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Bioremediation Journal

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Bioremediation via In Situ Electrotransformation

Delina Y. Lyon a; Jérémy Pivetal a; Laurine Blanchard a;Timothy M. Vogel a a Department of Environmental Microbial Genomics, Ecole Centrale de Lyon, Ecully, France Online publication date: 06 May 2010

To cite this Article Lyon, Delina Y. , Pivetal, Jérémy , Blanchard, Laurine andVogel, Timothy M.(2010) 'Bioremediation via

In Situ Electrotransformation', Bioremediation Journal, 14: 2, 109 — 119 To link to this Article: DOI: 10.1080/10889861003767092 URL: http://dx.doi.org/10.1080/10889861003767092

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Bioremediation Journal, 14(2):109–119, 2010 c 2010 Taylor and Francis Group, LLC Copyright  ISSN: 1088-9868 DOI: 10.1080/10889861003767092

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Bioremediation via In Situ Electrotransformation Delina Y. Lyon, ´ emy ´ Jer Pivetal, Laurine Blanchard, and Timothy M. Vogel Department of Environmental Microbial Genomics, Ecole Centrale de Lyon, Ecully, France

ABSTRACT Bioremediation of polluted sites relies on bacteria to degrade or transform contaminants into less noxious chemicals. To do so, bacteria require genes that encode the degradation enzymes and the capacity to properly express them, which may be lacking in indigenous bacteria. To increase the ability of indigenous bacteria to bioremediate a contaminated site, this research proposes the use of electrotransformation to facilitate bacterial uptake of exogenous degradation genes. As a proof of concept, a lindane degradation gene (linA) located on a broad host-spectrum expression plasmid (pBLN) was introduced into soil bacteria by electroporation both in vitro, in liquid media, and in situ, in soil. In both cases, the electrotransformed bacteria displayed an increase in lindane degradation and an increase in the linA gene copy number. The use of in situ electrotransformation could improve pollutant degradation rates and could provide another tool for bioremediation. KEYWORDS biodegradation, bioremediation, electrotransformation, gene bioaugmentation, γ -hexachlorocyclohexane, lindane

INTRODUCTION

Address correspondence to Delina Y. Lyon, Department of Environmental Microbial Genomics, Ecole Centrale de Lyon, 36, avenue Guy de Collongue Ecully, 69134 France. E-mail address: [email protected]

Bioremediation relies on the ability of microorganisms to degrade or transform a wide range of chemical compounds and, in so doing, clean up polluted sites. The key for effective bioremediation is to have the necessary genes encoding the functional enzymes in active bacteria at the polluted site. One way to add or increase the presence of degradation genes is to “bioaugment,” or to add bacteria known to express the enzymes of interest, to the site. Although bioaugmentation seems like a practical solution, the primary hurdle is survival of the inoculum in situ (Cases and de Lorenzo, 2005; Park et al., 2008). Inocula, even those enriched from the site of interest, are regularly out-competed, predated, or starved in their “new” environment by the well-adapted indigenous bacteria (Vogel, 1996; El Fantroussi and Agathos, 2005). Thus, the introduction of an enzymatic function through bioaugmentation is frequently insufficient because the bacteria carrying that function often cannot survive. To avoid the problem of inoculum survival, the degradation genes themselves could be introduced into indigenous bacteria and thus bestow the enzyme function. This has the additional potential advantage of conferring a further competitive advantage to the transformed bacteria. In nature, 109

