微藻高油脂的生物合成与膜基萃取研究
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摘要
利用微藻生产生物柴油,应对化石燃料日益枯竭和能源供应日趋紧张的同时,与环境二氧化碳的捕捉进行偶联,缓解温室效应,具有广阔的开发利用前景。但要微藻生物柴油实现商业化利用,真正替代石化燃料,还存在许多挑战,还有很长的路要走。特别是微藻油脂产量的进一步提高,油脂提取成本的进一步降低,如果可以在这两方面得到改进,无疑能为微藻生物柴油真正实现商业化打下坚实基础。针对这一问题,本论文以微藻Chlorella sp.为出发藻种,从微藻生物学的基因工程改造和微藻高浓二氧化碳的驯化培养两个角度强化微藻油脂富集,并且采用新型膜基萃取技术用于微藻油脂提取,以期解决提高微藻油脂产量和降低油脂提取成本等问题,主要内容包括以下五个方面：
(1)构建小球藻自养条件下核心及油脂积累的代谢网络模型,并对其进行分析。通过部分解释环境压力对代谢流分布的影响,淀粉合成途径与TAG合成途径的竞争,说明代谢网络模型较为可靠。通过TAG合成途径与生物质合成途径的对比,结合CASOP算法分析,均发现脂肪酸合成及Kennedy途径,特别是DGAT晦,对于TAG的合成有很高的重要性,为产油微藻的基因工程改造提供指导。
(2)对出发藻种Chlorella sp进行分离、纯化与鉴定。采用抗生素组合处理法和平板划线分离法分别去除细菌和霉菌,获得无菌海水小球藻藻株,建立海水小球藻无菌培养体系。对比三种小球藻基因组DNA提取方法,植物基因组DNA快速提取试剂盒法,操作过程相对简单,所需时间相对较少,而该方法和改良SDS-CTAB法,均能够在保证DNA纯度的同时,获得较大的DNA总量和DNA产率。PCR扩增rbcL基因并进行序列测定,鉴定实验室海水小球藻属于海水普通小球藻,并对其进行系统进化分析。
(3)采用农杆菌介导法转化普通小球藻,通过G418作为抗性标记进行转化子筛选,同时优化藻落PCR条件,将其应用于转化子的鉴定和稳定性监测。以PCR扩增获得酿酒酵母DGAT基因,构建pBI121-DGAT穿梭质粒,转化获得含有重组质粒的根癌农杆菌；以TAP作为藻菌共培养体系,根癌农杆菌转化普通小球藻,计算转化效率为127-148/106；优化藻落PCR条件,采用6%w/vChelex-100溶液,在温度100℃条件下,温浴10min,有利于提高藻落PCR效果；藻落PCR应用于转化子的鉴定和稳定性监测,发现转化不稳定问题。
(4)比较评价称重法、FT-IR法、尼罗红染色法三种油脂含量测定方法,采用气升式光生物反应器进行高浓二氧化碳驯化与油脂富集。大规模培养且需低频率监测时,称重法优先选用；实验室规模且需中频率监测时,FT-IR法和尼罗红染色法均可选用,FT-IR法可以同时对油脂、蛋白质、碳水化合物含量进行定量,尼罗红染色法可以同时对中性脂滴进行定位；实验室规模且需高频率监测时,尼罗红染色法优先选用。驯化培养藻体细胞浓度1.37g L-1,为普通小球藻藻种在烟道气等高浓二氧化碳环境中的应用提供了可能；驯化培养油脂产量589mg L-1和油脂含量47.2%,实现了在普通小球藻高浓二氧化碳驯化的同时,进行油脂的富集；1.0%C02情况下,C02固定速率达到最大值,为106.9mg L-1h-1。
(5)建立微藻油脂的膜基萃取技术。比较六种普通小球藻细胞破碎方法,高压均质为适宜的细胞破碎方法,处理3次后,细胞破碎效率为91.7%,油脂损失率为19.3%,释放油脂得率为74.0%；比较三种中空纤维膜材料,PVDF(细孔)为适宜的中空纤维膜材料,内外表面静态接触角分别为75.0°和77.1°,内表面为平整网状结构,外表面为粗糙且多孔结构,存在大量10-100nm圆形孔洞,断面为中间海绵层两端指状孔结构；比较两种有机萃取剂,乙酸乙酯为适宜的有机萃取剂,萃取率和总萃取率分别为56.2%、45.4%。优化操作条件,最佳水相流量为40mL min-1,折合膜外侧流速为0.28cm s-1,最佳有机相流量为21mL min-1,折合膜内侧流速为2.79cm s-1,萃取率和总萃取率分别为71.3%、57.5%；初步将其应用于浓缩藻液萃取,藻液浓缩2.5倍、5倍、15倍、30倍,萃取率和总萃取率分别为65.2%和52.9%、57.4%和46.8%、45.9%和37.8%、36.1%和30.1%。
Fossil energy crisis and global greenhouse effects are two major challenges of the21st century. Microalgae as an alternative and renewable feedstock for biodiesel production, has drawn wide attention. However, there are still many challenges for the commercial utilization of biodiesel from microalgae as the algal-oil price remained rather higher than that of fossil fuels. Particularly, the further improvements of microalgal lipid production, as well as the reduction of lipid extraction costs are two problematic areas for the algal biofuel production technology. In this study, two different aspects of genetic engineering strategy and high CO2concentration domestication strategy were used for the enhancement of microalgal lipid production with microalgae Chlorella sp., and a new membrane-based lipid extraction method was also proposed for the reduction of extraction costs. The main research of the thesis included the following five aspects:
1. The core metabolic network and lipid accumulation network of Chlorella sp. under autotrophic condition was constructed and analyzed. The reliability of the metabolic network model was proved by the partially explanation of the environmental pressures impact on metabolic flux distribution and the competition between starch and TAG synthesis pathways. Combination of metabolic network analysis with CASOP algorithm analysis showed that fatty acid synthesis and Kennedy pathway, especially the DGAT enzyme, was of great importance for the TAG accumulation.
