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Keywords:

  • Pseudomonas pseudoalcaligenes KF707;
  • chemotaxis;
  • polychlorinated biphenyls;
  • PCBs;
  • chlorobenzoic acids;
  • biofilm;
  • metals

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Pseudomonas pseudoalcaligenes KF707 is a polychlorinated biphenyls (PCBs) degrader, also tolerant to several toxic metals and metalloids. The work presented here examines for the first time the chemotactic response of P. pseudoalcaligenes KF707 to biphenyl and intermediates of the PCB biodegradation pathway in the presence and absence of metals. Chemotaxis analyses showed that biphenyl, benzoic acid and chlorobenzoic acids acted as chemoattractants for KF707 cells and that metal cations such as Ni2+ and Cu2+ strongly affected the chemotactic response. Toxicity profiles of various metals on KF707 cells grown on succinate or biphenyl as planktonic and biofilm were determined both in the presence and in the absence of PCBs. Notably, KF707 cells from both biofilms and planktonic cultures were tolerant to high amounts (up to 0.5 g L−1) of Aroclor 1242, a commercial mixture of PCBs. Together, the data show that KF707 cells are chemotactic and can form a biofilm in the presence of Aroclor 1242 and specific metals. These findings provide new perspectives on the effectiveness of using PCB-degrading bacterial strains in bioremediation strategies of metal-co-contaminated sites.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Numerous organic xenobiotics demonstrate susceptibility to microbial degradation, and in many cases, the catabolic pathways are characterized in different organisms (Ellis et al., 1999, 2006). Therefore, although challenges exist, organic polluted sites can be effectively restored through bioremediation strategies. On the other hand, the removal of metal ion pollutants is a particularly difficult task because metals cannot be degraded; only their chemical speciation can be changed (Hietala & Roane, 2009). Hence, bacteria-based organic pollutant bioremediation approaches require that microorganisms are active in the presence of the target contaminant, as well as other co-contaminants such as toxic metals (Sandrin & Hoffman, 2007; Hietala & Roane, 2009). In this respect, toxic metals are known to influence biological remediation processes under both aerobic and anaerobic conditions, posing serious limits to bioremediation efficacy (Sandrin & Maier, 2003; Sandrin & Hoffman, 2007). Unfortunately, the presence of both toxic metals and organic xenobiotics as co-contaminants is a widespread environmental problem that affects 40% of the hazardous waste sites worldwide (http://www.epa.gov/superfund; http://www.atsdr.cdc.gov/cercla; Sandrin & Maier, 2003). Organic pollutants, encompassing aromatic hydrocarbons and their chlorinated derivatives, can be found together with such metals as arsenic, cadmium, chromate, copper, lead, mercury, nickel and zinc. Thus, obtaining a better understanding of metal toxicity to strains with organic pollutant degradation capacities is compelling.

Bioremediation of soil contaminated with polychlorinated biphenyls (PCBs) is an attractive clean-up strategy due to the potential to mineralize the pollutants and its low cost when compared with alternative strategies (Ohtsubo et al., 2004). Burkholderia xenovorans LB400 (formerly known as Burkholderia cepacia or Burkholderia fungorum LB400) and Pseudomonas pseudoalcaligenes KF707 have been the most extensively studied species with respect to the degradation of PCBs (Fedi et al., 2001; Camara et al., 2004; Chávez et al., 2006). These two microorganisms show distinct differences in the ranges of PCB congeners that they can degrade. The number of congeners oxidized by LB400 enzymes is far wider that that oxidized by KF707, and LB400 enzymes also exhibit higher activity for di-para-substituted PCBs (Bopp, 1986; Gibson et al., 1993). Further studies with LB400 suggest that carbon source and growth state affect degradation ability (Parnell et al., 2010).

To date, the influence of co-contaminating metals on organic degradation has not yet been evaluated for the above PCB degraders. However, several studies of biodegradation of organic pollutants by a number of organisms with various metals are now available (Sandrin & Maier, 2003). There are a few specific studies that allude to different growth conditions that influence the bioremediation outcome. One such example is investigations into naphthalene biodegradation in the presence of several metals where media and growth state contributes to the degree of degradation inhibition by the metal (Hoffman et al., 2005). Understanding the correlation of the biogeochemistry and physiology of a microbial system is a challenge yet an integral part of developing successful bioremediation strategies.

As chemotaxis is a selective advantage to the bacteria in guiding them to locate food sources, the full knowledge of the chemotactic features of motile degraders towards pollutants is an a priori necessity to predict the success of any microbial remediation route (Chávez et al., 2006). In particular, the high toxicity of the many PCB congeners and their very low solubility and subsequent poor bioavailability are significant factors that might influence bacterial chemotaxis (Camara et al., 2004; Parnell et al., 2006; Gordillo et al., 2007). Biphenyl-utilizing bacteria are able to metabolize different PCBs into chlorobenzoic acids (CBAs) using biphenyl-catabolic enzymes via an oxidative route (Furukawa & Miyazaki, 1986). It has been reported that several motile biphenyl-degrading bacteria, Pseudomonas putida P106, Rhodococcus erythropolis NY05 and Pseudomonas sp. B4, show positive chemotactic responses towards biphenyl (Wu et al., 2003; Gordillo et al., 2007). Notably, intermediate metabolites of PCB degradation pathways such as 3- and 4-chlorobenzoates (CBAs) are attractants for P. putida PR2000, which was previously grown on benzoate or 4-hydroxybenzoate (Harwood, 1989; Harwood et al., 1990). Other studies demonstrated that a nonmotile mutant of Pseudomonas sp. B4 showed a clear disadvantage for growth on biphenyl when compared with motile wild type (Gordillo et al., 2007). Together, these data underline the importance of both motility and chemotaxis in the microbial degradation of PCBs.

