Organochlorinated pesticide degrading microorganisms isolated from contaminated soil

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    erRESEARCH PAPER New Biotechnology Volume 32, Number 1 January 2015

    Organochlorinated pesticide degradingmicroorganisms isolated fromcontaminated soilPetra Lovecka1, Iva Pacovska1, Petr Stursa1, Blanka Vrchotova1,Lucie Kochankova2 and Katerina Demnerova1

    1Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technicka 3, Prague 166 28, Czech Republic2Department of Environmental Chemistry, Institute of Chemical Technology, Technicka 3, Prague 166 28, Czech Republic

    Degradation of selected organochlorinated pesticides (g-hexachlorocyclohexane g-HCH,

    dichlorodiphenyltrichloroethane DDT, hexachlorobenzene HCB) by soil microorganisms was

    studied. Bacterial strains isolated from contaminated soil from Klatovy-Luby, Hajek and Neratovice,

    Czech Republic, capable of growth on the selected pesticides were isolated and characterised. These

    isolates were subjected to characterisation and identification by MS MALDI-TOF of whole cells and

    sequence analysis of 16S rRNA genes. The isolates were screened by gas chromatography for their ability

    to degrade the selected pesticides. Some isolates were able to degrade pesticides, and the formation of

    degradation products (g-pentachlorocyclohexane (g-PCCH), dichlorodiphenyldichloroethylene (DDE)

    and dichlorodiphenyldichloroethane (DDD)) observed in liquid culture confirmed their degradation

    capability. The isolates and DNA samples isolated from the contaminated soil were also screened for the

    bphA1 gene (encoding biphenyl-2,3-dioxygenase, the first enzyme in the PCB degradation pathway) and

    its occurrence was demonstrated. The isolates were also screened for the presence of linA, encoding

    dehydrochlorinase, the first enzyme of the HCH degradation pathway. The linA gene could not be found

    in any of the tested isolates, possibly due to the high specificity of the primers used. The isolates with the

    most effective degradation abilities could be used for further in situ bioremediation experiments with

    contaminated soil.IntroductionIncreased agricultural production to meet the ever increasing

    demand for food, has been achieved thanks to the widespread

    use of herbicides and insecticides. Thanks to these substances,

    several diseases caused by insect vectors have been eradicated [1].

    Throughout the 20th century organic pesticides such as organo-

    chlorinated substances were primarily used, even though they can

    survive in the environment for decades [2]. Organochlorinated

    pesticides (OCPs) are generally white crystalline substances which

    are poorly soluble in water, more soluble in organic solvents

    and highly soluble in fats. Due to their chemical resistance andCorresponding author:. Lovecka, P. ( 26 solubility in fats they accumulate in adipose tissue and subsequent

    biomagnification to higher trophic levels occurs. The basic repre-

    sentatives of OCPs include DDT, lindane, technical HCH (hexa-

    chlorocyclohexane), dieldrin, aldrin, heptachlor, chlordane,

    hexachlorobenzene (HCB) and others. These compounds were

    used primarily as insecticides, but nowadays are banned in most

    countries due to proven negative effects on human health and the

    environment [3]. They occur more and more frequently as con-

    taminants of soil, air and water sources, posing a risk both to

    humans and to the environment. Increased attention has been

    paid to persistent metabolites resulting from the partial degrada-

    tion of OCPs, which is carried out mainly by microorganisms.

    Factors affecting the rate of degradation include the compound

    1871-6784/ 2014 Published by Elsevier B.V.


  • New Biotechnology Volume 32, Number 1 January 2015 RESEARCH PAPER

    TABLE 1

    Values of soil contamination

    Soil sample Contaminant (mg/kg dry matter)

    DDT HCB Lindan (HCH) Zn Cu Pb As

    H1 1.52 0.03 0.05 4695 282.2 97.4 29.5

    H2 1.442 0.01 0.06 6205 250 123.6 18.5

    H3 11.8 1.03 96.5 344 818 110 7727.9

    HA 0.01 0.01 0.02 86.9 42.5

  • RESEARCH PAPER New Biotechnology Volume 32, Number 1 January 2015

    TABLE 2

    Identification of chosen isolated bacteria by MS MALDI-TOF and analysis of 16S rRNA gene

    Isolates Identification by MS MALDI-TOF Score valuea Identification by sequencing 16S rRNA gene RDP score

    NE6 Aeromonas sp. 2.26 Pseudomonas sp. 0.92

    H23 Rhodococcus sp. 2.29 Rhodococcus erythropolis 0.86

    H16 Bacillus sp. 1.89 Bacillus pumilus 0.98

    H1D7 Stenotrophomonas sp. 1.82 Stenotrophomonas sp. 0.99

    NE15 Unidentified Lysinibacillus fusiformis 0.99

    NE22 Bacillus sp Bacillus cereus 0.85

    HA1 Bacillus sp 2.14 Bacillus cereus 0.91

    a Range: 0.001.699: identification not reliable; 1.71.999: probable genus identification; 2.02.299: secure genus identification, probable species identification; 2.33.0: highly probable

    species identification.

