【作者】 贾爱萍；

【导师】 李永涛；

【作者基本信息】 华南农业大学 ， 土壤学， 2019， 博士

【摘要】 抗生素和重金属作为添加剂在养殖业生产中应用普遍,过量且未被利用的部分被动物排出体外,随废弃物的资源化利用过程不断输入农田土壤,长期累积造成土壤中污染物的浓度不断升高,对土壤质量、微生态环境及人类健康构成威胁。本研究以养殖业中应用最为普遍的铜和四环素为目标污染物,研究单一及复合污染对土壤氨氧化微生物影响,以及由此引起的氨氧化过程相关指标的响应变化,同时筛选出针对铜-四环素复合污染的抗性降解菌,并将其用于复合污染的修复机理研究,探索新兴复合污染的生物修复技术。研究主要得出以下结论:(1)针对养殖业带来的重金属-抗生素复合污染问题,本研究筛选出2株铜-四环素复合污染抗性降解菌ZFS-1X和ZFS-2M。经鉴定,两株菌分别为约氏不动菌Acinetobacter johnsonii和腐皮镰刀菌Fusarium solani,能以四环素为唯一碳源同时具有铜抗性,经富集培养后用于后续复合污染修复实验。(2)土壤中铜的形态分布与土壤性质和铜的浓度密切相关。单一及复合污染处理的供试土壤中可氧化态含量最高,抗性降解菌液修复处理的供试土壤中残渣态含量最高;低浓度铜污染以可还原态百分含量最大,中高浓度铜污染以酸溶态百分含量最大;酸溶态铜随污染物总量的增加而增加,随培养时间的延长而下降;低浓度四环素对铜有活化作用,中高浓度四环素和抗性降解菌对酸溶态铜含量影响不明显。(3)四环素在土壤中的降解规律与土壤性质有关,外源菌可以改变四环素的降解过程。单一及复合污染处理土壤中四环素的降解主要在前14d,铜离子对低浓度四环素降解无影响,但影响中、高浓度四环素的降解,影响规律不明显;筛选出的抗性降解菌可有效提高四环素的降解速度,降低残留率,降解效果为混合菌液>2M>1X。(4)氨氧化细菌(AOB)和氨氧化古菌(AOA)的基因丰度在不同土壤中的分布存在差异,污染胁迫对氨氧化微生物具有抑制作用。单一及复合污染处理供试土壤中AOB基因丰度百分含量高于AOA,抗性降解菌修复处理供试土壤中则是以AOA为优势菌群;单一及复合污染处理在培养过程中AOA基因丰度百分含量逐步上升并高于AOB,对照的转变时间为56d,污染处理的转变时间提前至14d或28d,各处理对AOB的抑制率在培养前中期较AOA大,后期较AOA小;抗性降解菌修复处理土壤中AOA基因丰度百分含量在培养过程有下降的趋势,但始终占主导地位;AOB对污染物的响应更为敏感,AOA耐受能力更强,二者在驱动氨氧化过程中互为补充,在环境胁迫时AOA的驱动作用更大。(5)单一及复合污染处理对土壤呼吸、土壤酶、铵态氮、硝态氮、AOA和AOB作用方向(抑制或促进)和强度各不相同,指标间的联动性也存在差异。铵态氮与AOB在不同的处理条件下都具有极显著正相关关系,可作为AOB的特征指示指标。外源菌对复合污染抑制AOA、AOB、硝态氮的效果和促进铵态氮、过氧化氢酶的效果都有强化作用,作用强度以混合菌和2M菌较大,对复合污染抑制蔗糖酶的效果无显著影响。外源菌在培养前中期使复合物污染抑制氨氧化的作用加剧,后期通过提高四环素的降解率缓解了复合污染对氨氧化的抑制作用。更多还原

