光生物反应器敏感性参数及小球藻异养—稀释—光诱导串联培养工艺中试研究
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摘要
微藻在能源、食品、饲料、环保及医药等领域具有广泛的应用,光生物反应器是微藻培养的核心装置,特别是微藻能源研发热潮的兴起,对光生物反应器优化与放大的需求尤为迫切。迄今,国内外对光生物反应器虽己开展了不少研究,但光生物反应器的优化仍然基本凭经验、且主要依靠传统的实验方法针对某一特定形式的反应器开展,工作量大且推广性差；近年来出现了采用CFD技术对光生物反应器进行优化,但仅局限于理论计算,很少经过微藻培养的验证；此外,对于光生物反应器的放大,不同的研究者往往采用不同的放大参数,哪种放大参数合适?目前尚不清楚。因此,光生物反应器的优化与放大尚没有一套系统、可靠且具有普适性的方法。究其原因,主要在于众多表征光生物反应器中混合及藻细胞受光特性参数对光生物反应器性能影响的重要性及敏感性,迄今尚不清楚。此外,笔者所在实验室在国内外首创的微藻异养—稀释—光诱导培养技术亟待进行中试放大研究,且光生物反应器也是微藻光诱导的核心装置,亟待研究。
鉴于上述情况,本文采用CFD方法与小球藻光诱导(部分为光自养)等热模实验相结合的方法,通过相关性分析确定影响各类光生物反应器性能的具有共性的敏感性因素,然后基于敏感性参数设计、优化了系列规模化的微藻光诱导培养系统,最后开展了小球藻异养—稀释—光诱导串联培养工艺的中试研究。
(1)优化并建立了蛋白核小球藻的光衰减模型并研究了藻细胞色素含量对光衰减的影响。Lambert-Beer光衰减模型仅适合于低藻细胞浓度,Hyperbolic光衰减模型对高藻细胞浓度更适合,Cornet光衰减模型适合于较宽藻细胞浓度范围。笔者所建立的M-Hyperbolic模型能较好地拟合藻细胞浓度小于2.68g/L条件下的光衰减过程。此外,在相同藻细胞浓度下,随着细胞中色素含量的增加,光的衰减程度加剧,而在高藻细胞浓度下,藻细胞色素含量对光衰减的影响较小。
(2)研究了入射光强、藻细胞浓度及通气量等对平板式光生物反应器中混合及藻细胞受光特性的影响并对不同放大准则进行了理论分析。随着藻细胞浓度增加,比光照速率(qi)和体积平均光强均(Iavv)呈指数趋势下降；随着入射光强增加,qi和Iavv呈线性增加；随着通气量增加,藻液湍动能(TE)及其耗散率(TED)呈线性增加,流体平均径向速率(Ur)呈幂函数增加,混合时间(tm)呈指数下降,藻细胞光暗循环周期(LDCT)呈幂函数下降,藻细胞时均光强(Iavt)和藻细胞在光区停留时间占循环时间的比例(LF)几乎不受影响。以I“v和qi相等为准则对平板式光生物反应器进行放大,外部光强相等时,15L反应器中藻细胞浓度分别仅为3L反应器中藻细胞浓度的0.26倍和0.36倍；初始藻细胞浓度相等时,15L反应器外部光强分别是3L反应器外部光强的3.7倍和2.81倍。以径向混合时间(Tr)、tm和LDCT相等为放大准则时,15L反应器的单位体积通气功率分别约是3L反应器的1倍、2倍和5.7倍。
(3)将室内3L平板式光生物反应器、户外70L槽式培养系统和50L鼓泡式培养系统中小球藻的光诱导结果(藻细胞死亡速率、藻细胞叶绿素和蛋白质含量的上升速率)与培养系统中的混合及藻细胞受光特性参数进行相关性分析,结果表明Iavv、Iavt和qi是影响藻细胞光诱导效果的主要参数。在相同外部光强,不同光程下,影响培养系统性能的主要是藻液的受光强度。在相同外部光强及相同的光程下,影响光生物反应器性能的主要因素是藻液的混合。采用相等Iavv、相等qi和相等LDCT为放大准则具有较好的效果,而采用相等Tr和tm为放大准则,效果较差。
(4)基于强化藻液在光照方向的混合设计了新型内部构件及相应的15L平板式光生物反应器。小球藻在新型平板式反应器中的光自养培养结果与反应器中混合及藻细胞受光特参数之间的相关性分析表明,流体在光照(光衰减)方向的速率(UL)及藻细胞的LDCT是影响反应器性能的主要敏感性参数。在此基础上,在15L鼓泡式(Bubble).隔板式(Split)及气升式(Airlift)平板反应器中对所确定的敏感性参数进行了验证,并首次系统地确定了这三种平板式光生物反应器的性能：Split平板光生物反应器优于Airlift平板光生物反应器,Bubble平板光生物反应器性能最差。在通气量1.0vvm条件下,类型a反应器中的最高藻细胞浓度和比生长速率分别比对照(Bubble反应器)提高了92.5%和28.0%,类型b反应器中的最高藻细胞浓度和比生长速率分别比对照提高了33.8%和25.6%,类型c反应器中的最高藻细胞浓度和比生长速率分别比对照提高了45.2%和29.8%。
(5)基于上述确定的敏感性参数对规模化的光生物反应器进行优化及验证。不同内部结构的1000L平板式光生物反应器及300L柱式立袋、不同操作条件下的中型跑道池及不同桨型下的圆池中微藻的光诱导或光自养培养结果均表明,反应器中流体在光衰减方向的速率(UL)及藻细胞光暗循环周期(LDCT)显著地影响反应器的性能,敏感性参数的准确性不仅得到了进一步验证,而且表明UL及LDCT这两个敏感性参数具有普适性。
