木薯酒精废糟培养小球藻工艺及灵芝生物转化小球藻的研究
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摘要
本论文以节能环保、清洁发酵、提高经济效益为目的,研究了木薯酒精废水培养蛋白核小球藻,二次废水回用酒精生产的废水处理工艺,研究了各项工艺条件和参数。为了提高小球藻的应用价值,增加废水处理的效益,研究用灵芝对小球藻进行生物转化,采用动物体内抗肿瘤试验,评价了转化发酵液的药效,并对发酵液中可能的增效成分进行了研究。
在250ml三角瓶中,用浓度为10-100%的木薯酒精废水培养钝顶螺旋藻、牟氏角毛藻、鱼腥藻、斜生栅藻和蛋白核小球藻。结果表明最适宜用此废水培养的微藻是蛋白核小球藻。用不加水稀释的木薯酒精废水培养该藻时,最适条件是初始pH6.0、温度27℃、光强3000 lux和光生物反应器中培养基循环速率110 ml min-1。
在最适条件下用管式光生物反应器分别进行了间歇培养,分割培养和连续培养工艺的研究。在间歇培养(pH6.0)的对数期,流加pH自然的废水(pH3.8)进行连续培养,当D=0.17 day-1时,反应器能达到平衡,并维持较好的稳定达30天以上。pH、COD去除率和藻浓度分别稳定在6.22-6.47, 72.21-76.32%和3.55-3.73 g l-1范围内,废水中乳酸、醋酸、柠檬酸和琥珀酸的去除率分别为96.42%、100%、95.89%和39.44%,并能收获具有高附加值的藻细胞。此工艺解决了废水回用酒精生产使用碱调节pH和废水中物质浓度高的问题,处理后的废水可以直接回用到酒精生产中去。
在灵芝基础培养基中添加不同浓度的小球藻粉,进行灵芝发酵,最适宜的添加量为10 g l-1,灵芝多糖等生物活性成分都有不同程度的增加。对比分析小球藻醇提物、灵芝基础培养基发酵液醇提物、灵芝生物转化小球藻发酵液中醇提物,组成成分的种类和含量的变化说明灵芝能对小球藻进行生物破壁并进行生物转化。生物转化发酵液以剂量为13 ml kg-1 d-1时,对小鼠肝癌的抑瘤效果最佳,达67.70%,与灵芝基础培养基发酵液(41.61%)和基础培养基发酵液配相同浓度小球藻水提液的抑瘤率(43.48%)相比,差异显著,体现了1+1>2的效果。
灵芝生物转化小球藻发酵液中的转化多糖能显著提高荷瘤小鼠免疫学功能,从而显著提高抑瘤效果,在最适剂量(0.2 g kg-1d-1)时,抑瘤率为76.2%。不但明显高于灵芝基础培养基发酵液粗多糖,明显高于小球藻粗多糖,而且明显高于基础培养基所产多糖和小球藻多糖两者的叠加效应。发酵液中多糖含量的增加和转化多糖抗肿瘤效果的提高是灵芝生物转化小球藻发酵液抗肿瘤效果提高的原因之一。
对灵芝生物转化小球藻发酵液中醇提物进行分析,保留时间为25.778min的组分增加量较高,经醇提、正丁醇萃取、大孔树脂柱分离、C-18柱精制,并经UV、LC/MS和IR图谱分析,是一种黄酮类化合物。黄酮类化合物通常具有抗肿瘤和其它生理活性功能,它的增加应该也是导致灵芝生物转化小球藻发酵液抗肿瘤效果提高的原因之一。
The dissertation was focused on the technics of using wastewater from cassava ethanol production to culture Chlorella pyrenoidosa, the determination of operation conditions and technological parameters, aimed to recycle the treated wastewater in ethanol fermentation to save energy, realize depurated fermentation, enhance economical benefit. In order to increase the applied value of C. pyrenoidosa, through anti-tumor experiment in vivo, the medicine effect of bioconversion ferment liquid was estimated after C. pyrenoidosa was bio-converted by Ganoderma lucidum. And the increased components of ferment liquid were also studied.
After Spirulina platensis, Chactoceros muelleri, Anabaena sp., Scenedesmus obliquus and C. pyrenoidosa were cultured in 250-ml conical flasks, with wastewater from cassava ethanol production of different concentration (from 10 to 100%), C. pyrenoidosa was the optimum algae cultured in the undiluted wastewater through selection. The results showed that the optimum cultivation conditions were initial pH of 6.0, temperature at 27oC,continuous illumination at 3000 lux and cycle speed of 110 ml min-1 in a cycle tubular photobioreactor, when the undiluted wastewater was used to culture the algae..
