Succinic acid production from HPAC-pretreated pine
Succinic acid has been produced using various microbes such as A. succinogenes , M. succiniciproducens , Y. lipolytica ,E. coli , and C. glutamicum (Table 3). Moreover, this process has been enhanced by engineering the genes associated with glucose metabolism (TCA cycle or glyoxylate cycle).[8] For example, the overexpression of a single gene encoding for pyruvate carboxylase (pyc) significantly increased succinic acid yields in a lactate dehydrogenase 1 knock-out mutant of C. glutamicum .[5] Nevertheless, unlike several gene knock-out mutants, the C. glutamicum wildtype can be used to produce succinic acid under anaerobic conditions.[48] Table 3 compares the succinic acid production yields of different recombinant C. glutamicum strains and other microbes. Interestingly, the production yields of succinic acid from the hydrolysates tended to be much lower than those achieved using pure glucose as a carbon source and showed a wide range of yield depending on the cell-dried weight (CDW, cell concentration) and fermentation time. These results suggest that the carbon sources and the cell concentration are the rate-limiting factors in the biosynthesis of succinic acid (Okino et al., 2008).[5]
The single knock-out mutant of the ldhA gene in C. glutamicum , ΔldhA-6 (10.15–21.19 g L-1 CDW), was incubated in 100 mL of 1–5% hydrolysate (Table 2), and the metabolites (succinic acid, lactic acid, and acetic acid) produced under semi-anaerobic condition were analyzed (Fig. 4). The glucose in the 1–4% hydrolysate was almost consumed by the ΔldhA-6 mutant after 9 h, and produced 3.64, 7.45, 8.49 and 13.77 g L-1 of succinic acid, without lactic acid. Simultaneously, the xylose consumption was entirely delayed at the same interval of time. The ΔldhA-6 mutant (CDW: 21.08 g L-1) in the 5% hydrolysate produced higher levels of lactic acid than in the other hydrolysates, the semi-anaerobic or anaerobic fermentation condition of which required to tightly retain to block surging lactic acid production. In some cases, there was a failure to retain the semi-anaerobic conditions, and lactic acid, which is the dominant metabolite, was produced 3.6 times higher than succinic acid in the 5% hydrolysate by the ΔldhA-6 mutant (data not shown). Although the ΔldhA-6 gene activity was lost in the ΔldhA-6mutant, lactic acid still tended to be produced under the semi-anaerobic condition in the 1–5% HPAC-pretreated hydrolysates over the 9 h period. It is estimated that a minor metabolic pathway related to lactic acid production was stimulated by some derivative in the hydrolysate of the HPAC-pine. Indeed, xylo-oligomers, cello-oligomers, xylose, mannose, and unidentified chemicals are identified as candidate materials responsible for this lactic acid production. However, additional research is required to confirm which material is responsible for lactic acid.
A comparison of the conversion rate to succinic acid (Fig. 5), illustrated that the best condition, among the hydrolysates, was to ferment the 4% hydrolysate with approximately 20 g L-1 CDW for 9 h, as it provided 1.58 g L-1 h-1 productivity with a 98% glucose consumption rate. Cell densities of 10.15 g L-1 for 1%, 15.72 g L-1 for 2%, and 16.08 g L-1 for 3% hydrolysate were required for a 9h complete consumption, while an 88% glucose consumption was shown in the fermentation of 5% hydrolysate. The correlations between cell concentration, succinic acid production, and glucose consumption are summarized in Fig. 6. A more efficient and economical production of succinic acid was attempted from the 4% hydrolysate using higher cell concentration (26.89 g L-1 CDW) than the previous experiment, which resulted in 14.82 g L-1 succinic acid production over 6 h, showing productivity of 2.47 g L-1 h-1 and an 86.2% glucose and a 20.0% xylose consumption. It is close to the 2.5 g L-1 h-1 value, which is the minimum productivity of succinic acid provided from corn-based sugar required to compete with the current market petrol-based succinic acid production.[1] There is still the potential to increase succinic acid production from 4% hydrolysate because remaining sugars available for the conversion include 15% glucose and 80% xylose. To increase efficiency of succinic acid production, we incorporated and overexpressed the succinic acid transporter gene,sucE , under the Psod promoter using the CRISPR/cpf1 gene editing system (Fig. 7). The co-expression transformant (Psod:sucE-ΔldhA, 10.00 g L-1 CDW) exhibited higher production of succinic acid in 4% pine hydrolysate compared to theΔldhA-6 mutant (10.94 g L-1 CDW). In comparison of succinic acid production of the 4, 5, and 10% hydrolysate (containing 27.45, 39.66, and 57.66 g L-1 reducing sugars, respectively), the optimal concentration of the hydrolysates in the fermentation with [Psod:sucE- ΔldhA ] transformant (28–30 g L-1 CDW) was found to be 4%, consistent with our previous results. The productivity of succinic acid was found to be 3.83 g L-1 h-1 for the 3 h fermentation and 2.95 g L-1 h-1 for the 6 h fermentation period. In the fed-batch with 4% hydrolysate, the first feeding was carried out after 24 h of the fermentation. This feeding consisted of 20 mL of 20% pine hydrolysate (155.08 g L-1), which was adjusted to be equivalent to the final concentration of the 4% hydrolysate. As a result, the amount of succinic acid produced was doubled, which reached to 39.67 g L-1. Yield of succinic acid from in-put reducing sugars is 56.71 %. The yield of succinic acid from glucose that is mainly consumed during the fermentation is ~84.4%. Small amount of acetic acid was measured at the time of feeding, while lactic acid was observed at the late stage of fermentation, furthermore, about half of the xylose remained in the solution. Based on the results of glucose consumption, we fed 20 mL of 20% pine hydrolysate three times at 6, 9, and 24 h. However, the efficiency of succinic acid production decreased immediately after each feeding. These findings emphasize the significance of maintaining a concentration of 4% hydrolysate to achieve high-efficiency of succinic acid production. It seems that xylose accumulation is a limiting factor during the fermentation over 4% hydrolysate, requiring further study on the retardation factors in high concentration of hydrolysate, which is technically important to improve succinic acid production using a fed-batch system.
Succinic acid production has been studied using diverse microbes. There are hitherto some rare cases of succinic acid production using hydrolysates from lignocellulosic biomasses. We conducted the overall process of succinic acid production from lignocellulosic biomass, and suggest an optimization condition in each process for succinic acid production. In this study, the conversion ratio of glucose to succinic acid was higher than in previous studies using engineered C. glutamicum strains in which several genes were knocked-out and/or overexpressed (Table 3). This indicates that the single knock-outΔldhA mutant and co-expression of sucE gene is a sufficient succinic acid producer from glucose. To achieve further economical production of succinic acid from HPAC-pretreated pine, future studies must focus on improving xylose consumption rate and fermentation efficiency particularly in high concentration of hydrolysate, which can deliver economic feasibility to succinic acid production from lignocellulosic biomass.