Isolation and Characterization of a Novel Imidacloprid-Degrading

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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]

1

ACS Paragon Plus Environment


1

ABSTRACT

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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.

11

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

12

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

13

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

14

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

15

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

16

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.

19

strains, biodegradation, HFERP.

20 2


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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,

25

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

26

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

27

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

28

transport, and transformation processes.3-5

29

The interaction of pesticides with different environmental abiotic and biotic

30

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

32

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

33

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

35

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

36

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

37

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

38

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

39

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

40

maintenance of soil quality.

3



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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-

49

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

28

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

4


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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

78

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

79

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

81 82 5


(DSN-


83

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. .

96 97

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.

111

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

114

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



125

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

130

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.

134 135

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

153

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.

158 159

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

163

(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

181

parameters

182

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

183

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

for

IMDA

degradation

were

calculated

using

the

exponential

184 185

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,

195

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,

206

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

207

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

210

alignment and phylogenetic trees generation to examine interrelationships among strains.

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41

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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

218

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

219

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

220

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

222

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

223

microorganisms capable of growing on IMDA.

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

225

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

226

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

228

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

229

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

232

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

233

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

234

time, a single degradate accumulated in the growth medium.

235

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

240

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

241

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

243

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

244

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

245

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

246

on IMDA.

247 248

Identification of the Unique Isolates Degrading Imidacloprid

249

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

250

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



251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262 263

Kinetics of Imidacloprid Biodegradation

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

265

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

266

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

267

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

268

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

269

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

270

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

271

concentration was reduced to traces (

Isolation and Characterization of a Novel Imidacloprid-Degrading

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