本文選題：荒漠土壤 + 石油污染��； 參考：《蘭州理工大學(xué)》2017年博士論文

【摘要】：石油開發(fā)和使用過程中造成的環(huán)境污染問題日益明顯,嚴(yán)重威脅人類健康。利用微生物降解來處理石油污染是環(huán)保有效、經(jīng)濟(jì)實(shí)用的方法之一。論文針對西北荒漠地區(qū)石油污染問題,進(jìn)行污染土壤微生物多樣性分析,篩選出了高效石油降解菌,并進(jìn)行降解菌生物學(xué)特性、降解相關(guān)基因和降解機(jī)制研究,將高效石油降解菌應(yīng)用于含油廢水生物處理,主要研究內(nèi)容如下:采用Illumina Miseq高通量測序分析了玉門石油污染荒漠土壤微生物多樣性,發(fā)現(xiàn)石油污染荒漠土壤中細(xì)菌類群豐富,具有明顯多樣性,包括33門,48綱,78目,179科和471屬。主要優(yōu)勢細(xì)菌類群為厚壁菌門(Firmicutes)、變形菌門(Proteobacteria)、擬桿菌門(Bacteroidetes)、放線菌門(Actinobacteria)、綠彎菌門(Chloroflexi)和梭桿菌門(Fusobacteria)。優(yōu)勢菌屬包括芽孢桿菌屬(Bacillus)、乳球菌屬(Lactococcus)、海洋桿菌屬(Oceanobacillus)、腸球菌屬(Enterococcus)、檸檬酸桿菌屬(Citrobacter)、類芽孢桿菌(Paenibacillus)、鏈球菌屬(Streptococcus)、普氏菌屬(Prevotella)、假單胞菌屬(Pseudomonas)、迪茨氏菌屬(Dietzia)、類諾卡氏菌屬(Nocardioides)、節(jié)細(xì)菌屬(Arthrobacter)、鏈霉菌屬(Streptomyces)、奈瑟氏菌屬(Neisseria)、韋永氏球菌屬(Veillonella)、微小桿菌屬(Exiguobacterium)、纖毛菌屬(Leptotrichia)、嗜血肝菌屬(Haemophilus)、梭菌屬(Fusobacterium)、羅氏菌屬(Rothia)和不動桿菌屬(Acinetobacter)等,其中包括了大部分常見的石油降解菌屬。采用富集培養(yǎng)和涂布平板法從石油污染土壤分離出能利用石油生長的細(xì)菌37株,結(jié)合細(xì)菌形態(tài)和16S r RNA序列分析發(fā)現(xiàn)分離的37株細(xì)菌分別屬于放線菌門(Actinobacteria)、γ變形菌綱(Gammaproteobacteria)、β變形菌綱(Betaproteobacteria)、芽孢桿菌門(Bacilli)和α變形菌綱(Alphaproteobacteria),歸屬于21屬的34種。優(yōu)勢菌屬為假單胞菌屬(Pseudomonas)、紅球菌屬(Rhodococcus)、微球菌屬(Micrococcus)、寡養(yǎng)單胞菌屬(Stenotrophomonas)、無色桿菌屬(Achromobacter)和葡萄球菌屬(Staphylococcus),占分離細(xì)菌總數(shù)的51.35%,有36株細(xì)菌對原油有明顯降解能力。在原油含量為1500 mg/L的基礎(chǔ)培養(yǎng)基中培養(yǎng)7 d,8株菌的降解率不低于30.55%,11株菌降解率介于10.05%~28.37%,18株菌降解率不高于8.05%。根據(jù)常見石油降解菌降解相關(guān)基因設(shè)計(jì)了特異性引物,利用特異性PCR擴(kuò)增,檢測了37株菌的降解相關(guān)基因,結(jié)果表明25株細(xì)菌含有烷烴單加氧酶基因,6株含芳烴雙加氧酶基因,6株菌含聯(lián)苯雙加氧酶基因,4株菌含萘雙加氧酶基因,3株菌含甲苯雙加氧酶基因,2株菌含鄰苯二酚雙加氧酶基因。并成功克隆出2種烷烴單加氧酶和1種芳烴雙加氧酶基因。對4株具有高效降解能力的菌株KB1、2182、JC3-47和1217進(jìn)行了生物學(xué)特性及石油降解能力的分析。經(jīng)細(xì)菌形態(tài)、生理生化及16S r DNA序列分析,鑒定為紅平紅球菌(Rhodococcus erythropolis)、馬紅球菌(Rhodococcus equi)、慶笙紅球菌(Rhodococcus qingshengii)和銅綠假單胞菌(Pseudomonas aeruginosa)。KB1、2182和JC3-47在溫度10~50℃、p H 3~9、0~5.0%Na Cl鹽度下生長良好,其中KB1和2182的最適生長溫度為35℃,JC3-47的最適生長溫度為30℃,KB1和2182還可在p H 2和9.0%鹽度(w/v)的極端條件下生長,菌株1217生長適應(yīng)性更強(qiáng),在溫度5~65℃、pH 2~10、0~9.0%NaCl鹽度下均可生長,最適生長溫度、p H和鹽度分別為35℃、p H 9和0%。4株菌能在以十二烷、十八烷、苯、甲苯、二甲苯和萘為唯一碳源培養(yǎng)基生長,對中鏈及長鏈烷烴都具有較強(qiáng)降解能力,其中KB1、2182和1217對十六烷具有較強(qiáng)適應(yīng)性,KB1和JC3-47還能在含芘培養(yǎng)基生長,1217能在鄰苯二酚的培養(yǎng)基中生長,均能產(chǎn)生表面活性劑,對十六烷具有一定粘附能力。研究了紅平紅球菌KB1在低溫條件下的生長特性和石油降解情況,結(jié)果表明菌株KB1在總烷烴濃度為10500 mg/L的基礎(chǔ)培養(yǎng)基中10℃培養(yǎng)時(shí)能較好生長,第5 d菌體數(shù)量達(dá)到最大值,GC-MS分析發(fā)現(xiàn)對總烷烴降解率為64.55%,其中正辛烷29.27%,正癸烷46.25%,正十二烷89.13%,正十四烷77.59%,正十六烷70.35%,正十八烷55.16%,正二十二烷57.38%,正二十四烷67.18%和二十八烷82.64%。進(jìn)行了紅平紅球菌KB1在10℃培養(yǎng)時(shí)降解混合烷烴的轉(zhuǎn)錄組分析,發(fā)現(xiàn)與30℃相比,表達(dá)差異明顯的基因有2957條,其中上調(diào)基因1584條,下調(diào)基因1373條。當(dāng)Log2比值5,上調(diào)基因99條,下調(diào)基因119條,其中33條基因存在于不同細(xì)胞組分中,108條具有分子功能,95條參與了生物學(xué)過程。結(jié)合Go功能富集和KEGG通路分析,發(fā)現(xiàn)石油烴降解相關(guān)差異表達(dá)基因201條,包括脂肪酸、芳香化合物、甲烷、萘、二甲苯、乙苯、甲苯和多環(huán)芳烴降解途徑中的差異表達(dá)基因,其中上調(diào)基因135條,下調(diào)基因66條,當(dāng)以Log2比值5為基準(zhǔn),參與脂肪酸、芳香化合物、甲烷、萘和乙苯降解的13條基因上調(diào)顯著,有3條基因下調(diào)明顯。