Production of Itaconic Acid Through Microbiological Fermentation of Inexpensive Materials

Itaconic acid (IA), also known as methylenesuccinic acid, is widely used in many fields such as water treatment, adhesive, synthetic resin and medicine (Yang and Li, 2009). In the report 'Top Value Added Chemicals from Biomass' released by the US Department of Energy (DOE) in 2004, the IA was selected as one of the 12 most promising platform chemicals that can be converted to value added chemicals (Steen et al., 2010), thereby contributing to meeting energetic and economic goals. The global production of the IA in 2011 was 41 400 t, and that number is projected to increase to 407 790 t by 2020, with a value of approximately 5.7×10⁸ US dollar (Weastra, 2013). At present, the IA is produced mainly via a bio- fermentation process by strains such as Aspergillus terreus with sugar or starch as raw materials. When the A. terreus is used, the yield of the IA from monosaccharides is higher than that from polysaccharides, from which glucose gives the highest yield (Kuenz et al., 2018). However, glucose-based media are too expensive for industrial production. From the perspective of raw material sources and environmental protection, the development of renewable raw materials with diverse sources and low prices to decrease costs is becoming a focus research on IA production through microbial fermentation.

Agriculture and forestry residues mainly include agricultural straw, agricultural processing residues, and forestry products and processing residues. In 2015, the annual number of major agricultural and forestry residues in China was approximately 1.11×10⁹ t, which is equivalent to 5.66×10⁸ t of standard coal, with the available resources of approximately 4.11×10⁸ t (Zhang, 2018). Although agricultural and forestry residues have many advantages, such as large quantities, broad sources, cleanliness, and sustainability, they have long been discarded, burned, or placed in landfill, thus not only wasting resources but also causing environmental pollution. In the presence of global energy crisis and environmental deterioration, chemicals from lignocellulosic resources have become a major topic in the development and utilization of biomass resources. With the increased governmental support for new renewable energy industries, as an important component of biomass resources, agricultural and forestry residues have great potential for conversion to biofuels and chemicals. These applications may be highly important in alleviating energy shortages, improving the environment, accelerating afforestation, and preventing land loss in China. In this paper, research progress on the IA production from alternative raw materials is reviewed. In particular, the current status of types of alternative raw materials as well as existing problems in the IA production, screening of strains capable of fermenting the alternative raw materials, and regulation of fermentation process are also reviewed.

Owing to an unsaturated double bond and two carboxyl groups (Fig. 1), the IA is highly reactive and can be self-polymerized or polymerized with other monomers such as acrylonitrile, and can also be subjected to esterification and addition (Karaffa et al., 2015). The IA is an important chemical raw material and widely applied to many fields, including water treatment, adhesives, synthetic resins, pharmaceuticals, pesticides, antibacterial agents, cosmetics, and optical materials (Delidovich et al., 2016; Bafana et al., 2018) in the form of itaconic sodium salt or esters (Blazeck et al., 2015). In case of the lower production cost by replacing sugar raw materials with renewable resources such as lignocellulose, the IA could be used to replace petrochemical-based polyacrylic acid and to produce methyl methacrylate (a thermoplastic material) and 3-methyltetrahydrofuran (a potential biofuel) (EI-Imam, 2014).

Figure 1.  The IA structure

According to a report from the US DOE, the production cost of the IA by fermentation would need to decrease to at least 0.5 dollar per kg from 1.8-2.0 dollar per kg to be economically competitive with petrochemical- base products. For organic acid products, raw material is a major factor causing high production cost. Currently, the market price of glucose is approximately 0.35-0.60 dollar per kg, and the price of sucrose is 0.45-0.72 dollar per kg (Choi et al., 2015). Therefore, seeking cheaper and non-food renewable raw materials is one of the best strategies to decrease the production cost of the IA. At present, alternative materials for the IA fermentation used in industry or research are shown in Fig. 2.

