造纸废水培养富油微藻的研究
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摘要
微藻制备生物柴油已越来越受到各国的重视,而成本过高是制约其发展的主要问题之一。本文研究了利用造纸废水培养富油微藻的可行性,以期为降低微藻柴油成本寻找一条新的途径。论文进行了微藻的筛选、紫外诱变和条件优化,对造纸废水进行了成分分析、水解及初步脱毒处理,最后考察了微藻对水解液的利用情况。
选取了6株微藻进行初筛。结果显示藻的含油量在17%～26%之间,其中栅藻的含油量最高,为25.77%。C16和C18为几株藻的主要脂肪酸组分。除小新月菱形藻外,其他藻异养能力均较强。六碳糖和二糖(麦芽糖)是较好的碳源,藻对五碳糖的利用情况较差。葡萄糖的加入可大大提高微藻的生长速率,兼养组小球-1,小球藻-2和栅藻收获时生长量分别是自养时的4.4,7.5,6.3倍。综合考虑生长量和含油量两个因素,筛选出栅藻进行下一步研究。
通过紫外诱变,筛选出一株栅藻编号为栅-1,其生长速率较未优化前增长了39%,含油量达到了28%,颜色深绿,生长旺盛。并通过2*(6-2)部分析因设计实验筛选得出氮源和温度为影响栅藻油脂产率的显著因素。通过L9 (33)的正交实验,确定出适于栅藻产油的最佳条件为氮源浓度为6.0mM,温度25℃,光照强度为4000lux,葡萄糖浓度10g/L,pH值5,在此条件下油脂产率可达到75.46 mg·L~(-1)d~(-1)。
对马尾松硫酸盐黑液进行了成分分析,结果表明:黑液中还原糖为1.92 g/L,半纤维素含量为35.69 g/L。在培养基中添加不同比例的黑液,随着培养基中黑液浓度的增大,黑液对栅藻的影响增大。在显微镜下观察发现在100%黑液中,栅藻形态发生了变化。说明黑液中存在着对微藻生长不利的物质。
对黑液进行水解。考察了不同水解方法下还原糖获得量。结果显示同酸水解相比,酶水解效率较高。脱木素后再酶水解,水解液中还原糖可达到9.18g/L。脱木素的最佳条件为pH值3.0,温度75℃,硫酸浓度6%,此时上清液中的半纤维素含量最高,达到47.61g/L。对水解液做液相色谱分析,发现水解液中主要成分为葡萄糖3.63 g/L,其次为纤维二糖1.81 g/L,并有少量阿拉伯糖和木糖,含量分别为0.02g/L和0.8g/L。同时检测出水解液中含有较多甲酸和乙酸,浓度分别为3.10 g/L,2.90 g/L。利用石灰过中和,减压真空浓缩和活性炭进行脱色脱毒处理,水解液变为浅黄色,透光率增大,还原糖损失率为5.64%。
利用脱毒处理后的黑液水解液培养栅藻-1,其生长量较未处理的黑液葡萄糖培养基大幅度提高,达到了1.23g/L,测定含油量为24.52%,油脂产率达到23.20 mg·L~(-1)d~(-1),栅藻-1的形态恢复正常。
将纤维素从黑液中提取出,水解培养微藻,结果表明在半纤维素水解液培养基中的栅藻-1,其生长量与处理后的黑液水解液培养基相比有所提高,通过调整培养基配方,半纤维素水解液中栅藻干重达到2.70 g/L,比葡萄糖培养基提高了20%,含油量为25.70%,油脂产率可达到53.37 mg·L~(-1)d~(-1)。
Biodiesel from micro algae has been gaining national attention. But high cost is one of the major problem restricting the development of algal biodiesel.The feasibility of using paper mill wastewater for oil-riched algae cultivation was studied, hoping to find a new way to reduce the cost of biodiesel from algae. Algae with high oil-content and growth rate was screened, mutagenized by ultraviolet and cutivitation conditions were optimized. Then the composition of wastewater was analyzed. After hydrolysis and detoxification, the hydrolysate was used for algae cultivation.
The oil content of six algae used was between 17%～26%. Scenedesmus obliquus was with the highest oil content of 25.77%.C16 and C18 were major fatty acid of these algae. Except Nitzschia closterium, the other five algae were with heterotrophic ability. Hexose and disaccharide(maltose) were good carbon source, but algae could not utilize pentose. Growth rate was highly increased when glucose was added in the medium. Mass growth of Chlorella sp-1,Chlorella sp-2 and S. obliquus cultivated mixtrophically was 4.4, 7.5 and 6.3 times higher than that of autotrophically. Based on growth rate and oil content, S. obliquus was screened for further research.
