小球藻CHLORELLA VULGARIS油脂积累特性及以污水为底物的油脂制备研究
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摘要
由于能源危机和温室效应的挑战，可再生且不增加温室气体排放的微藻能源近年来得到了广泛关注。微藻作为生物能源的原料，具有生长速度快，能量转换效率高，潜在生产规模大的优势。但是微藻培养和收集过程的营养物和能量消耗，提高了生产成本，限制了微藻能源的工业化生产，利用污水作为微藻培养的底物可以有效的降低微藻培养成本，并且同时对污水进行处理。
本论文以污水中常见的Chlorella vulgaris为研究对象，考察了C. vulgaris不同代谢类型时的生长特性，然后测定了藻细胞在不同培养条件下的脂含量变化，对C. vulgaris的混养代谢和油脂积累过程进行了分析。通过对化学混凝和电混凝的优化比较，确定了C. vulgaris的收集方法，并对C. vulgaris生物质在不同温度下的干燥结果进行了比较。在对以污水为底物的C. vulgaris油脂积累过程进行研究后，构建了C. vulgaris油脂制备系统，对系统运行的结果进行了分析和评价。
以BG11为基础培养基，通过有机碳源和光照的调节使C. vulgaris分别进行自养、异养和混养代谢。未曝气时，自养和异养代谢的比生长速率分别仅为0.0018h~(-1)和0.0083h~(-1)，而混养代谢则达到0.0204h~(-1)。与乙醇、乙酸钠相比，葡萄糖更适于作为C. vulgaris的有机碳源。曝气能够显著提高不同代谢类型的比生长速率，空气曝气时的混养代谢比生长速率提高到0.1008h~(-1)，而以20%CO_2为气源时，比生长速率则下降到0.0930h~(-1)。对C. vulgaris不同代谢类型的生长特性分析表明，在C. vulgaris的混养代谢过程中，光合自养和有机异养同时发生，并且两种代谢途径互相影响，共同决定藻细胞的组成和比生长速率。
调节培养液中的初始营养物浓度，考察了C. vulgaris的油脂积累特性。氮缺乏能够显著提高藻细胞的脂含量，3.75g/LNaNO_3时脂含量仅为7.59%，而0.1g/L NaNO_3时则达到22.58%。在氮缺乏基础上，磷含量对脂含量的影响不显著。葡萄糖对油脂的积累十分重要，5g/L葡萄糖的培养液，96h脂含量达到17.40%，此后由于葡萄糖不足，240h时脂含量下降到10.07%。对藻细胞在不同营养条件下脂含量变化的分析表明，当细胞由于营养限制而停止生长后，需要继续获取充足的光照或有机底物，才能够促进细胞油脂的积累。
通过对搅拌速度、曝气量、电流密度、初始细胞密度和pH等因素的研究，建立了C. vulgaris的电混凝收集方法，当初始细胞密度为0.241g/L， pH5.0，电流密度0.42mA/cm~2，50rpm持续时，电混凝15min时收集率达到97%，能耗仅为0.610kWh/kg。对不同培养条件的C. vulgaris培养液收集结果显示，与硫酸铝混凝相比，电混凝在收集率和油脂回收率方面均较高，20min时的收集率达到99%~100%，油脂回收率为99%~100%，但是出水中Al3+含量是硫酸铝混凝的1.97~2.33倍。较高的干燥温度会改变C. vulgaris生物质的组成，与-70°C冻干相比，105°C烘干时C. vulgaris的干重、脂含量和油脂中的酯含量分别降低了3.30%、7.73%和26.30%，而40°C烘干所得到的C. vulgaris生物质各项性质与冻干相近。
分别以人工配水和生活污水培养C. vulgaris，由于生活污水中污染物较为复杂，因此细胞密度由人工配水时的1.496g/L降低到生活污水的0.575g/L；灭菌后的生活污水中C. vulgaris生长较快，但是未灭菌组的污染物去除效率较高。曝气、光照和底物浓度均会对污水中C. vulgaris的油脂积累产生影响，适当提高CO_2浓度和曝气量能够提高C. vulgaris细胞密度和脂含量，光照强度和光照时间的增加也可以改善C. vulgaris的培养效果，通过向污水中投加葡萄糖提高培养液的COD:TN，能够显著的提高细胞密度和脂含量，当COD:TN为50:1时，细胞密度达到1.181g/L，脂含量为14.38%。
根据以上研究的结果，构建了C. vulgaris油脂制备系统，对生活污水进行抽滤、稀释预处理后，投加适量KH_2PO_4和葡萄糖使其COD:TN:TP达到300:6:1，灭菌后用于C. vulgaris的培养，5%CO_2曝气。在2000lx和7000lx的光照强度下，藻细胞密度分别达到0.954g/L和1.203g/L，脂含量为14.49%和15.75%，污水中的COD、TN和TP的去除率分别达到70%，90%和96%以上。成本分析结果表明，以生活污水为底物时，C. vulgaris油脂制备成本明显低于缺氮培养基，2000lx光照时的成本为71.57元/kg，仅是缺氮培养基的58.29%。如果以高浓度有机废水代谢葡萄糖、利用自然光提供光照并考虑污水处理收益后，则C.vulgaris油脂成本还可以进一步降低到22.86元/kg。
Due to the challenge of energy crisis and greenhouse effect, microalgae energywhich renewable and do not increase the greenhouse gas emissions received lots ofattentions recently years. Microalgae has some advantages to be used as bioenergymaterial, such as higher growth rate and energy conversion ratio, larger potentialproduction scale. However, the expensive cost of nutrient in medium and energyconsumption in collection process restrict the commercial production of microalgaeenergy. The production cost of microalgae would be decreased significantly ifwastewater was used as medium, and wastewater was treated simultaneously.
