| 题名 | 改性当地土壤技术去除藻华与降解藻毒素的研究 |
| 作者 | 李宏 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 潘纲 |
| 关键词 | 有毒水华 改性土壤技术 微囊藻毒素 高效液相色谱-电喷雾串联四极杆质谱 生物降解 toxic algal blooms modified local soil microcystins UPLC-MS/MS biodegradation |
| 其他题名 | Removal of harmful algal blooms and degradation of microcystins using modified local soils |
| 学位专业 | 环境工程 |
| 中文摘要 | 去除天然水体中蓝藻水华的方法常导致藻细胞的破裂及藻毒素（microcystins, MCs）的释放。本文在建立了超高效液相色谱-电喷雾串联四极杆质谱（UPLC-MS/MS）测定水体中MC-RR和MC-LR的方法的基础上，研究了壳聚糖改性土壤技术除藻对藻毒素释放的长短期影响，探索出一种利用天然培养基来分离藻毒素降解菌的方法，系统阐述了壳聚糖改性当地土壤技术除藻对藻毒素归趋的影响，考察了壳聚糖改性当地土壤絮凝结合微生物改性土覆盖对藻华去除和藻毒素降解的效果与机理，主要结果如下： 1）在所选定的色谱、质谱条件下，UPLC-MS/MS对MC-RR和MC-LR的检出限分别为2.0 ng/L和1.0 ng/L，定量限为6.0 ng/L和3.0 ng/L，回收率分别达98.9%-106.5%和98.4-101.5%。测定太湖梅梁湾水样时，当水体中MCs浓度达到UPLC-MS/MS方法定量限20倍以上时，可通过加入内标物亮氨酸脑啡肽，实现快速（不经过水样富集）且准确（相对标准偏差< 5.0%）的藻毒素测定。 2）壳聚糖改性土壤技术在除藻的同时，使胞内藻毒素随着藻细胞沉降到水体底部，进而可在短期内显著降低表层水体中溶解态藻毒素的浓度，但随着藻细胞的腐烂分解，水体中藻毒素浓度会升高。40天的柱实验结果表明，改性当地土壤絮凝技术将分散在水体中的蓝藻细胞聚集到水体底部，相比对照，使水体中溶解态MC-RR和MC-LR浓度分别降低了46%和37%。由于沉降蓝藻的衰亡分解，底泥-水界面的ORP显著降低。絮凝后采用土壤、载氧土壤、沙子进行覆盖时，由于覆盖层对溶解态藻毒素的吸附及对蓝藻絮体的封闭，水体中藻毒素的释放量显著减少，溶解态MC-RR和MC-LR浓度分别比对照降低了62%-86%和26%-68%。载氧土壤覆盖絮体时，底泥-水界面的ORP在6天内高于其他处理。 3）在利用天然培养基分离藻毒素降解菌的过程中，微生物菌落在10%的底泥水浸提液培养基上无法生长，而在以10%底泥水浸提液作为富集培养基、浓缩10倍后的底泥水浸提液作为分离培养基时，5类菌株（WC-1至WC-5）在培养基表面形成。利用传统的无机盐培养基所分离到的2株微生物（MS-1和MS-2）和利用上述天然培养基所分离到的5株微生物在无机盐培养基中能将初始浓度为207.57 μg/L的MC-RR和171.84 μg/L的MC-LR于6天内完全降解。在持续添加藻毒素的太湖水样中，菌株MS-1和MS-2需要6天的延滞期才能开始降解藻毒素，MC-RR和MC-LR在第6天的去除率分别为13%和15%，对于菌株WC-1至WC-5，培养第3天就出现了藻毒素的降解，MC-RR和MC-LR在第3天的去除率达44%和31%。菌株WC-1至WC-5在18天的实验期内对MC-RR的去除率达89%以上，对MC-LR的去除率达87%以上，并且在第18天时的降解速率相比第3天出现2至6倍的增长。通过对在天然水体中藻毒素降解效率较高的WC-4和WC-5的16S rRNA基因测序及进化关系分析可知，WC-4属于Pseudomonas sp An18，WC-5属于Acinetobacter sp. dcm5A。 4）在初始藻藻浓度为1.38×1010 cells/L，水体溶解态藻毒素MC-LR为0.84 μg/L，MC-RR为5.14 μg/L的富营养化水体中，壳聚糖改性土壤技术可使约90%的藻细胞及胞内毒素沉降到水体底部。在40天的培养实验中，壳聚糖改性土壤絮凝处理下水体表层溶解态MC-LR和MC-RR相比初始浓度增长了10倍，且由于藻絮体中藻华细胞的破裂，底泥-水界面藻毒素浓度显著高于水体表层。壳聚糖改性土壤絮凝+土壤覆盖处理加剧了藻华细胞的破裂，但相比对照，水体表层的MC-LR和MC-RR分别降低了30%和70%，通过分析柱实验体系内各部分（水体、覆盖层、絮体曾、底泥层、悬浮藻细胞）藻毒素的含量发现，壳聚糖改性土壤絮凝+土壤覆盖处理下体系中MC-RR总量及MC-LR总量相比对照、壳聚糖改性土壤絮凝处理均无显著差异（P>0.05）。当通过高效藻毒素降解菌Pseudomonas sp An18改性土壤并将其用为壳聚糖改性土壤絮凝除藻之后的覆盖材料时，底泥-水界面及水体表层中溶解态藻毒素浓度在40天的试验期内未出现明显变化，体系中总藻毒素量相比对照降低了90%。结果表明壳聚糖改性土壤絮凝使藻华细胞沉降到水体底部后，选择具有藻毒素降解功能的微生物改性土作为覆盖材料，一方面覆盖层抑制了释放的藻毒素从絮体向水体中的扩散，进而避免了溶解态藻毒素在水体中的稀释与扩散，另一方面负载了藻毒素降解菌的覆盖材料有效避免了菌体在水体中的稀释，这些因素共同作用下，实验体系中藻毒素实现了90%的去除。 |
| 英文摘要 | Many measures used for harmful algal blooms (HABs) removal often result in lysis of algal cells and release of microcystins (MCs). This study developed an ultra performance liquid chromatography electrospray ionization tandem triple quadrupole/mass spectrometry (UPLC-MS/MS) method for determination of microcystins (MC-RR and MC-LR) in water samples, then investigated the short term and long term effects of MLS technology on release of microscystins, established an alternative approaches for isolating MCs-degrading bacteria using natural media, studied the influence of midified local soil technology (MLS) plus varies capping material on the fate of MCs in column experiments, finally, we proposed a method for simultaneous removal of cyano-HAB and MCs using chitosan modified local soils (MLS) flocculation plus MCs-degrading bacteria modified soil capping. The main results were list as follows: 1) The limit of detection (LOD) for MC-RR and MC-LR using the developed ultra performance liquid chromatography electrospray ionization tandem triple quadrupole/mass spectrometry (UPLC-MS/MS) method was 2.0 ng/L and 1.0 ng/L, the limit of quantification (LOQ) was 6.0 ng/L and 3.0 ng/L, and the recoveries were in the range of 98.9%-106.5% and 98.4%-101.5%, respectively. When the method was applied for determination of MC-RR and MC-LR in water samples of Taihu Lake, the fast (without preconcentration of water sample) and accurate (RSD<5.0%) determination of MCs can be achieved when internal standard (leucine enkephalin) was added in the raw water when