Effects of Different Quinoid Redox Mediators on the Anaerobic

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Environ. Sci. Technol. 2002, 36, 1497-1504

Effects of Different Quinoid Redox Mediators on the Anaerobic Reduction of Azo Dyes by Bacteria JO ¨ RG RAU, HANS-JOACHIM KNACKMUSS, AND ANDREAS STOLZ* Institut fu ¨ r Mikrobiologie, Universita¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

The addition of quinoid redox mediators to anaerobically incubated cultures of various taxonomically different bacterial species resulted in significantly increased reduction rates for the azo dye amaranth. From different quinones tested, generally anthraquinone-2-sulfonate (AQS) and lawsone (2-hydroxy-1,4-naphthoquinone) caused the highest increase in the azoreductase activities. The effects of AQS and lawsone were studied in greater detail with Sphingomonas xenophaga BN6 and Escherichia coli K12. Both strains reduced the quinones under anaerobic conditions with significantly different relative activities. The chemically reduced forms of AQS, lawsone, and different other quinones were assayed for their ability to decolorize amaranth, and a good correlation between the redox potentials of the quinones and the reduction rates of the azo dyes was observed. The addition of AQS or lawsone also increased the ability of unacclimated sewage sludge to reduce azo dyes. Chemically pure lawsone could be replaced by the powdered leaves of the henna plant which contain significant amounts of lawsone.

Introduction Sulfonated azo compounds are widely used as dyes for textiles, food, and cosmetics. In conventional sewage treatment plants sulfonated azo dyes usually resist biodegradation and are either precipitated with the sewage sludge or released by the effluent into the rivers (1, 2). One possible strategy for the biological treatment of sulfonated azo compounds is an anaerobic/aerobic treatment. This strategy is based on the observation that various bacterial strains reduce azo dyes under anaerobic conditions to the corresponding amines. The latter may be mineralized in a subsequent aerobic process (3-12). The unspecific anaerobic reduction of the azo compounds in these systems usually proceeds rather slowly and an acceleration of these reactions would facilitate possible technical applications. It has been demonstrated recently that the rate for the anaerobic reduction of azo dyes by Sphingomonas xenophaga BN6 could be significantly increased by the addition of different quinones, such as anthraquinone-2-sulfonate or 2-hydroxy-1,4-naphthoquinone (13, 14). It was suggested that the quinones acted in this system as redox mediators which were enzymatically reduced by the cells of S. xenophaga BN6 to the corresponding hydroquinones which subsequently reduced the azo dyes in the culture supernatants in a purely chemical redox reaction (Figure 1). * Corresponding author phone: +49 711 6855489; fax: +49 711 6855725; e-mail: [email protected]. 10.1021/es010227+ CCC: $22.00 Published on Web 03/01/2002

 2002 American Chemical Society

During the last years evidence is accumulating that quinoid compounds (especially humic substances) can play important roles as redox mediators in anaerobic reduction processes (15, 16). Thus it was demonstrated that the addition of naphthoquinones and “natural organic matter“ (presumed humic substances) could significantly enhance the reduction rate of nitro aromatic compounds and hexachloroethan in the presence of “bulk reductants“ (e.g. H2S) (17-20). Furthermore, it had been recently shown that strictly anaerobic Fe(III)-reducing bacteria use the reduction of quinone moieties of humic substances (and also sulfonated anthraquinones) to transfer the reduction equivalents released during the anaerobic oxidation of organic substrates (21). Because the utilization of quinoid redox mediators should allow very unspecific reduction processes with various azo dyes (and also other xenobiotic compounds), in the present study the kinetics and possible applicability of this reaction for the treatment of azo compounds was analyzed in greater detail.

