IBE fermentation profile by the recombinant C. acetobutylicum XY16 harboring pSADH
After successful introduction of plasmid harboring pSADH into C. acetobutylicum XY16, batch fermentation without pH control was carried out to investigate the effects of sadh gene expression on solvent production and cell growth (Fig. 1). As expected, no residual acetone was detected in the culture medium after 44 h of fermentation. Instead, 1.13 g/L of isopropanol occurred after the expression of sadh in XY16 (pSADH) (Fig. 1a). During the fermentation process, pH values of the culture broth decreased from the initial value of 5.68 to around 4.23 in the acidogenesis phase, whereas the pH did not have a big fluctuation until the end of fermentation. It was observed that the maximum biomass reached 4.19 after 24 h of cultivation and then decreased rapidly. Further incubation did not increase the solvent production and glucose uptake with only 14.0 g/L of glucose consumed after 48 h of fermentation. The recombinant strain XY16 (pSADH) did not undergo a typical acid re-assimilation phase with the residual acetate and butyrate concentration reaching approximately 2.59 and 2.39 g/L, respectively. These results indicated that the metabolic profile of XY16 (pSADH) was dramatically affected by the expression of sadh gene. When entering the solventogenesis phase, cell growth began to decrease and IBE solvent production only reached 3.88 g/L, of which butanol, isopropanol, and ethanol concentrations were 2.13, 1.13, and 0.62 g/L, respectively.
To better understand the discrepancy shifting from ABE to IBE, the total levels of NADPH and NADH were further examined (Fig. 1b). As seen, when solvent production was initiated during 20–25 h, the NADPH levels of parent strain XY16 were kept at a stable level of about 0.15 μmol/g DCW, while those of the recombinant XY16 (pSADH) were dramatically decreased from the initial level of 0.17 μmol/g DCW to a lower level of 0.07 μmol/g DCW, indicating that the synthesis of isopropanol enlarged the demand of NADPH (Fig. 1b). This is also consistent with our expectation that more isopropanol synthesized, more NADPH consumed. On the other hand, the NADH levels showed a typical characteristic that continuously increased through the acidogenesis phase and decreased in the solventogenesis phase (Fig. 1c). However, the NADH level of XY16 (pSADH) was lower than that of XY16 and the total amount of NADPH and NADP+ within XY16 (pSADH) was increased (data not shown), suggesting that NADH was phosphorylated by NADK to support cell growth and solvent production. It is known that the reducing agent, NADPH, is an important coenzyme required in several biological reactions, especially for cell growth [7, 9]. The further driving force for isopropanol synthesis increased the demand of NADPH, resulting in low level of NADPH and NADH. To increase the solvent flux, it is crucial to provide constant NADH and NADPH to achieve a balanced redox status for cell growth and IBE production.
High IBE production by the recombinant C. acetobutylicum XY16 (pSADH) through pH regulation
Cofactor manipulation could potentially be a powerful and economical strategy for the improvement of overall IBE production [9]. pH has been reported as a critical factor for ABE fermentation, which greatly influences the production of solvents through regulation of the intracellular level of NAD(P)H. However, the optimal pH varied depending on the culture conditions and strains [1, 18]. Thus, the effect of various pH values on IBE production by the recombinant C. acetobutylicum XY16 (pSADH) was further investigated. Batch fermentations were performed at pH values of 4.6, 4.8, 5.0, 5.2, and 5.5 in the modified P2 medium. As shown in Fig. 2, cell growth and IBE production were significantly affected by pH values. Under these conditions, the growth of engineered strain recovered to that of the parent strain XY16 and showed the typical biphasic fermentation profile of C. acetobutylicum (Fig. 2a) [1]. When pH was maintained at low value of 4.6, the maximum cell density of 6.40 (OD600) was obtained at 24 h (Fig. 2a) and the final IBE concentration reached 14.36 g/L (Fig. 2c). However, when pH was maintained at 5.2 or 5.5, a decrease in IBE production was observed, and the metabolic flux shifted towards acids production rather than solvents, resulting in high acids production with 12.51 g/L of butyric acid and 7.23 g/L of acetic acid (Fig. 2g, h). Similar results have been reported in other studies, in which more acids were produced at higher pH values than lower ones [19,20,21]. When pH was controlled at 4.8, the maximal IBE production of 16.09 g/L was obtained, of which the concentration of butanol, isopropanol, and ethanol was 9.97, 4.98, and 1.14 g/L, respectively (Fig. 2d–f). Meanwhile, the recombinant strain XY16 (pSADH) utilized glucose more efficiently at pH 4.8 with a high glucose consumption rate of 0.86 g/L/h (Fig. 2b). It can be concluded that maintenance of optimal pH at 4.8 for recombinant strain XY16 (pSADH) not only favored cell growth during acidogenesis, but also improved IBE production during solventogenesis.
