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

Bioresource Technology 308 (2020) 123319

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Second-generation itaconic acid: An alternative product for biorefineries? Antonio Irineudo Magalhães Jr, Júlio Cesar de Carvalho , Juliano Feliz Thoms, Rafaeli Souza Silva, Carlos Ricardo Soccol ⁎

T

Federal University of Paraná, Department of Bioprocess Engineering and Biotechnology, P.O. Box 19011, ZIP Code 81531-990, Curitiba, Paraná, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Biorefinery Lignocellulosic biomass Inhibitory compounds Toxic effect Aspergillus terreus NRRL 1960

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.

1. Introduction The potential of itaconic acid (IA) produced by fermentation has been investigated and demonstrated by several authors as a renewable alternative to classic monomers in the petrochemical industry, such as acrylic and methacrylic acids (Okabe et al., 2009; Willke and Vorlop, 2001). IA can be polymerized by addition or condensation, leading to superabsorbents and specialty polymers, and can also be transformed into many other small molecules (Magalhães et al., 2017). These characteristics, together with the quest to manufacture environmentally friendly chemicals and the reduction of fossil resources, can expand the global itaconate market. IA is industrially produced by sucrose fermentation with the filamentous fungus Aspergillus terreus (Willke and Vorlop, 2001). Although higher yields are obtained when pure glucose is fermented, other substrates can be used to produce IA, such as hydrolyzed starch and xylose (Kautola, 1990; Petruccioli et al., 1999; Yahiro et al., 1997). However, productivity is quite variable, reaching 1.15 g L−1 h−1 for glucose fermentation (Hevekerl et al., 2014) and 0.25 g L−1 h−1 for xylose (Kautola, 1990). The main monomers of cellulose and hemicellulose are glucose and xylose, respectively. These components, together with lignin, constitute the lignocellulosic biomass from the solid residue of feedstock harvest and agroindustry (Magalhães et al., 2019). The production of IA from agricultural residues can be a viable route in biorefineries, but pretreatments and hydrolysis steps are necessary to convert cellulose and hemicellulose into fermentable sugars. Different treatments are ⁎

required to obtain monomeric sugars from lignocellulosic biomass, and the most promising methods are those with the lowest generation of fermentation inhibitors. Recalcitrant biomass material is deconstructed by thermochemical processes, such as acid hydrolysis, steam explosion, or Organosolv, and biochemical processes, such as enzymatic hydrolysis (Medina et al., 2016; Qing et al., 2017; Zhou and Xu, 2019). The primary sugar produced in the thermochemical step is xylose. Still, depending on the temperature, pressure, reaction time, and catalyst concentration, this pretreatment can generate inhibitors, such as furfural, 5-hydroxymethyl furfural (HMF), acetic acid, and other weak acids (Batista et al., 2019). In pretreatments with steam explosion with or without catalyst, for example, the temperatures can vary from 190 to 210 °C in 5 to 10 min, reaching 8 g L−1 of acetic acid, 2.7 g L−1 of formic acid, 2.9 g L−1 of furfural and 0.5 g L−1 of HMF (Bondesson et al., 2013; Zimbardi et al., 2007) while the cellulosic fraction can be extracted by well-established enzyme reactions with glucose production and no inhibitor formation (Gupta and Verma, 2015). Regardless of the biomass, treatment, and hydrolysis, the resulting broth will contain glucose, xylose, furfural, HMF, acetic acid, and other by-products in different concentrations. IA production from a broth containing all these components from treated biomass, without a detoxification process, can lead to a competitive process for biorefineries. A concentration method for the hydrolyzed broth is necessary to achieve an ideal titer of sugars, but that also increases the concentrations of microbial inhibitors. One of the bottlenecks in 2nd generation IA production is finding a balance between high-yield hydrolysis and a

Corresponding author. E-mail address: [email protected] (J.C. de Carvalho).

https://doi.org/10.1016/j.biortech.2020.123319 Received 28 January 2020; Received in revised form 2 April 2020; Accepted 3 April 2020 Available online 05 April 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.

