【摘要】：本研究利用前期已篩選的4株高效石油降解菌,采用響應(yīng)面法構(gòu)建混合菌并考察環(huán)境因素對(duì)石油降解混合菌降解石油效果的影響,分析石油降解單菌及混合菌降解產(chǎn)物,探討石油烴的降解機(jī)制,并為篩選微生物提供理論基礎(chǔ)。選取小麥秸稈、生物質(zhì)炭和活性炭為吸附載體,通過吸附-包埋法對(duì)混合菌進(jìn)行固定化用于室內(nèi)模擬修復(fù)石油污染土壤,比較不同固定化載體土壤修復(fù)效果和微生物群落結(jié)構(gòu)及分布特征。響應(yīng)面優(yōu)化混合菌配比結(jié)果表明,植生拉烏爾菌(Raoultella planticola)接種量為4%、粘質(zhì)沙雷氏菌(Serratia marcescens)接種量為2%、蠟狀芽孢桿菌(Bacillus cereus)接種量為2%、變棲克雷伯氏菌(Klebsiella variicola)接種量為2%,優(yōu)化后的混合菌對(duì)石油降解率可達(dá)68.83%�；旌暇鷮�(duì)鹽度和p H耐受范圍廣,最佳鹽度0.5%,最佳p H 7.5。同時(shí)發(fā)現(xiàn)混合菌在5%鹽度的條件下,5天后原油的降解率仍可達(dá)到40.37%,可以用于石油污染海水的修復(fù)。在以柴油為唯一碳源,等接種量培養(yǎng)條件下,單菌株對(duì)石油烴的降解率達(dá)到41.83%~54.87%,混合菌降解效率高于單菌株,達(dá)到64.27%。結(jié)合GC-MS分析結(jié)果,R.planticola和S.marcescens對(duì)C12~C16的直鏈烴碳源有降解作用;B.cereus和K.variicola對(duì)C12~C22的直鏈烴均有降解效果。4株單菌都可以在有氧條件下降解正十六烷,脫氫生成雙鍵并引入羰基。B.cereus、K.variicola和混合菌還能進(jìn)一步氧化十六烷生成不飽和羧酸酯并形成帶甲基的支鏈。雙鍵和羰基引入可能是正十六烷有氧條件下前期降解的一種新途徑。采用小麥秸稈、生物質(zhì)炭和活性炭3種載體對(duì)混合菌進(jìn)行吸附包埋固定,結(jié)果表明固定化混合菌比游離態(tài)混合菌對(duì)石油的去除效果顯著提高,可以在相對(duì)短的時(shí)間內(nèi)達(dá)到相對(duì)高的去除率。生物質(zhì)炭固定化混合菌對(duì)原油培養(yǎng)基5d降解效率高達(dá)99.76%。秸稈、生物質(zhì)炭和活性炭空白載體5d原油吸附量分別為49.08%、61.48%、63.12%。充分證明固定化混合菌小球類似于吸附降解一體的微型反應(yīng)器。掃描電鏡圖觀察到生物質(zhì)炭和活性炭小球內(nèi)部具有大量孔隙的骨架結(jié)構(gòu),微生物在載體內(nèi)部生長(zhǎng)良好。固定化混合菌模擬修復(fù)石油污染土壤實(shí)驗(yàn)結(jié)果表明,固定化混合菌對(duì)原油的降解效果比游離菌好,且不同固定化載體修復(fù)效果有差異。經(jīng)過42d的修復(fù),滅菌組中秸稈固定化混合菌修復(fù)效果最好,降解率為54.92%,比單純的游離混合菌提高了20%;未滅菌組中生物質(zhì)炭固定化混合菌修復(fù)效果最好,降解率為54.19%,比游離菌提高了近30%。說明在沒有土著菌群競(jìng)爭(zhēng)情況下,秸稈通過增強(qiáng)土壤養(yǎng)分含量而增加土壤脲酶活性,對(duì)于提高混合菌石油降解率是顯著影響條件;而在土著菌群存在的未滅菌組土壤中,生物質(zhì)炭可以提供固定化混合菌最適宜的生長(zhǎng)環(huán)境是顯著影響條件,比秸稈提高養(yǎng)分對(duì)于修復(fù)土壤更重要。滅菌組秸稈固定化修復(fù)處理后期脲酶活性最高,脲酶活性最大值0.75mg?g-1?24h-1為空白對(duì)照組的3.26倍;未滅菌組生物質(zhì)炭修復(fù)脲酶活性最高為1.15 mg?g-1?24h-1為空白對(duì)照組的2.05倍。土壤脲酶活性與固定化混合菌石油降解率呈正相關(guān)。土壤香儂-威納指數(shù)與微生物多樣性T-RFLP的分析表明,滅菌組固定化混合菌修復(fù)土壤種群多樣性呈遞減趨勢(shì),未滅菌組多樣性先升高再下降;石油降解率和微生物多樣性變化相吻合,石油降解率高的微生物多樣性高。固定化混合菌是由厚壁菌門和變形菌門組成,在石油生物降解過程中起重要作用。在滅菌組和未滅菌組,生物質(zhì)炭都是促進(jìn)厚壁菌門和變形菌門生長(zhǎng)的最優(yōu)載體,使二者總和占土壤比例分別高達(dá)5.37%、3.73%。同時(shí)發(fā)現(xiàn)滅菌條件下,秸稈固定化載體能促進(jìn)芽孢桿菌綱生長(zhǎng),使其占土壤比例高達(dá)1.35%;在未滅菌組,生物質(zhì)炭載體最能促進(jìn)?-變形菌綱生長(zhǎng),使其含量高達(dá)2.71%。對(duì)各樣本的T-RFLP譜圖進(jìn)行聚類分析,結(jié)果發(fā)現(xiàn)滅菌組秸稈和未滅菌組生物質(zhì)炭群落相似度高;生物質(zhì)炭和活性炭群落相似度高;而未滅菌組秸稈固定化土壤與其它5組距離較遠(yuǎn),群落相似度低。
[Abstract]:In this study, four strains of high-efficiency petroleum-degrading bacteria were used to construct the mixed bacteria by response surface methodology. The effects of environmental factors on the petroleum-degrading effect of the mixed bacteria were investigated. The degradation products of the single bacteria and the mixed bacteria were analyzed, and the mechanism of petroleum hydrocarbon degradation was discussed. Straw, biomass charcoal and activated carbon were used as adsorbent carriers to immobilize mixed bacteria for indoor simulated remediation of petroleum-contaminated soil. The effects of different immobilized carriers on soil remediation, microbial community structure and distribution characteristics were compared. Response surface methodology (RSM) was used to optimize the proportion of mixed bacteria. The Inoculated Amount of anticola, Serratia marcescens, Bacillus cereus, Klebsiella variicola and Klebsiella variicola was 4%, 2% and 2%, respectively. The oil degradation rate of the optimized mixed bacteria was 68.83%. The mixed bacteria had wide tolerance to salinity and P H, and the optimum salinity was 0.5% and the best was the highest. At the same time, it was found that under the condition of 5% salinity, the degradation rate of crude oil could still reach 40.37% after 5 days, which could be used for the remediation of oil-polluted