The Nitrogen Transformation Pathway By Pseudomonas Putida Y-9 Under Aerobic Conditions And Its Influencing Factors

Ammonium and nitrate are both common inorganic nitrogen in soil and wastewater and occupy the positions of the lowest nitrogen and the highest nitrogen in nature,respectively.Microorganisms transform these two kinds of different valence nitrogen through the nitrogen cycle,which could protect aquatic ecosystems from eutrophication.Due to the complexity and diversity of microbial communities,the microbial nitrogen-cycling network is complex and the new microbial nitrogen-transforming reactions and pathways have been reported.Many environmental factors?such as the common factors?temperature,salinity,nitrogen concentration,etc.?and metal ions as well as metal oxide nanoparticles that have only been concerned in recent years?was found to affect the nitrogen transformation of microorganism.However,the response mechanism of microorganisms to environmental factors is flexible.In our previous study,Pseudomonas putida Y-9,a cold-adapted bacterium,was isolated from the long-term flooded paddy soil.Further,strain Y-9 oxidized ammonium under aerobic conditions without the accumulation of hydroxylamine and reduced nitrate and nitrite accompany with an accumulation of ammonium.Nevertheless,previous studies showed that hydroxylamine is an inevitable intermediate during all the ammonium oxidation pathways under aerobic conditions,and dissimilatory nitrate reduction to ammonium?DNRA?only occurs under anaerobic conditions.The difference between the nitrogen transformation characteristics of strain Y-9 and the exiting nitrogen transformation pathways suggested that there might be new nitrogen cycling pathways in strain Y-9.In this study,the specific ammonium oxidation pathway and nitrate reduction pathway in strain Y-9 under aerobic conditions was explored by combining the analyzing of the transformation characteristics of ammonium,hydroxylamine and nitrate,the enzymology and molecular biological method?including enzyme activity analysis,gene amplification,whole genome scanning,etc?,as well as ¹⁵N isotope tracing technology.Furthermore,the effect and mechanism of the four common metal oxide nanoparticles on the ammonium oxidation pathway of strain Y-9 in the presence of Mg²⁺was explored.At the same time,the impact of nirBD gene on nitrogen transformation in strain Y-9 was studied.Finally,how to adjust the external conditions to achieve the large-scale removal of NO₃^-and the minimum loss of nitrogen in the system were discussed.The major results of this study are as follows:?1?Strain Y-9 was found to oxidize ammonium rapidly?the ammonium oxidation rate reached 100%?without the accumulation of hydroxylamine,nitrite,and nitrate under aerobic conditions.There are two possible reasons for this phenomenon.One is that the order of the relative enzyme activity was NiR>HAO>AMO,resulting in the fast conversion of hydroxylamine,nitrite,and nitrate.The other possible reason is that the ammonium oxidation pathway of strain Y-9 is different from previously reported pathways.Further study found that the AMO activity was undetectable,and the specific activity of AMO-a was far higher than those of HAO,NR,and NiR,which suggested that the ammonium oxidation process of strain Y-9 is not via hydroxylamine.The transformation characteristics of ammonium and hydroxylamine illustrated that there was no gas nitrogen during the transformation process of hydroxylamine,but nitric oxide and nitrous oxide were detectable during the ammonium oxidation process.The above results further proved that the ammonium oxidation of strain Y-9 does not use NH₂OH as an intermediate.Additionally,allylthiourea?a classical inhibitor of AMO?did not inhibit the oxidation of ammonium by strain Y-9.The gene amplification assay further demonstrated that the amoA gene doesn't exist in strain Y-9.These two phenomena indicated that there is not AMO in strain Y-9.All the results showed that the novel ammonium oxidation pathway of strain Y-9 may be NH₄⁺?NO?N₂O.Whether strain Y-9 can directly transform ammonium into NO or through other intermediates,which needs further study.?2?When the amount was greater than 1 mg/L,the nanoparticles all showed inhibition effect on ammonium removal of strain Y-9,the order of inhibition intensity was NiO NPs>CuO NPs>ZnO NPs>TiO₂ NPs.Interestingly,low content of CuO NPs?0.1,0.5 mg/L?and NiO NPs?0.1 mg/L?could enhance the growth and the ammonium removal efficiency of strain Y-9,the ammonium removal rate reached more than 87%.The concentration of metal ions released from different metal oxide nanoparticles was different.Moreover,a decrease in Mg²⁺levels was significantly positively correlated with the growth of strain Y-9 and its ammonium removal efficiency,while negatively correlated with the Ti²⁺,Zn²⁺,and Cu²⁺release of NPs.Further research on effect of metal oxide nanoparticles and their corresponding cations on ammonium removal revealed that four kinds of metal oxide nanoparticles affected Mg²⁺?the essential element for growth of strain Y-9?absorption in strain Y-9 via different routes,thus impacting NH₄⁺removal efficiency,i.e.,the effect of NiO NPs was caused by itself,TiO₂ NPs'impact was solely due to the release Ti⁴⁺,while the influence of CuO NPs and ZnO NPs was based on both the particles and released ions.?3?Strain Y-9 removed 82%of the