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Interactions of earthworms with indigenous and bioaugmented PCB-degrading bacteria Ekawan Luepromchai a , Andrew C. Singer b , Ching-Hong Yang b , David E. Crowley a;b; a

Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521, USA b Department of Environmental Sciences, University of California, Riverside, CA 92521, USA Received 19 February 2002; received in revised form 19 April 2002; accepted 2 May 2002 First published online 10 July 2002

Abstract Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soils has been achieved using bioaugmentation with PCBdegrading bacteria and earthworms. To further study the contribution of earthworms to bioremediation, an experiment was conducted in which the changes in indigenous and bioaugmented PCB-degrading bacteria were analyzed during treatment of contaminated soil using earthworms (Pheretima hawayana) alone or in combination with the PCB-degrading bacteria, Ralstonia eutrophus and Rhodococcus sp. ACS. Bacteria used for bioaugmentation were induced with carvone and salicylic acid in culture and were repeatedly applied every 3^4 days to the surface of unmixed, 20-cm long soil columns containing 100 ppm Aroclor 1242. After 9 weeks of treatment, the soil bacterial communities were analyzed using PCR primers for the bph genes. Results showed that approximately 50% of the PCBs were removed in the top 9 cm using a combination of earthworms and bioaugmentation, whereas bioaugmentation or earthworms applied alone were effective only for removing PCBs from the top 3 cm of the soil columns. Enhanced removal of PCBs caused by earthworms was associated with an increase in the population size of culturable, indigenous biphenyl-degrading bacteria, and an increase in the level of the bphA and bphC genes. The results suggest that earthworms facilitate PCB bioremediation by enhancing the dispersal of PCB-degrading bacteria in bioaugmented columns, as well as providing environmental conditions that favor the growth and activity of indigenous PCB-degrading bacteria. 8 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : bph gene probe; Bacterial community analysis ; PCB bioaugmentation; Earthworm

1. Introduction Bioremediation is an environmentally sound, and potentially low cost method to clean up soils that are contaminated with polychlorinated biphenyls (PCBs) [1]. Nonetheless, there is still concern over the reliability and general e⁄cacy of bioremediation that has precluded the commercial application of these methods [2^4], and there is a consensus that further improvements will require a better understanding of the biological processes that facilitate bioremediation [2,5,6]. Bioremediation can employ either bioaugmentation in which additional bacteria are added to the soil, or may be achieved by biostimulation of contaminant degrader populations within the indige-

* Corresponding author. Tel. : +1 (909) 7873785; Fax : +1 (909) 7873993. E-mail address : [email protected] (D.E. Crowley).

nous soil micro£ora. This can be accomplished by addition of supplemental nutrients, addition of cosubstrates that induce enzymes for degradation, the use of surfactants to increase the bioavailability of the contaminant, or by altering the soil physical and chemical conditions to enhance microbial activity. During bioaugmentation, indigenous bacteria may play a complementary role in the degradation process. In this regard, molecular techniques for microbial community analysis are particularly useful for monitoring changes in microbial populations during xenobiotic degradation processes and the response of bacterial communities to di¡erent bioaugmentation and biostimulation treatments [7^10]. In prior research in our laboratory, methods for bioremediation of PCBs have been developed that use repeated applications of PCB-degrading bacteria to contaminated soil. Since PCBs are not readily utilized as carbon sources for growth, their degradation is enhanced by addition of a cosubstrate such as biphenyl which induces expression of

0168-6496 / 02 / $22.00 8 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 2 9 4 - 5

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the bph genes encoding for biphenyl degradation. This can also be achieved using selected monoterpenes or salicylic acid that serve as cosubstrates for cometabolism of PCBs, but which are less toxic than biphenyl [11]. Another improvement in these methods has been the co-application of a surfactant that mobilizes PCBs and serves as a growth substrate for the soil inoculants and the indigenous bacteria [12]. Lastly, the most recent innovation has been the use of earthworms to enhance the dispersal of PCB-degrading bacteria that are applied to the surface of the contaminated soil. In prior research with earthworms [13], the disappearance of PCBs appeared to be due to a combination of e¡ects including enhanced dispersal of the inoculum, improved soil aeration and increased soil carbon and nitrogen supply. Interestingly, in control treatments, earthworms alone were found to promote PCB degradation, which suggested that earthworms also in£uence the indigenous biphenyl-degrading bacteria through their activities in the soil. To further study this phenomenon, the research reported here examines the interactions between earthworms and the PCB-degrading bacteria during bioremediation of a PCB-contaminated soil using bioaugmentation with Ralstonia eutrophus, Rhodococcus sp. ACS and their spent culture media that contains nutrients, inducing substrates, and surfactant. Changes in PCB-degrading bacterial populations were monitored by polymerase chain reaction (PCR) detection of the bphA and bphC genes and enumeration of bacteria that can use biphenyl as a growth substrate. The results suggest complex interactions in which earthworms stimulated both the bioaugmented and indigenous bacteria to degrade PCBs, and enhanced the overall e⁄cacy of removing PCBs below the soil surface by mixing the soil and bacteria.

