【作者】 鲍亦璐；

【导师】 宁正祥；

【作者基本信息】 华南理工大学 ， 食品科学， 2012， 博士

【摘要】 微藻利用光和CO₂合成蛋白质、糖类、脂类以及色素等大分子物质并放出O2，在人类食品、保健、医药、环保和生物炼制领域具有广阔的应用前景。本文针对目前微藻培养中存在的生产成本高、产率低的问题，主要从营养盐方面入手，通过分批培养研究了主要营养盐对螺旋藻、二形栅藻生长和产物积累的影响，以及培养中pH和无机碳源变化、氮源和磷源消耗、微藻生长三者之间的内在联系。在建立微藻生长与营养盐消耗相关模型的基础上，构建了三种微藻培养营养盐流加策略并进行比较评价，最终用于螺旋藻培养的营养盐优化和二形栅藻培养的油脂积累调控，就微藻培养中与营养盐有关的操作及成本进行了优化与控制。本论文的主要结果如下：（1）采用NaHCO₃作为碳源，后期培养液pH值过高（>11.0）会抑制螺旋藻生长，而流加CO₂作为碳源能够控制培养液pH值在适合螺旋藻生长的范围（8.0¹0.0）；充足的碳源、氮源、磷源有利于螺旋藻生长，促进螺旋藻蛋白质及叶绿素的积累，而碳源过量或氮源、磷源限制会影响螺旋藻生长，但有利于螺旋藻总糖的积累；尿素、NH₄NO₃、NH₄Cl和NH₄HCO₃能够替代NaNO₃作为螺旋藻培养的氮源，但会出现高浓度抑制螺旋藻生长的现象，其抑制作用由大到小的顺序依次为：NH₄HCO₃> NH₄Cl>NH₄NO₃>尿素。（2）在微藻培养中，当培养液营养盐浓度较低时，营养盐的比消耗速率由微藻比生长速率和细胞内相对应的营养元素份额共同决定；当培养液营养盐充足时，藻细胞内碳、氮、磷元素份额较为稳定，营养盐的比消耗速率主要由微藻比生长速率决定。此时维持微藻培养液pH值稳定的CO₂补加量、氮源消耗量和磷源消耗量都与微藻生物量增量存在较好的线性相关性，可以采用以下三种营养盐反馈流加策略来维持微藻培养液营养盐浓度稳定，分别为pH和硝酸根电极联用的反馈流加策略、基于pH的反馈流加策略和基于吸光值的反馈流加策略。（3）理论探讨了基于吸光值和基于pH值反馈流加营养盐，影响流加过程中营养盐浓度的一些可能因素，结果表明：采用基于吸光值的反馈流加策略，培养液中营养盐浓度的波动与吸光值步长、营养盐利用率和藻细胞内营养元素份额有关；采用基于pH的反馈流加策略，培养液中营养盐浓度的波动与培养液pH值、pH步长及培养液中游离CO₂浓度有关。通过螺旋藻分批补料培养对以上三种营养盐流加策略进行实验验证发现： pH和硝酸根电极联用的反馈流加策略，由于硝酸根电极会发生电位漂移，培养液中氮源、磷源浓度波动较大，不适合营养盐的长期在线监控；基于吸光值的反馈流加策略有利于培养液中氮源、磷源浓度控制；而基于pH的反馈流加策略有利于培养液中碳源浓度和pH值的控制。将基于pH和基于吸光值的反馈流加策略联用，能够更加准确地控制微藻培液中碳源、氮源、磷源的浓度，适用于营养盐的长期在线监控。（4）采用上述流加策略对螺旋藻培养的营养盐进行优化，基于pH反馈流加CO₂作为碳源，螺旋藻的最大生物量浓度可达3.30g/L，比NaHCO₃作为碳源的螺旋藻分批培养提高了92.30%；基于吸光值反馈流加NH₄HCO₃作为氮源，将培养液中NH3-N浓度控制在1mmol/L时，螺旋藻生长不受到氮源限制或抑制，与同样培养条件下恒速、变速及基于pH反馈流加NH₄HCO₃的培养相比，能够得到最大的生物量（2.98g/L）、氮源得率系数（7.32g/g）、蛋白质含量（64.11%）和叶绿素含量（13.40mg/g）。（5）将营养盐流加策略应用于螺旋藻大规模生产，与传统以NaHCO₃作为碳源、NaNO₃作为氮源、K2HPO4作为磷源、采收后根据经验补料的培养方式相比，采用CO₂作为碳源、NaNO₃和NH₄HCO₃作为混合氮源、H3PO4作为磷源、培养中采用基于吸光值或基于pH反馈流加营养盐，能够长期将培养液中碳源、氮源、磷源浓度控制在螺旋藻生长适合的范围，降低螺旋藻培养的营养盐成本约70%，同时单位面积产量提高20%以上。（6）二形栅藻分批培养结果显示：二形栅藻最适的培养pH值为7.5；与采用NaNO₃和尿素作为氮源相比，采用NH₄HCO₃作为氮源时，二形栅藻生长较快，油脂产率较高；高碳、低氮和低磷条件能够促进二形栅藻油脂积累，但氮源限制对二形栅藻油脂积累的促进作用大于磷源限制。基于以上结果，在二形栅藻分批补料培养中，通过pH反馈控制CO₂的流加将培养液pH值控制在7.5±0.3，基于吸光值反馈控制NH₄HCO₃和K2HPO4的流加，在氮磷源流加阶段，二形栅藻快速生长，氮磷源反馈流加停止后，培养液中的氮源和磷源能够迅速耗尽，促进二形栅藻油脂快速积累，其油脂产率与分批培养、氮源和磷源流加速率分别为0.8mmol/（L·d）和9.6μmol/（L·d）的恒速补料培养以及氮源和磷源流加速率分别为0.4mmol/（L·d）和4.8μmol/（L·d）的恒速补料培养相比，分别提高58.2%、53.3%和37.1%。更多还原

【Abstract】 Microalgae are capable of utilizing sunlight and carbon dioxide to synthesize organicsubstrates such as protein, carbohydrate and lipid, and oxygen is produced at the same time.Microalgae are widely used in food, health care, medicine, environment and bio-energy fields.Considering the problem of high production cost and low productivity in microalgaecultivations, the nutrient salts were chosen to be the main investigating objects. The influenceof the main nutrient on Spirulina platensis and Scenedesmus dimorphus growth andproduction were studied. The relationships among the changes of pH and inorganic carbonsource, the consumption of nitrogen and phosphorus sources and the biomass accumulationwere learned in this study. Based on the relationship between the microalgae growth andnutrient consumption, three kinds of nutrient feeding strategies were proposed and compared.Furthermore, the nutrient feeding strategies were applied for nutrient optimization inSpirulina platensis cultivation and regulation of lipid accumulation in Scenedesmusdimorphus cultivation. As a result, the operating and production cost related to nutrient inmicroalgae cultivations were optimized and controlled by using these nutrient feedingstrategies. Main results were as follows:（1） In Spirulina platensis cultivation, the pH value of