seawater. According to the results of GC-MS analysis, R. planticola and S. marcescens could degrade the carbon source of C12-C16, B. cereus and K. variicola could degrade the linear hydrocarbons of C12-C22. All the four strains could degrade n-hexadecane under aerobic conditions, dehydrogenate to form double bonds and introduce carbonyl. B. cereus, K. variicola and mixed bacteria could further oxygen. Hexadecane is converted into unsaturated carboxylic esters and methyl-branched chains are formed. The introduction of double bonds and carbonyl groups may be a new way of pre-degradation of n-hexadecane under aerobic conditions. The removal efficiency of crude oil by immobilized mixed bacteria was 99.76%. The adsorption capacity of crude oil by straw, biomass carbon and activated carbon was 49.08%, 61.48% and 63.12% respectively. Scanning electron microscopy (SEM) showed that there were a lot of pores in the biochar and activated carbon spheres, and microorganisms grew well in the carrier. Immobilized mixed bacteria simulated the remediation of oil-contaminated soil. The results showed that the immobilized mixed bacteria had better degradation effect on crude oil than free bacteria, and there was no significant difference between immobilized mixed bacteria and free bacteria. After 42 days of repair, the straw immobilized mixed bacteria had the best repair effect, the degradation rate was 54.92%, which was 20% higher than that of the pure free mixed bacteria; the biochar immobilized mixed bacteria had the best repair effect, the degradation rate was 54.19%, which was nearly 30% higher than that of the free bacteria. Under the competition of Aboriginal microflora, straw increased soil urease activity by increasing soil nutrient content, which had a significant effect on improving the oil degradation rate of mixed bacteria; while in the unsterilized soil of Aboriginal microflora, biomass char could provide the most suitable growth environment for Immobilized Mixed bacteria, which was significantly affected by straw extraction. The highest urease activity was 0.75 mg? G-1? 24h-1 in the sterilized group, which was 3.26 times higher than that in the blank control group, and 1.15 mg? G-1? 24h-1 in the non-sterilized group, which was 2.05 times higher than that in the blank control group. The Shannon-Wiener index and microbial diversity T-RFLP analysis showed that the biodiversity of the immobilized mixed bacteria in the sterilized group tended to decrease, the biodiversity of the unsterilized group increased first and then decreased, and the biodegradation rate and microbial diversity were consistent, and the biodiversity of the high biodegradation rate was diverse. Immobilized mixed bacteria are composed of thick-walled phylum and deformed phylum, which play an important role in the process of petroleum biodegradation. In both sterilized and non-sterilized groups, biomass char is the best carrier to promote the growth of thick-walled phylum and deformed phylum, making the total proportion of them in soil as high as 5.37% and 3.73% respectively. Straw immobilized carriers could promote the growth of Bacillus spp. and accounted for 1.35% of the soil, while biomass carbon carriers could promote the growth of Proteus spp. and the content of Proteus spp. was as high as 2.71% in the non-sterilized group. The community similarity of charcoal and activated carbon was high, while the immobilized soil of non-sterilized group was far from the other five groups, and the community similarity was low.
【學(xué)位授予單位】：陜西科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X53;X172

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