Chlorella vulgaris was used as model strain in this paper. The growthcharacteristics of C. vulgaris under various trophic mode were investigated.Thereafter, lipid content of algal cell with various nutrient concentration wasdetermined. Both mixotrophic mode and lipid accumulation of C. vulgaris wereanalyzed. Through the optimization and comparison of chemical coagulation andelectronical coagulation, the collection method for C. vulgaris was confirmed. Theeffect of drying temperature on biomass constitute was also investigated. Based onthe research of lipid accumulation in C. vulgaris with wastewater used as medium,C. vulgaris lipid production system was constituted. The results of system operationwas analyzed.
BG11was used as basal medium, C. vulgaris was cultivated underphototrophic, heterotrophic and mixotrophic mode with various organic carbon andlight condition. The specific growth rates of autotrophic and heterotrophic with noaeration were only0.0018h~(-1)and0.0083h~(-1), respectively. As for mixotrophic, itincreased to0.0204h~(-1). Compare to ethanol and sodium acetate, glucose was moresuitable to be used as organic carbon source for C. vulgaris cultivation. The specificgrowth rates could promote significantly by aeration. It increased to0.1008h~(-1)undermixotrophic with air aeration, while slightly decreased to0.0930h~(-1)when20%CO_2used as air source. Analysis of various trophic mode of C. vulgaris indicated thatphotosynthetic metabolism and heterotrophic metabolism were occurredsimultaneously in algal cell under mixotrophic mode. The pathways were interacteach other, and codetermine the constitute and specific growth rate of algal cell.
The characteristic of C. vulgaris lipid accumulation was investigated withvarious nutrient concentration in medium. Nitrogen deficiency would enhance lipidcontent significantly. The lipid content of algal cell increased from7.59%in3.75g/LNaNO_3to22.58%in0.1g/L NaNO_3. Phosphate limitation play a lessimportant role in lipid accumulation than nitrogen. Glucose play a very important role in lipid accumulation. When C. vulgaris was cultivated with5g/L glucose, thelipid content achieved17.40%at96h. However, only10.58%of lipid content wasobtained at240h due to glucose had been exhausted. The analysis of lipidaccumulate process under various nutrient condition indicated that nutrientdeficiency would restrict the growth rate of microalgae. If organic carbon and lightenergy were abundant in nutrient deficiency culture, then the major part ofmetabolic flux was converted into lipid accumulation, therefore lipid contentincreased.
