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题名	微藻培养多参数在线监控系统的构建与应用
作者	张东梅
学位类别	博士
答辩日期	2014-06
授予单位	中国科学院研究生院
导师	丛威
关键词	微藻培养   多参数监控   在线   生理参数   代谢流分析
其他题名	Development and application of microalgae culture on-line multi-parameter monitoring and control sys
学位专业	生物化工
中文摘要	微藻因具有光合效率高、生长速度快、细胞产品丰富等特点，被认为是未来生产能源和化学品的重要原料之一，已成为全球关注的研发热点。然而目前为止，微藻培养过程的优化还停留在简单的环境因子上，而细胞本身的耗碳速率、放氧速率、胞内组份变化等参数是真正与细胞生长、产物积累直接相关联的，通过在线计算并调控这些参数，将可能大幅度提高光合效率和目标物质产量。但目前微藻培养过程仍缺少必要的基础数据和相应的研究装备及方法。为此，本文首先研究了微藻培养过程重要参数的监测与控制方法，并基于理论分析提出了多个生理相关参数的计算方法。在此基础上，构建了一套微藻培养多参数在线监控系统，并将其应用于螺旋藻和拟微球藻培养体系，实现了培养过程多参数的在线监测控制和实时计算。 本文首先研究了培养基中无机碳源和无机氮源的原位在线监测方法。利用pH电极和DCO2电极联用的双电极法在线测量螺旋藻培养过程中培养液中HCO3-浓度，培养过程中利用自动反馈控制补加CO2将培养液pH控制在9.5 ± 0.1，双电极法可以准确测定培养液中HCO3-的浓度，误差基本小于10%。研究了利用硝酸根离子选择性电极在线测量和控制微藻培养基质中NO3-浓度的方法。通过测量不同浓度Cl-，HCO3-，NO3-混合溶液的硝酸根离子选择性电极电位值，获得了消除Cl-，HCO3-干扰的校正方程；校准时使标准曲线的溶液温度与工作溶液温度一致以消除温度的影响；设定修正系数消除离子强度的影响。其测量结果误差基本在10%以内。通过硝酸根离子选择性电极反馈控制补加NaNO3，实现恒氮源浓度培养。 在培养液中碳源和氮源监测方法的基础上，本文构建了一套微藻培养多参数在线监控系统，能够较全面地监测控制微藻培养过程环境参数，主要包括pH、DO、DCO2、温度、罐压、进气流量(空气、CO2、N2、O2)、尾气中CO2和O2含量等，并基于理论分析提出了多个生理相关参数的计算方法，主要包括：OER、SOER、CUR、SCUR、生物量、培养液中氮源含量、PQ (Photosynthesis quotient)，以及根据PQ实时计算藻细胞内主要组份。将此系统用于不同碳源和氮源浓度螺旋藻的培养，首次实现了原核蓝藻细胞培养过程中胞内蛋白、糖类、总脂含量的在线计算，利用在线计算方法能够很好地预测螺旋藻在缺氮条件下培养过程中胞内组分含量的变化。 进一步将微藻培养多参数在线监控系统用于最有潜力的生物柴油藻种之一真核类的拟微球藻。利用微藻培养多参数在线监控系统对拟微球藻进行全氮和限氮培养。在线获得培养过程参数，并将生理参数的计算方法应用到本实验中，实时获得藻生物量、CUR、SCUR、OER、SOER、培养液中氮源浓度、细胞内主要组分含量等重要生理参数。进一步研究光照条件下细胞线粒体呼吸速率，首次实现了在线计算微藻胞内碳流和能量流分配。发现拟微球藻在光合自养过程中，细胞吸收的碳中30%-60%用于合成细胞生物质，其余部分在细胞代谢反应中形成CO2排出体外。单位摩尔碳细胞生长需约5 mol ATP/mol C用于细胞物质合成和约0.4 mol ATP/mol C的生长相关维持能量(GAM)，且全氮和限氮培养结果相近，限氮培养时缺氮前后变不大。全氮培养的非生长相关维持能量(NGAM)约为2.2 mmol ATP/gh，而限氮培养中NGAM随细胞缺氮逐渐减少，基本在2.1-0.98 mmol ATP/gh范围内，结果与文献报道结果基本一致。针对拟微球藻限氮培养初步研究了其缺氮前后初级碳代谢流变化，获得重要节点流量，可为调控油脂合成提供参考。
英文摘要	Microalgae, well-known for its high photosynthetic efficiency, fast growth rate, various products, etc., have been considered as an important raw material for the production of energy and chemicals. Recently, microalgae culture has become a world-wide research hotspot. However, so far, microalgae culture process control still only focused on simple environmental factors, which is much simpler than the optimal control of fermentation process. If some physiological parameters could be calculated or regulated online, which could reflect cell growth and product accumulation directly such as carbon uptake rate, oxygen evolution rate, and metabolic changes of cells, the efficiency of the production of target products may be greatly improved. However, there is no appropriate equipment or methods for microalgae culture process control at present. Therefore, this study investigated the monitoring and control of some important process parameters and the computing methods of some physiological parameters in microalgae culture and developed a set of microalgae culture system, which was applied in cultures of Spirulina platensis and Nannochloropsis sp.. This work achieved the on-line monitoring and calculation of some