【作者】 王孟；

【导师】 王国英；

【作者基本信息】 太原理工大学 ， 建筑与土木工程， 2017， 硕士

【摘要】 随着社会工业化进程的加速,水污染问题日益加剧,水中氮素含量日益升高。水体富营养化问题困扰着人类的健康和水生植物的生长。因此,水体中氮素的去除一直以来都是水处理领域研究的热点问题。由于传统脱氮工艺技术存在着短板与不足,新型脱氮工艺和技术的研发一直是国内外学者的研究方向。研究发现,一些异养型硝化菌能够在有氧的情况下进行反硝化作用,由此诞生了异养硝化-好氧反硝化这种新型的生物脱氮技术,使硝化和反硝化能在同一装置中进行,同时去除污水中的碳和氮,并且将氮素转变为气态氮排出水体,从而解决了传统废水脱氮工艺在经济效益与处理效率两方面的矛盾。Diaphorobacter sp.PDB3从焦化废水处理系统的活性污泥中分离纯化得到,在前期的研究中已证实菌株PDB3具有异养硝化-好氧反硝化的性能。本文首先通过单因素试验研究了碳源种类、碳氮比、溶解氧、pH、培养温度等因素对菌株脱氮性能的影响,利用响应面法（RSM）优化了培养条件;在此基础上,研究了菌株的异养硝化以及好氧反硝化性能,通过酶活性分析、动力学分析、以及不同条件下的反硝化性能分析,对菌株的生长条件以及脱氮机制有了进一步的了解。单因素试验表明,Diaphorobacter sp.PDB3生长代谢所需的最佳碳源是琥珀酸钠,不能利用乙醇碳源。碳氮比、溶解氧、pH、培养温度会对菌体的异养硝化-好氧反硝化过程产生较为明显的影响。采用响应面法对影响菌体生长和氨氮降解率的因素进行研究,通过对拟合回归模型方差分析,对碳氮比和温度、碳氮比和摇床转速、温度和摇床转速之间对氨氮降解率的两两交互影响的分析,确定菌株降解氨氮的最佳工艺参数为:碳氮比10.64、温度28.69℃、摇床转速128.91r/min、ph值7.2、接种量5%、钙离子0.1mmol/l、镁离子0.1mmol/l、铁离子0.1mmol/l。验证试验表明,在优化后的培养基中氨氮降解率比优化前提高了8.68%。菌体在最优条件下的生长曲线表明,细胞浓度于21h达到最大值,此时od600达到1.439,比生长速率最大达到0.302h-1,在异养硝化过程中检测到少量的硝氮和亚硝氮的存在,之后逐渐降低。菌株PDB3在高浓度氨氮废水中生长良好,在菌体处于对数生长期时氨氮和总氮得到快速降解,氨氮的降解与菌体的生长有直接的关系;适当提高氨氮浓度可以促进菌体的生长并提高氨氮降解率。氮平衡分析表明,胞内氮由0.72mg/l增加到52.12mg/l,增长量占到氨氮去除量的49.41%;氮损失为45.09mg/l,占氨氮去除量的42.74%,这部分氮由气态氮的形式排出系统,由此可知,细胞同化作用和异养硝化-好氧反硝化作用为菌株脱氮的主要形式。酶活分析表明,菌株PDB3在生长过程中,羟氨氧化酶、硝酸盐还原酶和亚硝酸盐还原酶均可检测到,证明了菌株PDB3的硝化与反硝化途径是偶联的。结合整个硝化反硝化过程,可知diaphorobactersp.PDB3的脱氮途径为:NH₄^?-N→NH₂OH-N→NO₂^￣-N→NO₃^￣-N→N₂。菌株PDB3对氨氮的降解符合一级反应动力学模型,最大比生长速率max为0.365h-1,氨氮最大比降解率为1.724mg/（l·h）,ks为420.51mg/l。Diaphorobactersp.PDB3能够分别以亚硝氮和硝氮为唯一氮源进行菌体生长和反硝化作用,反应过程未检测到羟胺,均检测到氨氮,推测在反硝化过程中菌株将部分亚硝氮和硝氮转变为氨氮,供其生长代谢。当反硝化培养基中氮源浓度为200 mg/L,C/N为10时,菌株PDB3的反硝化降解率可达60%左右,且菌株可利用较高浓度的硝氮和亚硝氮进行生长代谢。菌株在进行反硝化时所需的碳氮比更高,这可能是由于菌体不能直接利用亚硝氮和硝氮作氮源,需先将其转变为氨氮,这个过程需要碳源来提供能量。随着碳氮比的升高,菌体反硝化作用越强,当碳氮比为15时,菌株对100 mg/L的亚硝氮降解率达到96.98%,可完全去除100 mg/L的硝氮。更多还原

