Organochlorinated pesticide degrading microorganisms isolated from contaminated soil

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<ul><li><p>Research</p><p>Pap</p><p>erRESEARCH PAPER New Biotechnology Volume 32, Number 1 January 2015</p><p>Organochlorinated pesticide degradingmicroorganisms isolated fromcontaminated soilPetra Lovecka1, Iva Pacovska1, Petr Stursa1, Blanka Vrchotova1,Lucie Kochankova2 and Katerina Demnerova1</p><p>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</p><p>Degradation of selected organochlorinated pesticides (g-hexachlorocyclohexane g-HCH,</p><p>dichlorodiphenyltrichloroethane DDT, hexachlorobenzene HCB) by soil microorganisms was</p><p>studied. Bacterial strains isolated from contaminated soil from Klatovy-Luby, Hajek and Neratovice,</p><p>Czech Republic, capable of growth on the selected pesticides were isolated and characterised. These</p><p>isolates were subjected to characterisation and identification by MS MALDI-TOF of whole cells and</p><p>sequence analysis of 16S rRNA genes. The isolates were screened by gas chromatography for their ability</p><p>to degrade the selected pesticides. Some isolates were able to degrade pesticides, and the formation of</p><p>degradation products (g-pentachlorocyclohexane (g-PCCH), dichlorodiphenyldichloroethylene (DDE)</p><p>and dichlorodiphenyldichloroethane (DDD)) observed in liquid culture confirmed their degradation</p><p>capability. The isolates and DNA samples isolated from the contaminated soil were also screened for the</p><p>bphA1 gene (encoding biphenyl-2,3-dioxygenase, the first enzyme in the PCB degradation pathway) and</p><p>its occurrence was demonstrated. The isolates were also screened for the presence of linA, encoding</p><p>dehydrochlorinase, the first enzyme of the HCH degradation pathway. The linA gene could not be found</p><p>in any of the tested isolates, possibly due to the high specificity of the primers used. The isolates with the</p><p>most effective degradation abilities could be used for further in situ bioremediation experiments with</p><p>contaminated soil.IntroductionIncreased agricultural production to meet the ever increasing</p><p>demand for food, has been achieved thanks to the widespread</p><p>use of herbicides and insecticides. Thanks to these substances,</p><p>several diseases caused by insect vectors have been eradicated [1].</p><p>Throughout the 20th century organic pesticides such as organo-</p><p>chlorinated substances were primarily used, even though they can</p><p>survive in the environment for decades [2]. Organochlorinated</p><p>pesticides (OCPs) are generally white crystalline substances which</p><p>are poorly soluble in water, more soluble in organic solvents</p><p>and highly soluble in fats. Due to their chemical resistance andCorresponding author:. Lovecka, P. (loveckap@vscht.cz)</p><p>www.elsevier.com/locate/nbt 26 solubility in fats they accumulate in adipose tissue and subsequent</p><p>biomagnification to higher trophic levels occurs. The basic repre-</p><p>sentatives of OCPs include DDT, lindane, technical HCH (hexa-</p><p>chlorocyclohexane), dieldrin, aldrin, heptachlor, chlordane,</p><p>hexachlorobenzene (HCB) and others. These compounds were</p><p>used primarily as insecticides, but nowadays are banned in most</p><p>countries due to proven negative effects on human health and the</p><p>environment [3]. They occur more and more frequently as con-</p><p>taminants of soil, air and water sources, posing a risk both to</p><p>humans and to the environment. Increased attention has been</p><p>paid to persistent metabolites resulting from the partial degrada-</p><p>tion of OCPs, which is carried out mainly by microorganisms.</p><p>Factors affecting the rate of degradation include the compoundhttp://dx.doi.org/10.1016/j.nbt.2014.07.003</p><p>1871-6784/ 2014 Published by Elsevier B.V.