生物电化学系统定向还原硝基苯及能量循环补偿模式研究
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摘要
硝基苯属于典型的难降解有机物，并且对人体有致突变、致畸和致癌的潜在危害，已经被列入我国公布的58种优先控制有机污染物中。由于硝基的“吸电子”效应造成苯环电子云密度的降低是硝基苯在传统好氧生物处理中难以有效去除的主要原因。通过还原法先将硝基苯转化为苯胺可以显著提高废水的可生化性。硝基苯的电化学还原方法是一种可控而高效的手段，然而传统电化学方法中需要采用特殊的电极材料或引入贵金属催化剂来实现硝基苯向苯胺的定向转化，这大大限制了其应用于废水处理。近年来发展起来的生物电化学系统（BES），由于微生物作为催化剂所具有绿色、低成本、可自我更新等优势，是颇具发展前景的环境友好型废水处理技术。本研究采用阴极作为唯一电子供体，证实了基于微生物作为催化剂的生物阴极具有催化硝基苯定向还原为苯胺的能力，并发现通过向阴极引入有机碳源可以进一步提升这种催化能力。在此基础上，为了实现BES还原硝基苯过程中的能量循环补偿，开展了利用生物阳极回收苯胺电子和能量的研究，揭示了氧气对回收苯胺电子和能量的重要作用及可能机制。
以阴极作为唯一电子供体条件下，阴极电位恒定在-0.4V时，生物阴极显著提高了硝基苯向苯胺转化的效率和速率，周期内生物阴极中苯胺生成效率达到93%，是非生物阴极的6.16倍，同时生物阴极在催化硝基苯还原为苯胺过程中降低了毒性中间产物亚硝基苯的积累，亚硝基苯最大积累量较非生物阴极下降38.7%。循环伏安分析显示与微生物相关的某种中点电位为-0.315V的氧化还原物质可能介导了阴极微生物胞外电子传递过程。表观一级动力学模型的构建与分析表明，生物阴极能够同时催化硝基苯和亚硝基苯还原，相应动力学常数分别较没有微生物存在时提高了62%和100%。基于焦磷酸测序的微生物群落分析结果表明，在阴极特殊环境的选择压力下，生物阴极微生物群落结构发生了明显的变化，生物阴极样品中优势菌属为Rhizobium sp.、Leucobactersp.、Achromobacter sp.、Mycobacterium sp.、Dysgonomonas sp.和Pseudomonassp.。这些微生物所具有的硝基苯定向还原、固碳以及可能的阴极胞外电子传递功能是生物阴极能够催化硝基苯定向还原为苯胺的关键。
通过向阴极引入有机碳源，能够驯化获得催化硝基苯定向还原性能更好的生物阴极。生物阴极在硝基苯还原为苯胺的过程中鲜有毒性中间产物亚硝基苯的积累。循环伏安分析表明，有机碳源存在时能够提升生物阴极的电催化活性，表现为硝基苯还原电位进一步正移70mV。16S rRNA基因克隆文库分析表明，有机碳源存在条件下驯化获得的生物阴极中，优势菌种（占文库比例为74.7%）的16S rRNA基因与Enterococcus aquimarinus LMG16607最为相似，Enterococcus异养生长的特性与有机碳源的存在有着密切关系。在生物阳极和生物阴极构建的生物电化学系统中，提升外加电压能够增加硝基苯还原的表观一级动力学常数（kNB），但同时也会增加硝基苯还原的比能耗并降低电流效率。增加有机碳源葡萄糖的浓度也能提高kNB，在葡萄糖浓度较低的范围内（≤200mg/L），单位葡萄糖浓度增量引起的kNB增量是其浓度较高范围内（≥200mg/L）的5.1倍。为避免过度增加体系中的COD，有机碳源的引入量控制在200mg/L左右较为适宜，此时在各外加电压条件下（0.15V~0.5V），苯胺的生成率均超过97%，硝基苯还原一级动力学常数较无有机碳源时平均提高52±6%。
将阳极暴露于空气中的操作方式可以促进阳极电化学活性微生物从苯胺回收电子和能量。通过不同供氧方式的实验，证实这种能力与氧气的存在密切相关。阳极室顶空氧气浓度对苯胺的电子和能量回收效能有着明显的影响。在考察的氧气浓度范围内（21%~100%），顶空氧气含量为70%的时候能够获得最高的输出电流（0.155mA）和最大功率密度（3.32±0.4W/m3）。但库伦效率随氧气浓度的增加从10.32±1.36%降低至4.61±1.11%。根据GC-MS对苯胺代谢产物的分析以及结合间歇性断路实验结果，作者推测了苯胺在生物阳极代谢并转化为电极电子的方式，即阳极液和阳极生物膜外层的微生物在有氧的条件下将苯胺转化为有机酸，阳极生物膜内层的电化学活性微生物在缺氧条件下利用产生的有机酸作为底物产电。通过循环伏安分析，表明阳极电化学活性微生物将电子传递给阳极的过程主要通过游离性电子中介体介导。推定的中介体在循环伏安曲线上表现出两对可逆的氧化还原峰，中点电位分别为-0.027V和0.063V。
根据本文中苯胺在生物阳极氧化和硝基苯在生物阴极还原的实验结果，并结合热力学以及化学计量学的分析，作者提出了利用苯胺回收电子部分反哺硝基苯还原的工艺模式。计算表明，不同操作条件下，利用苯胺回收电子反哺硝基苯还原可以节约22%~53%的额外电子供体需求，并内部循环补偿17%~40%的能量消耗。
Nitrobenzene (NB), one of typical recalcitrant organic compounds, is reportedas possible mutagens, teratogens or carcinogens and has been listed in58China’spriority control organic pollutants. As the electrophilic effect of nitryl decreases theelectron density of the benzene, NB is only to a limited extent degraded in aerobicbiological processes. An effective strategy is to transform NB to aniline (AN) first,which is considerably