黄土塬石油污染土壤的降解规律及生物修复优化研究
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摘要
石油类污染物在典型油区的生物降解规律和土壤修复技术的研究已成为国内外环境、生物学领域的热点。针对陇东黄土塬石油污染土壤的区域性和重要性,通过石油污染物在土壤中的吸附行为和自然生物降解规律的试验,选择本源土壤微生物中能够降解石油的菌株,优化构建了降油菌群及其环境适应因子,研究了翻耕、添加锯末、氮磷营养剂强化技术条件对修复过程的作用；通过混合菌中各菌株分子水平的鉴定与同源性分析,提出了不同菌种的保藏条件。主要研究成果如下：
(1)陇东黄土对原油吸附等温线很好地符合Langmuir和线性吸附方程,其吸附系数分别为0.013和0.012ml/g；5种介质吸附原油能力从大到小依次为石英砂、高岭土、黄绵土、高压灭菌土、硅藻土。水相pH、温度和石英砂含量的升高,不利于原油在土壤中的吸附,而盐度会加大原油的吸附量。模拟自然条件下生物降解土壤中的石油类污染物发现：未污染土壤中加0.15%、0.3%、0.5%石油降解的半衰期分别为17.2、18.2和23.7d,生物降解系数分别为0.040、0.038和0.029mg/d；在不同含油浓度下,石油降解率随添加量呈下降趋势,说明石油污染浓度过大会抑制微生物的自然降解效率。
(2)在陇东油田受石油污染的土壤中,以原油为唯一碳源,通过富集分离的方法筛选出13株菌株,各菌株均能在pH值为5-9的范围内生长,但在pH值为8左右的石油培养基里石油降解率最大,可达到95%以上。各菌降解石油表现出先降解高碳数正构烷烃为低碳数正构烷烃,高碳数正构烷烃中奇数碳向偶数碳正构烷烃演化的规律;各菌作用霍烷需要比正构烷烃更高的能量,对芳烃的作用最难,降解率也最低,主要表现形式是对稠环边上的甲基具有去甲基作用,这也是产生天然气的过程,其中F1、A5、D4表现更为突出。混合菌对于正构烷烃的降解率要高于各个单菌的作用；在正交配比优化实验中,假单胞菌属菌(A6)对维持混合菌株的高降解率起到重要的作用。在盐度为8g/1、pH为8、温度在30℃左右以及油浓度为400-600mg/1条件下,混合菌降解率达到最高,且稳定性和适应性比单株菌系好,能更好地适应不同条件下的土壤或水环境。
(3)向土壤中加入混合菌并结合翻耕、添加锯末技术,一方面从细菌数量、水分、氧气等方面改善了土壤的环境,另一方面有利于微生物分解得到的小分子代谢产物的挥发,从而大大加快残油的去除速度。加入混合菌使土壤中细菌含量提高一个数量级的条件下,土壤中石油的半衰期比自然土壤(不加菌)缩短了近4倍。土壤湿度为24.1%时,加入混合菌使土壤中细菌含量达到1.09×108个/kg土,治理5%油污土壤的半衰期是41d,治理62d后,向土壤中加入混合菌并结合翻耕技术的降解率达到了84.64%。添加膨松剂(锯末)提高了土壤的保水和通气能力,而且对于生物修复过程中土壤的脱氢酶活性有明显的促进作用。在每天翻耕的样品中,第62天测量时酶活性已经增长到4.0左右,比不添加膨松剂和翻耕频率较低的土壤高出2倍左右。混合菌剂与有机肥联合作用能够加强石油污染土壤的修复能力,添加2%、4%与8%菌剂和有机肥48天后的降解效率为68.01%、80.42%和78.47%,均大于CK的降解率,四种处理中4%菌剂和有机肥的修复效果最显著。添加的有机肥中氮磷的含量是影响石油降解率的主要因素,只有加适量的有机肥才能使降解效果达到最好,4%的有机肥添加量使降解效果达到最好。
(4)经分子鉴定,菌种A6为铜绿假单胞菌属、D4为蒙氏假单胞菌属、A5为鲁菲不动杆菌属、F1为黄色类诺卡氏菌属、F2为暗黑微绿链霉菌属。各菌种可以冻干粉或不同斜面培养基,于37℃培养24h后置于4℃冰箱备用。
The study of biological degradation and soil remediation technology to the petroleum pollutants in typical region has become focus in the domestic and international environment, biological field. In the light of regional and importance for Longdong Loess Plateau polluted by petroleum, the study of sorption behaviors of crude oil in soil and law of natural biodegradation of petroleum contaminated soil is applied. Then oil-degradation bacterial strain are selected from local microorganisms in soil, the oil-degradation mixed bacterium and adaptal factors has been optimized and constructed; and technology conditions of aggrandizeing bioremediation, such as plowing, added sawdust, nitrogen and phosphorus-nutrient agent are studied on repairing process, the role and influence of these measures are studied. Lastly, the mixed bacteria strains from the molecular level are identificated and analyzed from homology, put forward preservation conditions for every bacteria. The main study results are as follows:
(1) Static experiments for sorption behaviors of crude oil for loess soil in Longdong area are carried out, the adsorption isotherms of crude oil in loess soils are well described by the Langmuir and linear equation. The adsorption coefficients are0.013and0.012mg/1; The adsorption capacity of oil in the five media in turn is:Silica sand, Kaolin, Loessal soil, High pressure sterilized soil, Diatomite; The increase of pH, temperature and silica sand are not favorable to adsorption of crude oil in soil, increasing the salinity can increase adsorption of crude oil. Situ-biodegradation to depredate petroleum pollutants in soil under simulating natural conditions are applied,and find that the half-life of degradations of petroleum oil in no-polluted soil added0.15%,0.3%,0.5%petroleum are17.2,18.2,23.7d, the bio-degradation coefficients are0.0401,0.0388,0.0293mg/d; In different petroleum concentrations, petroleum degradation rate shows trend of decline with increase of petroleum's addition. This performance shows that excessive concentration of petroleum can inhibit microbial natural degradation efficiency.
