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Keywords:

  • motility;
  • chemotaxis;
  • polychlorobiphenyls;
  • bioremediation;
  • environmental pollution

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The polychlorinated biphenyl (PCB)-degrading Pseudomonas sp. B4 was tested for its motility and ability to sense and respond to biphenyl, its chloroderivatives and chlorobenzoates in chemotaxis assays. Pseudomonas sp. B4 was attracted to biphenyl, PCBs and benzoate in swarm plate and capillary assays. Chemotaxis towards these compounds correlated with their use as carbon and energy sources. No chemotactic effect was observed in the presence of 2- and 3-chlorobenzoates. Furthermore, a toxic effect was observed when the microorganism was exposed to 3-chlorobenzoate. A nonmotile Pseudomonas sp. B4 transformant and Burkholderia xenovorans LB400, the laboratory model strain for PCB degradation, were both capable of growing in biphenyl as the sole carbon source, but showed a clear disadvantage to access the pollutants to be degraded, compared with the highly motile Pseudomonas sp. B4, stressing the importance of motility and chemotaxis in this environmental biodegradation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bioremediation of soil contaminated with polychlorinated biphenyls (PCBs) is an attractive clean-up strategy due to its potential to mineralize pollutants and to be inexpensive (Ohtsubo et al., 2004). Many genetic, enzymological, and biochemical analyses of PCB-degradative pathways have provided the basis for the engineering of specific enzymes and have been used to modify genetically microorganisms to improve their performance in bioremediation of PCBs (Tiedje et al., 1993; Timmis et al., 1994; Pieper & Reineke, 2000). Burkholderia xenovorans LB400 (formerly known as Burkholderia fungorum LB400) is one of the most-studied and effective aerobic PCB degraders known (Denef et al., 2004; Goris et al., 2004) and many other biphenyl-utilizing microorganisms with capacity to degrade PCBs to different extents have been isolated (Bopp, 1986; Bartels et al., 1999). However, little is known about the physiological and ecological adjustments that help PCB-degrading bacteria to degrade contaminated soil-sorbed chemicals (Chávez et al., 2006).

Most motile bacteria can sense and respond to low concentrations of organic compounds in their environment by the process of chemotaxis. In the last few years, there have been several reports on bacterial species responding chemotactically to different environmental pollutants, many of which are considered to be serious ecological problems (Pandey & Jain, 2002; Parales & Harwood, 2002). This phenomenon may be widespread among other chemicals and have important significance for the potential of these microorganisms as biodegraders. There is also evidence that chemotaxis can not only enhance biodegradation but also promotes the formation of microbial consortia (Law & Aitken, 2003; Wu et al., 2003), presumably by rapidly bringing cells into close contact with degradable substrates. Other physiological properties such as high-affinity uptake systems, adhesion to solid surfaces, biosurfactant production and biofilm formation have been suggested to reduce the distances between cells and solid chemicals and thus enhance substrate bioavailability (Wick et al., 2002; Wu et al., 2003; Ohtsubo et al., 2004).

The objective of this study was to determine whether biphenyl (B), chlorobiphenyls (CBs), PCB congeners and some of the intermediates of these biodegradations such as benzoate and its chloroderivatives (CBAs), which are excreted by some PCB-degrading bacteria (Potrawfke et al., 1998), are recognized as chemotactic effectors by Pseudomonas sp. B4. The results reported here indicate that biphenyl, CBs, PCBs and benzoate are attractants for Pseudomonas sp. B4. However, a nonmotile Pseudomonas sp. B4 transformant was not able to access the same compounds in capillary assays. To our knowledge, this is the first report of biphenyl-utilizing bacterial chemotaxis toward CBs and PCBs. Our findings are of importance as bacterial strains with the ability to sense the presence of PCBs or their metabolic intermediates may have an increased growth and survival advantage that could contribute to the bioremediation of PCB-contaminated environments.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Chemicals

Biphenyl was purchased from Merck (Hohenbrunn, Germany), benzoic acid and chlorobenzoates were obtained from Sigma (St Louis, MO) and chlorinated biphenyls and PCBs from Accustandard Inc. (New Haven, CT).