culture medium exceeded11.0atthe late growth phase using NaHCO₃as carbon source, which would lead to growth inhibition.Feeding CO₂as carbon source could maintain the pH value of the culture medium at thesuitable level for Spirulina platensis growth （8.0¹0.0）. It was found that sufficient carbon,nitrogen and phosphorus sources were benefit for Spirulina platensis growth and high proteinand chlorophyll content could be obtained. But with excess carbon source, nitrogen-limitationor phosphorus-limitation, Spirulina platensis growth could be affected and the accumulationof carbohydrate was promoted. Urea, NH₄NO₃, NH₄Cl and NH₄HCO₃could instead ofNaNO₃as nitrogen source for Spirulina platensis cultivation, but the growth inhibition wouldtake place when the concentration of ammonium was at a relatively high level. The order ofthe inhibitory effect of these nitrogen sources was NH₄HCO₃> NH₄Cl>NH₄NO₃>urea.（2） When the nutrient concentration was relatively low in the culture medium, thespecific consumption rate of nutrient was not only depended on the specific growth rate ofmicroalgae, but also on the nutrient cell quota of microalgae. When the nutrient was relativelysufficient in the culture medium, the cell quota of carbon, nitrogen and phosphorus in themicroalgae cells were relatively stable, and the specific consumption rate of nutrient wasmainly depended on the specific growth rate of microalgae. In the latter situation, the amount of CO₂addition to keep the pH value of culture medium stable, the consumption of nitrogensource and the consumption of phosphorus source were linearly correlated to theaccumulation of biomass. Thus three different nutrient feeding strategies were proposed fornutrient concentrations control in microalgae cultivations. They were the pH and nitrateelectrodes based feedback feeding method, the pH-based feedback feeding method and theabsorbance-based feedback feeding method.（3） Some probable factors affecting the change of nutrients concentration when using theabsorbance-based feedback feeding method and the pH-based feeding method wereinvestigated. The results implied that the fluctuation range of the nutrient concentrations inthe culture medium depended on the absorbance-step, utilization of nutrient and nutrient cellquota using the absorbance-based feedback feeding method. And the fluctuation range ofnutrient concentrations in the culture medium depended on the pH-step, pH value of culturemedium and free CO₂concentration in the culture medium using the pH-based feedingmethod. The proposed nutrient feeding strategies were applied in the Spirulina platensisfed-batch cultivations. It was found that when the pH and nitrate electrodes based feedbackfeeding method was used, the nitrogen and phosphorus concentrations were fluctuant in arelatively wide range due to the potential drift of the nitrate electrode. It implied this feedingmethod was not fit for long-time nutrient monitoring and controlling on-line. Theabsorbance-based feedback feeding method was benefit for the nitrogen and phosphorusconcentration control, while the pH-based feedback feeding method was benefit for the pHand carbon concentration control. Combining the pH-based feedback feeding method with theOD-based feedback feeding method, the carbon, nitrogen and phosphorus concentrationswould be accurately controlled during the microalgae cultivations，which implied they werefit for long-time nutrient control on-line.