高油脂微藻种质的筛选、鉴定及新品种培育
摘要
石化燃料日趋枯竭和环境压力的日益增强,迫使人类急切的寻找一种新型的、可再生的洁净能源。单细胞微藻的光合效率是油棕、水稻等作物的数十倍,而且生长快,含油量高,不与农作物争地,是具有很大应用前景的新能源植物。由于良种培育及高密度、低成本养殖等关键技术至今尚未解决,能源微藻(即油藻)产业化成本一直居高不下。故而,筛选高产、稳产、高含油量等优良农艺性状的新藻种、探索低成本培养条件是能源微藻能否产业化的关键,同时也是国内外研究的一大热点。本研究旨在通过试验,筛选高生物量、高含油量藻株,探索优势藻株生长及油脂积累条件,同时利用育种的方法培育新型油藻,为藻类的开发利用、生物质能源的深入研究奠定基础。
本试验以尼罗红为染料,优化了尼罗红荧光染色检测微藻中油脂相对含量的条件,确定尼罗红染色质量浓度为1.0μg/ml,染色时间为6min,二甲基亚砜体积浓度为20%,待测藻液密度OD600=0.12～0.52时染色效果最佳,为大规模筛选高含油量微藻奠定了基础。
以含油量高、生产率高为选育目标,用尼罗红染色荧光显微镜检测法对本实验室496株微藻进行了初步筛选,获得了40株油脂含量相对较高的藻株,通过生长和油脂动态积累情况进行跟踪检测,比较同一藻液吸光值(OD=0.35)时脂荧光强度、最高脂荧光强度和比平均生长速率等指标,初步确定编号La4-37、La2-14、SA2-1、SA2-17、SA2-8的藻株具有含油量高、生产率高等优良性状,具有作为生物柴油原料的潜力。重点对编号为La4-37的藻株进行了研究。通过细胞形态观察发现,La4-37细胞成球形,色素体为单个,绿色,杯状,细胞大小为3～10um,以孢子生殖为主；扩增18S rDNA,测序获得大小1756bp的DNA序列,通过生物信息学分析,确定La4-37为绿藻门(Chlorophyta),原球藻纲(Trebouxiophycea),小球藻科(Chlorellaceae),小球藻属(Chlorella), Chlo rella sorokiniana种。为更清楚了解La4-37生长及油脂积累情况,对藻株生长和油脂情况进行动态跟踪,发现藻株油脂在迟滞期有一个短暂积累,在指数生长后期和平稳期大量积累,最后通过检测La4-37平稳期生物量(1.072g/1),比平均生长速率(O.26d-1),脂荧光强度(8.71×10-5/cell),并与其它产油微藻比较发现,筛选得到的藻株La4-37具有含油量高,生物量高、生长速率快的优良性状,具有作为能源微藻开发的潜力。
环境条件会严重影响微藻的生长和油脂产量,而氮浓度和初始pH值是影响微藻生长和油脂积累较关键的环境因子。不同氮浓度藻株生长和油脂积累情况跟踪试验发现：随着氮浓度的降低,藻细胞油脂积累能力增强,生长速度降低。La4-37的pH梯度试验表明：在初始pH值为7-8时生长最好,初始pH值较低的培养基中(pH值为4和5),藻株几乎不能生长,在初始pH值高达12的培养基中也有一定程度的生长(比平均生长速率为0.2d-1)；油脂积累随着pH的升高而增强,初始pH值为1 2时最高。
为进一步获得含油量和生长速率更高的藻株,采用紫外线辐射法对La4-37进行诱变处理,经过初步筛选,获得296株诱变藻株,通过尼罗红荧光检测法对获得的藻株脂进行荧光检测,并与同密度下原始藻株脂荧光强度比较,筛选获得相对含油量最大的诱变藻株M077和M040。通过对诱变株生长及脂荧光强度动态跟踪发现,诱变株生长周期和油脂积累时期基本一样,当达到平稳期时,诱变株油脂积累能力均有较大提高(脂荧光强度分别是原始藻株的6.2倍和1.7倍),但生物量略有下降,M077为原始藻株的49%, M040为原始藻株的93%。
A new and renewable clean energy was urgent to be searched because of the increasing depletion of fossil fuels and enhance of environmental stress. The photo synthetic efficiency of unicellular microalgae is several times of that of lipid palm, rice and other crops. The microalgae have been considered to be a ideal source of biodiesel for its fast growth and high lipid content. Because the key technologies of high-density and low-cost cultivation have not yet been resolved, the industrialization cost of the energy algae was very high. Therefore, screening new algae species with high yield and high lipid content and exploring low-cost culture condition are the key factors of the energy algae industry, which is a hotspot in the world. This study was designed to screen algae with high biomass production and lipid content, establish a core germplasm collection for energy microalgae, optimize the culture condition of preponderant algae and reduce the cost of industrial cultivation And the methods cultivating new lipid algae was the basic of algae utilization and biomass energy research.
