發(fā)布時(shí)間：2018-08-24 21:02

【摘要】：隨著海洋石油的不斷開采及遠(yuǎn)洋運(yùn)輸業(yè)的發(fā)展,海洋石油污染越來越突出,給海洋環(huán)境及人類的生存發(fā)展帶來了極大的困擾,同時(shí)也造成了資源的極大浪費(fèi)。治理石油污染勢在必行。與物理和化學(xué)處理方法相比,生物處理技術(shù)由于安全、高效、經(jīng)濟(jì)、無二次污染、不需大型設(shè)備等優(yōu)勢,得到人們的重視。因此,利用微生物處理石油污染已成為當(dāng)今世界的熱點(diǎn)。本文主要研究尋求一種適合海洋的環(huán)境友好型的固定化菌劑來修復(fù)海洋的石油污染,主要包括柴油/原油降解菌的篩選、降解條件的優(yōu)化、固定化載體的篩選、固定化菌群降解原油前后原油組分變化的分析和固定化菌群在實(shí)際海水中石油降解特性分析。得到的主要結(jié)論如下：(1)分別以柴油和原油為唯一碳源,從深圳市蛇口港和鹽田港海域中篩選到50余株高效降解菌。針對柴油和原油,各選擇5株石油降解率較高的菌株,通過多維復(fù)配試驗(yàn)篩選出降解率最高的1種組合,再通過正交試驗(yàn)優(yōu)化各菌的接種量,最終構(gòu)建了由C1-8、C2-10和C3-13組成的高效柴油降解菌群CQ1及由S1-38、S1-30和S2-13組成的高效原油降解菌群SQ1。通過單因素實(shí)驗(yàn)研究了環(huán)境因素對菌群降解柴油/原油的影響。結(jié)果表明：當(dāng)溫度、pH、轉(zhuǎn)速和石油濃度分別為30℃、7.6、220 r/min和20 g/L時(shí),菌群CQ1對柴油的降解率最高可達(dá)到60.5%,CQ1對柴油的降解隨培養(yǎng)時(shí)間的增加有所升高,在培養(yǎng)3-9 d內(nèi)增幅較為明顯,7d時(shí)降解率即達(dá)到60%以上,此后柴油的降解率仍有小幅增長,但變化不大。當(dāng)溫度、pH、轉(zhuǎn)速和原油濃度分別為30℃、7.6、200r/min和20 g/L時(shí),菌群SQ1對原油的降解率可達(dá)73.52%,菌群SQ1對原油的降解率隨培養(yǎng)時(shí)間的增加有所升高。(2)通過生理生化試驗(yàn)及16S rRNA基因序列分析對石油降解菌進(jìn)行了初步鑒定,以柴油為唯一碳源篩選到的菌株主要分布于微桿菌屬(Microbacterium)、假單胞菌屬(Pseudomonas)、棒桿菌屬(Corynebacterium)、蒼白桿菌(Ochrobactrum)以及劍菌屬(Ensifer)等。以原油為唯一碳源篩選到的菌株主要分布于副球菌屬(Paracoccus)、拉布倫茨氏菌屬(Labrenzia)、鞘氨醇盒菌屬(Sphingopyxis)、假單胞菌屬(Pseudomonas)以及迪茨氏菌屬(Dietzia)等。其中,構(gòu)成柴油降解菌群CQ1的3株菌C1-8鑒定為微桿菌(Microbacterium sp.)、C2-10劍菌(Ensifer sp.)C3-13變異棒桿菌(Corynebacterium sp.);構(gòu)成原油降解菌群SQl的3株菌S1-30鑒定為變異棒桿菌(Corynebacterium sp.),S1-38為迪茨氏菌(Dietzia sp.), S2-13為拉布倫茨氏菌(Labrenzia sp.)。(3)通過對最佳載體的篩選、載體的投加量及固定化的時(shí)間的優(yōu)化,初步研究了固定化SQ1對原油的降解特性。結(jié)果表明：秸稈和無紡布是菌群SQ1良好的固定化載體,秸稈和無紡布固定化SQ1對原油的降解率分別達(dá)到76.85%和73.08%,均高于單獨(dú)投加SQl菌液。秸稈固定化SQ1中最佳的載體投加量和固定化時(shí)間分別為15.0g/L(干重)和36 h;無紡布固定化SQ1中最佳的載體投加量和固定化時(shí)間分別為15.0g/L(干重)和36h。2種固定化菌劑對原油的降解效果均隨培養(yǎng)時(shí)間增加而有所升高,第9d時(shí)達(dá)到最高,分別為73.08%和76.85%。(4)通過施加不同的無機(jī)氮源(尿素、KNO3、(NH4) 2SO4、NH4Cl)和磷源(K2HPO3、KH2PO3),研究了固定化SQ1對實(shí)際海水中原油的去除效果及其降解特性。結(jié)果表明：對固定化SQ1(秸稈)降解原油促進(jìn)作用最為明顯的氮源為NH4Cl,磷源為KH2PO3,當(dāng)NH4C1投加量為5.08g/L,KH2PO3投加量為1.14g/L時(shí),原油降解率可達(dá)66.05%,高于不投加營養(yǎng)鹽的處理53.42%。(5)通過GC-FID/GC-MS法分析了柴油或原油生物降解后,各烷烴組分的殘留情況。結(jié)果表明,柴油生物降解體系中,菌群CQl能夠降解大部分C11-C27之間的正構(gòu)烷烴,其中對C21-C27等中鏈烷烴降解率可以達(dá)到100%,CQ1對各烷烴組分的去除情況顯著好于構(gòu)成菌群的單一菌株,表現(xiàn)出明顯的協(xié)同作用。石油降解生物體系中,3株細(xì)菌和固定化SQl都能夠降解C8-C35之間的正構(gòu)烷烴,其中對C20-C24和C26-C35等中鏈、長鏈烷烴降解率可以達(dá)到100%。相比游離菌的SQ1,固定化SQ1對烷烴的降解效果更好,固定化菌群SQl對原油總烷烴的去除率為97.71%。其中,除了Pr、Py和C18,固定化SQ1對其余正烷烴的降解率是100%,顯示出強(qiáng)大的烷烴降解能力。本研究為微生物法去除海洋油污的奠定了良好的理論基礎(chǔ),并提供了有效的菌株資源,其所制備的固定化菌劑在海洋溢油的生物修復(fù)中具備廣闊的應(yīng)用前景。
[Abstract]:With the continuous exploitation of offshore oil and the development of ocean transportation, the pollution of offshore oil is becoming more and more serious, which has caused great trouble to the marine environment and the survival and development of human beings, and also caused a great waste of resources. The advantages of high efficiency, economy, no secondary pollution and no need of large-scale equipment have attracted people's attention. Therefore, the use of microorganisms to treat oil pollution has become a hot spot in the world. The main conclusions are as follows: (1) Using diesel oil and crude oil as the sole carbon source, the crude oil was separately screened from Shekou Port and Yantian Port in Shenzhen. More than 50 strains of high-efficiency degrading bacteria