</p><p>mailto:loveckap@vscht.czhttp://dx.doi.org/10.1016/j.nbt.2014.07.003</p></li><li><p>New Biotechnology Volume 32, Number 1 January 2015 RESEARCH PAPER</p><p>TABLE 1</p><p>Values of soil contamination</p><p>Soil sample Contaminant (mg/kg dry matter)</p><p>DDT HCB Lindan (HCH) Zn Cu Pb As</p><p>H1 1.52 0.03 0.05 4695 282.2 97.4 29.5</p><p>H2 1.442 0.01 0.06 6205 250 123.6 18.5</p><p>H3 11.8 1.03 96.5 344 818 110 7727.9</p><p>HA 0.01 0.01 0.02 86.9 42.5 </p></li><li><p>RESEARCH PAPER New Biotechnology Volume 32, Number 1 January 2015</p><p>TABLE 2</p><p>Identification of chosen isolated bacteria by MS MALDI-TOF and analysis of 16S rRNA gene</p><p>Isolates Identification by MS MALDI-TOF Score valuea Identification by sequencing 16S rRNA gene RDP score</p><p>NE6 Aeromonas sp. 2.26 Pseudomonas sp. 0.92</p><p>H23 Rhodococcus sp. 2.29 Rhodococcus erythropolis 0.86</p><p>H16 Bacillus sp. 1.89 Bacillus pumilus 0.98</p><p>H1D7 Stenotrophomonas sp. 1.82 Stenotrophomonas sp. 0.99</p><p>NE15 Unidentified Lysinibacillus fusiformis 0.99</p><p>NE22 Bacillus sp Bacillus cereus 0.85</p><p>HA1 Bacillus sp 2.14 Bacillus cereus 0.91</p><p>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</p><p>species identification.</p><p>FIGURE 1</p><p>Viability of selected isolates in mineral medium with HCH (50 mg/mL) and 1%peptone measured by Bioscreen CW.</p><p>Research</p><p>Pap</p><p>erDegradation experiments and chemical analysis procedureThe content of tested pollutants was measured in MM with 1%</p><p>peptone and addition of 50 mg/L of DDT, g-HCH or HCB. The</p><p>concentration of bacterial cells in the samples was 107 cells/mL</p><p>(A560 = 0.2). One part of each sample was placed in the freezer as a</p><p>control and the second part was cultivated for 10 days at 208C(130 RPM). All samples were extracted into N-hexane (2:4) for</p><p>30 min. The extracted samples were analysed by gas chromatog-</p><p>raphy (HP 5890) with an ECD detector under the following con-</p><p>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-</p><p>periment the dead biomass of isolates NE6 (A560 = 0.2) was per-</p><p>formed as a control.</p><p>Detection of degradative genesThe degradation pathways of PCB and DDT are similar, hence the</p><p>isolates were screened for the presence of bphA1 gene (the first</p><p>enzyme of the PCB degradation pathway - biphenyl-2,3-dioxygen-</p><p>ase) [12]. The isolates were also screened for the presence of the</p><p>linA gene, encoding the first enzyme of the HCH degradation</p><p>pathway dehydrochlorinase. The presence of linA was also tested</p><p>for in samples of DNA isolated from the contaminated soil (from</p><p>1 g of soil by UltraCleanSoil DNA Isolation Kit (MO BIO, USA)).</p><p>Bacterial strain DNAs were isolated by Qiamp DNA Mini Kit. PCR</p><p>was used as the amplification method. Primers FwlinA2 and</p><p>RevlinA2 were used for detection of linA [13]: FwlinA2 (50 GGC</p><p>CGC GAT TCA GGA CCT CTA CT 30) and RevlinA2 (50 CGG CCA</p><p>GCG GGG TGA AAT AGT 30). For detection of bphA the following</p><p>primers were used [14]: F463 (50 CGC GTS GMW ACC TAC AAR G</p><p>30) and R674 (50 GGTACATGTCRCTGCAGAAYTGC 30), and degen-</p><p>erated base: R = A,G; Y = C,T; M = A,C; S = G,S; and W = A,Tr were</p><p>used. The PCR mix was: 5 mL buffer, 0.5 mL BSA, 1 mL dNTP</p><p>(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.</p><p>Results and discussionIsolation and identification of bacterial strainsBacterial strains (Table 2) which were able to grow on solid mineral</p><p>medium with pesticides as a sole carbon source were isolated from</p><p>contaminated soil. The seven chosen bacterial isolates which were</p><p>selected for degradation experiments are presented in Table 2.28 www.elsevier.com/locate/nbtIsolates H16, H23, H1D7 originated from DDT contaminated soil</p><p>(soil samples H1, H2 from Klatovy). Isolates NE6, NE15, NE22 were</p><p>from vegetation areas of Spolana Neratovice with lindane contam-</p><p>ination (soil sample NE) and HA1 was in sludge from Hajek (soil</p><p>sample HA). Samples from Spolana Neratovice were found to have</p><p>the highest contamination of all destinations examined. All the</p><p>soils used for the isolation of bacteria were highly contaminated by</p><p>heavy metals. One isolate could not be identified by MS MALDI-</p><p>TOF because the database of bacterial strains does not contain the</p><p>bacteria isolated from environmental samples (soil, waste water,</p><p>sediments) and some uncommon strains. With 16S rRNA sequenc-</p><p>ing all seven isolates were successfully identified. The most abun-</p><p>dant strain among the isolates was the genus Bacillus.