Study On Enhancement Of L-lactic Acid Production By Fermentation Coupled With Separation

L-lactic acid is an important organic acid. It is widely used in the field of food, medicine and chemical engineering. The most promising application of L-lactic acid is to be an important starting material for the synthesis of poly (lactic acid) which is a new biodegradable and environmental friendly material. At present, there are many issues such as high cost of material, low yield and long period of batch fermentation, and large amount of energy consumption and pollution in process, which hinder its development. In order to overcome these disadvantages, inexpensive yam tuber was used as the main raw material for the fermentation. The sectional control of culture conditions and cell recycle fermentation were applied to increase the yield and productivity of L-lactic acid and obtain the by-product diosgenin cleanly. The major goal was to reduce the production cost.(1) Strain Lactobacillus rhamnosus HG09, producing L-lactic acid with high optical purity, was isolated from nature. Then an excellent positive forward mutant L.rhamnosus HG09F5-27 was obtained trough cooperation mutagenesis of ultraviolet and mutagens and subsequent five times of recursive protoplast fusions (Genome shuffling). When the fermentation was carried out in the medium containing 160g/L of yam starch hydrolysis sugar and 0.35mol/L of dioscin, the yield and productivity of L-lactic acid reached 141.33±4.11g/L and 1.76±0.04g/L/h, which were 4.09 times and 4.40 times those of the wild strain, respectively.(2) In order to save grain for human, a new no-grain material, yam tuber was screened out and used as the carbon source of L-lactic acid production to replace the more expensive corn. The wheat bran hydrolyzate, obtained from low-cost agricultural byproducts, was used as the nitrogen source. The expensive yeast extract was replaced by cheap persimmon juice to provide growth factors. Factorial experiment and response surface methods were adapted to optimize the medium. As a result, the maximum L-lactic acid concentration reached 144.28±2.71g/L, with the corresponding dosages of bran hydrolyzate and persimmon juice reduced by 0.750 and 16.00%, compared with those before optimization, respectively.(3) Kinetic model equations of batch fermentation using yam starch hydrolysis sugar as the substrate for L.rhamnosus HG09F5-27 were established. It was found that L-lactic acid yield, conservation rate andμmax firstly increased and then decreased with the increase of initial glucose concentration. Ca(OH)2 was found to be the most excellent neutralizer for pH adjustment to relieve the product inhibition during the fermentation. The culture conditions such as pH, temperature and dissolved oxygen were controlled step by step, and the appropriate subsection controlling strategy was established. The yield and productivity of L-lactic acid production reached 150.11±3.47g/L and 2.67±0.10g/L/h, which were 4.77% and 30.88% higher than those of constant controlling condition fermentations, respectively.(4) 100 L of external membrane bioreactor was utilized to realize the semi-continuous fermentation of L-lactic acid with cell recycles. It was found that substrate inhibition could be effectively relieved by feeding medium fermentations and the productivity of L-lactic acid production was 32.07% higher than that of non-feeding medium fermentation. In the cell recycle fermentation, the more the living cells at the end of Nth round of fermentation, the higher the productivity and the shorter the fermentation period in the (N+1)th round. When the cell recycle fermentation coupled with feeding substrate was carried out, the yield, conservation rate and productivity of L-lactic acid production arose first and decreased later. In the 8th round of L-lactic acid fermentation, the yield was 157.26±3.06g/L, which increased 12.89% compared with that of batch fermentation; the productivity reached 8.73±0.17g/L/h, which were 4.11 times of that of batch fermentation.(5) In order to obtain diosgenin, the by-product of L-lactic acid production, the pretreatment of yam tuber by steam explosion and enzymatic hydrolysis was performed, resulting that more than 90% of dioscin was dissolved in the yam sugar solution. And then the cell recycle fermentation coupled with separation was carried out. The fermentation broth was concentrated by nanofiltration equipment to enrich the dioscin. As a result, diosgenin yield reached to 2.21±0.13g/100g, which was 6.76% higher than that of traditional acid hydrolysis method (2.07±0.10g/100g), while the waste water and residue were reduced more than 95%. Thus, a clean process for diosgenin production was established.The main novelties of our work were as follows:(i) a L-lactate and dioscin resistant positive mutant strain was isolated by traditional physiochemical mutagens and genome shuffling methods. The optical purity of L-lactic acid from the mutant strain was more than 98%, and the yield and productivity were 4.09 times and 4.40 times of those of the wild strain, respectively; (ii) yam tuber, a new no-grain material, was screened out for L-lactic acid production. The material cost of L-lactic acid production was reduced by half compared with that of the corn material; (iii) pulse-feeding medium and cell recycle fermentations were applied to enhance L-lactic acid production. As a result, the yield of L-lactic acid increased by 12.89% and the productivity increased 3-fold in the 12-batch of semi-continuous fermentations, compared with those of the batch fermentation; (iv) the two-grade fermentations coupled with membrane separation were used to produce L-lactic acid and its by-product, diosgenin. Meanwhile, the waste water and residue of L-lactic acid production were only one-fifth and one quarter of those of traditional calcium salt methods, respectively, and the dosage of H2SO4, discharge of waste water and the residue was reduced by 95% than those of the traditional acid-hydrolysis methods, indicating that the goals to decrease the energy consumption and pollution were achieved.The systems and methods of fermentation coupled with membrane separation might also be suitable for the fermentations using other bacteria and yeast as the producing strains. The yam tuber material might also be suitable for the production of other products such as succinic acid and ethanol. This investigation has provided a reference for the production of several products from one starting no-grain material.