Isolation and Characterization of a Novel Imidacloprid-Degrading

May 1, 2015 - Thus far, only a small number and types of bacteria with limited ability in degrading imidacloprid have been reported. Also, genes regul...
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Isolation and Characterization of a Novel ImidaclopridDegrading Mycobacterium sp. strain MK6 from an Egyptian Soil Mahrous M Kandil, Carmen Trigo, William C. Koskinen, and Michael J. Sadowsky J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00754 • Publication Date (Web): 01 May 2015 Downloaded from http://pubs.acs.org on May 7, 2015

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Journal of Agricultural and Food Chemistry

REVISED jf-2015-007547 Isolation and Characterization of a Novel Imidacloprid-Degrading Mycobacterium sp. strain MK6 from an Egyptian Soil

Mahrous M. Kandil1,*, Carmen Trigo2, William C. Koskinen2, and Michael J. Sadowsky2,3

1

Department of Soil and Water, Faculty of Agriculture, Alexandria University, Egypt

2

Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota

55108, USA 3

BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108, USA

*Correspondence: Dr. Mahrous M. Kandil, Department of Soil and Water, Faculty of Agriculture, Alexandria University, Egypt 21545; Email: [email protected]

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ABSTRACT

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Thus far, only a small number and types of bacteria with limited ability in degrading

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imidacloprid have been reported. Also, genes regulating imidacloprid (IMDA)

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degradation have yet to be discovered. To study this in more detail, an enrichment

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technique was used to isolate consortia and pure cultures of IMDA-degrading bacteria.

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Through this approach, we successfully isolated a novel bacterium capable of completely

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degrading IMDA as a sole nitrogen source. The bacterium was subsequently identified as

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Mycobacterium sp. strain MK6 (Genbank accession KR052814) by sequence analysis of

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its 16s rRNA gene. BLASTn searches indicated that 16s rRNA gene from

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Mycobacterium sp. strain MK6 was 99% identical to several Mycobacterium spp.

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Mycobacterium sp. strain MK6 transformed 99.7% of added imidacloprid (150 µg mL-1)

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in < 2 weeks (t1/2 = 1.6 d) to 6-chloronicotinic acid (6-CNA) as its major metabolite.

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Although the isolated strain and mixed bacterial consortia were able to degrade IMDA,

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they failed to grow further on 6-CNA indicating a lack of IMDA mineralization to carbon

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dioxide. Small amounts of the desnitro-olefin and desnitro degradates of imidacloprid

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were observed during the incubation, but did not accumulate in culture.

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KEY WORDS: Imidacloprid, 6-chloronicotinic acid, desnitro-Olefin, Mycobacterium sp.

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strains, biodegradation, HFERP.

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INTRODUCTION

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Insecticides are globally used in agriculture to combat the loss of crops due to attack by

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microbial and insect pests. Insect damage to plants is a severe problem and results in the

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annual use of 940 million pounds of active ingredients worldwide.1 It has been reported,

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however, that more than 99% of applied pesticides do not reach their target pests and may

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affect non-target species or contaminate the environment.2 Following their deposition,

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many factors control the fate of pesticides in the environment including retention,

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transport, and transformation processes.3-5

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The interaction of pesticides with different environmental abiotic and biotic

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factors results in the transformation of parent compounds; microorganisms use many

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pesticides as a source of carbon, nitrogen, and energy - often transforming them into

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carbon dioxide, water, and into diverse sets of metabolites.6-8 These compounds may be

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less, or sometimes, more toxic to target and non-target pests and organisms.6 For

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instance, to determine the effect of insecticides on soil microorganisms, various assays

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such as nitrification, denitrification or N2-fixation have been commonly used.9-11 Cycoń et

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al.12, used two culture-independent (PLFA and PCR-DGGE), and culture dependent

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(CLPP) approaches to show that imidacloprid (1-(6-chloro-3-pyridylmethyl)-N-

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nitroimidazolidin-2-ylideneamine) induced significant changes in the composition of

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microbial communities and their metabolic activity, which in turn can influence on the

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maintenance of soil quality.