Pseudomonas pseudoalcaligenes KF707 is a robust soil PCB degrader that is able to grow in the presence of various metals and metalloids (Tremaroli et al., 2009; V. Tremaroli, unpublished data). It has also been shown that KF707 planktonic cells and biofilms are almost equally susceptible to several metal(loid)s (Se<As<Ni<Cd<Al<Te). Yet the strategy to counteract metalloid toxicity in biofilms is different from planktonic cells (Tremaroli et al., 2008; Harrison et al., 2009), and may be dependent on the growth media conditions (Tremaroli et al., 2009; V. Tremaroli, unpublished data). Information regarding the PCB-degrading abilities of P. pseudoalcaligenes KF707 is limited to studies in planktonic cultures (Furukawa & Miyazaki, 1986; Fedi et al., 2001), although it is recognized that biofilms provide distinct physiological traits that enhance the microbial capability of adaptation and survival to environmental stresses (Chávez et al., 2006; Costerton, 2007; Harrison et al., 2007; Kubota et al., 2008). KF707 properties such as chemotaxis, surface adherence and biofilm formation have recently been addressed under different nutritional and growth conditions (V. Tremaroli, unpublished data). However, one aspect that has not been evaluated is the capacity of a biodegrader microorganism to move and attach to a solid surface and start growing as a biofilm not only in the presence of the organic pollutant, or degradation intermediates, but also in the presence of toxic metals. Indeed, although biofilms are widely considered as the bacterial default mode of growth in natural environments, still little is known about biofilm metal tolerance (Harrison et al., 2007, 2009; Workentine et al., 2008) and even less is known about the way biofilms are formed and survive in the presence of various types of contaminants. Here we investigate these issues using the PCB degrader strain KF707. We demonstrate that KF707 is endowed with the ability to form biofilms using biphenyl as carbon source even in the presence of a PCB congener mixture and/or various metal(loid)s. Notably, we observe that biphenyl acts as a chemoattractant for KF707 cells, and that some metal ions, such as Ni2+ and Cu2+, interfere with KF707 chemotactic response, thus providing a contributing mechanism of the inhibition of bacterial strains to remediate at polluted sites co-contaminated with metal pollutants.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Chemicals

Biphenyl, benzoic acid (BA), CBAs and metals [Al2(SO4)3, NaAsO2, CdCl2, CuCl2, K2Cr2O7, NiCl2, Pb(NO3)2, ZnCl2] were purchased from Sigma (Sigma Chemical Company, St. Louis, MO). The PCB congener mixture Aroclor 1242 was purchased from Supelco Inc. (Bellefonte, PA). Metal stock solutions were prepared in double-distilled water, syringe filtered and stored at room temperature. A PCB stock solution was prepared by dissolving Aroclor 1242 in hexane to a concentration of 2000 p.p.m.

Media and growth conditions of planktonic cultures

Pseudomonas pseudoalcaligenes KF707 (Furukawa & Miyazaki, 1986) was grown aerobically at 30 °C in 50 mL glass flasks containing 10 mL of minimal salt medium (MSM) supplemented with 5 mM succinate or 5 mM biphenyl (nominal concentration) as the sole carbon source. The bacterium's ability to utilize BA and CBAs as growth substrates was tested in 30 mL glass test tubes containing 5 mL of MSM supplemented with BA, 2-, 3- or 4-CBA at a nominal concentration of 1 mM. Bacterial growth in BA and CBAs was followed by measuring the total protein content using the Lowry method. Composition of MSM (in g L−1): K2HPO4, 4.4; KH2PO4, 1.7; (NH4)2SO4, 2.6; MgSO4·7H2O, 0.4; CaSO4·2H2O, 0.0031; MnSO4·H2O, 0.05; FeSO4·7H2O, 0.1.

Chemotaxis assays

Swim plates for the qualitative analysis of chemotaxis (Robleto et al., 2003) were prepared in MSM containing 0.2% Difco Bacto Agar. KF707 cells exponentially growing in 5 mM succinate (OD660 nm∼0.5) were washed and re-suspended in a saline solution (NaCl 9 g L−1) and a 20 μL drop of cellular suspension was spotted at the centre of the plate. Crystals of succinate, BA, CBAs or biphenyl were added as chemoattractants on the right side of the plate while a saline solution (3 × 20 μL drops) was added on the left side of the plate as a negative control. For the characterization of the chemotactic response in the presence of PCBs, 60 μL of a solution 2 g L−1 of Aroclor 1242 was spotted on the top of biphenyl crystals, while an equivalent amount of hexane was added to the left side of the plates as a negative control for the solvent.

