对硝基酚（PNP）降解菌在废水处理及土壤污染修复中应用
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摘要
近年来，环境污染和安全问题日益成为关注的焦点，如何有效地减轻避免污染物对环境及人类健康的损害已成为亟待解决的问题。随着化学工业的发展，大量有毒难降解有机物随工业废水的排放进入环境，这些物质能在自然环境中长期存在、积累和扩散，对动植物的生存及人类的健康造成不良影响。目前，对废水中有毒难降解有机物的控制是水污染防治中的重要课题之一。硝基酚类化合物是重要的有毒难降解有机物，被广泛用于农药、医药、染料、炸药以及橡胶工业生产中，生物修复（Bioremedition）技术主要是利用生物有机体，尤其是微生物的降解作用将污染物分解并最终无害化的过程，具有高效快速、经济实用、安全简便等优点。国内外许多研究证实，微生物降解法在废水处理和土壤生物修复等领域具有广阔的开发应用前景。
但是关于废水中硝基酚生物降解的研究，还存在多方面的缺陷，对于硝基酚的生物毒性和降解性不同，很多学者所得的结论也有较大差别，有些结论甚至相反。多数学者致力于研究硝基酚的生物降解，但将硝基酚降解菌分别加到废水及污染土壤中的报道确很少。本研究利用富集培养法，从吉化污水处理厂氧化沟的活性污泥中，筛选并获得多株能够有效降解对硝基酚（PNP）的细菌菌株，其中菌株Z-1具有较好的降解性能，研究内容如下：
（1）通过驯化纯化培养，分离筛选出多株能以PNP为唯一碳源和能源生长的细菌，最终确定细菌菌株Z-1为最适菌株，并将其通过PCR鉴定，据16S rDNA序列分析鉴定红球菌属。最佳降解条件的确定为：在以PNP为唯一碳源的条件下，接种量4%-8%、温度15~35°C、pH6~11、PNP浓度在100~500mg/L的条件下，菌株的降解效果较好，且菌株能够耐受PNP的浓度为1000mg/L。
（2）确定了高效降解菌的代谢途径，主要是通过两种方式，本文为偏苯三酚径代谢PNP。细菌Z-1对PNP的降解主要受以下环境因素影响：初始浓度，pH值和温度等因素。在最佳操作条件下，废水中PNP的去除率达到90%以上。
（3）采用PVA和海藻酸钠的混合物为载体，通过正交实验确定出制备降酚菌株Z-1固定化的最优条件为：PVA为90g/L，海藻酸钠质量浓度为1.0%，H3BO3+CaCl2质量浓度为4.0%+3.0%，包埋剂与菌液体积比为30:1。钙化时间为12h，按此条件制成的固定化小球具有良好的降解性能和机械强度。
（4）确定了固定化小球降解对硝基酚废水的最佳条件，即接菌8%、温度为30~35°C、pH为6~8，摇床转速为120~160r/min。在单因素影响下，固定化小球对对硝基酚废水（PNP）中PNP的降解率均高于游离降解菌。与未固定化的Z-1相比，在温度、盐度、pH值等各方面都有一定的耐受性。这表明固定化细胞的耐受程度较好，原因在于载体的表面积增大了降解菌与污染物的接触面积，而载体的加入又保护了降解菌，避免其在降解过程中的损失减少了环境的抑制作用，从而提高固定化小球的降解性能。
（5）固定化小球在富集培养基中培养3d后在恒温摇床上以120r/min速度连续振荡5h，小球完全没有破碎现象，固定化小球的机械强度比较高，说明能够承受一定的废水水流的冲击力。而将制备好的固定化小球分别放入一定量的孔雀蓝和结晶紫溶液中，孔雀蓝和结晶紫色素可以在30min就可以到达大部分小球中心，说明固定化小球还具有良好的传质性能。
（6）将筛选纯化的菌株Z-1投加到预先采集的土壤当中，室内模拟微生物降解土壤污染物环境，对于土壤中PNP微生物处理的初期，各浓度PNP对土壤中的Z-1产生明显的抑制作用。而30d后100mg/kg条件中土壤细菌Z-1对土壤氮源、碳源的利用能力都有所增强，Z-1明显加快了土壤中PNP的降解，PNP对土壤微生物的抑制也在逐步减缓，说明Z-1的加入则能恢复并提高土壤微生物多样性，平衡土壤中土著菌种和污染物的关系，接种细菌Z-1进行生物的强化是解决PNP污染土壤修复的一种有效的方法。但是，在土壤PNP降解过程中，500mg/kg土样中土壤酶的活性也随着PNP浓度的变化而呈现抑制趋势。微生物在土壤中PNP的降解过程中对于土壤环境及微生物的影响由于土壤污染物浓度的不同，土壤酶有不同的表达。
In recent years, environmental pollution and security issue have become the focus of theattention, how to effectively avoid pollutants that would cause damage of human healthdeposit into the environment has become an urgent problem that needs to be solved. As thedeveloping of the chemical industry, a large amount of toxic and refractory organics inindustrial wastewater have been discharged into the environment. The substances which canexist in a long-term in the natural environment have adverse impact on animal, plant andhuman health. At present, the control of toxic and refractory organics in wastewater is animportant issue of the Water-Pollution-Control. The nitro phenol (p-Nitrophenol, PNP) is oneof the important raw materials in industries, such as chemical, printing, dyeing, medicine andexplosives, and it is a long term residual pollutant in soil and water of the environment whichpose a threat to plants, animals and human health. The nitro phenol used in the productionprocess could be carried into environment with the discharge of industrial wastewater.Bioremedition technology is the mainly use biological organisms to the treat pollutants, andeventually the pollutants would be contaminated decomposition of harmless process, and theadvantage is about it is that it is quickly, efficiently, economical, practical, safety and simple.Some scholars are dedicated to the study of biological degradation of nitro phenol, but thePNP in the soil pollution reports really little. In this paper, enrichment culture method andsewage treatment plant oxidation ditch were used, and the efficient bacterial strains of PNPdegradation were screened from the activated sludge. There are many bacterial strains thatwere screened from the activated sludge can effectively degradation of nitro phenol (PNP),but among all the strains Z-1in all the strains had the best degradation performance, theresults are as follows:
(1) PNP as the only carbon source and energy of the bacteria growth, through thephysiological and biochemical characteristics, and ultimately the bacterial strain Z-1wasdetermined for optimal strain, and identified by PCR, according to16S rDNA sequenceanalysis, strain Z-1was identified as Rhodococcus sp.. The optimal degradation conditionswere inoculation amount4%~8%, temperature15~35°C, pH6~11, PNP concentration100~500mg/L. Under the optimal condition, the degradation effect was better, and the straincould tolerate the PNP concentration of1000mg/L.
(2) The metabolic pathway of high efficiency degradation bacterium was confirmed,mainly through two kinds of ways, and the4-nitro catechol way was conformed to degradePNP in this paper. The degradation performance of the immobilized bacterium Z-1mainlyeffected by environmental factors: the initial concentration, pH, temperature and other factors.Under the optimal operation conditions, the remove of the PNP in wastewater reached above90%.
(3) The mixture of PVA and sodium alginate was used as the carrier, and orthogonalexperiment was used to determine immobilized optimal conditions of bacterium Z-1. Theresult are as following: PVA was90g/L, sodium alginate concentration was1.0%, H3BO3+ CaCl2concentrations were4.0%+3.0%, the proportion of embedding agent and bacteria liquidwas30:1, calcification time was12h, the embedding balls were made by the conditions abovehad the optimal degradation effectiveness and mechanical properties.
(4) The degradation conditions of PNP wastewater for the embedding balls to wasdetermined. The optimal conditions were inoculation amount8%, temperature30~35°C,pH6~8, rotation peed120~160r/min. In the single factor effect, the degradation efficiency ofPNP wastewater by immobilized bacterium was higher than the free bacterium. Thissuggested that the tolerance of immobilized cells was better, compared to the free bacterium,in the salinity, temperature, pH and other conditions. The reason is that the surface of thecarrier increased the contact area, and also protected the bacterium in the embedding ballsfrom the toxicity of the substrate, so that the degradation performance of immobilized ballwas improved.
(5) Immobilized balls in enrichment medium trained after3d in constant temperature and120r/min speed continuous oscillation5h, the balls had completely no broken phenomenon,which indicated that the mechanical strength of the immobilized balls was high could affordthe impact of the wastewater streams. Putting the immobilized balls into the solution ofcertain amount of peacock blue and crystal violet, the peacock blue and crystal violet couldreach the centre of the balls in30min which also indicated that immobilized balls had anexcellent transfer performance of.
(6) The degradation of PNP in soil was studied under the simulating conditions. PNPcaused obvious inhibition of the microorganism in early stage. After30d of degradation, thecarbon source using ability was significantly enhanced. The degradation speed of Z-1of PNPin the soil was also accelerated. The PNP inhibition to soil microbes was eliminated. PNPcould reduce microbial diversity in the soil, but the addition of Z-1could restore andimproved soil microbial diversity, which indicated that adding Z-1to the soil environmentwas an effective way to restore the PNP polluted soil. The relationship of indigenous bacteriaand pollutants had a balance in the soil. Inoculated with bacteria Z-1was a solution to aneffective remediation method of PNP contaminated soil. In the degradation process, thedifferent soil enzyme activity was accompanied by changes in the PNP concentration.Meanwhile, the results showed a different trend of microorganisms in the soil during thedegradation of the PNP in the soil environment. The influence of micro-organisms played notonly the existence of a catalytic role, but also came with the inhibition.
