BackgroundPolylactic 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.ResultsIn 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.ConclusionsGiven 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.
Background: Optically pure d-lactic acid (≥ 99%) is an important precursor of polylactic acid. However, there are relatively few studies on d-lactic acid fermentation compared with the extensive investigation of l-lactic acid production. Most lactic acid producers are mesophilic organisms. Optically pure d-lactic acid produced at high temperature not only could reduce the costs of sterilization but also could inhibit the growth of other bacteria, such as l-lactic acid producers.Results: Thermophilic Bacillus coagulans is an excellent producer of l-lactic acid with capable of growing at 50 °C. In our previous study, the roles of two l-lactic acid dehydrogenases have been demonstrated in B. coagulans DSM1. In this study, the function of another annotated possible l-lactate dehydrogenase gene (ldhL3) was verified to be leucine dehydrogenase with an activity of 0.16 units (μmol/min) per mg protein. Furthermore, the activity of native d-lactate dehydrogenase was too low to support efficient d-lactic acid production, even under the control of strong promoter. Finally, an engineered B. coagulans D-DSM1 strain with the capacity for efficient production of d-lactic acid was constructed by deletion of two l-lactate dehydrogenases genes (ldhL1 and ldhL2) and insertion of the d-lactate dehydrogenase gene (LdldhD) from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 at the position of ldhL1. Conclusions:This genetically engineered strain produced only d-lactic acid under non-sterilized condition, and finally 145 g/L of d-lactic acid was produced with an optical purity of 99.9% and a high yield of 0.98 g/g. This is the highest optically pure d-lactic acid titer produced by a thermophilic strain.
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