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bacterial gene transfer occurs not only from mother cell to daughter cells, but also between cells and species via a process called horizontal gene transfer (HGT). HGT is an important process believed to be responsible for a significant portion of the genomes of various organisms, but is more notorious as a method for spreading antibiotic resistance genes (Zaneveld et al., 2008). HGT is usually mediated by transmissible nucleic acid– based mobile genetic elements (MGEs), such as plasmids, transposons, bacteriophage-related elements, and genomic islands (Top et al., 2002; Nojiri et al., 2004). The idea of manipulating HGT to introduce MGEs containing degradation genes is called “gene bioaugmentation.” There is an abundance of HGT methods and MGEs that could or have been used for gene bioaugmentation. Conjugation, or the transfer of plasmids between bacteria of a certain group, has been successfully tested in the laboratory for the transfer of degradation genes (Top et al., 1998). This technique has been explored for the enhancement of the xenobiotic degradative capacity of activated sludge in wastewater treatment (Bathe et al., 2005; Nancharaiah et al., 2008). The remediation of soil contaminated with 2,4-dichlorophenol and cadmium was accelerated after gene bioaugmentation with degradation genes on plasmids transferred into indigenous bacteria via conjugation (Roane et al., 2001; Pepper et al., 2002). A methyl-carbamate–degrading gene was transferred between donor organisms and other species in soil via conjugation and other nonspecified transfer methods (Desaint et al., 2003). However, these methods still rely on a bacterial inoculum to introduce the degradation genes into the soil, and because only certain types of bacteria can conjugate with each other, a limited number of bacteria would be involved. Our research focuses on the use of transformation, or the bacterial uptake of exogenous cell-free DNA from the environment. This can occur without external forces in a process called natural transformation when the bacteria are naturally competent. Natural transformation was used to introduce an atrazine-degradation gene on a plasmid into bacteria in a biofilm (Perumbakkam et al., 2006). However, natural transformation only occurs with competent bacteria, which are difficult to predict in a complex and heterogeneous system like soil (Johnsborg et al., 2007; Levy-Booth et al., 2007). To affect a larger number and variety of bacteria, we propose the use of electrotransformation to transfer cell-free plasmid DNA directly into bacteria in situ at D. Y. Lyon et al.

a field site. Electrotransformation uses a pulsed electric field (PEF) to encourage bacterial uptake of extracellular DNA. The brief application of an electric field causes electroporation, which is the formation of transmembrane pores (Weaver, 1995; Teissie et al., 2005). Extracellular DNA can either enter the cell through those pores or by physical association with the now-perturbed membrane (Tieleman, 2004). Electrotransformation is among the more powerful transformation methods, as it can be applied to all types of cells (Prasanna and Panda, 1997; Yuan, 2007). Commercially available electroporation instruments are able to deliver high-voltage electric pulses to small amounts of liquid medium. To deliver such a pulse to a volume of soil, we used a high-voltage generator, which had previously been used in our laboratory to demonstrate that simulated lightning can facilitate genetic transfer in soil bacteria, by electrotransformation, in an evolutionary context (Demaneche et al., 2001). In this pilot experiment, we tested the feasibility of using in situ electrotransformation to insert a plasmid encoding a pollutant-degrading gene into resident soil bacteria. The contaminant of interest was lindane, or γ -hexachlorocyclohexane, a widely used pesticide prior to a 2006 ban on its agricultural use. Lindane contamination is a relatively common problem due to its widespread agricultural application and its chemical stability. Despite its recent release into the environment (less than 60 years), several bacterial species have been found that are capable of degrading and mineralizing lindane (Lal et al., 2006). One such species, Sphingobium francense, contains a gene, linA, that encodes a protein that performs the first two dechlorination steps of lindane (Nagata et al., 1999). In this research, a broadhost-range plasmid containing the linA gene (pBLN) was added to a soil microbial community that had not previously been exposed to lindane. First, we verified that at least some of the soil bacteria were capable of taking up the plasmid by electrotransformation in a liquid medium and that these bacteria could express linA, as measured by lindane degradation. The second step was to add the plasmid to soil, apply a pulsed electric field (PEF) using a high-voltage generator, and monitor lindane degradation while also verifying the physical presence of the degradation gene in the soil bacteria. We found increased lindane degradation in soil samples that had been electrotransformed with pBLN. In general, the results indicate that it is possible to introduce a degradation gene into soil microbes via in situ 110

electrotransformation, opening new avenues for bioremediation efforts.

