高压CO_2相态下乙醇发酵及酵母菌结构变化的研究
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摘要
压缩或超临界流体作为良好的萃取溶剂,在食品加工与保藏、制药和生物材料加工等领域有广阔的应用前景。而超临界二氧化碳又具有无毒、不易燃、易获得和临界点温度温和等优点,使其在萃取发酵生物产品时具有明显的优势。
高压二氧化碳对乙醇发酵有不利影响,乙醇的产量随处理压力的升高而大幅度降低,在1MPa时对不同pH值条件下的乙醇产量进行分析,发现在pH值为8时乙醇的产量达到最大,而在4MPa的压力条件下乙醇的产量几乎为零。针对这种情况,采取了加大接种量浓度为原来的4-6倍,乙醇的产量有一定程度的提高,但当压力达到临界点以上时,乙醇的产量也只有常压发酵的6%左右。对各种不同压力条件下处理后菌的存活率分析发现在1MPa时有部分菌失活,到6MPa时菌几乎全部失活,对这些样品常压培养24h后,显微镜测量发现菌又恢复常压发酵时的存活率。
在不同的压力条件下,对发酵液进行短时间30分钟的高压处理后进行常压发酵24h其乙醇产量接近常压发酵的产量。在相同条件下同时对超临界压力(7.5MPa)下不同pH发酵液的乙醇产量进行研究发现其产量与常压发酵产量(4.82%)相差很小,基本与初始pH值无关。
扫描电镜、原子力显微镜及视差扫描量热分析仪分别对常压和高压(1、6、8MPa)条件下处理的酵母菌的结构进行了分析。扫描电镜是对菌细胞的表面结构进行分析的,其结果非常明显:常压的菌细胞非常饱满、表面光滑并呈圆形或椭圆形,随着压力的升高,菌细胞表面逐渐变得粗糙并且形状变得细长,到8MPa压力的处理条件时,菌细胞表面出现了明显的褶皱,并且有少量菌发生破裂。原子力显微镜只是对常压发酵后菌的结构进行了检测,其结果表现为三维的立体结构与二维的平面信息分析图相结合,为以后的研究提供了一个新的视角。热分析仪分别对这四种条件下得到的菌进行了分析,发现高压处理后菌的内部结构发生了明显的变化,对结果进行初步分析认为细胞膜、蛋白质、及细胞内的脂类等物质发生了变性。
Compressed or supercritical fluids are excellent extraction solvents. They havegot wide applications in food processing and storage, medicine making, as well asbiomaterial processing. As an environmentally benign solvent, supercritical carbondioxide possesses many advantages due to its low toxicity, nonflammability, highavailability and ambient critical temperature. It could be used as an alternative toreplace traditional solvents in the extraction of many fermentation products.
High-pressure carbon dioxide has the disadvantage effect on the ethanolproduction. With the pressure increase the ethanol concentration decreased veryquickly. At 1MPa pressure, when the pH value is 8 the ethanol concentration is thelargest. And when using the common inoculation amount (10% v/v) of yeasts, theethanol concentration is almost zero at 4MPa pressure. In order to solve this problem,the larger inoculation amount (4-6 times concentration of the common inoculationamount) was used. The ethanol production has been raised in some extent, but abovethe supercritical pressure the ethanol conenntration is also very low, only about 6% ofthat at the atmospheric pressure fermentation. The viability of yeast cells underdifferent pressure conditions was also investigated. We found that there were a part ofcells inactivated under 1MPa pressure disposed, but almost all of the cells inactivatedunder a higher pressure of 6MPa condition, and when the samples was cultivated for24h under atmospheric pressure nearly all of the inactivated cells became activated.
The ethanol production under different pressure conditions, 30 min short timedisposed and to ferment under atmospheric pressure for 24h is nearly equal to theconcentration of atmospheric fermentation. On the same disposed time, under asupercritical pressure (7.5MPa), we found that the ethanol concentration is also nearlyequal to the atmospheric fermentation and almost no concern to the pH value.
We also use the SEM (Scanning electron microscopy) AFM (atomic forcemicroscopy) and DSC (differential scanning calorimetry) to evaluate the structurechange of Saccharomyces cerevisiae cells in ethanol fermentation broth underatmospheric, 1, 6, and 8MPa pressure environment. SEM was used to detect thesurface changes of the yeast cells, and the results revealed that the cells in theatmospheric fermentation broth were smooth and full and cells figure were almostround and ellipse. But the coarseness and wrinkle formation on the cell surface and
became severe with pressure increase, when the pressure was 8MPa, this becamemore severe and there were a little amount of cells broken. AFM was used to detectthe structure changes of yeast cells in the atmospheric pressure fermentation broth, theresults represented a new way: the three-dimensional stereograph combined with theplanar ichnography of signal analysis. It gave us a new perspective for study of cellsstructure. DSC was used to evaluate the inner substance changes of yeast cells in thefour different fermentation broths, the results were analyzed, and it could beconcluded that cells membrane, protein, and the lipid in the cells had obviousdenaturation.
