The Study On Mechanism Of RP4 Plasmid Affecte Nitration Reaction Of Ammonia Oxidizing Bacteria

Antibiotic resistance in bacteria is an increasing challenge to public health.The excessive and inappropriate use of antibiotics has diminished the effectiveness of life-saving antibiotic therapies and led to widespread environmental contamination.Therefore,antibiotic resistance genes(ARGs)are correspondingly recognized as environmental pollutants.A great number of studies have detected ARGs in various environments including sediments and man-made wastewater treatment plants.Biological wastewater treatment is an important process,but it provides a perfect opportunity for high-density ARGs in wastewater to propagate via horizontal gene transfer among bacteria from different taxonomic and ecological groups that inhabit the sludge.Ammonia oxidizing bacteria(AOB)play an important role in reactors by removing ammonia from biological wastewater treatment systems.Ammonia monooxygenase(AMO)is the rate-limiting enzyme that converts ammonia to hydroxylamine,and hydroxylamine oxidase(HAO)then catalyses the conversion to nitrite,which is a particularly sensitive step in the ammonium oxidation processes.The regulation of amoA and hao that encode these enzymes is therefore crucial and underpins the water treatment performance.Importantly,the presence of ARGs may affect the performance of the wastewater treatment system: As reported in previous studies,the inoculation of donor strains with the RP4 plasmid can decline the treatment efficiency in a membrane bioreactor(MBR)and a granular sequencing batch reactor(GSBR).ARGs may affect AOB and then determined the treatment performance.To further investigate the possible mechanism,parallel-running lab-scale sequencing batch reactors(SBRs)were constructed and inoculated with a donor strain,and variation in the treatment performance in different reactors was monitored.Conjugation between the RP4 plasmid and activated sludge was observed using fluorescence in situ hybridisation(FISH).The diversity of the microbial community structure and variation in the predominant species was explored by denaturing gradient gel electrophoresis(DGGE).Quantitative PCR and reverse transcription PCR(RT-PCR)were used to quantify the abundance of the RP4 plasmid,the AOB species present and the expression of amoA and hao.The following parallel-running SBR conditions were tested in duplicate:(A)SBR dosed with E.coli K12 Rif;(B)SBR dosed with E.coli K12(RP4)Rif;(C)SBR dosed with RP4;(D)undosed SBR(control).The result discoveried that mixed liquor suspended solids(MLSS)of total sludge was 1235 ± 55.146 mg/L.The MLSS in effluent was 24.95 ± 0.91 mg/m L.SV30 was 24%.Synthetic wastewater served as influent with an initial chemical oxygen demand(COD)of 100 mg/L,total ammonia nitrogen(NH4+-N)of 25 mg/L,and the NH4+-N removal efficiency(90 min)of >99%,the nitrite nitrogen(NO2--N)was almost removed within 120 min..At the same time,a strain of AOB was purified from the control SBR and identified as Nitrosomonas europaea.Inoculation was performed at a fixed time during the feeding phase.Ammonia nitrogen,nitrite nitrogen,nitrate nitrogen(NO3--N)were tested during this cycle.Activated sludge was extracted in the following cycle,at 30 min after the feeding phase.For quantification of donor strains,water samples from the second day before the strain-inoculating cycle was used for plate counting with antibiotics.The MLSS in effluent was also measured during this cycle when the SBRs were drained.Inoculation lasted 7 days,and tests were performed over 28 days.The results showed that although the removal efficiency in the other three SBRs was essentially unchanged during the early stages,a significant decrease was observed in the SBR inoculated with the donor strain with RP4 plasmid.After inoculation,the efficiency diminished below 90% on day 12,and this lower efficiency lasted for 10 days and dropped to 71% on day 20,but recovered to 94% on day 23,9 days after ceasing inoculation.Peak NO2--N levels were lower than control,and the reduced formation of NO2--N balanced the total nitrogen content due to the slow increase in NO3--N.From typical cycles in day 1,10 and 20,it was obvious that the NH4 +-N removal efficiency decreased in the SBR dosed with E.coli K12(RP4)Rif,and NO2--N,and NO3--N formation rates were also affected(day 1 can be seen as control).Peak NO2--N level was lower than control,and the reduced formation of NO2--N balanced the total nitrogen content due to the slow increase in NO3--N.Furthermore,the MLSS of total sludge in the SBR dropped to 800 mg/L by the end of the operation,and the MLSS in effluent increased to an average of 29 mg/L after inoculation,but dropped to an average of 25 mg/m L during the final 7 days.ANOVA revealed that differences before and after inoculation in the SBR were statistically significant(P < 0.05),whereas differences before and after inoculation were not statistically significant in the other three SBRs(P > 0.05).The presence of the RP4 plasmid diminished the NH4+-N removal efficiency,affected the settling properties of sludge leading to sludge losses.It showed that the conjugation betwenn RP4 plasmid and AOB was the reason that NH4+-N removal efficiency was declined.Simple E.coli and RP4 plasmid cannot affect the efficiency.Further studies were conducted by DNA and cDNA to discover the mechanism of RP4 plasmid affecte nitration reaction of AOB.DGGE suggested that most bacteria were Nitrosomonas and ?-proteobacteria.Throughout the entire operation,the dominant Nitrosomonas strains remained relatively steady.At the same time,H index was unchanged(P > 0.05).The changes in SBR were not because of the variation of AOB in sludge.The cell density of the donor strain reached almost 105 cfu/mL during dosing,but suffered a decline as soon as inoculation was stopped.On day 28,the final concentration of the donor strain was 102 cfu/m L.The ratio of the RP4 plasmid to total bacteria was 10-7 initially,but this reached a peak of 10-2 during inoculation,then declined before reaching a final ratio >10-5.The average ratio of AOB to total bacteria was 10-4 before inoculation.During the early stages of operation,this ratio was essentially unchanged,but from day 20 the amount of AOB increased slightly(P < 0.05).FISH showed a consequence of the co-localization of blue(DAPI),green(FITC)and red(TAMRA)dyes,consistent with conjugation between the RP4 plasmid and AOB.Meanwhile,no co-localization of the three fluorescein dyes was observed in the control groups.The results confirmed conjugation between the RP4 plasmid and AOB in SBRs.During inoculation in the SBR dosed with donor strain,expression of amoA and hao was higher than in the other three reactors(P < 0.05).The hao gene was up-regulated later than was amoA,and the fold change was smaller.Thoughtout the 28 days test,the up-regulated amoA and hao were still remained the transcription level.RP4 plasmid up-regulated the expression of amoA and hao in AOB through feedback regulation may because of the as the quantity or(and)function of AMO was affected by the RP4 plasmid itself.According to results,(1)4 parallel-running SBRs were built while a strain of AOB was purified and identified as Nitrosomonas europaea;(2).Conjugation between the RP4 plasmid(from the heterotrophic bacteria)and AOB(the autotrophic bacteria)was confirmed in the running SBR;(3)The conjugated RP4 plasmid diminished the NH4+-N removal efficiency in SBR,affected the settling properties of sludge leading to sludge losses;(4)The AOB in SBR were Nitrosomonas and ?-proteobacteria.Throughout the entire operation,the dominant Nitrosomonas strains remained relatively steady.(5)The conjugated RP4 plasmid up-regulated the expression of amoA and hao in AOB.It mya be a feedback regulation as the quantity or(and)function of AMO was affected by the RP4 plasmid.