Materials and Methods Bacterial Strains and Media. Rhizobium radiobacter d3 (formerly Agrobacterium tumefaciens d3; DSM 9674), Bacillus subtilis (DSM 618), Escherichia coli K12, Hydrogenophaga palleronii (DSM 63), Ralstonia eutropha 335 (DSM 531), Rhodococcus erythropolis MP50 (DSM 9675), and Sphingomonas xenophaga BN6 (DSM 6383) were taken from the strain collection of the institute. Lactobacillus plantarum (DSM 20174), Flexibacter filiformis (DSM 527), and Halobacterium salinarum (DSM 670) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). R. radiobacter d3, S. xenophaga BN6, R. erythropolis MP50, R. eutropha 335, and B. subtilis were grown at 30 °C and E. coli at 37 °C in Nutrient Broth (NB) medium and H. palleronii at 30 °C in Luria-Bertani (LB) medium. L. plantarum and F. filiformis were cultivated at 30 °C and H. salinarum at 37 °C in the media proposed by the DSMZ (media no. 11, 49, and 97, respectively). Determination of the UV/Vis Spectra of the Oxidized and Reduced Forms of Different Redox Mediators in Aqueous Solutions under Physiological Conditions. The concentration of amaranth was routinely determined spectrophotometrically at its absorbance maximum (λ ) 520 nm, 520 nm ) 26.6 mM-1 cm-1). These measurements were disturbed in the presence of certain mediator compounds, such as anthraquinone-2-sulfonate (AQS), anthraquinone2,6-disulfonate (AQ-2,6-DS), or lawsone (LQ), because the oxidized or reduced forms of these compounds showed considerable absorbance in this region of the visible spectrum. Therefore, the molar extinction coefficients of these compounds were determined under physiological conditions (Figure 2). Consequently, the (chemical) reduction of amaranth by the reduced form of AQS was determined at λ ) 550 nm ( ) 20.5 mM-1 cm-1) and at λ ) 570 nm in the presence of LQ or the reduced form of AQ-2,6-DS ( ) 12.2 mM-1 cm-1). Effects of Different Quinones on the Anaerobic Reduction of Amaranth by Different Bacterial Strains. The cultures (100-250 mL) were grown aerobically under the conditions indicated above until they reached the late exponential growth phase. Cells were harvested by centrifugation (8000g), washed, and resuspended in Na/K-phosphate buffer (50 mM, pH 7.7) to an optical density (OD546 nm) of about 5-15. (This buffer was amended with 25% w/w NaCl for H. salinarum.) VOL. 36, NO. 7, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Proposed mechanisms for the redox mediator dependent reduction of azo dyes by S. xenophaga BN6.

FIGURE 2. Comparison of the UV/vis spectra of amaranth (A), AQS (B), and lawsone (C) and their respective reduction products. The molar extinction coefficients were determined in Na/K-phosphate buffer (50 mM, pH 7.7). The dye and the quinones were chemically reduced by hydrogenation, and the spectra of the reduced forms were determined under anaerobic conditions using a microtiter plate reader located in an anaerobic chamber. The UV/vis spectra of the original compounds are shown as solid lines, those of the reduction products as thin lines. These cell suspensions were transferred to serum bottles, and oxygen was removed by repeated evacuation and flushing with nitrogen gas. The serum bottles were transferred to an anaerobic incubation chamber (Toepfer Lab System, Go¨ppingen, Germany), and aliquots (usually 20 µL) were transferred under strictly anaerobic conditions to the wells of a 96-wells microtiter plate. The wells of the microtiter plates contained in the standard tests in a total volume of 200 µL of 50 mM Na/K-phosphate buffer (pH 7.7), 10 mM glucose, 0.1 mM amaranth, 0.1-1 mM of the respective quinone and cells (OD546 nm 0.5-1.5). The microtiter plates were transferred to a microtiterplate reader (Power Wave 340; Biotek Kontron, Neufahrn, Germany), which was located inside the anaerobic chamber, and the decrease in absorbance was determined at 520 nm for 30 min at 30 °C (using 1 min measuring intervals). Chemical Reduction of Quinoid Redox Mediators. To obtain standards for the quantitation of the reduced forms of the quinoid redox mediators and for the analysis of the redox reactions of the quinones with the dyes the quinones were reduced chemically under a hydrogen atmosphere in the presence of a hydrogenation catalyst (22). The UV/vis spectra of the solutions with the reduction products were measured under anaerobic conditions with a microplate reader located in an anaerobic chamber. Identification of the Rate-Limiting Step in the Reduction of Amaranth by S. xenophaga and E. coli in the Presence of the Redox Mediators Lawsone or AQS. In an anaerobic chamber cuvettes were prepared which contained in a total volume of 1 mL of 50 µmol Na/K phosphate buffer (pH 7.7), 10 µmol of glucose, 0.1 µmol of amaranth, and resting cells (OD546 nm ) 0.5) of S. xenophaga or E. coli. Two cuvettes were prepared for each strain, and one of them was additionally supplied with 0.25 µmol of AQS (for S. xenophaga BN6) or 0.25 µmol of lawsone (for E. coli). The cuvettes were closed with gastight rubber stoppers, and the decolorization of amaranth (570 nm ) 12.2 mM-1 cm-1) was measured spectrophotometrically (with a Cary 50 spectrophotometer) at 30 °C in the presence or absence of the respective quinone. 1498

