Breeding And EGFP Labeling Of High Efficient Anthracene-Degrading Bacteria

Since 1970s',bioaugmentation was used as a tool to remove refractory compound in soil and wastewater, and to promote the efficiency of activated sludgy process. It was also used to improve the situation that contamination-degrading microorganism lacked or its degradability was low. Bioaugmentation exert important function in the bioremediation system and its soul was planting high-efficient strain. . Therefore, how to obtain high-efficient strain attracted many researchers' attentions.Anthracene was the sole carbon source in the experiment. Handling the anthracene-degrading consortium, a strain was obtained. Then the strain was sequentially dealt with UV inducting, acclimatizing and screening, ions implanting, protoplast mutating, and EGFP reporter gene labeling the mutants, while detecting activity and survival ability of mutants. High-efficient exogenous degrading strain was obtained which had carried reporter gene and can be used in bioaugmentation system. The whole work includes:1. Separate anthracene-degrading strain was obtained by selecting and screening the anthracene-degradable consortia which was isolated from the oil-contaminated sludgy. Under the limited condition, by acclimating and detecting the degradability, strain AN1 that had high degradability, was selected. Then through UV induction mutant ANul was obtained, which grow well in the inorganic substrate that acted anthracene as sole carbon source.2. By orthogonal arrays, it was tested that the optimum growth conditions were initial pH8.0, Tween80 volume ratio at 0.4%,37 ℃, skater rotary speed at 150r/min, Glucose concentration at 100mg/L.Under the conditions mutant ANul was domesticated by methods such as batch hunger and progressively increased anthracene, concentration reached 200mg/L.3. Two mutants were obtained by ions implantation. By examined in shaking flasks with anthracene being the sole carbon source and its concentration being 200mg/L.At 37 ℃, rotary speed 150r/min, the two mutants were cultivated for 30hours . The anthracene-degrading rate hit 73%, 75% and anthracene-enduring concentration hit 400mg/L.4 .To monitor the fate and efficiency of degrading bacteria in the bioaugmentation system,the strain ANI315 was labeled with EGFP reporter gene. According to the character of plasmid pTnmod-Ocm containing Tn5 transposon and the nucleic sequence of EGFP, the restriction site (Kpnl and Nsil) was used as the site of upstream and downstream primer for the PCR of EGFP.The product of PCR of EGFP and pTnmod-Ocm was digested by Kpnl and Nsil respectively, and then linked by DNAHgase after purification. The recombinant plasmids were electro-transformed into ANI315.The resulting transformants were obtained with the over expression of EGFP and appeared bright green stimulated by exogenous light. Identified by PCR of genomic DNA and southern blotting, the transformed EGFP gene lied in chromosome of ANI315.The transformant was named ETANI315.5.Compared with the original strain ANI315, the anthracene-degrading activity and survival of transformants were stable. The result revealed that EGFP gene could be used as vivo reporter in the bioaugmengtation system.