(2) The soil samples come from Huaqing/XiFeng oil areas of Gansu province which have being polluted by oil for a long time. Using crude oil as the sole carbon source,13strains are screened and isolated with the enrichment and separation method, the strains can all grow up in the pH5-9, and in oil medium (0.5%standard oil), at the pH8, the oil degradation rates reach a maximum (more than95%). The degrading appears that after being treated with various bacteria and the mixed bacterium, firstly, the higher molecular weight n-alkanes are degraded into lower molecular weight n-alkanes, and the n-alkanes in odd-numbered carbon are degraded into n-alkanes in even-numbered carbon. Every bacterium degradating hopane needs higher energy than n-alkanes, Aromatic hydrocarbons are degradated most difficultly, the degradation rate is lowest, mainly form is demethylation effection for the side of methyl on fused ring, which is the process of natural gas. Several bacteria and mixed bacterium all have stronger demethylation effect for methyl on fused ring, among them F1, A5, D4's performance is more outstanding. Mixed bacterium's degradation rate is higher than that of each single bacterium for n-alkanes (except for a positive role:n-tridecane). In process of orthogonal and optimizational experiment, Pseudomonas (A6) plays an important role on maintenance of high degradation rate in mixed strains. The bacteria agent'environmental conditions of crude oil degradation are:salinity is8g/l, pH is8, at30℃,400-600mg/1oil concentration conditions, the mixed strains'degradation rate is highest, and the stability and adaptability are better than individual strains. Therefore, the mixed bacterium has stronger adaptability to different soil and water environment in the practical application.
(3) Joining the mixed bacterium into soil and tillage technology, on the one hand, the soil environment is improved at bacteria, water, oxygen and other aspects; on the other hand tillage is in favor of volatilization of the small molecule metabolite by microbial decomposition, so as to greatly accelerate the speed of removal of residual oil. Under the condition of bacterium content increasing one order of magnitude by adding mixed bacterium into the soil, the half-life reduces nearly4times than the natural soil (without bacteria).When soil moisture is24.1%, the mixed bacterium is added into the soil, bacteria content reachs1.09×10, the half-life of treatment of5%soil polluted by petroleum is41days, after61d, degradation rate reachs84.64%by adding mixed bacterium into soil and tilling technology. Adding leavening agents (sawdust) can increase water retention and ventilation capacity of soil, but also has advantageous to improve dehydrogenase activity in the bioremediation process. In the samples turned every day, add leavening agents, on the sixty-second day, measuring enzyme activity has increased to about4.0, is about2times higher than not added leavening agents. Remediation ability of petroleum contaminated soil with mixed bacterium and organic fertilizer jointly is improved:after48days, under oil contents of50g/kg in the contaminated soil, the oil degradation rate of2%,4%,8%bacterium reach68.01%,80.42%,78.47%, which are clearly improved the biodegrading efficiency higher than CK degradation rate. The4%mixed bacterium's degradation efficiency is most significant. In organic fertilizer, Nitrogen and Phosphorus content is the main factor affected oil degradation rate, only the amount needed to a certain range could have active degradation efficiency, the4%organic fertilizer can achieve the best degradation efficiency.