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Simultaneously, the concentrations of the reduced forms of AQS and lawsone were determined at λ ) 398 or 450 nm, respectively (398 nm,AQSred ) 6.7 mM-1 cm-1, 450 nm,LQ ) 2.8 mM-1 cm-1). The extinction values indicated were corrected for the changes in absorbance at the respective wavelength caused by the sedimentation of the cells, which were determined in control experiments with cells of the same optical density suspended in Na/K-phosphate buffer without dye and quinones. Polarography. The reduction potentials of the quinones (0.1 mM each) were measured in aqueous solution (10 mL, pH 7.0) in 0.1 M K-phosphate buffer plus 2% (v/v) methanol using an EG&G Princeton Applied Research Corp. 264A polarographic analyzer, which was equipped with a standard mercury hanging drop electrode (model 303A, PARC, Princeton, NJ). The instrument conditions were as follows: 4 min purge time (N2), 0 to -900 mV scan range (relative to Ag/AgCl), 50 mV pulse size, and 10 mV s-1 scan rate. The reactions were calibrated with different quinones with known reduction potentials (23). Experiments with Activated Sludge. The activated sludges were obtained from the aerobic parts of the sewage treatment plants of the University of Stuttgart (Bu¨snau) or the city of Waiblingen (near Stuttgart). Particulate material was collected by centrifugation (8500g, 10 min) and resuspended in Na/ K-phosphate buffer (50 mM, pH 7.7). The reactions were performed at 30 °C under anaerobic conditions in serum bottles which were contained in a total volume of 6 mL Na/ K-phosphate buffer (50 mM), glucose (10 mM), amaranth (0.5 mM), biomass (corresponding to 0.7-1.3 mg of protein ml-1), and different concentrations (0-1.0 mM) of the quinones. After different time intervals (10 min- 24 h) aliquots were taken from the serum bottles, the cells were removed by centrifugation, and the concentration of amaranth was determined spectrophotometrically at 570 nm. Determination of Protein Content. The protein content of whole cells and activated sludge was determined by a modification of the Biuret-assay (24) with bovine serum albumin as standard.

FIGURE 3. Chemical structures of different quinones used in the present study as potential redox mediators. AlQ alizarin; AQC anthraquinone2-carboxylate; AQS anthraquinone-2-sulfonate; AQ-1,5-DS anthraquinone-1,5-disulfonate; AQ-2,6-DS anthraquinone-2,6-disulfonate; LpQ lapachol; LQ lawsone; PQ plumbagin; MQ menadione; JQ juglone; 1,4-NQ 1,4-naphthoquinone; 1,2-NQ 1,2-naphthoquinone; 4-A-1,2-NQ 4-amino-1,2-naphthoquinone; 1,2-NQ-4-S 1,2-naphthoquinone-4-sulfonate; BQ 1,4-benzoquinone. Chemicals. Alizarine, juglone, lapachol, plumbagin, menadione, and the anthraquinonesulfonates were supplied by Sigma (Deisenhofen, Germany), and anthraquinone-2carboxylate and lawsone were supplied by Aldrich (Steinheim, Germany). Stock solutions of the quinones were prepared in water (AQS, AQ-1,5-DS, AQ-2,6-DS, 1,2-naphthoquinone4-sulfonate, 10 mM each), 2% (v/v) methanol (lawsone, 2.5 mM), or methanol (1,2- and 1,4-naphthoquinone, 4-amino1,2-naphthoquinone, 1,4-benzoquinone, lapachol, juglone, plumbagine, alizarin, and menadione, 10 mM each). The sources, generic names, and CI numbers of the azo dyes investigated were as follows: Acid Red 27 (Amaranth, CI 16185), Acid Orange 7 (Orange II, CI 15510), Acid Yellow 23 (Tartrazine, CI 19140) were obtained from Sigma, Acid Orange 20 (Orange I, CI 14600) from Fluka (Buchs, Switzerland), and Acid Black 1 (CI 20470) from Aldrich. Food Yellow 3 (Sunset Yellow, CI 15985), Acid Red 1 (CI 18050), Acid Red 14 (CI 14720), Acid Red 18 (CI 16255), and Food Black 1 (CI 28440) were kindly supplied by Warner-Jenkinson (Bielefeld, Germany). The sources of all other chemicals have been described before (6, 22, 25).