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Table 1 Effect of the substitution of glucose by xylose in different stages of itaconic acid production (sporulation, inoculation and fermentation) by Aspergillus terreus NRRL 1960. Treatment

Residual xylose (g L−1)

Itaconic acid produced (g L−1)

GGG GGX GXX XXX

– 11.40 ± 2.44ab 20.37 ± 3.25a 9.41 ± 6.45b

31.00 16.00 11.72 10.83

± ± ± ±

3.43H 2.33Ic 0.82Icd 2.10Id

Biomass Produced (g L−1)

pH

9.78 ± 0.35 J 11.03 ± 0.12Ke 11.60 ± 0.32KLef 12.66 ± 0.69Lf

1.71 1.66 1.69 1.60

± ± ± ±

0.04 0.05 0.09 0.02

M Mg Mg Mg

GGG (sporulation with glucose, inoculation with glucose, fermentation with glucose); GGX (glucose, glucose, xylose); GXX (glucose, xylose, xylose); XXX (xylose, xylose, xylose). a-g, H-M Different letters in same column indicate significant statistical differences (p < 0.05, Turkey’s test); H-M capital letters to compare all assay (GGG, GGX, GXX, and XXX); a-b lowercase letters to compare only xylose fermentation (GGX, GXX, and XXX).

2. Materials and methods 2.1. Microorganism and culture medium The strain used in this investigation was Aspergillus terreus NRRL 1960, kindly donated by the Agricultural Research Service, (Peoria, USA). Spores were cultured in Czapek DOX agar medium: sucrose 30 g L−1; NaNO3 3 g L−1; K2HPO4 1 g L−1; KCl 0.5 g L−1; Fe2(SO4)3 10 mg L−1; agar-agar 13 g L−1; at 30 °C for 10 days. The method of inoculum production consisted of adding 1.106 spores mL−1 to the adapted medium described by Kuenz et al. (2012): glucose 50 g L−1; (NH4)2SO4 1.25 g L−1; urea 1.15 g L−1; NH4Cl 1 g L−1; MgSO4·7H2O 1 g L−1; CaCl2·2H2O 0.5 g L−1; KH2PO4 0.1 g L−1; CuSO4·5H2O 13.1 mg L−1; ZnSO4·7H2O 8 mg L−1; FeCl3·6H2O 1.67 mg L−1; and pH 3 adjusted with HCl 10%. Inoculum production was performed in 250 mL Erlenmeyer flasks containing 50 mL of media, with agitation at 140 rpm at 33 °C for 48 h. The IA production was performed by adding 10% v/v of inoculum in the fermentation medium, which had the following standard composition (Kuenz et al., 2012): glucose 100 g L−1; CaCl2·2H2O 5 g L−1; (NH4)2SO4 1.25 g L−1; urea 1.15 g L−1; NH4Cl 1 g L−1; MgSO4·7H2O 1 g L−1; KH2PO4 0.1 g L−1; CuSO4·5H2O 13.1 mg L−1; ZnSO4·7H2O 8 mg L−1; FeCl3·6H2O 1.67 mg L−1; and pH 3 adjusted with 10% HCl. The fermentation was carried out under conditions like inoculum production, but with agitation at 120 rpm, for 10 days.

Fig. 1. Kinetics of itaconic acid production from xylose with curve fitting: (square and dashed line) xylose consumed; (circle and dash-dotted line) biomass produced; (diamond and solid line) itaconic acid produced.

limited generation of inhibitors, or economic detoxification of the hydrolysate to avoid inhibition of the fungus Aspergillus terreus in IA production. Despite the recent research involving the production of IA by hydrolyzed wheat chaff and the effect of the inhibition by biomass pretreatment byproducts (Krull et al., 2017a), little is known about the byproducts inhibitory concentrations, and eventual synergistic effects. In second-generation processes, using hydrolysates from lignocellulosic biomass, the concentrations of glucose, xylose, and inhibitors vary wildly. Hydrolysate composition depends on pretreatment, hydrolysis method, and feedstock. We surveyed literature data about hydrolyzed broths of different feedstock residues, obtained by different treatments, in order to determine the typical concentrations of the main components. Based on that survey, this experimental research aimed to evaluate IA production from the xylose-rich broths obtained from typical hydrolysis of lignocellulosic biomasses, using synthetic media to evaluate the effects of the metabolic inhibitors (acetic acid, furfural, and HMF) and the fermentable sugars (xylose and glucose).

