Removal of aqueous phenol catalysed by a low purity soybean peroxidase

Journal of Chemical Technology and Biotechnology

J Chem Technol Biotechnol 77:851±857 (online: 2002) DOI: 10.1002/jctb.646

Removal of aqueous phenol catalysed by a low purity soybean peroxidase Katia Wilberg, Cristhiane Assenhaimer and Jorge Rubio* Laborato´rio de Tecnologia Mineral e Ambiental, Escola de Engenharia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

Abstract: The application of a low purity soybean peroxidase (LP-SBP), obtained from wasted seed hulls, as catalyst for phenol polymerisation in aqueous solution in the presence of hydrogen peroxide is described. The polymers formed precipitate out from solution and may be readily separated by physico-chemical techniques. LP-SBP offers the advantage of reduced cost compared with horseradish peroxidase (HRP). The SBP activity in fresh hulls was greater than in aged hulls and was preserved at 10 °C. There was a linear correlation between initial phenol concentration (1, 2, 5 and 10 mmol dm 3) and the minimum dosage of LP-SBP required to precipitate 95% of the phenolic polymers. Polyethylene glycol (molecular weight, 6000) at 1000 mg dm 3 did not extend LP-SBP lifetime. At all phenol concentrations tested, a retention time of about 100 min was suf®cient to achieve yields of 95%. # 2002 Society of Chemical Industry

Keywords: low purity soybean peroxidase; soybean seed hulls extract; phenol removal

1 INTRODUCTION

Phenolic compounds are found in various industrial wastewaters including those from petroleum re®ning, metal casting and wood preservation. Most of these compounds are toxic and many are carcinogens. Environmental legislation de®nes the maximum discharge limit in rivers as about 0.1 mg dm 3 of total phenols.1 However, the concentrations of phenols found in ef¯uents may vary from hundreds to thousands of mg dm 3 and their degradation is usually very dif®cult.2 Thus, efforts to develop new ef®cient methods to remove phenolic compounds from wastewaters become more and more important. Enzyme-based treatment methods appear to present advantages over conventional biological and physicochemical processes. Biological processes using activated sludge, commonly used to reduce wastewater organic content, are not very effective in reducing toxic pollutants to low levels.3 Physico-chemical treatments have low selectivity for the target pollutants and their ef®ciency decreases as the pollutant concentrations increase. Chemical oxidation processes are expensive to treat high levels of contaminants but are particularly ef®cient for dilute wastewaters. Conversely, enzymebased processes act with high speci®city, are very ef®cient in removing targeted compounds and compared with microorganisms, enzymes are easier to handle and store.4

Processes with high selectivity for removing speci®c pollutant compounds are necessary to meet increasingly strict regulations or to facilitate further treatment. Enzyme-based treatments have these features3 and are being optimised to accomplish maximum technical and economic ef®ciencies. The use of enzyme-based techniques to remove organic compounds from aqueous solution was ®rstly proposed by Klibanov and colleagues5,6 and has been continuously improved since then. These authors reported the use of puri®ed horseradish peroxidase (HRP) to remove 30 different phenols and aromatic amines with ef®ciencies exceeding 99%. Activated by hydrogen peroxide, the enzyme catalyses the oxidation of aromatic compounds, forming free radicals which undergo spontaneous polymerisation. The polymerised product precipitates out from the solution and can be readily separated by physico-chemical techniques. In this reaction, HRP presents a relatively short catalytic lifetime due to interaction between the polymers produced and the enzyme's active site.5,6 In order to minimise this inactivation, Nakamoto and Machida7 suggested the addition of compounds such as polyethylene glycol (PEG) to decrease the adsorption of polymers onto the enzyme's active site. According to Wu and colleagues8,9 the addition of PEG greatly reduced the amount of enzyme required, thus increasing the competitiveness of the process.

* Correspondence to: Jorge Rubio, Laborato´rio de Tecnologia Mineral e Ambiental, Escola de Engenharia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil E-mail: [email protected] Contract/grant sponsor: Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) Contract/grant sponsor: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (Received 10 October 2001; revised version received 11 February 2002; accepted 18 February 2002)

# 2002 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/2002/$30.00

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K Wilberg, C Assenhaimer, J Rubio

HRP has been the most researched peroxidase but its high cost encourages the search for less expensive sources of this enzyme.2 Soybean seed hulls have been identi®ed as a rich source of a peroxidase, the soybean peroxidase (SBP), and being a by-product of the soybean food industry, they provide a cheap and abundant source of peroxidase.10 Medium purity soybean peroxidase (MP-SBP) has proven to be very effective in removing phenolic compounds from water11±14 and the effect of the addition of PEG in protecting the enzyme from inactivation has also been demonstrated.12,14