For swim plate assays examining the effect of metal ions on chemotaxis, salts of various metals were added uniformly to the plates. The concentrations used were at sublethal levels and were at least 25 times lower than the minimal inhibitory concentrations (MIC): Al2(SO4)3, 0.4 mM; NaAsO2, 0.25 mM; CdCl2, 0.05 mM; CuCl2, 0.05 mM; K2Cr2O7, 0.06 mM; NiCl2, 0.05 mM; Pb(NO3)2, 0.4 mM; and ZnCl2, 0.05 mM. Also, in this case, the molecules to be tested as chemoattractants were added to the right side of the plates and control solutions to the left side. In order to assess the effects of metals on KF707 ability to swim, swimming assays were performed by adding both succinate and metals uniformly to the plates. Single colonies of KF707 grown on MSM+succinate were inoculated at the centre of the plates and the swimming ring was measured after 24 and 48 h of incubation at 37 °C. Control plates contained succinate and no metal was added.

The quantitative analysis of KF707 chemotactic response was performed in modified capillary assays (Mazumder et al., 1999) according to Gordillo et al. (2007). KF707 cells exponentially growing in succinate, biphenyl and BA were washed and resuspended in chemotaxis buffer (10 mM Tris-HCl, pH 7.4). Hundred-microlitre aliquots of a cellular suspension (∼1 × 108 CFU mL−1) were placed in 200 μL pipette tips and a disposable 2-cm 25-gauge needle was attached to a 1-mL tuberculin syringe and was subsequently used as the chemotaxis capillary. Capillaries held 200 μL of the compound to be tested (1 and 10 mM succinate; 0.01 and 0.1 mM biphenyl; 1 mM BA and CBAs) dissolved in a chemotaxis buffer or, in the case of biphenyl, in a chemotaxis buffer with 1.4% hexane. Control assays contained buffer only or buffer and 1.4% hexane for assays in which chemotaxis to biphenyl was tested. Cells were incubated with the capillary at room temperature for 90 min and then the content of the syringe was serially diluted in saline solution. Aliquots of appropriate dilutions were spot plated onto Luria–Bertani (LB) agar plates and the accumulation of bacterial cells in the capillary was determined as the averageCFUs obtained from duplicate assays. Results were expressed as the mean of four to nine independent experiments. The relative chemotaxis response (RCR) was measured as the ratio of the bacteria that entered the test capillary over the number of bacteria in the control assay. An RCR ≥2 was considered as significant (Mazumder et al., 1999). Quantitative chemotaxis assays were also performed to measure the ability of metals to act as attractants or repellents. In this case, KF707 cells exponentially growing in succinate were washed and added to the capillaries as described above, but metal solutions were added to the syringes. The viability of KF707 cells exposed to the metals for the duration time of the chemotaxis assay was also assessed in order to verify that the metals were added at sub lethal concentrations.

Growth of P. pseudoalcaligenes KF707 as a biofilm

Pseudomonas pseudoalcaligenes KF707 biofilms were grown in the Calgary biofilm device (CBD, MBEC-P&G) as described by Ceri et al. (1999), Harrison et al. (2010) and the manufacturer (Innovotech, ED, Canada). From the cryogenic glycerol stock, KF707 cells were streaked out twice on LB agar plates and colonies from the secondary culture were suspended into MSM to match the optical density of a 1.0 McFarland standard. In order to produce a standardized inoculum containing approximately 1 × 107 CFU mL−1, the bacterial suspension was diluted 1 : 30 into either MSM containing 0.3% succinate or fresh MSM. Hundred and fifty aliquots of the standardized inoculum were added to the wells of a 96-well microtitre plate. The peg lid of the CBD was fitted on the top of the microtitre plate and the device was placed on a gyrorotary shaker at 100 r.p.m. at 30 °C and 95% humidity. Biphenyl was crystallized on the pegs of the CBD by immersing the pegs twice in a solution of biphenyl 20 g L−1 in hexane and allowed to dry. For the analysis of biofilm growth in the presence of PCBs, the lid of the CBD was immersed in a solution of biphenyl 20 g L−1 in hexane containing 0.1 g L−1 Aroclor 1242. Growth was monitored by viable cell counts (CFU per peg, biofilm growth; CFU mL−1, control planktonic growth). For biofilm growth, three pegs were taken from the CBD at each time point, rinsed in saline to remove loose planktonic cells and immersed in saline containing Tween-20 (0.1% v/v). Biofilms were disrupted by a 10-min sonication using an Aquasonic 250HT ultrasonic cleaner (VWR International, Mississauga, ON, Canada) set at 60 Hz. After sonication, the cellular suspensions were serially diluted in a saline solution. As a control, viable cell counts of planktonic cultures growing in the same wells as the collected biofilms were carried out by serial dilution in a saline solution. Twenty aliquots of biofilm and planktonic dilutions were plated onto LB agar medium and plates were incubated at 30 °C for 24 h.