In this test, microalgae lipid can be qualitatively and quantitatively analyzed by Nile Red fluorescence staining method, through fluorospectro photometer and multifunctional ELIASA. The detection conditions was optimized:Nile Red dye concentration 1μg/ml, the dyeing time 6 min, DMSO concentration 20%, and tested algae density OD600=0.12～0.52. Large-scale primary screening of lip id-rich microalgae was researched by the multi-functional ELIASA. At the same time, lipid accumulation of the same algae at different culture condition and different develop period was observed by fluorospectro photometer.
With highoil content and high productivity for the breeding goal,496 microalgae were screened preliminarily by Nile Red fluorescence staining method in our laboratory. And 40 strains with relatively high oil content were obtained.
Lipid fluorescence intensity, maximum fluorescence intensity and the average growth rate of microalgae were compared at the same density (OD=0.35) by the dynamic growth and lipid accumulation. Preliminarily, microalgae named La4-37, La2-14, SA2-1, SA2-17 and SA2-8, which had high lipid content and high productivity, were the potential biodiesel feedstock. Algae named La4-37 was chosen to be identified. The cell morphologic observation indicated that the cell of La4-37 was spherical and sporogony, chromatoplast was single, green and goblet. The cell size of La4-37 was 3-10μm.1756bp DNA sequences of La4-37 were obtained by amplified 18S rDNA. The result of cell morphologic observation and 18S rDNA evaluation was indicated that La4-37 was indentified as Chlorella sorokiniana, Chlorella, Chlorellaceae, Trebouxiophycea, Chlorophyta. The growth and lipid accumulation of La4-37 were observed. As a result, a transient lipid accumulation at lag phase, but abundant lipid accumulation at the late and stationary phase of exponential growth. Finally, the biomass (1.072g/l), the average growth rate (0.26 d-1) and lipid fluorescence intensity (8.71×10-5/cell) of La4-37 were detected. Compared to other oil-producing microalgae, La4-37 with high lipid content and high biomass and high growth rate was a potential energy microalgae.
Environmental condition would affect the microalgae growth and lipid production seriously. Growth and lipid accumulation of La4-37 indifferent nitrogen concentrations and different pH was tested. The results were showed that the lipid accumulation of La4-37 increased and the growth rate decreased with the reduction of nitrogen concentration The results of pH gradient test indicated that La4-37 grew best at pH 7～8, but couldn't grow barely at low pH (4-5). In addition, La4-37 could grow to a certain extent (the average growth rate 0.2 d-1) at pH 12. The lipid accumulation of La4-37 was increased with the increase of pH. When the pH was 12, the relative lipid content was the highest.
