耐冷菌低温诱导形成碳酸镍矿物的稳定性调控及其冷适应分子机理

The mining and smelting of nickel sulfide ores have caused double soil pollution problems of acidification and excess available nickel in cold region. It is urgent to develop green technology for the remediation of the acidified nickel-polluted soil adapted to low temperature. Our previous studies have proven that psychrotrophic carbonate mineralized bacteria have the ability to simultaneously inhibit soil acidification and stabilize available nickel in soil. However, the bacterial ability to adapt to low temperature and the stability of the mineral are the bottlenecks that affect its scale application. So, this project will carry out the following key research: ① screen indigenous beneficial psychrotrophic bacteria that can induce nickel carbonate mineral at low temperature; ② research the effects of environmental conditions on the crystal type, morphology and the stability of nickel carbonate mineral induced by psychrotrophic bacteria at low temperature. Thus, the key factors that drive the formation of high stable biomineralization products formed at low temperature and the main controlling factors for the stability of mineralization products in the soil will be found; ③ explore the key cold adapted proteins expression and their metabolic pathway response mechanisms in psychrotrophic bacteria under dual stress of low temperature and high nickel through proteomic and target metabonomics analysis. The expected research results will systematically elaborate the stability regulation mechanism of nickel carbonate mineral induced by microorganism at low temperature and the coupling mechanism between bacterial cold adaptations. Hopefully, the above research will provide theoretical basis for the construction of high efficiency genetically engineered bacteria, research on the biomineralization mechanism at low temperature and efficient remediation of nickel contaminated soil in cold region.

寒区硫化物镍矿开采及冶炼会造成酸化和有效态镍含量高的双重土壤污染问题，急需开发适应低温环境的酸化镍污染土壤绿色修复技术。课题组前期研究发现耐冷碳酸盐矿化菌在低温下可同步抑制土壤酸化和固定有效态镍，但菌体细胞低温适应能力和矿物稳定性是影响其规模化应用的瓶颈问题。因此本项目开展如下重点研究：①筛选出可低温诱导形成碳酸镍矿物的优势土著有益耐冷菌；②研究环境条件对低温生物矿化形成碳酸镍矿物晶型、形貌以及矿化产物稳定性的影响，从而发现驱动高稳定性低温生物矿化产物形成的关键因子和矿化产物在土壤中稳定性的主要控制因素；③通过蛋白质组和靶向代谢组分析，阐明低温和高镍双重胁迫下耐冷菌的关键冷适应蛋白表达及其代谢通路的响应机制。预期研究结果将系统阐述耐冷菌低温诱导形成碳酸镍矿物稳定性的调控机理及其与耐冷菌冷适应之间的耦合关系，为构建高效基因工程菌、探究低温生物矿化机理和实现寒区镍污染土壤高效修复提供理论依据。

耐冷碳酸盐矿化菌可同时解决寒区硫化物镍矿开采及冶炼会造成的土壤酸化和有效态镍含量高的双重污染问题，对镍污染土壤的绿色修复具有重要意义，但目前耐冷碳酸盐矿化菌资源匮乏、冷适应机理不明、低温生物矿化机理不明确，影响其规模化应用。本项目在筛选土著耐冷碳酸盐矿化菌的基础上，研究了其生长代谢、抗镍、诱导镍的碳酸盐沉淀的机理与因子响应机制、冷适应机理和对镍胁迫的代谢组学响应机制。结果表明，从不同的重金属污染土壤中分离获得了2株耐冷碳酸盐矿化菌，分别为蜡样芽孢杆菌D2和类谷氨酸杆菌DC1，两株菌在10℃下均具有较强的抗镍、生长、产脲酶、产碱和pH适应能力，在低温下均可诱导镍的碳酸盐沉淀，其中菌株D2可诱导形成呈纳米球形的Ni2CO3（OH）2•H2O矿物晶体，并且菌体细胞在矿物晶体形成过程中起关键作用。沉淀时间、共存阴离子和一价阳离子的种类、尿素添加量对矿物晶体的形貌和晶型具有一定的影响，其中48 h以内，在K+和SO42-共存和1.5%以上尿素添加量的条件下，矿物晶体峰更加清晰、数量更多、形貌更规则。另外，在与Ca2+共存时，菌株D2可单独诱导形成Ca和Ni的碳酸盐矿物CaCO3和Ni2CO3(OH)2•H2O。 两株菌均可将土壤中的交换态镍诱导形成碳酸盐结合态镍和增加土壤pH，从而增强了修复的稳定性。发现并构建了菌株D2关键冷休克蛋白Csp2和Csp6的Csp-pet-28a载体，其过表达能明显促进大肠杆菌在低温下的生长，而且还发现Csp2和Csp6中丙氨酸含量较高，并提出了在亮氨酸上形成α螺旋可能是关键冷休克蛋白的主要特征。在镍胁迫下，菌株D2细胞膜收到明显的损伤，415 种代谢产物有差异，其中差异极显著的代谢产物为氨基酸，主要影响了半胱氨酸、甲硫氨酸等氨基酸的代谢、ABC 转运蛋白的合成、丙酮酸代谢等多个代谢通路，而外源谷氨酸和甲硫氨酸等氨基酸的添加则能够明显增强菌株D2的抗镍能力。以上研究结果可为利用耐冷碳酸盐矿化菌修复酸化镍污染土壤提供理论依据。