Results Influence of Different Quinones on the Anaerobic Reduction of Amaranth by Whole Cells of Different Bacterial Strains. It was previously shown that the presence of different quinones such as anthraquinone-2-sulfonate (AQS), anthraquinone-2,6-disulfonate (AQ-2,6-DS), or 2-hydroxy-1,4naphthoquinone (lawsone, LQ) considerably increased the rate of the anaerobic azo dye reduction by S. xenophaga BN6 (14). It was therefore tested if these and other quinones (for structural formulas, see Figure 3) showed a similar effect on other bacterial strains. Actually it was found that the addition of different quinones significantly increased the rate of decolorization of the model dye amaranth (Acid Red 27) by various bacteria (for structural formulas of the azo dyes used in this study, see Figure 4). Surprisingly, the effects of different redox mediators varied significantly among different bacterial strains (Table 1). Thus, for strain BN6 the addition of AQS allowed higher decolorization rates than lawsone. In contrast, this relationship was inversed for E. coli, which in the presence of lawsone (0.5 mM) showed an azo reductase activity (24 µmol min-1 per g of protein), which was about five times higher than the activity of S. xenophaga BN6 after the addition

of the same concentration of AQS (5 µmol min-1 per g of protein). Analysis of the Chemical Reduction of Amaranth by the Reduced Forms of AQS and Lawsone. The proposed mechanism for the redox mediator dependent reduction of azo compounds encloses two independent reactions: first, the quinones are enzymatically reduced to the corresponding hydroquinones, and second, the hydroquinones cleave the azo dyes in a purely chemical reaction (Figure 1). Therefore, both reactions were analyzed separately. The incubation of a fixed concentration of amaranth with varying concentrations of the quinones demonstrated that the chemical reduction of amaranth by AQS or lawsone required two moles of the respective hydroquinones for the decolorization of one mole of amaranth. This suggested a complete reduction of the azo dye to the corresponding amines. The reactions showed first-order kinetics with respect to the quinones. Obviously only one quinone molecule was involved in the rate determining step. The reaction rates were clearly dependent from the redox potential of the respective quinones (Figure 5). This demonstrated that quinones with a rather positive redox potential such as menadione, 1,4naphthoquinone, or juglone are unsuitable as redox mediators for the anaerobic treatment of azo dyes. Furthermore, it became evident that the reduced form of AQS was chemically a better reductant for the decolorization of amaranth than the reduced form of lawsone. Reduction of AQS and Lawsone by Whole Cells of E. coli or S. xenophaga BN6. For the analysis of the enzymatic reduction of the quinones by the microorganisms, E. coli and S. xenophaga BN6 were incubated under anaerobic conditions with different concentrations of AQS and lawsone. The reduction of the quinones was measured spectrophotometrically. Thus it was found that cells of S. xenophaga BN6 preferentially reduced AQS in the relevant concentration range (0.05-1.0 mM) compared to lawsone. In contrast, under these conditions, cells of E. coli reduced lawsone with significant higher activities than AQS (Figure 6). Furthermore, it was observed that in the system E. coli/lawsone significantly higher specific activities for the reduction of the quinones could be found than with S. xenophaga and AQS. Identification of the Rate-Determining Steps in the Lawsone or AQS-Dependent Decolorization of Amaranth. AQS and lawsone have significantly different two-electron standard redox potentials (E′0 ) -225 mV and E′0 ) -137 mV, VOL. 36, NO. 7, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Structures of the azo dyes used in this study. A Acid Red 27 (amaranth); B Acid Orange 20; C Acid Orange 7; D Food Yellow 3; E Acid Red 18; F Food Red 17; G Acid Red 14; H Acid Red 1; I Acid Yellow 23; J Acid Black 1; K Food Black 1.