Pokora and Johnson15 reported a method for oxidising organic and/or heavy metal contaminants in wastewaters, sludges or soils using SBP. Soybean seed hulls in three different forms ± slurry in water, on a solid support or directly in the ground form ± provided the enzyme for the reaction. Table 1 presents some relevant contributions, in chronological order, related to the enzymatic technique under study. Improvements in process operation as well as estimation of costs for wastewater treatment are summarised. The objective of this study was the evaluation of the ef®ciency of soybean seed hulls extracts, as a source of

Table 1. Contributions, in chronological order, related to the enzymatic technique under study

Authors, year

Contribution

Klibanov et al, 1980

Use of a high purity analytical grade HRP for the removal of over 30 different phenols and aromatic amines from water.5

Alberti and Klibanov, 1981

Use of a low purity HRP ± a crude extract of horseradishes ± for the removal of phenols and aromatic amines from water and for the treatment of wastewater from a chemical plant. Rough estimation of costs for 1 m3 of water treatment containing 1 mmol dm 3 phenols, taking into account cost of horseradishes and H2O2, was US$0.69. Commercial Fenton process was estimated at US$0.84.16

Nakamoto and Machida, 1992 Use of additives such as PEG to minimise high purity HRP inactivation.7 Johnson et al, 1992

Use of SBP obtained from soybean seed hulls for preparing phenolic resins. SBP was used both as a slurry of hulls in water solution and as a partially puri®ed enzyme according to the described techniques.17

Pokora and Johnson, 1993

Use of soybean seed hulls in three different forms ± slurry in water, on a solid support or directly in the ground form ± for oxidising organic and/or heavy metal contaminants in wastewaters. Rough estimation of costs for 1 m3 of water treatment containing 1 mmol dm 3 phenols, taking into account the cost of only soybean seed hulls, was US$0.51. Using a medium purity-industrial grade HRP, costs were estimated at US$57.60.15

Wu et al, 1993

Determination of optimum conditions to remove phenol from water using a high purity ± analytical grade HRP ± in the presence of PEG.8

Cooper and Nicell, 1996

Use of a high purity HRP and a low purity HRP ± a crude extract of horseradishes ± in the presence of PEG for the treatment of wastewater from a foundry. COD reduction was achieved only with the high purity HRP due to the organic matter present in the extract. Rough estimation of costs for 1 m3 of a foundry wastewater treatment containing 3.5 mmol dm 3 phenols, taking into account the cost of horseradishes, PEG and H2O2, was US$49.70. Using a high purity-analytical grade HRP, costs were estimated at US$69.47. Commercial Fenton process was estimated at US$8.10.2

Pokora and Johnson, 1996

Recovering SBP from soybean seed hulls using a freeze±thaw technique.18

Wright and Nicell, 1999

Characterisation of a medium purity industrial grade SBP for the removal of phenol.13

Flock et al, 1999

Use of a puri®ed SBP ± a developmental preparation ± and raw soybean seed hulls for the removal of phenol and 2-chlorophenol from water.19

Caza et al, 1999

Determination of optimum conditions to remove phenolic compounds from water using a medium purity ± industrial grade SBP.12

Kinsley and Nicell, 2000

Determination of optimum conditions to remove phenol from water using a medium purity ± industrial grade SBP in the presence and absence of PEG.14

Wilberg et al, 2000

Use of a low purity HRP ± a crude extract of horseradishes ± and PEG for the removal of phenol from water. COD reduction of about 50% was achieved. Use of a ¯otation technique for separation of polymers from water.20

Ibrahim et al, 2001

Use of an Arthromyces ramosus peroxidase (ARP) ± a developmental preparation ± in the presence of PEG to remove phenol from a re®nery wastewater. Rough estimation of costs for 1 m3 of water treatment containing 2 mmol dm 3 phenol, taking into account cost of ARP, PEG and H2O2, was US$7.03. Taking into account capital and chemical costs, phenol removal was estimated at US$7.17 for this preparation of ARP, US$1.05 for activated sludge, US$1.31 for activated carbon and US$1.56 for a low purity SBP ± a crude extract of soybean seed hulls, (based simply on the cost of soybean seed hulls it would cost US$1.42).21

852

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Soybean peroxidase removal of aqueous phenol

low purity soybean peroxidase (LP-SBP), to catalyse phenol removal, optimising the main chemical and operating parameters.