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Imidacloprid (IMDA) is among one of the most widely used neonicotinoid insecticides in

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agriculture.13-17 While imidacloprid is mainly applied via foliar application for insect

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control, it is also used as a seed dressing or stem treatment.14 Imidacloprid is

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characterized by variable persistence in soil, with a half-life up to 229 days in the field.18

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In contrast, Wu et al.19 found under laboratory conditions that the degradation of

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imidacloprid was rapid, with half-lives of 4-5 d. The persistence of imidacloprid in water

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increased with increasing application rates and pH values. 20 In aquatic environments, it

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was found that imidacloprid was more persistent in sediment than in water, with half-

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lives of 160 and 30 d, respectively.21-24

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The persistence of IMDA depends on different physicochemical and biological

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parameters, such as organic matter, pH, temperature, crops, time, and microbial activity.

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Oi25 and Cox et al.26 established that sorption increased with contact time in soil, thereby

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decreasing potential availability to degrading organisms in soil. Decreased availability,

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would in turn, increase persistence in soil. Dissolved organic carbon reduces imidacloprid

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sorption by competing with the pesticide molecules for sorption sites on the soil surface,

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increasing availability for degradation by microorganism and for leaching of imidacloprid

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and potentially increasing ground water contamination.27

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The metabolism of IMDA has been studied mainly in plants and mammals and

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the proposed pathways for IMDA transformation have mostly been based on studies of

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Eukaryotes.

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pure or mixed cultures of soil microorganisms.29-32 It is crucial to isolate and identify

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However, a few studies have focused on the transformation of IMDA by

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microorganisms capable of degrading different pesticides for their potential use in

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bioremediation strategies either directly, or via use of their enzymes.

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The objectives of this study were to: 1) isolate mixed cultures of bacteria

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degrading imidacloprid from an Egyptian soil, 2) purify and characterize unique isolates

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with the ability to transform imidacloprid and 3) identify imidacloprid metabolites

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produced during the degradation process. Results of this study are crucial for our

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understanding of imidacloprid transformation in the environment and for the

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development of possible bioremediation strategies.

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MATERIALS AND METHODS

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Chemicals and Pesticide

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Analytical standards of Imidacloprid (IMDA; 99.5% purity), 6-chloronicotininc acid (6-

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CNA; 99.5% purity) were purchased from Sigma-Aldrich, (St, Louis, Mo, USA),

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desnitro-imidacloprid

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IMDA; >97% purity) , and desnitro-olefin imidacloprid 1-(6-chloro-pyridin-3yl-methyl)-

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N-nitro-1H-imidazol-2-imine (DSN-olefin-IMDA; >97% purity) were provided by Bayer

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Corporation, Stilwell, KS. Imidacloprid and metabolite stock solutions (50 mg mL-1)

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were prepared in HPLC grade acetonitrile to ensure full solubility and were stored in the

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dark at 4 oC until used.

1-(2-chloro-pyridin-5-yl-methyl)-2-imino-imidazolidine

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Soil Sample Collection

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Soil samples were collected from the Abis Agricultural Research Farm, Alexandria

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University, Alexandria, Egypt. Five surface (top 15 cm) soil samples of about 1 kg each

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were collected randomly from an area following a traditional agricultural rotation

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farming system of wheat and clover. The area was selected based on the farm's record

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showing no history of imidacloprid application for the 5 years prior to sampling.

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Samples were collected by using a bleached-sterilized hand shovel, placed in sterile

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polyethylene bottles, transferred into lab on ice, and stored at 4 °C until used. The soil

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samples were mixed to form a composite sample that was used within 24 h for

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imidacloprid enrichment cultures. Following standard methods of soil analysis,33

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composite soil sample was classified as sandy clay loam texture of 27.9% clay, 10.1%

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silt, and 62.0 % sand, pH 7.6; EC 1.52 mS cm-1; organic matter 0.75%; and CEC =33

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cmole(+) kg-1 soil. .

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Culture Medium and Growth Conditions

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An enrichment culture technique was used to isolate imidacloprid-degrading bacteria

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from the soil described above. Isolation and degradation experiments were conducted

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using R-salts minimal medium (RSM) as previously described.34 The liquid RSM

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medium consists of solutions 1) 67.0 mL of 1M KH2PO4 (PH 6.9), 2) 5 mL of R-salt

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mixture (16.0 g MgSO4.7H2O, 0.4 g FeSO4.7H2O, and 0.8 mL HCl per 200 mL H2O), 3)

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200 µL of 1M CaCl2.2H2O, and 4) 1.0 mL of trace elements solution (10.0 mg 6

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ZnSO4.7H2O, 3.0 mg MnCl2.4H2O, 30 mg H3BO4, 20.0 mg CoCl2.6H2O, 1.0 mg

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CuCl2.2H2O, 2.0 mg NiCl2.6H2O, 3.0 mg Na2MoO4.2H2O per liter). Glucose stock

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solution (20%) was prepared and filter sterilized by polytetrafluorethylene PTFE

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membrane filters (0.22 µm) and stored at 4oC. Stock solution (10%) of ammonium nitrate

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(NH4NO3) was prepared. These solutions were autoclaved separately at 121 oC for 20

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min and mixed after cooling to 60 oC in water bath as needed. Solutions of Glucose,

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NH4NO3, and IMDA were added by PTFE syringe filters as described below.