FIGURE 5. Correlation between the redox potential of different hydroquinone/quinone redox couples and the ability of the respective hydroquinones to chemically reduce the azo dye amaranth. The hydroquinones were chemically prepared by hydrogenation from the corresponding quinones and incubated under anaerobic conditions with amaranth. Those hydroquinones which did not decolorize amaranth are show as open symbols with their respective redox potentials. The standard redox potentials for alizarin (AlQ), anthraquinone-2-carboxylate (AQC), lapachol (LpQ), and plumbagin (PQ) were experimentally determined, and the other values were taken from Fultz and Durst (23) and Vienozˇinskis et al. (40). For the abbreviations used see Figure 3. respectively) (23). The corresponding reduction potential for amaranth has been described as E′0 ) -250 mV (26). It was therefore necessary to determine the rate-limiting steps for the microbial reduction of amaranth in the presence of both redox mediators. Thus, resting cells of S. xenophaga BN6 or E. coli were incubated under anaerobic conditions with amaranth and AQS (S. xenophaga BN6) or lawsone (E. coli). In the experiment with AQS as redox mediator only small concentrations of the reduced form of AQS were observed during the reduction of amaranth (Figure 7A). However, after the complete conversion of the azo dye, a significant increase in the concentration of the reduced form of AQS was found. In a control experiment without AQS only a much lower 1500

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FIGURE 6. Reduction of different concentrations of lawsone ([) and AQS (2) by resting cells of S. xenophaga BN6 (A) and E. coli (B) under anaerobic conditions. The bacteria were grown aerobically with NB, washed, and resupended in Na/K phosphate buffer (50 mM, pH 7.7) to an optical density (OD546 nm) of about 5. Oxygen was removed by repeated evacuation and flushing with nitrogen gas, and the cells were transferred in an anaerobic chamber to a microtiter plate. Different concentrations of the respective redox mediators and glucose (10 mM) were added and the increase in absorbance measured at 398 nm (for AQS; E ) 6.7 mM-1 cm -1) or the decrease in absorbance at 450 nm (for lawsone; E ) 2.8 mM-1 cm -1) determined using a microtiter plate reader. basal activity was found. These experiments suggested that the enzymatically reduced form of AQS reacted almost

form of lawsone was found during the reduction of amaranth (Figure 7B). This suggested that during the utilization of lawsone as redox mediator, the chemical reduction of the amaranth by the hydroquinone participated significantly in the rate-limiting step. Conversion of Various Azo Dyes by Cells of E. coli and S. xenophaga BN6 in the Presence of the Redox Mediators AQS or Lawsone. The addition of lawsone or AQS resulted in a significantly increased reduction rate for amaranth by E. coli, S. xenophaga, and various other bacteria. To demonstrate the general applicability of this system for the treatment of textile wastewaters, various sulfonated azo dyes (for structural formulas see Figure 4) were incubated under anaerobic conditions with E. coli and S. xenophaga in the absence or presence of the redox mediators. Thus it was demonstrated that the addition of the redox mediators increased the reduction rates of all azo dyes tested (Table 2). Furthermore, it became evident that with all dyes tested AQS was more effective than lawsone with strain BN6. In contrast, lawsone was a more effective redox mediator with cells of E. coli. The observed variations in the relative increase of the dye reduction rates for different dyes were presumably due to differences in the redox potentials of the individual azo compounds.

