Study On L-lactic Acid Production Using Rhizopus Oryzae With A High Volumetric Productivity By Semi-continuous Fermentation

L-lactic acid is a commonly occurring organic acid,which is valuable due to its wide use in food,medicare,agticulture,chemical and other fields.There is also an increasing interest in its application in the synthesis of polylactic acid,which can be used for biodegradable plastic manufacturing.L-lactic acid can be produced by bacteria and fungi.Compared to the bacteria, Rhizopus oryzae has advantages including its low nutrient requirements,amylolytic characteristics and high optical purity product.It has been recognized as promising candidate for L-lactic acid production.However,there are also some problems in L-lactic acid production by Rhizopus oryzae, such as lower productivity of L-lactic acid,lower utilization ratio of raw material,difficulty to control morphorlogy and so on.In this work,a semi-continuous fermentation of Rhizopus oryzae AS 3.819 for L-lactic acid has been developed with L-lactic acid yield and a high volumetric productivity.Research mainly includes:(1) Effects of culture condition on L-lactic acid production and morphology of Rhizopus oryzae were studied in flask and bioreactor.(2) Kinetics models of semi-continuous fermentation by Rhizopus oryzae were studied in bioreactor.(3) Growth of pellets during the semi-continuous fermentation was described in detail.(4) Fluid dynamics of the semi-continuous fermentation in bioreactor was investigated.The main results and conclusions obtained are as follows.(1) In flask culture,culture conditions for semi-continuous fermentation were optimized,the results showed that good pellets and high L-lactic acid yield were gained when the culture conditions were as the following:using optimal medium,50 mL production medium in 250 mL flask,inoculation size 10%of seed culture broth which cultured for 24 h at 32℃,fermentation temperature at 28～30℃from 0 h to 36 h and at 34～36℃from 36 h to 72 h,agitation speed 200 r/min in cycle 1 fermentation and cultured for 24 h in each repeated cycle fermentation.Under the optimal culture conditions,semi-continuous fermentation lasted for 30 cycles,size of pellets were 0.8～1.0 mm,and they had an good ability to produce L-lactic acid,the volumetric productivity were 1.58 g/(L·h) in cycle 1 fermentation,and 2.67～3.00 g/(L·h) in the repeated cycles fermentation.(2) In 3 L bioreactor,the effects of temperature,agitation speed and aeration on L-lactic acid production and morphology of Rhizopus oryzae in cycle 1 fermentation were investigated,the results showed that the volumetric productivity was 2.51 g/(L·h) when the control conditions were as the following:at 28～32℃from 0 h to 30 h,agitation speed 400 r/min,aeration 1.5 L/(L·min), and at 34～36℃from 30 h to 60 h,agitation speed 300 r/min,aeration 0.5 L/(L·min).The effects of glucose concentration,temperature,agitation speed and aeration on productivity in repeated cycles fermentation were studied,the results showed that the volumetric productivity was above 3.80 g/(L·h) in 3 L bioreactor when the control conditions were as the following:glucose concentration 100 g/L,agitation speed 300 r/min,aeration 0.5 L/(L·min),culture temperature 34～36℃,and fermentation period was 20 h for each cycle.(3) The effects of nitrogen source and inorganic salts in feeding medium on the L-lactic acid production in semi-continuous fermentation were studied.The results showed that the nitrogen source and inorganic salts were indispensable for cell growth and L-lactic acid synthesis in repeated Cycles fermentation,accumulating biomass in the bioreactor did not increase the volumetric productivity,some biomass could be removed periodically to keep DCW about 10 g/L,and the semi-continuous fermentation with a high volumetric productivity would be going on.Under the optimal conditions,25 cycles for L-lactic acid production were carried out in 7 L bioreactor,the final productivity was 2.47 g/(L·h) for cycle 1 and above 3.40 g/(L·h) for other 19 cycles.The process of L-lactic acid production by Rhizopus oryzae in semi-continuous fermentation with a high volumetric productivity was fulfilled.