Metal and PCB susceptibility testing

Susceptibility to metals of KF707 biofilm and planktonic cultures was assessed in the CBD according to the procedure described previously (Ceri et al., 1999; Harrison et al., 2005, 2010), with a slight modification. In each assay, five concentrations of four different metals were tested in quadruplicate against the biofilms grown to the stationary phase on the pegs of the CBD. Challenge medium was prepared by diluting the metal stock solutions to the desired highest concentration in 3-(N-morpholino)propanesulphonic acid (MOPS)-buffered MSM [MOPS, 4.18 g L−1 (20 mM); K2HPO4, 0.26 g L−1 (3 mM); (NH4)2SO4, 2.6 g L−1; MgSO4·7H2O, 0.4 g L−1; CaSO4·2H2O, 0.0031 g L−1; MnSO4·H2O, 0.05 g L−1; and FeSO4·7H2O, 0.1 g L−1]. MOPS-buffered salt medium was used instead of phosphate-buffered salt medium in order to avoid metal precipitation (Teitzel & Parker, 2003). Additionally, phosphate was added at the concentration of 3 mM in order to avoid starvation. Serial fourfold dilutions of challenge medium were prepared along the rows of a 96-microtitre plate to yield the so-called challenge plate. The first and the last column of the plate contained no metal and served as sterility and growth controls. Biofilms grown to the stationary phase (24 h after inoculation for succinate-grown biofilms and 48 h for biphenyl-grown biofilms) were rinsed in a saline solution and exposed to metals by inserting the peg lid into the challenge plate. Three sample pegs and three aliquots of planktonic cultures were collected for the determination of CFU per peg and CFU mL−1, respectively. The exposure was carried out at 30 °C and 95% relative humidity in a rotary shaker at 100 r.p.m. for 4 or 48 h. During the exposure time, planktonic cultures were inoculated with cells shed from the biofilm, in a way that reflects the duality of the bacterial life in the environment. After exposure, biofilms were rinsed twice in a saline solution and transferred into a clean microtitre plate containing recovery medium (LB broth, 0.1% Tween-20). Biofilms were immediately disrupted by sonication for 10 min in recovery medium and then serially diluted. The planktonic cultures grown in the challenge plate were also collected and serially diluted. Twenty microlitre-aliquots of biofilm and planktonic dilutions were spot plated onto LB agar plates. LB plates were incubated at 30 °C and colonies were counted after 48 h. The data were then presented as a percentage survival kill curve against toxin concentration. Likewise, KF707 susceptibility to PCBs was assayed in the CBD by exposing succinate and biphenyl-grown biofilms to increasing concentrations of Aroclor 1242 (up to 500 p.p.m.). For the analysis of KF707 susceptibility to metals in the presence of PCBs as co-contaminants, the challenge plate was set up as described above, except that PCBs were added to every well of the challenge plate for a final concentration of 100 p.p.m.

Confocal laser scanning microscopy (CLSM)

KF707 biofilms grown in the CBD to the stationary phase (72 h) were visualized by CLSM after staining with acridine orange (AO, 0.1% w/v). Pegs were taken from the CBD, rinsed in a saline solution for 1 min and stained with AO for 5 min in the dark at room temperature. Microscopic visualization of the biofilms was carried out using a Leica DM IRE2 spectral confocal and multiphoton microscope with a Leica TCS SP2 acoustic optical beam splitter (AOBS) (Leica Microsystem, Richmond Hill, ON, Canada). Line averaging (× 2) was used to capture images with reduced noise. A 63 × water immersion objective was used in all the experiments. Image capture, 2D projections of z-stacks and 3D reconstructions were performed using Leica Confocal Software (Leica Microsystem) as described elsewhere (Harrison et al., 2006).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Chemotaxic response of KF707 to biphenyl in the presence or absence of PCBs

Pseudomonas pseudoalcaligens KF707 is a motile bacterium with a single polar flagellum, which allows the cells to swim towards nutrient attractants, such as tryptone (V. Tremaroli, unpublished data). In Fig. 1, swim plates for the analysis of KF707 chemotactic response to biphenyl in the absence (A) or presence (B) of PCBs are shown. In this assay, a positive chemotactic response is represented by a swimming ring formed by the cells moving in the direction of the attractant. Crystals of biphenyl (4.45 mg L−1 water solubility at 20 °C) were added to one side of the plate as chemoattractants. On the top of biphenyl crystals, a solution of Aroclor 1242, a commercial mixture of PCBs (60 μL, 2000 p.p.m.), was spotted (Fig. 1b). KF707 showed a chemotactic response to biphenyl and chemotaxis was maintained in the presence of high levels of PCBs.

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Figure 1.  Swim plate chemotaxis assay for the analysis of KF707 chemotactic response to biphenyl in the absence (a) or presence (b) of PCB mixture Aroclor1242. KF707 cells were grown in MSM+succinate, washed and inoculated at the centre of the swimming plate. Crystals of biphenyl were added to the right side of the plate as chemoattractants and sterile saline was added to the left side as a control. A positive chemotactic response is represented by the formation of a swimming ring in the direction of the attractant.

