Microbial Mineralization of 2,4,5-Trichlorophenol in Soil

Apr 25, 1996 - After 60 days of incubation, soil mineralized 15−45% of 2,4,5-TCP when it was exposed to 1, 10, or 100 ppm of this compound. Minimal ...
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Environ. Sci. Technol. 1996, 30, 1472-1476

Microbial Mineralization of 2,4,5-Trichlorophenol in Soil VALERIA MATUS, MO Ä N I C A V AÄ S Q U E Z , M A T IÄ A S V I C E N T E , A N D B E R N A R D O G O N Z AÄ L E Z * Laboratorio de Microbiologı´a, Departamento de Biologı´a Celular y Molecular, Facultad de Ciencias Biolo´gicas, P. Universidad Cato´lica de Chile, Alameda 340, Casilla 114-D, Santiago, Chile

Chlorophenols may be present in soils by land application of biocides or chlorophenol- and chloroguaiacol-containing sludges from pulp bleaching effluent treatment. Polychlorophenols like 2,4,5-trichlorophenol (2,4,5-TCP) are priority pollutants resistant to biodegradation. In this work, the mineralization (14CO2 evolution) of [U-14C-2,4,5]-TCP in soil not previously exposed to chloroorganics was studied. After 60 days of incubation, soil mineralized 1545% of 2,4,5-TCP when it was exposed to 1, 10, or 100 ppm of this compound. Minimal half-lives for 2,4,5TCP of 35-170 days were estimated. Less than 2% of mineralization was observed in soil exposed to 500 ppm or in incubations with sterile soil spiked with 10 or 100 ppm of 2,4,5-TCP. Thirty days of preincubation of soil with 10 or 100 ppm of 2,4,5-TCP increased the rate of mineralization of an additional amount of this pollutant. On the other hand, the presence of a mixture of chloroguaiacols decreased the mineralization of trichlorophenol in this soil. No microorganisms were able to grow using 2,4,5-TCP as the sole carbon and energy source. However, the absence of mineralization in sterile soil, the effect of the amount of and the preincubation with 2,4,5-TCP, and the effect of the presence of chloroguaiacols strongly support the involvement of microorganisms in this degradative process.

Introduction Although chlorophenols are natural compounds (1), most chlorophenols of anthropogenic origin are environmental pollutants. They may be present in soils by application of chlorophenol-containing biocides (2) or as metabolic intermediates in the degradation of other chloroaromatics (3, 4). Leaching of chlorophenols to the soil during landfilling or landspreading of chlorophenol- and chloroguaiacol-containing sludge from pulp bleaching effluent treatment plants has been also proposed (5, 6). Several chlorophenols are degradable (7, 8), but polychlorophenols like 2,4,5-trichlorophenol (2,4,5-TCP) are * Author to whom correspondence should be addressed; telephone: 56-2-2224516, ext. 2845; fax: 56-2-2225515; e-mail: [email protected].

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resistant to biodegradation in aerobic and anaerobic systems (9-11) and, therefore, are considered priority pollutants (12). There are a few reports on bacteria that degrade 2,4,5-TCP. Pseudomonas cepacea AC1100 (13) and Nocardioides simplex 3E (14) grow on 2,4,5-trichlorophenoxyacetate and 2,4,5-TCP. On the other hand, 2,4,5-TCP is not degraded or poorly degraded by bacterial strains that are able to grow on pentachlorophenol or 2,4,6-TCP (7, 15-19). Mineralization of 2,4,5-TCP by the ligninolytic fungi Phanerochaete chrysosporium has been reported (20). Reports on microbial degradation of trichlorophenols in freshwater, sediments, and soil systems have been published (19, 21), indicating a limited ability of these systems to remove 2,4,5-TCP. In this work, the degradation of 2,4,5-TCP in soil not previously exposed to chloroorganics was studied. The effect of the amount of 2,4,5-TCP, the preincubation with 2,4,5-TCP and the simultaneous presence of a mixture of chloroguaiacols, and the effect on soil microorganisms were evaluated in aerobic incubations performed with sterile or nonsterile soil. Through this chlorophenol isomer, not properly examined in previous studies, the potential of pristine soils to self-decontaminate was assessed.