2 MATERIALS AND METHODS 2.1 Chemicals

Puri®ed catalase from bovine liver (EC 1.11.1.6, 1 300 000 U cm 3 suspension) was purchased from Merck (Germany). Polyethylene glycol with an average molecular mass of 6000 (PEG-6000) was obtained from Synth (Brazil). Hydrogen peroxide (50% w/w) was provided by Laborpeq (Brazil) and phenol (99%, loose crystals) from Nuclear (Brazil). All other chemicals used were of analytical grade. 2.2 Soybean seed hulls extracts

2.2.1 pH 6 phosphate buffer extraction Because SBP is water soluble, it can be easily extracted by homogenising soybean seed hulls in aqueous media.17 Ground soybean seed hulls (diameter >2.362 mm) obtained from Bunge Alimentos SA (South Brazil) were washed with toluene to remove organic-soluble impurities.17 One part of hulls and two parts of toluene (v/v) were put in contact in a ¯ask for 30 min using an orbital shaker. After being dried at 25 °C, the hulls were suspended (12.5 w/v) in phosphate buffer (0.1 mol cm 3) pH 6.0,22 and homogenised. Then, the hulls were ground for 5 min at room temperature using a blender and the extracts were ®ltered in a bench-scale ®lter press using non-woven fabrics as ®lter media. Peroxidase was recovered from the ®ltrate extracts by the freeze±thaw technique described below.18 2.2.2 Freeze±thaw technique Filtrate extracts were placed in the freezer ( 10 °C) until frozen (more than 6 h). Homogenates were thawed in a thermostatic bath at 25 °C. After 30 min thawing, the particles settled and were removed by centrifugation at 7500 rpm for 8 min (25 °C). For some experiments the supernatant ¯uid was vacuum-®ltered using ®lter paper to remove the remaining suspended particles. The resultant extracts were stored at 10 °C and used as an LP-SBP. Details of the freeze±thaw technique are reported elsewhere.18 2.3 Peroxidase activity assay

The peroxidase activity of freeze±thaw extracts was measured just before use, at 25 °C using phenol, 4-aminoantipyrine (4-AAP) and hydrogen peroxide (H2O2) as substrates.23 The assay mixture contained 1.5 cm3 of a 20 mmol dm 3 phenol solution, 0.75 cm3 of a 9.6 4-AAP solution, 0.3 cm3 of a 2 mmol dm 3 H2O2 solution, 0.15±0.45 cm3 of extract containing LP-SBP, and up to 0.3 cm3 phosphate buffer (0.1 mol cm 3) pH 7.4.22 All reagents were prepared using the same buffer. The assay mixture (3 cm3) was held in a 1 cm light path cuvette. The active concentration of SBP was proportional to the colour development rate J Chem Technol Biotechnol 77:851±857 (online: 2002)

measured at 510 nm, during a period in which the substrate concentration was not signi®cantly reduced. The colour development rate during this period was converted to activity using an extinction coef®cient of 7100 mol dm 3 cm 1 based on hydrogen peroxide. One unit of enzymatic activity (U) was de®ned as the amount of enzyme that converted 1.0 micromol of hydrogen peroxide per min at 25 °C and pH 7.4. The results presented are mean values of, at least, duplicate measurements.

2.4

Phenol concentration measurement Phenol concentration was measured in the supernatant solutions, after settling of the polymer aggregates, using a colorimetric assay with an analytical range of 0.03±0.12 mmol dm 3.23 For sequential phenol concentration analysis, samples drawn from the reaction mixtures were ®rst mixed with catalase to stop the reaction.24 Reagents were added in the following order to a 16 mm path round cuvette: 2 cm3 samples diluted with catalase, 1.6 cm3 of 0.25 mol dm 3 sodium bicarbonate and 0.45 cm3 of 20.8 mmol dm 3 4-AAP. After vigorous stirring, 0.45 cm3 of 83.4 mmol dm 3 potassium ferricyanide were added and mixed again. Absorbance was measured at 525 nm, 9 min after the ferricyanide addition and converted to phenol concentration using a calibration curve. Phenol removal ef®ciency was evaluated throughout the reaction residence time. The results presented are the mean values of duplicate measurements.

2.5

Chemical oxygen demand (COD) measurement The COD was measured colorimetrically using a Merck reagent test in the analytical range from 100 to 1500 mg dm 3. Samples were ®rstly digested using a Merck Thermoreactor TR 300 and the colorimetric determinations were performed at 585 nm. Results presented are the mean values of duplicate measurements.