FIGURE 7. Identification of the rate-limiting step in the reduction of amaranth by S. xenophaga and E. coli in the presence of the redox mediators lawsone or AQS. The oxygen free cuvettes contained in a total volume of 1 mL of 50 µmol Na/K phosphate buffer (pH 7.7), 10 µmol of glucose, 0.1 µmol of amaranth, and resting cells (OD546 nm ) 0.5) of S. xenophaga (A) or E. coli (B). Two cuvettes were prepared for each strain, and one of them was additionally supplied with 0.25 µmol of AQS (for S. xenophaga BN6) or 0.25 µmol of lawsone (for E. coli). The decolorization of amaranth was measured spectrophotometrically in the presence (b) or absence (O) of the respective quinone. Simultaneously, the concentrations of the reduced forms of AQS (2) and lawsone ([) were determined. instantaneously with amaranth, which resulted in a very low steady-state concentration of the reduced form of AQS as long as amaranth was present. Thus with AQS, the enzymatic reduction of the quinone occurs to be the rate-limiting step. In a similar experiment with lawsone as redox mediator a significant increase in the concentration of the reduced

Comparison of the Effect of AQS and Lawsone on the Ability of Aerobic Activated Sludge To Reductively Cleave Amaranth. The results presented above suggested that in different bacterial species either AQS or lawsone were more efficient as redox mediators for the reduction of azo dyes. To test the importance of these systems for a possible application, sewage sludges from two different wastewater treatment plants were incubated with AQS or lawsone. These experiments demonstrated that both redox mediators were also effective in combination with nonadapted communal sewage sludge and that AQS showed a slightly higher efficiency in these systems (Figure 8). Effect of Henna Leaves on the Anaerobic Reduction of Amaranth by Sewage Sludge. The experiments described above were performed with pure chemicals, which presumably are too expensive for a practical treatment of wastewaters. It is well-known that some quinoid compounds are synthesized by various plants. Therefore, leaves from the henna plant (Lawsonia inermis), which contain about 1% (dry weight) of lawsone glucosides (27), were tested for their ability to enhance the reduction rate of activated sludge for amaranth. Actually, also the addition of this material resulted in a significant increase in the reduction rates (Figure 9).

TABLE 1. Effects of Various Quinones on the Ability of Different Bacteria To Reduce the Azo Dye Amaranth under Anaerobic Conditionsa anaerobic reduction of amaranth in the presence of taxonomic group

strain

AQS

γ-Proteobacteria “FCB-Phylum”

Gram--Bacteria Sphingomonas xenophaga BN6 +++ Agrobacterium tumefaciens +++ Ralstonia eutropha 335 +++ Hydrogenophaga palleronii ++ Escherichia coli K12 ++ Flexibacter filiformis ++

low GC high GC lactic acid bacteria

Bacillus subtilis Rhodococcus erythropolis Lactobacillus plantarum

Gram+-Bacteria +++++ + +

Halobacteria

Halobacterium salinarum

Archaea -

R-Proteobacteria β-Proteobacteria

a

g-1

AQ-2,6-DS

lawsone

lapachol

plumbagin

++ + ++ +

+ ++ + + +++++ +

+ + + + ++ -

++ nd nd ++ ++

+++ ++ +

++++ +++ ++

++ + +

++ + nd

-

-

-

-

nd, not done. Estimated reduction rates for amaranth: - e 0.4; + 0.5-2.0; ++ 2.1-5; +++ 5.1-10; ++++ 10.1-18; +++++ > 18 (µmol min-1 protein). The basal activities of all strains in the absence of the quinoid compounds were 0.1-0.5 µmol min-1 g-1 protein.

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TABLE 2. Effects of the Redox Mediators Lawsone and Anthraquinone-2-sulfonate on the Reduction of Various Azo Dyes by S. xenophaga BN6 and E. coli K12a reduction rates (µmol of azo dye per min and g of protein)

S. xenophaga BN6

E. coli K12

dye

λ (nm)

-

+ LQ

+ AQS

-

+ LQ

+AQS

Acid Red 27 Acid Orange 20 Acid Orange 7 Food Yellow 3 Acid Red 18 Food Red 17 Acid Red 14 Acid Red 1 Acid Yellow 23 Acid Black 1 Food Black 1

570 480 480 480 530 530 530 530 400 618 570

0.1 (0.06) 0.1 (0.04) 0.3 (0.03) 0.1 (0.12) 0.1 (0.12) 0.1 (0.11) 0.2 (0.13) 0.1 (0.1) 0.1 (0.03) 0.3 (0.15) 0.2 (0.04)