但是关于废水中硝基酚生物降解的研究，还存在多方面的缺陷，对于硝基酚的生物毒性和降解性不同，很多学者所得的结论也有较大差别，有些结论甚至相反。多数学者致力于研究硝基酚的生物降解，但将硝基酚降解菌分别加到废水及污染土壤中的报道确很少。本研究利用富集培养法，从吉化污水处理厂氧化沟的活性污泥中，筛选并获得多株能够有效降解对硝基酚（PNP）的细菌菌株，其中菌株Z-1具有较好的降解性能，研究内容如下：
（1）通过驯化纯化培养，分离筛选出多株能以PNP为唯一碳源和能源生长的细菌，最终确定细菌菌株Z-1为最适菌株，并将其通过PCR鉴定，据16S rDNA序列分析鉴定红球菌属。最佳降解条件的确定为：在以PNP为唯一碳源的条件下，接种量4%-8%、温度15~35°C、pH6~11、PNP浓度在100~500mg/L的条件下，菌株的降解效果较好，且菌株能够耐受PNP的浓度为1000mg/L。
（2）确定了高效降解菌的代谢途径，主要是通过两种方式，本文为偏苯三酚径代谢PNP。细菌Z-1对PNP的降解主要受以下环境因素影响：初始浓度，pH值和温度等因素。在最佳操作条件下，废水中PNP的去除率达到90%以上。
（3）采用PVA和海藻酸钠的混合物为载体，通过正交实验确定出制备降酚菌株Z-1固定化的最优条件为：PVA为90g/L，海藻酸钠质量浓度为1.0%，H3BO3+CaCl2质量浓度为4.0%+3.0%，包埋剂与菌液体积比为30:1。钙化时间为12h，按此条件制成的固定化小球具有良好的降解性能和机械强度。
（4）确定了固定化小球降解对硝基酚废水的最佳条件，即接菌8%、温度为30~35°C、pH为6~8，摇床转速为120~160r/min。在单因素影响下，固定化小球对对硝基酚废水（PNP）中PNP的降解率均高于游离降解菌。与未固定化的Z-1相比，在温度、盐度、pH值等各方面都有一定的耐受性。这表明固定化细胞的耐受程度较好，原因在于载体的表面积增大了降解菌与污染物的接触面积，而载体的加入又保护了降解菌，避免其在降解过程中的损失减少了环境的抑制作用，从而提高固定化小球的降解性能。
（5）固定化小球在富集培养基中培养3d后在恒温摇床上以120r/min速度连续振荡5h，小球完全没有破碎现象，固定化小球的机械强度比较高，说明能够承受一定的废水水流的冲击力。而将制备好的固定化小球分别放入一定量的孔雀蓝和结晶紫溶液中，孔雀蓝和结晶紫色素可以在30min就可以到达大部分小球中心，说明固定化小球还具有良好的传质性能。
（6）将筛选纯化的菌株Z-1投加到预先采集的土壤当中，室内模拟微生物降解土壤污染物环境，对于土壤中PNP微生物处理的初期，各浓度PNP对土壤中的Z-1产生明显的抑制作用。而30d后100mg/kg条件中土壤细菌Z-1对土壤氮源、碳源的利用能力都有所增强，Z-1明显加快了土壤中PNP的降解，PNP对土壤微生物的抑制也在逐步减缓，说明Z-1的加入则能恢复并提高土壤微生物多样性，平衡土壤中土著菌种和污染物的关系，接种细菌Z-1进行生物的强化是解决PNP污染土壤修复的一种有效的方法。但是，在土壤PNP降解过程中，500mg/kg土样中土壤酶的活性也随着PNP浓度的变化而呈现抑制趋势。微生物在土壤中PNP的降解过程中对于土壤环境及微生物的影响由于土壤污染物浓度的不同，土壤酶有不同的表达。
In recent years, environmental pollution and security issue have become the focus of theattention, how to effectively avoid pollutants that would cause damage of human healthdeposit into the environment has become an urgent problem that needs to be solved. As thedeveloping of the chemical industry, a large amount of toxic and refractory organics inindustrial wastewater have been discharged into the environment. The substances which canexist in a long-term in the natural environment have adverse impact on animal, plant andhuman health. At present, the control of toxic and refractory organics in wastewater is animportant issue of the Water-Pollution-Control. The nitro phenol (p-Nitrophenol, PNP) is oneof the important raw materials in industries, such as chemical, printing, dyeing, medicine andexplosives, and it is a long term residual pollutant in soil and water of the environment whichpose a threat to plants, animals and human health. The nitro phenol used in the productionprocess could be carried into environment with the discharge of industrial wastewater.Bioremedition technology is the mainly use biological organisms to the treat pollutants, andeventually the pollutants would be contaminated decomposition of harmless process, and theadvantage is about it is that it is quickly, efficiently, economical, practical, safety and simple.Some scholars are dedicated to the study of biological degradation of nitro phenol, but thePNP in the soil pollution reports really little. In this paper, enrichment culture method andsewage treatment plant oxidation ditch were used, and the efficient bacterial strains of PNPdegradation were screened from the activated sludge. There are many bacterial strains thatwere screened from the activated sludge can effectively degradation of nitro phenol (PNP),but among all the strains Z-1in all the strains had the best degradation performance, theresults are as follows:
(1) PNP as the only carbon source and energy of the bacteria growth, through thephysiological and biochemical characteristics, and ultimately the bacterial strain Z-1wasdetermined for optimal strain, and identified by PCR, according to16S rDNA sequenceanalysis, strain Z-1was identified as Rhodococcus sp.. The optimal degradation conditionswere inoculation amount4%~8%, temperature15~35°C, pH6~11, PNP concentration100~500mg/L. Under the optimal condition, the degradation effect was better, and the straincould tolerate the PNP concentration of1000mg/L.
