| Atrazine,a triazine herbicide,is widely used in agriculture and forestry because of its low cost and high efficiency.At present,atrazine is the most widely used herbicide in maize cultivation in Northeast China..It is also an environmental endocrine substance with biological toxicity and long residue characteristics.The ecological and environmental problems caused by its migration and residue in the environment are becoming increasingly serious.Effective measures are urgently needed to reduce the residue of atrazine in the environment.In recent years,the use of functional microorganisms to degrade long residual organic pollutants has been widely recognized and concerned.In actual agricultural production,atrazine is often used in combination with the sulfonylurea herbicide nicosulfuron to enhance the weeding effect.However,it is not clear whether the coexistence of nicosulfuron interferes with the biodeg radation of atrazine.Therefore,we selected the early screening of efficient atrazine degrading bacteria Arthrobacter sp.DNS10.In this study the effect of nicosulfuron on the degradation efficiency of strain DNS10 was investigated.The structure,microbial oxidative stress response,and degradation function gene transcription level reveal the relevant influence mechanism,and the following research results are mainly obtained:（1）The effect of Nicosulfuron methyl on the growth and atrazine removal of s train DNS10showed that there was no significant difference in the growth(OD600 value)of strain DNS10between different treatments at the early stage of culture（12 h-24 h）.With the culture time（48 h）increase,the number of cells was also increased,The results of the effect of nicosulfuron on the growth of strain DNS10 and the removal of atrazine showed that there was no significant difference in strain growth(OD600 value)between the treatments in the early stage of culture（12h-24 h）.With the increase of the culture time（48 h）,the OD600 values of the culture solution treated with 1,5 and 10 mg·L-1nicosulfuron at 48 h were 0.224±0.002,0.167±0.005,0.147±0.001,and no smoke was added.The OD600value of sulfomeuron treatment was 0.253±0.002,indicating that the addition of nicosulfuron could inhibit the growth of the strain DNS10,and the addition of 10 mg·L-1 nicosulfuron had the most obvious inhibitory effect.Similarly,when the start concentration of atrazine was 100 mg·L-1,there was no significant difference in the degradation rate of atrazine among different treatments in the early stage of culture.When add 1,5 and 10mg·L-1 nicosulfuron at 48 h,the degradation rate of atrazine was not significantly different.Similarly,when the initial concentration of atrazine added was 100 mg·L-1,the degradation rate of atrazine did not reach a significant difference between the treatments in the early stage of culture,and 1,5 and 10 mg·L-1 smoke was added at 48 h.The degradation rate of atrazine when sulfuron-methyl was 63.9±0.769%,49.1±0.879%and 42.6±0.510%,without the addition of nicosulfuron,the degradation rate of atrazine was 76.0±3.400%.It shows that the addition of nicosulfuron able to reduce the atrazine degradation efficiency of stra in DNS10,and the addition of 10 mg·L-1 nicosulfuron had the largely effect.（2）The results of SEM was showed that compared with the treatment without nicosulfuron,the morphology of DNS10 cells treated with 1,5 and 10 mg·L-1 nicosulfuron still maintained the original rod shape.However,holes appeared on the surface of cell membrane,and the degree of damage increased with the nicosulfuron increase,which indicated that nicosulfuron could destroy the surface structure of bacterial cell membrane and damage the integrity of cells.The LDH content of strain DNS10 increased with the increase of Nicosulfuron concentration,which further indirectly indicated that Nicosulfuron could destroy the integrity of cell membrane of strain DNS10.（3）Flow cytometry was used to observe DNS10 cells double stained with PI and CFDA,.The study shows that:add 1,5 When co-cultured with 10 mg·L-1 nicosulfuron for 48 h,the percentages of cells stained by PI（defined as"dead cells"）in the culture system were 0.9%,34.6%and 72.4%,respectively,while no nicosulfuron Cells stained by PI accounted for 0.0%in the blank treatment with Long added.In addition,the percentages of cells（defined as"live cells"）stained by c FDA in the treatment with 1,5 and 10 mg·L-1nicosulfuron were 57.8%,12.4%and 0.2%,respectively,while there was no nicosulfuron.Cells stained by c FDA accounted for 70.8%in the blank treatment with long added.The above results indicate that as the concentration of nicosulfuron increases,the percentage of dead cells of the strain DNS10 in the culture system increases.In terms of strain zeta potential,the zeta potential of the treated strain DNS10 without nicosulfuron was-15.50±0.44 mv when it was cultured for 48 h,while the strain DNS10 treated with 1,5 and10 mg·L-1 nicosulfuron added The zeta potentials are-14.17±0.44,-12.09±0.37 and-11.07±0.38 mv,respectively,indicating that the addition of nicosulfuron can weaken the electronegativity of the strain DNS10.As a result,the amount of atrazine that can be degraded by the strain is reduced,which indirectly indicates that the addition of nicosulfuron causes the degradation of atrazine to decrease.（4）Through the measurement of the accumulation of atrazine in cells,treated with 1,5 and 10mg·L-1 nicosulfuron was 0.96,1.25 and 1.51 times of that without nicosulfuron,respectively.The above results indicate that with the increase of the concentration of nicosulfuron at 48 h,the accumulation of atrazine in the cells increases.Under the above condi tions,The expression of trz N degradation gene in DNS10 was further determined by q PCR.The results showed that there was no significant difference between the treatment with 1 mg·L-1 nicosulfuron and the treatment without nicosulfuron.On the contrary,in the treatment with 5 mg·L-1 and 10 mg·L-1nicosulfuron,the relative expression level of trz N in strain 10 was only 0.72 times and 0.52 times of the trz N gene expression in the blank treatment.These results indicated that nicosulfuron could decrease the expression of degradation gene of DNS10,which might lead to the failure of atrazine degradation in the strain and increase the accumulation of atrazine in the strain（5）The results showed that compared with the blank treatment,nicosulfuron(1,5 and 10mg·L-1)had the highest oxidative damage.The content of reactive oxygen species（ROS）and malondialdehyde（MDA）in DNS10 increased significantly.In addition,nicosulfuron methyl at the above concentration level could also induce the increase of the activities of SOD,CAT and POD in DNS10 strainFrom the above results,It can be seen from the above results,that the addition of nicosulfuron increased the degree of oxidative damage of the strain DNS10,destroyed the integrity of the cell membrane of the strain DNS10,resulting in membrane damage,and weakened the cell viability of the strain DNS10.The addition of nicosulfuron reduced the strain.The electronegativity of DNS10affects the binding ability of atrazine and the strain DNS10.In addition,nicosulfu ron can also reduce the strain’s ability to degrade atrazine by reducing the expression of the strain’s degradation gene trz N.Based on the above results,nicosulfuron may be a potential mechanism for the degradation of DNS10. |
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