Figure 2.  Alternative raw materials for IA fermentation

The optimal raw material for the IA fermentation is glucose. Other sugars such as xylose and sucrose can be utilized by microorganisms, but with relatively low yield and conversion rate. Saha et al. (2017) have evaluated the yield of the IA from different monosaccharides by 100 A. terreus strains, in which 20 strains can synthesize the IA from xylose and arabinose, and the A. terreus NRRL 1971 could produce 36.4 g/L IA from mannose. As alternatives to pure sugar, industrial wastes, such as glycerin and ethanol, are also used in the IA production (Fig. 2). Zambanini et al. (2017) reported that Ustilago vetiveriae TZ1 produced 34.7 g/L IA and 46.2 g/L malate from glycerinum. The results of Kim et al. (2017) showed that the activity of both aconitase and aconitase decarboxylase is enhanced by controlling the expression of multiple cadA genes in Escherichia coli, and 319.8 mmol/L (41.6 g/L) IA was produced from 500 mmol/L citrate without using any buffer system and additional cofactors. Dowlathabad et al. (2007) studied the influence of temperature, stirring speed, and pH on fermentation with Jatropha seeds as carbon source. After a 120-hour fermentation, the maximum yield of the IA was 24.45 g/L.

Starch is considered one of the best alternatives to glucose, given its high purity, low cost, and source stability. When corn starch was hydrolyzed by saccharification enzyme or acid, the IA yield can reach 0.35-0.36 g/g starch (Delidovich et al., 2016). Corn starch hydrolyzed by nitric acid can be directly used for the IA production. Yahiro et al. (1997) reported that 60 g/L IA was produced with A. terrqus TN-484 in flask fermentation using corn starch partially hydrolyzed by nitric acid as the raw materials, without any additional nitrogen source or nutrients. The yield was similar to that of glucose and was successfully reproduced in a 2.5-liter airlift reactor. Gnanasekaran et al. (2018) isolated Aspergillus niveus MG183809 from soil, which can directly synthetize the IA from corn starch, wheat flour, and sweet potato with a maximum yield of (15.65±1.75) g/L.

Lignocellulose is also an alternative raw material for the IA fermentation, given that it is broadly distributed, clean, sustainable, and renewable. Recent studies have been focused on the utilization of agricultural materials such as bran, wheat husk, corn cob, and straw. EI-Imam et al. (2015) used acid hydrolysate of sorghum bran for the IA fermentation, and found that the hydrolysate had no effect on the growth of A. terreus, but the yield of the IA was only one-quarter that of glucose. Kocabas et al. (2014) assessed the performance of A. terreus NRRL1960 in producing xylanase and the IA from corn cob, cotton stalk, and sunflower stalk. In a study from Sun (2016), sugar hydrolyzed from wheat bran by dilute acid hydrolysis was fermented to produce the IA, and 26.72 g/L IA was gained after 144-hour fermentation with 100 g/L concentrated sugar, which was further increased to (39.14±0.43) g/L by the optimal regulation in fermentation process. Saha et al. (2018) investigated the IA production by six A. terreus strains with dilute-acid hydrolysate (with 41 g/L glucose, 30 g/L xylose, and 4.4 g/L arabinose) of wheat straw, whereas all A. terreus were unable to grow in the hydrolysate or to produce any IA. When 80 g/L sugar mixture (glucose, xylose and arabinose) was added to the 10-fold diluted hydrolysate, the strains grew and consumed most of sugar, but no IA was produced. Only when the strains were cultured in 1000-fold diluted hydrolysate with additional sugar mixture, about half of the IA yield was gained. Jimenez-Quero et al. (2016) utilized 2 A. terreus strains to produce the IA in 10-fold diluted wheat bran and corn cob hydrolysate with glucose added, but no IA was obtained. Pedroso et al. (2017) reported that 1.9 g/L IA was synthesized with detoxified phosphoric acid hydrolysate of rice bran, with a yield of 49 mg/g sugar. In addition, Krull et al. (2017a) gained 0.6 g/L IA from hydrolysate of wheat husk pretreated with sodium hydroxide at room temperature followed by washing. And 27.7 g/L IA was subsequently produced from detoxified hydrolysate.