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Isolation of Imidacloprid-degrading Bacteria by Enrichment Technique

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All experiments were prepared under aseptic conditions. Imidacloprid, C9H10ClN5O2,

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contains carbon and nitrogen on its pyridine and imidazolidine rings and side chain which

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might be used as a source of carbon, nitrogen, or both for soil microorganisms. Therefore,

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various enrichment cultures with or without carbon and nitrogen sources were prepared to

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isolate bacteria using IMDA as a sole N or C source, or both including all possible

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control treatments. Nitrogen-free RSM medium was supplemented with 150 mg L-1

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IMDA as a sole nitrogen source and 2 g/L glucose (10 mL of 20% stock solution) as a

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carbon source. Carbon-free RSM medium was supplemented with 150 µg mL-1 IMDA as

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a carbon source and 1 g L-1 NH4NO3 (10 mL of 10% stock solution) as a nitrogen source.

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Enrichment cultures of treatments described above were conducted in 250-mL

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Erlenmeyer flasks containing 100 mL of respective RSM medium inoculated with 5 g

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fresh-weight soil, incubated in the dark at 27 °C on G24 (New Brunswick, NJ)

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environmental incubator shaker at 150 rpm for three week period for the first run. This 7

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period was implemented to increase the chance of creating a favorable condition for

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IMDA-degrading bacteria, while building a growth-limiting culture for other soil

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microorganisms after they consume soil organic matter and nutrients, thus ensuring the

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adaptability of IMDA degrading bacteria to IMDA and its degradation byproducts.

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Successive enrichment subcultures were renewed every two weeks by transferring 1 mL

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from the old culture into a fresh enrichment flask containing 100 mL of the

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corresponding treatment medium. This step was repeated five more times to eliminate

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any impurities of organic matter originating from the soil sample and to attempt to isolate

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a mixed culture of IMDA-degrading microorganisms.

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Isolation and Purification of Imidacloprid-degrading Bacterial Isolates

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A microbial consortium isolated on IMDA was examined for growth and IMDA

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degradation. After confirming the ability of the consortium to degrade IMDA, individual

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bacterial isolates were obtained by serial dilution and pour plating of the consortium onto

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RSM solid medium supplemented with 1.5% Noble agar and IMDA (150 µg mL-1) as the

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sole nitrogen source. Plates were incubated at 28 °C for 15 d. Based on morphological

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features; discrete unique bacterial colonies were picked, and further purified by

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successive streaking on the same medium. This process was repeated several times to

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ensure isolation of pure cultures and the ability of isolates to grow on IMDA as a sole N

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source. Pure cultures were preserved in 20% sterile glycerol and stored at -70°C until

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used. 8

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Imidacloprid Degradation Assay and its Kinetics

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The consortium, and later individual pure culture isolates, was tested for their ability to

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degrade 150 mg L-1 IMDA in RSM liquid medium. Growth was determined by

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measuring optical density at 600 nm (OD600) and degradation was determined from

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analysis of IMDA in solution. Initially, the IMDA concentration in supernatant of

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cultures was monitored spectrophotometrically at 270 nm28,35, followed by high

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performance liquid chromatography (HPLC) analysis under the conditions described

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below. For this assay, 250 mL flasks containing 100 mL of RSM liquid media were

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inoculated with individual isolates, or a consortium, and maintained for 15 d under the

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incubation conditions described above. Five mL aliquots were taken every two days

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under aseptic conditions for measuring bacterial growth by optical density (OD600) and

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IMDA concentration in the culture medium.