Methods Soil. The soil samples used in this study were taken from a farm located 20 km south of Santiago, Chile. This soil had not been exposed to chlorinated organics for (at least) 10 years. The soil was a silty clay loam type (18% sand, 28% clay, and 54% silt), pH (water) 7.01, water holding capacity (whc) 29.45%, and extractable organic halogen 2.6 mg kg-1. The soil was air-dried and sifted (1 mm) before use. Sterile soil was prepared by autoclaving three times, and 1 day later, 10-15-g portions were placed in 125-mL Erlenmeyer flasks, covered with aluminum foil, and autoclaved again. Sterilization conditions were 0.1 kg cm-1 at 121 °C for 30 min. Chemicals. 2,4,5-TCP (purity higher than 99%), [U-14C2,4,5]-TCP (4.4 mCi mmol-1; radiochemical purity: approximately 95%), 2,4-dichlorophenol, and guaiacol were purchased from Sigma (St. Louis, MO). 4-Chloroguaiacol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol, and tetrachloroguaiacol (purity higher than 95%) were purchased from Helix Biotech Corp. (Vancouver, BC, Canada). For soil incubations, aqueous solutions of 2,4,5-TCP (1 g L-1), 4-chloroguaiacol (1.6 g L-1), 4,5-dichloroguaiacol (1.9 g L-1), 4,5,6-trichloroguaiacol (2.3 g L-1), and tetrachloroguaiacol (2.6 g L-1), pH 8-8.8, were prepared and used immediately. Other chemicals were of analytical purity. Soil Incubation. Ten or fifteen grams of soil was placed in 50-mL glass bottles and mixed with variable amounts of the 2,4,5-TCP stock solution to obtain final concentrations (dry weight) of 1.0-500 mg kg-1 (ppm) of soil. Where indicated, 4000-20 000 dpm of [14C]-2,4,5-TCP stock solution (1.15 × 108 dpm mL-1) and/or 5-10 ppm of each chloroguaiacol were added. Sterile distilled water to complete 0.5 mL of H2O/g of soil was added for proper mixing. Plain soil was prepared for incubation by the addition of 0.5 mL of sterile distilled H2O/g of soil. In experiments to test the effect of water content, soil was mixed with 0.25, 0.5, 0.75, or 1.0 mL of H2O/g of soil. Bottles

0013-936X/96/0930-1472$12.00/0

 1996 American Chemical Society

for dehalogenation experiments were covered with pierced aluminum foil. Bottles for sterile incubations were covered with aluminum foil. Bottles for mineralization experiments were tightly covered with a rubber stopper. Each set of experiments (i.e., sterile soil, plain soil, soil spiked with 2,4,5-TCP or 2,4,5-TCP plus a mixture of chloroguaiacols, or preincubated soil) was run in quadruplicate (mineralization measurements) or triplicate (dehalogenation and microbial count determinations). The fourth set was used for gas chromatography analysis. Incubations were performed in a dark chamber kept at 28 °C, without humidity control. The samples for dehalogenation had 1 mL of distilled water added every 2 days, with the exception of experiments with sterile soil where no loss of water was observed. Soil Analysis. Soil aqueous extracts were prepared by mixing 10 g of soil with 25 mL of distilled water and shaken for 2.5 h (250 rpm). Chloride determinations were performed in aqueous extracts, following a procedure described elsewhere (22, 23). Chloride released was expressed as the percentage of the maximum theoretical organic chloride content (molar basis) of each condition. Soil incubations without the addition of 2,4,5-TCP and sterile soil incubated with 100 ppm of 2,4,5-TCP gave negligible net chloride released values. Gas chromatography analysis was carried out in a 5890 Series II Hewlett-Packard system, with a HP fused silica capillary column coupled to a 5972 HP mass detector, following a described procedure (6, 24). 14CO Evolution. 2

Bottles for mineralization experiments were flushed with sterile air for 10 min, and the 14CO2 was trapped in a solution containing methanol (40%), ethanolamine (10%), 2,5-diphenyloxazol (0.2%), and 2,2′-(pphenylene)bis(5-phenyloxazol) (0.005%) prepared in toluene. Radioactivity in aqueous samples was measured adding 20% of the detergent ARCOPAL to this cocktail. Radioactivity was determined in a liquid scintillation instrument Beckman LS5000 TD. Bacterial Counts. About 1 g of soil was weighted and resuspended in 10 mL of sterile distilled water. The slurry was agitated for 1 h, and dilutions (10-4-10-6) were prepared in sterile conditions. Aliquots (0.1 mL) of each dilution were plated in triplicate, and the plates were incubated at 25 °C for 2-5 days. Counts were determined in plates containing 20 g L-1 nutrient broth and were

TABLE 1

Chloride Contents of Aqueous Extracts of Soil Samples after 15 and 40 Days of Incubation 2,4,5-TCP (ppm)

day 15 (%)

day 40 (%)

20 100 200

90 ( 6 67.8 ( 9.7 44.5 ( 8.7

106.6 ( 5.8 103.6 ( 14.9 76.6 ( 9.2

a Values are averages ( standard deviations of, at least, three measurements of each triplicate. Chloride contents are expressed as the percentage (molar basis) of the chloride released with respect to the total chlorine in the trichlorophenol. Values at day 0 were negligible.

