Nitrogen Removal And Heavy-Metal Resistance Characteristics Of Heterotrophic Nitrifying-Aerobic Denitrifying Bacterium Pseudomonas Putida ZN1

The increasingly frequent production activities of humans have led to the discharge of a large amount of substandard nitrogen-containing wastewaters into natural water,causing increasingly serious water pollution.The biological methods have been widely used in the treatment of nitrogen-containing wastewater due to its high nitrogen removal efficiency and environmental friendliness.Heterotrophic nitrification-aerobic denitrification,a novel microbial nitrogen removal method,not only greatly improves the rate of nitrogen removal,but also effectively removes organic pollutants from water bodies.Therefore,research on heterotrophic nitrification-aerobic denitrifying bacteria has become a“hot spot”of nitrogen removal from wastewater.However,certain heavy metals are usually contained in actual industrial wastewater,for example,steel,printing and dyeing,tanning,electroplating and other industrial wastewaters contain heavy metals,such as Ni,Cr,Zn and Cu,in varying degrees.The presence of these heavy metals inhibits the metabolic capacity of the microorganisms and severely affects the efficiency of nitrogen removal.In this study,a highly efficient heterotrophic nitrifying and aerobic denitrifying bacteria resistant to heavy metals was screened from coking wastewater,the effects of different factors on the heterotrophic nitrification performance were systematically studied,and the nitrogen removal mechanism of the strains was presumed based on the utilization of different nitrogen sources,nitrogen balance experiments,enzyme activity tests and gene detection.Finally,the heavy metal tolerance of the strain was initially investigated,in order to better provide technical guidance for practical applications.The main conclusions are as follows:?1?A heterotrophic nitrifying-aerobic denitrifying bacterium resistant to heavy metals was screened,and BLAST analysis of the 16S rDNA sequence showed that the strain had 100%homology with Pseudomonas putida.The strain was initially identified as Pseudomonas putida,named as ZN1.?2?The single factor experiments showed that the optimal conditions for the growth and heterotrophic nitrification of ZN1 are:citrate as carbon source,C/N16,temperature 30°C,pH 7.0,shaking speed 120 rpm;the ammonium removal efficiencies dropped with increasing initial NH₄⁺–N concentration,but the cell growth was not vulnerable in high ammonium concentration.?3?ZN1 has excellent heterotrophic nitrification performance with ammonium as the sole nitrogen source,and the nitrogen removal efficiency is97.5%at 24 h;the aerobic denitrification experiments showed that ZN1 can effectively utilize nitrate and nitrite,and the maximum removal efficiencies of nitrate and nitrite are 86.1%and 71.6%,respectively,in 36 h.For simultaneous nitrification and denitrification experiments with NH₄⁺–N+NO₃^-–N?or NH₄⁺–N+NO₂^-–N?as the nitrogen sources,ZN1 preferentially utilized NH₄⁺–N in the mixed nitrogen sources,and the removal efficiencies of NH₄⁺–N and NO₃^-–N?or NH₄⁺–N and NO₂^-–N?are 94.4%and 94.8%?or 96.3%and 99.4%?,respectively.?4?When NH₄⁺–N was used as a nitrogen source,N₂ production was detected in the gas detection test.The nitrogen balance experiment showed that57.0%of the initial NH₄⁺–N was converted into intracellular nitrogen,and the loss of 34.5%of nitrogen was presumed to produce N₂ through denitrification.The activities of three key enzymes were detected under aerobic conditions with ammonium as nitrogen source,and the activities of HAO,NR and NIR were0.158,0.015 and 0.092 U/mg proteins,respectively.Finally,the denitrification genes were amplified by PCR,napA and nirS were successfully obtained with the sequence length of 841 bp and 901 bp,respectively,the denitrification performance of strain ZN1 was confirmed from the genetic level.?5?Based on the utilization of different nitrogen sources,gas production and nitrogen balance,enzyme activity test and gene detection,it was concluded that the possible nitrogen removal pathway of ZN1 is heterotrophic nitrification combined with aerobic denitrification to produce N₂?NH₄⁺?NH₂OH?NO₂^-?NO₃^-?NO₂^-?NO?N₂O?N₂?.?6?ZN1 has excellent heavy metal tolerance,and the inhibitory effect of heavy metals on the heterotrophic nitrification followed the order Ni²⁺>Cr⁶⁺>Zn²⁺>Cu²⁺.The copper resistance gene copB-I was successfully amplified from ZN1,explaining the extremely high copper resistance.