P.M. Fedorak, Department of Biological Sciences, Univerity of Alberta, Edmonton, AB T6G 2E9, Canada.
L.A. BEAUDETTE, O.P. WARD, M.A. PICKARD and P.M. FEDORAK.2000.Three white rot fungi were compared for their ability to attack polychlorinated biphenyl (PCB) congeners in the presence and absence of the non-ionic Triton X-100 or the anionic Dowfax 8390 surfactants at half their critical micelle concentrations. Neither surfactant affected PCB biodegradation monitored by gas chromatography but the release of 14CO2 from 2,4′,5-[U-14C]trichlorobiphenyl by Trametes versicolor was stimulated 12% by Triton X-100. Since mineralization is the complete metabolism of the congener and biodegradation was measured as substrate disappearance, Triton X-100 is proposed to aid intracellular solubilization of 2,4′,5-trichlorobiphenyl for complete oxidation by T. versicolor.
When added to an aqueous medium, surfactant molecules form micelle aggregates above the CMC, with hydrophilic polar head groups arranged in contact with the medium and the hydrophobic tail groups within the micelle. Thus above the CMC, hydrophobic molecules may be sequestered within the micelle, increasing solubility. Below the CMC, the surfactants remain as monomers and do not increase the solubility of hydrophobic compounds but they can bind to surfaces within the medium such as microbial cells and pollutant particles. It has been shown that Triton X-100 and Dowfax 8390 inhibit the adhesion of bacteria to contaminants and promote the removal of bacteria already attached to similar contaminants ( Stelmack et al. 1999 ). Both these surfactants, at half their CMC, also slow the growth of a Pseudomonas strain and a Mycobacterium strain on anthracene, but not on glucose, indicating that microbial interaction with the poorly soluble growth substrate is also affected ( Stelmack et al. 1999 ).
Studies on the use of surfactants in PCB degradation are limited ( Liu 1980; Rouse et al. 1994 ), as are PCB studies involving fungi and surfactant effects on fungal metabolism ( Volkering et al. 1998 ). We have previously reported the degradation of six PCB congeners and the mineralization of one 14C-labelled congener by 12 white rot fungi ( Beaudette et al. 1998 ). In that study, the most active fungal strains were Bjerkandera adusta UAMH 8258 and UAMH 7308, Trametes versicolor UAMH 8272 and Pleurotus ostreatus UAMH 7964. A strain often cited in the literature for PCB degradation, Phanerochaete chrysosporium ATCC 24725, gave the lowest degradation and mineralization activities of the fungi tested. To determine if surfactant concentrations below the CMC can influence the biodegradation and mineralization of PCB congeners by fungi, we examined the effect of Triton X-100, a non-ionic surfactant, and Dowfax 8390, an anionic surfactant (hexadecyl diphenyl oxide disulphonate; Rouse et al. 1993 ), on the degradation of six PCB congeners and the mineralization of one congener by three fungi, B. adusta UAMH 7308, T. versicolor UAMH 8272 and P. chrysosporium ATCC 24725. In this paper we present biodegradation results from measuring the decrease in PCB congener concentrations using gas chromatography (GC) analyses of organic solvent-extracted cultures and mineralization results from measuring the release of 14CO2 from a radiolabelled congener.
White rot fungi are known to produce a variety of extracellular enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP, Hatakka 1994). In particular, LiP has been shown to oxidize many polycyclic aromatic hydrocarbons ( Vazquez-Duhalt et al. 1994 ) and nitrogen-containing aromatic compounds ( Vazquez-Duhalt et al. 1995 ). These oxidations by white rot fungi have been suggested as a means of removing organic compounds from contaminated environments ( Hammel 1989; Morgan et al. 1991 ; Field et al. 1993 ). Thus, the effects of the two surfactants on the activities of LiP and MnP in vitro were also studied.