(6)基于敏感性参数设计了系列规模化的微藻培养系统,并对小球藻异养—稀释—光诱导串联培养技术开展了中试研究。蛋白核小球藻在6000L发酵罐中异养培养的藻细胞平均生长速率后期仍可达3.0g/L/h,与50L发酵罐中生长速率相当；温度显著影响小球藻光诱导效率,且河水可替代自来水进行小球藻光诱导。在上述规模化的中型、大型跑道池及圆池中,经10～20h光诱导后,藻细胞的叶绿素和蛋白质含量可达20mg/g和50%以上,达到小球藻质量标准,这与在小规模光诱导系统中的光诱导效率相当。
本文的上述研究结果不仅为光生物反应器的优化和放大提供了依据而且为小球藻的异养-稀释-光诱导培养技术的产业化奠定了良好的基础。
Microalgae are widely applied in the fields of energy, food, feed, environment as well as pharmaceuticals industries. Photobioreactors (PBRs) are the key devices for microalgae cultivation. It is urgent to optimize and scale up PBRs, especially with the upsurge of microalgae energy research. So far, there have been a lot of researches on PBRs, but these works almost rely on empirical approach, and thus traditional experimental method which needs heavy workload were mainly used to optimize specific type of PBR. Meanwhile, the results of these reasearches almost cannot be broadly applicable to other types of PBRs. Recently, the Computational Fluid Dynamics (CFD) technology is applied to the optimization of PBR. However, these researches only focus on theoretical computational results, few of which are validated by using microalgae cultivation experiments. Different parameters are used to scale up PBRs, and it is still not clear which of these parameters are most efficient when scaling up different PBRs. Therefore, there is not a systemic, reliable and universal method for optimization and scale up of PBRs as for it is not known which parameters of mixing and light regime characteristic are most important and sensitive to the performance of PBR. Additionally, the pilot-scale experiment for the technology of heterotrophy-dilution-photoinduction sequential cultivation invented in our laboratory is in progress. As the key devices for microalgae photoinduction, the PBR is urgently required to be investigated.
Accordingly, an approach which integrates the CFD method and photoinduction (part of photoautotrophic cultivation) of Chlorella was used. The universal key parameters for affecting performance of PBR were determined through sensitive analysis method. Then pilot-scale system for microalgae photoinduction was designed and optimized based on key parameters. The pilot research of heterotrophy-dilution-photoinduction sequential cultivation was carried out.