(2) The metabolic pathway of high efficiency degradation bacterium was confirmed,mainly through two kinds of ways, and the4-nitro catechol way was conformed to degradePNP in this paper. The degradation performance of the immobilized bacterium Z-1mainlyeffected by environmental factors: the initial concentration, pH, temperature and other factors.Under the optimal operation conditions, the remove of the PNP in wastewater reached above90%.
(3) The mixture of PVA and sodium alginate was used as the carrier, and orthogonalexperiment was used to determine immobilized optimal conditions of bacterium Z-1. Theresult are as following: PVA was90g/L, sodium alginate concentration was1.0%, H3BO3+ CaCl2concentrations were4.0%+3.0%, the proportion of embedding agent and bacteria liquidwas30:1, calcification time was12h, the embedding balls were made by the conditions abovehad the optimal degradation effectiveness and mechanical properties.
(4) The degradation conditions of PNP wastewater for the embedding balls to wasdetermined. The optimal conditions were inoculation amount8%, temperature30~35°C,pH6~8, rotation peed120~160r/min. In the single factor effect, the degradation efficiency ofPNP wastewater by immobilized bacterium was higher than the free bacterium. Thissuggested that the tolerance of immobilized cells was better, compared to the free bacterium,in the salinity, temperature, pH and other conditions. The reason is that the surface of thecarrier increased the contact area, and also protected the bacterium in the embedding ballsfrom the toxicity of the substrate, so that the degradation performance of immobilized ballwas improved.
(5) Immobilized balls in enrichment medium trained after3d in constant temperature and120r/min speed continuous oscillation5h, the balls had completely no broken phenomenon,which indicated that the mechanical strength of the immobilized balls was high could affordthe impact of the wastewater streams. Putting the immobilized balls into the solution ofcertain amount of peacock blue and crystal violet, the peacock blue and crystal violet couldreach the centre of the balls in30min which also indicated that immobilized balls had anexcellent transfer performance of.
(6) The degradation of PNP in soil was studied under the simulating conditions. PNPcaused obvious inhibition of the microorganism in early stage. After30d of degradation, thecarbon source using ability was significantly enhanced. The degradation speed of Z-1of PNPin the soil was also accelerated. The PNP inhibition to soil microbes was eliminated. PNPcould reduce microbial diversity in the soil, but the addition of Z-1could restore andimproved soil microbial diversity, which indicated that adding Z-1to the soil environmentwas an effective way to restore the PNP polluted soil. The relationship of indigenous bacteriaand pollutants had a balance in the soil. Inoculated with bacteria Z-1was a solution to aneffective remediation method of PNP contaminated soil. In the degradation process, thedifferent soil enzyme activity was accompanied by changes in the PNP concentration.Meanwhile, the results showed a different trend of microorganisms in the soil during thedegradation of the PNP in the soil environment. The influence of micro-organisms played notonly the existence of a catalytic role, but also came with the inhibition.
引文
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