Kanamycin Biodegradation And Reutilization Of Its Fermentation Residue

Kanamycin is an important member of aminoglycoside antibiotics. It has been widely used for it is cheap, efficient and little anaphylactic reactions. But the antibiotic resistant microbes would increased because of the residues of antibiotics in the environment, which may greatly threaten the human being’s health. Several aspects are included in this research as follows:Screened and studied the kanamycin degrading strains, as well as their growth pattern and degradation characteristics for biodegradation process; Investigated the reuse of bacteria residue as nitrogen source in fermentation medium by modifying bacteria residue with physical, chemical and biological methods; Enhancd the production of kanamycin by mutagenesis. The main content and results are listed as follows:1. Three degradation strains, namely A1, A2, and A3, were screened out through mineral salt media used kanamycin as sole carbon source from kanamycin contaminated soil and activated sludge. They are all gram negative and short stick strains. Strains A1 and A2 were identified as Paracoccus kondratievae, while strain A3 was identified as Aquamicrobium sp. through 16srDNA and biochemical characteristics.2. The strain A2 was the most efficient for kanamycin biodegradation. So, its growth pattern and degrading properties were been investigated. When A2 was culture in LB media, its lag phase was in 0-5 h,5h-llh for logarithmic phase and>11 h for stable phase, respectively. Using logistics equation to analyze the growing curves of A2 with 0,200 mg/L,1000 mg/L kanamycin concentration, it can be found that kanamycin only inhibited the growth of A2 at the beginning and showed the concentration effect. The optimum growing conditions of A2 are pH 6-9,37 ℃.1 mM of Cd2+greatly inhibits the growth of A2.1 mM and 10 mM of Mg2+ can weakly promote the growth of A2. At the concentration of 10 mM, Zn2+, Cu2+, Co2+, Ni2+, Fe3+, Ca2+, Ba2+, Cd2+ and Mn2+ can inhibit the growth of A2 to different extent. A2 can tolerate 100 mg/L streptomycin and gentamycin.When cultivated in mineral salt medium, strain A2 showed high tolerance of kanamycin at 16000 mg/L. Its optimum degrading temperature is 37℃. Additional 0.2% glucose, maltose, beef extract, yeast extract, starch, and peptone can promote the degrading ability of A2, while mannitol and sucrose can restrain the degrading ability. When adding 0.05% different metal irons, Fe3+ has no significant effect on degrading ability of kanamycin, while Mn2+, Co24, Cu2+, Ba2+, Ni2+, Cd2+, Zn2+and Al3+ can restrain the degrading ability.3. Studying the use of A2 in treating kanamycin waste water:The concentration of kanamycin waste water has significant effect on the time needed for degrading kanamycin residue completely. Kanamycin can be degraded in 24 h when waste water concentration is 25%. When the concentration is 100%, kanamycin cannot be degraded easily, and there’s still 3.21 mg/L kanamycin remained after 72 h. The degrading efficiency of A2 on unfiltered and filtered waste water was almost the same, but the former had higher biomass than the latter. This means that the other microorganisms in the waste water did not affect the degration of kanamycin.4. Using A2 to treat kanamycin bacteria residue. It can reach rather high degrading rate in 24 h. When inoculating doses vary from 10% to 70% of bacteria residue, more inoculating doses can have the better result, especially inoculating doses increase from 10% to 30%, the increasing of degrading rate is most significant, up to 34.15%. When adding water from 30% to 90% of bacteria residue, degrading rate increases significantly. When adding 90% and 120% water, the degrading rate only increased 1.62%. Adding 1% different carbon and nitrogen sources, glucose and beef extract could restrain the degrading of kanamycin, while peptone has no significant effect, yeast extract could promote the degradation.5. Then, compared the kanamycin degrading ability of A2 cell suspension, intracellular enzyme, extracellular enzyme and cell debris suspension. The enzyme was confirmed to a constitutive enzyme after further research. And intracellular enzyme extracting conditions was optimized through orthogonal experiment, the optimum ultrasonic broken conditions were at the power of 300 W, frequency ultrasound 2s with interval of 2s, and 20 min. The optimum degrading conditions of intracellular enzyme were pH 4.5-6.5,35℃.0.1 mM of Fe3- can promote the enzyme activity, while Mn2+, Zn2-, Co2+, Ni2+ and Cu2+ can restrain the enzyme activity and Cu2+ has the distinct inhibitory effect. The Michaelis constant was certificated as 371.65 mg/L through the enzymatic degradation of different concentration of kanamycin and Michaelis-Menten equation.6. It was found that using fermented bacteria residue to completely replace soybean cake powder in fermentation media did not work well. Even if treated with NaOH, the maximum titer could reach less than 50%. Using bacteria residue to replace soybean cake powder partly can get pretty well result, adding 0.5-2.5% bacteria residue (replacing 14.29% to 71.43% soybean cake powder in the origin fermentation media) can get more than 90% titer. The research found fresh bacteria residue co-fermented with long-term stacked residue, mixture ratio was 1:1 can get favorite modification result, when used to replace 57.14% soybean cake powder, the titer can reach more than 90%.7. Also, use the ethyleneimine to obtain mutant strains for high production of kanamycin. Finally three mutant strains with enhanced production were obtained. The strain Y9 had the enhanced production of about 13% increasing to that of the initial strain.