Assavasirijinda Nilnate, Ge Deyong, Yu Bo, Xue Yanfen, Ma Yanhe
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
Microb Cell Fact. 2016 Jan 12;15:3. doi: 10.1186/s12934-015-0408-0.
Polylactic acid (PLA) is one important chemical building block that is well known as a biodegradable and a biocompatible plastic. The traditional lactate fermentation processes need CaCO3 as neutralizer to maintain the desired pH, which results in an amount of insoluble CaSO4 waste during the purification process. To overcome such environmental issue, alkaliphilic organisms have the great potential to be used as an organic acid producer under NaOH-neutralizing agent based fermentation. Additionally, high optical purity property in D-lactic acid is now attracting more attention from both scientific and industrial communities because it can improve mechanical properties of PLA by blending L- or D-polymer together. However, the use of low-price nitrogen source for D-lactate fermentation by alkaliphilic organisms combined with NaOH-neutralizing agent based process has not been studied. Therefore, our goal was the demonstrations of newly simplify high-optical-purity D-lactate production by using low-priced peanut meal combined with non-sterile NaOH-neutralizing agent based fermentation.
In this study, we developed a process for high-optical-purity D-lactate production using an engineered alkaliphilic Bacillus strain. First, the native L-lactate dehydrogenase gene (ldh) was knocked out, and the D-lactate dehydrogenase gene from Lactobacillus delbrueckii was introduced to construct a D-lactate producer. The key gene responsible for exopolysaccharide biosynthesis (epsD) was subsequently disrupted to increase the yield and simplify the downstream process. Finally, a fed-batch fermentation under non-sterile conditions was conducted using low-priced peanut meal as a nitrogen source and NaOH as a green neutralizer. The D-lactate titer reached 143.99 g/l, with a yield of 96.09 %, an overall productivity of 1.674 g/l/h including with the highest productivity at 16 h of 3.04 g/l/h, which was even higher than that of a sterile fermentation. Moreover, high optical purities (approximately 99.85 %) of D-lactate were obtained under both conditions.
Given the use of a cheap nitrogen source and a non-sterile green fermentation process, this study provides a more valuable and favorable fermentation process for future polymer-grade D-lactate production.
聚乳酸（PLA）是一种重要的化学结构单元，是一种众所周知的可生物降解且具有生物相容性的塑料。传统的乳酸发酵工艺需要碳酸钙作为中和剂来维持所需的pH值，这在纯化过程中会产生大量不溶性硫酸钙废物。为了克服此类环境问题，嗜碱微生物在基于氢氧化钠中和剂的发酵过程中具有作为有机酸生产者的巨大潜力。此外，D-乳酸的高光学纯度特性目前正吸引着科学界和工业界越来越多的关注，因为通过将L-或D-聚合物混合在一起，它可以改善PLA的机械性能。然而，尚未研究嗜碱微生物结合基于氢氧化钠中和剂的工艺使用低价氮源进行D-乳酸发酵的情况。因此，我们的目标是通过使用低价花生粕结合基于非无菌氢氧化钠中和剂的发酵来证明新的简化的高光学纯度D-乳酸生产方法。
在本研究中，我们开发了一种使用工程化嗜碱芽孢杆菌菌株生产高光学纯度D-乳酸的工艺。首先，敲除天然的L-乳酸脱氢酶基因（ldh），并引入来自德氏乳杆菌的D-乳酸脱氢酶基因以构建D-乳酸生产者。随后破坏负责胞外多糖生物合成的关键基因（epsD）以提高产量并简化下游工艺。最后，在非无菌条件下进行补料分批发酵，使用低价花生粕作为氮源，氢氧化钠作为绿色中和剂。D-乳酸滴度达到143.99 g/l，产率为96.09%，总生产率为1.674 g/l/h，其中在16 h时最高生产率为3.04 g/l/h甚至高于无菌发酵。此外，在两种条件下均获得了高光学纯度（约99.85%）的D-乳酸。
鉴于使用了廉价氮源和非无菌绿色发酵工艺，本研究为未来聚合物级D-乳酸生产提供了一种更有价值且有利的发酵工艺。