Microorganisms and growth conditions

Pseudomonas sp. B4 was isolated from the Elbe River (Germany), contaminated with PCBs. It was initially characterized as a biphenyl-utilizing bacterium belonging to the Gamma Phylogenetic cluster and to the Pseudomonas Taxonomic cluster, being closest to Pseudomonas putida by a 97.7% 16S rRNA gene sequence similarity (Bartels et al., 1999). Burkholderia xenovorans LB400, Pseudomonas sp. B4 and its nonmotile polyphosphate-deficient derivative (Chávez et al., 2006) were grown aerobically at 30°C on Luria–Bertani (LB) rich medium and M9 minimal salts medium (Sambrook et al., 1989; Chávez et al., 2004) supplemented with 0.1% biphenyl, 1% glucose or 0.1% benzoate as the sole carbon sources. The strains were also grown using chlorinated biphenyls and PCBs (at a final 1 mm concentration). Biphenyl and its chloroderivatives are poorly soluble in water and therefore the indicated concentrations in the figures are only nominal, most of the compounds being most likely saturated at concentrations higher than their solubility limits. When these insoluble carbon sources were used, they were dissolved in iso-octane (0.2% final concentration in each test and control assay). The microorganisms did not grow in the presence of iso-octane alone, which was nontoxic and showed no chemotactic properties compared with the compounds tested in the swarming or capillary assays (see below).

Swarming assays

Qualitative measurement of motility was done essentially as described by Harwood et al. (1994) using plates consisting of M9 minimal medium containing 0.3% Noble agar (Difco) and the chemoattractant carbon source at a final concentration of 1 mM. To test for toxic effects of some compounds on the bacteria, we used the same swarming plates, combined with a chemical-in-plug system as initially described by Tso & Adler (1974). Plugs of 2% agar that contained the compound to be tested as a toxic (100 mm) were incubated at 30°C until the swarming rings reached the plugs containing the test compounds (72 h). The inhibitory effect was seen as a clear area around the plug.

Modified capillary chemotaxis assay

The capillary assay with the modifications described previously (Mazumder et al., 1999) was done using a disposable 200-μL pipette tip as a chamber for holding 100 μL of bacterial suspension (usually 1 × 107 cells−1) in chemotaxis buffer (10 mm Tris-HCl, pH 7.4). A 2-cm 25-gauge needle (Becton Dickinson) was used as the chemotaxis capillary and was attached to a 1-mL tuberculin syringe (Becton Dickinson) containing a 200-μL portion of the compound to be tested in chemotaxis buffer in the presence of 0.2% iso-octane or a control containing only the chemotaxis buffer and 0.2% iso-octane. After 90 min incubation at room temperature the needle syringe was removed from the bacterial suspension and the content diluted and plated in LB medium. Accumulation of bacteria in the capillaries was calculated as the average from the CFUs obtained in duplicate plates and the results were expressed as the mean of at least three separate capillary assays for each determination. The relative chemotaxis index (RCI) was calculated as the ratio of the bacteria that entered the test capillary to that in the control capillary. An RCI of 2 or greater has been described as significant with this method (Mazumder et al., 1999).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Motility assays in swarm plates

The spreading behaviour of the colonies of Pseudomonas sp. B4 in solid agar strongly suggested it was highly motile compared with B. xenovorans LB400. Although B. xenovorans LB400 was originally described as a motile microorganism (Goris et al., 2004), this microorganism was nonmotile as observed by phase contrast light microscopy (not shown). The same behaviour has been reported previously by other researchers (Nielsen et al., 2000). By using the qualitative motility assay in swarming plates, Pseudomonas sp. B4 showed a clear motility for casaminoacids compared with the nonmotile Pseudomonas sp. B4 (Chávez et al., 2006) or B. xenovorans LB400 (not shown).

