The whole-cell catalysis of primary diols (C2-C6) by G. oxydans
Dual-functional modules of hydroxyl acids alleviate their application prospects in many high-end fields . However, the traditional methods for hydroxyl acid preparation produce a large amount of waste and by-products, violating the principles of green chemistry and sustainable development. Hence, employing G. oxydans as a core catalyst to design a green and environmentally friendly synthesis method for hydroxyl acids is an efficient approach to solve the current industrial bottlenecks. To explore the reaction mechanism of G. oxydans whole-cell catalysis, five linear diols were selected as substrates for kinetic study with OD600=2. The bioprocesses for the catalysis of EG, 1,3-PG, and 1,4-BG (100 mmol/L) by G. oxydans are described in Figure 2. The final catalytic products were GA, 3-HPA, and 4-HBA, respectively, eliminating the formation of diacids. The average consumption rates of EG, 1,3-PG, and 1,4-BG were 10.89, 20.4, 24.8 mmol/L/h, respectively. Compared with the chemical method, the purity of products was satisfactory, meeting the core requirements of green chemistry, the reaction efficiency still needed to be improved to meet the requirements for industrial production.
Figure 2 shows the bioprocess for the catalysis of 1,5-PG and 1,6-HG (100 mmol/L) by G. oxydans, respectively. Surprisingly, when the carbon chain length was ≥ 5, the primary diols showed qualitative differences. 1,5-PG and 1,6-HG catalysis followed a step-by-step process, further catalyzing to GTA and AA when primary diols were oxidized to hydroxyl acids. Moreover, the catalytic rates for 1,5-PG and 1,6-HG were very similar. The substrate consumption time for 100 mmol/L concentration was less than 3 h and 4 h, while the average substrate consumption rates reached 31.22 and 45.50 mmol/L/h, respectively. Although the reaction efficiency for C5/C6 was promising, the product quality was not satisfactory due to the formation of by-products (diacids). At present, hydroxyl acids are receiving much attention due to their two functional modules, exhibiting excelling properties. Hence, we further explored the regulation of low-cost substrates primary diols oxidized by G. oxydans to lay a theoretical foundation for the selective regulation technology to prepare hydroxyl acids from primary diols.
The whole-cell catalysis of primary diols (C2-C4) by G. oxydans combined with SOS technology
Previous studies indicated that G. oxydans could bio-transform EG, 1,3-PG, and 1,4-BG into hydroxyl acids. Although the purity of the products met the green chemistry and industrial production requirements, the catalytic efficiency was lower to strengthen the production economy. G. oxydans take oxygen as the final electron receptor, so the whole-cell catalysis requires a lot of oxygen support to enhance the catalytic rate [44,45]. However, the high cost of unrestricted oxygen supply can not be borne by the industry. Therefore, in this study, SOS technology was employed to maximize oxygen utilization while ensuring high catalytic efficiency. Moreover, SOS technology safely controls the pressure in the bioreactor by adjusting the oxygen inlet speed, eliminating the potential safety hazards in industrial production .
As shown in Figure 3, 48 h whole-cell catalysis of EG, 1,3-PG, and 1,4-BG were carried out in SOS-BR with G. oxydans OD600=2. Results indicated that GA, in the 48 h bioprocess, 3-HPA and 4-HBA accumulated 75.3 g·L-1, 83.2 g·L-1, and 94.3 g·L-1 respectively. In terms of GA productivity, the highest quantity of GA obtained was 74.5 g·L-1 with a productivity of 1.49 g/L/h by Wei et al. , lower than 1.6 g/L/h obtained in this study. Sun et al. reported a recombinant Escherichia coli to produce 38.7 g 3-HPA with an average yield of 35% in 72 h fermentation , the productivity was lower than SOS technology and numerous by-products were found. For 4-HBA, Sang et al.  employed recombinant Escherichia coli, producing 103.4 g·L-1 under the microaerobic conditions with 0.844 g/L/h productivity. Although the production of 4-HBA was slightly higher than that of whole-cell catalysis, its productivity was only 43% of SOS technology. Furthermore, a lot of inhibitors such as acetic acid were generated in the fermentation broth of recombinant Escherichia coli, seriously affecting the product quality. Apparently, the whole-cell catalysis with G. oxydans, combined with SOS technology, attained a continuous and efficient preparation of high-purity hydroxyl acids (C2-C4), providing solid technical support for their industrial production.
Bioprocess for 5-HVA preparation with pH regulation and cell-recycling technology in SOS-BR
A previous study showed that 5-HVA could be produced by G. oxydans, its production level was rather unsatisfactory, and GTA was accumulated in the system as a by-product. Moreover, the traditional chemical methods cannot be employed for preparing high-quality 5-HVA due to their high cost, limiting their application in medicine, material science, and other advanced fields. Therefore, targeted regulation of selective catalysis of 1,5-PG is a promising approach to produce green 5-HVA at an industrial scale. As shown in Figure 4, the whole-cell catalysis of 1,5-PG was performed under different pH gradients, including pH=2.5, 3.5, 4.5, 5.5, 6,5. When pH≥5.5, GAT was not produced even if the substrate 1,5-PG was completely bio-oxidized to 5-HVA. However, when pH was less than 5.5, the whole-cell catalysis showed two-stage reactions; 1,5-PG generated 5-HVA in the first step, and then 5-HVA was catalyzed to GTA in the second step. It is noteworthy that the results at pH=2.5 were contrary to the law because normal physiological activity could not be maintained under extremely acidic conditions, and G. oxydans lost catalytic activity after 2 h. Therefore, the proposed scheme of pH-regulated whole-cell catalysis provided a green and high-quality approach for the industrial production of 5-HVA without any by-products. To directionally obtain an ultra-high titer of 5-HVA, we selected pH=5.5 for the bioreactor experiment.