(1) Optimization and establishment of light attenuation model of Chlorella pyrenoidosa and investigation of the effect of pigment content on light attenuation were implemented. Lambert-Beer light attenuation model is suitable only for low density of microalgae, and the Hyperbolic light attenuation model suit to high microalgae concentration. The Cornet light attenuation is applicable to wide range of microalgae concentration. M-Hyperbolic model established by us fits the light attenuation process better in the condition of low microalgae concentrations less than2.68g/L. Additionally, at the same cell concentration, the degree of light attenuation increased with the increase of pigment content of microalgal cell. However, the effect of pigment content on light attenuation is little when the cell concentration is high.
(2) The effects of incident light intensity, microalgal cell concentration and air flow rate on parameters of mixing and light regime characteristic in flat plate PBR was investigated, and theoretical analysis of different formulas for scaling up PBR was executed. With the increase of microalgae concentration, specific irradiation rate (q;) and volume averaged light intensity (Iavv) decrease exponentially; as the incident light intensity increase, q; and Iavv increase linearly; as the air flow rate increases, turbulence energy (TE) and turbulence energy dissipation (TED) of microalgae culture show linear increasing trend, the radial velocity of fluid (Ur) show increasing trend of power function, the mixing time (tm) decrease exponentially, the light dark cycle time of microalgal cell decreases quickly and the time averaged light intensity (Iav') of cell and the fraction of the light time in light dark cycle (LF) are almost not change. The microalgae concentration in15L PBR can only be respectively0.26times and0.36times of that in3L PBR with the same incident light intensity. When the PBR is scaling up based on formulas of equivalence of qi and Iav\incident light intensity for15L PBR is respectively3.7fold and2.81fold of that in3L PBR with the same cell concentration. The unit volume power of aeration in15L PBR approximately1times,2times and5.7times of that in3L PBR when formulas of equal radial mixing time (Tr), tm and LDCT are employed.
(3) The correlation analysis of photoinduction results of microalgae (the death rate of microalgal cell, the increasing rate of chlorophyll and protein content) with parameters of mixing and light regime characteristic in indoor3L PBR, outdoor70L open tank culture system and50L bubble basin was executed. The results show that Iavv, lav'as well as q; are the key parameters to photoinduction of microalgae. The irradiance level of microalgal culture is the main factor influencing the performance of system at the conditions of the same incident light intensity but different light path of PBRs. The mixing is the key factor for performance of PBR when PBRs have the same incident light intensities and same light paths. The good results are obtained when formulas of equal Iavv and q; are used, and the bad results appeared when formulas of equal Trand tm are employed to scale up the flat plate PBRs.
(4) The novel inner structures and relevant15L PBRs are deigned based on enhancing the microalgal culture along the light irradiance direction. The correlation analysis of photoautotrophic results of Chlorella pyrenoidosa with mixing and light regime characteristic parameters indicated that the velocity of fluid along the light attenuation direction (Ul) and LDCT of microalgal cell are the main sensitive parameters for affecting the performance of PBR. The validation of sensitive parameters was executed in three type of15L flat plate PBRs, i.e. Bubble, Split and Airlift. It is the first time to determine the performance of these three types of flat plate PBR:the Split PBR is better than Airlift PBR; and the Bubble PBR is the worst. The maximum cell concentration and specific growth rate of microalgae in type-a PBR are increased respectively92.5%and28.0%than that in Bubble PBR (the control), the maximum cell concentration and specific growth rate of microalgae in type-b PBR are increased respectively33.8%and25.6%than the control, and the maximum cell concentration and specific growth rate of microalgae in type-c PBR are increased respectively45.2%and29.8%than those in the control.
(5) Optimization of the large scale PBR based on sensitive parameters and the results validated by microalgae culture experiment were carried out. The results of microalgae photoinduction and photoautotrophic culture in1000L flat plate PBRs and300L column PBRs with different inner structures, middle raceway ponds with different operation conditions and large circular ponds with different type of impellers all show that UL and LDCT of microalgae significantly affect the performance of PBR. Not only the reliability of sensitive parameters is validated, but also it is demonstrated that UL and LDCT have the universality for different types of PBRs.