0.5 (0.04) 2.3 (0.4) 0.7 (0.02) 0.5 (0.2) 0.4 (0.2) 0.2 (0.04) 1.1 (0) 0.4 (0.04) 0.4 (0.13) 0.9 (0.09) 1.8 (0.02)

2.9 (0.03) 5.1 (0.6) 4.1 (0.4) 2.3 (0.4) 2.4 (0.4) 2.2 (0.3) 2.8 (0.13) 2.1 (0.02) 2.8 (0.03) 1.8 (0.4) 2.6 (0.4)

0.1 (0.04) 0.5 (0.11) 0.3 (0.12) 0.2 (0.06) 0.2 (0.05) 0.2 (0.06) 0.2 (0.04) 0.2 (0.06) 0.0 (0.17) 0.2 (0.02) 0.3 (0.03)

11 (0.3) 40 (2.6) 5.7 (0.6) 5.5 (0.2) 8.2 (0.2) 5.4 (0.04) 30 (4.5) 5.1 (0) 0.6 (0.3) 27 (2.7) 39 (4.9)

0.3 (0.03) 1.4 (0.08) 0.7 (0.24) 0.4 (0.05) 0.3 (0.04) 0.3 (0.04) 0.3 (0.01) 0.2 (0.06) 0.2 (0.2) 0.6 (0.02) 0.4 (0.05)

a The cells were grown and freed of oxygen as described for the standard test with amaranth in the materials and methods section. The cells were incubated in the microtiterplate assay with 0.1 mM of the respective dye and if appropriate with 0.25 mM of the respective quinones. The turnover of the dyes was measured for 1 h (2 min measuring intervals) at the indicated wavelengths. The tests were performed in duplicate, and the standard deviations are given in parentheses. The minimal activity that could be determined by this test was about 0.4 U g-1 protein.

FIGURE 8. Comparison of the effects of different concentrations of AQS and lawsone on the reduction of amaranth by sewage sludge under anaerobic conditions. The activated sludges were obtained from the aerobic parts of the sewage treatment plants in Bu1 snau (filled symbols) and Waiblingen (open symbols) located in the Stuttgart area. The assays were performed under anaerobic conditions in serum bottles in the presence of different concentrations of AQS (4, 2) or lawsone (], [) plus glucose (10 mM) as described in the Materials and Methods section.

FIGURE 9. Effect of henna leaves on the anaerobic reduction of amaranth by sewage sludge. Different amounts of powdered henna leaves (pharmaceutical grade) were incubated under the same conditions as described in Figure 8 with amaranth and sewage sludge (from Bu1 snau).

Discussion In the present manuscript, it was shown that the addition of different quinones allowed taxonomically diverse bacterial 1502

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strains to decolorize various azo dyes with significantly increased reduction rates. These observations confirmed some preliminary results which demonstrated that both activated sludge and pure cultures of strain BN6 showed very similar reduction rates for amaranth after the addition of AQS (14). Thus it became evident that in the presence of redox mediators many heterotrophic aerobic bacteria decolorize azo dyes under anaerobic conditions. Presumably, similar reactions are involved in the numerous reports about the unspecific reduction of azo dyes by anaerobically incubated activated sludge (28-32). The results obtained suggest that the addition of quinones will increase the ability of activated sludge from various sources to reduce azo dyes without any specific previous adaptation of the organisms for the aspired purpose. Because this reaction is also feasible with naturally existing, biological degradable, quinones (e.g. lawsone) (33), it appears that the application of such quinones could have some technical potential for the treatment of wastewaters containing azo dyes, which results in a significant decolorization without adding any environmentally problematic substances to the wastewater. The involvement of different low molecular weight redox mediators in the bacterial reduction of azo dyes has been repeatedly suggested (26, 34, 35). In the present manuscript, it was attempted for the first time to perform separate analysis of the (biological) reduction of the quinoid redox mediators and the chemical reduction of the azo dyes by the reduced forms of the quinones. The results obtained with different hydroquinones and amaranth as a model dye, clearly demonstrated that the standard redox potential (E′0) of the mediators should be lower than approximately -50 mV. This corresponds with the E′0 -values for different azo dyes, which have been estimated to vary between E′0 ) - 430 mV and 180 mV (26). From the data collected by Dubin and Wright (26), an E′0 of about -250 mV was calculated for amaranth. Furthermore, it is well-known that from all cofactors which are commonly involved in redox-reactions in aerobic heterotrophic bacteria, NAD(P)H has the lowest reduction potential with E′0 of about -320 mV. These values therefore set the limits for the redox mediators which can be used in this system, because redox mediators with a significantly more negative redox potential will not be reduced by the cells and redox mediators with a E′0 > -50 mV will not reduce the azo bond at sufficient rates. Quinones may undergo one-electron reduction processes to the corresponding hydroquinone radicals or two-electron reductions to the corresponding hydroquinones. Therefore,

