【作者】 徐佳；

【导师】 安强； 苟基佐；

【作者基本信息】 重庆大学 ， 工程（环境工程）（专业学位）， 2019， 硕士

【摘要】 随着城市化进程加快以及工业、农业的快速发展,人类在生产和生活活动中排出的含各种类型污染物的废水,如有机类污染、无机物类污染以及重金属类污染等,这些含污染物的废水排入水体极易形成复合污染。复合污染不仅破坏水体生态环境,还对人体健康带来巨大的危害。水体中硝酸盐氮形成的无机类污染和Cr（Ⅵ）形成重金属类污染是较为常见的复合污染之一,在现有污水处理过程中,分别针对硝酸盐氮和Cr（Ⅵ）污染的处理方法研究较多,传统的去除硝酸盐氮的方法有膜分离法、离子交换法、氧化还原法、催化还原法以及微生物法等,主要常见的去除Cr（VI）方法主要有吸附法、溶剂萃取分离法以及化学沉淀法等,但上述方法仅针对于某一类污染物引起的污染。如何利用有效的措施经济节能、安全环保的去除水体中硝酸盐氮和Cr（Ⅵ）带来的复合污染受到人们的极大关注。微生物法在处理环境污染问题具有巨大的潜力,本文重点探求一种能在好氧反硝化同时还原Cr（Ⅵ）的细菌,用于处理硝酸盐氮和Cr（Ⅵ）对环境造成的复合污染问题。本研究的目的是研究菌株同时还原Cr（Ⅵ）和硝酸盐的能力及其反应代谢机理,使分离筛选出的细菌在实际污水处理中发挥作用,为提高污水的硝酸盐和铬处理效率提供理论依据。本文涉及的好氧反硝化菌株分离自重庆市某污水处理厂氧化沟工艺二沉池的活性污泥,在实验室驯化培养后开展了如下研究:对菌株G12进行16S rRNA基因鉴定;研究初始Cr（Ⅵ）浓度对好氧反硝化菌脱氮效率的影响以及硝酸盐浓度对Cr（Ⅵ）还原的影响;研究各种影响因素,如碳源、氮源、接种量以及共存二元重金属离子等对菌株在硝酸盐和Cr（Ⅵ）还原过程的影响;利用FTIR、XRD以及XPS等仪器分析研究Cr（Ⅵ）及其还原产物,从而研究Cr（Ⅵ）还原与好氧反硝化同时代谢机理。研究结果表明:（1）在反硝化固体培养基上,菌落表面边缘整齐,表面呈光滑、湿润、不透明、浅黄胶状。利用16S rRNA基因测序,确定该菌株G12属于Pseudomonas aeruginosa strain（铜绿假单胞菌属）,并命名为Pseudomonas aeruginosa strain G12。将菌株G12以1%（v/v）的比例接种到含500 mg/L的硝酸盐氮、10 mg/L的Cr（Ⅵ）的好氧反硝化培养基中,再以30℃、120 rpm的条件连续培养96 h。菌株G12在48 h进入生长稳定期,其OD₆₀₀值达到最大为1.7左右。硝酸盐氮在72 h左右去除率达到98%,基本被完全去除,于96 h硝酸盐浓度降低到了10 mg/L;在96 h时,Cr（Ⅵ）的还原率达到93.32%,上清液中Cr（Ⅵ）的浓度约为0.668 mg/L。（2）在初始硝酸盐浓度为100、300、500、700 mg/L时,培养72 h以后,硝酸盐基本全部被还原,还原率分别为95.8%、99.5%、97.2%、70.3%;当硝酸盐浓度初始浓度为500 mg/L时,菌株G12对1、5、10、15、20 mg/L的Cr（Ⅵ）还原转化率分别为86.6%、94.9%、98.4%、99.1%、99.2%,相应的对硝酸盐氮的还原率分别为96%、96.5%、97.2%、96.7%、97.4%;Cr（Ⅵ）的还原效果与培养基中的硝酸盐浓度成正相关;Cr（Ⅵ）的还原和硝酸盐还原是同时进行的,集中在菌株G12的对数增长期。（3）不同的环境影响因子对菌株G12的代谢影响实验结果表明:（1）在不同碳源条件下,菌株G12的生长量以及对硝酸盐氮和Cr（Ⅵ）的去除效果不同,不同碳源对菌株G12代谢影响的排序分别为:琥珀酸钠﹥柠檬酸钠﹥葡萄糖﹥乙酸钠,柠檬酸三钠较琥珀酸钠而言更廉价易得,在不影响实验效果的情况下,选用柠檬酸钠作为碳源;（2）在分别以氨氮、硝酸盐氮和亚硝酸盐氮为唯一氮源的情况下,不同氮源对菌株G12代谢影响的排序分别为:硝酸盐氮﹥氨氮﹥亚硝酸盐氮,故选用硝酸盐作为氮源进行后续研究;（3）接种量分别为1%、5%和10%（v/v）,连续培养72 h后,菌株G12的OD₆₀₀值以及对硝酸盐氮和Cr（Ⅵ）的去除率基本相同,接种量对菌株G12的影响较小,故直接以1%（v/v）的比例接种培养;（4）在二元重金属离子Cu²⁺或Mn²⁺共存条件下,菌株G12生长达到稳定期的时间延迟,但其生长量以及对硝酸盐氮和Cr（Ⅵ）的还原效果没有影响,Cu²⁺或Mn²⁺的存在在一定程度上提高了Cr（Ⅵ）的还原率;（4）通过FTIR、XPS和XRD等仪器分析可知:（1）菌株G12表面主要存在羧基、羟基、氨基、酰胺基等官能团,这些官能团与Cr（VI）的去除有关;（2）在铬元素Cr 2p轨道的XPS图谱中,在结合能为577.36和583.01eV处主要代表Cr³⁺的2p^1/2和2p^3/2杂化轨道,Cr（Ⅵ）的还原产物价态主要是Cr（Ⅲ）,即菌株G12将Cr（VI）还原成了正三价态;（3）菌株G12在细胞内Cr（VI）的还原产物为Cr（Ⅲ）,以CrCl₃晶体结构形式存在。更多还原

【Abstract】 With the acceleration of urbanization and the speedy improvement of industry and agriculture,wastewater containing diverse varieties of pollutants discharged by way of human beings in manufacturing and living activities together with organic pollutants,inorganic pollutants and heavy metallic pollutants,etc.It is easy to form complex pollution into the water body.Combined pollution not most effective damages the ecological environment of the water,however additionally brings top-notch damage to human health.An inorganic pollutant due to nitrate nitrogen in water and heavy metallic pollution caused by Cr（VI）is one of the greater not unusual composite pollution.Inside the present sewage treatment method,the treatment methods for nitrate nitrogen and Cr（VI）pollutants respectively there are numerous researches.The techniques for casting off nitrate nitrogen