trogen, pH 7.7) spiked with 100 ppm Aroclor 1242 as previously described [13]. Once the columns were ¢lled, they contained 20 cm of soil that was packed to within 5 cm of the top of the columns. The soil columns were allowed to equilibrate for 4 weeks prior to starting the treatments. Two days before applying the earthworms and bioaugmented bacteria, each column was watered with 70 ml of deionized water and 70 ml of minimal salts (MS) medium [14] to establish a 12% soil water content. The weights of the soil columns were noted to allow for the determination of water loss over time. The soil moisture contents were adjusted gravimetrically to their initial levels every 3^4 days. 2.2. Bioremediation treatments The experiment consisted of a 2U2 factorial design : with and without earthworms and bioaugmentation. The treatment employing bioaugmentation used two PCB-degrading bacteria, R. eutrophus H850 and Rhodococcus sp. ACS, which were added along with the spent media from their cultures. The control treatment received MS medium only. In the soil columns with the earthworms, two adult Pheretima hawayana earthworms (approximately 0.55 g earthworm31 ) were placed in the soil, and were fed every 3^4 days with approximately 1 g of rolled oats. Similar amounts of oats were added to the columns without earthworms. Three replicate columns were prepared for each of the four treatments for a total of 12 columns. The soil columns were maintained in a plant growth chamber at 20‡C with 70% relative humidity, a light intensity of 500 WE m32 s31 and a 16/8 h day/night cycle. 2.3. Inoculum preparation

2. Materials and methods 2.1. Soil microcosm preparation Twelve 30-cm long, 5.08-cm diameter brass columns were prepared as soil microcosms. The columns were closed on the bottom end with an open weave fabric and were partially ¢lled with a layer of 140 g of uncontaminated soil that could be extracted and analyzed at the end of the experiment to monitor any movement of PCBs in water that leached from the upper layer of the contaminated soil. The pristine soil barrier also served to minimize gas di¡usion from below into the soil column, thereby more closely simulating oxygen concentrations found at depth in the ¢eld. A layer of glass wool and two pieces of copper mesh (1 mm mesh) were added to completely cover the surface of the lower layer of the soil and to preclude the movement of earthworms into the lower soil zone. Each tube was then ¢lled with 0.6 kg of PCB-contaminated soil that was prepared from Dello loamy sand (typic Psammaquent, sandy alluvium, 0.90% carbon, 0.08% ni-

Microbial cultures were grown in 250-ml Erlenmeyer £asks containing 100 ml of culture medium, and were grown on a rotary shaker at 250 rev min31 . The Rhodococcus sp. ACS culture medium contained 100 mg l31 carvone, and 1000 mg l31 sorbitan trioleate dissolved in MS medium ; whereas, the R. eutrophus H850 cultures contained 500 mg l31 salicylic acid and 1000 mg g31 sorbitan trioleate. After approximately 20 h of growth, an additional 50 Wl (500 mg g31 ) of sorbitan trioleate was added to the culture £asks to maintain a su⁄cient concentration of the surfactant for desorbing soil-bound PCBs and to provide the inoculum with additional nutrients following soil inoculation [12]. After the surfactant was added, the £asks were shaken for 15 min to allow the sorbitan trioleate to dissolve into the medium. The soil columns were inoculated with 15 ml of the bacteria suspensions (108 colony-forming units (CFU) ml31 ), which were applied to the soil surface as a soil drench. The inoculation of the soil was repeated biweekly to maintain a cell density of 106 CFU g31 soil, alternating R. eutrophus H850 and Rhodococcus sp. ACS cultures at each inocula-