2. Separation, purification and identification of Chlorella sp. were carried out. Antibiotic combination treatment and streak plate method were used to remove bacteria and mold, respectively, and the sterile cultivation system was established. The comparison of three genomic DNA extraction methods showed that the plant genomic DNA extraction kit was esay operated and short time consuming, and high DNA purity and DNA yield could be obtained together with the improved SDS-CTAB method. PCR amplification of rbcL gene indicated that the Chlorella sp. was further belonged to Chlorella vulgaris species, and the phylogenetic analysis was accomplished.
3. A diacylglycerol acyltransferase (DGAT) gene from Saccharomyces cerevisiae S288c was constructed to pBI121vector, and the Agrobacterium-mediated transformation of DGAT in Chlorella vulgaris was established with tris acetate phosphate (TAP) as the co-cultivation medium and G418as the selection antibiotic. The results showed that the transformation frequency was127-148per106cells, and the colony PCR could be adopted to monitor the stability of DGAT transformation in Chlorella vulgaris when6%w/v Chelex-100solution was used as the DNA extraction buffer for10min of the incubation time. Although this transformation was still found to be unstable, the colony PCR did show its application in screening of the large scale genetically-engineered transformants besides that in the general amplification of genomic DNA fragments.
4. The high CO2concentration domestication and lipid enrichment were carried out in the airlift photobioreactor, and three methods of gravimetric determination, FT-IR spectroscopy and Nile Red staining were evaluated for algal lipid content analysis. The gravimetric determination was preferable for large-scale cultivation with low-frequency monitoring, while FT-IR and Nile Red were suitable for general laboratory cultivation with medium-frequency monitoring, in particularly Nile Red was appropriate for small samples when high-frequency screening was required. Moreover, the FT-IR method can simultaneously analyze the carbohydrate and protein contents, while the Nile Red staining can locate the lipid droplets. The biomass concentration reached1.37g L-1with the lipid concentration and lipid content of589mg L-1and47.2%after domestication, respectively, and the CO2fixation rate reached the maximum of106.9mg L-1h-1under1.0%CO2condition.
5. Microalgal lipid extraction with membrane based technology was established. High pressure homogenization was suitable for cell fragmentation with efficiency of91.7%after manipulation three times. PVDF (small one) hollow fiber membrane material was suitable for membrane based extraction, with the static contact angle of inner and outer surfaces of75.0°and77.1°, respectively. The inner surface was flat mesh structure, and the outer surface was rough and porous structure which had a large number of10-100nm circular holes, combining finger-like structures at two sides of the cross-section with microporous sponge-like structures in the middle. Ethyl acetate was suitable organic extractant and the extraction efficiency was56.2%. The operating conditions were optimized with aqueous phase flow of40mL min-1(0.28cm s-1) and organic phase flow of21mL min-1(2.79cm s-1). and the maximum extraction efficiency reached71.3%. As for the algal liquds concentrated with2.5times,5times.15times and30times, the extraction efficiency was65.2%.57.4%.45.9%and36.1%, respectively.