高压二氧化碳对乙醇发酵有不利影响,乙醇的产量随处理压力的升高而大幅度降低,在1MPa时对不同pH值条件下的乙醇产量进行分析,发现在pH值为8时乙醇的产量达到最大,而在4MPa的压力条件下乙醇的产量几乎为零。针对这种情况,采取了加大接种量浓度为原来的4-6倍,乙醇的产量有一定程度的提高,但当压力达到临界点以上时,乙醇的产量也只有常压发酵的6%左右。对各种不同压力条件下处理后菌的存活率分析发现在1MPa时有部分菌失活,到6MPa时菌几乎全部失活,对这些样品常压培养24h后,显微镜测量发现菌又恢复常压发酵时的存活率。
在不同的压力条件下,对发酵液进行短时间30分钟的高压处理后进行常压发酵24h其乙醇产量接近常压发酵的产量。在相同条件下同时对超临界压力(7.5MPa)下不同pH发酵液的乙醇产量进行研究发现其产量与常压发酵产量(4.82%)相差很小,基本与初始pH值无关。
扫描电镜、原子力显微镜及视差扫描量热分析仪分别对常压和高压(1、6、8MPa)条件下处理的酵母菌的结构进行了分析。扫描电镜是对菌细胞的表面结构进行分析的,其结果非常明显:常压的菌细胞非常饱满、表面光滑并呈圆形或椭圆形,随着压力的升高,菌细胞表面逐渐变得粗糙并且形状变得细长,到8MPa压力的处理条件时,菌细胞表面出现了明显的褶皱,并且有少量菌发生破裂。原子力显微镜只是对常压发酵后菌的结构进行了检测,其结果表现为三维的立体结构与二维的平面信息分析图相结合,为以后的研究提供了一个新的视角。热分析仪分别对这四种条件下得到的菌进行了分析,发现高压处理后菌的内部结构发生了明显的变化,对结果进行初步分析认为细胞膜、蛋白质、及细胞内的脂类等物质发生了变性。
Compressed or supercritical fluids are excellent extraction solvents. They havegot wide applications in food processing and storage, medicine making, as well asbiomaterial processing. As an environmentally benign solvent, supercritical carbondioxide possesses many advantages due to its low toxicity, nonflammability, highavailability and ambient critical temperature. It could be used as an alternative toreplace traditional solvents in the extraction of many fermentation products.
High-pressure carbon dioxide has the disadvantage effect on the ethanolproduction. With the pressure increase the ethanol concentration decreased veryquickly. At 1MPa pressure, when the pH value is 8 the ethanol concentration is thelargest. And when using the common inoculation amount (10% v/v) of yeasts, theethanol concentration is almost zero at 4MPa pressure. In order to solve this problem,the larger inoculation amount (4-6 times concentration of the common inoculationamount) was used. The ethanol production has been raised in some extent, but abovethe supercritical pressure the ethanol conenntration is also very low, only about 6% ofthat at the atmospheric pressure fermentation. The viability of yeast cells underdifferent pressure conditions was also investigated. We found that there were a part ofcells inactivated under 1MPa pressure disposed, but almost all of the cells inactivatedunder a higher pressure of 6MPa condition, and when the samples was cultivated for24h under atmospheric pressure nearly all of the inactivated cells became activated.
The ethanol production under different pressure conditions, 30 min short timedisposed and to ferment under atmospheric pressure for 24h is nearly equal to theconcentration of atmospheric fermentation. On the same disposed time, under asupercritical pressure (7.5MPa), we found that the ethanol concentration is also nearlyequal to the atmospheric fermentation and almost no concern to the pH value.
We also use the SEM (Scanning electron microscopy) AFM (atomic forcemicroscopy) and DSC (differential scanning calorimetry) to evaluate the structurechange of Saccharomyces cerevisiae cells in ethanol fermentation broth underatmospheric, 1, 6, and 8MPa pressure environment. SEM was used to detect thesurface changes of the yeast cells, and the results revealed that the cells in theatmospheric fermentation broth were smooth and full and cells figure were almostround and ellipse. But the coarseness and wrinkle formation on the cell surface and
became severe with pressure increase, when the pressure was 8MPa, this becamemore severe and there were a little amount of cells broken. AFM was used to detectthe structure changes of yeast cells in the atmospheric pressure fermentation broth, theresults represented a new way: the three-dimensional stereograph combined with theplanar ichnography of signal analysis. It gave us a new perspective for study of cellsstructure. DSC was used to evaluate the inner substance changes of yeast cells in thefour different fermentation broths, the results were analyzed, and it could beconcluded that cells membrane, protein, and the lipid in the cells had obviousdenaturation.
引文
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