon-to-nitrogen ratio (C:N ratio), initial glucose concentration and oxygen availability, were also important variables affecting the performance of the strain to produce itaconic acid from cellulose pulp hydrolysate. By selecting appropriate process conditions (sterilization by membrane filtration, medium supplementation with 3 g/L (NH4)2SO4, 60 g/L of initial glucose concentration, and oxygen availability of 7.33 (volume of air/volume of medium)), the production of itaconic acid was maximized resulting in a yield of 0.62 g/g glucose consumed, and productivity of 0.52 g/L·h.

Exploring the Brazilian diversity of Aspergillus sp. strains for lovastatin and itaconic acid production

Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98.5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its biosynthesis.

Itaconic acid production from undetoxified enzymatic hydrolysate of bamboo residues using Aspergillus terreus

Itaconic acid (IA) production by fermentation of undetoxified hydrolysate of bamboo residues by Aspergillus terreus was demonstrated. Monosaccharides were obtained by pretreatment and enzymatic hydrolysis of bamboo residues. A. terreus could not grow and synthesize IA in the hydrolysate. The buffer was confirmed to be an inhibitor, and was successfully replaced by deionized water as the suspension, to release equivalent sugar and eliminate the inhibition. Corn steep liquor significantly improved the adaptability of A. terreus to the hydrolysate at 2.0 g/L. The IA titer obtained (19.35 g/L IA) was the highest to be reported for IA production from lignocellulose without detoxification. Simultaneous saccharification and fermentation and fed-batch fermentation increased the titer to 22.43 g/L and 41.54 g/L, respectively. Meanwhile, economic assessment proved that bamboo residues were potential substrates for IA production with economic effectiveness.

Deletion analysis of the itaconic acid biosynthesis gene cluster components in Aspergillus pseudoterreus ATCC32359.

The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clone-based transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), amitochondrial organic acid transporter (mttA) and anMFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays aregulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars inlignocellulosic biomass.

Accelerating itaconic acid production by increasing membrane permeability of whole-cell biocatalyst based on a psychrophilic bacterium Shewanella livingstonensis Ac10

Whole-cell biocatalysts have numerous advantages including ease of preparation and coenzyme recovery over purified industrially used enzymes. However, the cell membrane can occasionally hinder cytoplasmic diffusion of the substrate, resulting in reduced biotransformation efficiency. Psychrophiles can grow and reproduce at low temperatures; their cell membranes are highly flexible, and their permeability can be improved via heat treatment at a moderate temperature. The aim of this study was to generate a psychrophile-based simple biocatalyst (PSCats) using Shewanella livingstonensis Ac10. This biocatalyst contained two enzymes that were heterologously expressed and converted citric acid to itaconic acid, thereby serving as a potential platform replacing the petroleum-based counterparts. The efficiency of the biocatalyst was increased via heat treatment at 45 °C for 15 min, and itaconic acid productivity of the cells after heat treatment (1.41 g/L/h) was increased around 6-fold in comparison with those without heat treatment (0.22 g/L/h). A large part of the productivity remained (67.3 %) when the cells were reused for 5 times (10 h for each reaction). Therefore, the potential of this heat-permeabilized psychrophile host to increase the productivity of whole-cell biocatalyst was proved; however, further research is necessary to understand the underlying mechanism.