（4） The nutrient in the Spirulina platensis cultivation was optimized using the feedingstrategies above. Meanwhile, feeding CO₂as the carbon source using the pH-based feedbackfeeding method, the maximum biomass concentration of3.30g/L could be obtained, whichwas92.30%higher than that of using NaHCO₃as the carbon source. Feeding NH₄HCO₃asthe nitrogen source by the absorbance-based feedback feeding method to maintain theammonium concentration in the culture medium at1mmol/L, Spirulina paltensis growthwould not be affected by nitrogen source inhibition or limitation. As a result, the maximumbiomass concentration （2.98g/L）, nitrogen-to-cell conversion factor （7.32g/g）, and contentsof protein （64.11%） and chlorophyll （13.40mg/g） obtained by using absorbance-basedfeedback feeding method were higher than those of using the constant, variable and pH-based feedback feeding methods.（5） The nutrient optimization and feeding strategies proposed in the thesis were appliedin large-scale cultivations of Spirulina platensis in outdoor open raceway pond. In thetraditional semi-continuous cultivations, the carbon, nitrogen and phosphate sources weresodium bicarbonate, sodium nitrate and dipotassium hydrogen phosphate, respectively andnutrient feeding was based on experience after harvest. When the carbon, nitrogen andphosphate sources were substituted with the carbon dioxide, sodium nitrate and ammoniumcarbonate, and phosphate, respectively, and the feeding method was substituted withabsorbance-based or pH-based feedback feeding method in the outdoor large-scalecultivations, the carbon, nitrogen and phosphorus source concentrations in the culture mediumcould be maintained in the optimum range for Spirulina platensis growth. Meanwhile, thecost of culture medium decreased by about70%and the productivity per area of Spirulinaplatensis improved by more than20%.（6） The results of batch cultivations of Scenedesmus dimorphus showed that theoptimum pH for Scenedesmus dimorphus was7.5. Compared with the situation of usingNaNO₃and urea as the nitrogen source, a faster growth and higher lipid productivity could beobtained using NH₄HCO₃as the nitrogen source. In carbon-sufficient but nitrogen-orphosphorus-limited cultures, the lipid production of Scenedesmus dimorphus could beincreased. Furthermore, the promoting effect of nitrogen limitation on lipid accumulation wasmore than that of phosphorus limitation. Based on the batch cultivation results, CO₂wasadded using the pH-based feedback feeding method and NH₄HCO₃and K2HPO4were addedusing the absorbance-based feedback feeding method in the fed-batch cultivation ofScenedesmus dimorphus. The pH value of the culture medium could be maintained at7.5±0.3.And Scenedesmus dimorphus grew well in the nitrogen and phosphorus sources feeding stage.After the feeding end of nitrogen and phosphorus sources, nitrogen and phosphorus sourcesexhaustion quickly occurred and the accumulation of lipid was promoted at the same time.Compared with the batch cultivations, the constant feeding cultivations with nitrogen feedingrate of0.8mmol/（L·d） and phosphorus feeding rate of9.6μmol/（L·d）, and the constantfeeding cultivation with nitrogen feeding rate of0.4mmol/（L·d） and phosphorus feeding rateof4.8μmol/（L·d）, the lipid productivity in the fed-batch cultivations using the proposedfeeding method increased by58.2%,53.3%and37.1%, respectively.更多还原