TNT污染土壤的生物泥浆反应器修复机理研究
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摘要
曾在军事上广泛应用的TNT因具有毒性和三致作用,而被中国、美国等列入优先控制污染物的名单,因此TNT生产、加工、使用及相关设施的销毁过程中造成的土壤污染及其修复问题也倍受关注。本文以TNT污染土壤为研究对象,在优化土壤中TNT的提取方法、基本探明TNT对厌氧发酵系统微生物群落毒性及土壤中TNT的生物有效利用性和厌氧转化机理的基础上,研究和提出了一种利用生物泥浆反应器高效、安全地修复TNT重污染土壤并使其达到相关标准限值(17.2mgTNT/kg)的技术方法,填补了国内生物泥浆反应器系统修复TNT重污染土壤领域的空白,对于类似介质中硝基类化合物的修复研究也有一定的理论参考价值。本研究获得的主要成果及结论如下：
(1)优化了土壤中TNT的提取方法。
当提取剂为乙腈时,在液固比为20mL乙腈：1g土壤、振荡12h-超声5h的条件下,粘土和沙土及相应的泥浆(水土比为2：1)中TNT平均提取回收率均大于95%,土壤类型对TNT平均提取回收率没有显著影响；而添加水平的影响极为显著,当土壤中TNT浓度≥500mgTNT/kg(土壤)时,TNT提取回收率介于95%-105%之间,当土壤中TNT浓度为10mgTNT/kg(土壤)时,其回收率为70%-130%,但均满足相应的提取回收标准。不同土壤介质中TNT回收率变异系数均小于10%。准确度和精密度均达到分析要求。
(2)解析了土壤对TNT的吸附及解吸行为,明确了TNT的生物有效利用潜力。在液土比为2：1的条件下,土壤对TNT的吸附在24h内可达到平衡,其吸附动力学与Elovich方程拟合效果最好(相关系数为0.962),吸附过程为非均相扩散过程；与吸附与解吸等温线拟合较好的Freundlich方程其指数1/n表明TNT在土壤中存在迟滞现象(解吸迟滞指数为3.2)；吸附自由能表明,TNT在土壤中是以表面物理吸附为主；污染土壤中TNT解吸较为迅速,且随着污染强度的增大而显著增强,也即表明土壤中TNT的生物可利用潜力较高。
(3)探明了TNT对厌氧发酵系统中产甲烷菌的毒性抑制作用。
TNT对厌氧发酵系统中产甲烷菌毒性随着浓度的升高而增加,当TNT浓度低于100mg/L时为轻度抑制(RA≥77.62%),浓度高于100mg/L时为中度抑制(40% (4)基本探明了TNT厌氧转化机理。
在pH为6-7的模拟厌氧体系中TNT可以被硫化钠和作为新生态氢发生源的金属Zn还原转化,动力学均遵循准一级动力学规律；还原剂的浓度在TNT厌氧转化中起着重要作用,5倍化学当量的还原剂浓度是最合适的；既模拟pH又模拟发酵成分的B系统中TNT转化所需的反应时间要短于仅模拟厌氧发酵液pH的A系统；在pH为6-7、还原剂浓度为5倍化学当量的B系统中,硫化钠和作为新生态氢发生源的金属Zn均可在2h内快速转化90%TNT。由此可知,厌氧环境下因生物作用产生的非生物还原剂硫化物、新生态氢等在TNT快速转化中起着重要作用,且随着还原剂浓度的升高其转化速率也会相应升高。
LC-MS分析表明,模拟厌氧系统中硫化物既可以对TNT硝基进行还原转化又可以对TNT苯环进行脱硝转化,其中以硝基还原为主要的还原方式,而Zn主要是通过加氢脱硝的方式对TNT实现还原转化；此外,TNT在模拟厌氧系统中的还原产物还能相互作用生成聚合物。即表明在厌氧环境下,TNT在还原剂作用下可生成多种的还原产物,其代谢途径因还原剂不同而有所差异。
(5)开发了一种高效的TNT污染土壤生物泥浆反应器修复技术,明确了其工艺技术参数及控制条件,为生物泥浆反应器的推广应用奠定了技术基础。
以1000mgTNT、kg(土壤)的污染土壤为研究对象,通过批次静态试验获得的TNT厌氧降解优化条件为：mCOD/mTNT为4、水土比为2：1、接种量为5%(以土壤干重计)、不外加硫酸盐(土壤本身硫酸盐含量为2021mgSO42-/kg(土壤))、表面活性剂为1.5CMC的Tween80时间为厌氧处理5d-好氧补充处理2d。在上述优化条件下,1000mgTNT/kg(土壤)的污染土壤在厌氧处理5d后就能达到修复标准,相应的容积负荷为100g/(m3·d),2d的好氧补充处理可进一步实现TNT污染土壤的稳定化和腐殖化,其修复周期短于文献报道的修复周期；当TNT浓度低于1000mgTNT/kg土壤,即泥浆体系中TNT容积负荷不大于100g/(m3·d).mCOD/mTNT≥4时,在厌氧处理5d后污染土壤中TNT能达到修复标准；当TNT浓度高于1000mgTNT/kg土壤时,则可通过控制容积负荷和mCOD/mTNT来达到修复标准。
序批式生物泥浆反应器的动态运行结果表明,4个周期内,1000mgTNT/kg(土壤)的TNT污染土壤在上述优化条件下经生物泥浆反应器厌氧修复5d-好氧补充处理2d后,能达到17.2mgTNT/kg(土壤)的标准限值要求,且可以进一步实现TNT污染土壤的稳定化和腐殖化。该反应器动态运行性能稳定,且其修复周期短于文献报道的周期。
TNT, a pollutant with toxic, carcinogenic, and mutagenic, has been list in a category of priority pollutants in China and USA. Since it has been used widely in military activities, the TNT pollution was serious over the world. Thus, the TNT bioremediation attracted more and more attention. In this paper, the method for TNT extraction from soil was optimized and the potential of TNT bioavailability was analyzed. Also, the toxicity of TNT on the methane-producing systems was evaluated. Moreover, the mechanism of the rapid disappearance of TNT under anaerobic condition was analyzed. Finally, a safe and high efficient bioslurry reactor technology has been.developed to remove TNT in contaminated soil, with the aim to meet the remediation standard of 17.2mg TNT/kg soil. The results of this study would fill a vacancy of remediation of heavy TNT-contaminated soil. It is also of referential value for the remediation of nitroaromatic compounds in similar media. Major research results are summarized as follows:
(1) The extraction method of TNT from soil
When extraction solvent was acetonitrile, the optimum extraction condition of TNT from soil was:20 mL acetonitrile:1 g soil,12 h vibration-5 h sonication. The average of TNT recovery rates from clay soil, sandy soil, and corresponding soil slurry (2:1 water/soil ratio) were higher than 95%. It suggested that the type of soil had no effect on TNT recovery. However, effect of TNT concentration on TNT average recovery rate was significant. When TNT concentration of soil was higher than 500 mg TNT/kg (soil), the average recovery rate of TNT from soil was between 95% and 105%; when TNT concentration of soil and corresponding soil slurry was 10 mg TNT/kg (soil), the average recovery of TNT was between 70% and 130%. The coefficient of variability is less than 10%. In all, both the accuracy and degree of precision of TNT recovery met the analysis requirement.
(2) The sorption/desorption behavior of TNT and the potential of TNT bioavailability
Under the condition of the liquid to soil ratio of 2:1, the sorption of TNT on soil reached the equilibrium state within 24 h and the sorption kenitics fitting Elovich equation reached the best, with the correlation coefficient of 0.962, which indicated that the sorption was a non-homogeneous diffusion process The sorption/desorption isotherm fitted Freundlich equation very well, of which the constant 1/n indicated existence of hysteresis (the calculated index of hysteresis was 3.2). The free energy of sorption suggested the physical sorption was dominant for TNT sorption on soil. The desorption of TNT from contaminated soil was fast and increased with the increase of pollution level of soil, indicating that the potential of TNT bioavailability was high.
(3) The methane-producing toxicity of TNT
The methane-producing toxicity of TNT increased with the increase of TNT concentration. The relative activity was. more than 77.62% when TNT concentration was lower than 100 mg/L, suggesting the inhibition was light. Relative activity was between 75% and 40% when TNT concentration was higher than 100 mg/L, suggesting the inhibition was middle. Moreover, the rapid conversion of TNT eliminated the threat of lyses to methanogenic bacteria under anaerobic condition. It could be concluded that the toxicity of TNT to microbiologic population wasn't large and the technology of anaerobic bioremediation of TNT was feasible.