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HPLC Analysis for Imidacloprid and its Degradation Products

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Culture samples were centrifuged at 10,000 x g for 10 min to separate cell pellets from

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supernatant. IMDA was measured in the supernatant by HPLC using a Waters HPLC

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(Milford, MA, USA) with a reverse phase C-18 (RP18) symmetry shield column

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(Waters-Millipore) (3.9 mm × 150 cm) and an ultraviolet (UV) detector. A 1.0 mL

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aliquot of growth medium was taken under aseptic conditions, centrifuged to remove

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bacterial cells, and filtered through 0.2 µm sterile Millipore membrane. A 0.5 mL aliquot

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of filtered supernatant was diluted to 2.5 mL using ultrapure water prior to injection. The 9

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HPLC operational conditions used were as previously described31: 30 min gradient of

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HPLC grade acetonitrile (ACN) and acidified (pH 3) ultrapure water [(0 min) 20%:80%

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ACN:H2O; (7 min) 22%:78% ACN:H2O; (14 min) 30%:70% ACN:H2O; (21 min)

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40%:60% ACN:H2O; (30 min) 20%:80% ACN:H2O)], injection volume of 20 µL, flow

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rate of 0.6 mL min–1, and UV detection at 270 nm for IMDA and 220 and 247 for

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metabolites. The HPLC column temperature was maintained at 24°C. Analytical grade

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IMDA, DSN-IMDA, DSN-olefin-IMDA, IMDA-urea, and 6-CNA were used to prepare a

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series of standard solutions. Standards of IMDA and 6-CNA (0-100 µg mL–1) were run

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each time of analysis. Standards of metabolites were run only after corresponding peaks

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were observed in solution. Linear calibration curve for IMDA and 6-CNA were observed

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in the range 1-100 mg mL–1 with 0.99 correlations. Retention times were 7.50, 2.50, 2.19,

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6.4 and 5.51 min for IMDA, DSN-olefin-IMDA, DSN-IMDA, IMDA-urea, and 6-CNA,

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respectively. Peak area was plotted against its calibrated linear fitted standards (0-100 µg

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mL-1) to quantitatively measure IMDA concentration in culture flasks. The kinetic

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parameters

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formula C t = C o e kt , where Ct is the concentration of IMDA at a time (t), Co is its initial

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concentration, k is its decay constant, and t is its half-life time (t1/2).

for

IMDA

degradation

were

calculated

using

the

exponential

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Clustering and Selection of Unique IMDA-degrading Isolates by HFERP

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Twenty, morphologically distinct, pure culture-isolates were subjected to Horizontal

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Flurophore-Enhanced Rep-PCR (HFERP) DNA fingerprinting technique.36 Rep-PCR was 10

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performed using an MJ research PTC 100 (MJ Research, Waltham, Mass.) thermocycler

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according to a published described protocol.36 Electrophoresis was done at 4°C for 17 h

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at 70 V as previously described, with constant buffer recirculation for separating DNA

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fragments.37 All rep-PCR experiments contained a positive control (E. coli) and a

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negative control (no DNA). DNA fingerprints were captured using a Typhoon 8600

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variable mode imager (Molecular Dynamics/Amersham Biosciences, Sunnyvale, Calif.)

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and images were subjected to analysis using BioNumerics v.2.5 software (Applied Maths,

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Sint-Martens-Latem, Belgium) as previously described.36

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Amplification of 16S rRNA Gene for the Identification of Unique Isolates

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The taxonomic identity of unique bacterial isolates degrading IMDA was determined by

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sequencing near full length copies of 16S rRNA gene. Universal primers 27f (5`-

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AGAGTTTGATCMTGGCTCAG-3`) and 1492r (5`-GGTTACCTTGTTACGACTT-3`)

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(Integrated DNA Technologies, Coralville, Iowa) were used for 16S rRNA gene

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amplification as described.38 The amplified 16S rRNA fragment were extracted and

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purified following the protocol of QIAquick PCR purification kit (Qiagen, Valencia, CA,

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USA). PCR reaction conditions were done as described.39 The concentration of the

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purified DNA was determined by NanoDrop 2000 spectrophotometer (Thermo Scientific,

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Wilmington, DE, USA) and DNA was sequenced at the Genome Sequencing Center (St.

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Paul, MN), University of Minnesota. Sequences were assembled by using BioEdit

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software.40 Assembled contigs were compared with known 16S rRNA gene sequences in 11

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the GenBank database by BLASTn search. MEGA4 software was used for multiple

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alignment and phylogenetic trees generation to examine interrelationships among strains.

