【作者】 孟丹；

【导师】 贺玉龙； 谭周亮；

【作者基本信息】 西南交通大学 ， 环境工程（专业学位）， 2022， 硕士

【摘要】 污泥消化是实现剩余污泥减量化与资源化的重要途径,但消化过程面临水解速率低等问题,通过预处理可有效强化污泥消化效果。游离亚硝酸（FNA）可通过厌氧消化液原位制备,具有绿色经济环保的特点,其在促进剩余污泥破胞、提升消化效率方面显示出优异的性能。与此同时,新兴污染物抗生素抗性基因（ARGs）在剩余污泥中大量赋存,研究证实预处理亦可促进消化过程中ARGs的去除,然而目前关于FNA预处理组合消化工艺中ARGs的分布与潜在去除机制尚缺乏报道。因此,本研究利用FNA对剩余污泥进行预处理,基于胞外聚合物及ARGs丰度变化情况探究FNA预处理破胞条件;设置FNA预处理组合厌氧/好氧消化工艺,探究厌氧/好氧消化体系中ARGs的分布与去除,解析微生物群落结构与ARGs潜在宿主演替规律,为FNA预处理组合消化工艺中的ARGs污染控制提供理论依据。主要研究结论如下:（1）FNA预处理条件结果表明:FNA浓度越高（p H越低和NO₂^--N浓度越高）,剩余污泥破胞效果及ARGs去除越明显,本研究选择p H 5.3、NO₂^--N 753mg/L作为后续厌氧、好氧消化的FNA预处理条件;一类整合子（int I1）、16Sr DNA、ARGs丰度与FNA浓度呈显著负相关,说明FNA预处理可能通过减少微生物量以及抑制基因水平转移来控制ARGs的增殖。（2）厌氧消化正交实验结果:不同因素对消化效果的影响顺序为温度>p H>剩余污泥/预处理污泥,实验组中最佳厌氧消化条件为剩余污泥/预处理污泥=2,温度45℃,p H=10。污泥胞外聚合物（EPS）含量在0～24 d下降约13.79%～54.67%,温度越高蛋白质分解释放氨氮效果越好,同时也越利于ARGs的去除。在最佳厌氧消化条件下,总ARGs和int I1削减分别为86.48%和84.22%,sul1、sul2和tet A分别削减0.86、1.19和1.53 log。Pearson相关性分析表明,营养元素在影响微生物生长的同时影响ARGs的宿主组成,进而间接影响ARGs的分布。基因水平转移在厌氧消化过程中对ARGs的传播可能起到重要作用。（3）好氧消化正交实验结果:不同因素对消化效果的影响顺序为温度>剩余污泥/预处理污泥>p H,实验组中最佳好氧消化条件为剩余污泥/预处理污泥=2,温度45℃,p H=10。微生物死亡促使氨氮上升,内源代谢中大分子有机物的分解促使SCOD的上升。EPS随着时间逐渐下降,其蛋白溶解速率较厌氧消化更快。最佳好氧消化条件下总ARGs和int I1分别削减99.71%和98.62%,磺胺类ARGs中sul1和sul2分别削减3.35、2.35 log,四环素类ARGs中tet Q、tet W、tet A分别削减0.63、1.38、4.31 log。优化最佳条件下,好氧消化对ARGs和int I1的削减优于厌氧消化。Pearson相关性分析表明,高温和高p H下int I1丰度的变化与营养元素相关。ARGs的削减主要源自磺胺类ARGs,一方面由于有机物的消耗殆尽,微生物细胞裂解导致生物量降低,另一方面好氧消化过程阻断了基因水平转移,降低了ARGs增殖的风险。（4）通过对微生物群落结构分析发现,门水平上均占主导的微生物有Actinobacteriota、Chloroflexi、Patescibacteria、Firmicutes和Proteobacteria。Sperman相关性分析表明int I1、sul1和sul2与10类门水平下的微生物Patescibacteria、Gemmatimonadota、Nitrospirota、Desulfobacterota、Bacteroidota、NB1-j、Bdellovibrionota、Zixibacteria、Proteobacteria、Acidobacteriota呈显著正相关。网络共线性分析表明在6个菌门下的13个菌属被认定为5种目标基因（sul1、sul2、tet W、tet A、int I1）的潜在宿主。ARGs的削减一方面是由于ARGs的潜在宿主减少控制了垂直传播;另一方面int I1潜在宿主减少控制了水平传播。更多还原

