| 摘要 | 近年来，基因工程蓝藻产乙醇作为第三代光直接生物合成燃料乙醇的生产技术受到越来越多的关注。本研究以产乙醇基因工程集胞藻PCC6803 Syn-ZG25为研究对象，首先对影响基因工程集胞藻生长和乙醇产量的因素进行了较系统的研究与优化；随后在不同形式反应器中进行逐级放大培养，比较集胞藻的生长和乙醇产量变化；对培养过程中的微生物杂菌污染进行了初步评价，分离并鉴定出几株乙醇消耗菌，并考察了其对乙醇的消耗特性。获得的具体研究结果如下：（1）考察了微量元素溶液、温度、磷浓度、接种浓度对集胞藻生长和乙醇产量的影响，结果显示，0-2倍范围内的A5微量元素溶液（以BG11中A5浓度为1）不会对产乙醇基因工程集胞藻的生长和乙醇产量产生显著影响。集胞藻最适生长温度为30 ℃，它无法在低温下正常生长和合成乙醇，短期高温培养尚能适应，但长期高温对集胞藻造成伤害，对乙醇生产不利。磷的添加能显著促进基因工程集胞藻的生长，从成本核算角度考虑，以添加24.15 mg/L的磷为宜。接种浓度越高，藻细胞生长速度越慢，最佳接种浓度为OD730=2.0。（2）盐胁迫能显著提高基因工程集胞藻的乙醇产量。随培养液中 NaCl浓度提高，藻生长速率降低；盐胁迫损伤细胞光反应中心II 的活性，抑制细胞的光合作用。随盐浓度提高，集胞藻的内源性代谢产物乙醇产量显著提高，在 20 g/L NaCl 中培养，乙醇产量较对照提高 91.8%。 在盐胁迫条件下，基因工程集胞藻通过调节光合作用和呼吸作用效率、提高乙醇脱氢酶的活性以应对胁迫，同时提高乙醇产量。（3）气泡柱式反应器、平板式反应器、封闭式跑道池中培养的产乙醇基因工程集胞藻均生长良好，但是，随着培养规模的扩大，集胞藻的生长速度下降。培养两周后，0.2 L气泡柱式反应器和2.0 L平板式反应器中乙醇产量无明显差别，但55 L封闭式跑道池反应器的培养液中几乎无乙醇积累，这可能与其培养液中污染的乙醇消耗菌有关。（4）在放大培养过程中，培养液中易发生微生物杂菌污染导致培养后期的乙醇消耗。从染菌的集胞藻培养液中分离出四株能够代谢乙醇的菌，经鉴定分别为红酵母、季也蒙酵母、短波单胞菌和微杆菌。其中乙醇消耗能力最强的是红酵母，乙醇比消耗速率达到391 g/(1015 cfu·d)；其次为季也蒙酵母，乙醇比消耗速率为80.1 g/(1015 cfu·d)；短波单胞菌和微杆菌的乙醇比消耗速率远低于红酵母和季也蒙酵母。将分离出的菌株与产乙醇集胞藻共培养7 d后，污染红酵母、季也蒙酵母、短波单胞菌、微杆菌的实验组乙醇产量分别下降了53.8%、23.6%、40.7%、27.3%。当培养液中污染少量乙醇消耗菌或污染的杂菌乙醇消耗速率较低时，培养液中乙醇积累开始减缓；而当污染的杂菌量较大或污染乙醇消耗速率较快的杂菌时，培养液中乙醇浓度迅速下降。四株菌对基因工程集胞藻的生长无明显影响，均可通过直接消耗乙醇而导致产乙醇基因工程集胞藻乙醇产量降低。; In recent years, more and more concerns had been paid on producing ethanol by genetically engineered cyanobacteria, which could convert CO2 to ethanol directly by photosynthesis. In this research, we have systematically studied and optimized the factors influencing cell growth and ethanol production of genetically engineered ethanol producing cyanobacteria Synechocystis PCC6803 Syn-ZG25. Then we cultivated Syn-ZG25 in different kind of bioreactors, in which growth conditions and ethanol productions of Syn-ZG25 were detected and compared. Four ethanol consuming strains were isolated and identified from the contaminated culture medium and their ethanol consuming capability was evaluated.The detailed results are summarized as follows:First, the effects of microelements, temperature, concentration of phosphorus, initial cell concentration were evaluated. The results showed that concentration of microelements varying from 0 to 2 folds (concentration of microelements in BG11 were seted as 1 fold) had no significant effect on growth and ethanol production of Synechocystis PCC6803 Syn-ZG25. The most suitable growth temperature of Syn-ZG25 was 30 ℃. Temperature lower than 15 ℃ could completely inhibit algae growth and ethanol production completely, and temperature higher than 40 ℃may had negative effect on algae cell. High concentration of phosphorus could increase the growth rate of Syn-ZG25, but not efficient when more than 24.15 mg/L. Higher initial cell concentration could lead to lower growth rate, but more biomass, the most suiltable initial concentration may be OD730=2.0.Second, salt stress