Biocomposite Preparation By Cross-linking Of Chitosan And Engineered Pseudomonas Putida MB285Cells And Effect On Dye Decolorization

Bacterial cell-surface display system has been recognized as a new technology in protein application, which has shown promising prospects in many biotechnological fields. An engineered Pseudomonas putida expressing and displaying bacterial laccases onto cell surface, namely, MB285, has been previously constructed in this laboratory, and has been successfully used as regenerable biocatalyst for biodegrading synthetic dyes. For further improving the performance of this biocatalyst system in terms of decolorization efficiency, stability and applicability under large-scale or continuous processes, in the present study, by using chitosan as the matrix substrate, a biocomposite material with the covalent cross-linking of the the P. putida MB285cells and the chitosan-microbeads was prepared, and was applied to decolorize several industrial synthetic dyes. The decolorization effiency, stability, and capabilities of the prepared biocomposite, as well as that of the repeated use and regenerative performance under continuous operations were investigated.Through self-configuring instrument components, a facile and applicable chitosan-microsome preparation system was self-assembled initially, and successfully prepared five chitosan microbead materials (CTS-B) with different microsome sizes. Under the given working voltage of5.3kV, the system generated a minimal microbead, namely, CTS-B1, which exhibited a minimal particle-diameter by450μm and a maximum average specific surface-area by1.36×10-6m2/g over other prepared microbeads. The single factor and orthogonal trials on five prepared CTS-B materials was then respectively performed based on the assessments of total laccase activities and immobilized cell counts of the resultant composites, an optimized cross-linkage condition using CTS-B1as the matrix,8%glutaraldehyde,20℃and reaction time for6h, and an optimized cell-immobilization condition under4℃, reaction time for6h and using cell suspension at OD600of5.0were verified. Scanning electron microscopy confirmed the surface immobilization of MB285cells onto CTS-B materials. Fourier transform infrared spectroscopy further confirmed the covalent cross-linkage property of MB285cells into CTS-B materials. The storage stability of the biocomposite with CTS-B1and MB285cells (namely, IBCC-B1) was comparatively investigated under a storage of30d. It showed that the biocomposite IBCC-B1has apparently higher stability of total enzyme activity over the unimmolibized MB285cells, as the total laccase activity of IBCC-B1retained70.8%, whereas the latter retained only54%of this activity.The optimization experiments on decolorization of two industrial synthetic dyes "Acidic Green25"(AG25) and "Acidic Red18"(AR18) were performed through single factor trials. It showed that the highest decolorization efficiency towards two dyes reached, while the decolorization reactions were performed under the conditions included the reaction time of30min, pH of2.5,25℃, IBCC-B1loading of2.0g, and a shaking speed of180rpm. Under the equivalent cell loading and the same reaction conditions, the decolorization efficiencies of unimmobilized MB285cells towards AG25and AR18were only75.7%and66.8%, indicated the improved decolorization efficiency of IBCC-B1over unimmobilized MB285cells. Meanwhile, IBCC-B1was also applied to decolorize several other synthetic dyes, such as "Active Blue198"(RB198),"Active Blue220"(RB220), and "Direct Red243"(DR243), which showed all remarkable decolorization efficiency by96.3%,95.7%, and94.3%of RB198, RB220, and DR243, respectively.The repeated decolorization performance of IBCC-B1was investigated under a continuous decolorization process of10rounds. It showed that IBCC-B1retained a high decolorization efficiency of84.7%after the4-round of continuous decolorization towards AG25. Further, with a subsequent cell culturing regenerative process in each decolorization reaction, IBCC-B1was reasily retrieved in the decolorization activity. It exhibited a decolorization efficiency of92.8%towards AG25after the4-round of decolorization processes. Therefore, this study demonstrates the methodology and feasibility by which the biocomposte with the engineered P. putida cells and chitosan microbead was successfully used as efficient, repetitive and regenerative biomaterial for biodegrading synthetic dyes, thereby opening new perspectives in large-scale or continuous operations of industrial dye biotreatment.