深海和南极产油真菌筛

发布时间：2018-07-25 10:04

【摘要】：本论文的内容主要是研究大洋与南极样品的真菌多样性、采用尼罗红与氯化三苯基四氮唑(TTC)方法进行产油真菌的初筛、采用气相色谱方法对产油菌的脂肪酸成分进行分析及研究培养基成份对产油菌产油发酵的影响。主要结果如下:对中太平洋(6个站位)、南大西洋(2个站位)及西太平洋(22个站位)共30个站位的沉积物进行分离鉴定,一个获得75株真菌,通过ITS鉴定结果显示,这75株菌属于14个属,它们分别是:Aspergillus sp.(27株)、Penicillium sp.(21株)、Cladosporium sp.(8株)、Fusarium sp.(6株)、Alternaria(2株)、Exophiala sp.(2株)、Engyodontium sp.(2株)、Acremonium sp.(1株)、Nectria sp.(1株)、Tritirachium sp.(1株)、Aureobasidium sp.(1株)、Thielavia sp.(1株)、Lecythophora sp.(1株)、Sarocladium sp.(1株)。三个海区中菌株数量最多的为曲霉属的真菌,可见曲霉属真菌在这三个海区中为优势属,其次为青霉属。在太平洋中,枝孢属的真菌也有一定的分布。其余属均只有几株,表明在三个大洋中分布较少。同时分别使用尼罗红和TTC对大洋样品中分离得到的369株真菌进行产油初筛,一共获得61株产油菌,分别属于10个属:Aspergillus(21株)、Fusarium(13株)、Cladosporium(10株)、Penicillium(9株)、Engyodontium(2株)、Sarocladium(2株)、Alternaria(1株)、Aureobasidium(1株)、Lecythophora(1株)、Nectria(1株)。产油菌所占比例为18.70%。其中曲霉属菌株占产油菌比例最高,为30.43%,其次为镰刀属菌株、枝孢属菌株与青霉素菌株,分别占总产油菌属的18.84%、14.49%与13.04%。对中国第29次南极科学考察获得的南极普里兹湾海域23个站位的深海沉积物样品进行了真菌分离培养,共获得了11个属的21株真菌。分别是:Penicillium(9株)、Cladosporium(3株)、Acremonium(1株)、Aureobasidium(1株)、Botryotinia(1株)、Eurotiales(1株)、Exophiala(1株)、Fusarium(1株)、Nectria(1株)、Phoma(1株)、Sarocladium(1株)。南极普里兹湾海域的真菌多样性较高,其中青霉属菌株最多,占总菌株比例为42.85%,是普里兹湾真菌中的优势属,其次为枝孢属菌株,占总菌株比例为14.29%。分别用尼罗红和TTC对南极普里兹湾海域分离得到的21株真菌进行产油初筛,共获得8株产油菌,分别属于7个属:Penicillium(2株)、Aureobasidium(1株)、Cladosporium(1株)、Eurotiales(1株)、Exophiala(1株)、Fusarium(1株)、Sarocladium(1株)。产油菌占总菌株的比例为38%。对分离得到的产油菌进行脂肪酸分析,结果表明:这些产油菌所含的基本为C16-C18脂肪酸,不饱和脂肪酸含量较高,脂肪酸中具有较长的碳直链且没有碳支链,因此可以作为生物柴油制备的原料油。考虑到油脂生产成本,寻找可利用廉价底物高产油脂的微生物具有深远的意义。为了寻找能够直接利用廉价底料作为碳源产油的真菌菌株,本文使用含有TTC的糖蜜培养基分离南极沉积物样品,获得一株产油菌,为P7-07-M1,属名为:Nectria,该菌株可以直接利用糖蜜作为碳源发酵产油。对从南极产油菌中选择一株产油菌(P7-07-M1),从大洋产油菌中选择两株产油菌(JL7KGC01A-M7与MP4CTD-11-M5)进行培养基优化,结果显示:菌株P7-07-M1达到最高产油量的时间为第8天,最适氮源和最适碳源分别为蛋白胨与葡萄糖,最适C/N比为168,最佳Cu SO4.5H2O浓度为0.0001g/L,最佳Mn SO4.H2O浓度为0.004g/L,通过培养基优化,菌株P7-07-M1产油量提升了27.89%;菌株JL7KGC01A-M7达到最高产油量的时间为第7天,最适氮源和最适碳源分别为蛋白胨与葡萄糖,最适C/N比为140,最佳Cu SO4.5H2O浓度为0.0002g/L,最佳Mn SO4.H2O浓度为0.003g/L,通过培养基优化,菌株JL7KGC01A-M7的产油量提升了30.15%;菌株MP4CTD-11-M5达到最高产油量的时间为第7天,最适氮源和最适碳源分别为蛋白胨与葡萄糖,最适C/N比为140,最佳Cu SO4.5H2O浓度为0.0001g/L,最佳Mn SO4.H2O浓度为0.004g/L,通过培养基优化,菌株MP4CTD-11-M5的产油量提升了111.16%。菌株MP4CTD-11-M5培养基优化效果最显著。
[Abstract]:The main content of this paper is to study the fungal diversity of Oceanic and Antarctic samples. Using Nile red and three phenyl tetrazolium chloride (TTC) method to screen the oil producing fungi, the fatty acid composition of the oil producing bacteria was analyzed by gas chromatography and the influence of the medium on the production of oil producing bacteria was studied. The main results are as follows: The sediments of 30 stations in the central Pacific (6 stations), southern the Atlantic (2 stations) and the Western Pacific (22 stations) were isolated and identified, and 75 fungi were obtained. The results of ITS identification showed that the 75 strains belonged to 14 genera: Aspergillus sp. (27 strains), Penicillium sp. (21 strains), Cladosporium sp. (8 strains), Fusarium sp. (6 strains), Alternaria (2 strains), Exophiala sp. (2 strains), Engyodontium sp. (2 strains), Acremonium sp. (1 strains), Nectria sp. (1 strains), Tritirachium sp. (1 strains), Aureobasidium 1, 1 plants, 1 strains (1 strains). The largest number of strains of the genus Aspergillus in the three sea areas, visible Aspergillus fungi In the three sea areas, the second is Penicillium. In the Pacific, the fungi of the genus cladospora are also distributed. The other genera are only a few, indicating that there are only a few strains in the three oceans. At the same time, 369 strains of fungi isolated from the ocean samples were screened using Nile red and TTC respectively. A total of 61 strains of oil producing bacteria were obtained. Don't belong to 10 genera: Aspergillus (21 strains), Cladosporium (13 strain), Penicillium (10 strain), Penicillium (9 strain), Engyodontium (2 strain), Sarocladium (2 