TABLE 2 14CO Evolution 2 2,4,5-TCPa

in Soil Samples Incubated with

soil spiked with added TCP TCP plus (ppm) none none none none nonec nonec 10 ppm of TCPc 10 ppm of TCPc 100 ppm of TCPc 100 ppm of TCPc 500 ppm of TCPc 500 ppm of TCPc M1b M1b M1b M2b M2b

1 10 100 500 10 100 10 100 10 100 10 100 1 10 100 10 100

specific rate of evolution of 14CO2a

cumulative % of 14CO at day 90 2

0.43 ( 0.15 (1-20) 0.59 ( 0.13 (1-20) 1.06 ( 0.03 (5-40) 0.02 ( 0.05 (15-37) 0.77 ( 0.06 (2-11) 1.11 ( 0.03 (5-27) 1.81 ( 0.03 (1-7) 2.66 ( 0.05 (2-11) 1.34 ( 0.03 (1-7) 2.31 ( 0.02 (2-11) 0.64 ( 0.01 (24-41) 0.49 ( 0.01 (66-93) 0.31 ( 0.11 (5-50) 0.41 ( 0.12 (1-30) 0.68 ( 0.03 (12-70) 0.18 ( 0.01 (15-70) 0.27 ( 0.02 (30-91)

14.7 ( 1.5 19.8 ( 0.2 34.9 ( 1.2 0.7 ( 0.3 16.1 ( 1.9 39.2 ( 2.8 22.9 ( 2.3 49.4 ( 2.3 22.9 ( 2.1 45.5 ( 3.4 27.7 ( 4.7 22.7 ( 9.4 12.8 ( 1.1 17.9 ( 0.1 26.1 ( 4.1 11.3 ( 0.9 18.2 ( 1.8

a Values are averages from triplicates. Values are expressed as µg of 2,4,5-TCP day-1 per 10 ppm of added 2,4,5-TCP and were calculated at the days indicated in parentheses. b M1, 4,5-dichloroguaiacol + 4,5,6trichloroguaiacol (10 ppm each). M2, 4-chloroguaiacol + 4,5-dichloroguaiacol + 4,5,6-trichloroguaiacol + tetrachloroguaiacol (5 ppm each). c Soil preincubated for 30 days.

expressed as cfu g of soil-1. Counts in incubations with sterile soil were never observed. Growth of Soil Microorganisms in Organic Compounds. Growth using specific organic compounds as the sole energy and carbon source was tested in batch cultures with minimal salt medium, performed as previously

FIGURE 1. Evolution of 14CO2 from [14C-2,4,5]-TCP in (4) sterile soil and soil spiked with 1 (O), 10 (]), 100 (0) or 500 (×) ppm of 2,4,5-TCP. Values (mean ( standard deviation) are averages from triplicates. Sterile soil was spiked with 100 ppm of 2,4,5-TCP.

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FIGURE 2. Evolution of 14CO2 from [14C-2,4,5]-TCP in soil spiked with (0) 10 and (9) 100 ppm after 30 days of preincubation with 0 (a), 100 (b), or 500 (c) ppm of 2,4,5-TCP. Values (mean ( standard deviation) are averages of triplicates.

described (23). Substrates tested were (0.2-1.0 mM) guaiacol, 2,4-dichlorophenol, and 2,4,5-TCP. Controls without inoculation or without carbon source were performed. Cultures (1 mL) were inoculated with 0.1 mL from the clear portion of a soil slurry containing about 107 cfu g of soil-1. Cultures (triplicates) were incubated for up to 30 days. Putative microorganisms mineralizing 2,4,5-TCP were screened in incubations performed in 50-mL flasks, covered with a rubber stopper. The flasks contained 10 mL of a 0.1 mM solution of 2,4,5-TCP plus 4000 dpm of [14C-2,4,5]-TCP. Triplicates of dilutions (10-1-10-3) from different soil incubations were prepared and incubated for up to 30 days, and 14CO2 evolution was determined as indicated above.

