Construction Of The Genetically Engineered Flocculation Bacteria And Screening Of Microbial Flocculant-producing Strains

Microbial flocculant is a kind of bioactive polymers secreted by microorganisms, andcan flocculate the suspending particles in the water. Because of its non-toxic,environmentally-friendly, and biodegradable characteristics, microbial flocculant has beenwidely used in food and fermentation industries, drinking water and wastewater treatment,industrial downstream process and other fields, and has aroused extensive attention frommany researchers. It has aroused extensive attention from domestic and foreignresearchers.In this study, the genetically engineered flocculation bacteria were constructed andmicrobial flocculant-producing strains were screened. The main methods and rusults weredescribed as follows:1. The FLO5gene was amplified by PCR from genomic DNA extracted fromSaccharomyces cerevisiae, and then cloned into pMD18-T vector and the recombinedplamid was named pMD18-T-FLO5. After sequence conformation, FLO5was recombinedinto prokaryotic expression vectors pET-22b and pET-28a to form pET22b-FLO5andpET28a-FLO5, which were then transferred into E.coli JM109for amplification. Extractedrecombined plasmids pET22b-FLO5and pET28a-FLO5, transferred into E.coliBL21(DE3). The target protein was expressed in the genetically engineered flocculationbatcterium BL21/pET28a-FLO5by induction with IPTG. SDS-PAGE showed a band withthe expected size of FLO5protein suggesting the expression of gene FLO5. The tests offlocculation activity of flocculation recombinant bacterium BL21/pET22b-FLO5andBL21/pET28a-FLO5showed that their flocculation activity were increased by1.48and2.85times respectively compared with that of original strains.2. A stain of high effective bioflocculant-producing bacteria with stable flocculantionactivity, named A5, was isolated. According to morphological observation, physiologicaland biochemical tests,16S rDNA sequence alignment analysis, the strain was classifiedinto Sphingobacterium. Response surface methodology was used to optimize theconditions for A5culture, and three important factors for the strain to produce flocculantdetermined with Plackett-Burman experimental design, they were: the content of glucose,urea, and sodium chloride. Box-Behnken experimental design determined the content of glucose15.0g/L, urea0.88g/L, sodium chloride0.13g/L, and the strain showed itsmaximum flocculation rate reached up to83.87%. The highest flocculation rate ofBL21/pET28a-FLO5mixed with the strain A5at the ratio of1:2was32.949%, which was2to3times higher than that of BL21/pET22b-FLO5mixed with the strain A5.