土壤柴油污染物微生物降解强化技术研究
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摘要
由于石油类具有较高的生物学毒性、在环境中的残留时间较长、产量大,因此如何对受其污染的环境进行有效修复日益受到社会关注。生物修复具有费用省、可现场处理污染土壤或水体、能够最大限度地降低污染物浓度和环境负面影响小等多重优势,因而非常适合我国目前的实际需要。为此,本研究以柴油为典型污染物,从油污染土壤中分离筛选高效降解菌株,以最大程度提高菌株在降解体系中的活性及降解能力为导向,既研究内因――如何最大程度强化菌株自身的能力,也考虑外因――如何使污染物状况和环境条件更适合菌株降解,深入、系统探讨各影响因子、环境条件及强化技术等对微生物降解的影响。具体结果如下:
采用富集培养方法,从2份长期受油类污染的土样中分离得到3株高效柴油降解菌,命名为Q10、Q14和Q18。经形态特征及生理生化特征鉴定,菌株Q10、菌株Q14和菌株Q18依次归为假单胞菌属(Pseudomonas sp.)、黄杆菌属(Flavobacterium sp.)和红球菌属(Rhodococcus sp.)。其适宜生长的温度、pH和底物浓度分别为30℃、7.0和1000 mg -1L。其中菌株Q18具备一定的生物表面活性剂产生能力,可使表面张力下降31.9mN/m。通过紫外线照射和氯化锂诱变等方式,对菌株Q18进行诱变处理,得到2种柴油降解效果最好的诱变菌株QY1和QY4,其分别能够降解培养液中47.5%和52.2%的柴油。
为了组合高效降解菌群,实验依据柴油污染物组成情况,将分离出来的菌株分别以烷烃为唯一碳源、环烷烃为唯一碳源,以及以多环芳烃为唯一碳源进行驯化培育(经驯化培养后的菌株分别记为Q8ˊ、Q9ˊ、Q10ˊ、Q11ˊ、Q14ˊ、Q15ˊ、Q17ˊ、Q18ˊ和QY4ˊ),并结合GC-MS技术,考虑到了各菌株的优势互补,将有不同降解功能的菌株Q10ˊ、菌株Q14ˊ、菌株Q18ˊ和菌株QY4结合在一起组成的菌群4,其对柴油具有最高的降解率(62.1%)。而分别由菌株Q8ˊ Q14ˊ Q15ˊ和QY4ˊ以及菌株Q11ˊQ14ˊQ18ˊ和QY1ˊ任意组合成的菌群5和菌群6对柴油的降解并不理想,最低的甚至低于单一菌株,仅为33.7%,这可能是菌株对柴油某一组分竞争利用造成的。
采用鼠李糖脂、TW-80、TX-100、SDS作为生物强化剂对柴油进行了生物降解试验。通过测定柴油降解率和菌株数量,结果表明表面活性剂都能不同程度增强柴油的生物可利用性,拓宽菌株的降解范围,强化菌株降解柴油。作为化学合成的表面活性剂Tween-80具有很好的增溶和乳化作用,其毒性和临界胶束浓度也较小,是较为理想的表面活性剂。而生物表面活性剂鼠李糖脂尽管作用更加明显,但其较高的成本,可能会限制其的广泛应用。
通过在微生物降解体系中种植苜蓿、芥菜对模拟柴油污染土壤进行了微生物降解试验。结果表明,与菌株单独降解相比,复合植物苜蓿,芥菜后菌株Q10、Q14及Q18对土壤中柴油污染物降解能力均有不同程度的提高。通过植物过氧化氢酶活性、菌株数量以及PCR-DGGE等技术分析,表明在本实验条件下,苜蓿和芥菜都不能有效降解柴油污染物,苜蓿和芥菜可能主要起到改善土壤环境、活化菌株数量的作用。苜蓿和芥菜强化菌株降解能力上并不相同,在砂土中,苜蓿强化菌株Q18降解柴油的能力强于芥菜;而在砂壤土和粘土中,芥菜强化菌株Q18降解柴油的能力强于苜蓿。
Because this kind of diesel oil has relatively high biological toxity and the residue is relatively permanent, people show more and more attention on how to remediate the polluted environment. Bioremediation of pesticides-contaminated environment, which exploits the ability of organisms to degrade and/or detoxify organic contamination, has been established as an efficient, economic, versatile, and environmentally sound treatment. Decontamination of polluted sites in our country with bioremediation has received increasing interest recently.