consist of membrane separation method,ion exchange method,redox method,catalytic reduction method,and microbial method.The removal strategies of Cr（VI）specifically include adsorption method and solvent extraction separation method.Chemical precipitation techniques,etc.,but the above techniques are most effectively directed to pollutants due to a certain sort of pollutant.How to use effective measures competitively low cost,energy-saving,safe and environmentally friendly to remove the complex pollutants because of nitrate nitrogen and Cr（VI）in water has acquired extremely good attention.The microbial technique has great ability in dealing with environmental pollution problems.This paper focuses on a form of the microorganism which can simultaneously reduce Cr（VI）in aerobic denitrification for the treatment of complex pollution caused by due to nitrate nitrogen and Cr（VI）.The motive of this study is to observe the capability of strains to concurrently reduce Cr（VI）and nitrate and its reaction and metabolism mechanism,so that the isolated and decided on bacteria play a position in the actual sewage treatment,and offer a principle for enhancing the efficiency of nitrate and chromium treatment in sewage.According to the cardio denitrifying strains involved in this paper were isolated from the activated sludge of the oxidation ditch process secondary sedimentation tank of a sewage treatment plant in Chongqing.After acclimation and cultivation in the laboratory,the subsequent studies have been performed:16S rRNA gene identity of strain G12;initial Cr have a look at Cr（VI）The effect of concentration on the denitrification performance of aerobic denitrifying bacteria and the effect of nitrate concentration on Cr（VI）reduction;various influencing elements consisting of carbon source,nitrogen source,inoculum size,and coexisting binary heavy metal ions have been studied.The consequences of strains on nitrate and Cr（VI）reduction procedures;FT-IR,XRD,and XPS were used to analyze Cr（VI）and to study the simultaneous metabolism of Cr（VI）reduction and aerobic denitrification,a mechanism for the reduction of products.The consequences confirmed that:（1）At the denitrifying solid medium,the surface of the colony become neat,and the surface was smooth,moist,opaque and faded yellow.Using the 16S rRNA gene sequencing,it changed into showed that the pressure G12 belongs to Pseudomonas aeruginosa strain（Pseudomonas aeruginosa）and turned into named Pseudomonas aeruginosa stress G12.Stress G12 turned into inoculated at a ratio of 1%（v/v）to aerobic denitrification medium containing 500 mg/L of nitrate nitrogen and 10 mg/L of Cr（VI）,after which at 30℃,120 rpm.The situations had been continuously cultured for 96 h.The strain G12 entered a strong stable growth at 48 hours,and its OD600 value reached a maximum of approximately 1.7.Nitrate nitrogen becomes absolutely removed in about 72 h,the elimination rate reached 98%,and the nitrate-nitrogen concentration became approximately 10 mg/L after 96 h.At 96h,the reduction rate of Cr（VI）reached 93.32%and Cr in the supernatant.The concentration of Cr（VI）is about 0.668 mg/L.