MATERIALS and METHODS
Chemicals and enzymes
Six PCB congeners, 2,3-dichlorobiphenyl (2,3-DCB), 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl and 2,2′,4,4′,5,5′-hexachlorobiphenyl, were purchased from Accustandard (New Haven, CT, USA). Labelled 2,4′,5-[U-14C]TCB (22 mCi mmol−1; >99% purity) was obtained from the Department of Environmental Chemistry, Stockholm University (Stockholm, Sweden). All other chemicals used in this study were reagent grade or better. Triton X-100 was from Rohm and Haas (West Hill, ON, Canada) and Dowfax 8390 from Dow Chemical Company (Midland, MI, USA). The CMC of each surfactant was determined in low-nitrogen medium using a tensiometer (model 70545; Central Scientific Company, Chicago, IL, USA). Commercial preparations of partly purified LiP and MnP were purchased from Intech One-Eighty (North Logan, UT, USA).
Bjerkandera adusta UAMH 7308 and T. versicolor UAMH 8272 were from the University of Alberta Microfungus Collection and Herbarium culture collection (Devonian Botanical Garden, University of Alberta, Edmonton, AB, Canada). Phanerochaete chrysosporium ATCC 24725 was from the American Type Culture Collection (Manassas, VA, USA). Working slants of each fungus were grown on potato dextrose agar (Difco, Detroit, MI, USA) for 10 d at 28 or 37 °C and then stored at 4 °C.
Inocula were prepared by removing mycelia from agar plates and adding these to 200 ml of a liquid nutrient-rich medium in 500-ml Erlenmeyer flasks ( Beaudette et al. 1998 ). Fungi that tended to grow as pellets were homogenized for 15 s in a 50-ml stainless steel homogenizer (Omni-Mixer; Sorvall, Norwalk, CT, USA). Growth was for 4 d at 28 or 37 °C on a rotary shaker at 200 rev min−1. The experimental medium was a low-nitrogen liquid medium ( Beaudette et al. 1998 ) which contained glucose (10 g l−1) and yeast extract (Difco; 0·2 g l−1). For the nutrient-rich medium, 2% malt extract was added to the low-nitrogen medium. Media were buffered to pH 4·5 by the addition of 2,2-dimethylsuccinic acid ( Tien & Kirk 1988).
After 7 d of aerobic growth, fungi were harvested by centrifugation at 10 000 g, washed with 100 ml low-nitrogen medium and homogenized if necessary. The wet weight was determined after centrifugation at 1800 g for 10 min and decanting the supernatant fluid. Triton X-100 and Dowfax 8390 were prepared as sterile aqueous 10-fold concentrates.
Biodegradation of a mixture of polychlorinated biphenyl congeners
The experimental design was essentially as described by Beaudette et al. (1998) . Low-nitrogen (10 ml) medium with or without surfactant was added to 158-ml serum bottles sealed with Teflon-lined stoppers (The West Company, Lionville, PA, USA). For each experiment, there were three groups of serum bottles: control live cultures containing 0·6 g (wet weight) biomass that received surfactant-free medium and PCBs (100 μg of each congener); experimental live cultures with medium containing surfactants and PCBs; and killed controls which received surfactant-free medium, PCBs and 1 ml 7% (v/v) perchloric acid. Each experiment was performed in triplicate. Cultures of B. adusta and T. versicolor were incubated at 28 °C while P. chrysosporium was incubated at 37 °C on a rotary shaker at 100 rev min−1. After 5 d, each serum bottle was filled with filter-sterilized O2 and then, after 21 d, each culture received 1 ml 7% perchloric acid and was extracted with 30 ml hexane.
The hexane-extracted PCBs were analysed by GC with an atom emission detector using the same method as that outlined by Kropp et al. (1997) with the following exceptions. The helium carrier gas flow was 4 ml min−1 and the oven temperature was initially held at 80 °C for 2 min and then programmed at 10 °C min−1 to 280 °C. The chlorine channel was monitored at 479 nm. External standard mixtures of the congeners were prepared to quantify the congeners in the samples. Biodegradation was calculated by subtracting the concentration of a congener in the acid-killed controls from the concentration in the corresponding live cultures with and without surfactant.