【Abstract】 Antibiotics and heavy metals have been widely used as the additives in the livestock and poultry breeding industry,while which would be released to the animal wastes if their dosage exceeded the normal requirement.With the long-term addition of animal wastes as a kind of organic manure to soils,excessive accumulation of antibiotics and heavy metals has been commonly observed,likely imposing threat to the soil quality and the healthy of micro-ecological environments and human being.Due to the enormous application,copper and tetracycline were chosen as the representatives to assess their individual and combined effect on the ammonia-oxidizing process together with how the related parameters varied with the changes in this process.Meanwhile,efforts have also been made to screen the resistant bacteria for joint pollution of copper and tetracycline and reveal the underlying resistance mechanisms,with the aim to develop a new bio-remediation strategy for this emerging environment problem.The primary conclusions are as the following:(1)Two strains of resistant bacteria for joint pollution of copper and tetracycline were obtained,namely ZFS-1X and ZFS-2M,which were further identified as Acinetobacter johnsonii and Fusarium solani,respectively.Since they were proved to use tetracycline as the sole carbon source and poss tolerance to copper toxicity,we use them in the following remediation experiments after enrichment culture.(2)The distribution of copper species in soil is closely related to soil properties and its concentration.The content of oxidizable form was the highest in the soil treated with single and combined pollution,while the content of residual form was the highest in the soil treated with resistant bacteria.The reducible form was predominant in soil with low addition of copper,while the content of acid soluble form was highest in medium and highly copper contaminated soil.The percentage of acid-soluble copper increased with the increase in the addition of copper,and decreased with the prolongation of culture time.The activity of copper would be increased with the low concentration of tetracycline,and no observable effect on the content of acid-soluble copper was found in the presence of medium and high concentration of tetracycline and resistant bacteria.(3)The degradation of tetracycline in soil is related to soil properties,and exogenous bacteria will change the degradation process of tetracycline.The degradation of tetracycline in the single and combined treatment occurred mainly in the first 14 days.Copper ions had no impact on the degradation of tetracycline of low concentration,but affect degradation of medium and high concentration of tetracycline with inconsistent trend.The screened resistant bacteria can effectively promote the degradation of tetracycline and reduce the residual rate,with the order of efficiency decreasing as the following: Mixed bacterial solution >2M>1X.(4)The distribution of ammonia-oxidizing archaea(AOA)and ammonia-oxidizing bacteria(AOB)varied with the physical and chemical properties of soil,and pollution stress has inhibitory effect on ammonia-oxidizing microorganisms.The AOB is the dominate bacteria in the single and combined treatment,whereas the AOA is the dominant flora in soil treated with the exogenous bacteria.The abundance of AOA gene gradually increased and exceeded that of AOB during the process of cultivation in the single and compound treatment.This change could be observed after 56 d in the control experiments,and only 14 d and 28 d were needed in single and combined pollution treatment.The rate of AOB inhibited in corresponding pollution treatment was larger than AOA in the early and middle of the culture period,and was smaller than that of AOA in the later stage.In the exogenous bacteria treatment,the percentage of AOA gene abundance exhibited a downward trend in the process of culture,while it was always dominant.We find that AOB is more sensitive to pollutants and AOA is more tolerant,and they will complement with each other in driving ammonia oxidation.The driving effect of AOA is greater than that of AOB under environmental stress.(5)The net effect(inhibition or promotion)of single and combined pollution of copper and tetracycline on soil respiration,soil enzymes,ammonium nitrogen,nitrate nitrogen,AOA and AOB differs with their concentrations.The correlation among these indicators was different.Highly positive correlation between ammonium nitrogen and AOB were found,which can be used as the characteristic indicator of AOB.The exogenous bacteria would enhance the inhibition of the combined pollution on AOA,AOB and nitrification and promotion on ammonification and the activity of catalase.This effect was more evident in the mixed bacteria or 2M bacteria,while they had no significant effect on the activity of sucrase.The inhibitory effect of compound pollution on ammonia-oxidizing was intensified by exogenous bacteria in the early and middle stage of incubation,and the inhibitory effect of compound pollution on ammonia-oxidizing was alleviated through improving the degradation of tetracycline by exogenous bacteria in the later stage.更多还原