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Results and Discussion Mineralization of 2,4,5-Trichlorophenol in Soil. The degradation of 2,4,5-TCP in this uncontaminated soil was initially detected because chloride was released (Table 1). The ability of this soil to mineralize different amounts of 2,4,5-TCP was then studied. Figure 1 shows the kinetics of 14CO evolution in soil spiked with 1, 10, 100, or 500 ppm 2 of 2,4,5-TCP. Higher mineralization rate was observed at 100 ppm (Table 2, top). A 5-day lag period before maximum mineralization rate was detected at 100 ppm. After 50 days of incubation, the rate of 14CO2 evolution decreased reaching a plateau at day 100 (Figure 1). At 1 and 10 ppm of 2,4,5TCP, the plateau was also reached after 100 days of incubation. In these two conditions, the lag period was

FIGURE 3. Evolution of 14CO2 from [14C-2,4,5]-TCP in soil spiked with 1 (O) 10 (]) or 100 (0) ppm of 2,4,5-TCP in the presence of a mixture of 4,5-dichloroguaiacol and 4,5,6-trichloroguaiacol (10 ppm each). Values (mean ( standard deviation) are averages of triplicates.

shorter and maximum specific rates of mineralization were about two times lower than for soils spiked with 100 ppm (Table 2, top). At the higher level of 2,4,5-TCP tested (500 ppm), the mineralization rate was negligible and not different from sterile soil amended with 100 ppm (Figure 1) or 10 ppm (not shown). Minimal half-lives of 35-170 days were estimated from the rates of 14CO2 evolution in soil incubated with 1-100 ppm of 2,4,5-TCP. These values are similar to estimated half-lives of 23-690 days for 2,4,5TCP, reported previously (11). The effect of the water content in the mineralization of 2,4,5-TCP was studied in soil amended with 100 ppm of 2,4,5-TCP, at a whc of 85%, 170%, 250%, or 340%. After 40 days of incubation, 14CO2 evolution was 4-5 times higher in soil at a whc of 85% or 170% than with a whc of 250% or 340% (data not shown). Also, a 5-day lag was detected with 0.25 or 0.5 mL of H2O/g of soil, whereas at 0.75 or 1.0 mL H2O/g of soil, mineralization started only after 20 days (data not shown). Although chloride was completely released after 40 days, carbon evolved as CO2 was (as a maximum) 45-49%, even upon prolonged incubation. Several reasons may explain this apparent contradiction. First, it is known that most dehalogenation reactions take place at initial steps of microbial metabolism (3, 4, 7), preceding biochemical transformations that produce most of evolved CO2 (tricarboxylic acid cycle). Second, with the exception of true cometabolism, microbial metabolism of organic compounds never transforms all the available carbon into CO2, since carbon may be incorporated into the cell biomass and/or remain as chloride-free dead-end metabolites (3, 4, 7). Finally, these dechlorinated intermediates could react with the soil matrix and become unavailable for further biotransformation. Aqueous extracts of incubated nonsterile and sterile soil contained about 5% and 20% of the initially added label, respectively. The latter suggests that most labeled material remained sorbed to the soil matrix. Unfortunately, volatilization of the label after organic solvent extraction did not allow us to perform mass balances for this material. Preincubation of Soil with 2,4,5-TCP. When plain, unspiked soil was preincubated for 30 days and then spiked with 10 or 100 ppm of 2,4,5-TCP, mineralization proceeded at similar rates as those experiments performed without

preincubation (compare top and middle of Table 2). A 5-day lag phase was detected when these soil samples were amended with 10 or 100 ppm (Figure 2a). Specific rates of mineralization for the second addition of 2,4,5-TCP were two times higher in preincubated soil spiked with 10 or 100 ppm of 2,4,5-TCP than in unspiked preincubated soil (Table 2, middle). Mineralization of 2,4,5-TCP showed a shorter lag phase when preincubated soil (with 10 or 100 ppm of 2,4,5-TCP) was amended with an additional 10 (data not shown) or 100 ppm of 2,4,5-TCP (Figure 2b). In contrast, when soil was preincubated with 500 ppm of 2,4,5-TCP, evolution of 14CO2 started only after 20 days of the addition of 10 or 100 ppm of 2,4,5-TCP (Figure 2c). Maximum removal rates after consecutive additions of 90 ppm of 2,4-dichlorophenol have been reported during soil acclimation to degrade this chlorophenol (25). On the other hand, a 10-day period of adaptation in the mineralization of 100 ppm of pentachlorophenol have been reported in a soil containing initially 4 ppm of this chloroaromatic (26). In the same study, a substantial reduction of pentachlorophenol removal was found in a soil containing 320 ppm of this chlorophenol, suggesting that the concentration of chloroorganics in the previous exposure is important in the adaptation period. The results of the present report support this statement. Effect of Chloroguaiacols in 2,4,5-TCP Mineralization. Chlorophenols and chloroguaiacols are present in effluents from chlorine bleaching of pulp (27). Because they may, eventually, end up in soil after disposal of the solids from the effluent treatment (28), it was interesting to know if chloroguaiacols affect the degradation of 2,4,5-TCP. The rate of mineralization in incubation with 10 or 100 ppm of 2,4,5-TCP was 30-35% lower in the presence of a mixture (M1) of 4,5-dichloroguaiacol and 4,5,6-trichloroguaiacol (10 ppm each) than in the absence of these chloroguaiacols (Table 2, bottom). Longer lag phases for soil incubated with 100 ppm of 2,4,5-TCP were observed (Figure 3). Maximum mineralization levels (Table 2, bottom) were less affected by the addition of this mixture of chloroguaiacols. However, if the mixture (M2) consisted of 4-chloroguaiacol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol, and tetrachloroguaiacol (5 ppm each), a 70-75% decrease in the rate and a 37-44% decrease in the final level of mineralization was observed (Table 2, bottom). Lag phases were

