摘要
L-乳酸是一种重要的有机酸,广泛应用于食品、医药、农业和化工中,利用L-乳酸制备的聚L-乳酸是一种新型的可生物降解材料。细菌发酵和真菌发酵是发酵生产L-乳酸的主要方法,其中米根霉具有营养要求简单、可进行同步糖化发酵、产酸光学纯度高等优点,是工业化生产最有潜力的菌株之一。但是,目前米根霉发酵生产L-乳酸仍然存在发酵强度低、原料利用率不高、菌体形态难以控制等问题。
本文以米根霉AS3.819为生产菌株,以提高L-乳酸产率及发酵强度为主要目标,研究米根霉半连续高强度发酵产L-乳酸的关键技术。通过优化摇瓶发酵条件,进行米根霉菌丝球半连续发酵生产L-乳酸和罐发酵试验,建立其动力学模型,分析发酵中菌丝球的生长过程,并采用计算流体动力学(CFD)模拟技术对其发酵条件进行模拟优化。主要研究结论如下:
(1)通过对种子培养条件及半连续发酵条件的优化,获得摇瓶中米根霉菌丝球半连续高强度发酵生产L-乳酸的控制模式:首批发酵中250 mL三角瓶装50 mL培养基,接种量10%,种子液32℃培养24 h,摇床转速200 r/min,发酵前期(0～36 h)温度为28～30℃,发酵后期(36～72 h)温度为32～34℃;重复批次发酵中每批发酵24 h。该发酵条件下,半连续稳定发酵30批,首批发酵菌丝球直径稳定控制为0.8～1.0 mm,其产酸能力较高,发酵强度为1.58g/(L·h),重复批次发酵强度2.67～3.00 g/(L·h)。
(2)研究3L发酵罐中发酵温度、搅拌转速及通气量对米根霉首批发酵产L-乳酸的影响,建立了米根霉菌丝球高强度发酵产L-乳酸的控制模式:发酵0～30h温度32～34℃、搅拌转速400 r/min、通气量1.5 L/(L·min),30～60 h温度34～36℃、搅拌转速300 r/min、通气量0.5 L/(L·min);该条件下,菌丝球直径稳定控制为0.9～1.2 mm,其产酸能力较高,发酵强度为2.51g/(L·h)。对重复批次发酵中葡萄糖质量浓度、温度、搅拌转速及通气量对发酵强度的影响进行研究,得到3L发酵罐中重复批次发酵生产L-乳酸的控制模式:补料培养基中葡萄糖质量浓度100 g/L,温度34～36℃、搅拌转速300 r/min、通气量0.5 L/(L·min),半连续发酵5批,每批发酵周期为20 h,发酵强度大于3.80g/(L·h)。
(3)补料培养基中N源及无机盐质量浓度对半连续发酵过程影响的研究表明,N源和无机盐是半连续发酵稳定进行不可缺少的因素:生物量在一定范围内增加,不能提高其发酵强度,在半连续发酵中间歇取出部分菌体保持DCW为10 g/L左右,可以维持高强度发酵。采用以上优化发酵条件,7 L发酵罐中米根霉半连续发酵生产L-乳酸25批,首批发酵强度为2.47 g/(L·h),第2～20批发酵强度3.40～3.85 g/(L·h),实现了米根霉半连续高强度稳定发酵生产L-乳酸。
(4)对米根霉半连续发酵产L-乳酸菌体生长、产物生成及底物消耗的动力学进行研究,发现重复批次发酵中每批产物生成及底物消耗曲线接近于直线。选择Logistic及Luedeking-Piret方程作为米根霉发酵产L-乳酸的动力学方程,运用Matlab软件将试验数据进行拟合,对方程中关键参数求解,建立米根霉半连续发酵动力学模型,并对模型进行验证。其中首批发酵动力学模型中X_m为7.6164 g/L、μ_m为0.1802 h~(-1)、α为3.2631、β为0.5980、p为1.9875、q为1.0232;重复批次发酵过程中,对初始菌体量及L-乳酸产量采用“零处理”方法,进行动力学方程求解,其中第2批发酵动力学模型中X_m为8.246 g/L、μ_m为0.0087h~(-1)、α为0.9610、β为0.4929、p为3.2438、q为1.0326,δ为98.63%;半连续发酵第2批到第10批,每批的生物量逐渐增加,菌体最大比生长速率逐渐减小,菌体重复利用效率逐渐降低但仍大于90%,最大比产物合成速率逐渐减小。验证试验表明建立的动力学模型能够很好的描述发酵过程(R~2≥0.98)。
(5)对摇瓶及发酵罐中菌体生长过程进行分析,菌丝球的形成过程为:孢子萌发成菌丝,菌丝凝聚在一起形成菌丝元,菌丝元生成具有致密菌核的菌丝球;发酵后期菌丝球内部逐渐形成中空核,且该中空核及菌丝球直径越来越大;重复利用多次的菌丝球逐渐破裂成菌片,菌片表面菌丝生长,大部分形成具有菌毛层的菌片,极少部分形成游离菌丝;菌片中心菌丝自溶衰亡形成具有中空核的菌囊;随着发酵的进行,菌囊由于其内部菌丝衰亡及流体剪切作用破碎成菌片。半连续发酵液中规则菌丝球、菌片、菌囊3种不同形态的菌丝球相互转变,其中规则菌丝球百分比逐渐降低,菌片及菌囊百分比逐渐增加;它们都具有较高的L-乳酸脱氢酶(LDH)活性,共同维持了半连续高强度发酵的进行。通过石蜡切片观察发现,具有致密菌核的菌丝球菌体代谢产酸能力较强;具有中空核的菌丝球壁外缘菌体细胞代谢活力旺盛。
(6)菌丝球大小对发酵强度影响比较明显,直径过大或过小的菌丝球产酸能力较低,直径为0.9～1.2 mm的菌丝球,产酸能力较高,有利于高强度发酵生产L-乳酸。
(7)采用CFD模拟技术对3 L发酵罐内不同转速的流体动力学进行模拟,建立3 L发酵罐中发酵工艺的CFD调控模式:首批发酵中剪切速度范围为0.009～1.582 m/s,重复批次发酵中剪切速度范围为0.009～1.192 m/s;根据该控制模式,优化7 L发酵罐中发酵条件,采用该优化发酵条件米根霉半连续高强度发酵生产L-乳酸20批,其中首批发酵强度为2.51g/(L·h),重复批次发酵强度为3.58～4.01g/(L·h);对50 L放大发酵罐的CFD模拟,确定了放大发酵条件,实际50 L放大发酵罐发酵试验中发酵产酸为94.56 g/L。
利用米根霉菌丝球半连续高强度发酵生产L-乳酸,增加了菌体利用率,节约了原材料、缩短了重复批次发酵周期、提高了发酵强度,为实现米根霉半连续高强度发酵生产L-乳酸的工业化奠定了良好的基础。
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 R~2≥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.
引文
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