【Abstract】 Sludge digestion is an important way to realize the reduction and recycling of waste activated sludge（WAS）,but it faced the low digestion hydrolysis rate.Pretreatment can effectively strengthen the effect of sludge digestion.Free nitrous acid（FNA）pretreatment has shown excellent performance in promoting residual sludge cell breakage to enhance digestion efficiency.Its main advantages are green,economically and environmental friendly,it can be prepared in situ by anaerobic digestate.Antibiotic resistance genes（ARGs）,as novel contaminants,are widely enriched in WAS and are potentially risky.Studies have confirmed that pretreatment can also promote the removal of ARGs in the digestion process.However,few studies have been conducted to investigate the distribution and removal of ARGs through FNA pretreatment combined digestion process.In this study,FNA was used to pretreat WAS,and the cell breaking conditions of FNA pretreatment were explored based on the change of extracellular polymeric substances（EPS）and ARGs.The distribution and removal of ARGs under different digestion conditions were investigated with the optimal FNA pretreatment combination anaerobic/aerobic digestion.The correlation analysis of ARGs,int I1 and sludge physicochemical properties was performed.The potential hosts of ARGs were analyzed by correlating ARGs with microbial community structure.The main conclusions are as follows:（1）FNA pretreatment conditions:Higher concentration of FNA（lower p H and higher NO₂^--N concentration）,the more obvious WAS cell breaking effect and ARGs removal.In this experiment,p H 5.3 and NO₂^--N 753 mg/L were selected as the FNA pretreatment conditions for subsequent anaerobic and aerobic digestion.Int I1,16Sr DNA,ARGs abundance and FNA concentration were significantly negatively correlated,indicating that FNA pretreatment may control ARGs proliferation by reducing microbial load and inhibiting gene level transfer.（2）The optimization of anaerobic digestion conditions:The influencing factors of temperature>p H>sludge ratio,the best conditions were sludge ratio=2,temperature45°C,p H=10.The EPS of sludge decreased by 13.79%-54.67%from 0 to 24 d.Higher temperature of anaerobic digestion,the better effect of protein decomposition to release NH₄⁺-N,and the better removal of ARGs.The optimal anaerobic digestion conditions resulted in 86.48%and 84.22%reduction of total ARGs and int I1,sul genes（sul1 and sul2）and tet genes of tet A were reduced by 0.86,1.19 and 1.53 log respectively.Pearson correlation analysis showed that nutrient elements indirectly influenced the propagation of ARGs by affecting microbial growth and thus the host composition of ARGs.Horizontal gene transfer plays an important role in the propagation of ARGs during anaerobic digestion.（3）The optimization of aerobic digestion conditions:The influencing factors of temperature>p H>sludge ratio,the best conditions were sludge ratio=2,temperature45°C,p H=10.Microbial death promoted the increase of NH₄⁺-N,the rise of SCOD due to the decomposition of macromolecular organic matter during endogenous metabolism.EPS decreased gradually with time and its proteolysis rate was faster than that of anaerobic digestion.Total ARGs and int I1 were decreased by 99.71%and98.62%,sul genes（sul1 and sul2）,tet genes（tet Q,tet W,and tet A）were reduced by 3.35,2.35,0.63,1.38,and 4.31 logs,respectively.Under optimal conditions,the reduction of ARGs and int I1 by aerobic digestion was better than that by anaerobic digestion.Pearson correlation analysis showed that the changes in int I1 abundance at high temperature and high p H were correlated with nutrient elements.Reduction of ARGs is mainly caused by sul genes.On the one hand,biomass is reduced due to microbial cell lysis due to exhaustion of organic matter;on the other hand,horizontal gene transfer is blocked by aerobic digestion process,which reduces the risk of ARGs proliferation.（4）Analysis of the microbial community structure revealed that the microorganisms that dominated both at the phylum level were Actinobacteriota,Chloroflexi,Patescibacteria,Firmicutes,and Proteobacteria.Sperman correlation showed that int I1,sul1,and sul2 were associated with 10 microorganisms under the class phylum level Patescibacteria,Gemmatimonadota,Nitrospirota,Desulfobacterota,Bacteroidota,NB1-j,Bdellovibrionota,Zixibacteria,Proteobacteria,and Acidobacteriota were significantly and positively correlated.Network covariance analysis showed that 13 genus level under 6 phylum level were identified as potential hosts for the 5 target genes（sul1,sul2,tet W,tet A,int I1）.更多还原