【作者】 曲春波；

【导师】 史贤明；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2009， 博士

【摘要】 磷是培养小球藻（Chlorella）常用Basal培养基中的大量成分,其含量以及磷与碳的比例直接关系到小球藻生长繁殖和代谢产物合成,若供应不足,则会成为生长限制因子,但若供应超量,则是资源浪费并容易引起环境富营养化。利用啤酒废水异养培养小球藻是一种废水资源化处理方式。本论文主要围绕上述问题开展了以下几个方面的研究工作:（1）采用初始葡萄糖浓度为10 g/l的Basal培养基,通过测定小球藻异养培养生长参数,从五株小球藻中,筛选到两株适宜高效异养培养的藻株,分别为Chlorella pyrenoidosa 15-2070和Chlorella vulgaris 15-2075,其最高生物量分别为5.3 g/l、5.2 g/l,最高生物量出现时间均在第3天,最大比生长速率分别为0.98 /d、0.96 /d,糖对细胞转化率分别为0.54 g/g、0.52 g/g。（2）用筛选到的Chlorella pyrenoidosa 15-2070用于啤酒废水的资源化处理。利用啤酒综合废水100 %替代纯水配制Basal培养基,含10 g/l葡萄糖的培养液中获得5.3 g/l藻细胞,结果表明,啤酒综合废水对小球藻生长没有抑制作用,可以用于小球藻高密度异养培养。采用本文设计的几种啤酒综合废水预处理方式,几种主要污染物最高去除率为:CODcr,92.2 %;BOD5,95.1 %;NO3--N,98.5 %;NH4+-N,92.3 %;这表明异养小球藻能有效净化啤酒综合废水。在本文设计的几种啤酒洗糟废水预处理方式下,啤酒洗糟废水资源化处理过程中小球藻生物量增加量为0.3 - 0.6 g/l,几种主要污染物最高去除率为:CODcr,52.4 %;BOD5,24.7 %;总氮,30.0 %;总磷,9.8 %,这表明异养小球藻能去除啤酒洗糟废水中的部分污染物,但培养工艺有待进一步优化。（3）在培养基初始PO₄^3--P浓度为0 - 284.5 mg/l之间的变量试验条件下,初始葡萄糖浓度为10 g/l时,异养培养条件下小球藻生长对C/P的需求范围是206/1 - 2060/1,单位生物量的磷吸收量（P/B）的范围是0.8 - 8.1 mg/g;混养培养条件下对C/P的需求范围是103/1 - 2060/1,P/B的范围是0.8 - 16.1 mg/g;初始葡萄糖浓度为40 g/l时,异养和混养培养条件下,小球藻对C/P的需求范围都是206/1 - 2060/1,P/B的范围都是0.8 - 8.1 mg/g;自养培养条件下,小球藻P/B的范围是0.8 - 30.0 mg/g。温度、溶氧、pH、接种量都影响小球藻生长,但不影响P/B。试验结果表明,小球藻能耐受高浓度的磷酸盐且生长良好。Basal培养基中PO₄^3--P浓度为284.5 mg/l,采取自养、混养和异养培养模式培养小球藻时,该值都超出小球藻所能吸收利用的最大磷量;初始葡萄糖浓度为10 g/l时,异养培养小球藻时Basal培养基中的PO₄^3--P超出量最高达62.5倍,培养结束时至少有84.0 %的磷酸盐残留在培养废液中,若直接排放到环境中,将是发生富营养化的隐患。（4）在培养基初始PO₄^3--P浓度为0 - 284.5 mg/l之间的变量试验条件下培养小球藻,通过离子色谱法检测小球藻细胞内总磷含量,葡萄糖浓度为10 g/l时,异养培养条件下,细胞内总磷含量为1.0 - 10.0 mg/g,小球藻能吸收的最大磷量是满足生长所需最低磷量的10倍;混养培养条件下,细胞内总磷含量为1.0 - 20.0 mg/g,小球藻能吸收的最大磷量是满足生长所需最低磷量的20倍。小球藻细胞中多余的磷一般是以多聚磷的形式储存在细胞内。通过荧光显微镜和透射电镜观察异养培养小球藻多聚磷形成状况,发现多聚磷主要分布在液泡中以及细胞壁和细胞膜的间隙。将含多聚磷的小球藻细胞转移到无磷培养基中继续培养,小球藻可以利用胞内储存的多聚磷继续生长,直到细胞内总磷含量为1.0 mg/g时,生长基本停止。（5）在培养基初始NO3--N浓度范围为0 - 311.9 mg/l之间的变量试验条件下,初始葡萄糖浓度为10 g/l时,异养培养条件下小球藻生长对C/N的需求范围是20/1 - 44/1,单位生物量的氮吸收量（N/B）的范围是17.6 - 37.1 mg/g;混养培养条件下的C/N范围是18/1 - 44/1,N/B的范围是17.6 - 43.3 mg/g;自养培养条件下,小球藻N/B的范围是17.6 - 75.0 mg/g。Basal培养基中NO3--N浓度为173.3 mg/l,异养和混养培养条件下,减少44.0 %硝酸盐用量可以获得相同的生物量;自养培养模式下,该值超出小球藻所能吸收利用最大氮量（75.0 mg/l）2.3倍。在满足小球藻生长的氮量范围内,小球藻叶绿素（包括叶绿素a和叶绿素b）含量随着硝酸氮含量的增加而增加;但若硝酸氮含量超出小球藻所能吸收利用的最大氮量,叶绿素含量不再增加。在满足小球藻生长的PO₄^3--P浓度范围内,PO₄^3--P含量对小球藻叶绿素含量没有影响。更多还原