one-electron and two-electron potentials can be used to compare the relative tendency of different quinones to take up reduction equivalents. The redox potentials used throughout the present manuscript were the two-electron potentials. This is somehow in contrast to previous work about the reduction of nitroaromatic compounds in anaerobic environments which demonstrated that the relative reduction rates of the nitroaromatic compounds could be predicted from the one-electron potentials. It was suggested that in this system the first reductive step from the nitro compound to the nitroaromatic radical anion is in most cases the rate determining step and thus determine the velocity of the complete reductive process from the nitro- to the aminogroup (18, 19). From the (scarce) available literature it appears that the rate limiting step during the reduction of azo compounds is somehow different from the situation with nitro compounds, because it has been suggested for the polarographic reduction of azobenzene in DMF that the reduction to the anion radical would occur by a fast oneelectron transfer reaction, which was followed by a second slow electron transfer process leading to a stable dianion (36-38). Different types of reduction processes for nitro- or azo-groups may also be expected from the reaction stoichiometries because in the case of nitroaromatic compounds the second subsequent one-electron reduction step from the nitroaryl anion radical to the nitroso-compound requires the release of a water molecule, which is not relevant for the formation of a hydrazo-derivative from an azo-compound. The relative importance of the two-electron potentials for the reduction of azo compounds in our system is also suggested from literature data which demonstrated that in aqueous solutions at neutral pH-values during the chemical reduction of azobenzenes by the reduced form of anthraquinone-2-sulfonate semiquinones are kinetically not important (39). There are also some practical motives which suggest to use the two-electron potentials for a first estimate if a quinone may be useful as a redox mediator for the decolorization of azo dyes, because far more tabulated two-electron potentials are found in the literature for the relevant quinones than one-electron potentials and it is also much easier to determine the two-electron potentials of new quinones by polarographic methods. Thus, also we cannot exclude from the literature or from our experimental work that the one-electron potentials are relevant for the reactions studied here, at the present state of our knowledge it seems that it is practically useful and from a theoretical stand-point at least allowed to use the two-electron potentials. The analysis of the reaction kinetics suggested that only one mole of the redox mediators reacted with one mol of amaranth as the rate determining step. This is reasonable because this reaction is a simple bimolecular reaction, and already the reduction of the azo group to the hydrazo derivative will result in a pronounced decolorization. The incubation of a fixed concentration of amaranth with varying concentrations of the quinones demonstrated that the chemical reduction of amaranth by AQS or lawsone required two moles of the respective hydroquinones. This stoichiometry suggested a complete reduction of the azo dye to the corresponding amines and confirmed our previous results that cells of strain BN6 reduced different azo dyes under anaerobic conditions in the presence of AQS to stoichiometric amounts of the corresponding aromatic amines (14). It was previously suggested that in S. xenophaga BN6 the (membrane-bound) NADH:ubiquinone oxidoreductase of the respiratory chain is responsible for the reduction of AQS (and thus the azo-reductase activity) (14). The differences that were observed in the present study for the reduction of

lawsone and AQS by S. xenophaga and E. coli (and also other bacteria) suggested that in both strains either one enzyme activity with different substrate specifities or different enzymatic systems may be responsible for the enzymatic reduction of the relevant quinones. We are currently analyzing the molecular basis for the ability of E. coli to reduce lawsone.

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Received for review September 6, 2001. Revised manuscript received January 4, 2002. Accepted January 15, 2002. ES010227+