(6) A Serie of large scale systems for microalgae cultivation were designed based on sensitive parameters. And the pilot study of heterotrophy-dilution-photoinduction sequential cultivation was carried out. The growth rate of Chlorella pyrenoidosa in6000L fermenter can still achieved about3.0g/L/h at late stage of the heterotrophic culture. This growth rate of microalgal cell is comparative with that in50L fermenter. Temperature remarkably affects efficiency of Chlorella pyrenoidosa photoinduction, and the river water can replace tap water for Chlorella pyrenoidosa photoinduction. The chlorophyll and protein content of microalgal cell can reach over20mg/g and50%respectively after10～20h photoinduction in large scale microalgal culture system. The efficiency of photoinduction in these large scale systems is almost the same with that in small scale systems such as50L bubble basins.
In thispresent study, our results can not only provide basis for optimization and scale up of PBRs, but also load good foundation for industrialization of heterotrophy-dilution-photoinduction sequential cultivation of Chlorella pyrenoidosa.
鉴于上述情况,本文采用CFD方法与小球藻光诱导(部分为光自养)等热模实验相结合的方法,通过相关性分析确定影响各类光生物反应器性能的具有共性的敏感性因素,然后基于敏感性参数设计、优化了系列规模化的微藻光诱导培养系统,最后开展了小球藻异养—稀释—光诱导串联培养工艺的中试研究。
(1)优化并建立了蛋白核小球藻的光衰减模型并研究了藻细胞色素含量对光衰减的影响。Lambert-Beer光衰减模型仅适合于低藻细胞浓度,Hyperbolic光衰减模型对高藻细胞浓度更适合,Cornet光衰减模型适合于较宽藻细胞浓度范围。笔者所建立的M-Hyperbolic模型能较好地拟合藻细胞浓度小于2.68g/L条件下的光衰减过程。此外,在相同藻细胞浓度下,随着细胞中色素含量的增加,光的衰减程度加剧,而在高藻细胞浓度下,藻细胞色素含量对光衰减的影响较小。
(2)研究了入射光强、藻细胞浓度及通气量等对平板式光生物反应器中混合及藻细胞受光特性的影响并对不同放大准则进行了理论分析。随着藻细胞浓度增加,比光照速率(qi)和体积平均光强均(Iavv)呈指数趋势下降；随着入射光强增加,qi和Iavv呈线性增加；随着通气量增加,藻液湍动能(TE)及其耗散率(TED)呈线性增加,流体平均径向速率(Ur)呈幂函数增加,混合时间(tm)呈指数下降,藻细胞光暗循环周期(LDCT)呈幂函数下降,藻细胞时均光强(Iavt)和藻细胞在光区停留时间占循环时间的比例(LF)几乎不受影响。以I“v和qi相等为准则对平板式光生物反应器进行放大,外部光强相等时,15L反应器中藻细胞浓度分别仅为3L反应器中藻细胞浓度的0.26倍和0.36倍；初始藻细胞浓度相等时,15L反应器外部光强分别是3L反应器外部光强的3.7倍和2.81倍。以径向混合时间(Tr)、tm和LDCT相等为放大准则时,15L反应器的单位体积通气功率分别约是3L反应器的1倍、2倍和5.7倍。
(3)将室内3L平板式光生物反应器、户外70L槽式培养系统和50L鼓泡式培养系统中小球藻的光诱导结果(藻细胞死亡速率、藻细胞叶绿素和蛋白质含量的上升速率)与培养系统中的混合及藻细胞受光特性参数进行相关性分析,结果表明Iavv、Iavt和qi是影响藻细胞光诱导效果的主要参数。在相同外部光强,不同光程下,影响培养系统性能的主要是藻液的受光强度。在相同外部光强及相同的光程下,影响光生物反应器性能的主要因素是藻液的混合。采用相等Iavv、相等qi和相等LDCT为放大准则具有较好的效果,而采用相等Tr和tm为放大准则,效果较差。
(4)基于强化藻液在光照方向的混合设计了新型内部构件及相应的15L平板式光生物反应器。小球藻在新型平板式反应器中的光自养培养结果与反应器中混合及藻细胞受光特参数之间的相关性分析表明,流体在光照(光衰减)方向的速率(UL)及藻细胞的LDCT是影响反应器性能的主要敏感性参数。在此基础上,在15L鼓泡式(Bubble).隔板式(Split)及气升式(Airlift)平板反应器中对所确定的敏感性参数进行了验证,并首次系统地确定了这三种平板式光生物反应器的性能：Split平板光生物反应器优于Airlift平板光生物反应器,Bubble平板光生物反应器性能最差。在通气量1.0vvm条件下,类型a反应器中的最高藻细胞浓度和比生长速率分别比对照(Bubble反应器)提高了92.5%和28.0%,类型b反应器中的最高藻细胞浓度和比生长速率分别比对照提高了33.8%和25.6%,类型c反应器中的最高藻细胞浓度和比生长速率分别比对照提高了45.2%和29.8%。
(5)基于上述确定的敏感性参数对规模化的光生物反应器进行优化及验证。不同内部结构的1000L平板式光生物反应器及300L柱式立袋、不同操作条件下的中型跑道池及不同桨型下的圆池中微藻的光诱导或光自养培养结果均表明,反应器中流体在光衰减方向的速率(UL)及藻细胞光暗循环周期(LDCT)显著地影响反应器的性能,敏感性参数的准确性不仅得到了进一步验证,而且表明UL及LDCT这两个敏感性参数具有普适性。
(6)基于敏感性参数设计了系列规模化的微藻培养系统,并对小球藻异养—稀释—光诱导串联培养技术开展了中试研究。蛋白核小球藻在6000L发酵罐中异养培养的藻细胞平均生长速率后期仍可达3.0g/L/h,与50L发酵罐中生长速率相当；温度显著影响小球藻光诱导效率,且河水可替代自来水进行小球藻光诱导。在上述规模化的中型、大型跑道池及圆池中,经10～20h光诱导后,藻细胞的叶绿素和蛋白质含量可达20mg/g和50%以上,达到小球藻质量标准,这与在小规模光诱导系统中的光诱导效率相当。