Based on the optimization of string rate, aeration rate, current density, initialcell density and pH, electronical coagulation (EC) for C. vulgaris collection wasestablished. The97%of collection efficiency was achieved at15min by EC, withinitial cell density0.241g/L, pH5.0, current density0.42mA/cm~2and string rate50rpm. The energy consumption was only0.610kWh/kg. The C. vulgaris cultureunder different nutrient condition were collected by optimized aluminum sulfatecoagulation and EC, respectively. EC achieved higher collection efficiency and lipidrecovery efficiency than aluminum sulfate coagulation.99%~100%of collectionefficiency and99%~100%of lipid recovery efficiency were obtained by EC in20min. However, the Al3+concentration in effluent of EC was1.97~2.33times higherthan chemical coagulation. Constitute of algal cell would be changed under variousdrying temperature. Dry weight, lipid content and ester content of algal cell dryingin105°C decreased by3.30%,7.73%and26.30%compare to-70°C freeze drying.As for40°C drying, the constitute of algal cell was similar with freeze drying.
C. vulgaris was cultivated in artificial wastewater and domestic wastewater,respectively. Due to the pollutants in domestic wastewater were more complex, thecell density decreased from1.496g/L in artificial wastewater to0.575g/L indomestic wastewater. The specific growth rates of C. vulgaris in sterilized domesticwastewater was higher than in non-sterilized wastewater, while pollutant removalefficiency was contrary. The lipid accumulation of C. vulgaris would be effect byaeration, light and nutrient concentration in wastewater. Cell density and lipidcontent of C. vulgaris would enhance with suitable CO_2concentration and aerationrate. Moreover, the increase of light intensity and time were also improved C.vulgaris cultivation. Cell density and lipid content of C. vulgaris also increasedalong with the increase of COD:TN. When COD:TN was50:1,1.181g/L of celldensity and14.38%of lipid content was obtained.
Based on the results of previous research, the C. vulgaris lipid productionsystem was consisted. The domestic wastewater was pretreatment by filtration anddiluted, and then COD:TN:TP was adjusted to300:6:1by KH_2PO_4and glucoseaddition. After sterilization, the culture was used for C. vulgaris cultivation with5%CO_2aeration. When2000lx and7000lx light intensity was supplied, cell density reached0.954g/L and1.203g/L respectively, lipid content achieved14.49%and15.75%. As for COD, TN and TP, the removal efficiency were over70%,90%and96%, respectively. Cost analysis of the system indicated that the production cost ofC. vulgaris lipid with domestic wastewater used as medium was significantly lowerthan N-deficient medium. It was71.57yuan/kg with2000lx light intensity, only58.29%of N-deficient medium. If glucose was replaced by high organicconcentration wastewater, and sunlight used as light source, the profit of wastewatertreatment also calculated, the cost of C. vulgaris lipid could reduce to22.86yuan/kg.
