Metabolic patterns of A. niger M13 on acetic acid
Acetic acid makes the fermentation broth acidic. It can inhibit the growth of fermentation microorganisms. Undissociated organic acid molecules are lipid-soluble and can freely diffuse and penetrate into cells. They further dissociate into H+ in the cells, affecting intracellular acid-base balance. To maintain stable intracellular pH, microorganisms have to actively transport H+ from inside the cell to the outside, which is an energy-consuming process. This process requires a large amount of intracellular ATP consumption, leading to a lack of ATP and inhibition of normal microbial growth. In severe cases, it can even cause cell apoptosis[19]. The effects of different initial concentrations of acetic acid on the growth and fermentation of A. niger M13 are shown in Figure 1. When the initial concentration of acetic acid is less than 5.3 g/L, M13 consumes acetic acid in the fermentation broth within 72 h, with the highest consumption rate in 48-72 h. When the initial concentration of acetic acid is around 7.5 g/L, M13 completely consumes the acetic acid in 192 h. As shown in Figure 1B, under the conditions of fermentation for 12 days with acetic acid concentrations of 2.2, 3.1, 5.3, and 7.5 g/L, the utilization rate of glucose decreased by 16.18%, 26.86%, 32.39%, and 38.97%, respectively, compared with the control group. Meanwhile, when acetic acid was present, the efficiency of citric acid production was slow. As shown in Figure 1C, the rate of citric acid production increased with the rapid depletion of acetic acid after 48-72 h. In the control group, the fermentation produced about 17.50 g/L of citric acid in 12 days. The production rate of citric acid in the media containing the initial acetic acid concentration of 2.2, 3.1, 5.3, and 7.5 g/L was 13.55, 13.63, 12.05, and 6.73 g/L, respectively. Compared with the control group, the yield of citric acid decreased by 22.58%, 22.12%, 31.13%, and 61.51%, respectively. With the increase in initial acetic acid concentration, the ability of M13 to ferment and produce citric acid gradually decreased. Citric acid fermentation of M13 was severely inhibited in the medium with 7.5 g/L initial acetic acids, and after 8 days of cultivation, the fermentation of citric acid began. When the initial concentration of acetic acid is less than 5.3 g/L, the rate of citric acid production in the middle of the fermentation is slightly higher than that of the control group, which may be due to the cell simultaneously consuming acetic acid and glucose for the accumulation of citric acid. When the acetic acid concentration is 7.5 g/L, the growth rate of citric acid in the early stage is very low, and M13 does not produce citric acid. After acetic acid is completely metabolized 8 days later, the concentration of citric acid increases significantly, indicating that the function of M13 to produce citric acid is significantly inhibited under the inhibitory effect of a higher concentration of acetic acid. When there is no acetic acid, the biomass concentration of M13 is the highest, reaching 14 g/L. With the increase of initial acetic acid concentration, the biomass concentration of cells gradually decreases, indicating that the growth and reproduction of cells are inhibited. Especially under the initial acetic acid concentration of 7.5 g/L, the biomass concentration of M13 is the lowest, about 5.8 g/L, only 55.77% of the biomass concentration of the control group.
The concentration of acetic acid in the hydrolysate of lignocellulosic biomass is usually between 4-10 g/L[20] and inhibits the growth and fermentation of A. niger. Compared to other microorganisms, A. niger has a lower tolerance for acetic acid. For instance, in the presence of 4 g/L acetic acid, the ethanol production rate of C. glycerinogenes UG-21 is 2.37 g/L/h, and increasing the concentration of acetic acid to 6 g/L has almost no effect on its ethanol production[21]. This is because the inhibitory effect of acetic acid comes from undissociated molecules, which becomes more intense when the pH of the fermentation broths decreases due to the accumulation of organic acids.