easier mineralized than NB. The electrochemical reduction ofNB is an effieicent and controlable approach, however, the conventional processsuffer from the requirement of special electrode materials or noble metal catalysts toachieve the selective transformation of NB to AN, which limited its application inpractice. Recent develped bioelectrochemical system (BES) is proposed as aprespective process in wastewtaer treatment, since bacteria, as catalyst, hold theinherent advantages of low-cost, self-regeneration and evironment-friendly. Thepresent study demonstrated bacteria can use the cathode as the sole electron donor toselective reduce NB to AN. The introduction of organic carbon to biocathode furtherenhanced this selective transformation capability. Based on above findings, thestudy went on developing novel process that enabled energy internal loopcompensation by recovery energy from aniline at bioanode. The improtant role ofoxygen in the process was demonstrated and the involved mechanism was thandiscussed.
When cathode was served as sole electron donor, the transformation efficiencyand rate from NB to AN was dramatically increased with microbial catalysis.93%of AN formation efficiency was achieved in biocathode at the cathode potential of-0.4V, which was6.16times as high as that obtained in abiotic cathode. In addition,the maximum accumulation of toxic nitrosobenzene (NOB), the intermediate duringNB reduction to AN, was decreased by38.7%in biocathode compared to that inabiotic cathode. Cyclic voltammetry (CV) revealed that an unidentified redoxcompound with the midpoint potential around-0.315V could be responsible for theelectron transfer from cathode to bacteria. Based on the apparent first-order kineticmodel, biocathode was suggested to both catalyze the reduction of NB and NOB.The corresponding apparent first-order kinetic constant was increased by62%and100%, respectively. Moreover, the community of the inoculum and the biocathodesample was analyzed based on the pyrosequencing. The results indicated microbialcommunity was changed dramatically under the evolutionary pressure provied bycathode severved as sole electron donor. The predominant bacteria in biocathodecommunity consisted of Rhizobium sp., Leucobacter sp., Achromobacter sp., Mycobacterium sp., Dysgonomonas sp. and Pseudomonas sp.. The functions ofselective nitrobenzene reduction, carbion fixiation and the possible catodicextracellular electron tranfer in the microbial community might feature the catalyzedselective reduction of NB to AN in biocathode.