MATERIALS AND METHODS

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Bacterial Strains and Plasmids The 8.5-kb plasmid, pBLN, was constructed from the broad-host-range plasmid pBBR1-MCS3 (GeneBank ref.: XXU25059), which is able to replicate in many different types of bacteria (Kovach et al., 1995), with the addition of the gene linA from S. francense (Ceremonie et al., 2006) and the gene nptII (kanamycin resistance). The plasmid was maintained in Escherichia coli DH5α in Luria Bertani (LB) broth with 25 µg ml−1 kanamycin and 25 µg ml−1 tetracycline. It was extracted, when needed, using the NucleoSpin Plasmid Extraction kit (Macherey Nagel). Pseudomonas N3, a naturally electrocompetent bacterium, and S. francense Sp+ were used as positive controls for transformation and lindane degradation, respectively, and were maintained in LB without antibiotics (Ceremonie et al., 2006).

In Vitro Electrotransformation of Soil Microbes in Liquid Media Two types of noncontaminated soils from France were used in this research: a sandy loam from Cote ˆ Saint Andr´e (CSA) (Is`ere, France) and a prairie soil from Montrond (MON) (La Batie-Divisin, Is`ere, France). Indigenous microbes were extracted from the soil by grinding 1 g of soil in 10 ml of sterile 0.2% sodium hexametaphosphate in a prechilled Waring blender for 3 min (Bertrand et al., 2005). The large soil particles were removed via centrifugation at 100 ×g for 15 min. One milliliter of the supernatant was used to inoculate 250 ml of LB containing 200 µg ml−1 cycloheximide, to control unwanted fungal growth. The soil microbes were grown at 29◦ C for 48 h before being harvested by centrifugation at 8000 ×g for 10 min. The cell pellets were washed twice with 50 ml of sterile distilled water to remove residual salts before being resuspended in 150 µl sterile distilled water. Fifty microliters of the microbe suspension were then mixed with 500 ng of pBLN and electroporated in a 0.2 cm gap cuvette at 12.4 kV cm−1 , 200 ohms, and 25 µF (Gene Pulser II; Bio-Rad, Hercules, CA, USA). This setting was chosen using the criteria that an effective electroporation, maximizing the amount of DNA that enters cells, results in 60% cell mortality (Drury, 1994). The cells were resus111

pended in 1 ml SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 , 10 mM MgSO4 , 20 mM glucose) for 2 h at 29◦ C. Samples included a negative control of soil microbes with lindane (CSA-l, MON-l), a negative control of electroporated soil microbes with lindane (CSA-le, MON-le), electroporated soil microbes with lindane and pBLN (CSA-lpe, MON-lpe), soil microbes with pBLN to control for natural transformation (CSA-lp, MON-lp), and S. francense (Sfr) as a positive control for lindane degradation. Five hundred microliters of the transformed cell suspension were added to 7 ml of a minimal salts MS medium (0.5 g NaNO3 , 0.65 g K2 HPO4 , 0.17 g KH2 PO4 , 0.1 g MgSO4 , 5.60 mg FeSO4 per liter) supplemented with 1 mg ml−1 lindane (from a stock of 50 mg lindane ml−1 DMSO), 25 µg ml−1 kanamycin, 25 µg ml−1 tetracycline, and 100 µg ml−1 cycloheximide. The cells were incubated at 29◦ C for 5 days shaking at 120 rpm before assessing lindane degradation and linA presence.