采用間歇式活性污泥處理工藝,研究了添加紅平紅球菌KB1在不同溫度下對石油廢水的處理效果,發(fā)現(xiàn)處理20 d后,在10℃和30℃條件下添加KB1的對照組及實(shí)驗(yàn)組出水水質(zhì)的COD分別為58.50、49.32、55.68和45.73 mg/L,NH4+-N含量為7.78、5.52、7.64和6.01 mg/L,TP含量為0.187、0.164、0.232和0.196 mg/L,均達(dá)到石油煉制工業(yè)污染物直接排放要求(GB 31570-2015)。在10℃時(shí)實(shí)驗(yàn)組出水中原油含量為4.27 mg/L,石油去除率為97.40%,不添加KB1的對照組出水中原油含量為18.75 mg/L,石油去除率為88.58%。30℃條件下實(shí)驗(yàn)組出水中原油含量為3.17mg/L,石油去除率為98.07%,而對照組出水中原油含量為15.38 mg/L,去除率為90.63%。添加石油降解菌對原油去除有明顯促進(jìn)作用,在低溫條件下仍然有明顯去除效果。以改性和未改性花生殼為吸附原料,研究了其對廢水重金屬離子的吸附作用,發(fā)現(xiàn)改性花生殼對廢水中重金屬吸附效果更明顯,其吸附率在一定范圍內(nèi)與溶液p H、吸附時(shí)間、初始離子濃度和吸附劑量呈正相關(guān)。未改性花生殼在酸性條件下吸附率較高,而改性花生殼在堿性條件下吸附率更高,吸附15 min后達(dá)最大吸附率的88%,在50 min時(shí)吸附達(dá)到最大,吸附過程符合Lagergren的二級化學(xué)反應(yīng)動力學(xué)模型。
[Abstract]:The environmental pollution caused by oil development and use is becoming more and more obvious, which seriously threatens human health. The use of microbial degradation to treat oil pollution is one of the effective and economical methods. The paper analyzes the microbial diversity of contaminated soil in the northwest desert area, and selects the efficient oil. Biodegradation bacteria, biological characteristics of degrading bacteria, degradation related genes and degradation mechanisms, high efficiency petroleum degrading bacteria are applied to the biological treatment of oily wastewater. The main contents are as follows: Illumina Miseq high throughput sequencing was used to analyze the microbiological diversity of Yumen oil polluted desert soil and to discover bacteria in the oil polluted desert soil The group is rich and diverse, including 33 doors, 48 classes, 78 orders, 179 families and 471 genera. The dominant bacterial groups are Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Fusobacteria. The dominant bacteria include Bacillus sp. Genus (Bacillus), Lactococcus, marine bacilli (Oceanobacillus), Enterococcus (Enterococcus), citric acid (Citrobacter), Bacillus cereus (Paenibacillus), Streptococcus (Streptococcus), pricococcus (Prevotella), Pseudomonas (Pseudomonas), Dietz's (Dietzia), Nocardia (Nocardioide) S), Streptomyces (Arthrobacter), Streptomyces (Streptomyces), Neisseria (Neisseria), Neisseria genus (Veillonella), microbacter (Exiguobacterium), ciliated genus (Leptotrichia), Haemophilus Haemophilus (Haemophilus), Clostridium (Fusobacterium), rosiella (Rothia), and Acinetobacter (Acinetobacter), etc. Most common petroleum degrading bacteria were isolated from petroleum contaminated soil by enrichment culture and coated plate method, 37 strains of bacteria that could be used for oil growth were isolated, combined with bacterial morphology and 16S R RNA sequence analysis, the 37 strains isolated were classified as actinomycetes (Actinobacteria), gamma deforma (Gammaproteobacteria), and beta deformia Betaproteobacteria, Bacilli and Alphaproteobacteria, belonging to 34 species of 21 genera. The dominant genus are Pseudomonas (Pseudomonas), red coccyx (Rhodococcus), Micrococcus (Micrococcus), oligotrophomonas (Stenotrophomonas), acrobacterium (Achromobacter) and Staphylococcus (Staphyl). Ococcus), accounting for 51.35% of the total number of isolated bacteria, 36 bacteria have obvious degradation ability to crude oil. 7 d is cultivated in the base medium with 1500 mg/L crude oil content, 8 strains are less than 30.55%, 11 strains are 10.05%~28.37%, and the degradation rate of 18 strains is not higher than that of the common petroleum degrading bacteria. Specific primers were used to detect the degradation related genes of 37 strains of bacteria by specific PCR amplification. The results showed that 25 strains contained alkane monooxygenase genes, 6 aromatic dioxygenase genes, 6 strains containing biphenyl dioxygenase gene, 4 bacteria containing naphthalene dioxygenase gene, 3 bacteria containing toluene dioxygenase gene, and 2 bacteria containing catechol. 2 alkane monooxygenase and 1 aromatics bioxygenase genes were cloned successfully. The biological characteristics and oil degradation ability of 4 strains with high degradation ability KB12182, JC3-47 and 1217 were analyzed. By bacterial morphology, physiological and biochemical and 16S R DNA sequence analysis, it was identified as erythroping erythrococcus (Rhodococcus erythro). Polis), Rhodococcus equi, Rhodococcus qingshengii (Rhodococcus qingshengii) and Pseudomonas aeruginosa (Pseudomonas aeruginosa).KB12182 and JC3-47 grow well at the temperature 10~50 C, P H, and the optimum growth temperature is 35 degrees C, and the optimum growth temperature is 30, and 2182 is also available. Under the extreme conditions of P H 2 and 9% salinity (w/v), strain 1217 grows more adaptable, can grow at 5~65 C, pH 2~10,0~9.0%NaCl salinity, optimum growth temperature, P H and salinity at 35, P H 9 and 0%.4 strains can grow with twelve alkanes, eighteen alkanes, benzene, toluene, xylene and naphthalene as the sole carbon source. And long chain alkanes have strong degradation ability, of which KB12182 and 1217 have strong adaptability to sixteen alkanes. KB1 and JC3-47 can grow in pyrene medium, 1217 can grow in the culture medium of catechol. All of them can produce surfactant and have certain adhesion energy to sixteen alkanes. The KB1 of erythropycia Rhodococcus is studied under low temperature conditions. The growth characteristics and petroleum degradation showed that strain KB1 could grow well at 10 C in the base medium with total alkane concentration of 10500 mg/L, and the number of fifth D bacteria reached the maximum. GC-MS analysis found that the total alkane degradation rate was 64.55%, including n-octane 29.27%, n-decane 46.25%, twelve alkane 89.13%, and fourteen alkane 77.59%. Sixteen alkanes 70.35%, positive eighteen alkane 55.16%, positive twenty-two alkane 57.38%, positive twenty-four alkanes 67.18% and twenty-eight alkanes 82.64%. were used to degrade mixed alkanes at 10 C at 10 C. It was found that there are 2957 genes with significant difference in expression compared with 30 C, which up-regulated gene 1584 and lowered gene 1373. When Log2 ratio is the ratio. 