(1)构建小球藻自养条件下核心及油脂积累的代谢网络模型,并对其进行分析。通过部分解释环境压力对代谢流分布的影响,淀粉合成途径与TAG合成途径的竞争,说明代谢网络模型较为可靠。通过TAG合成途径与生物质合成途径的对比,结合CASOP算法分析,均发现脂肪酸合成及Kennedy途径,特别是DGAT晦,对于TAG的合成有很高的重要性,为产油微藻的基因工程改造提供指导。
(2)对出发藻种Chlorella sp进行分离、纯化与鉴定。采用抗生素组合处理法和平板划线分离法分别去除细菌和霉菌,获得无菌海水小球藻藻株,建立海水小球藻无菌培养体系。对比三种小球藻基因组DNA提取方法,植物基因组DNA快速提取试剂盒法,操作过程相对简单,所需时间相对较少,而该方法和改良SDS-CTAB法,均能够在保证DNA纯度的同时,获得较大的DNA总量和DNA产率。PCR扩增rbcL基因并进行序列测定,鉴定实验室海水小球藻属于海水普通小球藻,并对其进行系统进化分析。
(3)采用农杆菌介导法转化普通小球藻,通过G418作为抗性标记进行转化子筛选,同时优化藻落PCR条件,将其应用于转化子的鉴定和稳定性监测。以PCR扩增获得酿酒酵母DGAT基因,构建pBI121-DGAT穿梭质粒,转化获得含有重组质粒的根癌农杆菌；以TAP作为藻菌共培养体系,根癌农杆菌转化普通小球藻,计算转化效率为127-148/106；优化藻落PCR条件,采用6%w/vChelex-100溶液,在温度100℃条件下,温浴10min,有利于提高藻落PCR效果；藻落PCR应用于转化子的鉴定和稳定性监测,发现转化不稳定问题。
(4)比较评价称重法、FT-IR法、尼罗红染色法三种油脂含量测定方法,采用气升式光生物反应器进行高浓二氧化碳驯化与油脂富集。大规模培养且需低频率监测时,称重法优先选用；实验室规模且需中频率监测时,FT-IR法和尼罗红染色法均可选用,FT-IR法可以同时对油脂、蛋白质、碳水化合物含量进行定量,尼罗红染色法可以同时对中性脂滴进行定位；实验室规模且需高频率监测时,尼罗红染色法优先选用。驯化培养藻体细胞浓度1.37g L-1,为普通小球藻藻种在烟道气等高浓二氧化碳环境中的应用提供了可能；驯化培养油脂产量589mg L-1和油脂含量47.2%,实现了在普通小球藻高浓二氧化碳驯化的同时,进行油脂的富集；1.0%C02情况下,C02固定速率达到最大值,为106.9mg L-1h-1。
(5)建立微藻油脂的膜基萃取技术。比较六种普通小球藻细胞破碎方法,高压均质为适宜的细胞破碎方法,处理3次后,细胞破碎效率为91.7%,油脂损失率为19.3%,释放油脂得率为74.0%；比较三种中空纤维膜材料,PVDF(细孔)为适宜的中空纤维膜材料,内外表面静态接触角分别为75.0°和77.1°,内表面为平整网状结构,外表面为粗糙且多孔结构,存在大量10-100nm圆形孔洞,断面为中间海绵层两端指状孔结构；比较两种有机萃取剂,乙酸乙酯为适宜的有机萃取剂,萃取率和总萃取率分别为56.2%、45.4%。优化操作条件,最佳水相流量为40mL min-1,折合膜外侧流速为0.28cm s-1,最佳有机相流量为21mL min-1,折合膜内侧流速为2.79cm s-1,萃取率和总萃取率分别为71.3%、57.5%；初步将其应用于浓缩藻液萃取,藻液浓缩2.5倍、5倍、15倍、30倍,萃取率和总萃取率分别为65.2%和52.9%、57.4%和46.8%、45.9%和37.8%、36.1%和30.1%。
Fossil energy crisis and global greenhouse effects are two major challenges of the21st century. Microalgae as an alternative and renewable feedstock for biodiesel production, has drawn wide attention. However, there are still many challenges for the commercial utilization of biodiesel from microalgae as the algal-oil price remained rather higher than that of fossil fuels. Particularly, the further improvements of microalgal lipid production, as well as the reduction of lipid extraction costs are two problematic areas for the algal biofuel production technology. In this study, two different aspects of genetic engineering strategy and high CO2concentration domestication strategy were used for the enhancement of microalgal lipid production with microalgae Chlorella sp., and a new membrane-based lipid extraction method was also proposed for the reduction of extraction costs. The main research of the thesis included the following five aspects:
1. The core metabolic network and lipid accumulation network of Chlorella sp. under autotrophic condition was constructed and analyzed. The reliability of the metabolic network model was proved by the partially explanation of the environmental pressures impact on metabolic flux distribution and the competition between starch and TAG synthesis pathways. Combination of metabolic network analysis with CASOP algorithm analysis showed that fatty acid synthesis and Kennedy pathway, especially the DGAT enzyme, was of great importance for the TAG accumulation.