Batch, fed-batch and continuous culture was operated and researched in the cycle tubular photobioreactor under above optimum conditions. After the logarithmic phase of batch operation with wastewater of pH 6.0, the reactor could be continuously operated with pH natural (3.8) wastewater as feed solution. By a dilution ratio of 0.17 day-1, it could be operated stably for over 30 days in continuous operation. pH, removal rate of chemical oxygen demand and biomass (cell dried weight) concentration ranged from 6.22 to 6.47, 72.21 to 76.32 % and 3.55 to 3.73 g l-1, respectively. The removal rate of lactic acid, acetic acid, citric acid and succin acid in the wastewater were 96.42 %, 100 %, 95.89% and 39.44 %, respectively. And the algae which have high value were harvested. After treatment, the wastewater could be used again in the process of ethanol fermentation.
C. pyrenoidosa powder was appended by different concentration into the basic medium of G. lucidum, the G. lucidum fermentation was studied. The optimum concentration was 10 g l-1, and the output of polysaccharides and other bioactivity matters in ferment liquid was enhanced significantly. The ethanol extraction from C. pyrenoidosa, from ferment liquid of G. lucidum in basic madium, from ferment liquid of bioconverted C. pyrenoidosa by G. lucidum were contrasted and analyzed by HPLC. The changes of component kinds and their concentration illustrated that G. lucidum could bio-broke the cell wall of C. pyrenoidosa and bioconversion was achieved. The high dose group (13 ml kg-1d-1) of bioconversion ferment liquid had the highest tumor inhibition (67.70%) which was higher than that of ferment liquid with basic media(41.61%) and that of mixture liquid(43.48%)contained ferment liquid with basic media and water extraction of C. pyrenoidosa at same concentration, significantly. The result showed 1+1>2.
The bioconverted polysaccharides had the best activity of anti-tumor and immunological function. The anti-tumor inhibition was 76.2% at the optimum dose (0.2 g kg-1d-1). The polysaccharides from ferment liquid of bioconverted C. pyrenoidosa by G. lucidum had higher medicine effect not only than polysaccharides from ferment liquid of G. lucidum in basic madium, from C. pyrenoidosa, but also than the mixed polysaccharides. The increase of bioconversion polysaccharides in ferment liquid and the increase of its tumor inhibition was one of the reasons which resulted in the increase of anti-tumor of bioconversion ferment liquid.
The increase of component whose retention time was 25.778 min was high. The component might play an important role and advanced action in the bioactivity of conversation ferment liquid, was separated from the crude butanol extraction of culture liquid and purified by macroporous resin HPD-400 and octadecyl silane column. It was likely conjectured as a flavone according to UV, HPLC/MS and IR. Flavone compound usually had anti-tumor and other bioactivity. The increase of flavone in ferment liquid was also one of the reasons which resulted in the increase of anti-tumor of bioconversion ferment liquid.
在250ml三角瓶中,用浓度为10-100%的木薯酒精废水培养钝顶螺旋藻、牟氏角毛藻、鱼腥藻、斜生栅藻和蛋白核小球藻。结果表明最适宜用此废水培养的微藻是蛋白核小球藻。用不加水稀释的木薯酒精废水培养该藻时,最适条件是初始pH6.0、温度27℃、光强3000 lux和光生物反应器中培养基循环速率110 ml min-1。
在最适条件下用管式光生物反应器分别进行了间歇培养,分割培养和连续培养工艺的研究。在间歇培养(pH6.0)的对数期,流加pH自然的废水(pH3.8)进行连续培养,当D=0.17 day-1时,反应器能达到平衡,并维持较好的稳定达30天以上。pH、COD去除率和藻浓度分别稳定在6.22-6.47, 72.21-76.32%和3.55-3.73 g l-1范围内,废水中乳酸、醋酸、柠檬酸和琥珀酸的去除率分别为96.42%、100%、95.89%和39.44%,并能收获具有高附加值的藻细胞。此工艺解决了废水回用酒精生产使用碱调节pH和废水中物质浓度高的问题,处理后的废水可以直接回用到酒精生产中去。
在灵芝基础培养基中添加不同浓度的小球藻粉,进行灵芝发酵,最适宜的添加量为10 g l-1,灵芝多糖等生物活性成分都有不同程度的增加。对比分析小球藻醇提物、灵芝基础培养基发酵液醇提物、灵芝生物转化小球藻发酵液中醇提物,组成成分的种类和含量的变化说明灵芝能对小球藻进行生物破壁并进行生物转化。生物转化发酵液以剂量为13 ml kg-1 d-1时,对小鼠肝癌的抑瘤效果最佳,达67.70%,与灵芝基础培养基发酵液(41.61%)和基础培养基发酵液配相同浓度小球藻水提液的抑瘤率(43.48%)相比,差异显著,体现了1+1>2的效果。
灵芝生物转化小球藻发酵液中的转化多糖能显著提高荷瘤小鼠免疫学功能,从而显著提高抑瘤效果,在最适剂量(0.2 g kg-1d-1)时,抑瘤率为76.2%。不但明显高于灵芝基础培养基发酵液粗多糖,明显高于小球藻粗多糖,而且明显高于基础培养基所产多糖和小球藻多糖两者的叠加效应。发酵液中多糖含量的增加和转化多糖抗肿瘤效果的提高是灵芝生物转化小球藻发酵液抗肿瘤效果提高的原因之一。
对灵芝生物转化小球藻发酵液中醇提物进行分析,保留时间为25.778min的组分增加量较高,经醇提、正丁醇萃取、大孔树脂柱分离、C-18柱精制,并经UV、LC/MS和IR图谱分析,是一种黄酮类化合物。黄酮类化合物通常具有抗肿瘤和其它生理活性功能,它的增加应该也是导致灵芝生物转化小球藻发酵液抗肿瘤效果提高的原因之一。
The dissertation was focused on the technics of using wastewater from cassava ethanol production to culture Chlorella pyrenoidosa, the determination of operation conditions and technological parameters, aimed to recycle the treated wastewater in ethanol fermentation to save energy, realize depurated fermentation, enhance economical benefit. In order to increase the applied value of C. pyrenoidosa, through anti-tumor experiment in vivo, the medicine effect of bioconversion ferment liquid was estimated after C. pyrenoidosa was bio-converted by Ganoderma lucidum. And the increased components of ferment liquid were also studied.