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tion. Non-inoculated columns that served as the control treatment were amended with 15 ml of the MS medium. When the amount of the amendment was not su⁄cient to replenish the water loss between amendment applications, additional deionized water was applied. Soil sampling was conducted 3 days after the 18th amendment (total 9 weeks treatment). The soils were sampled at three depths by removing the soil from the columns and dividing them into three fractions taken from 0^3 cm, 3^9 cm, and 9^20 cm below the soil surface. Soil collected from each depth was thoroughly mixed, and subsampled for use in bacterial community and chemical analyses. 2.4. Bacterial community analyses 2.4.1. Biphenyl utilizer enumeration The population densities of the bacteria that could utilize biphenyl as a carbon source were enumerated using agar plates. Enumeration was performed by plating 10fold serial dilutions of soil samples onto the surface of agar plates containing MS Bacto agar with biphenyl crystal vapor as the sole carbon source. The plates were incubated at room temperature and numbers of the colonies were counted after 2 weeks. 2.4.2. Soil DNA extraction At the end of the experiment, the total DNA of the actively growing bacteria was immunolabeled with bromodeoxyuridine (BrdU), which is a thymidine nucleotide analog that can be used for DNA synthesis by bacteria. To label the DNA, 4 g soil was amended with 40 Wl of 100 mM BrdU and allowed to incubate at room temperature for 3 days. The total DNA was then extracted from 1 g soil samples using a FastDNA SPIN Kit for soil (Bio 101, Vista, CA, USA). The amount of DNA was estimated visually after electrophoresis in a 1% agarose gel. The BrdU-labeled DNA was isolated from the soil DNA using the immunocapture method previously described by Borneman [15]. 2.4.3. PCR detection of bphA and bphC genes PCR primers for the bphA and bphC genes were selected for detection of the genes encoding for enzymes in the PCB-degradation pathway. Biphenyl dioxygenase is the ¢rst enzyme in this pathway and is the product of the bphA gene, whereas 2,3-dihydroxybiphenyl dioxygenase is encoded by the bphC gene. Detection of the bphC gene followed the method by Park et al. [16]. The primer sequences for the bphC gene were 5P-CTGCACTGCAACGAACGCCAC-3P (primer 1) and 5P-GACACCATGTGGTGGTTGGT-3P (primer 2). The PCR primers for the bphA gene were designed from a conserved region that has been identi¢ed in several well-studied PCB degraders, including Pseudomonas pseudoalcaligenes KF707, Comamonas testosteroni, Pseudomonas sp. KKS102, Pseudomonas sp. B4, Burkhoderia sp. LB400, Rhodococcus sp.

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M5, Rhodococcus globerulus P6, and Rhodococcus sp. RHA1. The sequences were retrieved from GenBank and aligned with the software program, PileUp (Genetic Computer Group, Madison, WI, USA). The primer sequences were A13-gc (5P-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGATGTTCGGCCAGCACATGACG-3P (GC clamp underlined)) and A1r (5PGTCAAGAGCGGCAGCAGGAC-3P), which targeted a 230-bp fragment on the N-terminal sequence of the bphA1 gene coding for the iron sulfur protein in biphenyl dioxygenase. The GC clamp was attached to primer A13 for the denaturing gradient gel electrophoresis analysis. Each PCR reaction contained 40 ng DNA, 5 pmol of each primer, a Ready-To-Go PCR bead (Pharmacia Biotech, Piscataway, NJ, USA), and sterilized distilled water to a ¢nal volume of 25 Wl. The PCR ampli¢cation was performed on a PTC-200 thermocycler (MJ Research, Inc., Watertown, MA, USA) with a touchdown program to increase the speci¢city of the target. The temperature pro¢le was 5 min at 95‡C followed by ampli¢cation for 30 cycles with a 0.5‡C decrease in the annealing temperature after each cycle. The starting cycle consisted of 1 min at 94‡C, 30 s at 65‡C, and 1 min at 72‡C. Twenty cycles of the same program were conducted with a ¢xed annealing temperature of 50‡C, and a ¢nal extension for 6 min at 72‡C. The identi¢cations of the correct PCR products from bphA primers were con¢rmed by DGGE analysis with denaturing gradients ranging from 20 to 60%. The samples with a PCR product at the same position as the inoculated bacteria were considered positive. Di¡erences in the amount of the PCR products between the samples were estimated visually and scored from 0 to 4. 2.5. Chemical analyses PCBs were extracted from 20-g soil samples that were taken at each soil depth and from the lower uncontaminated soil layer that was used to collect water leachate. The samples were placed in 40-ml vials and extracted by the addition of 10 ml hexane:acetone (9:1 v/v) and 5 ml of 3% sodium dodecyl sulfate solution. The vials were sealed with Te£on tape and shaken on a horizontal shaker for 24 h. The soils were then centrifuged (10 000Ug for 20 min), after which they were frozen at 320‡C to separate the water from the organic supernatant. The organic supernatant was removed and analyzed by gas chromatography using £ame ionization detection (GC-FID) (Hewlett-Packard Co., Palo Alto, CA, USA) as previously described [13]. 2.6. Statistical analyses Statistical analyses of the di¡erences in PCB concentration were carried out by analysis of variance (ANOVA) with repeated measure design using SAS 8.0 for Windows (SAS Institute, Inc., Cary, NC, USA). Least signi¢cance

