Study On The Biomethylation Formation Mechanism Of Earthy-musty Haloanisoles In Drinking Water Distribution Systems

In recent years,T&O issues originated from drinking water distribution systems(DWDSs)have obtained more and more attentions,which become one of the significant scientific questions which need to be firstly resolved in order to guarantee the drinking water quality.Haloanisole(HA)is an emerging earthy and musty odorant,which is mainly produced via microbial O-methylation in DWDSs.However,the deep mechanism of HA formation in distribution pipelines is still unclear.Therefore,the main objective of this study is to evaluate the rule and mechanism of HA formation in DWDSs,which can provide important support for the establishment of HA control technology in the future.In this study,a pilot-scale DWDS and an annular reactor(AR)were used to investigate the effects of different influencing factors on 2,4,6-TCA formation,and the production of five HAs with different molecular structures as well as the microbial variations during O-methylation;the HA-producing abilities,O-methylation characteristics and genera distributions in practical DWDSs of different bacterial strains were explored;the effects of chlorine and heavy metals on the distribution of intracellular and extracellular HAs,and the relevant mechanisms were studied;based on macro transcriptome technology,the categories of potential halophenol O-methyltransferase(HPOMT)in bioiflms were screened.The results showed that:(1)The maximum 2,4,6-trichloroanisole(2,4,6-TCA)was produced in ductile iron pipe,with the concentration of 385 ng/L;the increases of temperatures(20℃ to 30 ℃)and bulk water velocities(0.1 m/s to 1.4 m/s)could both promote the formation of 2,4,6-TCA;the inhibition rate of 3 mg/L chlorine towards 2,4,6-TCA formation was 38.8%～47.1%.The production of five HAs in AR followed an order of 2,3,6-trichloroanisole(2,3,6-TCA)>2,4-dichloroanisole(2,4-DCA)>2,4,6-tribromoanisole(2,4,6-TBA)～2,4,6-TCA>2-monochloroanisole(2-MCA).During HP O-methylation,the biomass and community structure in AR were changed significantly.There was a good linear relationship between initial total biomass and HA formation potential([HA]amx):[HA]max=3.854×10-8CFU-0.5810.(2)Among eight bacterial strains(Z001～Z008),Z001 and Z002(Sphingomonas ursincola)mainly produced 2,3,6-TCA,2,4,6-TCA and 2,4,6-TBA,while Z006 an d Z007(Pseudomonas moraviensis)preferred to produce 2,4-DCA and 2-MCA.The HPOMTs in Z001-Z004 were inducible enzymes,while those in Z005 and Z008 were constitutive enzymes.S-adenosine methionine(SAM)was the main methyl donor of eight strains.The potential T&O risk degrees of five HAs followed an order of 2,4,6-TBA>2,4,6-TCA>2,3,6-TCA>2,4-DCA>2-MCA.In the consideration of T&O control,a new recommending 2,4,6-TCP standard was proposed:0.003 mg/L～0.07 mg/L.(3)For Z001,the intracellular 2,3,6-TCA consisted 87.4%～96.0%of the total 2,3,6-TCA production.Low levels of chlorine(0.05 mg/L～0.5 mg/L)could inhibit the formation of total HA,but increase the extracellular 2,3,6-TCA;1 mM and 10 mM of Ca2+or Mg2+could promote 2,3,6-TCA formation;low level of Fe3+or Zn2+(0.01 mM)could promote 2,3,6-TCA formation,while high levels of Fe3+and Zn2+(0.1 mM and 1 mM)could inhibit O-methylation;0.01 mM to 1mM of Cu2+and Mn2+ could hinder 2,3,6-TCA formation.Apart from HPOMT activity,metal ions might influence the membrane permeability and SAM cycle in cells to affect 2,3,6-TCA formation and cellular distribution.(4)Based on macro transcriptome technology,five phenolic compound O-methyltransferases(PCOMTs)were discovered.Through phylogeic analysis and comparative sequence analysis,ten Caffeoyl-CoA O-methyltransferases,three O-demethylpuromycin O-methyltransferases(dmpMOMT),one(3,5-dichloro-2,4,6-trihydroxyphenyl)hexan-1-one O-methyltransferase and one Ubiquinone biosynthesis O-methyltransferase were found having relatively high possibilities to O-methylate HP.Except for dmpMOMT,the expressions of most PCOMTs were higher in HP group than those in control group.PCOMTs were mainly from Chryseobacterium cucumeris,Pseudomonas nitroreducens and Enterobacter asburiae.The expression of SAM synthetase was promoted in HP group.