nitrate accompanied by an accumulation of ammonium and a little decrease of total nitrogen?21.08%?.This suggested that strain Y-9 could simultaneously assimilate and dissimilate nitrate.Ammonium inhibited nitrate transformation?removal efficiency was 22.65%?,illustrating that nitrate assimilation exists in strain Y-9.However,there was a light decrease of NO₃^-?11.68mg/L?when NH₄⁺was 92.75 mg/L?at 1 d?,indicating that strain Y-9 might also carry out the weak dissimilatory NO₃^-reduction.The detectable ammonium in the supernatant during the nitrate reduction process came from intracellular locations in strain Y-9.The nirBD that encodes nitrite reductase showed an important role in strain growth and ammonium production.These results suggested that the assimilation then mineralization process of nitrate or DNRA might be the source of ammonium in the supernatant.A ¹⁵N isotope experiment demonstrated that strain Y-9 can conduct dissimilatory nitrate reduction to ammonium?DNRA?and nirBD controls this process,the specific pathway was NO₃^-?NO₂^-?NH₄⁺.This further indicated that the loss of total nitrogen is due to denitrification.All results highlighted that strain Y-9 performs simultaneous nitrate assimilation,DNRA,and denitrification under aerobic conditions and nirBD controls the assimilation and DNRA process.?4?The stable hydrophilic protein NirBD that found in strain Y-9 had no transmembrane domain.Moreover,it had the nitrite and sulfite reductases iron-sulfur/siroheme-binding site that is associated with the reduction of nitrite to ammonium catalyzed by nitrite reductase.The?nirBD strain showed higher ammonium removal efficiency?90.70%?than wild strain?76.09%?,but lower total nitrogen removal ability?15.73 mg/L?than wild strain?17.74 mg/L?after 2 d of cultivation.The results combined with the ammonium oxidation pathway in strain Y-9proved that knocking out nirBD sequence enhances the ammonium assimilation.Most of the nitrate and nitrite was removed through nitrate assimilation and DNRA by strain Y-9 catalyzed by nirBD gene.After knocking out the nirBD genetic sequence,the nitrite that came from nitrate could not further be reduced to ammonium,resulting in a large accumulation of nitrite in the system,finally inhibiting the growth of strain Y-9and its denitrification ability.Nitrate had little effect on ammonium transformation of strain Y-9 whether knocking out nirBD genetic sequence or not,and the removal rate of ammonium in the system was still over 90%after 4 d cultivation.Nitrite significantly inhibited the ammonium transformation of strain Y-9?the removal rate was 44.77%?but had no impact on the ammonium conversation of?nirBD strain Y-9?the removal efficiency was 97.49%?.These might because that knocking out nirBD genetic sequence promoted ammonium assimilation,thus counteracting the toxicity of nitrite.Our results demonstrated that the nirBD gene in P.putida Y-9 could stimulate the process of NO₃^-assimilation and DNRA,and also showed impact on the ammonium oxidation.?5?The effect of carbon source,C/N,pH,dissolved oxygen on nitrate reduction by strain Y-9 was explored.The results showed that adding glucose could achieves the remarkable removal of nitrate?removal rate reached 89.79%?through stimulating the expression of nirBD gene in strain Y-9 to promote DNRA and nitrate assimilation,and inhibiting the denitrification?the loss of nitrogen in the system was only 22.77 mg/L?.Under aerobic conditions,the nirBD gene in strain Y-9 could be expressed effectively in different C/N ratios medium,and the highest expression was achieved in the medium that the C/N was 9.Strain Y-9 mainly removed nitrate?the removal efficiency was81.78%?through DNRA and nitrate assimilation when C/N ratio was 9,the denitrification process?the decrease of total nitrogen was 14.06 mg/L?was significantly weaker than that of C/N ratio 12 and 15?the total nitrogen decreased by35.41 and 22.77 mg/L,respectively?.Neutral and weak alkaline conditions?pH 7-9?promoted the three reduction pathways of nitrate in strain Y-9.Among them,the highest expression of nirBD was found when the initial pH was 7,which results in the highest nitrate removal?81.78%?and the lowest nitrogen loss?14.06 mg/L?.With the increase of shaking speeds,the expression of nirBD gene in strain Y-9 gradually increased the concentration of nitrate showed the same tendency.However,the decrease of total nitrogen in the system showed a trend of first rising and then declining?the maximum decrease of total nitrogen?34.58 mg/L?was achieved at 50 rpm?.Our results showed that using glucose as carbon source,controlling C/N ratio as 9,pH as 7and shaking speed as 150 rpm could promote the nitrate assimilation and DNRA by strain Y-9,weaken the denitrification,finally achieve the significant removal of nitrate and minimum loss of nitrogen in the system.In summary,there are two novel nitrogen transformation pathways in strain Y-9.Moreover,strain Y-9 had excellent ammonium and nitrate removal efficiency under aerobic conditions nearly without the accumulation of the harmful intermediate?nitrite?.The results suggested that strain Y-9 has a broad application prospect in nitrogen pollution wastewater treatment.Besides,the effects of external conditions on the three reduction pathways of nitrate in strain Y-9 provided theoretical support for technical research on nitrate removal and nitrogen retention in soil.