(4) Through molecular identification, strain A6is Pseudomonas aeruginosa genus, D4is Montessori Pseudomonas, Acinetobacter lwoffii species as A5, F1is yellow class of Nocardia genus, F2is dark green streptomyces. Every bacteria Can be freezed dried powder or be made from various slant of culture medium, at37℃, after24h cultured, conserved in4℃refrigerator spare.
(1)陇东黄土对原油吸附等温线很好地符合Langmuir和线性吸附方程,其吸附系数分别为0.013和0.012ml/g；5种介质吸附原油能力从大到小依次为石英砂、高岭土、黄绵土、高压灭菌土、硅藻土。水相pH、温度和石英砂含量的升高,不利于原油在土壤中的吸附,而盐度会加大原油的吸附量。模拟自然条件下生物降解土壤中的石油类污染物发现：未污染土壤中加0.15%、0.3%、0.5%石油降解的半衰期分别为17.2、18.2和23.7d,生物降解系数分别为0.040、0.038和0.029mg/d；在不同含油浓度下,石油降解率随添加量呈下降趋势,说明石油污染浓度过大会抑制微生物的自然降解效率。
(2)在陇东油田受石油污染的土壤中,以原油为唯一碳源,通过富集分离的方法筛选出13株菌株,各菌株均能在pH值为5-9的范围内生长,但在pH值为8左右的石油培养基里石油降解率最大,可达到95%以上。各菌降解石油表现出先降解高碳数正构烷烃为低碳数正构烷烃,高碳数正构烷烃中奇数碳向偶数碳正构烷烃演化的规律;各菌作用霍烷需要比正构烷烃更高的能量,对芳烃的作用最难,降解率也最低,主要表现形式是对稠环边上的甲基具有去甲基作用,这也是产生天然气的过程,其中F1、A5、D4表现更为突出。混合菌对于正构烷烃的降解率要高于各个单菌的作用；在正交配比优化实验中,假单胞菌属菌(A6)对维持混合菌株的高降解率起到重要的作用。在盐度为8g/1、pH为8、温度在30℃左右以及油浓度为400-600mg/1条件下,混合菌降解率达到最高,且稳定性和适应性比单株菌系好,能更好地适应不同条件下的土壤或水环境。
(3)向土壤中加入混合菌并结合翻耕、添加锯末技术,一方面从细菌数量、水分、氧气等方面改善了土壤的环境,另一方面有利于微生物分解得到的小分子代谢产物的挥发,从而大大加快残油的去除速度。加入混合菌使土壤中细菌含量提高一个数量级的条件下,土壤中石油的半衰期比自然土壤(不加菌)缩短了近4倍。土壤湿度为24.1%时,加入混合菌使土壤中细菌含量达到1.09×108个/kg土,治理5%油污土壤的半衰期是41d,治理62d后,向土壤中加入混合菌并结合翻耕技术的降解率达到了84.64%。添加膨松剂(锯末)提高了土壤的保水和通气能力,而且对于生物修复过程中土壤的脱氢酶活性有明显的促进作用。在每天翻耕的样品中,第62天测量时酶活性已经增长到4.0左右,比不添加膨松剂和翻耕频率较低的土壤高出2倍左右。混合菌剂与有机肥联合作用能够加强石油污染土壤的修复能力,添加2%、4%与8%菌剂和有机肥48天后的降解效率为68.01%、80.42%和78.47%,均大于CK的降解率,四种处理中4%菌剂和有机肥的修复效果最显著。添加的有机肥中氮磷的含量是影响石油降解率的主要因素,只有加适量的有机肥才能使降解效果达到最好,4%的有机肥添加量使降解效果达到最好。
(4)经分子鉴定,菌种A6为铜绿假单胞菌属、D4为蒙氏假单胞菌属、A5为鲁菲不动杆菌属、F1为黄色类诺卡氏菌属、F2为暗黑微绿链霉菌属。各菌种可以冻干粉或不同斜面培养基,于37℃培养24h后置于4℃冰箱备用。
The study of biological degradation and soil remediation technology to the petroleum pollutants in typical region has become focus in the domestic and international environment, biological field. In the light of regional and importance for Longdong Loess Plateau polluted by petroleum, the study of sorption behaviors of crude oil in soil and law of natural biodegradation of petroleum contaminated soil is applied. Then oil-degradation bacterial strain are selected from local microorganisms in soil, the oil-degradation mixed bacterium and adaptal factors has been optimized and constructed; and technology conditions of aggrandizeing bioremediation, such as plowing, added sawdust, nitrogen and phosphorus-nutrient agent are studied on repairing process, the role and influence of these measures are studied. Lastly, the mixed bacteria strains from the molecular level are identificated and analyzed from homology, put forward preservation conditions for every bacteria. The main study results are as follows:
(1) Static experiments for sorption behaviors of crude oil for loess soil in Longdong area are carried out, the adsorption isotherms of crude oil in loess soils are well described by the Langmuir and linear equation. The adsorption coefficients are0.013and0.012mg/1; The adsorption capacity of oil in the five media in turn is:Silica sand, Kaolin, Loessal soil, High pressure sterilized soil, Diatomite; The increase of pH, temperature and silica sand are not favorable to adsorption of crude oil in soil, increasing the salinity can increase adsorption of crude oil. Situ-biodegradation to depredate petroleum pollutants in soil under simulating natural conditions are applied,and find that the half-life of degradations of petroleum oil in no-polluted soil added0.15%,0.3%,0.5%petroleum are17.2,18.2,23.7d, the bio-degradation coefficients are0.0401,0.0388,0.0293mg/d; In different petroleum concentrations, petroleum degradation rate shows trend of decline with increase of petroleum's addition. This performance shows that excessive concentration of petroleum can inhibit microbial natural degradation efficiency.