本文的上述研究结果不仅为光生物反应器的优化和放大提供了依据而且为小球藻的异养-稀释-光诱导培养技术的产业化奠定了良好的基础。
Microalgae are widely applied in the fields of energy, food, feed, environment as well as pharmaceuticals industries. Photobioreactors (PBRs) are the key devices for microalgae cultivation. It is urgent to optimize and scale up PBRs, especially with the upsurge of microalgae energy research. So far, there have been a lot of researches on PBRs, but these works almost rely on empirical approach, and thus traditional experimental method which needs heavy workload were mainly used to optimize specific type of PBR. Meanwhile, the results of these reasearches almost cannot be broadly applicable to other types of PBRs. Recently, the Computational Fluid Dynamics (CFD) technology is applied to the optimization of PBR. However, these researches only focus on theoretical computational results, few of which are validated by using microalgae cultivation experiments. Different parameters are used to scale up PBRs, and it is still not clear which of these parameters are most efficient when scaling up different PBRs. Therefore, there is not a systemic, reliable and universal method for optimization and scale up of PBRs as for it is not known which parameters of mixing and light regime characteristic are most important and sensitive to the performance of PBR. Additionally, the pilot-scale experiment for the technology of heterotrophy-dilution-photoinduction sequential cultivation invented in our laboratory is in progress. As the key devices for microalgae photoinduction, the PBR is urgently required to be investigated.
Accordingly, an approach which integrates the CFD method and photoinduction (part of photoautotrophic cultivation) of Chlorella was used. The universal key parameters for affecting performance of PBR were determined through sensitive analysis method. Then pilot-scale system for microalgae photoinduction was designed and optimized based on key parameters. The pilot research of heterotrophy-dilution-photoinduction sequential cultivation was carried out.
(1) Optimization and establishment of light attenuation model of Chlorella pyrenoidosa and investigation of the effect of pigment content on light attenuation were implemented. Lambert-Beer light attenuation model is suitable only for low density of microalgae, and the Hyperbolic light attenuation model suit to high microalgae concentration. The Cornet light attenuation is applicable to wide range of microalgae concentration. M-Hyperbolic model established by us fits the light attenuation process better in the condition of low microalgae concentrations less than2.68g/L. Additionally, at the same cell concentration, the degree of light attenuation increased with the increase of pigment content of microalgal cell. However, the effect of pigment content on light attenuation is little when the cell concentration is high.