5, 99 and 119 down regulated genes were up-regulated, of which 33 genes were found in different cell components, 108 had molecular functions and 95 were involved in biological processes. 201 of the differential expression genes related to petroleum hydrocarbon degradation, including fatty acids, aromatic compounds, methane, naphthalene, dimethylbenzene, ethylbenzene, and methylene, were found in 108 of these genes and 95 were involved in the biological process. The differentially expressed genes in the degradation pathways of benzene and polycyclic aromatic hydrocarbons, in which 135 genes were up-regulated and 66 were down regulated, when the 13 genes involved in fatty acids, aromatic compounds, methane, naphthalene and ethylbenzene were up regulated by Log2 ratio 5, and 3 genes were down regulated obviously. The batch activated sludge treatment process was used to study the addition of red flat red. The treatment effect of KB1 at different temperatures on oil wastewater was found. After 20 D treatment, the COD of the control group adding KB1 at 10 and 30 centigrade was 58.50,49.32,55.68 and 45.73 mg/L respectively, NH4+-N content was 7.78,5.52,7.64 and 6.01 mg/L, TP content was 0.187,0.164,0.232 and 0.196 mg/L, all reached petroleum refining. The direct emission requirements of industrial pollutants (GB 31570-2015). The crude oil content in the experimental group was 4.27 mg/L at 10 C, the oil removal rate was 97.40%, the crude oil content in the control group without KB1 was 18.75 mg/L, and the oil removal rate was 3.17mg/L and the oil removal rate was 98.07% under the condition of 88.58%.30. The content of crude oil in the water is 15.38 mg/L, and the removal rate of 90.63%. adding petroleum degrading bacteria has obvious promotion effect on the removal of crude oil, and still has obvious removal effect at low temperature. The adsorption of heavy metal ions on wastewater is studied with modified and unmodified peanut shell as the adsorption material, and it is found that the modified peanut shell is heavy in the wastewater. The adsorption efficiency of metals is more obvious. The adsorption rate is positively correlated with the P H, the adsorption time, the initial ion concentration and the adsorption dose. The unmodified peanut shell has a higher adsorption rate under the acidic condition, and the modified peanut shell has a higher adsorption rate under the alkaline condition. The adsorption rate is 88% of the maximum adsorption rate after 15 min, and the adsorption is reached at 50 min. The adsorption process accords with the two level chemical reaction kinetics model of Lagergren.
【學(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：X172

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 車曉軍;屈撐囤;郭敏俊;;含油污泥堆肥處理的研究[J];廣州化工;2016年12期

2 王春;姜巖;岳希權(quán);;原油污染土壤生物修復(fù)研究進(jìn)展[J];應(yīng)用化工;2015年12期

3 劉文超;李曉森;劉永民;張占民;;石油污染土壤修復(fù)技術(shù)應(yīng)用現(xiàn)狀[J];油田化學(xué);2015年02期

4 劉志培;劉雙江;;我國污染土壤生物修復(fù)技術(shù)的發(fā)展及現(xiàn)狀[J];生物工程學(xué)報(bào);2015年06期

5 張勤;孫永明;;海上溢油圍油欄研究現(xiàn)狀[J];綠色科技;2015年04期

6 張紅巖;房蕾;王e，

本文編號：1972963