2.2. Effect of xylose in itaconic acid production The first stage of this investigation was to analyze the effect of the presence of xylose on IA production, from agar culture to inoculum to fermentation stage, to determine the best production sequence. The assay was performed by replacing glucose at different levels of the process to evaluate the adaptation of A. terreus to IA production from xylose. Thus, four treatments were carried out using as carbon source in the respective stages of sporulation, inoculum production, and fermentation, respectively: glucose-glucose-glucose (GGG treatment);

Table 2 Comparison of itaconic acid production from mixtures of xylose and glucose by Aspergillus terreus NRRL 1960. Initial xylose (g L-1)

Initial glucose (g L−1)

Residual xylose (g L−1)

Residual glucose (g L−1)

Total sugar consumed (g L−1)

Biomass produced (g L−1)

YX/S (%)

Itaconic acid produced (g L−1)

YP/S (%)

pH

25 25 25 50 50 50 75 75 75

25 50 75 25 50 75 25 50 75

1.19 0.93 5.80 1.14 25.55 30.10 25.90 41.10 51.98

0.00 0.00 0.00 0.00 0.23 1.35 0.00 1.08 6.27

48.81 74.07 94.20 73.86 74.22 93.56 74.10 82.81 91.76

11.06 12.28 11.21 12.37 12.41 12.17 12.47 11.79 12.52

22.65 16.57 11.90 16.75 16.72 13.00 16.83 14.24 13.64

6.42 18.84 11.90 19.29 19.56 17.99 17.09 18.12 16.99

13.14 25.43 12.64 26.12 26.35 19.23 23.07 21.88 18.51

2.20 2.03 2.02 1.97 2.12 2.06 2.03 2.03 2.03

2

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Fig. 2. Frequency chart of broth concentrations of (A) xylose; (B) acetic acid; (C) furfural; and (D) HMF, in hydrolysates of different lignocellulosic biomasses using different pre-treatments: (solid line) normal distribution of all data showed; (dotted line) normal distribution of the data showed disregarding the extreme results (acetic acid ≤ 5.2 g L−1, furfural ≤ 2.7 g L−1, HMF ≤ 1.6 g L−1).

glucose-glucose-xylose (GGX); glucose-xylose-xylose (GXX); xylose-xylose-xylose (XXX). The conditions were as described in section (2.1). Mixed carbon sources (glucose and xylose) are expected to be found in hydrolysates from different biomasses or processes. Therefore, the ability to produce IA with variable concentrations of xylose and glucose was analyzed. A complete factorial design (32) was investigated with different concentrations of glucose and xylose (25, 50, and 75 g L−1). The other components of the medium, as well as the fermentation conditions, followed the methodology described in Section (2.1).

experiment were determined by high-performance liquid chromatography (HPLC - Shimadzu Corp., Japan) using an Aminex HPX-87H column (BioRad, Germany) using a column temperature at 25 °C, 5 mM H2SO4 as mobile phase at a net flow rate of 0.06 mL min−1, a refractive index detector, and a UV detector (210 nm).

2.3. Effect of inhibitors in itaconic acid production

A biomass concentration analysis of A. terreus at different stages – sporulation, inoculum production, and fermentation – was performed to identify the carbon source effect on IA production. Although the literature does not make clear which is the ideal composition of medium for A. terreus growth before xylose fermentation, glucose is generally used in sporulation and inoculum production steps (Krull et al., 2017a; Saha et al., 2017). The results of replacing glucose with xylose in the stages of sporulation, inoculum production, and fermentation are shown in Table 1, with statistical analysis of variance, using Turkey’s test (p < 0.05), among the xylose remaining concentration after fermentation, IA production, biomass growth, and pH. The IA production from glucose (GGG treatment) reached approximately 31 g L−1, whereas for xylose fermentation (GGX, GXX, and XXX treatments), the results were between 10 and 16 g L−1, always lower than with glucose. Among xylose fermentations, GGX is better than XXX, while GXX has an intermediate production. These results show that the use of glucose during sporulation is better for IA production than replacing the carbon source with xylose in all stages. IA production, biomass growth, and xylose consumption by A. terreus NRRL 1960 are shown in Fig. 1. The variations in data points are considered typical to A. terreus, with standard deviations that can vary

3. Results and discussion 3.1. Itaconic acid production from xylose

The primary by-products of the pretreatment of lignocellulosic biomass are acetic acid, furfural, and HMF. The average concentrations of these byproducts from acid hydrolysis or steam explosion was determined from a literature survey. From this information, three trials were done with growing concentrations of furfural, HMF, and acetic acid from 0 to, respectively, 380, 700, and 1100 mg L−1. After inoculation, fermentation, and determination of the biomass and IA produced, a sigmoid function was fitted to the results, indicating limiting concentrations of the three by-products. This equation aims to determine the possible concentration values of each inhibitor that causes from the maximum to the reduction of half of the IA production. Values within this range were then chosen for evaluating the synergic effect between acetic acid, furfural, and HMF, using a complete factorial design (23) with one genuine replication. 2.4. Analytical procedures The results were analyzed using the software Statistica 7.0. The glucose and organic acids concentration of the samples from each 3