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Table 1 Population sizes of biphenyl-degrading bacteria in treated soil (U107 CFU g31 )a Depths

Soil columns No earthworm BIO

0^3 cm 3^9 cm 9^20 cm

b

Earthworm MS

24.3aA 6.7bA 0.6cAC

c

4.0aB 1.3bA 0.2cA

BIO

MS

140aC 7.5bA 5.2bB

31aA 7.9bA 0.7cC

a

Comparisons within a soil column between depths are signi¢cantly different (LSD, P 6 0.05) if marked with di¡erent lowercase letters. Comparisons within a depth between soil columns are signi¢cantly di¡erent (LSD, P 6 0.05) if marked with di¡erent uppercase letters. b BIO: Bioaugmentation treatment. c MS: MS medium treatment.

di¡erence (LSD) tests were performed to determine signi¢cant di¡erences between the means (P = 0.05) of the treatments. The cell densities of the biphenyl-utilizing bacteria, the colony count data were log-transformed prior to statistical analysis.

3. Results 3.1. Biphenyl-utilizing bacterial population densities The cell densities of culturable, biphenyl-utilizing bacteria and their distribution in soil columns were enumerated using agar plate assays that do not di¡erentiate between bioaugmented bacteria and the indigenous biphenyl-degrading bacteria. Total cell densities of biphenyl-degrading bacteria were a¡ected by the presence of earthworms and bioaugmentation, but only in the top 3 cm of the soil. Bioaugmentation alone increased the number of biphenyl utilizers 5-fold in the 0^3 cm depth, such that biphenyl utilizers had an average population size of 4U107 CFU g31 in non-bioaugmented columns and 24U107 CFU g31 in the bioaugmented columns (Table 1). The addition of earthworms signi¢cantly increased the number of biphenyl utilizers in the top 3 cm by 6- and 8-fold in bioaugmented and non-bioaugmented columns, respectively. In soil below this zone at the 3^9 cm depth, the population size of biphenyl-degrading bacteria ranged from 1.3 to 7.9U107

Fig. 1. PCR products ampli¢ed from bph gene speci¢c primers on agarose gels. Template DNA used in the ampli¢cation was as follows : lane 1, Burkhoderia sp. LB400; lane 2, Rhodococcus globerulus P6; lane 3, Ralstonia eutrophus H850; lane 4, Rhodococcus sp. ACS; lane 5, nonsterile soil amended with Ralstonia eutrophus H850; lane 6, non-sterile unamended soil ; and lane 7, no template. Bands corresponding to bphA and bphC genes were 310 bp and 182 pb long, respectively. The lower bands correspond to the primers used in the ampli¢cation.

CFU g31 soil, and there were no signi¢cant di¡erences between treatments. At the 9^20 cm depth, the population densities decreased another order of magnitude in all of the treatments with the exception of the bioaugmented columns with earthworms, which maintained 5U107 biphenyl degraders per g of soil. The densities of biphenyl utilizers at the 9^20 cm depth in the non-bioaugmented soils and treatments without earthworms ranged from 0.2U107 to 0.7U107 CFU g31 soil. 3.2. Detection of the bphA and bphC genes in soil The upper pathway of aerobic PCB degradation involves four enzymes that are encoded by the bphABCD genes, which transform individual PCB congeners to their

Table 2 PCR detection of bphA and bphC gene in treated soila Treatment

Earthworm bphA gene bphC gene No earthworm bphA gene bphC gene a

Mineral salts medium

Bioaugmentation

0^3 cm

3^9 cm

9^20 cm

0^3 cm

3^9 cm

9^20 cm

3 +++

3 +++

3 ++++

+ +

+++ ++

++ +++

3 3

3 3

3 3

++ ++

+++ ++

3 +++

3, no ampli¢cation; + to ++++, ampli¢cation, with the strength of the ampli¢cation signal increasing from + to ++++.