physiological parameters such as biomass concentrations, intracellular components contents, and intracellular carbon and energy partition. Firstly, the monitoring of inorganic carbon source and nitrogen source in medium on-line was studied. A two-electrode method, which coupled pH electrode and DCO2 electrode, was developed to determine the HCO3- content in medium. In the culture of Spirulina platensis, the errors were lower than 10%, when the pH of medium was controlled at 9.5 ± 0.1 by automatic injection of CO2. The nitrate ion selective electrode (NISE) was utilized for monitoring NO3- concentration in medium and control of feeding NaNO3. The mixed solution with different concentrations of Cl-, HCO3-, NO3- was measured with NISE to develop a calibration equation for the interference of Cl-, HCO3-. Keeping temperature of calibration solution the same as temperature of culture process could eliminate the effect of temperature. The effect of ionic strength could be eliminated by the correction factor. The errors of NISE were controlled within 10%, generally. Based on the monitoring of carbon source and nitrogen source in medium, a more comprehensive microalgae culture multi-parameter on-line monitoring and control system was developed. The related process parameters included: pH, DO, DCO2, temperature, pressure, the flow rate of inlet (air, CO2, N2, O2), contents of CO2 and O2 in off-gas. The physiological parameters included: the OER, SOER, CUR, SCUR, biomass concentration, PQ (photosynthesis quotient), and cell composition. The system was applied on cultivation of Spirulina platensis. For the first time, the contents of protein, carbohydrate and lipid of Spirulina platensis, one of the representative prokaryotic cyanobacteria, were calculated on-line. This method could predict the dynamic changes of cell composition in the process of nitrogen depletion. Eukaryotic microalga was also been studied to extend the application of the system. Nannochloropsis sp., one oleaginous microalga, as the potential strain for biodiesel production, was chosen. The system was applied in nitrogen-replete culture and nitrogen-limited culture of Nannochlorpsis sp.. The process and physiological parameters was obtained and the trends of these parameters were similar with those of Spriulina platensis. Furthermore, the light respiration of Nannochlorpsis sp. was investigated and the intracellular carbon and energy partition were calculated on line for the first time. In the photoautotrophic culture of Nannochlorpsis sp., 30%-60% of carbon fixed in Calvin cycle was used for biomass compounds, and the rest was expelled out of cells in metabolic reactions. For energy flux, cell growth acquired around 5 mol ATP/mol C for biopolymers synthesis, and 0.4
语种	中文
公开日期	2015-07-08
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/15541]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	张东梅. 微藻培养多参数在线监控系统的构建与应用[D]. 中国科学院研究生院. 2014.

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