Biocathode enrichied with the present of organic carbon (glucose) showedbetter performance on selective reduction of NB, in which NOB was rarelyaccumulated. Cyclic voltammetry revealed NB reduction peak was positively shiftedby70mV with the present of organic carbon, indicating the enhanced performancewas not only caused by the extra electrons donation from glucose but also theimprovement of bioelectrocatalytic activity.16S rRNA based analysis of the biofilmon the cathode indicated that the cathode was dominated by an Enterococcus species(occupied74.7%of the library) closely related to Enterococcus aquimarinus LMG16607. In BES coupled with bioanode and biocathode, the apparent first-orderkinetic constant (kNB) was increased with the increase of applied voltage, butconsumed more energy and decreased the current efficiency. kNBwas also increasedby introducing more glucose in to catholyte, kNBincrement caused by net incrementof glucose in lower concentration range (≤200mg/L) was5.1times as high as thatin higher glucose concentration range (≥200mg/L). To avoid the excess increase ofCOD in wastewater, the suggested concentration of added organic carbon was200mg/L, which resulted in AN formation efficiency over97%at all tested appliedvoltages (0.15V~0.25V) and averagely increased the apparent first-order kineticconstant by52±6%compared to that without organic carbon.
The recovery of electron and energy from AN was found to be facilitated byexposing bioanode to air, which was then further demonstrated to be depending onthe present of oxygen in limited oxygen donation test. Oxygen content in the anodehead space was found to impact AN degradation and the elelctrons recoverysignificantly. The highest output current (0.155mA) and maximum power density(3.32±0.4W/m3) was observed when70%of gas in anode headspace was composedby oxygen at the beginning. However, columbic efficiency decreased with theoxygen concentration increasing from10.32±1.36%to4.61±1.11%in the testedinitial oxygen content conditions (21%~100%). Based on the results of GC-MS andthe intermittent open circuit experiment, the possible mechanism using AN as thesole electron donor in present of oxygen was suggested as following. AN was firstaerobicly converted to organic acids by microbes in the anolyte and located at theouter layer of anodic biofilm, which was then uptook by electrochemical activebacteria located at the inner layer of anodic biofilm and produce current underanoxic condition. CV indicated that anodic extracellular electron tranfer wasfulfilled by certain microbial secreted soluble electron mediator, which has tworeversible redox peaks with the mid-point potentials of-0.027V and0.063V, respectively.
To achieve the energy internal loop compensation, the process of NB reductionwith electrons partial fed-back from AN oxidation was proposed. The saved externalelectron donor and internal loop compensated energy was estimated as22%~53%and17%~40%, respectively, based on the experiment results and the thermodynamicand stoichiometric calculation.
