荒漠土壤石油降解菌多样性、生物学特性及低温降解机制

发布时间：2018-06-03 13:34

本文选题：荒漠土壤 + 石油污染　；参考：《兰州理工大学》2017年博士论文

【摘要】：石油开发和使用过程中造成的环境污染问题日益明显,严重威胁人类健康。利用微生物降解来处理石油污染是环保有效、经济实用的方法之一。论文针对西北荒漠地区石油污染问题,进行污染土壤微生物多样性分析,筛选出了高效石油降解菌,并进行降解菌生物学特性、降解相关基因和降解机制研究,将高效石油降解菌应用于含油废水生物处理,主要研究内容如下:采用Illumina Miseq高通量测序分析了玉门石油污染荒漠土壤微生物多样性,发现石油污染荒漠土壤中细菌类群丰富,具有明显多样性,包括33门,48纲,78目,179科和471属。主要优势细菌类群为厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)和梭杆菌门(Fusobacteria)。优势菌属包括芽孢杆菌属(Bacillus)、乳球菌属(Lactococcus)、海洋杆菌属(Oceanobacillus)、肠球菌属(Enterococcus)、柠檬酸杆菌属(Citrobacter)、类芽孢杆菌(Paenibacillus)、链球菌属(Streptococcus)、普氏菌属(Prevotella)、假单胞菌属(Pseudomonas)、迪茨氏菌属(Dietzia)、类诺卡氏菌属(Nocardioides)、节细菌属(Arthrobacter)、链霉菌属(Streptomyces)、奈瑟氏菌属(Neisseria)、韦永氏球菌属(Veillonella)、微小杆菌属(Exiguobacterium)、纤毛菌属(Leptotrichia)、嗜血肝菌属(Haemophilus)、梭菌属(Fusobacterium)、罗氏菌属(Rothia)和不动杆菌属(Acinetobacter)等,其中包括了大部分常见的石油降解菌属。采用富集培养和涂布平板法从石油污染土壤分离出能利用石油生长的细菌37株,结合细菌形态和16S r RNA序列分析发现分离的37株细菌分别属于放线菌门(Actinobacteria)、γ变形菌纲(Gammaproteobacteria)、β变形菌纲(Betaproteobacteria)、芽孢杆菌门(Bacilli)和α变形菌纲(Alphaproteobacteria),归属于21属的34种。优势菌属为假单胞菌属(Pseudomonas)、红球菌属(Rhodococcus)、微球菌属(Micrococcus)、寡养单胞菌属(Stenotrophomonas)、无色杆菌属(Achromobacter)和葡萄球菌属(Staphylococcus),占分离细菌总数的51.35%,有36株细菌对原油有明显降解能力。在原油含量为1500 mg/L的基础培养基中培养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%。根据常见石油降解菌降解相关基因设计了特异性引物,利用特异性PCR扩增,检测了37株菌的降解相关基因,结果表明25株细菌含有烷烃单加氧酶基因,6株含芳烃双加氧酶基因,6株菌含联苯双加氧酶基因,4株菌含萘双加氧酶基因,3株菌含甲苯双加氧酶基因,2株菌含邻苯二酚双加氧酶基因。并成功克隆出2种烷烃单加氧酶和1种芳烃双加氧酶基因。对4株具有高效降解能力的菌株KB1、2182、JC3-47和1217进行了生物学特性及石油降解能力的分析。经细菌形态、生理生化及16S r DNA序列分析,鉴定为红平红球菌(Rhodococcus erythropolis)、马红球菌(Rhodococcus equi)、庆笙红球菌(Rhodococcus qingshengii)和铜绿假单胞菌(Pseudomonas aeruginosa)。KB1、2182和JC3-47在温度10~50℃、p H 3~9、0~5.0%Na Cl盐度下生长良好,其中KB1和2182的最适生长温度为35℃,JC3-47的最适生长温度为30℃,KB1和2182还可在p H 2和9.0%盐度(w/v)的极端条件下生长,菌株1217生长适应性更强,在温度5~65℃、pH 2~10、0~9.0%NaCl盐度下均可生长,最适生长温度、p H和盐度分别为35℃、p H 9和0%。4株菌能在以十二烷、十八烷、苯、甲苯、二甲苯和萘为唯一碳源培养基生长,对中链及长链烷烃都具有较强降解能力,其中KB1、2182和1217对十六烷具有较强适应性,KB1和JC3-47还能在含芘培养基生长,1217能在邻苯二酚的培养基中生长,均能产生表面活性剂,对十六烷具有一定粘附能力。研究了红平红球菌KB1在低温条件下的生长特性和石油降解情况,结果表明菌株KB1在总烷烃浓度为10500 mg/L的基础培养基中10℃培养时能较好生长,第5 d菌体数量达到最大值,GC-MS分析发现对总烷烃降解率为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%。进行了红平红球菌KB1在10℃培养时降解混合烷烃的转录组分析,发现与30℃相比,表达差异明显的基因有2957条,其中上调基因1584条,下调基因1373条。当Log2比值5,上调基因99条,下调基因119条,其中33条基因存在于不同细胞组分中,108条具有分子功能,95条参与了生物学过程。结合Go功能富集和KEGG通路分析,发现石油烃降解相关差异表达基因201条,包括脂肪酸、芳香化合物、甲烷、萘、二甲苯、乙苯、甲苯和多环芳烃降解途径中的差异表达基因,其中上调基因135条,下调基因66条,当以Log2比值5为基准,参与脂肪酸、芳香化合物、甲烷、萘和乙苯降解的13条基因上调显著,有3条基因下调明显。采用间歇式活性污泥处理工艺,研究了添加红平红球菌KB1在不同温度下对石油废水的处理效果,发现处理20 d后,在10℃和30℃条件下添加KB1的对照组及实验组出水水质的COD分别为58.50、49.32、55.68和45.73 mg/L,NH4+-N含量为7.78、5.52、7.64和6.01 mg/L,TP含量为0.187、0.164、0.232和0.196 mg/L,均达到石油炼制工业污染物直接排放要求(GB 31570-2015)。在10℃时实验组出水中原油含量为4.27 mg/L,石油去除率为97.40%,不添加KB1的对照组出水中原油含量为18.75 mg/L,石油去除率为88.58%。30℃条件下实验组出水中原油含量为3.17mg/L,石油去除率为98.07%,而对照组出水中原油含量为15.38 mg/L,去除率为90.63%。添加石油降解菌对原油去除有明显促进作用,在低温条件下仍然有明显去除效果。以改性和未改性花生壳为吸附原料,研究了其对废水重金属离子的吸附作用,发现改性花生壳对废水中重金属吸附效果更明显,其吸附率在一定范围内与溶液p H、吸附时间、初始离子浓度和吸附剂量呈正相关。未改性花生壳在酸性条件下吸附率较高,而改性花生壳在碱性条件下吸附率更高,吸附15 min后达最大吸附率的88%,在50 min时吸附达到最大,吸附过程符合Lagergren的二级化学反应动力学模型。
[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.
【学位授予单位】：兰州理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X172

【参考文献】