（2）While the preliminary nitrate concentration is 100,300,500,700 mg/L,the nitrates are all decreased after 72 h of culture,and the elimination rate are 95.8%,99.5%,97.2%,70.3%,respectively;whilst the preliminary concentration of nitrate concentration turned into 500 mg/L,the reduction conversion rates of Cr（VI）of strain G12 to at least 1,5,10,15,and 20 mg/L had been 86.6%,94.9%,98.4%,99.1%,and 99.2%,respectively.%,the corresponding elimination rate of nitrate nitrogen had been 96%,96.5%,97.2%,96.7%,and 97.4%,respectively;the reduction impact of Cr（VI）changed into definitely correlated with the nitrate concentration within the medium;Cr（VI）The reduction and nitrate reduction were carried out simultaneously,focusing at the logarithmic growth phase of strain G12.（3）The outcomes of different environmental impact elements at the metabolism of strain G12 confirmed that:1 below distinctive carbon assets,the growth of strain G12 and the elimination impact of nitrate nitrogen and Cr（VI）had been distinctive,and one of a kind carbon source pairs The primary and secondary order of the consequences of strain G12 metabolism have been:1 sodium citrate>sodium succinate>glucose>sodium acetate,so sodium citrate changed into chosen because the carbon source;2 Inside the case of ammonia nitrogen,nitrate nitrogen,and nitrite nitrogen respectively.inside the case of a single nitrogen source,the primary and secondary ordering effects of various nitrogen sources on the metabolism of strain G12 had been:nitrate nitrogen>ammonia nitrogen>nitrite nitrogen,so nitrate changed into used as nitrogen source for subsequent research;3 After 1%,5%and10%（v/v）,the OD₆₀₀ value of stress G12 and the removal fee of nitrate nitrogen and Cr（VI）had been basically the equal after 72 hours of continuous culture.The impact of inoculum on strain G12 turned into higher.Small,so immediately inoculated in a ratio of 1%（v/v）;4 in the presence of binary heavy metal ions Cu²⁺or Mn²⁺coexistence,the growth of strain G12 reached a stable time frame,but its increase and the nitrate nitrogen and The reduction impact of Cr（VI）is not Influence,the presence of Cu²⁺or Mn²⁺improves the elimination rate of Cr（VI）to a certain extent;（4）it can be seen through instrumental analysis which include FTIR,XPS and XRD:1 the primary floor of G12 is carboxyl,hydroxyl,amino,amide,etc.Functional groups,these Functional groups are associated with the removal of Cr（VI）;2 within the XPS spectrum of the Cr2p orbitals of chromium,the 2p^1/2 and 2p^3/2 hybrid orbitals,which especially represent Cr³⁺,at binding energies of 577.36 and 583.01 eV,Cr The valence state of the decreased production of（VI）is in particular Cr（Ⅲ）,this is,the strain G12 reduces Cr（VI）to a positive trivalent state;3 the reduction product Cr（Ⅵ）of the strain G12 inside the cell is trivalent chromium,The crystal form of CrCl₃ exists.更多还原