In Situ Electrotransformation of Soil Bacteria in Soil The soils were prepared in large batches by sieving through a 2-mm mesh, crushing with a mortar and pestle to achieve a fine texture, and then adjusting the humidity to 9%. The following samples were prepared of each CSA and MON: soil with lindane (CSA-sl, MON-sl), soil with lindane and PEF (CSA-sle, MONsle), soil with lindane and pBLN (CSA-slp, MON-slp), soil with lindane and pBLN and PEF (CSA-slpe, MONslpe), a positive control for transformation of soil with simulated lightning with Pseudomonas N3 and pBLN (Montrond soil only, MON-slpen), a positive control for lindane degradation with soil and S. francense (CSAsl+, MON-sl+), and a negative control of soil without lindane (CSA-s, MON-s). Each sample consisted of 30 g of soil with 25 µl of 100 ng µl−1 pBLN, where needed. The applied plasmid concentration is similar to what has been successfully used previously with our highvoltage generator (Demaneche et al., 2001). Once the 30 g soil samples were prepared, they were pressed into plastic Petri dishes that were lined with aluminum foil, wired to facilitate transmission of electricity (Figure 1). A high-voltage generator was used to deliver an electric pulse of between 4 and 7 kV via a brass electrode (2.5 cm in diameter, 3.5 cm thick; Figure 1b). After the sample was exposed to the PEF, it was emptied into a Bioremediation via In Situ Electrotransformation

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FIGURE 1 (a) High-voltage generator, arrow marks where the sample is placed; (b) rear and front view of modified Petri dishes lined with aluminium for soil electrotransformation.

ultraviolet (UV)-sterilized plastic jar and incubated for 1 h at 29◦ C. A 1 g sample of each soil was taken in triplicate to perform a DNA extraction. Then 300 mg of lindane was added to each of the jars of soil after the electric shock for all samples except the negative controls without lindane. The high lindane concentrations are within the range found in contaminated sites and were chosen to facilitate observation of lindane degradation and overcome any bioavailability constraints (Pereira et al., 2008). The jars were incubated at 29◦ C for 3 weeks for MON or for 2 months for CSA, with periodic shaking and air intake. The samples were then analyzed for lindane degradation, and their DNA was again extracted to test for linA presence via quantitative polymerase chain reactions (qPCR). D. Y. Lyon et al.

Detection of Lindane Degradation The linA gene encodes an enzyme that performs the first two dechlorinations of lindane (γ -hexachlorocyclohexane); the resulting 1,3,4,6tetrachloro-1,4-cyclohexadiene spontaneously dechlorinates to 1,2,4-trichlorobenzene for a total of three chlorines released per lindane molecule. Lindane degradation was measured via an increase in chloride concentration using an Orion 710A+ Advanced ISE/pH/mV/ORP meter (Thermo Electron). For the liquid electrotransformation samples, 2 ml of the culture with lindane in MS medium were centrifuged at 12,000 × g for 5 min. The supernatant was transferred into another tube, and the chloride concentration was 112

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measured. For soil electrotransformation samples, 10 g of soil was measured in triplicate from each jar and vortexed for 30 min in 5 ml of sterile MilliQ water to release and solubilize the chloride ions. The sample was then centrifuged at 18,000 × g for 10 min, the supernatant was recovered, and the chloride concentration was measured.

GCT GG-3 for an expected amplicon of about 200 bp. The cycling parameters were 95◦ C for 10 min, followed by 40 cycles of 95◦ C for 15 s, 53◦ C for 30 s, and 72◦ C for 60 s, followed by melting curve analysis.

DNA Extraction

Lindane was extracted from soil by vortexing 0.5 g soil with 1.5 ml methanol for 1 h. The sample was then filtered using a syringe equipped with a 0.2-µm filter. The samples were analyzed using an Agilent Technologies 6850 Network GC System with a 5975 VL mass spectrometer equipped with a HP-5MS fused silica capillary column. The GC was kept at 100◦ C for 2 min, then increased at 15◦ C min−1 to 325◦ C, and maintained at this temperature for 3 min. The injector was set at 250◦ C, and helium was used as the carrier gas at 0.5 ml min−1 constant flow.