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fermentation conditions, although with highly controlled agitation and aeration: Saha et al. (2017) obtained about 35 g L−1 of IA using A. terreus NRRL 1960 in 125 mL flasks (25 mL/33 °C/200 rpm); Krull et al. (2017a) reported 16.4 g L−1 of IA with A. terreus DSM 23,081 in microtiter plates (100 μL/33 °C/950 rpm); Kautola et al. (1985) obtained 30 g L−1 of IA with A. terreus NRRL 1960 in stirred tank reactor (10 L/ 36 °C/0.7 g L−1 min−1); Maassen et al. (2014) achieved 27.5 g L−1 of IA with Ustilago maydis MB215 in stirred tank reactor of 3 or 6 L (2 or 2.5 L/30 °C/dissolved oxygen at 80 ± 10%). The effects of using culture media with mixed carbon sources on the IA production, biomass growth, yields, and pH were evaluated through an experimental design with glucose and xylose at a three-level concentration (25, 50, and 75 g L−1). The results are shown in Table 2. There were no significant differences (p < 0.05) in the conditions tested for IA production. It was expected that an increase in both sugars would result in an increase in production yield, which does not occur. According to the data shown in Table 2, sugar concentrations (xylose + glucose) above 75 g L−1 tend to IA production between 17.0 and 19.6 g L−1. The data demonstrated that there is a preference in the fermentation of glucose by A. terreus NRRL 1960, and the presence of xylose induces a decrease in IA production. When only glucose was used in the fermentation (Table 1), IA production reached 31.00 ± 3.43 g L−1 on ten days of fermentation, while for all other tests using xylose, the highest yield was 25.96 ± 1.79 g L−1 (Fig. 1). 3.2. Itaconic acid production with inhibitor compounds A survey of literary data on hydrolyzed broths from various lignocellulosic biomasses (sugarcane bagasse, EFB, straw of wheat and corn) treated by steam explosion or acid hydrolysis was compilated in a frequency graph, shown in Fig. 2, with concentrations of xylose, acetic acid, furfural, and HMF (Alriksson et al., 2011; Bari et al., 2009; Brugnago et al., 2011; de la Torre et al., 2017; Hamzah et al., 2011; Ibrahim et al., 2011; Medina et al., 2016; Oliveira et al., 2013; Rocha et al., 2012; Sakdaronnarong et al., 2017; Sapci et al., 2013; Wanderley et al., 2013; You et al., 2016; Zhu et al., 2011). The average concentration of acetic acid (2.8 ± 1.6 g L−1), HMF (0.6 ± 1.0 g L−1), furfural (0.8 ± 0.6 g L−1) and xylose (14.3 ± 7.1 g L−1) were determined. According to the normal distribution curve of Fig. 2, the concentrations of acetic acid, furfural, and HMF can range up to 7.5, 2.5, and 3.0 g L−1, respectively. If the extreme values of the data are disregarded (acetic acid ≤ 5.2 g L−1, furfural ≤ 2.7 g L−1, HMF ≤ 1.6 g L−1), the expected concentration range for the hydrolyzed broth is of 0–7 g L−1 for acetic acid, 0–1.8 g L−1 for furfural and 0–1.1 g L−1 for HMF. The evaluation of acetic acid, furfural, and HMF concentration on the production of IA and biomass is shown in Fig. 3, with sigmoidal curves that describe the inhibitory behavior of these biomass hydrolysis by-products. Within the range chosen, acetic acid and furfural showed the highest inhibition of IA production. According to Fig. 3A, the maximum inhibition of A. terreus growth and IA production occur when the concentration of acetic acid is 3000 mg L−1. The inhibitory effect of acetic acid is mainly due to the intracellular pH alteration during the

Fig. 3. Inhibition of itaconic acid production with different compounds: (A) acetic acid; (B) furfural; (C) HMF; (squared) itaconic acid; (circle) biomass; (solid line) model sigmoidal of itaconic acid; (dashed line) model sigmoidal of biomass.