Low-nitrogen medium (10 ml) with and without surfactant was added to 158-ml serum bottles, sealed with Teflon-lined stoppers and autoclaved. Each serum bottle received 0·6 g (wet weight) of biomass and an atmosphere of pure O2. Each experiment consisted of triplicate acid-killed controls and live cultures. After 5 d of incubation at 28 or 37 °C (depending upon the inoculum), each culture received a mixture of 100 μg unlabelled 2,4′,5-TCB and 1 μg 2,4′,5-[U-14C]TCB in acetone (200 000 d.p.m. bottle−1). Cultures were returned to the incubator and, at various times over a 30-d period, flushed with filter-sterilized O2 to remove 14CO2. The 14CO2 trapping and counting methods are outlined by Beaudette et al. (1998) .
Lignin peroxidase activity was determined by the method of Tien & Kirk (1988) using veratryl alcohol as the substrate. Reaction mixtures contained 0·02 U LiP. Dowfax 8390 was tested at concentrations from 0 to 0·5 CMC. Triton X-100 was used at 0·5 CMC.
Manganese peroxidase activity was determined by the method of Kuwahara et al. (1984) using phenol red as the substrate. Reaction mixtures contained 0·02 U MnP and one of the surfactants at 0·5 CMC.
Determination of the surfactant critical micellar concentrations
In the low-nitrogen medium, the CMC of Triton X-100 was 0·29 mmol l−1 and that of Dowfax 8390 15 μmol l−1. For in-vivo use the surfactants were used at half their CMCs (0·14 mmol l−1 for Triton X-100 and 8 μmol l−1 for Dowfax 8390).
Biodegradation of polychlorinated biphenyl congeners
The extent of biodegradation of the six PCB congeners in the three fungal cultures that received no surfactant was essentially the same as reported by Beaudette et al. (1998) . By GC analysis, little or no PCB biodegradation was observed in the P. chrysosporium cultures, with most of the PCB removal in these cultures attributed to adsorption to biomass. Trametes versicolor and B. adusta were more active cultures, with the former fungus biodegrading between 15 and 65% of the six congeners.
Table 1 summarizes the extent of degradation of two congeners, 2,3-DCB and 2,4′,5-TCB, used in this study; 2,3-DCB was more extensively biodegraded. Data for 2,4′,5-TCB are given for comparison of results with 2,4′,5-[U-14C]TCB.
Table 1. Biodegradation (%) of 2,3-dichlorobiphenyl(DCB) and 2,4′,5-trichlorobiphenyl(TCB) at 21 d incubation*
Between 50 and 65% biodegradation of 2,3-DCB was observed with both B. adusta and T. versicolor ( Table 1), while the more highly chlorinated 2,4′,5-TCB was biodegraded by between 9 and 37% (after subtraction of the acid-killed cell controls). Statistically, there was no significant difference in the degradation of the two PCB congeners in the presence and absence of either surfactant by these two fungi. The data in Table 1 illustrate that there was essentially no removal of these congenes by P. chrysosporium that could be attributed to biodegradation. The negative values resulted when the removal in the acid-killed controls (through adsorption to the biomass) was greater than the removal in the viable cultures.
Mineralization of 2,4′,5-[U-14C]trichlorobiphenyl
Figure 1 shows 14CO2 release from 2,4′,5-[U-14C]TCB by the three fungi in the presence and absence of surfactants. Most of the 14CO2 was released during the first 15 d. At day 30, T. versicolor, B. adusta and P. chrysosporium produced 19 ± 2·5%, 5·9 ± 0·6% and 4·3 ± 1·4%14CO2, respectively, in the presence of Triton X-100 as compared with 7·0 ± 1·0%, 4·2 ± 0·6% and 2·5 ± 0·1% in the absence of Triton X-100, thus showing a 12–1·7% increase in mineralization in the presence of Triton X-100.
Stimulation of mineralization was most significant in T. versicolor where there was an increase in the 30-d accumulative mineralization of 2,4′,5-[U-14C]TCB to 14CO2 caused by Triton X-100. This effect was most noticeable during the period of 5–15 d. The effect of Dowfax, although not statistically significant after 14 d, did show a trend towards stimulation of mineralization in this fungus in the early stages. In the other two fungi, insignificant increases in mineralization were caused by the surfactants. The absence of 14CO2 in the killed controls with and without surfactant indicated that mineralization was a result of fungal metabolism.