A strain named S.obliquus -1 was screened by UV mutagenesis, the growth rate of which increased by 39% than that of pre-mutagenesis. The strain was with an oil content of 28%. Conditions for lipid productivity were optimized in two stages: fractional factorial design (FFD) and orthogonal experiments. Nitrogen and temperature were selected as significant factors. And the optimal conditions occurred at nitrogen 6.0mM, temperature 25℃,illumination 4500 lux, glucose 10g/L and pH 5.0, with the highest lipid productivity of 75.46mg L~(-1)d~(-1).
The component of pine kraft black liquor was analyzed. Result shows that there were reducing sugar 1.92 g/L and hemicellulose 35 g/L. Dilute acid hydrolysate mainly contained glucose and some cellobiose. S obliquus -1 was cultivated in medium with different proportion of black liquor.Result show that with the increase concentration of black liquor, the influence of black liquor on algae increased. It was observed that the morphology of S.obliquus -1 changed, which means that the black liquor maybe contains some toxic substances for algae.
The black liquor was hydrolyzed and reducing sugar content was investigated using different hydrolysis methods. Result shows that compared with acid hydrolysis, enzymatic hydrolysis was with higher reducing sugar content. A reducing sugar content of 9.18g/L was obtained by enzymatic hydrolysis after delignification. And the optimum conditions for delignification was pH 3.0, temperature 75℃,sulfuric acid concentration 6%, when the highest hemicellulose content of 47.61 g/L was obtained in the wupernatant residual.
The main sugar in hydrolysate was glucose 3.63 g/L, followed by cellobiose 1.81 g/L, xylose 0.8 g/L and arabinose 0.2 g/L. Meanwhile, 3.10 g/L formic acid and 2.90 g/L acetic acid was detected. After overliming by Ca(OH)2, decompression vacuum concentration and detoxification of activated carbon bleaching, hydrolysate became light yellow and light transmission rate increased. Reducing sugar loss rate was 5.64%.
When S.obliquus -1was cultivated in hydrolysate after treatment, its growth yield reached to 1.23 g/L, greatly increased than that of in black liquor medium with glucose, oil content was 24.52 % and lipid productivity was 23.20 mg L~(-1)d~(-1) , the morphology of S.obliquus -1 returned to normal.
Hemicellulose was extracted from black liquor and hydrolyzed. The hydrolysate was used to cultivate S.obliquus -1. It was found that growth yield of S.obliquus -1 greatly increased than that of black liquor medium. By adjusting medium formula, growth yield of S.obliquus -1 increased to 2.7g/L, an increase of 20 % than that of in glucose medium, oil content was 25.7%, the final lipid productivity reached 53.37mg L~(-1)d~(-1).
选取了6株微藻进行初筛。结果显示藻的含油量在17%～26%之间,其中栅藻的含油量最高,为25.77%。C16和C18为几株藻的主要脂肪酸组分。除小新月菱形藻外,其他藻异养能力均较强。六碳糖和二糖(麦芽糖)是较好的碳源,藻对五碳糖的利用情况较差。葡萄糖的加入可大大提高微藻的生长速率,兼养组小球-1,小球藻-2和栅藻收获时生长量分别是自养时的4.4,7.5,6.3倍。综合考虑生长量和含油量两个因素,筛选出栅藻进行下一步研究。
通过紫外诱变,筛选出一株栅藻编号为栅-1,其生长速率较未优化前增长了39%,含油量达到了28%,颜色深绿,生长旺盛。并通过2*(6-2)部分析因设计实验筛选得出氮源和温度为影响栅藻油脂产率的显著因素。通过L9 (33)的正交实验,确定出适于栅藻产油的最佳条件为氮源浓度为6.0mM,温度25℃,光照强度为4000lux,葡萄糖浓度10g/L,pH值5,在此条件下油脂产率可达到75.46 mg·L~(-1)d~(-1)。
对马尾松硫酸盐黑液进行了成分分析,结果表明:黑液中还原糖为1.92 g/L,半纤维素含量为35.69 g/L。在培养基中添加不同比例的黑液,随着培养基中黑液浓度的增大,黑液对栅藻的影响增大。在显微镜下观察发现在100%黑液中,栅藻形态发生了变化。说明黑液中存在着对微藻生长不利的物质。
对黑液进行水解。考察了不同水解方法下还原糖获得量。结果显示同酸水解相比,酶水解效率较高。脱木素后再酶水解,水解液中还原糖可达到9.18g/L。脱木素的最佳条件为pH值3.0,温度75℃,硫酸浓度6%,此时上清液中的半纤维素含量最高,达到47.61g/L。对水解液做液相色谱分析,发现水解液中主要成分为葡萄糖3.63 g/L,其次为纤维二糖1.81 g/L,并有少量阿拉伯糖和木糖,含量分别为0.02g/L和0.8g/L。同时检测出水解液中含有较多甲酸和乙酸,浓度分别为3.10 g/L,2.90 g/L。利用石灰过中和,减压真空浓缩和活性炭进行脱色脱毒处理,水解液变为浅黄色,透光率增大,还原糖损失率为5.64%。
利用脱毒处理后的黑液水解液培养栅藻-1,其生长量较未处理的黑液葡萄糖培养基大幅度提高,达到了1.23g/L,测定含油量为24.52%,油脂产率达到23.20 mg·L~(-1)d~(-1),栅藻-1的形态恢复正常。
将纤维素从黑液中提取出,水解培养微藻,结果表明在半纤维素水解液培养基中的栅藻-1,其生长量与处理后的黑液水解液培养基相比有所提高,通过调整培养基配方,半纤维素水解液中栅藻干重达到2.70 g/L,比葡萄糖培养基提高了20%,含油量为25.70%,油脂产率可达到53.37 mg·L~(-1)d~(-1)。