(4) The mechanism of the rapid disappearance of TNT in an anaerobic solution
Under Simulated Anaerobic Conditions at pH 6-7, TNT could be reduced by both Na2S and Zn0 which could produce nascent hydrogen, and both of TNT reduction kinetics followed the pseudo-first order rate law. The reductant concentration played a very important role in reduction of TNT, and a 5-fold increase in reductant concentration above the theoretical stoichiometric concentration was optimum. The reaction time for the conversion of TNT in system B which both controlled pH and simulated components of an anaerobic solution was much shorter than that in system A which only controled pH. About 90% TNT in system B could be reduced by both Na2S and Zn0 within 2h at pH 6-7, when the reductants concentration was increased 5-fold. It could be concluded that abiotic reductants including sulfide and nascent hydrogen, which were produced by biological action, played a very important role in the rapid disappearance of TNT under anaerobic condition, and the conversion of TNT increased with reductants concentration increasing.
The LC/MS results of intermediates indicated that, both nitroreduction and denitration of TNT could be initiated by sulfide simultaneously, but the former was dominant over the latter. However, only denitration could be initiated by Zn0. Moreover, intermediates of both pathways may form polymer. It could be concluded that TNT could be transformed into many products by reductants under anaerobic condition and the reduction pathways varied with different reductants.
(5) The high efficient bioslurry reactor technology for remediation of heavy TNT-contaminated soil
Based on the results of the batch static experiments for remediation of 1000 mg TNT/kg (soil) TNT-contaminated soil, the optimum condition were obtained as follows:4 of mCOD/mTNT, 2 of the ratio of liquid to solid,5% of inoculum,1.5 fold CMC of Tween 80,5 d anaerobic treatment and 2 d aerobic operation. Under the optimal conditions, 1000mg TNT/kg (soil) TNT-contaminated soil reached the remediation standard (17.2 mg TNT/kg (soil)) after 5 d anaerobic treatment and the volume loading was 100 g/(m3-d);the subsequent 2 d aerobic treatment could further realized the stabilization and humification; the whole remediation period was shorter than that of literature. Also, under the optimal conditions, all polluted soil, with volume loading less than 100 g/(m3·-d)and mCOD/mTNT more than 4, could be remediated after 5 d anaerobic treatment; while the polluted soil with volume loading more than 100 g/(m3-d) could reach the remediation standard by controlling volume loading and mCOD/mTNT
The results from dynamic operation of sequence boi-slurry reactor suggested that TNT concentration of 1000 mg TNT/kg (soil) soil met the remediation standard (17.2 mgTNT/kg (soil)) after 5 d anaerobic treatment-2 d aerobic treatment during four operation periods on the basis of sufficient stir (200 rpm) and under the above optimum condition. Moreover it realized stabilization and humification of contaminated soil.The operation performance of bio-slurry reactor was steady and remediation period was shorter than that of literature.
(1)优化了土壤中TNT的提取方法。
当提取剂为乙腈时,在液固比为20mL乙腈：1g土壤、振荡12h-超声5h的条件下,粘土和沙土及相应的泥浆(水土比为2：1)中TNT平均提取回收率均大于95%,土壤类型对TNT平均提取回收率没有显著影响；而添加水平的影响极为显著,当土壤中TNT浓度≥500mgTNT/kg(土壤)时,TNT提取回收率介于95%-105%之间,当土壤中TNT浓度为10mgTNT/kg(土壤)时,其回收率为70%-130%,但均满足相应的提取回收标准。不同土壤介质中TNT回收率变异系数均小于10%。准确度和精密度均达到分析要求。