Using diesel oil as unique carbon source, take soil sample from sewage control factory and gas station, compare change of diesel oil content in medium, microbe having the capacity of desulphurization was selected and named as Q10, Q14 and Q18 with efficiency of 36.0%, 39.3% and 42.1%, respectively. After managed with ultraviolet radiation, the highest desulphurization efficiency QY1 and QY4 was gained with efficiency of 47.5% and 52.2%. After isolation, purification this microbe as well as detection of its appearance, physiological and biochemical characteristics, growth characteristics, it was identified Pseudomonas sp., Flavobacterium sp. and Rhodococcus sp. according to bacteriological analytical manual (FDA) and taxology of microorganism. The reduction in surface tension was observed in the experiments, strain Q18 may be a potential biosurfactant producer.
In order to construct highly effective microbial consortium, 9 bacteria were selected by initial oil test. Their functions in the process of degrading petroleum were detected by GC-MS analysis and by which we construct some rational consortiums. The consortium 2, consisted of strain Q10, Q14, Q18 and QY4, was more efficient in diesel oil degradation than the individual isolates with efficiency of 62.1%. But the consortium consisted by random was less efficient in diesel oil degradation. The soil culture conditions for the biodegradation of diesel by mixed bacteria were also investigated. The optimal culture conditions were: cultivated temperature 30℃, initial pH value 7.0 and the percentage of diesel concentration 1%.
By the means of research the influence of surfactants on the growth of mixed bacteria and test the effects of surfactants to the biodegradation of diesel, we can make some conclusions that: the influence of surfactants on the growth of bacteria may correlate to the concentration of surfactants. With the increased of the concentration of surfactants, the delay of logarithmic growth phase extended. In our experiment, the inhibition to the growth of mixed bacteria was anion surfactant SDS>nonionic surfactant Tween-80>nonionic surfactant TX-100. In the presence of the anion surfactants, the degradation of diesel was not increased. But adding the nonionic surfactants can increase the degradation of diesel. If the concentration of Tween-80 and TX-100 under 100mg L-1, with the concentration of the nonionic surfactants increased, the degradation efficiency of diesel enhanced.
And the plants, alfalfa and Indian mustard, do improve the effectiveness of diesel oil biodegradation. Both of them along with bacteria exhibited more effective degradation of diesel oil than isolates alone. The diesel oil biodegradation due to combination of the plants and the bacteria go up to 69.2%. Indian mustard shows more effective in the improvement of the biodegradation of diesel oil than alfalfa. The strain Q18 had the greatest degradation ability for diesel oil in sandy loam. Both alfalfa and mustard along with bacteria was more effective in degradation of diesel oil than bacteria alone. Mustard resulted in the maximum diesel oil reduction in sandy loam and clay, but alfalfa resulted in the maximum diesel oil reduction in sandy.
采用富集培养方法,从2份长期受油类污染的土样中分离得到3株高效柴油降解菌,命名为Q10、Q14和Q18。经形态特征及生理生化特征鉴定,菌株Q10、菌株Q14和菌株Q18依次归为假单胞菌属(Pseudomonas sp.)、黄杆菌属(Flavobacterium sp.)和红球菌属(Rhodococcus sp.)。其适宜生长的温度、pH和底物浓度分别为30℃、7.0和1000 mg -1L。其中菌株Q18具备一定的生物表面活性剂产生能力,可使表面张力下降31.9mN/m。通过紫外线照射和氯化锂诱变等方式,对菌株Q18进行诱变处理,得到2种柴油降解效果最好的诱变菌株QY1和QY4,其分别能够降解培养液中47.5%和52.2%的柴油。
为了组合高效降解菌群,实验依据柴油污染物组成情况,将分离出来的菌株分别以烷烃为唯一碳源、环烷烃为唯一碳源,以及以多环芳烃为唯一碳源进行驯化培育(经驯化培养后的菌株分别记为Q8ˊ、Q9ˊ、Q10ˊ、Q11ˊ、Q14ˊ、Q15ˊ、Q17ˊ、Q18ˊ和QY4ˊ),并结合GC-MS技术,考虑到了各菌株的优势互补,将有不同降解功能的菌株Q10ˊ、菌株Q14ˊ、菌株Q18ˊ和菌株QY4结合在一起组成的菌群4,其对柴油具有最高的降解率(62.1%)。而分别由菌株Q8ˊ Q14ˊ Q15ˊ和QY4ˊ以及菌株Q11ˊQ14ˊQ18ˊ和QY1ˊ任意组合成的菌群5和菌群6对柴油的降解并不理想,最低的甚至低于单一菌株,仅为33.7%,这可能是菌株对柴油某一组分竞争利用造成的。
采用鼠李糖脂、TW-80、TX-100、SDS作为生物强化剂对柴油进行了生物降解试验。通过测定柴油降解率和菌株数量,结果表明表面活性剂都能不同程度增强柴油的生物可利用性,拓宽菌株的降解范围,强化菌株降解柴油。作为化学合成的表面活性剂Tween-80具有很好的增溶和乳化作用,其毒性和临界胶束浓度也较小,是较为理想的表面活性剂。而生物表面活性剂鼠李糖脂尽管作用更加明显,但其较高的成本,可能会限制其的广泛应用。