After Spirulina platensis, Chactoceros muelleri, Anabaena sp., Scenedesmus obliquus and C. pyrenoidosa were cultured in 250-ml conical flasks, with wastewater from cassava ethanol production of different concentration (from 10 to 100%), C. pyrenoidosa was the optimum algae cultured in the undiluted wastewater through selection. The results showed that the optimum cultivation conditions were initial pH of 6.0, temperature at 27oC,continuous illumination at 3000 lux and cycle speed of 110 ml min-1 in a cycle tubular photobioreactor, when the undiluted wastewater was used to culture the algae..
Batch, fed-batch and continuous culture was operated and researched in the cycle tubular photobioreactor under above optimum conditions. After the logarithmic phase of batch operation with wastewater of pH 6.0, the reactor could be continuously operated with pH natural (3.8) wastewater as feed solution. By a dilution ratio of 0.17 day-1, it could be operated stably for over 30 days in continuous operation. pH, removal rate of chemical oxygen demand and biomass (cell dried weight) concentration ranged from 6.22 to 6.47, 72.21 to 76.32 % and 3.55 to 3.73 g l-1, respectively. The removal rate of lactic acid, acetic acid, citric acid and succin acid in the wastewater were 96.42 %, 100 %, 95.89% and 39.44 %, respectively. And the algae which have high value were harvested. After treatment, the wastewater could be used again in the process of ethanol fermentation.
C. pyrenoidosa powder was appended by different concentration into the basic medium of G. lucidum, the G. lucidum fermentation was studied. The optimum concentration was 10 g l-1, and the output of polysaccharides and other bioactivity matters in ferment liquid was enhanced significantly. The ethanol extraction from C. pyrenoidosa, from ferment liquid of G. lucidum in basic madium, from ferment liquid of bioconverted C. pyrenoidosa by G. lucidum were contrasted and analyzed by HPLC. The changes of component kinds and their concentration illustrated that G. lucidum could bio-broke the cell wall of C. pyrenoidosa and bioconversion was achieved. The high dose group (13 ml kg-1d-1) of bioconversion ferment liquid had the highest tumor inhibition (67.70%) which was higher than that of ferment liquid with basic media(41.61%) and that of mixture liquid(43.48%)contained ferment liquid with basic media and water extraction of C. pyrenoidosa at same concentration, significantly. The result showed 1+1>2.
The bioconverted polysaccharides had the best activity of anti-tumor and immunological function. The anti-tumor inhibition was 76.2% at the optimum dose (0.2 g kg-1d-1). The polysaccharides from ferment liquid of bioconverted C. pyrenoidosa by G. lucidum had higher medicine effect not only than polysaccharides from ferment liquid of G. lucidum in basic madium, from C. pyrenoidosa, but also than the mixed polysaccharides. The increase of bioconversion polysaccharides in ferment liquid and the increase of its tumor inhibition was one of the reasons which resulted in the increase of anti-tumor of bioconversion ferment liquid.
The increase of component whose retention time was 25.778 min was high. The component might play an important role and advanced action in the bioactivity of conversation ferment liquid, was separated from the crude butanol extraction of culture liquid and purified by macroporous resin HPD-400 and octadecyl silane column. It was likely conjectured as a flavone according to UV, HPLC/MS and IR. Flavone compound usually had anti-tumor and other bioactivity. The increase of flavone in ferment liquid was also one of the reasons which resulted in the increase of anti-tumor of bioconversion ferment liquid.
引文
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