In addition, several studies have investigated the IA fermentation with forest residues as substrate. In 1978, Kobayashi (1978) first proposed that wood waste hydrolysate could be used to produce the IA. With 13 g/L xylose and lower than 2 g/L glucose released from hemicellulose hydrolysate of beech wood, Ustilago maydis produced 0.36 g/L IA after 10-h lag period; and when 30 g/L additional glucose was added, the lag period was shortened to 6 h and the yield increased to 4.1 g/L. The study demonstrated that comprehensive utilization of cellulose and hemicellulose in beech wood for the IA production was feasible (Klement et al., 2012). In addition, A. terreus can also synthesize the IA with xylose and arabinose (Saha et al., 2017), which laid a foundation for the full utilization of both hemicellulose and cellulose part in the lignocellulosic materials. Tippkötter et al. (2014) found that A. terreus NRRL 1960 can not grow in the enzymatic hydrolysate of beech wood pretreated with organic solvent without detoxification. Yang (2018) has investigated the feasibility of producing the IA from bamboo wastes and found that bamboo hydrolysate processed by pretreatment and enzymatic hydrolysis could not be directly used for the growth and IA production by A. terreus.

Utilization of alternative materials other than pure sugars may introduce unintended negative effects. For example, the inherent phosphorus in potato starch can prolong the growth cycle of A. terreus and hence decrease the yield of the IA (Petruccioli et al., 1999). Similarly, the inherent nitrogen in plant-derived hydrolysate may affect the IA production by low nitrogen-demanding U. maydis (Klement et al., 2012). For lignocellulosic materials, cellulose and hemicellulose are usually degraded to fermentable sugars by pretreating and hydrolyzing before further conversion into the IA. Nevertheless, the pretreatment may produce multiple inhibitors such as formic acid, acetic acid, furfural, hydroxymethylfurfural (HMF), and levulinic acid, which influence to some extent the microbial growth and IA production. Moreover, enzyme (Krull et al., 2017a) and buffer solution (Yang, 2018) used in the hydrolysis, manganese and other metal ions, as well as certain salts in the hydrolysate could also bring unfavorable effects (Karaffa et al., 2015). Krull et al. (2017a) found that 0.8 g/L acetic acid strongly inhibited the IA production by A. terreus NRRL 1972, 1.0 g/L acetic acid fully inhibited the growth of A. terreus DSM23081, and formic acid, the HMF as well as furfural, even at a low concertation (0.1 g/L), could still suppress the strain growth and metabolism. In the study from Saha et al. (2018), enzymatic hydrolysate of wheat straw pretreated with dilute acid contained 2 g/L acetic acid, 1.6 mmol/L HMF, and 9.7 mmol/L furfural, as well as calcium, magnesium, sodium, potassium, sulfur, aluminum, iron, manganese, and copper, etc. And it was found that 4.0 mmol/L HMF, 15.0 mmol/L furfural, 0.35 g/L magnesium and 0.05 g/L copper ions could partially inhibit the growth and IA production of all six A. terreus strains, and 2.5 g/L acetic acid, 0.05 g/L iron and 0.0001 g/L manganese completely inhibited the IA production. Meanwhile, the fermentable sugar is usually a mixture of glucose, xylose, arabinose and galactose, nevertheless glucose is preferred by A. terreus and pentose is subsequently consumed only when glucose is almost exhausted. Thus, besides for the inhibition factors in the lignocellulosic hydrolysate, how to efficiently use hexose and pentose must also be addressed (Yang, 2018).

At present, the strategies to alleviate the inhibited factor in lignocellulosic hydrolysate include acid-base neutralization, protein denaturation by heating, metal ion removal by ion exchange, activated carbon adsorption, and other detoxification methods. Nevertheless, the IA yield is still not comparable to that of produced from sugar and the production cost will be increased simultaneously. To address this issue, further studies may be focused on breeding resistance strains for lignocellulose hydrolysate and developing more economical and effective fermentation technologies.