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longer (15 and 30 days) for incubations with 10 and 100 ppm of 2,4,5-TCP, respectively. The removal of 2,4,6-trichlorophenol in agricultural soil also incubated with tri- and tetrachloroguaiacol has been reported before (29). Previous work has also shown that soil is able to transform chloroguaiacols with the transient formation of chlorocatechols (5). As chlorocatechols are toxic for soil microorganisms (23, 30), their formation may explain the decrease in mineralization of 2,4,5-TCP reported here. However, GC analysis of appropriate soil incubations only showed complete removal of chlorophenol andchloroguaiacols, suggesting that chlorocatechols were not accumulated (data not shown). Effect of 2,4,5-TCP on Soil Microorganisms. The absence of mineralization in sterile soil, the lag phase observed in several experimental conditions, the effect of the water content, the amount of 2,4,5-TCP, the presence of chloroguaiacols, and the preincubation with 2,4,5-TCP strongly support the involvement of microorganisms in this degradative process. The ability of soil microorganisms to mineralize and dechlorinate 2,4,5-TCP in uncontaminated soils has not been reported previously. Degradation of 2,4,5-TCP and other chlorophenols has been studied in relatively pristine lake water (31, 32), where humic, phenolic substances are present. In that system, about 10% of 2,4,5TCP (1 ppm) was mineralized, which is in the same range of our study. On the other hand, the possible effect of 2,4,5-TCP in the adaptation of organochlorine-degrading soil microorganisms during incubation was followed in two ways. First, the isolation through batch enrichment of 2,4,5-TCP, 2,4dichlorophenol, or guaiacol degraders was attempted. Except for guaiacol, no microorganisms able to grow using such compounds as the sole carbon and energy source could be isolated. Second, the detection of microorganisms mineralizing 2,4,5-TCP was attempted in plain soil and in two soil samples exhibiting high levels of mineralization (100 ppm of 2,4,5-TCP, 30 days of incubation). Despite a prolonged incubation (up to 30 days), 14CO2 evolution could not be observed. The failure to isolate 2,4,5-TCP degraders is not surprising. Bacterial strains able to grow on 2,4,5TCP have not been isolated directly. Pseudomonas putida AC1100 was obtained after “plasmid assisted breeding” (13) and Nocardiodes simplex came from a soil exposed for long time to 2,4,5-trichlorophenoxyacetate (14). On the other hand, Lang et al. obtained a mixed culture that proliferates using the 2,4,5-TCP as the sole carbon source from a soil sample contaminated with several other chloroorganics (33). Our inability to isolate 2,4,5-TCP degraders could be also explained by the toxicity of this compound to bacterial strains (33, 34). Alternatively, the microbial population that mineralizes 2,4,5-TCP could be nonculturable (as is reported in ref 35), or it requires a growth substrate or growth factor in addition to 2,4,5-TCP to grow on minimal media. On the other hand, exposure of microbiota to 2,4,5-TCP or their degradation products did not affect total bacterial counts and, probably, putative 2,4,5-TCP degraders. Previous work has shown no correlation between cell counts and the presence of 2,4,5-TCP and other contaminants in several soil samples (33).

Grants FONDEF FI-17 and FONDECYT 0558/93 from CONICYT-CHILE. We are grateful to Jaime Eyzaguirre for a careful reading of the manuscript.

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Received for review June 5, 1995. Revised manuscript received October 23, 1995. Accepted January 9, 1996.X ES950381U

Acknowledgments This work was supported by Grant 92-021 RG/BIO/LA from the Third World Academy of Sciences and, partially, by

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Abstract published in Advance ACS Abstracts, March 15, 1996.