通过在微生物降解体系中种植苜蓿、芥菜对模拟柴油污染土壤进行了微生物降解试验。结果表明,与菌株单独降解相比,复合植物苜蓿,芥菜后菌株Q10、Q14及Q18对土壤中柴油污染物降解能力均有不同程度的提高。通过植物过氧化氢酶活性、菌株数量以及PCR-DGGE等技术分析,表明在本实验条件下,苜蓿和芥菜都不能有效降解柴油污染物,苜蓿和芥菜可能主要起到改善土壤环境、活化菌株数量的作用。苜蓿和芥菜强化菌株降解能力上并不相同,在砂土中,苜蓿强化菌株Q18降解柴油的能力强于芥菜;而在砂壤土和粘土中,芥菜强化菌株Q18降解柴油的能力强于苜蓿。
Because this kind of diesel oil has relatively high biological toxity and the residue is relatively permanent, people show more and more attention on how to remediate the polluted environment. Bioremediation of pesticides-contaminated environment, which exploits the ability of organisms to degrade and/or detoxify organic contamination, has been established as an efficient, economic, versatile, and environmentally sound treatment. Decontamination of polluted sites in our country with bioremediation has received increasing interest recently.
Using diesel oil as unique carbon source, take soil sample from sewage control factory and gas station, compare change of diesel oil content in medium, microbe having the capacity of desulphurization was selected and named as Q10, Q14 and Q18 with efficiency of 36.0%, 39.3% and 42.1%, respectively. After managed with ultraviolet radiation, the highest desulphurization efficiency QY1 and QY4 was gained with efficiency of 47.5% and 52.2%. After isolation, purification this microbe as well as detection of its appearance, physiological and biochemical characteristics, growth characteristics, it was identified Pseudomonas sp., Flavobacterium sp. and Rhodococcus sp. according to bacteriological analytical manual (FDA) and taxology of microorganism. The reduction in surface tension was observed in the experiments, strain Q18 may be a potential biosurfactant producer.
In order to construct highly effective microbial consortium, 9 bacteria were selected by initial oil test. Their functions in the process of degrading petroleum were detected by GC-MS analysis and by which we construct some rational consortiums. The consortium 2, consisted of strain Q10, Q14, Q18 and QY4, was more efficient in diesel oil degradation than the individual isolates with efficiency of 62.1%. But the consortium consisted by random was less efficient in diesel oil degradation. The soil culture conditions for the biodegradation of diesel by mixed bacteria were also investigated. The optimal culture conditions were: cultivated temperature 30℃, initial pH value 7.0 and the percentage of diesel concentration 1%.
By the means of research the influence of surfactants on the growth of mixed bacteria and test the effects of surfactants to the biodegradation of diesel, we can make some conclusions that: the influence of surfactants on the growth of bacteria may correlate to the concentration of surfactants. With the increased of the concentration of surfactants, the delay of logarithmic growth phase extended. In our experiment, the inhibition to the growth of mixed bacteria was anion surfactant SDS>nonionic surfactant Tween-80>nonionic surfactant TX-100. In the presence of the anion surfactants, the degradation of diesel was not increased. But adding the nonionic surfactants can increase the degradation of diesel. If the concentration of Tween-80 and TX-100 under 100mg L-1, with the concentration of the nonionic surfactants increased, the degradation efficiency of diesel enhanced.
And the plants, alfalfa and Indian mustard, do improve the effectiveness of diesel oil biodegradation. Both of them along with bacteria exhibited more effective degradation of diesel oil than isolates alone. The diesel oil biodegradation due to combination of the plants and the bacteria go up to 69.2%. Indian mustard shows more effective in the improvement of the biodegradation of diesel oil than alfalfa. The strain Q18 had the greatest degradation ability for diesel oil in sandy loam. Both alfalfa and mustard along with bacteria was more effective in degradation of diesel oil than bacteria alone. Mustard resulted in the maximum diesel oil reduction in sandy loam and clay, but alfalfa resulted in the maximum diesel oil reduction in sandy.
引文
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