to almost 1 g L−1 (Saha et al., 2017). The results show that it took 10 days of fermentation to reach the maximum yield of IA, about 25 g L−1. The biomass production remained close to 14 g L−1, while the residual xylose varied around 10 g L−1. The data obtained in this investigation similar with other studies to those shown by other studies, with a final IA titer between 16 and 35 g/L, under different

Table 3 Comparison of itaconic acid production by Aspergillus terreus NRRL 1960 from glucose with acetic acid, furfural, and 5-hydroxymethylfurfural. Initial acetic acid (mg L−1)

Initial furfural (mg L−1)

Initial HMF (mg L−1)

Residual glucose (g L−1)

Itaconic acid produced (g L−1)

Biomass produced (g L−1)

pH

1100 0 1100 0 1100 0 1100

0 380 380 0 0 380 380

0 0 0 700 700 700 700

45.02 25.34 47.91 23.80 51.22 19.62 47.91

6.73 ± 3.60 9.27 ± 2.37 3.89 ± 0.94 14.34 ± 2.95 4.84 ± 1.36 10.83 ± 4.60 3.83 ± 0.36

10.34 ± 0.20 9.88 ± 0.39 9.47 ± 1.11 9.73 ± 0.03 12.20 ± 1.93 11.04 ± 0.49 9.53 ± 0.01

3.16 2.62 3.27 2.58 3.36 2.53 3.40

± ± ± ± ± ± ±

5.31 6.12 12.06 4.06 2.07 6.62 1.05

4

± ± ± ± ± ± ±

0.35 0.22 0.08 0.07 0.08 0.14 0.05

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diffusion of the undissociated acid through the plasma membrane, deviating from ideal conditions and affecting enzymatic activities (Axe and Bailey, 1995; Pampulha and Loureiro-Dias, 1989; Peláez et al., 2012; Stratford et al., 2009). IA production decreases by 55% with the addition of 375 mg L−1 acetic acid and increasing 72% with 750 mg L−1 of inhibitor concentration, and then the yield dropped again. This decrease/increase effect of low concentration of weak acids is also described by Krull et al. (2017), that investigated the effect of the products of pretreatment degradation of different lignocellulose sugars on A. terreus DSM 23081 production on microtiter plates at 33 °C for 3.8 days at 950 rpm. Acetic acid, formic acid, levulinic acid, furfural, and HMF were investigated at the concentration of 0 to 2000 mg L−1 The addition of 100 mg L−1 of acetic acid resulted in an increased IA concentration of 32.4 to 45.2 g L−1. However, the concentration dropped to 19.1 g L−1 when 500 mg L−1 of acetic acid was added and totally inhibited the production when the concentration was higher than 1000 mg L−1. The decrease/increase effect for IA production can be explained by the accumulation of low concentration of weak acids in the cytoplasm. The production of IA typically begins with pH 3.1 and ends with pH ranging from 1.8 to 2.2 (Krull et al., 2017b). Acetic acid, pKa = 4.76, is undissociated under these conditions. Acetic acid, or other weak acids, are liposoluble to the cell membrane in their undissociated form and insoluble when they are dissociated (Maiorella et al., 1983). The acid diffuses into the cytoplasm and gets ionized due to the pH inside the cell. The typical cytoplasmic pH of fungal mycelia was reported to be close to neutrality, pH 7.0–7.5 (Stratford et al., 2009). Acetate is accumulated in the cytoplasm because it is not liposoluble, causing the reduction of intracellular pH (Axe and Bailey, 1995; Peláez et al., 2012; Stratford et al., 2009). This accumulation is neutralized by extracellular pumping of protons by active transport requiring ATP expenditure (Larsson et al., 1999). This increases cellular metabolism by increasing the production of IA and cell growth. However, the accumulation of a high concentration of acetic acid and the pumping of protons can generate the exhaustion of the energetic reserves of the cell (Russell, 1992). Thus, this results in catastrophic acidification of the cytoplasm, inhibiting various enzymes from metabolism and leading to cell death (Krebs et al., 1983; Pampulha and Loureiro-Dias, 1989; Stratford et al., 2009). In the case of furfural (Fig. 3B), the culture begins to be affected by 1500 mg L−1 and is totally inhibited at concentrations of 2800 mg L−1, for both biomass and IA production. The HMF assays used lower concentrations than the acetic and furfural acid tests (0 to 700 mg L−1). This may have contributed to the fact that HMF did not inhibit cell growth (Fig. 3C). However, fermentation was reduced when the HMF concentration reached 700 mg L−1. In the work of Krull et al. (2017), HMF and furfural had similar effects on IA production with a decrease 70% of IA concentration when 100 mg L−1 of any of these inhibitors were added. Concentrations of 2000 mg L−1 of furfural resulted in only 2.8 g L−1 of IA produced. A. terreus did not grow in the presence of HMF at these concentrations. The main inhibitory effect of furfural and HMF is on the reduction of pyruvate dehydrogenase activity, which may impair the acetyl-CoA pathway in the citric acid cycle of metabolism of A. terreus and reduce IA production (Krull et al., 2017a; Modig et al., 2002). The adapted sigmoid equation that best fit the data for inhibition of x IA production was the Gompertz function ( f (x ) = e e ), where , , 2 , and R for acetic acid, furfural and HMF were, respectively: (38.458; 0.492; 7.462 10−4; 0.82); (7.577 108; 17.127; 1.077 104; 0.93); (28.112; 1.102 10−6; 0.019; 0.80). From this curve it was possible to determine that the concentration of acetic acid, furfural and HMF with potential to inhibit 50% of IA production was 1178, 368 and 702 mg L−1, respectively. The synergistic effect between the inhibitory compounds in the composition of the culture medium, analyzed from the data generated by the sigmoid in Fig. 3, for IA production, biomass growth, and pH are