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isolated strains. The 16s rRNA gene sequence of isolated IMDA degrading MK6 strain

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was deposited in the NCBI GenBank database under accession number KR052814.

The sequence of 16S rRNA gene was used for assigning taxonomic status to the

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RESULTS AND DISCUSSIONS

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Enrichment of Imidacloprid-degrading Bacteria

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An enrichment culture was used to isolate consortia, and eventually pure cultures of

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bacteria, that were capable of transforming IMDA (150 mg L-1) when used as a sole N

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source for growth. The enrichment technique has an advantage over direct isolation

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methods in its high probability of success in isolation due to acclimatization and

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providing favorable conditions for some species while suppressing the growth of

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others.42-44 After 5 cycles of enrichment renewal, we obtained a mixed culture of

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microorganisms capable of growing on IMDA.

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The addition of glucose as the sole source of carbon produced considerable

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microbial growth through the 5 rounds of sub-culturing indicating that IMDA was mostly

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likely being used as a nitrogen source. As shown in Figure 1A, growth (OD600) of the

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mixed culture supplemented with both IMDA and glucose was much greater than other

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treatments, including those supplemented with both IMDA and nitrogen. Other

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treatments provided limited and often insignificant growth. As a consequence, the 12

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biodegradation of IMDA as a nitrogen source was further investigated throughout this

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study. HPLC analyses were used to follow IMDA concentration in mixed culture growth

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in medium where IMDA served as the sole N source. Results in Figure 1B show that as

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the quantity of IMDA decreased in the glucose-supplemented culture significantly with

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time, a single degradate accumulated in the growth medium.

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Isolation and Purification of Imidacloprid-degrading Bacteria from Mixed Culture

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A dilution series of the consortium was done to isolate individual bacterial isolates

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capable of IMDA degradation. About 20 morphologically distinct colony isolates were

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selected and subjected to HFERP DNA fingerprint analysis to select for unique clones.

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Results in Figure 2 show the HFERP DNA fingerprints of these isolates. All 20 isolates

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could be divided into seven distinct groups that individually degraded IMDA. Strains in

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each DNA fingerprint group were individually tested for their ability to degrade IMDA.

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Results in Figure 3 show the growth of the IMDA-degrading consortium and individual

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isolates grown in minimal medium containing IMDA as a sole nitrogen source. One of

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these isolates (isolate MK6) showed rapid growth and had IMDA degradation rates

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similar to that seen with the consortium, while the other isolates showed slower growth

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on IMDA.

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Identification of the Unique Isolates Degrading Imidacloprid

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Isolate MK6 was confirmed for IMDA transformation ability and was further identified

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by analysis of 16S rRNA gene sequence. Sequence similarities were determined by 13

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BLASTn analysis and indicated that the near full length 16S rRNA gene sequence of

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isolate MK6 was 99.9% identical to that of Mycobacterium cosmeticum, strain LTA-388.

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To our knowledge, this is the first discovery of an IMDA-degrading Mycobacterium sp.

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strain. In addition, BLASTn searches showed that sequence of 16S rRNA gene of this

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strain was 98-99% identical to those of Mycobacterium neoaurum VKM Ac-1815D

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(CP006936.2), Mycobacterium rhodesiae NBB3 (CP003169.1), Mycobacterium gilvum

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Spyr1 (CP002385.1), Mycobacterium smegmatis str. MC2 155 (CP000480.1),

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Mycobacterium smegmatis strain INHR2 (CP009496.1), and Mycobacterium gilvum

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PYR-GCK (CP000656.1), whose full genomes were completely sequenced. A

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phylogenetic tree (Fig. 4) of the imidacloprid degrading strain was constructed by using

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MEGA4 software (http://www.megasoftware.net/mega4/mega41.html).41

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Kinetics of Imidacloprid Biodegradation

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Results in Figure 5A show that the reduction of concentration of IMDA in liquid was

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concomitant with a continuously rising growth (growth rate, µ=0.375 day-1) of isolate

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MK6. Based on HPLC analysis, more than 99% of the initial concentration of IMDA

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(150 mg L-1) was degraded within 2 wk by this strain at 27 oC, making it among the most

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efficient IMDA degrading bacterium reported to date. For instance, a Klebsiella sp. strain

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was only reported to degrade 70% of IMDA.45 Figure 5B shows the percentage of IMDA

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degradation and the concurrent production of its major metabolite. While the IMDA

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concentration was reduced to traces (