以阴极作为唯一电子供体条件下，阴极电位恒定在-0.4V时，生物阴极显著提高了硝基苯向苯胺转化的效率和速率，周期内生物阴极中苯胺生成效率达到93%，是非生物阴极的6.16倍，同时生物阴极在催化硝基苯还原为苯胺过程中降低了毒性中间产物亚硝基苯的积累，亚硝基苯最大积累量较非生物阴极下降38.7%。循环伏安分析显示与微生物相关的某种中点电位为-0.315V的氧化还原物质可能介导了阴极微生物胞外电子传递过程。表观一级动力学模型的构建与分析表明，生物阴极能够同时催化硝基苯和亚硝基苯还原，相应动力学常数分别较没有微生物存在时提高了62%和100%。基于焦磷酸测序的微生物群落分析结果表明，在阴极特殊环境的选择压力下，生物阴极微生物群落结构发生了明显的变化，生物阴极样品中优势菌属为Rhizobium sp.、Leucobactersp.、Achromobacter sp.、Mycobacterium sp.、Dysgonomonas sp.和Pseudomonassp.。这些微生物所具有的硝基苯定向还原、固碳以及可能的阴极胞外电子传递功能是生物阴极能够催化硝基苯定向还原为苯胺的关键。
通过向阴极引入有机碳源，能够驯化获得催化硝基苯定向还原性能更好的生物阴极。生物阴极在硝基苯还原为苯胺的过程中鲜有毒性中间产物亚硝基苯的积累。循环伏安分析表明，有机碳源存在时能够提升生物阴极的电催化活性，表现为硝基苯还原电位进一步正移70mV。16S rRNA基因克隆文库分析表明，有机碳源存在条件下驯化获得的生物阴极中，优势菌种（占文库比例为74.7%）的16S rRNA基因与Enterococcus aquimarinus LMG16607最为相似，Enterococcus异养生长的特性与有机碳源的存在有着密切关系。在生物阳极和生物阴极构建的生物电化学系统中，提升外加电压能够增加硝基苯还原的表观一级动力学常数（kNB），但同时也会增加硝基苯还原的比能耗并降低电流效率。增加有机碳源葡萄糖的浓度也能提高kNB，在葡萄糖浓度较低的范围内（≤200mg/L），单位葡萄糖浓度增量引起的kNB增量是其浓度较高范围内（≥200mg/L）的5.1倍。为避免过度增加体系中的COD，有机碳源的引入量控制在200mg/L左右较为适宜，此时在各外加电压条件下（0.15V~0.5V），苯胺的生成率均超过97%，硝基苯还原一级动力学常数较无有机碳源时平均提高52±6%。
将阳极暴露于空气中的操作方式可以促进阳极电化学活性微生物从苯胺回收电子和能量。通过不同供氧方式的实验，证实这种能力与氧气的存在密切相关。阳极室顶空氧气浓度对苯胺的电子和能量回收效能有着明显的影响。在考察的氧气浓度范围内（21%~100%），顶空氧气含量为70%的时候能够获得最高的输出电流（0.155mA）和最大功率密度（3.32±0.4W/m3）。但库伦效率随氧气浓度的增加从10.32±1.36%降低至4.61±1.11%。根据GC-MS对苯胺代谢产物的分析以及结合间歇性断路实验结果，作者推测了苯胺在生物阳极代谢并转化为电极电子的方式，即阳极液和阳极生物膜外层的微生物在有氧的条件下将苯胺转化为有机酸，阳极生物膜内层的电化学活性微生物在缺氧条件下利用产生的有机酸作为底物产电。通过循环伏安分析，表明阳极电化学活性微生物将电子传递给阳极的过程主要通过游离性电子中介体介导。推定的中介体在循环伏安曲线上表现出两对可逆的氧化还原峰，中点电位分别为-0.027V和0.063V。
根据本文中苯胺在生物阳极氧化和硝基苯在生物阴极还原的实验结果，并结合热力学以及化学计量学的分析，作者提出了利用苯胺回收电子部分反哺硝基苯还原的工艺模式。计算表明，不同操作条件下，利用苯胺回收电子反哺硝基苯还原可以节约22%~53%的额外电子供体需求，并内部循环补偿17%~40%的能量消耗。
Nitrobenzene (NB), one of typical recalcitrant organic compounds, is reportedas possible mutagens, teratogens or carcinogens and has been listed in58China’spriority control organic pollutants. As the electrophilic effect of nitryl decreases theelectron density of the benzene, NB is only to a limited extent degraded in aerobicbiological processes. An effective strategy is to transform NB to aniline (AN) first,which is considerably easier mineralized than NB. The electrochemical reduction ofNB is an effieicent and controlable approach, however, the conventional processsuffer from the requirement of special electrode materials or noble metal catalysts toachieve the selective transformation of NB to AN, which limited its application inpractice. Recent develped bioelectrochemical system (BES) is proposed as aprespective process in wastewtaer treatment, since bacteria, as catalyst, hold theinherent advantages of low-cost, self-regeneration and evironment-friendly. Thepresent study demonstrated bacteria can use the cathode as the sole electron donor toselective reduce NB to AN. The introduction of organic carbon to biocathode furtherenhanced this selective transformation capability. Based on above findings, thestudy went on developing novel process that enabled energy internal loopcompensation by recovery energy from aniline at bioanode. The improtant role ofoxygen in the process was demonstrated and the involved mechanism was thandiscussed.