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In Fig. 2, swim plates for the analysis of KF707 chemotactic response to biphenyl and intermediates of biphenyl and PCB metabolism, such as BA and CBA (2-, 3-, 4-CBA), are shown. Plates were incubated at 30 °C for 96 h before pictures were taken, a time chosen based on the growth curves in Fig. 2b. In the case of BA, the migration of KF707 cells stopped before the crystals were reached, suggesting that BA is toxic or produces a repulsive response at high concentrations. No chemotactic response was detected for KF707 in this plate assay for 2-, 3- and 4-CBA, although cell growth curves indicated that KF707 is able to grow on 2- and 3-CBA as carbon sources (Fig. 2b). Additionally, no swim plate chemotactic response to major carbon source compounds such as tryptone was observed for a cheA mutant, suggesting that the observations are true chemotaxis (not shown).

image

Figure 2.  (a) Swim plate chemotaxis assay for the analysis of KF707 response to biphenyl and intermediates of biphenyl and PCB metabolism. Crystals of the test attractant were added to the right side of the plate and sterile saline was added to the left side as a control. Attractants: Succ (succinate), biphenyl, BA, 2-, 3- and 4-CBA. (b) Growth curves of KF707 grown on PCB metabolic intermediates. Cell growth was evaluated by measuring the cell protein content per volume.

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To further evaluate chemotaxis in KF707, a chemotaxis capillary assay for the quantitative analysis of KF707 response to biphenyl, BA and CBAs is reported in Fig. 3. It is known that the enzymes for biphenyl catabolism are induced by biphenyl in KF707. Hence, we tested the chemotactic response of KF707 cells to biphenyl under two conditions: (1) in cells grown to the exponential phase using succinate as a carbon source, where the catabolic pathway would not be induced, and (2) in cells grown using biphenyl. Cells grown in succinate were chemotactic towards the lowest concentration of biphenyl used in this study, while cells grown in biphenyl showed positive chemotaxis to high concentrations of biphenyl (Fig. 3a). Figure 3b shows the response of KF707 to intermediates of biphenyl and PCB metabolism, such as BA and CBAs. Succinate-grown cells did not show chemotaxis to benzoate and CBAs, while both benzoate- and biphenyl-grown cells displayed a positive response to BA, 2- and 3-CBA, but not to 4-CBA. These results are in agreement with the ability of KF707 cells to use BA, 2- and 3-CBA, but not 4-CBA as carbon sources for cell growth (Fig. 2b). Thus, the results presented in Fig. 3 show that the chemotactic response of KF707 to biphenyl is constitutive, while the response to BA and CBAs requires induction by previous growth in benzoate.

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Figure 3.  Chemotaxis capillary assay for the quantitative analysis of KF707 response to biphenyl, BA and CBAs. (a) KF707 cells grown on either succinate or biphenyl and tested for chemotaxis towards these two compounds. (b) KF707 cells grown in succinate, biphenyl and BA tested for chemotaxis towards BA, 2-, 3- and 4-CBA (all benzoates at 1 mM). Error bars in (a) and (b) indicate the SE. Numbers on the top of each bar indicate the RCR [the ratio of the bacteria in the test capillary over the number of bacteria in the control (CTR)]. *Significant differences in comparison with the control (P<0.05, Student&apos:s t-test).

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Chemotaxis response to succinate or biphenyl in the presence of metals

To investigate the chemotaxis to succinate and/or biphenyl in the presence of metals, KF707 cells were grown in succinate and inoculated at the centre of the swimming plates. In Fig. 4, swim plates with no metals (control, CTR) or subinhibitory concentrations of metal cations and anions are shown. The swim plates demonstrate that Cu2+, Cd2+ and Ni2+ cations interfered with KF707's attraction to succinate (Fig. 4a) and biphenyl (Fig. 4b), whereas the other metals tested (Al3+, AsO2, CrO42−, Pb2+ and Zn2+) displayed little to no apparent effect. The ability of KF707 to swim in the presence of metals was tested in swimming plates in which both the carbon source (succinate) and the metals were added to the medium. Swimming halos were measured after 24 and 48 h and decreased swimming was observed only in the case of Ni (not shown). In addition, we tested whether the metals used in this study were able to act as chemorepellents for KF707 cells. Modified capillary assays were performed by adding metals to the syringe at the same concentrations as those used in the chemotaxis plate assays. In no case was a significant negative chemotactic response observed (not shown).

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Figure 4.  Swim plate chemotaxis assays towards succinate (a) and biphenyl (b) in the presence of various metal ions. Succinate grown KF707 cells were washed and inoculated at the centre of the swimming plates. Metals were added to the swim medium while succinate and biphenyl crystals were added to the right side of the plates and saline to the left.