In order to obtain algal strains with higher oil content and growth rate, La4-37 was treated with ultraviolet irradiation.296 mutagenic microalgae were obtained after preliminary screening. Two mutagenic microalgae M077 and M040 with relatively lipid content were isolated through the lipid fluorescence detection and the lipid fluorescence intensity at the same algal density by Nile Red fluorescence staining method
The growth and lipid dynamic fluorescence intensity of mutagenic microalgae was showed that the growth cycle of mutant strains and the period of lipid accumulation were the same. When the mutagenic microalgae grew to the stationary phase, the lipid accumulation had improved greatly (the lipid fluorescence intensity of M077 and M040 was 6.2 and 1.7 times as that of La4-37, respectively). But, the biomasses of mutagenic microalgae were decreased. The biomass of M077 was 49% of La4-37, and the biomass of M040 was 93% of La4-37.
本试验以尼罗红为染料,优化了尼罗红荧光染色检测微藻中油脂相对含量的条件,确定尼罗红染色质量浓度为1.0μg/ml,染色时间为6min,二甲基亚砜体积浓度为20%,待测藻液密度OD600=0.12～0.52时染色效果最佳,为大规模筛选高含油量微藻奠定了基础。
以含油量高、生产率高为选育目标,用尼罗红染色荧光显微镜检测法对本实验室496株微藻进行了初步筛选,获得了40株油脂含量相对较高的藻株,通过生长和油脂动态积累情况进行跟踪检测,比较同一藻液吸光值(OD=0.35)时脂荧光强度、最高脂荧光强度和比平均生长速率等指标,初步确定编号La4-37、La2-14、SA2-1、SA2-17、SA2-8的藻株具有含油量高、生产率高等优良性状,具有作为生物柴油原料的潜力。重点对编号为La4-37的藻株进行了研究。通过细胞形态观察发现,La4-37细胞成球形,色素体为单个,绿色,杯状,细胞大小为3～10um,以孢子生殖为主；扩增18S rDNA,测序获得大小1756bp的DNA序列,通过生物信息学分析,确定La4-37为绿藻门(Chlorophyta),原球藻纲(Trebouxiophycea),小球藻科(Chlorellaceae),小球藻属(Chlorella), Chlo rella sorokiniana种。为更清楚了解La4-37生长及油脂积累情况,对藻株生长和油脂情况进行动态跟踪,发现藻株油脂在迟滞期有一个短暂积累,在指数生长后期和平稳期大量积累,最后通过检测La4-37平稳期生物量(1.072g/1),比平均生长速率(O.26d-1),脂荧光强度(8.71×10-5/cell),并与其它产油微藻比较发现,筛选得到的藻株La4-37具有含油量高,生物量高、生长速率快的优良性状,具有作为能源微藻开发的潜力。
环境条件会严重影响微藻的生长和油脂产量,而氮浓度和初始pH值是影响微藻生长和油脂积累较关键的环境因子。不同氮浓度藻株生长和油脂积累情况跟踪试验发现：随着氮浓度的降低,藻细胞油脂积累能力增强,生长速度降低。La4-37的pH梯度试验表明：在初始pH值为7-8时生长最好,初始pH值较低的培养基中(pH值为4和5),藻株几乎不能生长,在初始pH值高达12的培养基中也有一定程度的生长(比平均生长速率为0.2d-1)；油脂积累随着pH的升高而增强,初始pH值为1 2时最高。
为进一步获得含油量和生长速率更高的藻株,采用紫外线辐射法对La4-37进行诱变处理,经过初步筛选,获得296株诱变藻株,通过尼罗红荧光检测法对获得的藻株脂进行荧光检测,并与同密度下原始藻株脂荧光强度比较,筛选获得相对含油量最大的诱变藻株M077和M040。通过对诱变株生长及脂荧光强度动态跟踪发现,诱变株生长周期和油脂积累时期基本一样,当达到平稳期时,诱变株油脂积累能力均有较大提高(脂荧光强度分别是原始藻株的6.2倍和1.7倍),但生物量略有下降,M077为原始藻株的49%, M040为原始藻株的93%。
A new and renewable clean energy was urgent to be searched because of the increasing depletion of fossil fuels and enhance of environmental stress. The photo synthetic efficiency of unicellular microalgae is several times of that of lipid palm, rice and other crops. The microalgae have been considered to be a ideal source of biodiesel for its fast growth and high lipid content. Because the key technologies of high-density and low-cost cultivation have not yet been resolved, the industrialization cost of the energy algae was very high. Therefore, screening new algae species with high yield and high lipid content and exploring low-cost culture condition are the key factors of the energy algae industry, which is a hotspot in the world. This study was designed to screen algae with high biomass production and lipid content, establish a core germplasm collection for energy microalgae, optimize the culture condition of preponderant algae and reduce the cost of industrial cultivation And the methods cultivating new lipid algae was the basic of algae utilization and biomass energy research.