Total DNA was extracted from microbes in liquid media or from soil using a phenol:chloroform extraction protocol (David et al., submitted). Prior to DNA extraction from soil, nonincorporated plasmid and other extracellular DNA were destroyed using DNase. In triplicate, 0.5 g of each soil sample was incubated in 1 ml DNase buffer (40 mM Tris-HCl, 8 mM MgCl2 ) with 400 units DNase (Roche, Basel, Switzerland) at 37◦ C for 1 h. The reaction was stopped with 20 mM EDTA, and the soil samples were centrifuged at 12,000 × g for 5 min. For the long-term CSA soil studies, an UltraClean Soil DNA Extraction kit (MoBio) was used to extract DNA from 1 g of soil. No DNase treatment was performed. All extracted DNA was quantified spectrophotometrically using an Implen NanoPhotometer.

Quantitative Polymerase Chain Reaction (qPCR) qPCR was performed in a Rotor-Gene 6000 realtime rotary system (Corbett Life Science) using the SensiMixPlus SYBR kit (Quantace, London, United Kingdom). The following primers were used to detect the linA gene: forward (nested-linA-F1) 5 -GCT CAT TGC CGT AGA CAA-3 and reverse (nested-linA-R1) 5 -GCT CAT ACT CAT CCG TGA AG-3 , with an expected product of 296 bp. The cycling parameters were 95◦ C for 10 min, followed by 45 cycles of 95◦ C for 15 s, 60◦ C for 20 s, and 72◦ C for 20 s, followed by a melting curve analysis that verified the specificity of the amplification. To further verify the identity of the amplicons, a normal PCR was performed using Illustra Hot Start Mix RTG PCR beads (GE Healthcare), and the products were sequenced. DNA samples were diluted to the same concentration (between 5 and 10 ng µl−1 ) prior to qPCR. To ensure that equal quantities of DNA were being analyzed, a qPCR of 16S rDNA was performed using the following primers (Fierer et al., 2005): forward (Eub338) 5 -ACT CCT ACG GGA GGC AGC AG-3 , and reverse primer (EUB518) 5 -ATT ACC GCG GCT 113

Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

Statistical Treatment of the Results All experiments were performed at least in triplicate. The data shown is the mean with error bars indicating standard error of the mean. Statistical significance was determined using Student’s t test where p ≤ .05.

RESULTS AND DISCUSSION Electroporation of Soil Microbes The goal of this research was to evaluate whether a biodegrading enzyme function, such as lindane degradation, could be introduced into a soil microbial community via electrotransformation. This was done by introducing an MGE (pBLN) containing a lindanedegradation gene (linA) into artificially contaminated soil (there was no lindane detectable by GC-MS in CSA). Before the experiments were performed in soil, we verified that linA could be incorporated and expressed in soil microbes. The soil microbial communities extracted and cultured from CSA and MON were electroporated with pBLN at 988 V (4.94 kV cm−1 ) for about 10 ms. After 5 days, the incorporation and quantity of linA were assessed by extracting the DNA from the bacteria and performing a qPCR targeting linA. The copy numbers of linA in each sample were compared to the copy numbers in the soil microbe control MON-l or CSAl (Table 1). In both soil microbial extracts, there was Bioremediation via In Situ Electrotransformation

TABLE 1 Comparison of the Copy Numbers of linA in Soil Microbes Extracted from Two Types of Soil and Electroporated with pBLN

Montrond soil microbes

Fold change

MON-le MON-lp MON-lpe Ps. N3 + control∗

0.44 ± 0.14 0.28 ± 0.15 3.21 ± 2.2 352 ± 273

CSA soil microbes

Fold change

CSA-le CSA-lp CSA-lpe∗

1.26 ± 0.7 1.82 ± 0.82 4682 ± 3648

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Note. The fold change is the quotient of the linA copy number in the sample divided by the copy number in the negative control (MON-l or CSA-l). e = electropotated; p = pBLN. ∗ Significantly different results with a p ≤ .05.