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Pseudomonas pseudoalcaligenes KF707 can form biofilms with biphenyl as the sole carbon source and were unaffected by PCBs

In previous works (Tremaroli et al., 2008; V. Tremaroli, unpublished data), we have shown that P. pseudoalcaligenes KF707 is able to grow as a biofilm on the polystyrene surface of the Calgary Biofilm Device (CBD), using standard carbon sources. However, biofilm growth in the presence of biphenyl and/or PCBs was not evaluated. CLSM was used for the visualization of KF707 biofilms grown on the CBD (Fig. 5). Biofilms were visualized after 72 h of growth in either biphenyl (Fig. 5a) or biphenyl with PCBs (Fig. 5b). Observations showed that KF707 was able to colonize the pegs of CBDs and form biofilms in the presence of PCBs. As KF707 was able to form a biofilm on the CBD; this incubation method was used to evaluate the susceptibility of KF707 biofilm and planktonic cells to PCBs. Under biphenyl-induced degradation conditions, biofilm and planktonic cells were observed to be highly tolerant to PCBs up to the highest concentration used (Supporting Information, Fig. S1). As a control for the intrinsic toxicity of PCBs, KF707 was also grown on succinate, a condition in which the biphenyl metabolism was not induced. In the case of succinate-grown cultures, a significant decrease in culture viability was observed only for planktonic cells at the highest concentration used (0.5 g L−1 of Aroclor 1242). These data demonstrate that growth in biofilms confers protection against the toxicity of PCBs as succinate-growing biofilms are more tolerant than succinate-growing planktonic cells.

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Figure 5.  CLSM for the visualization of KF707 biofilms grown on the CBD. Biofilms were grown for 72 h on biphenyl (a) and biphenyl with PCBs (b). KF707 biofilms were stained with AO.

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Tolerance of P. pseudoalcaligenes KF707 to metals growing on biphenyl and PCB

By culturing KF707 in the CBD, we assessed the survival of biphenyl- or succinate-grown cultures during exposure to metal cations and anions for 4 and 48 h (Fig. 6). KF707 cells were grown either in biphenyl (PCB degradation conditions) or in succinate (control for nondegradation conditions) and then exposed to increasing concentrations of metals. KF707 biofilms were observed to be generally more tolerant than planktonic cells. Generally, decreased survival was found with increased exposure times as observed for other organisms (Harrison et al., 2004).

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Figure 6.  Metal susceptibility testing of KF707 planktonic and biofilm cells. KF707 cells were grown either in biphenyl or in succinate, and then exposed to increasing concentrations of metals for either 4 h (filled symbols) or 48 h (open symbols) as indicated in the legend insert of panels a and i. Panel (1) presents data for As (a, b), Cr (c, d), Cd (e, f) and Pb (g, h) and panel (2) for Al (i, j), Cu (k, l), Ni (m, n) and Zn (o, p). The kill curves are normalized at zero metal concentration to 100% survival.

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KF707 showed a strong resistance to AsO2 and to Ni2+, with some increased toxicity at the high concentrations and long exposure times. Tolerance was observed towards CrO42−, Cd2+, Pb2+ and Zn2+ when KF707 was grown on succinate and as a biofilm. However, considerable toxicity was observed for Al3+, Cd2+, Cu2+, CrO42−, Pb2+ and Zn2+ with long-term exposure (48 h) when grown on biphenyl. These observations remained essentially unchanged for identically performed experiments with the concomitant exposure of cultures to metals and PCBs (Aroclor 1242, 0.1 g L−1) (Fig. S2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The degradation of Ps can be facilitated by biphenyl-utilizing bacteria. However, the bioavailability of PCBs relative to biphenyl and their toxicity to the organisms in the contaminated environment is also a variable to be considered (Chávez et al., 2006). Furthermore, it is recognized that metals can reduce the rate of biodegradation of organic chemicals (Sandrin & Hoffman, 2007). Thus, the behaviour of an organism in an environment co-contaminated by organic pollutants and metals is worth examining, in order to evaluate for any synergistic toxicities or inhibition of the desired bioprocess.

Here, we show that P. pseudoalcaligenes KF707 is tolerant to the PCB congener mixture Aroclor 1242 and its chemotaxis is not affected by the presence of PCBs. Parnell et al. (2010) found that the expression patterns of genes involved in PCB degradation in B. xenovorans LB400 were growth phase dependent and were upregulated in the stationary phase. Because it is suggested that planktonic stationary phase shows similarities in physiology to organisms in a biofilm (Spoering & Lewis, 2001), we explored biofilm growth of KF707. We found that KF707 is able to form biofilms growing on biphenyl as well as in the presence of PCBs.

Intermediates of the PCB degradation pathway, such as CBAs, are typically toxic for most bacteria (Chávez et al., 2006). Burkholderia xenovorans LB400, one of the most extensively studied species with respect to PCB degradation, cannot grow on CBAs, and the PCB degradation was inhibited by these intermediate compounds (Chávez et al., 2006). Similarly, the growth of Pseudomonas sp. B4, also a PCB degrader, was inhibited by 3-CBA. Additionally, no chemotaxis was observed for this strain in the presence of 2- and 3-CBAs (Gordillo et al., 2007). Our work here demonstrated that KF707 has a constitutive chemotactic response to biphenyl, while the response to BA and CBAs required previous growth in BA or biphenyl. Experiments also demonstrated that KF707 is not inhibited by the CBA intermediates and in fact can grow on 2- and 3-CBA as well as BA, thus demonstrating a clear advantage of KF707 over LB400 in this regard.