(2)解析了土壤对TNT的吸附及解吸行为,明确了TNT的生物有效利用潜力。在液土比为2：1的条件下,土壤对TNT的吸附在24h内可达到平衡,其吸附动力学与Elovich方程拟合效果最好(相关系数为0.962),吸附过程为非均相扩散过程；与吸附与解吸等温线拟合较好的Freundlich方程其指数1/n表明TNT在土壤中存在迟滞现象(解吸迟滞指数为3.2)；吸附自由能表明,TNT在土壤中是以表面物理吸附为主；污染土壤中TNT解吸较为迅速,且随着污染强度的增大而显著增强,也即表明土壤中TNT的生物可利用潜力较高。
(3)探明了TNT对厌氧发酵系统中产甲烷菌的毒性抑制作用。
TNT对厌氧发酵系统中产甲烷菌毒性随着浓度的升高而增加,当TNT浓度低于100mg/L时为轻度抑制(RA≥77.62%),浓度高于100mg/L时为中度抑制(40%
在pH为6-7的模拟厌氧体系中TNT可以被硫化钠和作为新生态氢发生源的金属Zn还原转化,动力学均遵循准一级动力学规律；还原剂的浓度在TNT厌氧转化中起着重要作用,5倍化学当量的还原剂浓度是最合适的；既模拟pH又模拟发酵成分的B系统中TNT转化所需的反应时间要短于仅模拟厌氧发酵液pH的A系统；在pH为6-7、还原剂浓度为5倍化学当量的B系统中,硫化钠和作为新生态氢发生源的金属Zn均可在2h内快速转化90%TNT。由此可知,厌氧环境下因生物作用产生的非生物还原剂硫化物、新生态氢等在TNT快速转化中起着重要作用,且随着还原剂浓度的升高其转化速率也会相应升高。
LC-MS分析表明,模拟厌氧系统中硫化物既可以对TNT硝基进行还原转化又可以对TNT苯环进行脱硝转化,其中以硝基还原为主要的还原方式,而Zn主要是通过加氢脱硝的方式对TNT实现还原转化；此外,TNT在模拟厌氧系统中的还原产物还能相互作用生成聚合物。即表明在厌氧环境下,TNT在还原剂作用下可生成多种的还原产物,其代谢途径因还原剂不同而有所差异。
(5)开发了一种高效的TNT污染土壤生物泥浆反应器修复技术,明确了其工艺技术参数及控制条件,为生物泥浆反应器的推广应用奠定了技术基础。
以1000mgTNT、kg(土壤)的污染土壤为研究对象,通过批次静态试验获得的TNT厌氧降解优化条件为：mCOD/mTNT为4、水土比为2：1、接种量为5%(以土壤干重计)、不外加硫酸盐(土壤本身硫酸盐含量为2021mgSO42-/kg(土壤))、表面活性剂为1.5CMC的Tween80时间为厌氧处理5d-好氧补充处理2d。在上述优化条件下,1000mgTNT/kg(土壤)的污染土壤在厌氧处理5d后就能达到修复标准,相应的容积负荷为100g/(m3·d),2d的好氧补充处理可进一步实现TNT污染土壤的稳定化和腐殖化,其修复周期短于文献报道的修复周期；当TNT浓度低于1000mgTNT/kg土壤,即泥浆体系中TNT容积负荷不大于100g/(m3·d).mCOD/mTNT≥4时,在厌氧处理5d后污染土壤中TNT能达到修复标准；当TNT浓度高于1000mgTNT/kg土壤时,则可通过控制容积负荷和mCOD/mTNT来达到修复标准。
序批式生物泥浆反应器的动态运行结果表明,4个周期内,1000mgTNT/kg(土壤)的TNT污染土壤在上述优化条件下经生物泥浆反应器厌氧修复5d-好氧补充处理2d后,能达到17.2mgTNT/kg(土壤)的标准限值要求,且可以进一步实现TNT污染土壤的稳定化和腐殖化。该反应器动态运行性能稳定,且其修复周期短于文献报道的周期。
TNT, a pollutant with toxic, carcinogenic, and mutagenic, has been list in a category of priority pollutants in China and USA. Since it has been used widely in military activities, the TNT pollution was serious over the world. Thus, the TNT bioremediation attracted more and more attention. In this paper, the method for TNT extraction from soil was optimized and the potential of TNT bioavailability was analyzed. Also, the toxicity of TNT on the methane-producing systems was evaluated. Moreover, the mechanism of the rapid disappearance of TNT under anaerobic condition was analyzed. Finally, a safe and high efficient bioslurry reactor technology has been.developed to remove TNT in contaminated soil, with the aim to meet the remediation standard of 17.2mg TNT/kg soil. The results of this study would fill a vacancy of remediation of heavy TNT-contaminated soil. It is also of referential value for the remediation of nitroaromatic compounds in similar media. Major research results are summarized as follows:
(1) The extraction method of TNT from soil
When extraction solvent was acetonitrile, the optimum extraction condition of TNT from soil was:20 mL acetonitrile:1 g soil,12 h vibration-5 h sonication. The average of TNT recovery rates from clay soil, sandy soil, and corresponding soil slurry (2:1 water/soil ratio) were higher than 95%. It suggested that the type of soil had no effect on TNT recovery. However, effect of TNT concentration on TNT average recovery rate was significant. When TNT concentration of soil was higher than 500 mg TNT/kg (soil), the average recovery rate of TNT from soil was between 95% and 105%; when TNT concentration of soil and corresponding soil slurry was 10 mg TNT/kg (soil), the average recovery of TNT was between 70% and 130%. The coefficient of variability is less than 10%. In all, both the accuracy and degree of precision of TNT recovery met the analysis requirement.
