Isolation And Identification Of A Heterotrophic Nitrification-Aerobic Denitrification Bacterium Comamonas Testosteroni Y62 And Its Simultaneous Nitrification And Denitrification Mechanism

With the rapid development of fishery industries in China towards high-density aquaculture,which has led to the heavy accumulation of ammonia and nitrite in aquaculture ponds.Excessive ammonia and nitrite can be toxic to aquatic animals and accelerate the deterioration of aquatic ecosystems.However,the traditional biological denitrification technology seperated the nitrification and denitrification process,which brought about the expensive cost and longer operation time.Heterotrophic nitrification-aerobic denitrification bacteria are newly found in recent years,which could perform simultaneous nitrification and denitrification?SND?,broking through the traditional understanding on nitrogen removal theory.HN-AD bacteria have attracted more and more attention.Up to now,bacteria with this capacity have been widely repoted,such as Acinetobacter sp.,Pseudomonas sp.,and Klebsiella sp.etc.Although many researches have documented that thses bateria are sensitive to dissolved oxgen concentration,their nitrogen removal capacity would be strongly inhibited when the level of dissolved oxgen exceeded 6 mg·L^-1.Additionly,there are still few studies on the simultaneous nitrification and denitrification capacity of HN-AD bacteria in domestic.Therefore,it is of great importance to study on the simultaneous nitrification and denitrification performance of HN-AD bacterium in high dissolved oxgen condition.?1?Sreening and Identification of HN-AD bacteriumAccording to BTB plates screen,and HNM and DM re-screen test,we isolated an HN-AD bacterium termed as Y62 capable of high dissolved oxygen tolerance capacity from aquaculture pond water sampling in the Binhai Breeding Base?Pudong,Shanghai,China?.Strain Y62 exhibited efficient nitorgen removal,with NH₄⁺-N and NO₂^--N removal rate of 65.71%and 97.1%in 24 h,respectively.Under microscopic observation,the strain was gram-negative,short rod-shaped.And the colonies of strain Y62 were creamy white,round shaped with smooth surfaces and regular edges,sticky on the LB agar plate.BLAST analysis of the 16S r DNA sequence of strain Y62 showed that this strain had99.61%homology with Comamonas testosteroni.Together with its these results,strain Y62 was initially identified as Comamonas testosteroni,named as C.testosteroni Y62.The biosecurity test showed that strain Y62 was secure.Zebrafish could survive in river water with strain Y62 suspensions of 1.2×10⁸ CFU·m L^-1.And there was not hemolytic phenomenon on blood agar plates when strain Y62 grew.This is the first report on C.testosteroni strain capable of heterotrophic nitrification-aerobic denitrification capacity in domestic.?2?Simultaneous nitrification and denitrification mechanism of strain Y62Ammonium and nitrite were used as the mix nitrogen source of SND media to study the simultaneous nitrification and denitrification capacity of strain Y62.The results showed strain Y62 had excellent simultaneous nitrification and denitrification performance.NH₄⁺-N was used as the nitrogen source in nitrification process,with the NH₄⁺-N removal rate of 76.46%.And NO₂^--N was used in the later denitrification process,with the NO₂^--N removal rate of 97.1%in 48 h.The NO₃^--N accumulation was totally removed in 12 h.The successfully amplification of amo A,nir K and nap A genes also confirmed the SND capacity of strain Y62 from the genetic level,which suggested that the nitrogen removal by strain Y62 via the complete simultaneous nitrification and NO?N₂O.?3?Effects on the growth and SND performance of strain Y62The single factor experiments indicated that strain Y62 had excellent oxygen tolerance capacity,when the DO was 6.62 mg·L^-1,the NH₄⁺-N and NO₂^--N removal rate reached 77.50%and 97.62%,the OD₆₀₀ reached 1.138.The carbon source,C/N ratio,p H and temperature has significant effect on the simultaneous nitrification and denitrification performance of strain Y62?P<0.05?.The optimal conditions for the growth and SND performance of strain Y62 were:sodium citrate as carbon source,C/N ratio 10,temperature 30°C,p H 7.0.?4?Immobilization of strain Y62 for aquaculture water nitrogen removalStrain Y62 was immobilized on glass pumice with physical adsorption method to remove nitrogen from aquaculture water.The NH₄⁺-N removal were obvious enhanced by immobilized Y62,with NH₄⁺-N removal rate of 53.16%.Meanwhile,immobilization techonology weakened the inhibition of high concentration of NO₂^--N and NO₃^--N on the denitrification of strain Y62,which improved the nitrogen removal efficiency of strain Y62.In summary,C.testosteroni strain Y62 exhibited high oxygen tolerance capacity,and its simultaneous nitrification and denitrification was efficient,which make it a promising candidate for the application for aquaculture water treatment.