It has recently been described that chemotaxis of P. putida G7 towards naphthalene dissolved in a nonaqueous-phase liquid substantially increases the rates of mass transfer and degradation of these hydrophobic pollutants (Law & Aitken, 2003). To study the importance of motility in accessing some of the pollutants that Pseudomonas sp. B4 can transform or use as carbon sources, we started checking the motility of these microorganisms in soft agar swarm plates of M9 medium supplemented with different CBs. A clear swarm response was seen (Fig. 1) in plates containing biphenyl (B), 2-CB, 3-CB or 4-CB. These results were in agreement with the capacity of these cells to grow on these compounds as the sole carbon sources.

image

Figure 1.  Chemotactic swarming responses of Pseudomonas sp. B4 to different chlorobiphenyls. Cells grown in minimal medium were inoculated in plates containing the same medium supplemented with biphenyl (B) and different CBs (2-CB, 3-CB and 4-CB) as the sole carbon sources by stabbing them at a point corresponding to the centre of the swarm ring and incubated for 72 h at 30°C. The negative print of the photographs is shown for better contrast of the swarming rings.

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Quantitative chemotaxis responses of Pseudomonas sp. B4 to different compounds

The capillary assays showed that Pseudomonas sp. B4 cells accumulated in capillaries containing increasing biphenyl concentrations with a maximum number of cells apparently obtained at 1 mm biphenyl nominal concentration inside the capillary, as higher concentrations gave results with low reproducibility (Fig. 2a). As Pseudomonas sp. B4 is able to grow in biphenyl and several CBs (Chávez et al., 2004), it may be possible that previous growth of the microorganism in biphenyl would induce a chemotactic response towards these compounds. Chemotaxis of Pseudomonas sp. B4 toward biphenyl or CBs (Figs 2b and 3) was not induced by previous growth of the cells in these substrates compared with that seen by growing previously the cells in glucose and therefore can not be considered an ‘inducible type’ (Parales & Harwood, 2002). On the other hand, chemotaxis of Pseudomonas sp. strain NCIB 9816–4 and P. putida G7 towards naphthalene is known to be induced by previous growth in the chemoeffector (Grimm & Harwood, 1997).

image

Figure 2.  (a) Chemotactic response of Pseudomonas sp. B4 towards increasing biphenyl concentration. Cells grown to the mid-exponential phase in 1% glucose were collected by centrifugation and resuspended in chemotaxis buffer. After centrifugation, the chemotactic response of Pseudomonas sp. B4 towards biphenyl was examined. (b) Effect of growth medium in chemotaxis of Pseudomonas sp. B4 toward different compounds. Cells were grown to the mid-exponential phase in minimal medium containing 1 mm biphenyl (white bars), 1% glucose (light grey bars) as the sole carbon source or in LB medium (dark grey bars). The capillary assay was done as in (a). The bacterial cells attracted by the indicated compounds were determined after duplicate plating in LB medium. Error bars indicate the SDs based on three different replicated experimental values. Numbers on top of each bar indicate the relative chemotactic indexes.

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image

Figure 3.  Chemotaxis of Pseudomonas sp. B4 towards several organochlorine compounds. Cells were grown to the mid-exponential phase in minimal medium containing 1 mm biphenyl (white bars) or 1% glucose (grey bars) as the sole carbon sources. For the capillary assay, cells were collected by centrifugation and resuspended in chemotaxis buffer. The bacterial cells attracted by biphenyl, 2-CB, 3-CB, 4-CB and 2, 3-CB were determined after plating the cells in LB medium. Error bars indicate the SDs for three replicated experimental values. Numbers on top of each bar indicate the relative chemotactic indexes.

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Cells were attracted to glucose and biphenyl when previously grown in defined media. Conversely, cells previously grown in rich medium (LB) did not show significant chemotactic responses (Fig. 2b). This may be due to the presence of an excess of reserve nutrients that prevent the chemical attraction by the tested carbon sources.