(2) The soil samples come from Huaqing/XiFeng oil areas of Gansu province which have being polluted by oil for a long time. Using crude oil as the sole carbon source,13strains are screened and isolated with the enrichment and separation method, the strains can all grow up in the pH5-9, and in oil medium (0.5%standard oil), at the pH8, the oil degradation rates reach a maximum (more than95%). The degrading appears that after being treated with various bacteria and the mixed bacterium, firstly, the higher molecular weight n-alkanes are degraded into lower molecular weight n-alkanes, and the n-alkanes in odd-numbered carbon are degraded into n-alkanes in even-numbered carbon. Every bacterium degradating hopane needs higher energy than n-alkanes, Aromatic hydrocarbons are degradated most difficultly, the degradation rate is lowest, mainly form is demethylation effection for the side of methyl on fused ring, which is the process of natural gas. Several bacteria and mixed bacterium all have stronger demethylation effect for methyl on fused ring, among them F1, A5, D4's performance is more outstanding. Mixed bacterium's degradation rate is higher than that of each single bacterium for n-alkanes (except for a positive role:n-tridecane). In process of orthogonal and optimizational experiment, Pseudomonas (A6) plays an important role on maintenance of high degradation rate in mixed strains. The bacteria agent'environmental conditions of crude oil degradation are:salinity is8g/l, pH is8, at30℃,400-600mg/1oil concentration conditions, the mixed strains'degradation rate is highest, and the stability and adaptability are better than individual strains. Therefore, the mixed bacterium has stronger adaptability to different soil and water environment in the practical application.
(3) Joining the mixed bacterium into soil and tillage technology, on the one hand, the soil environment is improved at bacteria, water, oxygen and other aspects; on the other hand tillage is in favor of volatilization of the small molecule metabolite by microbial decomposition, so as to greatly accelerate the speed of removal of residual oil. Under the condition of bacterium content increasing one order of magnitude by adding mixed bacterium into the soil, the half-life reduces nearly4times than the natural soil (without bacteria).When soil moisture is24.1%, the mixed bacterium is added into the soil, bacteria content reachs1.09×10, the half-life of treatment of5%soil polluted by petroleum is41days, after61d, degradation rate reachs84.64%by adding mixed bacterium into soil and tilling technology. Adding leavening agents (sawdust) can increase water retention and ventilation capacity of soil, but also has advantageous to improve dehydrogenase activity in the bioremediation process. In the samples turned every day, add leavening agents, on the sixty-second day, measuring enzyme activity has increased to about4.0, is about2times higher than not added leavening agents. Remediation ability of petroleum contaminated soil with mixed bacterium and organic fertilizer jointly is improved:after48days, under oil contents of50g/kg in the contaminated soil, the oil degradation rate of2%,4%,8%bacterium reach68.01%,80.42%,78.47%, which are clearly improved the biodegrading efficiency higher than CK degradation rate. The4%mixed bacterium's degradation efficiency is most significant. In organic fertilizer, Nitrogen and Phosphorus content is the main factor affected oil degradation rate, only the amount needed to a certain range could have active degradation efficiency, the4%organic fertilizer can achieve the best degradation efficiency.
(4) Through molecular identification, strain A6is Pseudomonas aeruginosa genus, D4is Montessori Pseudomonas, Acinetobacter lwoffii species as A5, F1is yellow class of Nocardia genus, F2is dark green streptomyces. Every bacteria Can be freezed dried powder or be made from various slant of culture medium, at37℃, after24h cultured, conserved in4℃refrigerator spare.
引文
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