Mutation Breeding And Laboratory Evolution Of Thermotolerant Bacillus Coagulans For L-Lactic Acid Production

Bacillus coagulans is a spore-forming gram-positive bacteria, and widely used to produce L-Lactic acid at higher temperature (>50℃) with the superiorities of effectively controlling the risk of contamination, as well as significantly reducing the amount of cooling water for saving the costs. In this work, two strategies were conducted to further improve the temperature tolerance of Bacillus coagulans:1) Breeding of Bacillus coagulans mutants basing on Atmospheric and Room Temperature Plasma (ARTP) mutation method and high-throughput screening (HTS) platform;2) Laboratory evolution of Bacillus coagulans through gradually increasing the cultivation temperature.A strain named HL5was chosen from six Bacillus coagulans strains as the parent strain through a series of tests processes, including plate screening, multi-temperature (from50to55℃) shake flask cultures, and5L batch fermentations, with general consideration of cell growth, lactic acid production and glucose consumption. Subsequently, the fermentation performances of HL5with three low-cost biomass-based saccharified solutions (corn meal, cassava flour and cellulose) as carbon sources were investigated. The results demonstrated that all of these carbon sources could be utilized by HL5, and their productivities were4.19g/L/h,3.75g/L/h,5.00g/L/h, respectively, indicating that HL5could be a potential lactic acid producer for industrial scale.With the aim of obtaining high-performance mutants, high-throughput screening system for Bacillus coagulans was established.48deep-well microtiter plate (MTP) cultivation was adopted using bromocresol green as an indicator for screening. The good correlation of fermentation results between different-scale cultivations confirmed the feasibility of applying48-MTP instead of shake flasks for culturing Bacillus coagulans. Total750mutants were obtained after ARTP mutagenesis and plate screening, and a thermotolerant (55℃) mutant1H6showed the best performance on cell growth, lactic acid production and glucose consumption. Moreover, another strain named C was obtained via laboratory evolution method. In5L bioreactor at55℃, the L-lactic acid titers and productivities of1H6and C reached112.5g/L and4.00g/L/h,115.0g/L and4.42g/L/h, respectively, which were12.5%and29%,15.0%and42.6%higher than those of parent strain HL5.Finally, we studied the fermentation performances of parent strain HL5, mutants1H6 and C with high initial glucose concentration (200g/L). The tolerance to high concentration of glucose of the mutants1H6and C were enhanced, and L-lactic acid titers at52h were125g/L,100g/L, rspectively, while the titer with HL5was only90g/L.