Capillary assays showed that Pseudomonas sp. B4 cells accumulated in capillaries containing 1 mm of biphenyl, 2-CB, 3-CB, 4-CB and 2,3-CB with RCI values higher than 2 in most cases (Fig. 3), confirming in general the results obtained by swarming towards these compounds (Fig. 1). In the case of 4-CB and 2,3-CB, the RCI values were not always higher than 2, probably due to their high toxicity and lower solubility in the chemotaxis buffer.

During biodegradation of PCBs, different chlorobenzoates are generated as intermediates (Potrawfke et al., 1998). Some of these compounds are toxic to the biodegrading bacteria (Camara et al., 2004; Parnell et al., 2006) and therefore it was considered of importance to determine whether Pseudomonas sp. B4 was chemotactically stimulated by these derivatives. Figure 4a shows the chemotactic response to benzoate, reaching a maximum number of cells inside the capillary when the concentration was 0.1 mm, a value similar to that reported for other microorganisms (Harwood et al., 1990). Cells previously grown in glucose (grey bars, Fig. 4b) showed a chemotactic effect to benzoate but not to its chloroderivatives. On the other hand, cells previously grown on biphenyl (Fig. 4b, white bars) or benzoate (Fig. 4b, hatched bars) were chemotactic towards benzoate but not its chloroderivatives. It is clear that previous growth of the cells on biphenyl or benzoate stimulated the chemotactic response of Pseudomonas sp. B4 towards benzoate, suggesting an inducible type of response. Previous growth on benzoate showed a stimulation of chemotaxis of the cells to 4-CBA. This effect was not seen in the case of 3-CBA, most likely due to a toxic effect of 3-CBA on the cells (see below). Pseudomonas sp. B4 used as growth substrates biphenyl, 2-CB, 3-CB, 4-CB and 2,3-CB but it did not grow on 2-CBA, 3-CBA and 4-CBA. 4-CBA was an attractant in spite of the lack of growth of Pseudomonas sp. B4 on it, suggesting that the response to this compound was not an energy taxis response.

image

Figure 4.  Chemotactic response of Pseudomonas sp. B4 towards benzoate and chlorobenzoates. (a) Cells were grown to the mid-exponential phase in minimal medium containing 1% glucose as the sole carbon source. After centrifugation, the chemotactic response of Pseudomonas sp. B4 towards increasing benzoate concentration was examined. (b) Effect of growth medium in chemotaxis of Pseudomonas sp. B4 toward different CBAs. Cells were grown to the mid-exponential phase in minimal medium containing 1% glucose (grey bars), 0.1% biphenyl (white bars) or 0.1% benzoate (hatched bars) as the sole carbon sources. The capillary assay was done as in (a). The bacterial cells attracted by the indicated compounds were determined after duplicate plating in LB medium. Error bars indicate the SDs based on three different replicated experimental values. Numbers on top of each bar indicate the relative chemotactic indexes.

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Figure 5a shows that Pseudomonas sp. B4 has a chemotactic swarming response towards benzoate, being capable of metabolizing the attractant compound. No swarming assay was done with 3-chlorobenzoate as Pseudomonas sp. B4 did not grow on it and this assay requires that cells grow on the attractant to obtain a response. We explored the toxic effect of these chlorinated benzoates and found only 3-CBA to be toxic at a concentration of 0.1 mm (Fig. 5b and c). Nevertheless, we cannot exclude the possibility that 3-CBA may also be a repellent for Pseudomonas sp. B4. On the other hand, 2-CBA and 4-CBA did not interfere with the swarming in a benzoate-supplemented plate.

