The Role Of Quorum Sensing On The Denitrification Of Pseudomonas Aeruginosa And Its Potential Application

Nitrate is one kind of common and typical pollutants in wastewater,and the characteristics of nitrate wastewater are wide source and large emission.When plenty of nitrate wastewater is discharged into the water body,it will not only cause serious pollution to the surface water,but also cause groundwater pollution.It will be likely to cause lesions,such as methemoglobin and cancerization,if people drink groundwater which contains excessive nitrate for long time.A large amount of nitrate wastewater discharged into lakes and seas could easily cause eutrophication or water bloom.Therefore,it is urgent to find an efficient method to remove nitrate.In this paper,the effects of quorum sensing on denitrifying behavior of facultative bacteria P.aeruginosa under aerobic and anaerobic conditions were investigated,and the similarities and differences between quorum sensing regulated aerobic and anaerobic denitrification of P.aeruginosa were obtained.In addition,we selected one aerobic activated sludge(Hangzhou seven sewage treatment plant,China)as inoculum to explore the role of quorum sensing in the bioaugmentation of P.aeruginosa under aerobic conditions.After successful colonization of strain P.aeruginosa,quorum quenching agent(Acylase I from porcine kidney)was added to explore the effect of quorum quenching on aerobic denitrification in P.aeruginosa.The main conclusions are as follows:(1)Under anaerobic conditions,P.aeruginosa single mutant AlasI and Arhll have higher nitrate removal abilities and could produce more N2(the percentage of N2 in ?lasI and ?rhll were 29.6%and 28.5%,while wild strain was only 19.9%).Adding correspond exogenous signal molecule(s)to?lasI and Arhll mutant strain would repress the reduction of nitrate which was similar to that in wide type.Enzyme and molecular biology data further proved that quorum sensing system of las and rhl had inhibitory effect on anaerobic denitrification of strain.2?M 3-oxo-C12=HSL has obvious effect on anaerobic denitrification in double mutant ?lasI?rhlI,while 10?M C4-HSL was not.In conclusion,the anaerobic denitrification activity in P.aeruginosa was regulated by 3-oxo-C12-HSL and C4-HSL.Regulation by the las quorum sensing system was dependent on the rhl quorum sensing system;anaerobic denitrification was regulated by quorum sensing at the transcriptional level.(2)Under aerobic conditions,At the late log phase or the stabilization period,wide type strain could reduce 144 mg/L nitrate,while the removal of nitrate in strains with a deletion in las or rhl quorum sensing system were as high as 164 mg/L and 163 mg/L.In addition,the reduction of nirate in double quorum sensing systems mutant was similar to that in single quorum sensing deletion strain.After we added 2 ?M 3-oxo-C12-HSL or 10?M C4-HSL to its corresponding mutant,the denitrification efficiency in mutants decreased to the level of wild type strain.That means las and rhl quorum sensing systems could inhibit aerobic denitrification in P.aeruginosa.The activities of denitrifying enzymes NAP and NIR and expression of denitrifying functional genes showed that the regulation of aerobic denitrification by quorum sensing was accomplished the regulation of QS on expression level of genes.Quorum sensing signal quencher kidney acylase was useful to improve the removal efficiency of nitrate in P.aeruginosa,and this result also reflected that quorum sensing has inhibitory effect on aerobic denitrification in P.aeruginosa.(3)P.aeruginosa and quorum quenching agent were added to acclimated activated sludge to explore the effect of quorum sensing on P.aeruginosa colonization,the results shown that quorum quenching has negative effect on the colonization process.On the basis of bioaugmentation,we explored the role of quorum sensing on the removal of nitrate,reduction of nitrate and concentration of signals shown that quorum quenching can improve the removal of nitrate,it's just because acylase I decreased the concentration of 3-oxo-C12-HSL and C4-HSL.(4)QS has negative effects on both anaerobic and aerobic denitrification,but las QS system regulated anaerobic denitrification mainly dependent on rhl QS system,while it was not the same regular under aerobic denitrification process.