strain), Alternaria (1 strain), Aureobasidium (1 strain), Lecythophora (1 strain), Nectria (1 strain). The proportion of oil producing bacteria is 18.70%., and the proportion of Aspergillus is the highest, 30.43%, and second is sickle. Spore strains, cladospora strains and penicillin were 18.84%, 14.49% and 13.04%., respectively, and 14.49% of the deep-sea sediment samples from 23 stations in the Antarctic Bay of Antarctica were isolated and cultured by 13.04%., and 21 fungi of 11 genera were obtained, respectively: Penicillium (9 strains), Cladospo Rium (3 strains), Acremonium (1 strain), Aureobasidium (1 strain), Botryotinia (1 strain), Eurotiales (1 strain), Exophiala (1 strain), Fusarium (1 strain), Nectria (1 strain), Phoma (1 strain), Sarocladium (1). The fungal diversity in the sea area of Antarctica is higher, among which Penicillium is most, and the proportion of total strains is 42.85%. It is the dominant genera of the Prem Bay fungi. The second was cladospora, and the proportion of total strains was 14.29%., 21 strains of fungi isolated from the sea area of Antarctica were screened with Nile red and TTC respectively, and 8 strains of oil producing bacteria were obtained, which belong to 7 genera: Penicillium (2 strains), Aureobasidium (1 strains), Cladosporium (1 strains), Exophiala (1 strains), Fusarium (1 strains), S, S, S. Arocladium (1 strains). The ratio of the oil producing bacteria to the total strains was 38%. to analyze the fatty acid of the isolated oil producing bacteria. The results showed that the oil producing bacteria contained basically C16-C18 fatty acids, high content of unsaturated fatty acids, long carbon chain and no carbon branching in fatty acids, so they could be used as raw materials for biodiesel. Oil. Considering the cost of oil production, it is of great significance to find the microorganisms which can make use of cheap substrates to produce high yield oil. In order to find a fungal strain which can be used as a carbon source directly using cheap substrate, this paper uses a molasses medium containing TTC to separate Antarctic sediment samples and obtain a strain of P7-07-M1, named Nectria. The strain can be fermented with molasses as a carbon source to produce oil. A strain of oil producing bacteria (P7-07-M1) from the Antarctic oil producing bacteria (P7-07-M1) is selected, and two strains of oil producing bacteria (JL7KGC01A-M7 and MP4CTD-11-M5) are selected from the marine oil producing bacteria to optimize the medium. The results show that the strain P7-07-M1 reaches the maximum oil yield for eighth days, the optimum nitrogen source and the optimum. The optimum C/N ratio was 168, the optimum Cu SO4.5H2O concentration was 0.0001g/L and the optimum Mn SO4.H2O concentration was 0.004g/L. The oil production of strain P7-07-M1 increased by 27.89% by the optimization medium, and seventh days for the strain JL7KGC01A-M7 to reach the highest oil production, and the optimum nitrogen source and the optimum carbon source were peptone and Portuguese. The optimum C/N ratio was 140, the optimum Cu SO4.5H2O concentration was 0.0002g/L, the optimum Mn SO4.H2O concentration was 0.003g/L, the oil production of strain JL7KGC01A-M7 increased by 30.15%, the strain MP4CTD-11-M5 reached the maximum oil production time seventh days, the optimum nitrogen source and the optimum carbon source were peptone and glucose, and the optimum C/N ratio was 140. The optimum Cu SO4.5H2O concentration is 0.0001g/L and the optimum Mn SO4.H2O concentration is 0.004g/L. The optimization of the oil production of the strain MP4CTD-11-M5 improves the optimization effect of the MP4CTD-11-M5 medium of 111.16%. strain by optimizing the medium of the strain MP4CTD-11-M5.
【学位授予单位】：国家海洋局第三海洋研究所
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ929;Q93;TE667

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