【Abstract】 In Basal medium, phosphorus is the major nutrients for the cultivation of Chlorella. Phosphorus concentration and the ratio of C/P in this medium have direct effects on the growth of Chlorella and the synthesis of metabolites The phosphorus contents will become a limiting factor for the growth of Chlorella if they are less than that needed, while eutrophication will be caused if their contents excess. Beer wastewater generally contains nontoxic materials such as N, P, and they can cause eutrophication if it was discharged directly. On the other hand, the cultivation of Chlorella consumes a large amount of pure water. Therefore, the combination of these two processes was proposed, and the main results are shown as follows.（1） Five Chlorella strains were screened on Basal medium containing 10 g/l glucose, it was shown that Chlorella pyrenoidosa 15-2070 and Chlorella vulgaris 15-2075 were suitable for heterotrophic mass cultivation according to the growth parameters. The maximum biomass was 5.3 g/l and 5.2 g/l, their maximum biomass appeared at the 3rd day, the specific growth rate was 0.98 /d and 0.96 /d, the cell growth yield on glucose was 0.54 g/g and 0.52 g/g for C. pyrenoidosa and C. vulgaris, respectively.（2） Beer wastewater was used for the cultivation of C. pyrenoidosa. The mixed beer wastewater was used to completely replace distilled water for the preparation of Basal medium containing 10 g/l glucose, on which Chlorella growth had not been restrained and 5.3 g/l biomass was achieved in this medium. It was shown that the mixed beer wastewater could be used as a major component for the heterotrophic of Chlorella. In addition, with appropriate preprocessing to the mixed wastewater, it was shown that the highest removal efficiencies were CODcr, 92.2 %; BOD5, 95.1 %; NO3--N, 98.5 %; NH4+-N, 92.3 % were achieved during the process. For the saccharifying process of wastewater, with appropriate preprocessing to the saccharifying process of wastewater, the increased biomass were 0.3 - 0.6 g/l, and it was shown that the highest removal efficiencies were CODcr,52.4 %;BOD5,24.7 %;total-N,30.0 %;total-P,9.8 %, during the growth of Chlorella, it can deplete some nutriment, but culture and treatment processing need to be optimized further。（3） The effects of various initial PO₄^3--P concentrations from 0 to 284.5 mg/l were investigated on the growth of Chlorella. The results indicated that optimal ratios of carbon-to-phosphorous （C/P） were 206/1 - 2060/1, the cellular assimilation of phosphorus per Biomass Unit （P/B） were 0.8 - 8.1 mg/g for heterotrophic cultivation, and C/P were 103/1 - 2060/1, P/B were 0.8 - 16.1 mg/g for mixotrophic cultivation of C. pyrenoidosa with initial 10 g/l glucose; while C/P were 206/1 - 2060/1, P/B were 0.8 - 8.1 mg/g for both heterotrophic and mixotrophic cultivation with initial 40 g/l glucose; and P/B were 0.8 - 30.0 mg/g for autotrophic cultivation. Temperature, dissolved oxygen, pH and inocula have only effects on the growth of Chlorella, whereas no effects on P/B. Chlorella could be tolerant to high concentrations of phosphate and grows well. The PO₄^3--P concentration in Basal medium was 284.5 mg/l, and this value was in excess of the maximum phosphorus concentration that C. pyrenoidosa absorbed under heterotrophic, mixotrophic and autotrophic cultivation. Thus, the concentration of phosphorus was 62.5-fold higher than that of required for the heterotrophic cultivation with 10 g/l glucose, and there were at least 84.0 % phosphate remained at the end of cultivation.（4） The total cellular phosphorus （TP） was determined using Ion Chromatography when the medium containing various initial PO₄^3--P concentrations from 0 to 284.5 mg/l. TP were at the range of from 1.0 to 10.0 mg/g, the maximum absorbed phosphorus was 10-fold higher than the lowest concentration phosphorus of that required for growth under heterotrophic cultivation; and TP were at the range from 1.0 to 20.0 mg/g, the maximum absorbed phosphorus was 20-fold higher than the lowest concentration phosphorus of that required for growth under mixotrophic cultivation with initial 10 g/l glucose. The excessive phosphorus beyond the required of growth were commonly stored in the cells in the form of polyphosphate. The distribution and accumulation of polyphosphate were investigated by fluorescent microscopy and transmission electron microscope（TEM）. The polyphosphate granules were observed in vacuole, and the clearance between cell wall and cell membrane. Chlorella cells containing polyphosphate grew in phosphate-free Basal medium, and the total cellular phosphorus content was 1.0 mg/g at the end of the cultivation.（5） The effects of various initial NO3--N concentrations from 0 to 311.9 mg/l on Chlorella growth were investigated. The results showed that the optimal ratios of carbon-to-nitrogen （C/N） were 20/1 - 44/1, the cellular assimilation of nitrogen （N/B） were 17.6 - 37.1 mg/g for heterotrophic cultivation; and C/N were 18/1 - 44/1, N/B were 17.6 - 43.3 mg/g for mixotrophic cultivation of C. pyrenoidosa with initial 10 g/l glucose; while N/B were 17.6 - 75.0 mg/g for autotrophic cultivation. When the NO3--N concentration of Basal medium was 173.3 mg/l, the reduction on 44.0 % NO3--N amount was able to obtain the same biomass for heterotrophic and mixotrophic cultivation, meanwhile 173.3 mg/l was 2.3-fold higher than the maximum nitrogen concentration that C. pyrenoidosa absorbed for the autotrophic cultivation. When nitrogen concentration was in the range that Chlorella absorbed, chlorophyll （including a and b） content increased with more NO3--N concentration in the medium. Chlorophyll content did not increase if nitrogen concentration was above the maximum absorbed amounts. Phosphorus concentration in the range enough for Chlorella growth had no effect on the chlorophyll content.更多还原