Dr R.J. Grant IFAB, Department of Biology, University of York, Heslington, York., YO10 5 DD, UK (e-mail: email@example.com).
Aims: To isolate, select, identify and assess the potential for the biodegradation of synthetic pyrethroids (SPs) in sheep dips.
Methods and Results: SP-degrading bacteria were isolated from a mixed soil sample consisting of garden soil and soils from farms where SPs had been used. The two largest in size were then identified using microscopy, biochemical and genetic techniques to be members of the genera Pseudomonas and Serratia. By comparing the 16S rRNA gene sequences, the Pseudomonas sp. discovered was shown to group within the Pseudomonas fluorescens intrageneric cluster. The Serratia isolated was closely related to Serratia plymuthica. Cell growth and degradation was greatest in the Pseudomonas sp. culture where there was breakdown of 60 mg l−1 to 6 mg l−1 technical cypermethrin in 20 days. Tolerance to the SPs was greater in the Pseudomonas sp. but was found to depend on the availability of other carbon sources and nutrients.
Conclusions: The bacteria characterized show the potential to be used in a bioremediation application for the treatment of SP residues.
Significance and Impact of the Study: The SP-degrading bacteria may have use in the disposal of used SP residues and with further research could lead to an alternative route of disposal for use in agriculture or industry.
Current methods of disposal for used sheep dips are either by contractor or by spreading onto waste land. Before disposal, used dip can be treated with other chemicals to degrade insecticides, but the waste must still be removed as above (Armstrong and Phillips 1998; Morrison 1998). Thus, biodegradation is a practical and applicable solution for detoxifying SPs in a range of varying conditions, whether it be in a dip trough, river or soil.
Previous biodegradation studies have isolated organisms capable of degrading different SPs when present as the sole carbon source. The biggest reductions were of permethrin from approximately 40 mg l−1 to 20 mg l−1 after 4 weeks in anaerobic, dark, non-shaken cultures at 75°C (Maloney et al. 1997). These conditions would not be suitable for use in the environment. As sheep dips are formulations and not solely SP, instead of using high concentrations of SPs as the sole carbon source, concentrations of the formulated and technical compounds were used.
Thus, this research centres on the isolation, identification and degrading ability of micro-organisms for SPs in real-life conditions.
MATERIALS AND METHODS
Chemicals and media
Technical cypermethrin compound (72·6% pure) was provided by Zeneca Agrochemicals (Yalding, Kent, UK). ‘Ecofleece’ non-OP sheep dip (contains 10% cypermethrin) was provided by Bimeda (Liverpool, UK). ‘Bayticol’ scab and tick dip (contains 6% flumethrin) was provided by Bayer (Leverkusen, Germany). The mineral salts medium used was Bushnell Haas (BH) broth composed of (l−1 deionized water): K2HPO4, 1 g; KH2PO4, 1 g; NH4NO3, 1 g; CaCl2, 0·02 g; MgSO4, 0·2 g; FeCl3, 0·05 g, taken to pH 7 with NaOH.
Nutrient broth (NB), nutrient agar and technical agar were obtained from Oxoid.
Technical cypermethrin was prepared by dissolving it into 10 times its volume of absolute alcohol.
Soil was obtained from the land immediately surrounding a sheep dip trough where SP sheep dips had been previously used, and mixed with garden soil.
Isolation and culture conditions
The isolation of all micro-organisms was performed using the BH medium. Technical agar (15 g) was added to 1 litre BHB for a solid plate medium. After autoclaving, the synthetic pyrethroid was added at the concentrations stated below.
Soil (1–5 g) was placed into 50 ml BHB and SP in 150 ml conical flasks. One set contained Bayticol at 1000 mg l−1 and a second set contained a mixture of 10 000 mg l−1 Bayticol, 6000 mg l−1 and Ecofleece (820 mg l−1 technical cypermethrin compound in 1 ml absolute alcohol). The first set was left in darkness for 12 weeks and the second was shaken at 100 rev min−1 for 10 days at 25°C in darkness. There were eight replicate condition cultures in Set one and two in Set two.