image

Figure 5.  Chemotactic swarming responses and toxic effect of 3-chlorobenzoate in Pseudomonas sp. B4. Cells grown in minimal medium were inoculated in plates containing the same medium supplemented with 1 mm benzoate (a) as the sole carbon source by stabbing them at a point corresponding to the center of the swarm ring and were incubated for 72 h at 30°C. (b) Toxic effect of different CBAs in a swarming assay combined with a chemical-in-plug method. Cells grown in minimal medium were inoculated in plates containing the same medium supplemented with 1 mm benzoate as the sole carbon source as in (a), except that agarose plugs containing 100 mm of each of the indicated compounds were present on the plate at the beginning of the incubation. (c) Effect of different 3-CBA concentrations on cell viability of Pseudomonas sp. B4. Exponential phase suspensions of Pseudomonas sp. B4 previously grown in 1% glucose were exposed to different concentrations of 3-CBA and samples were taken at 0 (white bars) and 90 min (hatched bars) to determine the number of CFU.

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Chemotaxis toward benzoate and chlorobenzoates has been studied before in P. putida PRS2000 (Harwood et al., 1990). Pseudomonas sp. B4 behaved similarly to P. putida PRS2000 when confronted with benzoate (attractant in both microorganisms) or to 2-CBA (nonattractant in both cases). On the other hand, both Pseudomonas strains responded differently towards 3-CBA. The capacity of a compound to elicit a different chemotactic response is sometimes related to its nutritional properties. The same compound can act as an attractant for a microorganism capable of utilizing it as a growth substrate or as a repellent for a bacterium for which the compound is toxic. 3-CBA was very toxic for Pseudomonas sp. B4 and it could not grow on it (Fig. 5). In contrast, P. putida PRS2000 can grow on 3-CBA and it is an attractant for this bacterium (Harwood et al., 1990).

Synchronized regulation of bacterial chemotaxis to many xenobiotic compounds, with their respective degradation and/or transformation, indicates that this phenomenon might be an integral feature for degradation (Pandey & Jain, 2002; Chávez et al., 2006). It is clear that the efficiency of bioremediation of a contaminated field with organochlorine compounds will depend not only on the metabolic capacities and microbial chemotactic behaviour but also on the toxicity of the intermediates formed during the process and the commensal relationships with other members of the microbial consortium that is able to degrade these intermediates. For example, in a model consortium consisting of two organisms, Burkholderia sp. LB400 (nonmotile) and Pseudomonas sp. B13(FR1) (motile), the organisms apparently interact metabolically because Pseudomonas sp. B13(FR1) can reach and metabolize the CBA produced by Burkholderia sp. LB400 when grown on chlorobiphenyl (Nielsen et al., 2000).