    FIGURE 1

    Viability of selected isolates in mineral medium with HCH (50 mg/mL) and 1%peptone measured by Bioscreen CW.



    erDegradation experiments and chemical analysis procedureThe content of tested pollutants was measured in MM with 1%

    peptone and addition of 50 mg/L of DDT, g-HCH or HCB. The

    concentration of bacterial cells in the samples was 107 cells/mL

    (A560 = 0.2). One part of each sample was placed in the freezer as a

    control and the second part was cultivated for 10 days at 208C(130 RPM). All samples were extracted into N-hexane (2:4) for

    30 min. The extracted samples were analysed by gas chromatog-

    raphy (HP 5890) with an ECD detector under the following con-

    ditions: column HP-5MS 60 m, 0.25 mm, 0.55 mm, carrier gas N21 mL/min, temperature program: 508C 1 min, 258C/min to 1958C,18C/min to 2058C, 5 min, 38C to 2808C 5 min, isobaric condition,evaluation ClarityTM, DataApex s.r.o. CR. For each pesticide ex-

    periment the dead biomass of isolates NE6 (A560 = 0.2) was per-

    formed as a control.

    Detection of degradative genesThe degradation pathways of PCB and DDT are similar, hence the

    isolates were screened for the presence of bphA1 gene (the first

    enzyme of the PCB degradation pathway - biphenyl-2,3-dioxygen-

    ase) [12]. The isolates were also screened for the presence of the

    linA gene, encoding the first enzyme of the HCH degradation

    pathway dehydrochlorinase. The presence of linA was also tested

    for in samples of DNA isolated from the contaminated soil (from

    1 g of soil by UltraCleanSoil DNA Isolation Kit (MO BIO, USA)).

    Bacterial strain DNAs were isolated by Qiamp DNA Mini Kit. PCR

    was used as the amplification method. Primers FwlinA2 and

    RevlinA2 were used for detection of linA [13]: FwlinA2 (50 GGC

    CGC GAT TCA GGA CCT CTA CT 30) and RevlinA2 (50 CGG CCA

    GCG GGG TGA AAT AGT 30). For detection of bphA the following

    primers were used [14]: F463 (50 CGC GTS GMW ACC TAC AAR G

    30) and R674 (50 GGTACATGTCRCTGCAGAAYTGC 30), and degen-

    erated base: R = A,G; Y = C,T; M = A,C; S = G,S; and W = A,Tr were

    used. The PCR mix was: 5 mL buffer, 0.5 mL BSA, 1 mL dNTP

    (10 mM), 2 0.1 mL primers (100 mM), 40.8 mL redistilled water,0.5 mL Taq polymerase DNAzyme II and 2 mL sample DNA.

    Results and discussionIsolation and identification of bacterial strainsBacterial strains (Table 2) which were able to grow on solid mineral

    medium with pesticides as a sole carbon source were isolated from

    contaminated soil. The seven chosen bacterial isolates which were

    selected for degradation experiments are presented in Table 2.28 H16, H23, H1D7 originated from DDT contaminated soil

    (soil samples H1, H2 from Klatovy). Isolates NE6, NE15, NE22 were

    from vegetation areas of Spolana Neratovice with lindane contam-

    ination (soil sample NE) and HA1 was in sludge from Hajek (soil

    sample HA). Samples from Spolana Neratovice were found to have

    the highest contamination of all destinations examined. All the

    soils used for the isolation of bacteria were highly contaminated by

    heavy metals. One isolate could not be identified by MS MALDI-

    TOF because the database of bacterial strains does not contain the

    bacteria isolated from environmental samples (soil, waste water,

    sediments) and some uncommon strains. With 16S rRNA sequenc-

    ing all seven isolates were successfully identified. The most abun-

    dant strain among the isolates was the genus Bacillus.

    Isolate viability in the presence of pesticidesAC abiotic control of bacterial strains, in the presence of toxins,

    was evaluated by monitoring the growth curves (Bioscreen C1

    apparatus). The best results were achieved by growing isolates on

    mineral medium with pesticides (50 mg/mL) and 1% peptone.

    Growth curves of isolates H16, HA1 and H23 are presented in

    Fig. 1. In Table 3 specific growth rates for seven selected isolates on

    all three tested pesticides are shown.

    Degradation of HCH, DDT and HCB by bacterial isolatesAll seven isolates obtained from contaminated soils from Klatovy,

    Neratovice, and Hajek showed the ability to degrade HCH, DDT or

    HCB. The identification of those isolates was performed by MS

    MALDI-TOF. Pesticide degradation by the best isolates was tested

    in mineral medium with 50 mg/mL pesticide and 1% peptone

  • New Biotechnology Volume 32, Number 1 January 2015 RESEARCH PAPER