Each culture was spread (50 μl) onto BH agar plates containing the same concentrations of SP as the set they came from. These were incubated for 3 days or until visible growth appeared thereafter at a temperature of 30°C. The largest colonies were selected from the plates and re-streaked onto individual plates. From these plates, colonies were picked and used to inoculate flasks containing one of either 1000 mg l−1 Bayticol, 600 mg l−1 Ecofleece or 82 mg l−1 technical cypermethrin. (Note: The amounts of each chemical provided approximately equal concentrations of SP in mg l−1.) These bacteria were stored long-term on porous beads in cryopreservative fluid at −20°C, and short-term at 4°C, in order to be used for the degradation and toxicity experiments, in each of which the starting number of cells was approximately 2000 ml−1.
Gram staining and the appropriate API strip (bioMerieux, Lyon, France), either 20E or 20NE, were performed to assess basic physiology. Scanning electron microscopy (SEM) was used to examine the cells and their surfaces. For the SEM, a fresh concentrated cell suspension was fixed and dried before examination in a Hitachi S-2400 scanning electron microscope.
Primary PCR was performed, using the Y1 and Y3 primers, as described in Haukka (1997). PCR product was purified using the CONCERT PCR purification kit (GibcoBRL, Paisley, UK) and sequenced, on both strands, using an ABI 377 sequencer and BigDye terminator cycle sequencing kit with AmpliTaqFS DNA polymerase (ABI Perkin Elmer, California), and Y1, Y3, IntF (GCTYAACSTGGGAACTGC) and IntR (TTTACRGCGTGGACTACC) as sequencing primers. CLUSTALX (Thompson et al. 1997) was used for multiple sequence alignment and phylogenetic analysis. Clustal uses the Neighbour-Joining algorithm (Saitou and Nei 1987) with the Kimura 2-parameter model. Alignments were adjusted manually using GeneDoc (Nicholas et al. 1997) and trees were drawn using Treeview (Page 1996). Sequences from type cultures used in Anzai et al. (2000) for Pseudomonas (sensu stricto) and Sproer et al. (1999) for Serratia were obtained from the databases and used as standards.
Biodegradation and toxicity testing of synthetic pyrethroids
Cell numbers were monitored by plate counting serial dilutions of each culture. Degradation was measured using a novel bioassay consisting of fruit flies in a universal tube, three repeats per culture. Tests have previously shown that the time taken for half of the flies to die relates to the concentration of chemical present (Grant 2001). The pH was measured when sampling each culture.
Microbial toxicity was assessed purely to check that the bacteria could survive and proliferate in the concentrations of SP present in the experiment, and if these concentrations were higher than expected. Cell growth was again measured by plate counts of dilutions of each culture. Cultures of 10 000 mg l−1 Bayticol + 6000 mg l−1 Ecofleece + 820 mg l−1 technical cypermethrin in BHB and NB (giving approximately 1800 mg l−1 total SP), and 1000 mg l−1 Bayticol + 600 mg l−1 Ecofleece + 82 mg l−1 technical cypermethrin in BHB (giving approximately 180 mg l−1 total SP), with each organism were set up at 25°C, shaking at 100 rev min−1.
Control cultures with no SPs, and with 10 ml l−1 absolute alcohol to check for alcohol toxicity (as in the experiments with cypermethrin), were also carried out.
Isolation of micro-organisms
After 3 days, Set one yielded over 300 colonies per plate of around 50 organisms with different colony morphologies. These ranged in size from 0·5 to 2 mm of their approximate diameters. From the colonies, the 10 largest were selected and cultured separately. Size was used as the deciding factor because it shows the biggest reaction, even if in response to stress. Only two of these showed any significant growth and they were selected for further investigation.
After 3 days, Set two gave tens of thousands of colonies per plate. All appeared to be of similar colony morphology.
Identification of micro-organisms
Gram-staining and oxidase-testing of the selected micro-organisms provided basic information so that the appropriate API strip could be chosen. Their morphological appearance on agar plates led to their distinguishing names, ‘Circle’ and ‘White’. ‘Circle’, from Set one, was a Gram-negative oxidase-positive rod. ‘White’, from Set two, was a Gram-negative oxidase-negative rod.
For the Circle organism, an API20NE test strip was used and for White, an API20E was used. These gave the following profiles: Circle=1557555 (API20NE); White =120 (6 or 7) (3 or 1) 63 (API20E).