</p><p>Isolate viability in the presence of pesticidesAC abiotic control of bacterial strains, in the presence of toxins,</p><p>was evaluated by monitoring the growth curves (Bioscreen C1</p><p>apparatus). The best results were achieved by growing isolates on</p><p>mineral medium with pesticides (50 mg/mL) and 1% peptone.</p><p>Growth curves of isolates H16, HA1 and H23 are presented in</p><p>Fig. 1. In Table 3 specific growth rates for seven selected isolates on</p><p>all three tested pesticides are shown.</p><p>Degradation of HCH, DDT and HCB by bacterial isolatesAll seven isolates obtained from contaminated soils from Klatovy,</p><p>Neratovice, and Hajek showed the ability to degrade HCH, DDT or</p><p>HCB. The identification of those isolates was performed by MS</p><p>MALDI-TOF. Pesticide degradation by the best isolates was tested</p><p>in mineral medium with 50 mg/mL pesticide and 1% peptone</p></li><li><p>New Biotechnology Volume 32, Number 1 January 2015 RESEARCH PAPER</p><p>TABLE 3</p><p>Specific growth rate (hour1) of isolates for cultivation on mineral medium with peptone and individual pesticides (concentration50 mg/mL)</p><p>Isolates Identification Specific growth rate</p><p>with DDT (hour1)</p><p>Specific growth rate</p><p>with HCB (hour1)</p><p>Specific growth rate</p><p>with HCH (hour1)</p><p>NE6 Pseudomonas sp. 0.5 0.4 0.2</p><p>H23 Rhodococcus erythropolis 0.5 0.5 0.5</p><p>H16 Bacillus pumilus 0.1 0.2 0.2</p><p>H1D7 Stenotrophomonas sp. 0.3 0.2 0.2</p><p>NE15 Lysinibacillus fusiformis 0.5 0.4 0.2</p><p>NE22 Bacillus cereus 0.4 0.4 0.5</p><p>HA1 Bacillus cereus 0.4 0.4 0.4</p><p>FIGURE 2</p><p>Chromatogram of metabolite products for DDT degradation by</p><p>Stenotrophomonas sp.</p><p>FIGURE 3</p><p>Chromatogram of metabolite products for HCH degradation by Bacillus</p><p>pumilus g-pentachlorocyclohexane peak with retention time 13.6 min.</p><p>ResearchPap</p><p>er(Table 4). The HA1 (Bacillus cereus 21.3%) and H16 (Bacillus</p><p>pumilus 27%) isolates showed the best degradative ability for</p><p>HCH. Isolates NE15 (Lysinibacillus fusiformis 32.7%), NE22 (Ba-</p><p>cillus cereus 36.7%) and H1D7 (Stenotrophomonas sp. 26.7%)</p><p>have shown the ability to degrade DDT and the best isolate for</p><p>HCB degradation was H1D7 Stenotrophomonas sp. (34.9%). The</p><p>concentration of pesticide in abiotic incubation was the same as</p><p>the beginning of the experiment. Degradation products of DDT,TABLE 4</p><p>Degradation of organochlorinated pesticides by selected isolates in individual pesticides in sample)</p><p>Isolates Identification Degradation HCH (</p><p>NE6 Pseudomonas sp. 18.8 </p><p>H23 Rhodococcus erythropolis 4.6 </p><p>H16 Bacillus pumilus 27 </p><p>H1D7 Stenotrophomonas sp. 17.3 </p><p>NE15 Lysinibacillus fusiformis 19.4 </p><p>NE22 Bacillus cereus </p><p>HA1 Bacillus cereus 21.3 </p><p>AC Abiotic control 1.2 </p><p>AC abiotic control experiment with death biomass of NE6.dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyl-</p><p>dichloroethane (DDD) were detected in samples of isolate H1D7</p><p>during the experiment with this pesticide (Fig. 2). g-Pentachlor-</p><p>ocyclohexane was detected during the cultivation of isolate H16</p><p>with g-HCH (Fig. 3).