【Abstract】 With the acceleration of social industrialization process,the problem of water pollution become more severe,and the nitrogen content in water is increasing.Water eutrophication problems plague the health of human beings and the growth of aquatic plants.Therefore,the removal of nitrogen in water has always been a hot issue in the field of water treatment.Due to the shortcomings of traditional denitrification technology,the research and development of new denitrification technology has been a direction of domestic and foreign scholars.Studies have found that some heterotrophic nitrifying bacteria can be denitrified in the presence of oxygen.The new biological denitrification technology,which is heterotrophic nitrification-aerobic denitrification,was developed.This allows nitrification and denitrification to be carried out in the same device while removing carbon and nitrogen from the wastewater and converting nitrogen into gaseous nitrogen.Thus solving the conflict of economic efficiency and processing efficiency in the traditional waste water denitrification process.Diaphorobacter sp.PDB3 was isolated and purified from the activated sludge of the coking wastewater treatment system.It was confirmed that the strain PDB3 had heterotrophic nitrification-aerobic denitrification.In this paper,the effects of carbon source,carbon nitrogen ratio,dissolved oxygen,pH and culture temperature on the denitrification were studied by single factor experiment.The culture conditions were optimized by response surface methodology（RSM）.On this basis,the nitrification and aerobic denitrification properties of the strain were analyzed.The enzymatic activity analysis,kinetic analysis and denitrification performance analysis under different conditions were used to understand the growth conditions and denitrification mechanism of the strain.Single factor test showed that the optimum carbon source for the growth and metabolism of Diaphorobacter sp.PDB3 is sodium succinate.The strain can not use ethanol as carbon source.The C/N,dissolved oxygen,pH and culture temperature had a significant effect on the heterotrophic nitrification-aerobic denitrification process.The factors affected the cell growth and ammonia nitrogen degradation rate were studied by response surface methodology.The influence of C/N and temperature,C/N and shaking speed,temperature and shaking speed on the degradation rate of ammonia nitrogen were analyzed by the variance analysis of the fitting regression model.The optimum process parameters were as follows: C/N 10.64,temperature 28.69 ℃,shaking speed 128.91 r/min,pH 7.2,inoculation 5%,calcium ion 0.1 mmol/L,magnesium ion 0.1 mmol/L and iron ion 0.1 mmol/L.The results showed that the degradation rate of ammonia in the optimized medium was 8.68% higher than that before optimization.The growth curve of the cells under the optimal conditions showed that the cell concentration reached the maximum at 21 h,and the OD600 reached 1.439.The maximum growth rate was 0.302 h-1.A small amount of nitrate and nitrite nitrogen were detected and then gradually reduced during heterotrophic nitrification.The strain PDB3 grew well in the high concentration of ammonia nitrogen wastewater,and the ammonia nitrogen and total nitrogen were rapidly degraded when the cells were in the logarithmic growth phase.The degradation of ammonia nitrogen was directly related to the growth of the bacteria.The proper increase of ammonia concentration could promote the cell growth and increase the ammonia nitrogen degradation rate.Nitrogen balance analysis showed that intracellular nitrogen increased from 0.72 mg/L to 52.12 mg/L,the growth accounted for 49.41% of ammonia nitrogen removal.The nitrogen loss was 45.09 mg/L,accounting for 42.74% of ammonia removal,which was the gaseous nitrogen overflowing the system.It can be seen that cell assimilation and heterotrophic nitrification-aerobic denitrification are the main forms of strain denitrification.According to the enzyme analysis,hydroxylamine oxidase,nitrate reductase and nitrite reductase could be detected during the growth of strain PDB3.The nitrification and denitrification pathways of the strain PDB3 were also confirmed.Combined with the nitrification and denitrification process,it can be seen that the denitrification pathway of Diaphorobacter sp.PDB3 is: NH₄?-N→NH₂OH-N→NO₂?-N→NO₃?-N→N₂.The degradation of ammonia nitrogen by strain PDB3 was in accordance with the first order reaction kinetics model.The maximum specific growth rate μmax was 0.365 h-1,the maximum specific degradation rate of ammonia nitrogen was 1.724 mg/（L·h）,and the KS was 420.51 mg/L.Diaphorobacter sp.PDB3 could grow and denitrify with nitrite nitrogen and nitric nitrogen as the sole nitrogen source.No hydroxylamine was detected during the reaction,and ammonia nitrogen was detected,witch was presumed that the nitrite and nitric nitrogen in the denitrification process will be converted into ammonia nitrogen for its growth and metabolism.When the nitrogen concentration in the denitrification medium was 200 mg/L and the C/N was 10,the denitrification rate of the strain PDB3 was about 60%,and the strain can grow and metabolize with the higher concentration of nitrate and nitrite nitrogen.The C/N ratio required for denitrification is higher.This may be due to the fact that the cells can not directly utilize nitrite and nitrate nitrogen as nitrogen sources and need to be converted to ammonia nitrogen.This process requires a carbon source to provide energy.With the increase of C/N ratio,the degradation rate of 100 mg/L nitrite was 96.98%,and 100 mg/L nitrite was completely removed when the C/N ratio was 15.更多还原