Bioaugmentation Of The Herbicide MCPA By Donor Strains Harboring Plasmid PDOM In Soil

A typical phenoxyalkanoic acid herbicide,2-methy-4-chlorophenoxyacetic acid?MCPA?,has been widely used to control broadleaf weeds and sedge in agricultural practice.The massive and frequent use of this herbicide causes soil residual pollution which harms the ecological environment,threatening the safety of agricultural products and human health.Microbial remediation is an important way to eliminate soil pesticide pollution.In this study,an enhanced green fluorescent protein?EGFP?segment was tagged into the donor strain Cupriavidus gilardii T1.The donor strain harboring plasmid?pDOM?-mediated bioaugmentation was conducted both under incubation condition with repeated application and in field,to evaluate the restrictive factors for the pDOM plasmid transfer in soil ecosystem and to explore the dynamics of tfd A gene copy number following the repeated application using quantitative analysis of the tfd A gene with q PCR.High-throughput sequencing of soil microbial16S r RNA gene and ITS?Internal Transcribed Spacer?gene were performed to reveal the effects of pDOM plasmid on soil microbial community structure and functions under the incubation and field condition.This study aims to develop a plasmid-mediated bioaugmentation method that could maintain a persistent capacity for the degradation of pesticide in soil,in order to provide a foundation for bioremediation of soil pollutants.Main results of this study were as follows:?1?The EGFP-tagged C.gilardii T1::p TR-EGFP was directly obtained from E.coli DH5??p TR-EGFP?and E.coli DH5??p RK2013?by triparental matings using p RK2013 as helper plasmid.Confocal laser scanning microscope?CLSM?and the tfd A gene magnification results,along with the degradation experiment,confirmed that C.gilardii T1::p TR-EGFP was successfully constructed and had the same capacity as C.gilardii T1 to degrade MCPA.?2?The MCPA degradation capacity by donor strains harboring plasmid pDOM remained the same in sterilized and unsterilized soils.The DT₅₀of MCPA by C.gilardii T1 were 4.33 days in sterilized soil and 3.65 days in unsterilized soil.The DT₅₀of MCPA by C.gilardii T1::p TR-EGFP were 2.95 in sterilized soil and 2.87 in unsterilized soil days.?3?Repeated application of MCPA was conducted to study the persistent capacity of MCPA degradation by donor strains harboring plasmid pDOM in soil and the effects on soil microbial community.Four rounds of repeated applications resulted in a DT₅₀of 2.20,3.01,2.67 and 4.25 days,respectively,and tfd A gene copies ranging from 3.33×10⁶to 1.60×10⁷copies/g soil.A general CLSM detection of EGFP expression in all soil samples after four repeated application indicated that the donor strains harboring plasmid pDOM resulted in a persistent degradation capacity of MCPA.The addition of pDOM was beneficial to maintain a stable soil microbial community structure by reducing the influence of pesticides on indigenous bacteria.The growth of C.gilardii T1::p TR-EGFP and its capability to degrade MCPA was likely coordinated by the metabolic process of Methylotenera.?4?Restrictive factors of pDOM plasmid transfer were investigated by adding C.gilardii T1::p TR-EGFP to uncultivated farmland soil in the suburb of Hefei.The DT₅₀of MCPA was 6.3,1.9,and 1.1 days following treatments with cell density of 10⁵,10⁶,and 10⁷CFU/g in soil,respectively;whereas it was 0.30,3.30 and 3.15 days with an initial MCPA application rate of 1,10 and 50 mg/kg,respectively.Under different temperature and water-holding capacity,the capacity of C.gilardii T1::p TR-EGFP to degrade MCPA followed a sequence of 30?>35?>25?and 50%WHC>40%WHC>30%WHC.The EGFP expression was detectable with CLSM in all soil treatments.?5?The dynamics of MCPA degradation by donor strains harboring plasmid pDOM in soil and the effects on soil microbial community were studied under field condition.The maximum degradation rate of MCPA appeared at 3 days following C.gilardii T1 treatment?0.30 mg/?kg·d??and 2 days following C.gilardii T1::p TR-EGFP treatment?0.20 mg/?kg·d??,respectively.The degradation rates of MCPA by C.gilardii T1 and C.gilardii T1::p TR-EGFP reached 51.43%and 48.19%,respectively,after 45 days.The EGFP expression was detectable in soil samples treated with plasmid pDOM throughout day 0 to 45,indicating that the donor strain was able to survive in soil.High-throughput sequencing of 16S r RNA and ITS genes revealed that applying donor strain into soil had a potential of effectively repairing MCPA pollution and positively regulating soil microbial community structure shifted by the MCPA application.