SEM observations showed that Circle organisms were fairly smooth rods (Fig. figr rid="f1">1) whereas the White organisms were rough rods with a crumpled texture (Fig. 2).
Phylogenetic analysis of 16S rRNA sequence demonstrated that Circle was a Pseudomonas species with support for inclusion in the Pseudomonas fluorescens group as defined by Anzai et al. (2000) (Fig. 3); Ps. Circle accession number is AJ417370.
Analysis of sequences indicated that White was a Serratia species with high 16S sequence similarity to Serratia plymuthica (Fig. 4); Ser. White accession number is AJ417371.
The bacteria isolated showed different tolerances to the SP chemicals dependant upon the availability of other more preferred growth substrates. This has been previously shown with such chemicals as phenol and 4-chlorophenol (Loh and Wang 1998). Figure 5 shows that with nutrient broth, the micro-organisms can withstand and grow in the higher concentration of 1800 mg l−1 total SPs, whereas the same concentrations with BH broth proved fatal to all. However, with 180 mg l−1 total concentration of SPs in BHB, the micro-organisms would grow; for 1 ml−1 of culture, Circle control and Circle with SPs grew to approximately 6 × 106, White control to approximately 3 × 105 and White with SPs to approximately 1 × 104. The low growth with White, however, was not found with the individual chemical cultures. The alcohol control showed that at the concentrations used, this had no affect on the growth of either organism, with cfu approximating those with BHB only.
Biodegradation of synthetic pyrethroids by micro-organisms
Under the culture conditions, the isolated Pseudomonas and Serratia spp. degraded the SPs by at least 50% after 20 days (Fig. 6a); in each case degradation was greater than the natural breakdown (Fig. 6b). The technical cypermethrin compound was degraded the most by the Circle organism, to around 6 mg l−1. Cell growth (Fig. 7) showed a huge increase in cell numbers after 7 days, with a decline thereafter. This happened with all the SPs, including the technical cypermethrin.
The pH changed very little over the experimental period, staying at 7·0 ± 0·1, except for Circle with technical cypermethrin which rose to 7·4 after 20 days.
The degrading bacteria examined were isolated from mixed soil originating from SP-contaminated farmland surrounding a sheep dipping facility and garden soil. Previous isolations have yielded a variety of different micro-organisms, including methanogens and novel thermophilic anaerobes from a geothermal hot spring (Maloney et al. 1997), but the Circle and White organisms appear to bear no resemblance to these.
The pattern of cell growth (Fig. 7) differed depending on which compound was used, and the growth in the bacteria control cultures was greater than some cultures containing SP. This may be due to a component in the formulated dips as growth with the technical cypermethrin was far greater than the controls. The cell growth graph (Fig. 7) also shows a large increase in viable cells immediately, before a tailing off. This may be due to the decreasing availability of SP present causing competition between cells and thus, cell death, or, possibly, to the lack of available minerals or substrate causing the cells to become weak and more susceptible to the SP toxicity (Loh and Wang 1998). Another possibility is the toxicity of the SP metabolites, although their toxicity is negligible (Extoxnet 1999). Growth in the cultures containing technical cypermethrin shows the same pattern although at much higher cell numbers. The factor of the non-SP parts of the formulations supporting growth seems unlikely (see the discussion on the SP toxicity above).
Identification of the bacteria using the 16 s rRNA sequences showed White to be very closely related to Serratia plymuthica, although this is based solely on the sequence of one gene. Circle was identified to be a member of the Pseudomonasfluorescens lineage, but there is low bootstrap support to be able to reliably separate this group further to place Circle against a single species.
With regard to the aims of the work, to biodegrade SP insecticides for disposal, the micro-organisms isolated show that it is possible, although real-world sites would have variable temperatures, mixing and nutrient content. However, these problems could potentially be overcome.
With regard to the bioassay used for the measurement of SP concentration, it has been shown that the matrix of the SP cultures with bacteria is a testable solution without requiring any additional modification or alteration (Grant 2001).
In conclusion, two organisms from different genera were shown to degrade the SPs when present in technical or formulated forms. The increased tolerance when alternative carbon sources were available indicates that there may be a co-metabolism effect. Further work examining the degradation and primary/secondary utilization under different nutrient conditions may improve upon the current rates of degradation, leading to a practical degradation system for SP-containing insecticides.
Present address: Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.