Metabolic patterns of A. niger M13 on furfural
Furfural can destroy cell membranes and DNA, thereby inhibiting sugar fermentation and the TCA cycle. Furfural metabolism also consumes NADPH, which is necessary for cell biosynthetic reactions[22]. Figure 2 shows the effect of different initial concentrations of furfural on the growth and fermentation of an A. niger M13 strain. During the initial fermentation stage, M13 can completely consume furfural with concentrations ranging from 0.43 to 1.81 g/L in the medium. When the initial furfural concentration is below 1.34 g/L, M13 can consume most of the furfural within 12 h. However, it takes 48 h for cells to completely consume 1.81 g/L of furfural (Figure 2A). Figures 2A and 2B also show that M13 hardly consumes glucose when furfural is present in the medium. During the 10-day fermentation period in medium containing 0.43, 0.88, 1.34, and 1.81 g/L of furfural, glucose utilization rates decrease by 17.72%, 18.40%, 26.62%, and 31.92%, respectively, compared to the control group (Figure 2B). Furfural has a strong inhibitory effect on the growth of M13 and citric acid accumulation, and citric acid cannot be detected until furfural is completely consumed. Compared with the control group, when the initial furfural concentration is 0.43 g/L, the citric acid yield decreases by 61.28% (Figure 2C). As the initial furfural concentration increases, cell growth is severely inhibited. Figure 2D shows that the maximum biomass concentration of cells in the control group is 10.4 g/L. With the increase of initial furfural concentration, the biomass concentration of cells gradually decreases. When the initial furfural concentration is 1.81 g/L, the biomass concentration of the M13 strain is only 49.04% of that in the control group. In summary, Furfural strongly inhibits cell growth and citric acid accumulation. Even after furfural is completely metabolized, cell growth and citric acid production rates cannot be restored.
Metabolic patterns of A. niger M13 on HMF
HMF exhibited similar inhibitory effects as furfural on microorganisms by disrupting their cell membrane, thereby increasing membrane permeability and inhibiting microbial growth. However, according to reports, S.cerevisiae and Pichia stipitis could largely reduce HMF into 2,5-dihydroxymethylfuran, which has lower toxicity, under the action of dehydrogenases, and the continuous accumulation of 2,5-dihydroxymethylfuran did not affect cell growth and ethanol production[23]. In this study, A. niger could metabolize HMF. As the initial concentration of HMF in the medium increased from 0.24 g/L to 1.02 g/L, the metabolic rate of HMF increased (Figure 3A). Unlike the situation in the presence of furfural, when HMF was present, the cells could consume glucose and HMF together, and only the rate of glucose consumption was relatively slow during the early stage of fermentation. After 48 hours of fermentation, HMF was completely consumed, and the rate of glucose consumption was almost the same as that in the control group. Before HMF was completely consumed, A. niger hardly produced citric acid. However, it was found that furfural had a significantly greater inhibitory effect on cell growth and citric acid fermentation than HMF, as shown by the comparison of Figure 2 and Figure 3.
Detoxification and fermentation of corncob dilute acid hydrolysate by A. niger M13
A. niger M13 mycelium biodetoxification of corncob dilute acid hydrolysate
M13 mycelium was inoculated into corncob dilute acid hydrolysate medium at different inoculum levels (Figure 4). M13 mycelium degraded significant amounts of acetic acid, furfural, and HMF. However, different broth volumes did not have significant variations in detoxifying inhibitors in corncob dilute acid hydrolysate (Figure 4). M13 mycelium completely degraded furfural within 96 hours, HMF in 144 hours, and acetic acid in 192 hours, while the detoxification rate was 100% for these inhibitors in the hydrolysate (Figure 4A). Under the presence of three inhibitors simultaneously, M13 mycelial pellets preferred to degrade furfural and HMF, and then acetic acid after furfural was completely degraded (Figure 4A). Meanwhile, within the first 96 hours of inoculation, M13 mycelial pellets did not use any of the carbon sources in the media. Glucose was consumed after 96 hours, followed by xylose and then arabinose (Figure 4B). In the presence of furfural, A. niger M13 mycelial pellets barely produced citric acid, indicating that furfural prevented the production of citric acid during fermentation. The rate of citric acid production increased substantially from the moment furfural was completely degraded, resulting in a maximum of 6.0 g/L citric acid yield (Figure 4C).
A. niger M13 spores biodetoxification of corncob dilute acid hydrolysate
To investigate the effect of different spore inoculum sizes on detoxification efficiency, 0.2, 0.5, 1.0, and 1.5 ml of M13 spore solutions were inoculated into 20 ml of maize cob dilute acid hydrolysate medium, which contained approximately 0.181×108, 0.4525×108, 0.905×108, and 1.3575×108 spores, respectively.
