Characteristic And Sulfamethoxazole From Aqueous Solution Degradation Mechanism Of Pseudomonas Psychrophila HA-4

Sulfamethoxazole (SMX) is an antibiotic with nitrogen(N) heterocycle structure, and it is suggested to be persistent to biodegradation. Therefore, it could not be removed effectively by the traditional wastewater treatment. Nowadays, with extensive use of SMX, it is ubiquitously present in surface water with a high detected frequency and it has become a newly pollutant. However, low temperature often affects the removal of SMX. So increasing the removal rate of SMX has been an urgent issue. Microbial pure culture technology was adopted to isolate a newly psychrophilic high efficient SMX-degrading bacterium (strain HA-4) for this problem. Moreover, strain identification and the growth and biodegradation characteristic of strain HA-4were explored. The metabolic pathway of SMX by strain HA-4was proposed. Based on genic level, the biodegradation mechanism of strain HA-4was also discussed. The research results are of strategic significance to develop inoculant of high efficient SMX-degrading microorganism and control SMX pollution in environment.Psychrophile HA-4, isolated in this research, can utilize SMX as the sole sources of carbon. It was identified as genus Pseudomonas. The optimal growth and degradation conditions for HA-4were as follows: pH of6.0, a range of temperatures within10-15°C, rotation speed of150r/min and inoculation volume of15%(V/V). The maximal removal rate of SMX by strain HA-4was34.30%after192h under this condition. Strain HA-4also had the certain of biodegradation capability for some common pharmaceutical and personal care products (PPCPs) and aromatic compounds, such as carbazole, carbamazepine, aniline, and benzidine and so on.The peptone could accelerate SMX degradation and the increase of removal rate reach to above16%. When the concentrations of Ca2+, Fe3+, Al3+, Cd2+, Co2+, Cu2+and Zn2+reached to1mmol/L in the medium, it had an inhibitory effect on strain HA-4’s ability to degrade SMX. The percentages of unsaturation and short chain strain fatty acids reached to23.03%and3.58%respectively, which were the maximal proportions. It demonstrated one of the cold-adapted strategies for strain HA-4. Haldane equation had shown the growth kinetics of strain HA-4on the inhibitory substrate (SMX), which correlated well with the Haldane model. The kinetics parameter values were determined to be μmax=0.01h1, Ks=20.91mg/L and Ki=170.60mg/L.The intermediates from SMX biodegradation were identified as aniline,3-amino-5-methylisoxazole,4-aminothiophenol, sulfanilamide and4-amino-N-hydroxybenzenesulfonamide by GC-MS, and HR-MS analysis. SMX was partly mineralized by strain HA-4. During the process of SMX mineralization, N atoms and S atom of SMX were transformed into NH4+and SO42-respectively, which leaded to the releases of NH4+and SO42-to solution. Based on above analysis results, a newly degradation pathway of SMX by strain HA-4was proposed. Firstly, SMX was hydrolyzed at its linear chain end along the two paths, that is, N-C bond cleavage and S-N bond cleavage according to the co-occurrence of3-amino-5-methylisoxazole and4-amino-N-hydroxybenzenesulfonamide. Due to unstabitily, simultaneously and selectively removing sulfur (C-S bond cleavage) and amino-group from4-amino-N-hydroxybenzenesulfonamide may lead to the formation of aniline. Simultaneously, SO42-and NH4+were released into the solution. Aniline is then further degraded and mineralized by strain HA-4. Finally, the degradation intermediates were mineralized to H2O and CO2.The whole genomes of Pseudomonas psychrophila HA-4was sequenced with Solexa paired-end sequencing technology. After annotation by RAST annotation server, the result indicated the genome size and GC content of strain HA-4were5,235,696bp and56.4%. As functional genes annotated were fell into28categories.155encoding genes,167genes, and75genes were related to Stress Response, Fatty acid, Lipid, and Isoprenoid metabolism, and Metabolism of Aromatic Compounds respectively. Six cold shock protein-encoding genes were annotated in the whole genomes of strain HA-4, containing cspA1, cspA2, cspA3, cspC, cspD and cspG. They belongs to cspA family, and the first three genes are cspAs. It was supposed that the cold shock protein-encoding genes were helpful for strain HA-4to adapt to low temperature environment.The S-(hydroxymethyl) glutathione dehydrogenase and29antibiotic-resistant genes from the annotation may contribute to tolerating SMX in certain concentrations for strain HA-4. The S-(hydroxymethyl) glutathione dehydrogenase may participate in biotransformation.The oxidordeuctase may have significance for SMX biodegradation. Based on bioinformatics analysis, SMX was transformed into aniline by catalysis of several enzymes, such as hydrolase, dibenzothiophene desulfurization enzyme and so on. Aniline was then further metabolized by catechol branch of β-ketoadipate pathway. Aniline biodegradation was controlled by catabolic pathways of meta position. Firstly, aniline was degraded into catechol as the intermediate by dioxygenase. Then catechol was further degraded to succinic acid and acetyl-CoA of citric acid cycle intermediates via multi step reactions. Finally, the degradation intermediates were mineralized to H2O and CO2via TCA cycle. It confirms that strain HA-4has ability of mineralizing SMX, which makes a lot of sense for environment pollution control.