The resistance of P. pseudoalcaligenes KF707 towards several metals was evaluated using succinate or LB broth media as planktonic and biofilm (Tremaroli et al., 2008). It was demonstrated that under these two growth conditions, KF707 showed only minor changes in resistance to metal cations and oxyanions. As expected, biofilms of KF707 tended to be more tolerant to exposure to antimicrobials (Costerton, 2007; Tremaroli et al., 2008; Turner et al., 2008). In the present study, kill curves were generated for eight metal(loids) for both planktonic growth and biofilms for two different exposure times (4 and 48 h). Comparing the sensitivity of KF707 to our previous work performed in a similar fashion on Pseudomonas fluorescens ATCC 13525 (Workentine et al., 2008), Escherichia coli JM109, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC29213 (Harrison et al., 2004), we find that as a whole, biofilm growth of KF707 using succinate media leads to similar resistance to Ni2+ as P. fluorescens and E. coli, but less than that of the other organisms. KF707 demonstrated the same level of tolerance to AsO2, but showed slightly greater resistance to Cd2+ as the other species. KF707 was found to be far more sensitive to Al3+ and Cu2+, and was slightly more sensitive to Zn2+ and CrO42− than those bacteria evaluated in our previous studies.

Upon moving away from succinate as the carbon source, overall, the sensitivity to the metals was greater when biphenyl was used. This sensitivity upon growth on biphenyl indicates different physiological susceptibility using this carbon source, which may be related to oxidative stress as suggested previously by Chávez et al. (2004) and effects on membrane structure (Yamada et al., 2006). Redox and reactive oxygen species stress are proposed modes of toxicity for some metals (Stohs & Bagchi, 1995; Silver & Phung Le, 2005; Harrison et al., 2007, 2009). The combination of the two stressors would lead to a serious survival challenge for PCB-degrading microorganisms.

The analysis of the impact that the presence of metals has on swimming and chemotaxis is crucial to understand their effect on the microorganism's potential for bioremediation under co-contaminated conditions. Cd2+, and particularly Cu2+ and Ni2+, were shown to interfere with chemotaxis towards both succinate and biphenyl. Alone, these metals did not act as chemorepellents and, apart from Ni2+, did not interfere with KF707 swimming abilities. Although found to be quite toxic to KF707 in comparison with other organisms, no effect of the presence of Al3+ or CrO42− was found on chemotaxis. Overall, there appears to be no correlation between the toxicity of the metal to KF707 growing as a biofilm and the effect of metals on chemotaxis to the carbon source, whether succinate or biphenyl. A likely explanation for this phenomenon is the chemotaxis assay being performed with cells having physiological features more similar to log-phase planktonic than biofilm cultures.