Biodiesel from micro algae has been gaining national attention. But high cost is one of the major problem restricting the development of algal biodiesel.The feasibility of using paper mill wastewater for oil-riched algae cultivation was studied, hoping to find a new way to reduce the cost of biodiesel from algae. Algae with high oil-content and growth rate was screened, mutagenized by ultraviolet and cutivitation conditions were optimized. Then the composition of wastewater was analyzed. After hydrolysis and detoxification, the hydrolysate was used for algae cultivation.
The oil content of six algae used was between 17%～26%. Scenedesmus obliquus was with the highest oil content of 25.77%.C16 and C18 were major fatty acid of these algae. Except Nitzschia closterium, the other five algae were with heterotrophic ability. Hexose and disaccharide(maltose) were good carbon source, but algae could not utilize pentose. Growth rate was highly increased when glucose was added in the medium. Mass growth of Chlorella sp-1,Chlorella sp-2 and S. obliquus cultivated mixtrophically was 4.4, 7.5 and 6.3 times higher than that of autotrophically. Based on growth rate and oil content, S. obliquus was screened for further research.
A strain named S.obliquus -1 was screened by UV mutagenesis, the growth rate of which increased by 39% than that of pre-mutagenesis. The strain was with an oil content of 28%. Conditions for lipid productivity were optimized in two stages: fractional factorial design (FFD) and orthogonal experiments. Nitrogen and temperature were selected as significant factors. And the optimal conditions occurred at nitrogen 6.0mM, temperature 25℃,illumination 4500 lux, glucose 10g/L and pH 5.0, with the highest lipid productivity of 75.46mg L~(-1)d~(-1).
The component of pine kraft black liquor was analyzed. Result shows that there were reducing sugar 1.92 g/L and hemicellulose 35 g/L. Dilute acid hydrolysate mainly contained glucose and some cellobiose. S obliquus -1 was cultivated in medium with different proportion of black liquor.Result show that with the increase concentration of black liquor, the influence of black liquor on algae increased. It was observed that the morphology of S.obliquus -1 changed, which means that the black liquor maybe contains some toxic substances for algae.
The black liquor was hydrolyzed and reducing sugar content was investigated using different hydrolysis methods. Result shows that compared with acid hydrolysis, enzymatic hydrolysis was with higher reducing sugar content. A reducing sugar content of 9.18g/L was obtained by enzymatic hydrolysis after delignification. And the optimum conditions for delignification was pH 3.0, temperature 75℃,sulfuric acid concentration 6%, when the highest hemicellulose content of 47.61 g/L was obtained in the wupernatant residual.
The main sugar in hydrolysate was glucose 3.63 g/L, followed by cellobiose 1.81 g/L, xylose 0.8 g/L and arabinose 0.2 g/L. Meanwhile, 3.10 g/L formic acid and 2.90 g/L acetic acid was detected. After overliming by Ca(OH)2, decompression vacuum concentration and detoxification of activated carbon bleaching, hydrolysate became light yellow and light transmission rate increased. Reducing sugar loss rate was 5.64%.
When S.obliquus -1was cultivated in hydrolysate after treatment, its growth yield reached to 1.23 g/L, greatly increased than that of in black liquor medium with glucose, oil content was 24.52 % and lipid productivity was 23.20 mg L~(-1)d~(-1) , the morphology of S.obliquus -1 returned to normal.
Hemicellulose was extracted from black liquor and hydrolyzed. The hydrolysate was used to cultivate S.obliquus -1. It was found that growth yield of S.obliquus -1 greatly increased than that of black liquor medium. By adjusting medium formula, growth yield of S.obliquus -1 increased to 2.7g/L, an increase of 20 % than that of in glucose medium, oil content was 25.7%, the final lipid productivity reached 53.37mg L~(-1)d~(-1).
引文
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