some basal presence of the gene linA. The negative controls MON-le, CSA-le, MON-lp, and CSA-lp showed no significant increase in linA copy number. However, although the increase observed in MON-lpe linA copy number was not statistically significant, there was a significant increase in linA copy number in CSA-lpe, indicating that the electrotransformation was able to introduce a significant amount of linA into the soil microbes. This electrotransformation would only assist in bioremediation if the incorporated gene was functional. Thus, in parallel with measuring linA incorporation, the expression of linA was also assessed via lindane degradation as measured by increases in chloride concentration (Figure 2). Congruent with the qPCR data, there is no increase in chloride concentration in the negative controls MON-le, CSA-le, MON-lp, and CSA-lp, indicating a lack of natural transformation. However, in MON-lpe and CSA-lpe, there was a significant increase in chloride concentration,

indicating that lindane was being degraded. Assuming that chloride is well solubilized in the medium and taking into account the three potential chloride ions produced per lindane molecule degraded by LinA, MON-lpe and CSA-lpe each degraded about 9% of the lindane in the medium over 5 days. When comparing the chloride concentrations with the linA copy numbers, it is clear that soil microbes are capable of incorporating the vector pBLN carrying linA and expressing the gene. While serving to examine the feasibility of electrotransformation in soil, this experiment also demonstrates the use electrotransformation to bioremediate aqueous media such as sediments and aquifers. The next step of this research was to attempt in situ electrotransformation of bacteria in soil through application of a PEF.

In Situ Electrotransformation of Soil Microbes An in situ electrotransformation was performed with both microbial communities present in the MON and CSA soils. The soil samples were exposed to a 20 kV pulse, resulting in currents between 8.12 and 11.25 amps and potentials between 6.25 and 7.81 kV. The soils were exposed to a similar voltage as used in the liquid electroporation experiment (2.2 kV cm−1 in the soil and 4.94 kV cm−1 in the liquid medium). The soils were incubated with 10 mg lindane per g of soil for 3 weeks to allow sufficient time for noticeable lindane degradation, which was evaluated by resuspending the soils in water and measuring chloride ion concentrations.

FIGURE 2 Chloride concentrations indicative of lindane degradation in low-salt media with soil microbes electroporated with pBLN. The standard error of the mean is shown in the error bars. Results significantly different (p < .05) from microbe-only controls CSA and MSA are marked with ∗ . l = lindane; e = electroporated; p = pBLN; Sfr = S. francense positive control; medium = MS medium with lindane. D. Y. Lyon et al.

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FIGURE 3 (A ) Lindane degradation as evidenced by increases in chloride concentration in Montrond and CSA soils that had been electrically shocked with pBLN. Chloride concentrations were measured after 3 weeks in water. (B) Chloride concentration in CSA soils that had been incubated for 2 months after electrotransformation with pBLN. The soil shocked with pBLN showed an increase in lindane degradation, as measured by chloride concentration. Error bars indicate standard error of the mean. Values significantly different from the control (p < .05) are indicated with ∗ . l = lindane; e = electrotransformed; p = pBLN; + = S. francense.

After 3 weeks in the CSA soil, the natural transformation control (CSA-slp), the experimental sample (CSA-slpe), and the positive controls (CSA-slpen, CSAsl+) all showed significant increases in lindane degradation as compared to the negative control with lindane, CSA-sl (Figure 3A). The increases in CSA-slp could be due to natural transformation, which was not seen during the liquid electrotransformation test. In that experiment, the soil microbes were cultured beforehand, which may have selected against the naturally competent bacteria in the soil. CSA-slpe, which was higher but not significantly different from CSA-slp, degraded about 0.11% of the lindane in the soil, although this 115

was not as high as the positive controls CSA-slpen and CSA-sl+, which degraded 0.25% and 1.4% of the lindane, respectively. The amount of chloride released by CSA-sl+ is disproportionately high, since each lindane molecule degraded by S. francense generates six chloride ions instead of three. Overall, it appears that the in situ electrotransformation increased the lindane-degrading capacity of the CSA soil, although not significantly more than natural transformation ( p = .075 for onetailed Student’s t test with unequal variance). For the Montrond soil, the only significant increase in lindane degradation was in the positive control MON-sl+ (Figure 3A). Both MON-slpe and the Bioremediation via In Situ Electrotransformation