本论文以污水中常见的Chlorella vulgaris为研究对象，考察了C. vulgaris不同代谢类型时的生长特性，然后测定了藻细胞在不同培养条件下的脂含量变化，对C. vulgaris的混养代谢和油脂积累过程进行了分析。通过对化学混凝和电混凝的优化比较，确定了C. vulgaris的收集方法，并对C. vulgaris生物质在不同温度下的干燥结果进行了比较。在对以污水为底物的C. vulgaris油脂积累过程进行研究后，构建了C. vulgaris油脂制备系统，对系统运行的结果进行了分析和评价。
以BG11为基础培养基，通过有机碳源和光照的调节使C. vulgaris分别进行自养、异养和混养代谢。未曝气时，自养和异养代谢的比生长速率分别仅为0.0018h~(-1)和0.0083h~(-1)，而混养代谢则达到0.0204h~(-1)。与乙醇、乙酸钠相比，葡萄糖更适于作为C. vulgaris的有机碳源。曝气能够显著提高不同代谢类型的比生长速率，空气曝气时的混养代谢比生长速率提高到0.1008h~(-1)，而以20%CO_2为气源时，比生长速率则下降到0.0930h~(-1)。对C. vulgaris不同代谢类型的生长特性分析表明，在C. vulgaris的混养代谢过程中，光合自养和有机异养同时发生，并且两种代谢途径互相影响，共同决定藻细胞的组成和比生长速率。
调节培养液中的初始营养物浓度，考察了C. vulgaris的油脂积累特性。氮缺乏能够显著提高藻细胞的脂含量，3.75g/LNaNO_3时脂含量仅为7.59%，而0.1g/L NaNO_3时则达到22.58%。在氮缺乏基础上，磷含量对脂含量的影响不显著。葡萄糖对油脂的积累十分重要，5g/L葡萄糖的培养液，96h脂含量达到17.40%，此后由于葡萄糖不足，240h时脂含量下降到10.07%。对藻细胞在不同营养条件下脂含量变化的分析表明，当细胞由于营养限制而停止生长后，需要继续获取充足的光照或有机底物，才能够促进细胞油脂的积累。
通过对搅拌速度、曝气量、电流密度、初始细胞密度和pH等因素的研究，建立了C. vulgaris的电混凝收集方法，当初始细胞密度为0.241g/L， pH5.0，电流密度0.42mA/cm~2，50rpm持续时，电混凝15min时收集率达到97%，能耗仅为0.610kWh/kg。对不同培养条件的C. vulgaris培养液收集结果显示，与硫酸铝混凝相比，电混凝在收集率和油脂回收率方面均较高，20min时的收集率达到99%~100%，油脂回收率为99%~100%，但是出水中Al3+含量是硫酸铝混凝的1.97~2.33倍。较高的干燥温度会改变C. vulgaris生物质的组成，与-70°C冻干相比，105°C烘干时C. vulgaris的干重、脂含量和油脂中的酯含量分别降低了3.30%、7.73%和26.30%，而40°C烘干所得到的C. vulgaris生物质各项性质与冻干相近。
分别以人工配水和生活污水培养C. vulgaris，由于生活污水中污染物较为复杂，因此细胞密度由人工配水时的1.496g/L降低到生活污水的0.575g/L；灭菌后的生活污水中C. vulgaris生长较快，但是未灭菌组的污染物去除效率较高。曝气、光照和底物浓度均会对污水中C. vulgaris的油脂积累产生影响，适当提高CO_2浓度和曝气量能够提高C. vulgaris细胞密度和脂含量，光照强度和光照时间的增加也可以改善C. vulgaris的培养效果，通过向污水中投加葡萄糖提高培养液的COD:TN，能够显著的提高细胞密度和脂含量，当COD:TN为50:1时，细胞密度达到1.181g/L，脂含量为14.38%。
根据以上研究的结果，构建了C. vulgaris油脂制备系统，对生活污水进行抽滤、稀释预处理后，投加适量KH_2PO_4和葡萄糖使其COD:TN:TP达到300:6:1，灭菌后用于C. vulgaris的培养，5%CO_2曝气。在2000lx和7000lx的光照强度下，藻细胞密度分别达到0.954g/L和1.203g/L，脂含量为14.49%和15.75%，污水中的COD、TN和TP的去除率分别达到70%，90%和96%以上。成本分析结果表明，以生活污水为底物时，C. vulgaris油脂制备成本明显低于缺氮培养基，2000lx光照时的成本为71.57元/kg，仅是缺氮培养基的58.29%。如果以高浓度有机废水代谢葡萄糖、利用自然光提供光照并考虑污水处理收益后，则C.vulgaris油脂成本还可以进一步降低到22.86元/kg。
Due to the challenge of energy crisis and greenhouse effect, microalgae energywhich renewable and do not increase the greenhouse gas emissions received lots ofattentions recently years. Microalgae has some advantages to be used as bioenergymaterial, such as higher growth rate and energy conversion ratio, larger potentialproduction scale. However, the expensive cost of nutrient in medium and energyconsumption in collection process restrict the commercial production of microalgaeenergy. The production cost of microalgae would be decreased significantly ifwastewater was used as medium, and wastewater was treated simultaneously.
Chlorella vulgaris was used as model strain in this paper. The growthcharacteristics of C. vulgaris under various trophic mode were investigated.Thereafter, lipid content of algal cell with various nutrient concentration wasdetermined. Both mixotrophic mode and lipid accumulation of C. vulgaris wereanalyzed. Through the optimization and comparison of chemical coagulation andelectronical coagulation, the collection method for C. vulgaris was confirmed. Theeffect of drying temperature on biomass constitute was also investigated. Based onthe research of lipid accumulation in C. vulgaris with wastewater used as medium,C. vulgaris lipid production system was constituted. The results of system operationwas analyzed.