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respective chlorobenzoic acids [17]. We selected the bphA and bphC genes to monitor the inoculated and indigenous PCB-degrading bacteria because these genes exhibit high homology among many known PCB degraders [18]. PCR fragments corresponding to the bphA and bphC genes were ampli¢ed from genomic DNA of known PCB-degrading bacteria (i.e. Burkhoderia sp. LB400, R. globerulus P6, R. eutrophus H850, and Rhodococcus sp. ACS) and from soil DNA that was inoculated with R. eutrophus H850 (Fig. 1). The bphA gene PCR products were further analyzed by DGGE. All DGGE bands from the PCB degraders had the same relative position at about 50% denaturant (data not shown). At the end of the experiment, the bphA gene was detected only in soil DNA samples from bioaugmented soil (Table 2). Within the bioaugmented treatments, distribution of the bphA gene at di¡erent soil depths was in£uenced by the presence of earthworms. The bphA gene product was produced by PCR ampli¢cation of DNA extracted from soil at all depths in the bioaugmented treatments with earthworms, and in the bioaugmented soil columns at 0^3 and 3^9 cm depths, but not at the 9^20 cm depth in bioaugmented columns without earthworms. The bphC gene product distribution did not correspond with the distribution of the bphA gene, and was detected at high levels throughout the soil pro¢le in non-bioaugmented soils with earthworms. Lesser quantities of the bphC gene were detected in the bioaugmented soils, irrespective of the presence of earthworms. As with the bphA gene, the bphC gene was not detected in control columns without earthworms.

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ment employing bioaugmentation with PCB-degrading bacteria and earthworms resulted in signi¢cant disappearance of PCBs at all depths (23%, 52%, and 61% recovery at 0^3 cm, 3^9 cm, and 9^20 cm, respectively). In contrast, PCB disappearance in the bioaugmented soil columns without earthworms was signi¢cantly less as compared to bioaugmented columns with earthworms. In the treatment employing bioaugmentation with PCB-degrading bacteria without earthworms, approximately 50% of the PCBs were recovered in the top 3-cm layer of soils, which increased to 79 and 71% PCB recovery in the 3^9 and 9^20 cm depths, respectively. These values at the soil depths below 3 cm were not signi¢cantly di¡erent from those obtained in the non-bioaugmented control treatment without earthworms. Earthworms alone without bioaugmentation promoted PCB disappearance in the upper 3 cm, although not to the same extent as the combined treatment using both bioaugmentation and earthworms (Table 3). PCB recovery in non-bioaugmented columns with earthworms was 53% at the 0^3 cm depth, but increased to 76 and 72% at the 3^9, and 9^20 cm depths, respectively. In the control treatment without bioaugmentation or earthworms, the di¡erences in PCB recovery between depths were not signi¢cant. PCB recoveries in these columns ranged from 78 to 80%. A consistently small amount of PCBs were recovered from the pristine, leachate collection soil, and averaged 12.5 ppm across all treatments. Calculations of the mass of PCBs that leached from the upper soil pro¢le into the uncontaminated soil showed that there was an approximate 3% loss of the PCBs from the soil columns due to leaching.

3.3. PCB recovery Di¡erences in PCB recovery were used to determine PCB losses due to biodegradation and any other loss mechanisms that may be associated with earthworms and bioaugmentation (Table 3). After 9 weeks, the treat-

Table 3 Percent PCB recovery from soil after bioaugmentation in the presence or absence of earthwormsa Depths

Soil columns No earthworm BIO

0^3 cm 3^9 cm 9^20 cm Leachate soild (ppm PCB) a

b

51aA 79bA 71abA 14A

Earthworm

c

BIO

MS

78aB 82aA 80aA 12A

23aC 52bB 61cA 12A

53aA 76bA 72bA 12A

MS

Comparisons within the soil columns between depths are signi¢cantly di¡erent (LSD, P 6 0.05) if marked with di¡erent lowercase letters. Comparisons within a depth between soil columns are signi¢cantly different (LSD, P 6 0.05) if marked with di¡erent uppercase letters. b BIO: Bioaugmentation treatment. c MS: MS medium treatment. d PCB extracted from uncontaminated soil layer at 20^25 cm depth.

