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.