Our study here has evaluated the effect of carbon source and the toxicity of metabolic intermediates, growth state (planktonic vs. biofilm), effect of the presence of various metals and chemotactic ability of P. pseudoalcaligenes KF707. Our work shows that although the presence of most metals does not alter the response of KF707 chemotaxis and growth on biphenyl and the chlorinated mixture Aroclor 1242, the presence of specific metal ions as co-contaminants could impair KF707s capacity to colonize and degrade PCB-polluted soils. This is a major concern in considering the use of this organism in polluted sites heavily co-contaminated with PCBs and metals, particularly Al3+ and Cu2+, shown here to affect KF707 growth and chemotaxis, respectively.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by discovery grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to R.J.T. and H.C. D.Z. and S.F. were funded by MIUR (Prin-2008). CLSM was made possible by a Canadian Foundation for Innovation (CFI) Bone and Joint Disease Network grant to H.C. The CBD plates used in this study were kindly donated by Innovotech Inc. We are grateful to C. Stremick and Dr J.J. Harrison for excellent technical assistance and expert advice.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • 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.
  • Ceri H, Olson ME, Stremick C, Read RR, Morck D & Buret A (1999) The calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37: 17711776.
  • 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 Microb 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.
  • Costerton JW (2007) The Biofilm Primer. Springer Press, Berlin.
  • Ellis LB, Hershberger CD & Wackett LP (1999) The university of Minnesota Biocatalysis/Biodegradation database: specialized metabolism for functional genomics. Nucleic Acids Res 27: 373376.
  • Ellis LB, Roe D & Wackett LP (2006) The university of Minnesota biocatalysis/Biodegradation database: the first decade. Nucleic Acids Res 34: D517D521.
  • Fedi S, Carnevali M, Fava F, Andracchio A, Zappoli S & Zannoni D (2001) Polychlorinated biphenyl degradation activities and hybridization analyses of fifteen anaerobic strains isolated from a PCBs-contaminated site. Res Microbiol 152: 583592.
  • Furukawa K & Miyazaki T (1986) Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes. J Bacteriol 166: 392398.
  • Gibson DT, Cruden DL, Haddock JD, Zylstra GJ & Brand JM (1993) Oxidation of polychlorinated biphenyls by Pseudomonas sp. strain LB400 and P. pseudoalcaligenes KF707. J Bacteriol 175: 45614564.
  • Gordillo F, Chavez FP & Jerez CA (2007) Motility and chemotaxis of Pseudomonas sp. B4 towards polychlorobiphenyls and chlorobenzoates. FEMS Microbiol Ecol 60: 322328.
  • Harrison JJ, Ceri H, Stremick CA & Turner RJ (2004) Biofilm susceptibility to metal toxicity. Environ Microbiol 6: 12201227.
  • Harrison JJ, Turner RJ & Ceri H (2005) High throughput metal susceptibility testing of microbial biofilms. BMC Microbiol 5: 53.
  • Harrison JJ, Ceri H, Yerly J, Stremick CA, Hu Y, Martinuzzi R & Turner RJ (2006) The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary biofilm device. Biol Proc Online 8: 194215.
  • Harrison JJ, Ceri H & Turner RJ (2007) Multimetal resistance and tolerance in microbial biofilms. Nat Rev Microbiol 5: 928938.
  • Harrison JJ, Tremaroli V, Stan MA, Chan CS, Vacchi-Suzzi C, Heyne BJ, Parsek MR, Ceri H & Turner RJ (2009) Chromosomal antioxidant genes have metal ion-specific roles as determinants of bacterial metal tolerance. Environ Microbiol 11: 24912509.
  • Harrison JJ, Stremick CA, Turner RJ, Allan ND, Olson ME & Ceri H (2010) Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening. Nature Protoc 5: 12361254.
  • Harwood CS (1989) A methyl-accepting protein is involved in benzoate taxis in Pseudomonas putida. J Bacteriol 171: 46034608.
  • Harwood CS, Parales RE & Dispensa M (1990) Chemotaxis of Pseudomonas putida toward chlorinated benzoates. Appl Environ Microb 56: 15011503.
  • Hietala KA & Roane TM (2009) Microbial remediation of metals in soils. Advances in Applied Bioremediation. Soil Biology, Vol. 17 (SinghA, KuhadRC & WardOP, eds), pp. 201220. Springer, Berlin.
  • Hoffman DR, Okon JL & Sandrin TR (2005) Medium composition affects the degree and pattern of cadmium inhibition of naphthalene biodegradation. Chemosphere 59: 919927.
  • Kubota H, Senda S, Nomura N, Tokuda H & Uchiyama H (2008) Biofilm formation by lactic acid bacteria and resistance to environmental stress. J Biosci Bioeng 106: 381382.
  • 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.
  • Ohtsubo Y, Kudo T, Tsuda M & Nagata Y (2004) Strategies for bioremediation of polychlorinated biphenyls. Arch Microbiol 65: 250258.
  • Parnell JJ, Park J, Denef V, Tsoi T, Hashsham S, Quensen J III & Tiedje JM (2006) Coping with polychlorinated biphenyl (PCB) toxicity: physiological and genome-wide responses of Burkholderia xenovorans LB400 to PCB-mediated stress. Appl Environ Microb 72: 66076614.
  • Parnell JJ, Denef VJ, Park J, Tsoi T & Tiedje JM (2010) Environmentally relevant parameters affecting PCB degradation: carbon source- and growth phase-mitigated effects of the expression of the biphenyl pathway and associated genes in Burkholderia xenovorans LB400. Biodegradation 21: 147156.
  • Robleto EA, Lopez-Hernandez I, Silby MW & Levy SB (2003) Genetic analysis of the AdnA regulon in Pseudomonas fluorescens: nonessential role of flagella in adhesion to sand and biofilm formation. J Bacteriol 185: 453460.
  • Sandrin TR & Hoffman DR (2007) Bioremediation of organic and metal co-contaminated environments: effects of metal toxicity, speciation, and bioavailability on biodegradation. Environmental Bioremediation Technologies (SinghSN & TripathiRD, eds), pp. 134. Springer, Berlin.
  • Sandrin TR & Maier RM (2003) Impact of metals on the biodegradation of organic pollutants. Environ Health Persp 111: 10931101.
  • Silver S & Phung Le T (2005) A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J Ind Microbiol Biot 32: 587605.
  • Spoering AL & Lewis K (2001) Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol 183: 67466751.
  • Stohs SJ & Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radical Bio 18: 321336.
  • Teitzel GM & Parker MR (2003) Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl Environ Microb 69: 23132120.
  • Tremaroli V, Fedi S, Turner RJ, Ceri H & Zannoni D (2008) Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions. Arch Microbiol 190: 2939.
  • Tremaroli V, Workentine ML, Weljie AM et al. (2009) Metabolomic investigation of the bacterial response to a metal challenge. App Environ Microbiol 75: 719728.
  • Turner RJ, Zannoni D, Hynes MF & Ceri H (2008) Antimicrobial agents and biofilms; resistance/tolerance is multifactorial. Chemistry Today 26: 2022.
  • Workentine ML, Harrison JJ, Stenroos PU, Ceri H & Turner RJ (2008) Pseudomonas fluorescens' view of the periodic table. Environ Microbiol 10: 238250.
  • Wu G, Feng Y & Boyd SA (2003) Characterization of bacteria capable of degrading soil-sorbed biphenyl. Bull Environ Contam Toxicol 71: 768775.
  • Yamada T, Shimomura Y, Hiraoka Y & Kimbara K (2006) Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation. Appl Microbiol Biot 73: 452457.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Susceptibility testing of KF707 biofilm (labeled Biof) and planktonic (Plank) cells to PCBs.

Fig. S2. Metal susceptibility testing of KF707 planktonic and biofilm cells in the presence of PCBs.

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