(4) The kinetic equations for describing microbial growth,substrate consumption and L-lactic acid production had been presented.In repeated cycles fermentation the curves of L-lactic acid production and substrate consumption are nearly linear relation.Logistic equation and Luedeking-Piret equation were chosen as the kinetics models,and the key parameters were calculated by Matlab.X_m,μ_m,α,β,p and q was 7.6164 g/L,0.1802 h^-1,3.2631,0.5980,1.9875 and 1.0232 respectively in cycle 1 fermentation.In repeated cycles fermentation the L-lactic acid and biomass were initialized as zero,and the key parameters were found in cycle 2,X_m,μ_m,α,β,p and q was 8.246 g/L,0.0087 h^-1,0.9610,0.4929,3.2438 and 1.0326 respectively.From cycle 2 to cycle 10,biomass increased,whileμ_m,the largest specific production and the reutilize efficiency decreased,but the reutilize efficiency still above 90%.The models were found to represent the fermentation process fairly well with R²â‰¥0.98.(5) The growth of pellets filamentous Rhizopus oryzae in flask and bioreactor were surveyed. The results showed the growth process of pellets in submerge culture was as the following:spores germinated into mycelia,mycelia aggregated into loose packed hypha,in the earlier stage "fluffy" pellets with a tightly packed core were formed,then the pellets with a center contains no recognizable mycelia were occurred and become bigger,the center size of the pellets also increased during the semi-continuous fermentation.These pellets reutilized for many times were broken up into pieces and most of the pieces become "fluffy" pieces with the growth of outer mycelia.The vesicles with a center contains no recognizable mycelia were formed because of the cell lyses inside the pieces.And the vesicles were broken up into pieces later because of cell lyses and shearing force of the broth.Uniform pellets,pieces and vesicles were named pellets,and they changed to each other in the broth.Percent of uniform pellets decreased,while percents of pieces and vesicles increased in the semi-continuous fermentation.And all the pellets with a good activity of LDH actualized L-lactic acid production in the semi-continuous fermentation.The inner structure of pellets was investigated with microscope by the paraffin section technology,the results showed that "fluffy" pellets with a tightly packed core gained in earlier stage had a higher activity to produce L-lactic acid,while the outer mycelia had more vitality to produce L-lactic acid than the inner mycelia for pellets with no recognizable mycelia core.(6) Effects of pellets size on the volumetric productivity were investigated,only uniform pellets with size of 0.9～1.2 mm had advantages for L-lactic acid production with a high volumetric productivity in fermentation.(7) Fluid dynamics in a 3 L bioreactor was studied at different agitation speed,with the method of computational fluid dynamics(CFD).The simulation analysis showed that the good conditions for high L-lactic acid yield and pellets forming were as the following:the shearing velocity 0.009～1.582 m/s for cycle 1 fermentation,0.009～1.192 m/s for repeated cycles fermentation.In terms of the results above,the optimal agitation speed was also obtained by the simulation analysis in 7 L bioreactor.Under the optimal condition in 7 L bioreactor the semi-continuous fermentation was carried out,the productivity was 2.51 g/(L·h) for cycle 1 and 3.58～4.01 g/(L·h) for other 19 cycles.The L-lactic acid production by Rhizopus oryzae was carried out with the optimal agitation speed basing on the results simulated by CFD in 50 L bioreactor,and the L-lactic acid concentration was 94.56 g/L.Those results above proved that the CFD simulated method offers a technological means to optimize the operating system in scaled-up fermentation.Mycelia could be repeatedly used for many times,utilization ratio of fungi was increased, fermentation time in each circle was shortened,and the volumetric productivity was improved during the semi-continuous fermentation.The above results can provide some theoretical basis for commercial process of L-lactic acid production using Rhizopus oryzae with a high volumetric productivity in semi-continuous fermentation.