Tolerance And Mechanism Of Heterotrophic Nitrifying Bacteria Pseudomonas Putida Strain LX1 To Heavy Metal Ions

Usually,a large number of nitrogen-containing industrial wastewater will contain a certain amount of heavy metals at the same time,causing compound pollution of heavy metals and nitrogen,damaging the ecological balance of the water environment,and posing a major threat to public health and biodiversity.Heterotrophic nitrification-aerobic denitrification（HN-AD）,as an efficient new type of nitrogen-containing wastewater treatment technology,can not only remove organic carbon compounds in water,but also realize simultaneous nitrification and denitrification,and some heterotrophic nitrifying bacteria also have the ability to tolerate heavy metals.In this study,a heterotrophic nitrifying strain with strong tolerance to heavy metals was screened from the activated sludge stressed by high concentration of nano nickel oxide in the laboratory for a long time.K₂Cr O₄、Cr₂O₃、Ni O and Pb Cl₂were used as experimental materials to systematically study the growth and metabolism process and interaction mechanism of the experimental strain,clarify the molecular mechanism of heavy metal tolerance of the strains,and provide scientific guidance for the practical application of heterotrophic nitrifying bacteria.The main results are as follows:（1）A heterotrophic nitrifying strain LX1 with tolerance to heavy metals was screened from the activated sludge stressed by heavy metals for a long time in the laboratory.The test showed that it also had aerobic denitrification performance.16S r DNA partial gene sequence sequencing identified strain LX1 as Pseudomonas putida.The optimum growth and denitrification conditions of strain LX1 were sodium succinate as carbon source,C/N was 10,r=160 r/min,p H=7,T=30°C.Strain LX1 could grow and remove nitrogen normally when NH₄⁺-N and NO₃^--N were used as the sole nitrogen source,and the maximum removal rates of NH₄⁺-N and NO₃^--N could reach93.55%and 81.68%within 48 hours.（2）The effects of different concentrations of Cr₂O₃、K₂CrO₄、NiO and Pb Cl₂stress on the growth and denitrification characteristics of strain LX1 were quite different.It was found that K₂Cr O₄had a great impact on the heterotrophic nitrification of strain LX1.The NH₄⁺-N removal rate gradually decreased with the increase of K₂Cr O₄concentration.The maximum NH₄⁺-N removal rates were 93%,90.3%and 83.3%when the concentration was 5,10 and 25 mg/L,but the strain almost stopped growing when the concentration was greater than 50 mg/L.In addition,although Cr₂O₃,Ni O and Pb Cl₂had certain inhibitory effects,strain LX1 has a good ability to restore ammonia oxidation.Under the stress of 100 mg/L,the maximum NH₄⁺-N removal rates were still as high as 90.3%,89.25%and 90.86%respectively,showing good tolerance.The heterotrophic nitrification process showed that after the addition of K₂Cr O₄,Ni O and Pb Cl₂,there was almost no accumulation of intermediate products such as nitrate and nitrite in the process.It may be that strain LX1 can directly use the generated nitrate and nitrite as denitrifying substrate.With NO₃^--N as the sole nitrogen source,the aerobic denitrification results showed that there was no nitrite accumulation in the whole culture process,which may be because the nitrite produced in the process was immediately removed by nitrite reductase.（3）Although K₂CrO₄、NiO and PbCl₂affect the growth and nitrification of the strain by inhibiting the activity of key enzymes,the antioxidant effect maintained the normal growth and metabolism to a certain extent,and the tolerance of strain LX1 to Ni O and Pb Cl₂was better than that of K₂Cr O₄.According to the three-dimensional fluorescence spectrum of EPS,the growth and metabolic function of strain LX1 was damaged,the protein like substances decreased and the humus content increased under K₂Cr O₄stress;Under Ni O stress,the content of protein like substances increased significantly with the increase of concentration;Under the stimulation of low concentration of Pb Cl₂,the content of protein like substances increased significantly,while under the stimulation of high concentration of Pb Cl₂,the content of protein like substances decreased and the content of humus increased.（4）FTIR spectra showed that the spectral shape of the infrared spectra of the three heavy metals was consistent with that of the control group,almost unchanged,but it brought varying degrees of changes to the intensity of the peak.XRD patterns showed that the position of characteristic peaks was not affected after being stressed by heavy metals K₂Cr O₄、Ni O and Pb Cl₂,and the sharpness changed to a certain extent according to different metal types.The results showed that heavy metals were adsorbed on the surface of strain LX1,but no new substances were produced.The species and lattice constants of substances did not change with the addition of heavy metals.（5）The transcriptome analysis of the strain stressed by heavy metal chromium and the original strain showed that Go enrichment showed that the most significant differences were mainly in phosphatidylamide binding,modified amino acid binding,active transmembrane transporter activity,response to oxidative stress,cell membrane and extracellular membrane,indicating that the transport function,oxidative stress and cell membrane composition played an important role in the response of strain LX1 to heavy metal stress.KEGG analysis showed that the enrichment was significant mainly in biofilm formation,bacterial secretion system,protein export,styrene degradation,tyrosine metabolism and other pathways.These differences may enhance the permeability of the cell membrane of strain LX1,affect the utilization of various elements in the culture medium and the activity of related enzymes in the growth process of strain LX1,and then make the cell growth and metabolic process different.As a differentially expressed gene related to Cr metabolism,the up regulation of glycerol phospholipid metabolic pathway was closely related to the resistance of strain LX1 to heavy metal chromium stress.ABC transporters were considered to play an important role in the ability of microorganisms to resist stress,could transport inorganic salt ions,sugars,amino acids and so on.The significant down-regulation of heat shock proteins（HSPs 20）also indicated that HSPs played an important role in organism’s response to stress.Strain LX1 responded to the stress mechanism of heavy metals by regulating the expression of these differential genes.