could increase the ethanol production of Syn-ZG25 significantly. The cell growth rate, photosystem II (PS II) activity and photosynthesic rate decreased with increasing of salt concentration. For Syn-ZG25 growing in BG11 with more than 10 g/L NaCl, dark respiration rate increased. With increasing of salt concentration, catabolism of endogenous carbohydrate increased, and ethanol excretion rate increased accordingly. The ethanol yield of Syn-ZG25 growing in 20 g/L NaCl was increased substantially by 91.8% to that in the control medium without NaCl. These results indicated that the Synechocystis sp. PCC 6803 Syn-ZG25 could regulate photosynthesis and respiration rates, presumably enhance catabolism of endogenous carbohydrate by enhancing activity of alcohol dehydrogenase, as the response to salt stress, and then resulting in the increase of ethanol yield.Third, Synechocystis sp. PCC 6803 Syn-ZG25 was cultivated in bubble column photobioreactor (0.2 L), flat-panel photobioreactor (2.0 L), closed raceway pond (55 L). In all three photoreactors, Syn-ZG25 could grow well. With increase of the bioreactor’s volume, the algae growth rate decreased. The ethanol productions in bubble column photobioreactor and flat-panel photobioreactor were almost the same after two weeks’ cultivation. There was nothing ethanol accumulated in the closed raceway pond, which might be result from contaminated microorganisms.Fourth, microorganism contaminations which could consume ethanol offen occur in the end of cultivation. Four ethanol consuming microorganisms were isolated from contaminated culture of genetically engineered Synechocystis sp. PCC6803 Syn-ZG25. The ethanol consuming capabilities and impacts on ethanol production of infectious microorganimsms were evaluated. Based on their morphological characteristics, 16S rDNA gene sequence and 26S rDNA gene sequence, the strains GR13, GW13, GT17, GY22 were identified as Rhodotorula sp., Meyerozyma guilliermondii, Brevundimonas sp. and Microbacterium sp. respectively. Rhodotorula sp.had the highest specific ethanol consumption rate, reach to 391 g/(1015 cfu·d). That number for Meyerozyma guilliermondii was 80.1 g/(1015 cfu·d). By contrast, the ethanol consuming capabilities of Brevundimonas sp. and Microbacterium sp. were much smaller. After co-cultuered with ethanol consuming microorganisms for 7 d in the BG11-sp medium, ethanol productions of the genetically engineered Synechocystis sp. contaminated by Rhodotorula sp., Meyerozyma guilliermondii, Brevundimonas sp.and Microbacterium sp. were decreased by 53.8%, 23.6%, 40.7%, 27.3% respectively. These four microorganisms had insignificant influences on growth of Synechocystis sp., yet they significantly decreased the ethanol production by consuming ethanol directly. |
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