</p><p>In soils, the initial attack on DDT appears to be centred on the</p><p>aliphatic trichloroethyl group of the molecule and proceeds in</p><p>either one of two directions, depending on the prevailing envi-</p><p>ronmental parameters. Under aerobic conditions, DDT undergoes</p><p>dehydrochlorination to yield DDE. Under anaerobic conditions</p><p>transformation of DDT to DDD by reductive dechlorination</p><p>is considered the dominant reaction [15]. Pentachlorocyclohexeneconcentration 50 mg/mL (% decrease of original concentration of</p><p>%) Degradation DDT (%) Degradation HCB (%)</p><p>19.6 22.3</p><p>26.7 34.9</p><p>32.7 7.4</p><p>36.6 </p><p>12.8 7.9</p><p>1.4 1.2</p><p>www.elsevier.com/locate/nbt 29</p></li><li><p>RESEARCH PAPER New Biotechnology Volume 32, Number 1 January 2015</p><p>FIGURE 4</p><p>Detection of linA DNA by agarose gel electrophoresis (1% gel). 1: 100 bp</p><p>standard, 2: Negative control, 3: Positive control Sphingobium francense, 4:</p><p>DNA of contaminated soil from Neratovice, 5: DNA of isolates NE15 Lisinibacillus fusiformis.</p><p>FIGURE 5</p><p>Detection of bphA DNA by agarose gel electrophoresis (1% gel). 1: H23 </p><p>Rhodococcus erythropolis, 2: NE6 Pseudomonas sp., 3: H1D1 </p><p>Stenotrophomonas sp., 4: HA1 Bacillus cereus, 5: Positive control Pseudomonas sp. JAB1, 6: negative control, 7: 100 bp marker</p><p>Research</p><p>Pap</p><p>erwas formed as the central metabolite during the aerobic</p><p>degradation of isomer g-HCH by Sphingomonas, which suggests</p><p>that a common pathway of degradation exists for all the HCH</p><p>isomers [16]. These findings confirm reports of g-HCH degradation</p><p>by Pseudomonas sp. and P. paucimobilis UT26 [17].</p><p>Detection of degradation genesDetection of linA</p><p>Bacterial isolates and DNA isolated from the contaminated soil</p><p>were tested for the presence of linA (the first enzyme of the HCH</p><p>degradation pathway) and the results of the PCR analysis are</p><p>shown in Fig. 4. The gene was detected only in DNA obtained</p><p>from contaminated soil from Neratovice (NE). Individual isolates</p><p>were assessed for the presence of linA, the first enzyme of the HCH</p><p>degradation pathway. Isolates were subjected to PCR with specific</p><p>primers. The absence of linA amplicon in isolate DNA was probably</p><p>caused by the specificity of the primers, which were taken from the</p><p>literature [13] and were designed for Sphingobium francense. None</p><p>of these isolates were related to this strain. linA was found in</p><p>contaminated soil and it can be assumed that this strain is present</p><p>in the soil, but was not isolated [17].</p><p>Detection of bphAThe presence of bphA, encoding enzyme biphenyl-2,3-dioxygen-</p><p>ase, was also studied. This enzyme catalyses the first step in the</p><p>degradation of PCBs and the first step in the degradation pathway</p><p>of DDT is catabolised by enzyme dioxygenase (DDT 2,330 www.elsevier.com/locate/nbtdioxygenase). Therefore, all isolates were subjected to PCR with</p><p>specific primers. Some isolates which were able to grow on both</p><p>PCB and DDT as a sole carbon source were selected for investiga-</p><p>tion of bphA gene presence (Fig. 5). The presence of bphA was</p><p>confirmed only in isolate NE6, Pseudomonas sp. The isolation and</p><p>characterisation of Pseudomonas sp. capable of aerobic degrada-</p><p>tion of DDT to 4-chlorobenzoic acid has been previously de-</p><p>scribed [18].</p><p>ConclusionOrganochlorinated pesticides other than DDTs, HCHs and HCB</p><p>have never been used in large quantities in the Czech Republic.</p><p>Thus our studies were oriented towards these three pesticides.</p><p>Previous results [19] indicate that pesticide residues persist in</p><p>the top layer of soils even 20 years after they were banned.</p><p>Biodegradation of these pesticide residues can proceed in soil,</p><p>albeit at a slow rate. To enhance degradation in situ several strate-</p><p>gies have been proposed. They include the addition of DDT-</p><p>metabolising microbes to contaminated soils and/or the manipu-</p><p>lation of environmental conditions to enhance the activity of</p><p>these microbes [20,21]. Isolated...</p></li></ul>

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