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positive control MON-slpen showed slight but statistically insignificant increases in chloride concentration. The lindane-degradation activity seems to have functioned better in the CSA soil. The S. francense positive controls in the CSA soil had an order of magnitude higher chloride concentration than in the Montrond soil, translating to a 1.4% reduction in the introduced lindane concentration by CSA-sl+ compared to 0.1% by MON-sl+. This could be due to lower levels of pBLN electrotransformation in the Montrond soil, differences in nutrient availability leading to slower growing or performing microbes, or soil chemistry. Due to the perceived success with the CSA soil, this soil was chosen for a longer-term experiment. A longer soil electrotransformation experiment was performed to see if lindane degradation would increase over time. The CSA soil was prepared as in the previous experiment, and the soil was incubated for 2 months at 29◦ C, with the chloride concentration results shown in Figure 3B. As expected, CSA-sl+ results in a much higher level of lindane degradation as compared to the CSA-sl+ after 3 weeks shown in Figure 3A. The CSAslpe after 2 months also showed a significant increase in chloride concentration, representing about 0.19% lindane originally in the sample. But although the percent of lindane degraded after 2 months in CSA-sl+ (3.4%) increased 1.4-fold compared to the amount degraded after 3 weeks (1.4%), the amount of lindane degraded by CSA-slpe increased only 0.72-fold (0.19% versus 0.11%) after 2 months. The increase in lindane degradation by CSA-slpe is not commensurate with what was expected after 2 months. This could be explained by a loss of the plasmid in this system. In this pilot experiment, pBLN provides no in situ selective benefit to the microbes carrying and expressing the encoded genes, e.g., the microbes cannot use any new carbon sources nor do they have a selective advantage over other bacteria. If anything, the expression of linA is a metabolic burden and results in the microbes acidifying their local microenvironment. These two factors could lead to the loss of the plasmid in the system, either through microbes losing the plasmid or the plasmid-carrying microbes being out-competed (De Gelder et al., 2007).

linA Gene Detection in Soil Microbes after In Situ Electrotransformation After the in situ soil electrotransformation of MON and CSA, the DNA from the microbes in the soil was D. Y. Lyon et al.

extracted, and a qPCR was performed to detect linA both directly after the electrotransformation and after 3 weeks of incubation. Directly after the electrotransformation, the linA fragment targeted by the qPCR is found in all of the MON and CSA samples (Figure 4A). This concurs with the presence of linA seen in the electroporation studies (Table 1). However, it does not appear to be a functional form of the gene, judging from the lack of lindane degradation in the MON-sl and CSA-sl negative controls in Figure 3. At time 0 in the CSA samples, the linA levels are fairly consistent in all of the samples, even though it would be expected that the electrotransformation would introduce linA into some bacteria. The background level of a nonfunctional linA fragment in the native community might be masked by any increases due to the electrotransformation. For the MON samples, the linA level increases in both MON-slp and MON-slpe (with MON-slpe showing a higher copy number of linA), implying the involvement of both natural transformation and electrotransformation. After 3 weeks of incubation with lindane, the linA gene fragment is still detected in all tested samples from both soils (Figure 4B). Compared to time 0 (Figure 4A), MON-sle shows an augmentation in linA copy number that could be due to electrical stimulation of the microbial community (Hunt et al., 2009) or selective pressure exerted by the presence of lindane. There was no correlating increase in lindane degradation in the MON-sle sample, but the positive MON-sl+ control did not display a high degree of degradation either (Figure 3A). Interestingly, at 3 weeks, there is a decrease in linA in CSA-slpe and MON-slpe. As discussed in the section above, this could be a reflection of the metabolic burden exerted by the plasmid on its host bacterium, leading to loss of the plasmid. The bacteria that may have taken up the gene appeared to have either died or lost the plasmid in the 3 weeks since the electrotransformation and that the majority of the degradation activity had already occurred (Figure 3). The same CSA samples in Figure 3C that had been incubated for 2 months underwent DNA extraction and qPCR targeting linA (Figure 4C). Although these results cannot be directly compared with the previous results in Figure 4A and B, since they are with different samples and with different DNA extraction protocols, they continue the trend seen in Figure 4B, with the loss of the linA gene in CSA-slpe compared to CSA-sle and CSA-slp. The samples in Figure 4C are not significantly 116