Effects of surfactants on lignin peroxidase and manganese peroxidase activities
Lignin peroxidase and MnP were affected differently by the two surfactants. Inhibition of LiP activity by Dowfax 8390 occurred at low surfactant levels ( Fig. 2). This inhibition was observed with as little as 0·01 CMC Dowfax 8390 and no activity was observed at 0·05 CMC. Lignin peroxidase activity was unaffected by Triton X-100 at a concentration of 0·5 CMC and MnP activity was unaffected by either surfactant at a concentration of 0·5 CMC.
For bioremediation of poorly soluble contaminants, such as PCBs, low solubility or bioavailability is rate-limiting for microbial growth and metabolism and thus limits removal of the target compound ( Volkering et al. 1995 ). While surfactant use above the CMC enhances the solubility of poorly soluble target compounds, it has variable effects on bioremediation. Few studies have been performed at surfactant concentrations below the CMC. Such studies allow solubility effects to be separated from interactions of the surfactant with the microbe and target compound as investigated here.
The CMC of 0·29 mmol l−1 for the non-ionic surfactant Triton X-100 was essentially the same in the pH 4·5 low-nitrogen medium used in this study and the pH 7 phosphate-buffered medium (CMC = 0·24 mmol l−1) used by Stelmack et al. (1999) . In contrast, the CMC for the anionic surfactant Dowfax 8390 was reduced 53-fold from 0·8 mmol l−1 in the pH 7 phosphate-buffered medium ( Stelmack et al. 1999 ) to 15 μmol l−1 in the low-nitrogen medium at pH 4·5. Differences in pH would have a greater influence on the CMC of an anionic surfactant than a non-ionic surfactant.
In the present study, the three fungi mineralized 2,4′,5-[U-14C]TCB and biodegraded the two PCB congeners in the absence of surfactants to the same extent as previously reported ( Beaudette et al. 1998 ). Trametes versicolor was slightly more active than B. adusta, while P. chrysosporium exhibited little or no activity of either type.
At concentrations of half the CMC, Triton X-100 and Dowfax 8390 were generally without effect on biodegradation as measured by substrate removal using GC methods ( Table 1). However, they stimulated mineralization, as measured by 14CO2 evolution ( Fig. 1), significantly in T. versicolor. For example, Triton X-100 stimulated the release of 14CO2 from 2,4′,5-[U-14C]TCB threefold at 20 d from 5 to 17% and by 2·5-fold at 30 d from 7 to 19%. The effect of Dowfax 8390 on T. versicolor mineralization was less significant but demonstrable and no effect was noted with either surfactant on B. adusta mineralization. Thus, at a concentration below that at which PCB solubility is affected, a surfactant can stimulate mineralization; neither surfactant caused inhibition of mineralization or biodegradation by either active fungus and there were no obvious effects on fungal growth as noted visually or by mycelial mass measurements.
This positive effect of low surfactant concentrations on overall cellular metabolism in fungi is in contrast to the negative effect on bacterial adhesion to non-aqueous phase liquids and bacterial growth on anthracene ( Stelmack et al. 1999 ). This does not indicate any difference in bacterial and fungal metabolism but relates to the comparison of metabolic and physical parameters under investigation in the two studies. The stimulus of Triton X-100 on mineralization but not substrate disappearance in T. versicolor may be related to their relative rates of activity. Mineralization is always less than metabolism, the difference being the intra- or extracellular metabolite pool. It appears that sub-CMC surfactant concentrations do not stimulate fungal uptake or early metabolic steps but may, in some way, enhance the later metabolic enzymes in respiration. Investigation of these possibilities is the next stage of our studies. Elmayergi et al. (1973) found that the respiration of Aspergillus niger was enhanced when 3 g l−1 of the anionic polymer Carbopol-934 was in the growth medium. This effect was attributed to an increased interfacial surface area of cell clusters in the liquid medium.
Finally, the in vitro experiments with MnP and LiP demonstrate that care must be taken in selecting a surfactant to stimulate attack of a contaminant by an extracellular enzyme. The severe inhibition of LiP activity in the presence of a low concentration of Dowfax 8390 illustrates that a surfactant may be incompatible with an extracellular enzyme, thereby rendering it inactive.
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. The authors thank Åake Bergman, Stockholm University, for supplying the radiolabelled congener and Atsumi Hashimoto for technical assistance.