were selected for diesel oil and crude oil. Five strains with high petroleum degrading rate were selected respectively. One combination with the highest degrading rate was screened out by multidimensional compound test. The inoculation quantity of each strain was optimized by orthogonal test. Finally, the high-efficiency diesel degrading bacteria CQ1 composed of C1-8, C2-10 and C3-13 and the groups of S1-38, S1-30 and S2-13 were constructed. The results showed that when the temperature, pH, rotational speed and petroleum concentration were 30, 7.6, 220 r/min and 20 g/L respectively, the highest degradation rate of CQ1 to diesel oil could reach 60.5%, and the degradation rate of CQ1 to diesel oil increased with the culture time. When the temperature, pH, rotational speed and crude oil concentration were 30, 7.6, 200 r/min and 20 g/L respectively, the degradation rate of crude oil by SQ1 could reach 73.52%, and the degradation rate of crude oil by SQ1 could reach 73.52%. (2) Petroleum-degrading bacteria were preliminarily identified by physiological and biochemical tests and 16S rRNA gene sequence analysis. The strains isolated from diesel oil were mainly distributed in Microbacterium, Pseudomonas, Corynebacterium and Ochrobactrum. The strains isolated from crude oil were mainly distributed in Paracoccus, Labrenzia, Sphingopyxis, Pseudomonas and Dietzia. Among them, the three strains of CQ1, C1-8, were identified as micro-organisms. Microbacterium sp., Ensifer sp., Corynebacterium sp. C3-13, three strains of SQl were identified as Corynebacterium sp., S1-38 as Dietzia sp., S2-13 as Labrenzia sp. (3) By screening the best carrier, the carrier was identified as Corynebacterium sp. The results showed that straw and non-woven fabrics were good immobilization carriers for SQ1, and the degradation rates of SQ1 immobilized on straw and non-woven fabrics were 76.85% and 73.08% respectively, which were higher than that of SQl immobilized on straw. The optimum carrier dosage and immobilization time were 15.0 g/L (dry weight) and 36 h, respectively. The optimum carrier dosage and immobilization time were 15.0 g/L (dry weight) and 36 h. The effect of immobilized SQ1 on the removal of crude oil from seawater was studied by applying different inorganic nitrogen sources (urea, KNO3, (NH4) 2SO4, NH4Cl) and phosphorus sources (K2HPO3, KH2PO3). When the dosage of KH2PO3 was 1.14g/L and 8g/L, the degradation rate of crude oil reached 66.05%, which was higher than that of the treatment without nutrients (53.42%). Among them, the degradation rate of C21-C27 and other medium-chain alkanes can reach 100%. The removal of each alkane component by CQ1 is significantly better than that by a single strain, showing obvious synergistic effect. Compared with free bacteria SQ1, immobilized SQ1 had better effect on the degradation of alkanes. The removal rate of total alkanes from crude oil by immobilized SQl was 97.71%. Among them, except Pr, Py and C18, the degradation rate of immobilized SQ1 was 100%, showing a strong ability to degrade alkanes. The immobilized microbial agents have broad application prospects in the bioremediation of marine oil spills.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X172;X55

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本文編號：2202029