the toxins level were above 20-fold of the LOQ. 2) Algal cells together with intracellular MCs were mitigated into sediment using chitosan modified local soil technology, through which the dissolved MC-RR and MC-LR in the surface water in a short term in closure experiment were remarkably reduced, however, long term studies indicated the dissolved MCs may increase with the decomposition of sunk algal cells. In column experiment, flocculation of cyanobacterial cells use chitosan modified soil led to the transfer of potential microcystins from the water column to sediment, and reduced 46% and 37% of dissolved MC-RR and MC-LR in comparison to control during 40 days incubation. The ORP value was significantly reduced with the flocculation of cyanobacterial cells using chitosan modified soil attributed to the decomposition of cyanobacterial cells. The reduction of dissolved MC-RR and MC-LR reached 62%-86% and 26%-68%, respectively, when the flcos were capped with local soil, oxygen loaded soil and sand due to the blocking of cyanobacterial as well as adsorption of microcystins. When the oxygen saturated soil was used to cap the flocs, the ORP value was remarkablely high than other treatments within 6 days. 3) Direct application of 10% water extracts of sediment as media led to a failure of bacterial growth, while five colony species (WC-1 toWC-5) appeared using water extracts of sediment as accumulation media and concentrated water extracts of sediment as isolation media, respectively, in comparison to two species (MS-1 andMS-2) isolated from artificial media .With initial concentration of 207.57 μg/L MC-RR and 171.84 μg/L MC-LR in mineral salts medium, the above mentioned 7 strains of bacteria capable of eliminating MCs within 6 days. In the simulated biodegradation test in Lake Taihu water with continuous supply of MCs, a lag phase of 6 days was required for MS-1 and MS-2 to degrade 13% and 15% of the added MC-RR and MC-LR, respectively, whereas the lag phase was 3 days with approximately 44% and 31% removal of MC-RR and MC-LR by WC-1 toWC-5. During the 18 days experiment, degradation of MC-RR and MC-LR by WC-1 toWC-5 reached up to 89% and 87%, respectively, with 2 to 6-fold enhancement of removal rate. 16S rRNA gene sequences and phylogenetic analysis indicated the potential to amplify species of MCsdegrading bacteria when natural media were employed. 4) With initial concentration of 1.38×1010 algae cells/L, 0.84 μg/L dissolved MC-LR and 5.14 μg/L MC-RR, more than 90% algal cells together with intracellular MCs were converged in the sediment using chitosan-MLS technology. During the 40 days’ incubation, dissolved MC-LR and MC-RR in surface water showed 10-fold increase in the flocculation only systems, and the toxins levels in sediment-water interface were remarkablely higher then surface water. The maximum concentration of MC-LR and MC-RR in water was reduced by 30% and 70 % in local soil capping treatments, however, the total content of MCs in the whole sediment-water columns (including that in water, capping layer, flocs layer, sediment layer and suspended algal cells) remained similar with that in control systems and chitosan-MLS only systems (P>0.05). In contrast, more than 90% reduction of both dissolved and total MC-LR and MC-RR was observed in Pseudomonas sp An18 modified soil capping treatments. The high performance of toxin decomposition was due to the combined mechanism of flocculation and embedded bacteria in the capping material, which prevent dilution of added bacteria, confine the released toxins, and strengthen the biodegradation in the capping layers. |
| 公开日期 | 2015-06-16 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/13479]  |
| 专题 | 生态环境研究中心_环境水质学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 李宏. 改性当地土壤技术去除藻华与降解藻毒素的研究[D]. 北京. 中国科学院研究生院. 2014. |
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