Metabolism Of The Nicotinic Insecticide Nitenpyram By Rhodococcus Ruber

Nitenpyram is a highly effective and low-toxic chloropyridine insecticide.A series of ecological problems caused by the widespread use of NIT have attracted more and more attention.Microbial remediation has the advantages of low cost,rapidness,good effect,no secondary pollution,not breaking ecology balance,and simple technology.Furthermore only the fungus Phanerochaete sordida YK-624 was reported to degrade NIT,therefore the mechamism of microbial degradation of NIT is still unclear.In this paper,a strain named D188 with the ability of high-efficient NIT degradation was isolated from the activated sludge for treatment of the wastewater produced from NIT production.Through morphological observation and 16S rRNA gene sequence analysis,D188 was identified as Rhodococcus ruber.This strain is now deposited in China General Microbiological Culture Collection Center,CGMCC,under the number of CGMCC 17550.High performance liquid chromatography（HPLC）and liquid chromatography mass spectrometry（LC-MS）analysis showed that R.ruber CGMCC 17550 converts NIT into three hydroxylated products through a new hydroxylation pathway,3-OH-NIT,8-OH-NIT and 14-OH-NIT.Nuclear magnetic resonance（NMR）analysis confirmed that 3-OH-NIT was the product of hydroxylation at the C3 site on the 6-chloropyridine ring of NIT,In addition,a product NIT-1 by the NIT methylene bridge cleavage was also detected.The above results indicated that R.ruber CGMCC 17550 has both NIT hydroxylation and methylene bridge cleavage.R.ruber CGMCC 17550 resting cells at OD₆₀₀ of 1 degraded NIT from an initial concentration of 432.94μmol/L to 345.75μmol/L in 72 h,with a degradation rate of 22.59%and a half-life of 6.64 d.The NIT degradation rate increased with the increasing biomass.R.ruber CGMCC 17550resting cells at OD₆₀₀ of 9 degraded NIT from an initial concentration of 432.94μmol/L to 7.24μmol/L in 72 h,The NIT degradation rate was 98.38%,and the half-life was1.52 d.The above results indicated that R.ruber CGMCC 17550 resting cells had strong NIT degradation capability.R.ruber CGMCC 17550 degradated NIT with addition of cometabolic substrate indicated that when 0.2%glucose and fructose were added as cometabolism substrates,R.ruber CGMCC 17550 resting cells degraded 53.38%and 52.16%of NIT（the initial concentration was 100 mg/L）in 3 d,respectively.The NIT degradation rate was 1.55and 1.51 times higher than that of the control group without carbohydrate addtion respectively.With the addition of 0.2%sodium pyruvate and sodium succinate as cometabolic substrates,R.ruber CGMCC 17550 resting cells degraded 49.41%and41.18%of the NIT at the initial concentration of 100 mg/L within 3 d,respectively,Which was 1.43 and 1.19 times higher than that of the control group without organic acid addition,In addition,the addition of 0.2%sucrose had no effect on the metabolism of NIT by R.ruber CGMCC 17550.The effect of 100μmol/L metal ions on the metabolism of NIT by R.ruber CGMCC 17550 indicated that Zn²⁺had a slight promotion effect on the metabolism of R.ruber CGMCC 17550,and the promotion rates were 2.78%.Co²⁺,Cu²⁺,Ni²⁺and Mo²⁺had inhibitory effects on the metabolism of NIT by R.ruber CGMCC 17550,and the inhibition rates were 78.49%,73.73%,48.21%and 9.58%,respectively.Al³⁺,Ca²⁺,Mg²⁺and Mn²⁺have no effect on the metabolism of NIT by R.ruber CGMCC 17550.The effects of NIT transforamtion mixture volume of 1 mL,2 mL,5 mL and 10 mL on the conversion of NIT by R.ruber CGMCC 17550 were explored,and the results showed that the degradation rates were 48.36%,49.54%,49.16%and 43.21%,respectively.The results showed that R.ruber CGMCC 17550 with bottling volumes of 1 mL,2 mL,and 5 mL were not sensitive to dissolved oxygen during the metabolism of NIT.The resting cells of R.ruber CGMCC 17550 with a transforamtion mixture volume of 10 mL were sensitive to dissolved oxygen during the process of NIT metabolism.Without adding cometabolic substrate,the degradation of NIT by R.ruber CGMCC 17550 free cells in surface water showed that the concentration of NIT was degraded from 99.58μmol/L to 37.98μmol/L after incubation for 8 d,the degradation rate was 61.86%and the half-life was 6.47 d.The results of NIT conversion by R.ruber CGMCC 17550 immobilized cells in surface water showed that the content of NIT was reduced from 389.88μmol/L to 48.19μmol/L after 8 d incubation,the degradation rate was 84.26%and the half-life was 4.74 d.These results indicated that both free cells and immobilized cells of R.ruber CGMCC 17550 have a strong NIT degradation ability in surface water.The genome data indicated that strain CGMCC 17550 has a circular chromosome with a length of 5,364,592 bp a high GC-content of 70.58%and has no plasmid.Among 5198 annotated genes from R.ruber CGMCC17550,2718 genes are related to the KEGG pathway.There are 18 cytochrome P450 genes among the annotated genes.The cytochrome P450 enzyme inhibitor 1-ABT was used to explore its effect on the degradation of NIT by R.ruber CGMCC 17550.With the addition of 0.5 mmol/L 1-ABT,the NIT degradation of R.ruber CGMCC 17550 was 113.83μmol/L,while the control group was 220.61μmol/L.The results showed that 1-ABT inhibited 52.38%of the degradation of NIT.Meanwhile the content of NIT-1 by 1-ABT inhibition was 7.09μmol/L,while the control group was 35.34μmol/L,which indicating that 1-ABT inhibited the production of NIT-1 by 79.93%.The content of 3-OH-NIT by 1-ABT inhibition was 17.64μmol/L,while the control group was 116.51μmol/L,which indicating that 1-ABT inhibited the production of 3-OH-NIT by 84.85%.1-ABT inhibits the degradation of NIT and the production of hydroxylation products,indicating that P450 enzyme may be involved in the NIT hydroxylation by R.ruber CGMCC 17550.In conculsion,R.ruber CGMCC 17550 can degradate neonicotinoid insecticide NIT via three different NIT hydroxylation pathways.R.ruber CGMCC 17550 free cells and immobilized cells have strong NIT degradation ability in surface water.The results of this study will help to understand the environmental metabolism and degradation mechanism of NIT,and help to guide the development of NIT bioremediation agents.