In 2019, Keiichi et al. employed over-supported platinum catalysts and obtained a 62% yield of 5-HVA, δ-valerolactone, and methyl 5-hydroxyvalerate . The results revealed a low yield and presence of abundant derivatives, seriously affecting the purity of products. Moreover, in 2021, Hee et al. reported a fermentative production of 5‑HVA by metabolically engineered Corynebacterium glutamicum, and 55 g·L-1 5-HVA and 10 g·L-1 GTA were produced after 28 h fermentation . Apparently, their designed bioprocess was not satisfactory for industrial-scale production of 5-HVA. Hence, we conducted whole-cell catalysis for 5-HVA bio-production in SOS-BR with pH regulation (Figure 5A). Results showed that 102.3 g·L-1 of 5-HVA was accumulated without the formation of diacids during 48 h whole-cell catalysis with average productivity of 2.1 g/L/h. Simultaneously, the production in the first 10 h was 58.9 g·L-1 and the productivity was 5.9 g/L/h, exceeding the highest level of 5-HVA (55 g·L-1 during 28 h). The broth contained only 0.2 g·L-1 substrates, without any diacid production at 48 h, and 5-HVA yield was as high as 99.8%. Additionally, SOS-BR maintained a high dissolved oxygen (DO) level during the whole-cell catalysis process, meeting the oxygen demand for G. oxydans. At the same time, the cost of oxygen utilization was greatly saved due to the strict sealing environment of the entire system, improving the economy of the entire bioprocess. Moreover, we successfully performed cell-recycling technology with 6 rounds in SOS-BR (Figure 5B). During 48 h whole-cell catalysis, a total of 274.1 g 5-HVA was accumulated, and the production of each round was 52.5 g·L-1, 47.8 g·L-1, 46.7 g·L-1, 44.8 g·L-1, 40.2 g·L-1, and 39.4 g·L-1, respectively. In conclusion, combined with pH control and SOS-BR technology, we successfully synthesized high-quality 5-HVA with an ultra-high titer, providing a promising avenue for the industrialization of 5-HVA and hydroxyl acids.
Bioprocess for 6-HCA synthesis with pH regulation and cell-recycling technology in SOS-BR
Compared to other hydroxyl acids, the technology for industrial production of 6-HCA is currently unavailable. As 6-HCA is an intermediate, hydroxyl and carboxyl groups often undergo oxidation or reduction during synthesis, generating 1,6-HG, AA, and other by-products. A little literature is available on the preparation of 6-HCA. In 1999, Fischer et al. employed metal catalysts to prepare 6-HCA at high temperature (100~300℃) and high pressure (10~300 bar), but the products were accompanied by abundant 1,6-HG and esters . Therefore, it was significant to develop a green and efficient method for 6-HCA synthesis by G. oxydans. Results revealed that the pH of the broth showed an obvious regulatory effect on the whole-cell catalytic process (Figure 6). The whole-cell catalysis of 1,6-HG was divided into two stages at pH ≤ 6. The first step was the oxidation of 1,6-HG to an intermediate product, 6-HCA, which was converted to AA. Surprisingly, when the pH of broth was adjusted to 7, the conventional two-stage catalysis was regulated at the first stage. The results suggested that when 1,6-HG was oxidized to 6-HCA, it did not further react to form AA, improving the product quality of 6-HCA and eliminating the formation of by-products. According to previous research results, the weak acidic environment was more suitable for the physiological and biochemical capacity of G. oxydans, hence, pH=7 was selected for 6-HCA biopreparation.
Based on the findings of the pH regulation experiment, the whole-cell catalysis for preparing 6-HCA was performed at pH=7 (Figure 7A). In fed-batch mode, 48.8 g·L-1 of 6-HCA was accumulated within 48 h, and the productivity was 1.01 g/L/h, which was the highest reported level for 6-HCA. The kinetic curve showed that the inhibition effect was evident after the formation of hydroxyl acids and the productivity decreased from 4.5 g/L/h to 0.3 g/L/h. When the catalysis was performed for 8 h, the productivity decreased to 2 g/L/h, 50% lower than that of the initial level. Therefore, we performed the cell-recycling experiment in 8 rounds for single batch catalysis, and the results are shown in Figure 7B. During 48 h of whole-cell catalysis, the cell-recycling experiment was successfully implemented for 6 rounds, and the last round still maintained 75% capacity. Finally, 129.3 g of 6-HCA was accumulated in 6 rounds, with average productivity of 2.7 g/h, 1.6 times higher than that of a single batch. The importance of 6-HCA underscores its social demand; however, the existing technologies cannot support the market demand. Therefore, the method for preparing 6-HCA proposed in the study, overcomes the disadvantages of traditional methods, demonstrating promise for the industrial production of 6-HCA in the future.