4. Discussion This research examined the interactions of earthworms with indigenous and bioaugmented PCB-degrading bacteria. Through the inevitable modi¢cation of the soil environment via burrowing and comminution, earthworms appeared to increase inoculum dispersal and the population size of indigenous PCB-degrading microorganisms, which may have contributed to the observed increase in PCB biodegradation. Earthworm-amended soils that were bioaugmented with PCB-degrading bacteria contained higher levels of biphenyl-degrading bacteria in the top 3 cm of the soil, and contained increased levels of the bphA and bphC genes that could be detected in the top 9 cm of the soil using PCR-based methods. The combined treatment with earthworms and bioaugmentation was by far the best treatment and resulted in signi¢cantly enhanced disappearance of PCBs, with approximately 50% of the PCBs being removed in the top 9 cm of the soil columns after 9 weeks of treatment. In comparison, bioaugmentation with PCBdegrading bacteria in the absence of earthworms, or the addition of earthworms without bacterial bioaugmentation

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was e¡ective for increasing the removal of PCBs only in the top 3 cm of the soil columns. Aerobic mineralization of PCBs requires the activity of two di¡erent populations of bacterial species, the ¢rst consisting of bacterial species that contain the bph genes encoding the upper pathway for degradation of chlorinated biphenyls to chlorobenzoates. Microorganisms containing the lower pathway genes may then degrade the resulting chlorobenzoates, which can be used as carbon substrates for growth. Alternatively, the chlorobenzoates may be fortuitously metabolized during growth-linked metabolism on other carbon substrates [1,19]. Although most soils contain high population densities of bacteria that carry genes for the upper pathway encoding the enzymes for biphenyl degradation, these bacteria are not usually induced to degrade PCBs unless they are grown on a cosubstrate that induces the expression of the bph genes. In addition to biphenyl, several naturally occurring substrates including monoterpenes and salicylic acid have been identi¢ed as potential cometabolites, putting in question whether these are the ‘natural’ substrates for PCB-degrading enzymes. The ¢nding that earthworms alone also promote PCB removal is intriguing since no cometabolites were added in the control treatment. This suggests that earthworm casts (i.e. egested soil from the earthworm) may contain certain compounds that serve as cometabolites for induction of the bph genes. Addition of earthworms alone to the soil columns was associated with an 8-fold increase in the population size of culturable, indigenous biphenyl-degrading bacteria. Compounds originating from earthworm activity, such as urea, have been speculated as potential stimulants of PCB degradation [13], however, they are unlikely to be related to bph induction. Biphenyl-degrading bacteria vary greatly in their ability to degrade di¡erent PCB congeners. Previously, increasing the population size of biphenyl-utilizing bacteria, either through bioaugmentation with selected PCB-degrading bacteria, or by biostimulation with cosubstrates that induce the bph genes has been shown to enhance PCB degradation [11,12,20^24]. In this research, earthworm activity was associated with an increase in the level of the bphC gene throughout the entire 20-cm soil pro¢le of columns without bacterial bioaugmentation. Curiously, this was not accompanied by a concomitant increase in the level of detection in the bphA gene, which suggests that the earthworms a¡ected bacteria other than those carrying the bph operon that normally contains both genes. The bphA gene is involved in dioxygenation of the biphenyl ring at the 2,3 position with formation of dihydrodiol, whereas the bphC gene codes for a dioxygenase that is involved in ring ¢ssion of phenylcatechol [25]. The independent detection of bphC, but not the bphA gene suggests that indigenous bacteria may exist that have an alternate gene coding for the ¢rst step. Alternatively, they may carry a gene similar to bphC that is contained in bacteria that are enriched by earthworm activity, but which may or

may not be involved in PCB degradation. For example, the increased detection of bph genes may re£ect the presence of homologous dioxygenase genes that are responsible for degradation of naphthalene, toluene, and benzoate [17]. Analyses of the ways in which earthworms a¡ect microbial consortia that are involved in PCB degradation, and the distribution of di¡erent genes encoding for PCBdegrading enzymes will require better knowledge of the diversity of these enzymes and the development of appropriate probes for monitoring their quantity and expression during bioremediation of contaminated soils.

Acknowledgements We thank James Borneman for providing valuable advice in nucleic acid extraction, Sam Alvey for his assistance on data analysis, and Katechan Jampachaisri for help with the statistical analysis. Funding for Ekawan Luepromchai was provided by Chulalongkorn University, Thailand.

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FEMSEC 1379 7-8-02