As shown in Figure 5, M13 spores significantly degraded acetic acid, furfural, and HMF in the corncob dilute acid hydrolysate, and the removal efficiency of the inhibitory substances increased significantly with increasing inoculum size. Particularly, with an inoculum size of 1.3575 × 108 spores, M13 completely degraded 1.35 g/L furfural in 12 h, 0.37 g/L HMF in 24 h, and 7.5 g/L acetic acid in 48 h, achieving a removal rate of 100% for the three inhibitory substances. M13 spores preferentially degraded furfural, followed by HMF, and lastly, acetic acid, and acetic acid consumption occurred simultaneously with sugar consumption. When inhibitory substances were present in the medium, A. niger M13 barely produced citric acid. However, once inhibitory substances were completely degraded, the production rate of citric acid by A. niger M13 significantly increased. At an inoculum size of 1.3575 × 108 spores, 4.14 g/L citric acid was produced. Interestingly, in contrast to the lowest inoculum size condition, which yielded 5.5 g/L citric acid, it is possible that in the cases where a larger inoculum size was used, while a significant amount of carbon sources were consumed by A. niger for cell growth and inhibitory substance degradation, there was insufficient carbon source remaining for citric acid production during fermentation (Figures5C and 5D).
After pretreatment with dilute acid, we conducted a biological detoxification study using A. niger M13 spores with some lignocellulosic raw materials (Table 2), the main components of which were listed in Table 1. After 24 hours of detoxification, furfural and HMF in the hydrolysate were completely removed, and about 32% of acetic acid was removed. For fermentation systems that use acetic acid and sugar as substrates, A. niger M13 spores are preferred for detoxification for 24 hours to remove furfural and HMF. In addition, studies have shown that a small amount of acetic acid has a promoting effect on microbial fermentation. Wu et al. found that as the concentration of acetic acid in the medium increased from 0 to 30 g/L, the yield and productivity of 2,3-butanediol gradually increased, and the yield of 2,3-butanediol reached 0.4 g/g at 30 g/L acetic acid concentration[24]. Qian et al. used Yarrowia lipolytica W29 to produce citric acid and malic acid in crude glycerol. The maximum concentration of citric acid was 9.87 g/L with the addition of 10 g/L acetic acid salt solution in 50 g/L glycerol, which was 14.7% higher than that without acetic acid salt solution[25]. After 48 hours of detoxification, furfural, HMF, and acetic acid were completely removed from the lignocellulosic dilute acid hydrolysate, which is suitable for fermentation systems using sugar as a substrate. Using A. niger M13 spores for detoxification is a highly feasible option. These spores can completely remove the main inhibitory substance from the hydrolysate within 1-2 days, while retaining the carbon source for subsequent fermentation production.
Currently, many bacteria and fungi have been identified as having the ability to degrade and inhibit the growth of inhibitors in hydrolysates (Table 3). Issatchenkia orientalis S-7 is capable of degrading both acetic acid and furfural in hemicellulose hydrolysate. After 80 hours of cell culture, the removal rates of acetic acid and furfural were 100% when the hydrolysate contained 4 g/L acetic acid and 0.4 g/L furfural[26]. Fonseca et al. used Issatchenkia occidentalis (CCTCC M 206097) to detoxify the hydrolysate of sugarcane bagasse. The detoxification efficiency was 66.67% for eugenol, 73.33% for ferulic acid, 62% for furfural, and 85% for HMF within 24 hours[27]. Aspergillus nidulans FLZ10 was able to detoxify steam-exploded corn stover fiber, with removal rates of 75% formic acid, 54% acetic acid, and 100% of both HMF and furfural after 72 hours of cultivation[28]. Compared to the strains currently used for biodegradation, A. niger M13 spores can degrade inhibitors in hydrolysate with a removal rate of 100% within 48 hours and can maintain a high removal rate even in the presence of higher levels of inhibitors, at rates of 0.1566 g/L/h for acetic acid, 0.1125 g/L/h for furfural, and 0.015 g/L/h for HMF. The order of inhibitor degradation by A. niger M13 is furfural, HMF, and then acetic acid. Therefore, for fermentation systems that use acetic acid and sugars as substrates, M13 spores can be used to remove furfural and HMF as the priority.