shown in Table 3. The results are evaluated with the experimental design of furfural, HMF, and acetic acid in a concentration of three levels from 0 to, respectively, 380, 700, and 1100 mg L−1. Analysis of statistical data (p < 0.05) showed that the interaction between acetic acid and furfural affects A. terreus cell growth. The production of biomass and itaconic acid were the worst results close to 3.8 g L−1 and 9.5 g L−1, respectively, in the presence of acetic and furfural acid. The synergy between these hydrolyzed compounds shows that the presence, even in small concentrations, will inhibit the IA production and A. terreus metabolism. However, the presence of HMF favors biomass growth. The consumption of glucose and IA production were inhibited by the presence of acetic acid under the conditions tested. This experiment corroborates the data showed in Fig. 3, demonstrating that only HMF, at concentrations below 600 mg L−1, does not inhibit IA production and that acetic acid and furfural must be detoxified from the broth prepared with hydrolyzed biomass. In the investigation of Roque et al. (2019), the global removal efficiency of acetic acid in detoxified hydrolysate by liquid–liquid extraction were close to 90%. Santana et al. (2018) achieved excellent results in the reduction of the concentration of furfural and HMF by adsorption with activated carbon, the removal was greater than 99%. Other physical, chemical, and biological methods, such as ultrafiltration, neutralization, and adsorption with ion-exchange resins, can be used to remove or minimize the effect of inhibitory compounds (Kumar et al., 2019). In this study, the IA production was reduced in the acetic acid, furfural, and HMF concentrations above 188 mg L−1, 175 mg L−1, and 700 mg L−1, respectively. However, the recommended limit concentrations of a hydrolyzed biomass broth for the IA production are at 0 mg L−1 acetic acid, 90 mg L−1 furfural and 350 mg L−1 HMF by the experimental data, or smaller than 600 mg L−1 HMF by the sigmoid projection. 4. Conclusion The use of xylose to produce itaconic acid (IA) by Aspergillus terreus NRRL 1960 may be recommended, but glucose fermentation is better. The mix of both sugars is not adequate, because xylose decreases the final IA titer from glucose. A. terreus is tolerant up to 350 mg L−1 of 5hydroxymethylfurfural, but concentrations about 200 mg L−1 of acetic acid and furfural reduces the IA yield, with synergistic effect between these two inhibitory compounds over the microbial metabolism. This indicates that separated processes of glucose from xylose with detoxification treatment may be essential for the success to produce secondgeneration itaconic acid. CRediT authorship contribution statement Antonio Irineudo Magalhães Júnior: Conceptualization, Methodology, Investigation, Formal analysis. Júlio Cesar de Carvalho: Conceptualization, Methodology, Formal analysis, Supervision, Project administration. Juliano Feliz Thoms: Investigation, Formal analysis. Rafaeli Souza Silva: Investigation, Formal analysis. Carlos Ricardo Soccol: Resources, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was supported by the National Council of Technological and Scientific Development (CNPq), grants 442271/ 2017-4 and Coordination for the Improvement of Higher Education Personnel (CAPES), PNPD Program. 5

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