基于组学的废渣堆场重金属抗性菌株响应机理及吸附与浸出特性研究

Discharge of smelting waste contaminated with heavy metals and related elements is known to have an adverse effect on the environment and solving this problem has for long been presented as a challenge. The biosorption-sequestration and bioleaching of heavy metals by resistant microbes can be a prominent part of the solution owing to unique metabolic interactions between heavy metals and resistant microbes. However, biosorption or bioleaching by microbes needs a long period process and often shows a decline in process efficiency as time goes on, which are mostly attributed to inadequate knowledge about the metabolic response mechanism of heavy metals in resistant microbes, and lack of timely effective regulation specific to rate-limiting steps. In order to overcome the problem mentioned above, in this project, Illumina high-throughput sequencing is used for the transcriptome comparative analysis of resistant microbial strain. Combined with data obtained from deep genomic sequencing, expression differences at mRNA level are exploited to construct the metabolic network, thereby revealing the detoxification mechanism of heavy metals in cells. Furthermore, the expression levels of critical metabolites that are involved in detoxification of heavy metals will be up-regulated through two aspects, namely, the optimization of culture conditions and the over-expression of endogenous genes by genetic modification. And then, composites of magnetic nanoparticles combined with resistant microbial strains are used for detecting the biosorption behavior of heavy metals in water waste, and bioleaching properties of heavy metals by resistant microbial strains in smelting slag soil are also analyzed. The establishment of microbial heavy metal resistance metabolic network provides theoretical basis for efficiency improvement of microbial remediation and resource recycling of heavy metals.

利用环境微生物自身特有的生理代谢活动来应对冶炼生产造成的重金属环境污染具有独特的优越性和广阔的应用前景。然而，在重金属的生物吸附或浸出处理过程中存在周期长、效率低等问题，其主要障碍是未完全掌握抗性菌株在重金属胁迫下的代谢规律，以及缺少针对反应控制步骤做出及时有效的优化调控手段。针对这一问题，本申请项目提出采用Illumina高通量测序技术分析优良高效菌种在重金属胁迫下的转录组学变化，从mRNA水平阐述表达差异规律，并结合基因组样品的深度测序，构建其整体代谢网络，揭示抗性菌株的重金属离子解毒机理。在此基础上，通过菌种培养条件的优化和利用基因工程手段对菌种自身进行内源过表达这两个方面，有针对性地提高参与重金属代谢活动的关键中间产物的表达。经过磁性纳米颗粒负载抗性菌株，分析复合物在废水中吸附重金属的行为，以及对抗性菌株浸出土壤重金属的特性进行分析，为废弃重金属的资源化利用提供理论依据。

利用环境微生物自身特有的生理代谢活动来应对冶炼生产造成的重金属环境污染具有独特的优越性和广阔的应用前景。本项目在两种环境介质中（水体和土壤），分别研究了经化学改性的真菌菌丝在水体中吸附重金属的效率、Mn(II)氧化细菌浸出土壤重金属的效率，以及各自相关的吸附机制。并且，进一步着重探讨了Mn(II)氧化细菌应对锰胁迫时的生理代谢调控机制。结果表明：（1）合成的“淡紫拟青霉菌-纳米二氧化硅-海藻酸钙”（P. lilacinus-SN-Cal-Alg）微球复合材料水溶液中对Pb(II)有较好的吸附效果，最大吸附量为282.49 mg/g；（2）合成的多壁碳纳米管真菌菌丝复合材料（FM-CNTs）对Cu(II)具有高效的吸附能力，吸附过程符合Langmuir模型，FM-CNTs在水体中对Cu(II)的最大吸附量为342.22 mg/g；（3）细菌Providencia sp.LLDRA6能够适应Mn(II)高浓度的生长环境。在Mn(II)存在情况下，LLDRA6细胞表面形成了Mn元素层状聚集物，这些聚集物中有Mn(III)和Mn(IV)的存在，说明细菌LLDRA6具有氧化、产生锰氧化物的能力。采用联合该菌与其产生的锰氧化物的策略对污染土壤进行淋洗，发现相比纯菌液，联合淋洗策略浸出土壤重金属的效率分别提高了：Pb (6.05%)，Cr (26.4%)，Cd (19.56%)，Cu (10.82%)，Mn (5.17%)，Zn (1.68%)；（4）细菌Providencia sp. LLDRA6在受到Mn(II)胁迫时，负责苯乙酸分解代谢的操纵子会被转录激活，这是因为在Mn(II)胁迫下，细菌产生了大量的活性氧，这些活性氧可参与形成高活高毒性的oxepins，而oxepins又是苯乙酸分解代谢通路中关键的中间底物，因此导致苯乙酸分解代谢被激活。