Study On Characteristics And Mechanism Of Simultaneous Bisphenol F Degradation,heterotrophic Nitrification And Aerobic Denitrification By Pseudomonas Sp.HS-2

Bisphenol F(BPF)as a substitute of Bisphenol A has been widely used in the production of polycarbonate and epoxy resins,resulting in the production of wastewaters containing high concentration of BPF.Moreover,the low concentration of BPF has been detected in domestic sewage.As BPF is of biotoxicity and endocrine disruption,the biodegradation of BPF has drawn extensive attention.To date,many aerobic BPF-degrading bacteria have been isolated.These bacteria generally use ammonium as a nitrogen source for growth and metabolism.Traditional denitrification occurred under anaerobic conditions.However,BPF could be rarely degraded and mineralized under anaerobic conditions.The discovery of aerobic denitrification bacteria make it possible to simultaneously remove BPF and ammonium.Based on the background above,a HN-AD strain Pseudomonas sp.HS-2 utilizing bisphenol F(BPF)as a sole carbon source was isolated.The characteristics of BPF degradation and ammonium removal by strain HS-2 were studied,and their transformation pathways were analyzed,respectively.A novel HN-AD bacterium capable of simultaneous BPF degradation and ammonium removal was isolated from the bisphenols-contaminated soil and identified as Pseudomonas sp.(Genbank accession number KU512758).The colony of strain HS-2 was brownish white and circular in shape with a central apophysis on a mineral salt agar plate.SEM analysis showed that strain HS-2 is a rod shaped bacterium.And gram staining experiment showed that strain HS-2 is negative.The optimal carbon source for ammonium removal was sodium citrate.This strain could completely degrade BPF up to 500 mg/L using BPF as a sole carbon source.When the ability of strain HS-2 to degrade BPF and remove ammonium began to degenerate,additional sodium citrate was used along with the increase of BPF concentration for rejuvenating its ability.The results showed that the cells capable of simultaneous BPF degradation and ammonium removal was successfully rejuvenated and renamed as strain HAW6.The characteristics of BPF degradation and ammonium removal by strain HS-2 were studied.The effects of C/N of 5-25,pH 5-9,tempreture of 20-40 °C and shaking speeds of 50-200 rpm on the removal of the two compounds were investigated,respectively.The results showed that the optimal conditions were C/N=20,35 °C,pH 7 and 150 rpm,when strain HS-2 utilized BPF and ammonium as sole carbon and nitrogen sources,respectively.Under the optimal conditions,the removal efficiencies of BPF and NH4+-N reached 99% and 96%,respectively,along with the removal of 90% total organic carbon and 94% total nitrogen.These studies indicated that strain HS-2 could use BPF for removing NH4+-N via HN-AD.Sodium citrate could be used as the co-metabolism substrate by strain HS-2 for accelerating simultaneous BPF degradation and ammonium removal.The presence of NO3--N could also promote the HN process of strain HS-2,that is,NO3--N accelerated NH4+-N removal along with BPF degradation.GC-MS was used for the identification of four metabolites,and the pathway of BPF degradation was proposed.Firstly,BPF was hydroxylated to bis(4-hydroxyphenyl)methanol,further oxidized to 4,4'-dihydroxybenzophenone,and then followed by 4-hydroxyphenyl-4-hydroxybenzoate and 1,4-hydroquinone produced by Baeyer-Villiger and oxidation reactions,finally mineralized to CO2.Ammonia monooxygenase inhibitors allylthiourea and octyne could completely inhibit BPF degradation and NH4+-N removal.The results implied that ammonia monooxygenase in strain HS-2 might be involved in initial hydroxylation of BPF during the ammoxidation.It was the first report to our knowledge that ammonia monooxygenase was involved in aerobic degradation of BPF.Using spectrophotometer and GC,the pathway of NH4+-N transformation was analyzed.The present study showed that ammonium was transformed to nitrate by nitrification,then transformed to N2 by denitrification.No N2 O was detected during the reaction process.Nitrogen balance analysis demonstrated that the transformation efficiency from NH4+-N to N2 could reach 47.7%,and up to 41.5% of initial NH4+-N was converted to intracellular nitrogen by strain HS-2 after 48 h.The nitrate reductase gene napA(861 bp,KY316375)and the nitrite reductase gene nirS(1237 bp,KY386892)were successfully amplified using PCR.The above studies showed that BPF and its metabolites could be used as carbon and energy sources for NH4+-N removal via HN-AD by strain HS-2.