【作者】 王娟；

【导师】 张玉；

【作者基本信息】 大连理工大学 ， 环境科学与工程， 2021， 硕士

【摘要】 硝酸盐是目前水体中最常见的污染物之一。过量的NO₃^-进入水体,会对自然环境和人类健康产生巨大威胁。自养反硝化技术在低C/N、营养成分匮乏的水体脱氮处理过程中有着较高的应用价值。硫铁基质自养反硝化作为一种新型高效的脱除贫营养水体中硝酸盐的方法受到研究者青睐。为探究在硫铁共存体系中,Fe（II）对体系中NO₃^-降解的生物和化学过程贡献,本研究采用Fe²⁺和S₂O₃^2-做电子供体,分别在纯菌和混菌的条件下探究微生物的硫自养、铁自养、以及硫铁共基质自养反硝化的作用规律和机理,主要研究结果如下:实验分离纯化出一株具有反硝化功能的兼性厌氧菌,16S r DNA序列分析其为柠檬酸杆菌属,命名为Citrobacter sp.ZY630。对该菌株利用Fe（II）还原NO₃^-过程探究,结果表明,在无外加有机物的情况下,菌株ZY630可以以Fe²⁺为电子供体还原NO₃^-。经过120 h反应,Fe²⁺+菌组约1.1 mmol·L^-1的NO₃^-完全降解,产物包含NO₂^-、N₂和N₂O。通过对照分析,发现体系中NO₃^-到NO₂^-的转化是由生物作用主导。同时发现6 mmol·L^-1Fe²⁺+菌组较3 mmol·L^-1Fe²⁺+菌组反硝化程度进行更彻底,表明Fe²⁺有助于促进该体系反硝化过程。进一步探究菌株ZY630利用Fe²⁺降解NO₂^-过程,发现120 h后,加菌加Fe²⁺体系的Fe²⁺氧化总量与NO₂^-还原总量的摩尔比约为2.22,较只加Fe²⁺体系的摩尔比略高,表明NO₂^-和Fe²⁺之间化学反应占优。所以,菌株ZY630厌氧Fe²⁺氧化NO₃^-还原是一个生物化学耦合过程。在探究菌株ZY630硫自养反硝化功能的过程中,发现菌株ZY630虽然可以发生硫自养反硝化,但是其需要有机物来维持菌株长期的正常生长。菌株ZY630在以Fe²⁺和S₂O₃^2-作电子供体时,不能获得足够的能量维持菌株生长和反硝化反应的进行。实验以S₂O₃^2-为唯一外加电子供体进行了硫自养反硝化污泥（混菌）的驯化,经过35天培养,污泥中富集了以Sulfurimonas和Thiobacillus属为主要组成的硫自养菌群。通过序批式实验验证驯化污泥中微生物的硫自养反硝化性能,发现其在48 h内可利用S₂O₃^2-将约1 mmol·L^-1的NO₃^-全部转化为N₂,生成SO₄^2-积累在体系中。实验同时探究了驯化污泥菌群的铁自养反硝化功能,发现驯化污泥中的微生物可以以Fe²⁺为电子供体进行铁自养反硝化。在证实了驯化污泥中微生物可以以单独的S₂O₃^2-和Fe²⁺为电子供体进行自养反硝化的基础上,将污泥接种于硫铁共存的体系,发现在S₂O₃^2-含量不足以将所有的NO₃^-还原为N₂的情况下,微生物可以利用Fe²⁺作为电子供体继续反硝化。因此,驯化的硫自养污泥可以利用S₂O₃^2-和Fe²⁺作电子供体还原NO₃^-,具有硫铁共基质自养反硝化功能。更多还原

【Abstract】 Nitrate is one of the most common pollutants in water today.Excessive NO₃^-enters the water and poses a huge threat to the natural environment and human health.Autotrophic denitrification technology has high application value in the denitrification process of water bodies with low C/N and lack of nutrients.Sulfur and iron substrate autotrophic denitrification is favored by researchers as a novel and efficient method to remove nitrate from nutrient-poor waters.To investigate the contribution of Fe（II）to the biological and chemical processes of NO₃^-degradation in the system of sulfur and iron coexistence,this study used Fe²⁺and S₂O₃^2-as electron donors to investigate the action patterns and mechanisms of sulfur autotrophy,iron autotrophy,and sulfur-iron co-substrate autotrophic denitrification of microorganisms under pure and mixed bacterial conditions,respectively,and the main findings are as follows.A facultative anaerobes strain with denitrification function was isolated and purified in this experiment.By 16S r DNA sequence analysis,it was Citrobacter genus,named Citrobacter sp.ZY630.The strain was probed for their ability to reduce nitrate by ferrous oxidation,and the results showed that the strain ZY630 could reduce NO₃^-with Fe²⁺as the electron donor in the absence of additional organic matter.After 120 h of reaction,about 1.1mmol·L^-1of NO₃^-in the Fe²⁺+bacterias group was completely degraded,and the products included NO₂^-,N₂O and N₂.The control analysis revealed that the conversion of nitrate to nitrite in the system was dominated by biological action.It was also found that the 6 mmol·L^-1Fe²⁺+bacterias group carried out denitrification more completely than the 3 mmol·L^-1Fe²⁺+bacterias group,indicating that Fe²⁺helped to promote the denitrification process in this system.Further studies on the biological and chemical pathways in the NO₂^-degradation process by strain ZY630 using Fe²⁺.It was found that the molar ratio of total amount of Fe²⁺oxidation to total amount of NO₂^-reduction was about 2.22 after 120 h,which was slightly higher than that of the system with only Fe²⁺,indicating that the chemical reaction between NO₂^-and Fe²⁺was dominant.The anaerobic ferrous oxidative nitrate reduction by strain ZY630 is a biochemically coupled process.During the investigation of the sulfur autotrophic denitrification process of the strain ZY630,it was found that although the strain ZY630 can undergo sulfur autotrophic denitrification,it requires organic matter to maintain the long-term normal growth of the strain.Strain ZY630 could not obtain enough energy to sustain the growth of the strain and the denitrification reaction when using Fe²⁺and S₂O₃^2-as electron donor.Sulfur autotrophic denitrifying sludge（mixed bacteria）was domesticated with S₂O₃^2-as the only applied electron donor,and after 35 days of incubation,the sludge is enriched with sulfur autotrophic bacteria group mainly composed of Sulfurimonas and Thiobacillus.Sequencing batch experiments were conducted to verify the sulfur autotrophic denitrification performance of microorganisms in the domesticated sludge.It was found that S₂O₃^2-could be used within 48 h to convert about 1 mmol·L^-1of NO₃^-into N₂and form SO₄^2-accumulate in System.At the same time,the iron autotrophic denitrification function of the bacteria in the domesticated sludge was investigated.It was found that the microorganisms in the domesticated sludge could use Fe²⁺as electron donor for iron autotrophic denitrification.Based on the confirmation that microorganisms in domesticated sludge can perform autotrophic denitrification with S₂O₃^2-and Fe²⁺alone as electron donors,the sludge was inoculated in a sulfur-iron coexistence system,and it was found that microorganisms can continue denitrification using Fe²⁺as electron donor when the S₂O₃^2-content is not sufficient to reduce all NO₃^-to N₂.Therefore,the domesticated sulfur autotrophic sludge can use S₂O₃^2-and Fe²⁺as electron donors and has sulfur-iron co-substrate autotrophic denitrification.更多还原