At present, physicochemical mutagenesis, protoplast fusion, and genetic engineering are major breeding strategies for high-yield IA-producing strains. However, most studies mainly take glucose as substrate directly, and only few researchers studied the breeding using low-cost alternatives materials. Reddy et al. (2002) isolated A. terreus SKR10 from garden waste, which could produce the IA from fruit scraps and starch, with a yield of 20 g/L, 28.5 g/L (acid hydrolyzed corn starch as substrate), and 31 g/L (enzymatic hydrolyzed corn starch as substrate), respectively. With nitrosoguanidine, colchicine, and sodium azide mutagenesis, the yield from fruit scrapes reached 31-32 g/L and further increased to 46 g/L and 50 g/L from acid hydrolyzed and enzymatic hydrolyzed corn starch, respectively. The A. terrqus T730 was treated with 20 keV low-energy hydrogen ion beam and nitrogen ion beam for 50-150 s. As a result, a strain with resistance to high concentration of sucrose and high IA yield was isolated, which could produce 9.5% and 11.5% IA from 15% and 20% sucrose respectively when cultured in shaking flasks for 4-5 d, and got a stable IA yield in a 10-L fermentation tank (Yang et al., 2003). To obtain strains for the IA production from starch, Kirimura et al. (1997) performed protoplast fusion of A. terrqus and Aspergillus usamii, and got a stable fusant F-112 which was similar to A. terreus in morphology and could produce 35.9 g/L IA with 120 mg/mL soluble starch, which was five times higher than that of A. terrqus. Zhu (2003) obtained a stable fusant F3 by protopast fusion of A. terrqus T-730 and citric acid-producing Aspergillus niger Ni-5K. The strain directly produced 40.9 g/L IA from 10% original starch, with a conversion rate of 40.9%. Li et al. (2016) screened an A. terreus strain by plasma mutagenesis under normal temperature and pressure, which produced 19.30 g/L IA from the hydrolysate of undetoxified corn straw pretreated with steam explosion, whereas the yield decreased to 0.54 g/L by the wild strain.

Genetic engineering is another effective means for improving the IA yield and environmental adaptability of strains. Recent studies consider that in A. terreus, the IA is formed from aconitate through cis-aconitate decarboxylase (CAD) in Tricarboxylic acid cycle with pyruvate as the primary precursor (Klement et al., 2013), which is supported by the isolation of the CAD and the identification of associated genes (Fig. 3) (Li et al., 2011). These studies provide the basis for the breeding of the IA high-yield strain by genetic engineering. Hossain et al. (2016) inserted the cadA gene of A. terreus into AB1.13 to obtained low yield of the IA, which was further increased by five times when both mttA and mfsA genes were overexpressed in AB1.13. The results confirmed that the IA synthesis was associated with these three gene pairs. Given the high ability to synthesize citric acid precursor of the IA, A. niger and Yarrowia lipolytica (Blazeck et al., 2015) are usually used as starting strains for genetic engineering. Meanwhile, Saccharomyces cerevisiae (Blazeck et al., 2014) and E. coli (Harder et al., 2016; Jeon et al., 2016), were also applied to genetically engineered for the IA production because of the clear genetic background. By the heterogeneous expression of key genes, the IA was synthesized in these microorganism, yet with relative low yield. When the modified pfkA gene from A. niger was inserted into A. terreus, the IA yield increased from 13.5 g/L to 31 g/L in the screened transformant (Tevž et al., 2010). Usually, microbes are unable to synthesize the IA directly from lignocellulose. However, Zhao et al. (2018) heterologously expressed the CAD gene of A. terreus in Neurospora crassa, directly synthesized (20.414±0.674) mg/L IA from cellulose, and the IA yield from corn stalk and switchgrass reached (10.4±0.4) mg/L and (8.6±0.2) mg/L, respectively. In addition, the study also demonstrated that the secreted cellulase was key to the direct conversion of cellulose to the IA. For U. maydis, cyp3-encoded P450 monooxygenase catalyzes the synthesis of the IA byproducts. When the cyp3 gene was deleted, and the ria1 gene regulator group was overexpressed, the IA yield increased by 3.5 times over that of the wild strain, and with no byproducts produced (Geiser et al., 2016).