Detection of NADH and NADPH levels in the recombinant C. acetobutylicum XY16 (pSADH)
The previous studies have shown that the introduction of sadh cannot completely convert acetone to isopropanol in some solventogenic Clostridium sp. For example, although the transformant C. acetobutylicum ATCC 824 (pFC007) after overexpression of ctfA/B genes along with sadh showed high capacity for conversion of acetone into isopropanol (> 95%), however, the residual acetone of 0.9 g/L was still detected [8]. Different from those studies, acetone could be completely converted into isopropanol by the recombinant strain XY16 (pSADH), but the total IBE production was only 3.88 g/L without pH control. Only through pH control strategy, the maximum IBE production was increased up to 16.09 g/L. To elaborate the underlying mechanisms, the intracellular levels of NADPH and NADH were investigated. As shown in Fig. 3, during acidogenesis, the intracellular NADH increased rapidly with cell growth (Figs. 2a, 3b). NADH has been reported as one of the major contributing factors for solvent production [1]. Supplementation of NADH precursors could efficiently improve solvent production. For example, addition of nicotinamide (VB3) could obviously improve both s-ADH and butanol dehydrogenase (BDH) activities and improve solvent production [22]. As shown in Fig. 3b, after 20 h of fermentation, NADH as the main reducing power was consumed to synthesize the solvents and regeneration of NAD+. Thus, plenty of NADH was created by consuming large amount of glucose, accompanied with a dramatic increase in the cell biomass and solvent production (Fig. 2b, c). Compared to other pH conditions, the NADH level at pH 4.8 did not increase obviously, due to the fact that the produced NADH was mostly consumed for the solvent production. Levels of NADPH in the pH-controlled fermentation were much higher than those in the extract from the control (Fig. 1b). In addition, the increase of NADPH during solventogenesis phase indicated that more reducing equivalents were produced in the forms of NADPH. This trend also clearly indicated by the increase of isopropanol concentration. Therefore, the increased solvent production was attributed to the improved availability of intracellular NADH and NADPH.
High IBE production by the recombinant C. acetobutylicum XY16 (pSADH) through supplementation of calcium carbonate
NADPH can be generated from NADP+ and NADK is the sole enzyme catalyzing the generation of NADP+ from NAD+ [23]. It has been reported that NADK can be activated by the factors of calcium ions [24, 25]. Hence, CaCO3 was added into the fermentation medium, which may play dual roles for pH adjustment and activation of NADK. Accordingly, various amounts of calcium carbonate (0, 2, 4, 6, 8, 10, and 12 g/L) were supplemented into the medium and batch fermentations were carried out for 72 h using the recombinant C. acetobutylicum XY16 (pSADH) (Fig. 4a). In the control batch without CaCO3, the biomass (OD600) reached 0.47 and only 2.13 g/L of butanol was produced when cultured in anaerobic bottles. When the dosage of calcium carbonate increased to 2 and 4 g/L, the butanol production increased to 4.19 and 7.56 g/L, respectively. As shown in Fig. 4a, a small quantity of CaCO3 led to the rapid cell growth of strain XY16 (pSADH). Meanwhile, the glucose utilization by strain XY16 (pSADH) was also increased in P2 medium supplemented with CaCO3. Both cell growth and final IBE concentration increased along with the increase of CaCO3 concentration. The highest IBE production of 17.77 g/L and cell density of 8.10 were achieved in a serum bottle medium spiked with 10 g/L CaCO3. In addition, no significant difference in glucose utilization and IBE production was observed when XY16 (pSADH) was cultivated in P2 medium containing above 10 g/L CaCO3.
To further investigate solvent production profiles by strain XY16 (pSADH) under optimal CaCO3 concentration, batch fermentation in 5 L fermentor was carried out (Fig. 4b). When 10 g/L of calcium carbonate was supplemented, the initial pH value of the fermentation medium was 5.82. With the increase of fermentation duration, the pH value decreased to around 4.9, which is the optimal pH for IBE fermentation (Fig. 2). In addition, it is worth noting that the IBE concentration increased dramatically from 3.07 to 17.77 g/L, with a glucose consumption rate of 0.99 g/L/h. Cell growth was also significantly improved in the presence of CaCO3, and the biomass of XY16 (pSADH) increased up to 9.10. Hence, supplementation of CaCO3 provides a favorable pH range for the growth of XY16 (pSADH), which also contributed to the increase of IBE production.
In addition to acting as the buffering agent, CaCO3 could also increase the NADPH availabilities (Fig. 3). Compared with pH control strategy, the level of NADPH was increased to the highest (Fig. 3a). When CaCO3 was added, the driving force from Ca2+ could enhance the reaction from NAD(H) to NADP(H), and consequently, the isopropanol concentration reached 6.06, which was 22% higher than that at pH 4.8. It is also worth noting that total solvent production was increased by 10% from 16.09 to 17.77 g/L with 10.51, 6.02, and 1.24 g/L of butanol, isopropanol, and ethanol, respectively (Fig. 4). Meanwhile, high solvent yield of 0.312 g/g was obtained compared with at pH 4.8. In our current study, it suggests that improving the availability of NAD(P)H is an efficient approach for increasing IBE solvent production.