(2) The effects of incident light intensity, microalgal cell concentration and air flow rate on parameters of mixing and light regime characteristic in flat plate PBR was investigated, and theoretical analysis of different formulas for scaling up PBR was executed. With the increase of microalgae concentration, specific irradiation rate (q;) and volume averaged light intensity (Iavv) decrease exponentially; as the incident light intensity increase, q; and Iavv increase linearly; as the air flow rate increases, turbulence energy (TE) and turbulence energy dissipation (TED) of microalgae culture show linear increasing trend, the radial velocity of fluid (Ur) show increasing trend of power function, the mixing time (tm) decrease exponentially, the light dark cycle time of microalgal cell decreases quickly and the time averaged light intensity (Iav') of cell and the fraction of the light time in light dark cycle (LF) are almost not change. The microalgae concentration in15L PBR can only be respectively0.26times and0.36times of that in3L PBR with the same incident light intensity. When the PBR is scaling up based on formulas of equivalence of qi and Iav\incident light intensity for15L PBR is respectively3.7fold and2.81fold of that in3L PBR with the same cell concentration. The unit volume power of aeration in15L PBR approximately1times,2times and5.7times of that in3L PBR when formulas of equal radial mixing time (Tr), tm and LDCT are employed.
(3) The correlation analysis of photoinduction results of microalgae (the death rate of microalgal cell, the increasing rate of chlorophyll and protein content) with parameters of mixing and light regime characteristic in indoor3L PBR, outdoor70L open tank culture system and50L bubble basin was executed. The results show that Iavv, lav'as well as q; are the key parameters to photoinduction of microalgae. The irradiance level of microalgal culture is the main factor influencing the performance of system at the conditions of the same incident light intensity but different light path of PBRs. The mixing is the key factor for performance of PBR when PBRs have the same incident light intensities and same light paths. The good results are obtained when formulas of equal Iavv and q; are used, and the bad results appeared when formulas of equal Trand tm are employed to scale up the flat plate PBRs.
(4) The novel inner structures and relevant15L PBRs are deigned based on enhancing the microalgal culture along the light irradiance direction. The correlation analysis of photoautotrophic results of Chlorella pyrenoidosa with mixing and light regime characteristic parameters indicated that the velocity of fluid along the light attenuation direction (Ul) and LDCT of microalgal cell are the main sensitive parameters for affecting the performance of PBR. The validation of sensitive parameters was executed in three type of15L flat plate PBRs, i.e. Bubble, Split and Airlift. It is the first time to determine the performance of these three types of flat plate PBR:the Split PBR is better than Airlift PBR; and the Bubble PBR is the worst. The maximum cell concentration and specific growth rate of microalgae in type-a PBR are increased respectively92.5%and28.0%than that in Bubble PBR (the control), the maximum cell concentration and specific growth rate of microalgae in type-b PBR are increased respectively33.8%and25.6%than the control, and the maximum cell concentration and specific growth rate of microalgae in type-c PBR are increased respectively45.2%and29.8%than those in the control.
(5) Optimization of the large scale PBR based on sensitive parameters and the results validated by microalgae culture experiment were carried out. The results of microalgae photoinduction and photoautotrophic culture in1000L flat plate PBRs and300L column PBRs with different inner structures, middle raceway ponds with different operation conditions and large circular ponds with different type of impellers all show that UL and LDCT of microalgae significantly affect the performance of PBR. Not only the reliability of sensitive parameters is validated, but also it is demonstrated that UL and LDCT have the universality for different types of PBRs.
(6) A Serie of large scale systems for microalgae cultivation were designed based on sensitive parameters. And the pilot study of heterotrophy-dilution-photoinduction sequential cultivation was carried out. The growth rate of Chlorella pyrenoidosa in6000L fermenter can still achieved about3.0g/L/h at late stage of the heterotrophic culture. This growth rate of microalgal cell is comparative with that in50L fermenter. Temperature remarkably affects efficiency of Chlorella pyrenoidosa photoinduction, and the river water can replace tap water for Chlorella pyrenoidosa photoinduction. The chlorophyll and protein content of microalgal cell can reach over20mg/g and50%respectively after10～20h photoinduction in large scale microalgal culture system. The efficiency of photoinduction in these large scale systems is almost the same with that in small scale systems such as50L bubble basins.
In thispresent study, our results can not only provide basis for optimization and scale up of PBRs, but also load good foundation for industrialization of heterotrophy-dilution-photoinduction sequential cultivation of Chlorella pyrenoidosa.
引文
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