BG11was used as basal medium, C. vulgaris was cultivated underphototrophic, heterotrophic and mixotrophic mode with various organic carbon andlight condition. The specific growth rates of autotrophic and heterotrophic with noaeration were only0.0018h~(-1)and0.0083h~(-1), respectively. As for mixotrophic, itincreased to0.0204h~(-1). Compare to ethanol and sodium acetate, glucose was moresuitable to be used as organic carbon source for C. vulgaris cultivation. The specificgrowth rates could promote significantly by aeration. It increased to0.1008h~(-1)undermixotrophic with air aeration, while slightly decreased to0.0930h~(-1)when20%CO_2used as air source. Analysis of various trophic mode of C. vulgaris indicated thatphotosynthetic metabolism and heterotrophic metabolism were occurredsimultaneously in algal cell under mixotrophic mode. The pathways were interacteach other, and codetermine the constitute and specific growth rate of algal cell.
The characteristic of C. vulgaris lipid accumulation was investigated withvarious nutrient concentration in medium. Nitrogen deficiency would enhance lipidcontent significantly. The lipid content of algal cell increased from7.59%in3.75g/LNaNO_3to22.58%in0.1g/L NaNO_3. Phosphate limitation play a lessimportant role in lipid accumulation than nitrogen. Glucose play a very important role in lipid accumulation. When C. vulgaris was cultivated with5g/L glucose, thelipid content achieved17.40%at96h. However, only10.58%of lipid content wasobtained at240h due to glucose had been exhausted. The analysis of lipidaccumulate process under various nutrient condition indicated that nutrientdeficiency would restrict the growth rate of microalgae. If organic carbon and lightenergy were abundant in nutrient deficiency culture, then the major part ofmetabolic flux was converted into lipid accumulation, therefore lipid contentincreased.
Based on the optimization of string rate, aeration rate, current density, initialcell density and pH, electronical coagulation (EC) for C. vulgaris collection wasestablished. The97%of collection efficiency was achieved at15min by EC, withinitial cell density0.241g/L, pH5.0, current density0.42mA/cm~2and string rate50rpm. The energy consumption was only0.610kWh/kg. The C. vulgaris cultureunder different nutrient condition were collected by optimized aluminum sulfatecoagulation and EC, respectively. EC achieved higher collection efficiency and lipidrecovery efficiency than aluminum sulfate coagulation.99%~100%of collectionefficiency and99%~100%of lipid recovery efficiency were obtained by EC in20min. However, the Al3+concentration in effluent of EC was1.97~2.33times higherthan chemical coagulation. Constitute of algal cell would be changed under variousdrying temperature. Dry weight, lipid content and ester content of algal cell dryingin105°C decreased by3.30%,7.73%and26.30%compare to-70°C freeze drying.As for40°C drying, the constitute of algal cell was similar with freeze drying.
C. vulgaris was cultivated in artificial wastewater and domestic wastewater,respectively. Due to the pollutants in domestic wastewater were more complex, thecell density decreased from1.496g/L in artificial wastewater to0.575g/L indomestic wastewater. The specific growth rates of C. vulgaris in sterilized domesticwastewater was higher than in non-sterilized wastewater, while pollutant removalefficiency was contrary. The lipid accumulation of C. vulgaris would be effect byaeration, light and nutrient concentration in wastewater. Cell density and lipidcontent of C. vulgaris would enhance with suitable CO_2concentration and aerationrate. Moreover, the increase of light intensity and time were also improved C.vulgaris cultivation. Cell density and lipid content of C. vulgaris also increasedalong with the increase of COD:TN. When COD:TN was50:1,1.181g/L of celldensity and14.38%of lipid content was obtained.
Based on the results of previous research, the C. vulgaris lipid productionsystem was consisted. The domestic wastewater was pretreatment by filtration anddiluted, and then COD:TN:TP was adjusted to300:6:1by KH_2PO_4and glucoseaddition. After sterilization, the culture was used for C. vulgaris cultivation with5%CO_2aeration. When2000lx and7000lx light intensity was supplied, cell density reached0.954g/L and1.203g/L respectively, lipid content achieved14.49%and15.75%. As for COD, TN and TP, the removal efficiency were over70%,90%and96%, respectively. Cost analysis of the system indicated that the production cost ofC. vulgaris lipid with domestic wastewater used as medium was significantly lowerthan N-deficient medium. It was71.57yuan/kg with2000lx light intensity, only58.29%of N-deficient medium. If glucose was replaced by high organicconcentration wastewater, and sunlight used as light source, the profit of wastewatertreatment also calculated, the cost of C. vulgaris lipid could reduce to22.86yuan/kg.
引文
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