Production Study Of L-lactic Acid By Immobilized Rhizopus Oryzae From Corn Stalk Hydrolyzate

Biomaterial polylactide has been widely paid attention in the world. Polylactide, abiocompatible and well-degraded polymeric material, is polymerized from the lacticacid. If the lactic acid can be produced from cheap biomass, that has great significancein medicine development, environment protection and resources saving. The lactic acidis basic organic acid and platform chemical. The biorefineries production of lactic acidwill be important industrial components of bio-based economies. To achievebiorefineries of lactic acid, several technical bottlenecks need to be overcome. One isthe low-cost and high-efficiency transformation of lignocellulose into small-moleculecarbohydrates, and the other is the conversion of carbohydrates into platform chemicalswith high-conversion ratio. Currently available technologies cannot fully meet theserequirements for large-scale industrial production and generate considerable economicbenefits. This research focused on the efficient sugar hydrolysis process andimmobilized fermentation of Rhizopus oryzae for L-lactic acid production.In a large agricultural country such as China, crop straw especially corn stover iswidely distributed and serves as a renewable resource. The efficiency of thesaccharification hydrolysis of corn stover must be enhanced for bio-refineries toproduce high-quality fuels and platform chemicals. In the process of the enzymatichydrolysis of corn stover cellulose to produce glucose, the supra-molecular structure ofcellulose crystalline regions is a major obstacle for the enzymatic hydrolysis of theβ-1,4-glycosidic bond. This study uses steam explosion technology and water/alcoholtreatment technology to destroy this structure and increase the efficiency of enzymaticcellulose hydrolysis.The steam explosion pretreatment of corn stalk was optimized with uniform design.The structures of untreated/treated corn stalk were detected by Scanning ElectronMicroscope (SEM) and X-ray diffraction (XRD), while their chemical composition wasalso investigated. Comparing with the untreated corn stalk, reducing sugar yield oftreated corn stalk was increased by97%after hydrolyzing. The relative content ofhemicelluloses and solvend decreased, while cellulose and lignin increased by29.73%and19.65%, respectively; the compact structure of corn stalk was remarkably destroyed.The optimum steam exploded process condition: operating pressure2.2MPa,solid-liquid ratio1:1(mL/g), reaction time9min and materials size40-60meshes. The enzymatic hydrolysis of cellulose treated by water/ethanol extraction aftersteam explosion was investigated. The substrate concentration, cellulase dosage, andreaction time were optimized with single-factor experiments and the response surfacemethodology. The reducing sugar yield reached672.36mg/g when the substrateconcentration was53.8g/L, cellulase dosage was53.32FPU/g, and reaction time was60.45h. Compared with the untreated and steam-exploded corn stover, the reducingsugar yield increased by170.46%and28.97%, respectively. The chemical compositionsof the untreated/treated corn stover were investigated and their structures werecharacterized by SEM. The results showed that the relative content of cellulose in cornstover treated by water/ethanol extraction increased significantly and the structurebecame fluffy due to the improved cellulose hydrolysis.To overcome such shortcomings as mycelium fracture injury and limited transferof oxygen and nutrients, the immobilization fermentation of Rhizopus oryzae wasinvestigated, including the choice of immobilization interface material, mountingbracket design, and the evaluation of the effectiveness of immobilization. Fermentationwith corn stover hydrolyzate as the main carbon source was conducted to screening theapplicable R. oryzae. Immobilized fermentation was performed in a shake flask and thereactor fermentation production of lactic acid was also studied to optimize thefermentation process.Comparison with a variety of R. oryzae-and Lactobacillus-fermented lactic acidproduction revealed that Rhizopus strain3.3462was the optimal fermentation strain. ForR. oryzae immobilized fermentation, the new support matrix was developed with thefiber material and geometry of the support matrix optimized. A comparative analysis ofthe fixing effects, cell growth, and L-lactid acid production showed that cotton fabricwas the optimal fiber surface for the support matrix and the hexagram w the mostsuitable geometry. Spore fixing kinetic studies revealed that R. oryzae spores cancompletely fix on the carrier, and that the adsorption rate sharply increased followingmycelium growth. By the mathematical fitting of the kinetic curves of spore adsorption,spore adsorption was found to follow first-order reaction kinetics. The optimal matrixsize, matrix number, reactor capacity, and spore inoculum density were determined by100mL shake flask experiments. The best culture condition was as follows: matrixdiameter of2.5cm, three matrices, and106spores/mL.The immobilization technique was used for R. oryzae3.3462L-lactic acidproduction from corn stalk hydrolyzate. To couple the saccharification and fermentation process, the simultaneous saccharification and fermentation technology was studied.Fermentation kinetics curve analysis showed that glucose depletion occurred after48hof fermentation, the lactic acid concentration reached51g/L, and the ethanol productionduring the entire fermentation process was low. Using the xylose of the stoverhydrolyzate as the carbon source resulted in a very low xylose consumption rate; thus,xylose was not suitable as the sole carbon source. Using the corn stover hydrolyzate asthe carbon source, R. oryzae underwent a48h adaptation period, after which glucoseand xylose were rapidly consumed and the maximum level of production of lactate was176h. The entire process was generated byproducts such as ethanol and fumaric acid.The simultaneous saccharification and fermentation process suggested that the initialsaccharification time and cellulase were the key factors. The uniform design showedthat saccharification for4h and45FPU/g cellulase dosage yielded better simultaneoussaccharification and fermentation results in shake flask experiments.An airlift bioreactor designed by our group was used to study the amplificationprocess of L-lactic acid production by immobilized R. oryzae from corn stalkhydrolyzate. The curve of absorption and germination of the spores in the reactorshowed that all spores germinated after6h under ventilation. However, ventilationinterfered with spore absorption and needed10h. Oxygen transmission becomeschallenging after amplifying the culture medium volume. The gassing out measurementshowed that the oxygen uptake and transmission rates of the corn stalkhydrolyzate-containing medium were much larger than those of the glucose-containingmedium. Thus, the corn stalk hydrolyzate-containing medium can be used for R. oryzaefermentation in the airlift bioreactor. A comparison between immobilized and freefermentation revealed that the lactic acid conversion rate reached72%in the former andonly40.4%in the latter, which suggested that immobilized fermentation wassignificantly advantageous over free fermentation. To test the stability of immobilizedfermentation, a second batch fermentation as well as simultaneous saccharification andfermentation were conducted. Repeated batch fermentation can produce nineconsecutive batches, and the lactic acid conversion rate remained above40%to70%. Insimultaneous saccharification and fermentation, the lactic acid conversion rate reached57%.In summary, to transform the cellulose of corn stover into L-lactic acid, thephysical and chemical pretreatment of corn stover, as well as bio-enzymaticsaccharification were systematically studied. The technology for producing reducing sugar was established based on steam explosion, water/alcohol treatment, and cellulasehydrolysis. For the purpose of R. oryzae fermentation for L-lactic acid production fromcorn stover hydrolysis, a Rhizopus strain with a high lactic acid production capacity wasscreened. A support matrix for immobilized fermentation was then designed and thefermentation technology optimized. A higher L-lactic acid yield was obtained and thetechnology was found be stable for repeated batch fermentations as well assimultaneous saccharification and fermentation.