2. Separation, purification and identification of Chlorella sp. were carried out. Antibiotic combination treatment and streak plate method were used to remove bacteria and mold, respectively, and the sterile cultivation system was established. The comparison of three genomic DNA extraction methods showed that the plant genomic DNA extraction kit was esay operated and short time consuming, and high DNA purity and DNA yield could be obtained together with the improved SDS-CTAB method. PCR amplification of rbcL gene indicated that the Chlorella sp. was further belonged to Chlorella vulgaris species, and the phylogenetic analysis was accomplished.
3. A diacylglycerol acyltransferase (DGAT) gene from Saccharomyces cerevisiae S288c was constructed to pBI121vector, and the Agrobacterium-mediated transformation of DGAT in Chlorella vulgaris was established with tris acetate phosphate (TAP) as the co-cultivation medium and G418as the selection antibiotic. The results showed that the transformation frequency was127-148per106cells, and the colony PCR could be adopted to monitor the stability of DGAT transformation in Chlorella vulgaris when6%w/v Chelex-100solution was used as the DNA extraction buffer for10min of the incubation time. Although this transformation was still found to be unstable, the colony PCR did show its application in screening of the large scale genetically-engineered transformants besides that in the general amplification of genomic DNA fragments.
4. The high CO2concentration domestication and lipid enrichment were carried out in the airlift photobioreactor, and three methods of gravimetric determination, FT-IR spectroscopy and Nile Red staining were evaluated for algal lipid content analysis. The gravimetric determination was preferable for large-scale cultivation with low-frequency monitoring, while FT-IR and Nile Red were suitable for general laboratory cultivation with medium-frequency monitoring, in particularly Nile Red was appropriate for small samples when high-frequency screening was required. Moreover, the FT-IR method can simultaneously analyze the carbohydrate and protein contents, while the Nile Red staining can locate the lipid droplets. The biomass concentration reached1.37g L-1with the lipid concentration and lipid content of589mg L-1and47.2%after domestication, respectively, and the CO2fixation rate reached the maximum of106.9mg L-1h-1under1.0%CO2condition.
5. Microalgal lipid extraction with membrane based technology was established. High pressure homogenization was suitable for cell fragmentation with efficiency of91.7%after manipulation three times. PVDF (small one) hollow fiber membrane material was suitable for membrane based extraction, with the static contact angle of inner and outer surfaces of75.0°and77.1°, respectively. The inner surface was flat mesh structure, and the outer surface was rough and porous structure which had a large number of10-100nm circular holes, combining finger-like structures at two sides of the cross-section with microporous sponge-like structures in the middle. Ethyl acetate was suitable organic extractant and the extraction efficiency was56.2%. The operating conditions were optimized with aqueous phase flow of40mL min-1(0.28cm s-1) and organic phase flow of21mL min-1(2.79cm s-1). and the maximum extraction efficiency reached71.3%. As for the algal liquds concentrated with2.5times,5times.15times and30times, the extraction efficiency was65.2%.57.4%.45.9%and36.1%, respectively.
引文
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