We can conclude that PCB-degrading microorganisms to be used for bioremediation of soils contaminated with these compounds should have the capacity to detect and move through the soil particles towards a gradient of the PCB to be metabolized, having this way a more efficient microbe– substrate interaction. Conversely, nonmotile strains of PCB-degraders, such as the model strain B. xenovorans LB400, would be at a clear disadvantage to access these soil-sorbed organic pollutants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We are very grateful to J. Tiedje for the supply of B. xenovorans LB400 and B. Hofer and K.N. Timmis for the Pseudomonas sp. B4 strain. This research was supported by ICM project P-05-001-F. FG and FPCh were the recipients of CONICYT and DAAD Ph.D. scholarships, respectively.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  • Bartels F, Backhaus S, Moore ERB, Timmis KN & Hofer B (1999) Occurrence and expression of glutathione-S-transferase encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl utilizing bacteria. Microbiology 145: 28212834.
  • Bopp LH (1986) Degradation of highly chlorinated PCBs by Pseudomonas strain LB400. J Ind Microbiol 1: 2329.
  • Camara B, Herrera C, Gonzalez M, Couve E, Hofer B & Seeger M (2004) From PCBs to highly toxic metabolites by the biphenyl pathway. Environ Microbiol 6: 842850.
  • Chávez FP, Lünsdorf H & Jerez CA (2004) Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate. Appl Environ Microbiol 70: 30643072.
  • Chávez FP, Gordillo F & Jerez CA (2006) Adaptive responses and cellular behaviour of biphenyl-degrading bacteria toward polychlorinated biphenyls. Biotechnol Adv 24: 309320.
  • Denef VJ, Park J, Tsoi TV, Rouillard JM, Zhang H, Wibbenmeyer JA, Verstraete W, Gulari E, Hashsham SA & Tiedje JM (2004) Biphenyl and benzoate metabolism in a genomic context: outlining genome-wide metabolic networks in Burkholderia xenovorans LB400. Appl Environ Microbiol 70: 49614970.
  • Goris J, De Vos P, Caballero-Mellado J, Park J, Falsen E, Quensen III JF, Tiedje J & Bañadme P (2004) Classification of the PCB- and biphenyl degrading strain LB400 and relatives as Burkholderia xenovorans sp. nov. Int J Syst Evol Microbiol 54: 16771681.
  • Grimm AC & Harwood CS (1997) Chemotaxis of Pseudomonas sp. to the polycyclic aromatic hydrocarbon, naphthalene. Appl Environ Microbiol 63: 41114115.
  • Harwood CS, Parales RE & Dispensa M (1990) Chemotaxis of Pseudomonas putida toward chlorinated benzoates. Appl Environ Microbiol 56: 15011503.
  • Harwood CS, Nichols NN, Kim MK, Ditty JL & Parales RE (1994) Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate. J Bacteriol 176: 64796488.
  • Law AMJ & Aitken MD (2003) Bacterial chemotaxis to naphthalene desorbing from nonaqueous liquid. Appl Environ Microbiol 69: 59685973.
  • Mazumder R, Phelps TJ, Krieg NR & Benoit RE (1999) Determining chemotactic responses by two subsurface microaerophiles using a simplified capillary assay method. J Microbiol Meth 37: 255263.
  • Nielsen AT, Tolker-Nielsen T, Barken KB & Molin S (2000) Role of commensal relationships on the spatial structure of a surface-attached microbial consortium. Environ Microbiol 2: 5968.
  • Ohtsubo Y, Kudo T, Tsuda M & Nagata Y (2004) Strategies for bioremediation of polychlorinated biphenyls. Appl Microbiol Biotechnol 65: 250258.
  • Pandey G & Jain RK (2002) Bacterial chemotaxis toward environmental pollutants: role in bioremediation. Appl Environ Microbiol 68: 57895795.
  • Parales RE & Harwood CS (2002) Bacterial chemotaxis to pollutants and plant-derived aromatic molecules. Curr Opin Microbiol 5: 266273.
  • Parnell JJ, Park J, Denef V, Tsoi T, Hashsham S, Quensen III J & Tiedje JM (2006) Coping with PCB toxicity: the physiological and genome-wide response of Burkholderiaxenovorans LB400 to PCB (polychlorinated biphenyl)-mediated stress. Appl Environ Microbiol 72: 66076614.
  • Pieper DH & Reineke W (2000) Engineering bacteria for bioremediation. Curr Opin Biotechnol 11: 262270.
  • Potrawfke T, Löhnert TH, Timmis K & Wittich RM (1998) Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes. Appl Microbiol Biotechnol 50: 440446.
  • Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  • Tiedje JM, Quensen JF, Chee-Sanford J, Schimel JP & Boyd SA (1993) Microbial reductive dechlorination of PCBs. Biodegradation 4: 231240.
  • Timmis KN, Steffan RJ & Unterman R (1994) Designing microorganisms for the treatment of toxic wastes. Annu Rev Microbiol 48: 525557.
  • Tso WW & Adler J (1974) Negative chemotaxis in Escherichia coli. J Bacteriol 118: 560576.
  • Wick LY, Ruiz de Munain A, Springael D & Harms H (2002) Responses of Mycobacterium sp. LB501 T to the low bioavailability of solid anthracene. Appl Microbiol Biotechnol 58: 378385.
  • Wu G, Feng Y & Boyd SA (2003) Characterization of bacteria capable of degrading soil-sorbed biphenyl. Environ Cont Toxicol 71: 768775.