When cathode was served as sole electron donor, the transformation efficiencyand rate from NB to AN was dramatically increased with microbial catalysis.93%of AN formation efficiency was achieved in biocathode at the cathode potential of-0.4V, which was6.16times as high as that obtained in abiotic cathode. In addition,the maximum accumulation of toxic nitrosobenzene (NOB), the intermediate duringNB reduction to AN, was decreased by38.7%in biocathode compared to that inabiotic cathode. Cyclic voltammetry (CV) revealed that an unidentified redoxcompound with the midpoint potential around-0.315V could be responsible for theelectron transfer from cathode to bacteria. Based on the apparent first-order kineticmodel, biocathode was suggested to both catalyze the reduction of NB and NOB.The corresponding apparent first-order kinetic constant was increased by62%and100%, respectively. Moreover, the community of the inoculum and the biocathodesample was analyzed based on the pyrosequencing. The results indicated microbialcommunity was changed dramatically under the evolutionary pressure provied bycathode severved as sole electron donor. The predominant bacteria in biocathodecommunity consisted of Rhizobium sp., Leucobacter sp., Achromobacter sp., Mycobacterium sp., Dysgonomonas sp. and Pseudomonas sp.. The functions ofselective nitrobenzene reduction, carbion fixiation and the possible catodicextracellular electron tranfer in the microbial community might feature the catalyzedselective reduction of NB to AN in biocathode.
Biocathode enrichied with the present of organic carbon (glucose) showedbetter performance on selective reduction of NB, in which NOB was rarelyaccumulated. Cyclic voltammetry revealed NB reduction peak was positively shiftedby70mV with the present of organic carbon, indicating the enhanced performancewas not only caused by the extra electrons donation from glucose but also theimprovement of bioelectrocatalytic activity.16S rRNA based analysis of the biofilmon the cathode indicated that the cathode was dominated by an Enterococcus species(occupied74.7%of the library) closely related to Enterococcus aquimarinus LMG16607. In BES coupled with bioanode and biocathode, the apparent first-orderkinetic constant (kNB) was increased with the increase of applied voltage, butconsumed more energy and decreased the current efficiency. kNBwas also increasedby introducing more glucose in to catholyte, kNBincrement caused by net incrementof glucose in lower concentration range (≤200mg/L) was5.1times as high as thatin higher glucose concentration range (≥200mg/L). To avoid the excess increase ofCOD in wastewater, the suggested concentration of added organic carbon was200mg/L, which resulted in AN formation efficiency over97%at all tested appliedvoltages (0.15V~0.25V) and averagely increased the apparent first-order kineticconstant by52±6%compared to that without organic carbon.
The recovery of electron and energy from AN was found to be facilitated byexposing bioanode to air, which was then further demonstrated to be depending onthe present of oxygen in limited oxygen donation test. Oxygen content in the anodehead space was found to impact AN degradation and the elelctrons recoverysignificantly. The highest output current (0.155mA) and maximum power density(3.32±0.4W/m3) was observed when70%of gas in anode headspace was composedby oxygen at the beginning. However, columbic efficiency decreased with theoxygen concentration increasing from10.32±1.36%to4.61±1.11%in the testedinitial oxygen content conditions (21%~100%). Based on the results of GC-MS andthe intermittent open circuit experiment, the possible mechanism using AN as thesole electron donor in present of oxygen was suggested as following. AN was firstaerobicly converted to organic acids by microbes in the anolyte and located at theouter layer of anodic biofilm, which was then uptook by electrochemical activebacteria located at the inner layer of anodic biofilm and produce current underanoxic condition. CV indicated that anodic extracellular electron tranfer wasfulfilled by certain microbial secreted soluble electron mediator, which has tworeversible redox peaks with the mid-point potentials of-0.027V and0.063V, respectively.
To achieve the energy internal loop compensation, the process of NB reductionwith electrons partial fed-back from AN oxidation was proposed. The saved externalelectron donor and internal loop compensated energy was estimated as22%~53%and17%~40%, respectively, based on the experiment results and the thermodynamicand stoichiometric calculation.
引文
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