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FIGURE 4 (A ) Copies of the linA gene in CSA and Montrond soils electrotransformed with pBLN directly after electrotransformation (time zero) and (B) after 3 weeks. (C) Number of linA gene copies in CSA soil after 2 months incubation. N.A., data not available; N.D., signal not detected. Error bars indicate standard error of the mean. ∗ denotes samples that were significantly different (p < .05) from the soil controls CSA-sl/MON-sl or shocked soil control for time 0 Montrond samples, MON-sle. l = lindane; e = electrotransformed; p = pBLN; n = Pseudomonas N3; + = S. francense.

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different from each other ( p = .05), but it appears either that linA has not been conserved in the soil or it is not easily detected. The disappearance of linA from the CSA-slpe sample mirrors the decrease in lindane degrading activity seen at 2 months as compared to the positive CSA-sl+ sample (Figure 3B).

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In Situ Electrotransformation as a Bioremediation Tool This pilot experiment demonstrates that in situ electrotransformation to perform gene bioaugmentation could serve as a bioremediation tool. The liquid electrotransformation experiments were more successful than the in situ soil experiments, which is a common theme in the literature due to the complex nature of soil (Desaint et al., 2003). The system chosen in this research, lindane degradation via pBLN transformation, was merely a demonstration of the feasibility of this method, but it was not the best solution for a longterm degradation system. In future attempts, the MGE chosen should confer an advantage to the bacteria to enhance its persistence in the bacterial community. Although the stability and maintenance of MGEs has previously been shown to be difficult while attempting gene bioaugmentation (Desaint et al., 2003), the stability of the degradation gene in the system can be improved by locating the gene in a transposon, which could be integrated into the chromosome of the host organism (Springael and Top, 2004; Shintani et al., 2005). The addition of a PEF facilitates the uptake of these elements into microbes that might not ordinarily participate in HGT. This method could be particularly useful when attempting to remediate xenobiotics, or relatively newly manufactured compounds with entirely novel chemical structures, for which the degradation genes are not widely spread or even available (Leisinger, 1983; Timmis et al., 1994). Degradation genes could also be engineered for higher efficiency and/or better control and then transformed into indigenous bacteria. Regardless of the source of the genes, the use of this technology raises the safety issue of using genetically modified organisms (GMOs) (Sayre and Seidler, 2005), although some of these risks may be mitigated by engineering control systems into the GMO (Davison, 2005). Another hurdle is the actual application of this technology in the field. We propose two methods: in situ current generation or the application of current to smaller soil samples that are then replaced into the D. Y. Lyon et al.

field. There are existing technologies for in situ current generation, like PEF used for disinfecting foods in food packaging plants (Wouters and Smelt, 1997). This technology would have to be developed to work in field situations. The second proposal, the use of microcosms to reinoculate the soil, would be more feasible with existing technologies. Soil samples taken from the site to be remediated could be electrotransformed with the degradation genes of interest and then reinoculated into the site. This still presents an advantage over bioaugmentation, since indigenous bacteria are more adapted to the site than any foreign inoculum. Future experiments could also incorporate electroporation of extracted soil bacteria into water and reinoculation of soil with the electrotransformed cells.

ACKNOWLEDGMENTS The authors would like to thank Dr. H´el`ene C´er´emonie for the plasmid pBLN, Dr. Sandrine Deman`eche for her guidance with the electrotransformation, and Guillaume Libaude for operating the highvoltage generator. This research was supported in part by the Rhone-Alpes Region and the French Minister of Education.

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Bioremediation via In Situ Electrotransformation