Figure 3.  Biosynthetic routes of IA in Aspergillus terreus (Li et al., 2011)

Compared with the high yield (360 g/L) of citric acid, the low yield of the IA is a major constraining factor for its industrialization. Nutrient, fermentation method and conditions all affect the IA synthesis. As a strictly aerobic process, 1.5 mol oxygen is typically required to produce 1 mol IA, and hypoxia can decrease the adenosine triphosphate (ATP) concentration and further disrupt the balance between reduced form of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide (NAD⁺), decreasing the activities of citrate synthase and phosphofructokinase, and ultimately decreasing the IA yield (Zhao et al., 2018). When hemoglobin gene related to the oxygen transport was expressed in the IA producing strain, the intracellular oxygen transport capacity was improved, the IA yield could be increased while corresponding byproducts was decreased (Lin et al., 2004; Li et al., 2013).

Opinions differ regarding the influence of pH on the IA synthesis. Hevekerl et al. (2014) suggested that pH could affect the spore-germination rate. When the pH was lower than 3.0, the spore germination rate decreased and further affected the IA synthesis. In the fermentation process, the cells consume ammonium ions, and protons were released, thus resulting in the decrease in pH (Mattey, 1992). Larsen et al. (1955) argued that the enzyme system associated with the IA synthesis was activated in lower pH, and the IA can not be synthesized at pH 6.0. A study on the effects of sugar composition of the simulated lignocellulosic hydrolysate on the IA synthesis by A. terreus suggested that the synthesis process was favored at a pH between 2.0 and 3.1, in which the cell biomass and sugar utilization were relatively high (Saha et al., 2019). With increasing pH, the dissociation form of the IA changes from H₂IA to HIA^-, and eventually became IA2^-. And the HIA^- was associated with the final yield of the IA. When the pH was adjusted to 3.4 for the synthesis phase of the IA, the highest yield of 160 g/L was finally produced (Krull et al., 2017b). In industrial production, lower pH helps prevent contamination of the fermentation broth and inhibits the formation of byproducts.

In terms of nutrients, besides carbon source, the requirement for other nutrient ingredients slightly differs among microorganisms. Nitrogen is one of the most important nutrients, and ammonium nitrate and ammonium sulfate are the most common used. One Papagianni, (2007) suggested that a high carbon-nitrogen ratio prevented phosphofructokinase from being inhibited by high-energy charge and hence facilitates the IA synthesis. However, Vassilev et al. (1992) reported that in the fermentation process of fixed cells, the highest production rate of the IA appeared in continuous cultivation without nitrogen sources. The A. terreus required an optimal concentration range of nitrogen for the IA production, and the substrate utilization and the IA yield were decreased when exceeded. In contrast, U. maydis favored nitrogen- limited conditions, which would synthesize the IA only when nitrogen was exhausted (Klement et al., 2012). Kuenz et al. (2012) found that during the exponential growth phase of microbe, excessive phosphorus concentration increased intracellular ATP levels and energy charge, which would result in accelerated glycolysis, vigorous growth of bacteria and dense mycelium, thus leading to high medium viscosity. While proper phosphorus favored bacterial growth and the IA production. Meanwhile, as auxiliary nitrogen source, corn starch could improve the synthetic rate of the IA mainly due to the promotion of cell growth and IA production in advance by biotin (Xu et al., 1981), and hence the fermentation period was shortened. And Yang et al. (2018) found that appropriate corn syrup could enhance the adaptive capacity of A. terreus toward bamboo lignocellulose hydrolysate.

Global interests in renewable materials have accelerated the development of new technologies related to functional platform chemicals and fuels, and industrial manufacture driven by biotechnology need to adapt to new challenges. The IA production at higher yield with cheaper and alternative biomass materials has been recognized as a feasible strategy for the industrialization of the IA. Presently, compared with citric acid (> 200 g/L), the lower yield of the IA is one of the factors restricting its industrialization. Further studies could be focused on the inherent inhibition mechanism of the IA synthesis by genome, transcriptome, proteome, metabolome as well as synthetic biology approaches. In addition, the IA yield can be enhanced by improving key metabolic enzymes, blocking byproduct metabolic pathways, and increasing the carbon flux of the IA metabolic pathways. Moreover, cheap and renewable non-food raw materials could be adopted to lower the production cost of the IA. Finally, optimizing the process of converting lignocellulose into sugar, minimizing the influence of toxic substances in the hydrolysate, and screening strains with high tolerance should be focused in the future research.