(2) The sorption/desorption behavior of TNT and the potential of TNT bioavailability
Under the condition of the liquid to soil ratio of 2:1, the sorption of TNT on soil reached the equilibrium state within 24 h and the sorption kenitics fitting Elovich equation reached the best, with the correlation coefficient of 0.962, which indicated that the sorption was a non-homogeneous diffusion process The sorption/desorption isotherm fitted Freundlich equation very well, of which the constant 1/n indicated existence of hysteresis (the calculated index of hysteresis was 3.2). The free energy of sorption suggested the physical sorption was dominant for TNT sorption on soil. The desorption of TNT from contaminated soil was fast and increased with the increase of pollution level of soil, indicating that the potential of TNT bioavailability was high.
(3) The methane-producing toxicity of TNT
The methane-producing toxicity of TNT increased with the increase of TNT concentration. The relative activity was. more than 77.62% when TNT concentration was lower than 100 mg/L, suggesting the inhibition was light. Relative activity was between 75% and 40% when TNT concentration was higher than 100 mg/L, suggesting the inhibition was middle. Moreover, the rapid conversion of TNT eliminated the threat of lyses to methanogenic bacteria under anaerobic condition. It could be concluded that the toxicity of TNT to microbiologic population wasn't large and the technology of anaerobic bioremediation of TNT was feasible.
(4) The mechanism of the rapid disappearance of TNT in an anaerobic solution
Under Simulated Anaerobic Conditions at pH 6-7, TNT could be reduced by both Na2S and Zn0 which could produce nascent hydrogen, and both of TNT reduction kinetics followed the pseudo-first order rate law. The reductant concentration played a very important role in reduction of TNT, and a 5-fold increase in reductant concentration above the theoretical stoichiometric concentration was optimum. The reaction time for the conversion of TNT in system B which both controlled pH and simulated components of an anaerobic solution was much shorter than that in system A which only controled pH. About 90% TNT in system B could be reduced by both Na2S and Zn0 within 2h at pH 6-7, when the reductants concentration was increased 5-fold. It could be concluded that abiotic reductants including sulfide and nascent hydrogen, which were produced by biological action, played a very important role in the rapid disappearance of TNT under anaerobic condition, and the conversion of TNT increased with reductants concentration increasing.
The LC/MS results of intermediates indicated that, both nitroreduction and denitration of TNT could be initiated by sulfide simultaneously, but the former was dominant over the latter. However, only denitration could be initiated by Zn0. Moreover, intermediates of both pathways may form polymer. It could be concluded that TNT could be transformed into many products by reductants under anaerobic condition and the reduction pathways varied with different reductants.
(5) The high efficient bioslurry reactor technology for remediation of heavy TNT-contaminated soil
Based on the results of the batch static experiments for remediation of 1000 mg TNT/kg (soil) TNT-contaminated soil, the optimum condition were obtained as follows:4 of mCOD/mTNT, 2 of the ratio of liquid to solid,5% of inoculum,1.5 fold CMC of Tween 80,5 d anaerobic treatment and 2 d aerobic operation. Under the optimal conditions, 1000mg TNT/kg (soil) TNT-contaminated soil reached the remediation standard (17.2 mg TNT/kg (soil)) after 5 d anaerobic treatment and the volume loading was 100 g/(m3-d);the subsequent 2 d aerobic treatment could further realized the stabilization and humification; the whole remediation period was shorter than that of literature. Also, under the optimal conditions, all polluted soil, with volume loading less than 100 g/(m3·-d)and mCOD/mTNT more than 4, could be remediated after 5 d anaerobic treatment; while the polluted soil with volume loading more than 100 g/(m3-d) could reach the remediation standard by controlling volume loading and mCOD/mTNT
The results from dynamic operation of sequence boi-slurry reactor suggested that TNT concentration of 1000 mg TNT/kg (soil) soil met the remediation standard (17.2 mgTNT/kg (soil)) after 5 d anaerobic treatment-2 d aerobic treatment during four operation periods on the basis of sufficient stir (200 rpm) and under the above optimum condition. Moreover it realized stabilization and humification of contaminated soil.The operation performance of bio-slurry reactor was steady and remediation period was shorter than that of literature.
引文
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