钙信号调控木质素降解青霉菌丝球形成的机制研究

Fungal Pellets has been applied considerably in removing the degradation-resistant pollutant, decoloring the dyestuff, adsorbing the heavy metal ion and so on, with a characteristic of good mechanical strength, fast sedimentation velocity and easy collection. However, the molecular mechanism of pellets formation is not yet clear. In the earlier work, an efficient lignin-degrading fungal strain isolated and screened from sludge, which chronically contaminated by papermaking black liquor. And it was found that calcium ion had a surprisingly positive role on the pellets formation and waste treatment. Based on this, the proposal is proposed to carry out the below work centering on the mechanism of pellets formation regulated by calcium signal. The physiochemical change (morphology, mechanical strength) is to be studied firstly. Then the downstream signal pathway that is involved in pellets formation regulated by calcium signal will be identified at gene-protein expression levels with using of RT-PCR or polyacrylamide gel electrophoresis, in combination with the inhibitor treatments of calmodulin, calmodulin dependent protein kinase, and cAMP / MAPK pathways. Key genes related to pellet formation will be screened through analyzing on dynamic regulation of gene expression profile in different (primary-, middle-, and late-) stages of pellet formation by using RNA array, thereby molecular mechanism of pellets formation regulated by calcium signal will be revealing. This research will illuminate the molecular mechanism of forming mycelia pellet of Penicillium regulated by calcium, and also provide theoretical and experimental basis for applying pellet in practice.

真菌菌丝球在去除难降解物质、进行染料脱色、吸附重金属离子等方面具有广泛的应用价值，目前对于成球的分子机制尚不清楚。本项目前期获得的高效木质素降解青霉，该菌株在应用于造纸黑液的处理过程中，钙离子对其成球具有积极的促进作用，因此本项目拟围绕钙信号调控青霉菌丝球形成的机制进行下述研究：钙信号对菌丝球形成过程的形态调控作用；利用RT-PCR或聚丙烯酰胺凝胶电泳技术，结合钙调蛋白、钙调蛋白依赖的蛋白激酶、cAMP通路或MAPK通路的抑制剂处理，考察钙信号对下游通路中的基因表达水平和蛋白水平的影响，鉴定参与钙信号调控菌丝成球的信号通路；利用RNA array技术，分析钙信号对青霉菌丝球形成的早期、中期和后期基因表达谱的动态调控，进而筛选参与菌丝球形成的关键基因，最终揭示钙信号调控该菌株形成菌丝球的分子机制。研究结果不仅将阐明钙信号在调控青霉形成菌丝球的内在机制，也将为菌丝球应用于生产实践奠定理论基础。

本研究从造纸厂采集的污泥样本中分离筛选获得真菌菌株G-13，经18srDNA鉴定为烟曲霉菌（Aspergillus fumigatus）。以烟曲霉菌G-13形成的菌丝球为研究对象，通过探究不同的氮源、碳源、培养基的pH值、接种量、转速和温度对G-13成球的影响，得到了最佳成球条件。在最佳条件下培养72h，得到的菌丝球的球径最大，约为3.4mm。此时的菌丝球球径适中，表面光滑，弹性好，可以作为生物固定化载体。对最佳成球条件下的菌丝球运用扫描电镜观察其形成过程，发现菌丝成球会经历从非极性生长到极性的菌丝尖端延长、分枝菌丝形成菌丛等过程，最终形成一种由菌丝缠绕而成的中空的微生物颗粒--菌丝球。.以共固定化菌丝球的产酶能力作为指标，将木质素降解菌烟曲霉菌G-13和纤维素降解菌蜡样芽孢杆菌X10-1-2进行共固定，构建了一种菌丝球自固定化细胞体系，形成兼具木质素和纤维素降解能力的复合菌菌丝球。探讨了纤维素降解菌的接入时间、接种量、培养基的pH值、摇床的转速和温度5个因素对双菌种共固定化菌丝球成球及产木质素酶和纤维素酶活性的影响，获得了共固定化菌丝成球的最佳条件。以复合菌菌丝球为研究对象，考察了对造纸废水、重金属废水和染料废水的处理能力。复合菌菌丝球对于木质纤维素的降解、重金属离子和染料的吸附，较单一真菌菌丝球的吸附率分别高出10%、15%和20%以上，表明复合菌菌丝球在实际应用中具有更加优越的性能且该复合菌菌丝球不仅能处理造纸废水，还可用于处理染料废水和重金属离子废水。研究结果还显示，该复合菌菌丝球的应用范围广泛，可用于高温和强碱的体系中。而且经过3批次的废水处理，木质素、纤维素、半纤维素的降解率仍可达到20%以上，说明复合菌菌丝球可较长时间维持活性，能够持续地处理造纸废水，为工业污水处理提供了一个新的途径。