In this test, microalgae lipid can be qualitatively and quantitatively analyzed by Nile Red fluorescence staining method, through fluorospectro photometer and multifunctional ELIASA. The detection conditions was optimized:Nile Red dye concentration 1μg/ml, the dyeing time 6 min, DMSO concentration 20%, and tested algae density OD600=0.12～0.52. Large-scale primary screening of lip id-rich microalgae was researched by the multi-functional ELIASA. At the same time, lipid accumulation of the same algae at different culture condition and different develop period was observed by fluorospectro photometer.
With highoil content and high productivity for the breeding goal,496 microalgae were screened preliminarily by Nile Red fluorescence staining method in our laboratory. And 40 strains with relatively high oil content were obtained.
Lipid fluorescence intensity, maximum fluorescence intensity and the average growth rate of microalgae were compared at the same density (OD=0.35) by the dynamic growth and lipid accumulation. Preliminarily, microalgae named La4-37, La2-14, SA2-1, SA2-17 and SA2-8, which had high lipid content and high productivity, were the potential biodiesel feedstock. Algae named La4-37 was chosen to be identified. The cell morphologic observation indicated that the cell of La4-37 was spherical and sporogony, chromatoplast was single, green and goblet. The cell size of La4-37 was 3-10μm.1756bp DNA sequences of La4-37 were obtained by amplified 18S rDNA. The result of cell morphologic observation and 18S rDNA evaluation was indicated that La4-37 was indentified as Chlorella sorokiniana, Chlorella, Chlorellaceae, Trebouxiophycea, Chlorophyta. The growth and lipid accumulation of La4-37 were observed. As a result, a transient lipid accumulation at lag phase, but abundant lipid accumulation at the late and stationary phase of exponential growth. Finally, the biomass (1.072g/l), the average growth rate (0.26 d-1) and lipid fluorescence intensity (8.71×10-5/cell) of La4-37 were detected. Compared to other oil-producing microalgae, La4-37 with high lipid content and high biomass and high growth rate was a potential energy microalgae.
Environmental condition would affect the microalgae growth and lipid production seriously. Growth and lipid accumulation of La4-37 indifferent nitrogen concentrations and different pH was tested. The results were showed that the lipid accumulation of La4-37 increased and the growth rate decreased with the reduction of nitrogen concentration The results of pH gradient test indicated that La4-37 grew best at pH 7～8, but couldn't grow barely at low pH (4-5). In addition, La4-37 could grow to a certain extent (the average growth rate 0.2 d-1) at pH 12. The lipid accumulation of La4-37 was increased with the increase of pH. When the pH was 12, the relative lipid content was the highest.
In order to obtain algal strains with higher oil content and growth rate, La4-37 was treated with ultraviolet irradiation.296 mutagenic microalgae were obtained after preliminary screening. Two mutagenic microalgae M077 and M040 with relatively lipid content were isolated through the lipid fluorescence detection and the lipid fluorescence intensity at the same algal density by Nile Red fluorescence staining method
The growth and lipid dynamic fluorescence intensity of mutagenic microalgae was showed that the growth cycle of mutant strains and the period of lipid accumulation were the same. When the mutagenic microalgae grew to the stationary phase, the lipid accumulation had improved greatly (the lipid fluorescence intensity of M077 and M040 was 6.2 and 1.7 times as that of La4-37, respectively). But, the biomasses of mutagenic microalgae were decreased. The biomass of M077 was 49% of La4-37, and the biomass of M040 was 93% of La4-37.
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