2.6

Phenol removal procedure Duplicate tests were performed in 250 cm3 Erlenmeyer ¯asks at room temperature. The reaction medium was prepared by adding phenol, extract containing LP-SBP and H2O2 to phosphate buffer solution (0.1 mol cm 3), pH 6.0.22 When used, PEG with an average molecular weight of 6000 (PEG-6000) was added to a concentration of 1000 mg dm 3. Synthetic solutions were prepared with phenol concentrations of 1, 2, 5 and 10 mmol dm 3. The reaction was initiated by H2O2 addition in a 2:1 molar ratio of initial H2O2 to phenol. An orbital shaker at 120 rpm agitated the reacting mixtures for 3 h. This residence time was chosen to assure maximum phenol conversion.13,14 853

K Wilberg, C Assenhaimer, J Rubio

3 RESULTS AND DISCUSSION 3.1 SBP activity in soybean seed hulls

Press ®ltration of an homogenate of hulls resulted in losses of extract of about 28% by volume. These losses were not considered when calculating SBP activity in soybean seed hulls. Three different samples of soybean seed hulls (A, B and C) were used in this study over 1 year. SBP activity was measured in extracts resulting from the freeze± thaw technique as U per mass of hulls (U g 1). Figure 1 shows that initial SBP activity was different in each of the samples, being much lower in samples A (30 U g 1) and B (20 U g 1), than in sample C (85 U g 1). This is due to the fact that samples A, B and C were respectively 10, 12 and 4 months old at the time the initial activity was measured. This is in agreement with Pokora and Johnson15 who reported that SBP concentration in aged hulls was less than in fresh ones. Figure 1 also shows the preservation of SBP activity during storage time. Samples were received and stored, protected from light and humidity, under different temperature conditions. Samples A and B were stored at room temperature, while sample C was stored at 10 °C. In contrast to samples A and B, SBP activity in sample C was preserved over storage time. Thus, hulls stored at low temperatures prevent SBP from inactivation and avoid the loss of activity observed in aged hulls. Extracts of hulls used as an LP-SBP source were also kept at 10 °C and also preserved their SBP activity (data not shown). The peroxidase activity measured in sample C was 3±4 times higher than that usually found in horseradishes,2,16,20 and the use of fresher hulls results in even higher activities. However, SBP has a lower rate

Figure 1. Preservation of SBP activity in samples of soybean seed hulls during storage time. Sample A (*); sample B (); sample C (~). Samples A, B and C were 10, 12 and 4 months old respectively at the time the initial SBP activity was measured. All samples were protected from light and humidity during storage. Samples A and B were stored at room temperature and sample C was stored at 10 °C.

854

of catalysis than HRP, requiring a larger dosage to remove a given quantity of phenolic substrate.13 3.2 Removal of aqueous phenol by a low purity SBP (LP-SBP)

Enzyme-based wastewater treatments need to offer the bene®ts of low operating costs. LP-SBP extracts have potential to substitute for high cost puri®ed enzymes, although they do add organic matter to the system and so enhance the overall COD in the discharge. Hence the dosage of LP-SBP needs to be minimised to reduce COD values in the treated wastewater. LP-SBP dosage studies were performed using seed hulls from sample C and 1, 2, 5 and 10 mmol dm 3 phenol solutions. The selected condition was de®ned as the minimum dosage required to achieve 95% phenol removal. This ef®ciency was used only for comparison purposes, although greater ef®ciencies can be achieved by the technique. Three-hour batch experiments were conducted in phosphate buffer (0.1 mol cm 3), pH 6.022 using a 2:1 molar ratio of initial H2O2 to phenol. The pH value was chosen based on the data of Wright and Nicell13 who found a maximum MP-SBP activity at pH 6.4. The 2:1 molar ratio of initial H2O2 to phenol and the retention time were chosen according to the work of Kinsley and Nicell14 who performed experiments with an MP-SBP. According to Fig 2, a linear relationship with a slope of 0.8 U cm 3 mmol dm 3 was found between minimum LP-SBP dosages required to achieve 95% phenol removal and initial phenol concentrations. Experiments in the presence of 1000 mg dm 3 PEG-6000 were performed to assess the protective

Figure 2. Minimum LP-SBP dosages which are required to accomplish 95% phenol removal as a function of initial phenol concentration (1, 2, 5 and 10 mmol dm 3). Reactions were performed using a 2:1 molar ratio of initial H2O2 to phenol and a 3 h retention time.

J Chem Technol Biotechnol 77:851±857 (online: 2002)

Soybean peroxidase removal of aqueous phenol

effect of this additive over LP-SBP. According to Kinsley and Nicell,14 only PEGs having molecular weights greater than 1500 contribute to MP-SBP protection, with protection increasing with increasing PEG molecular weights. However, under the reaction conditions studied here, the protective effect of PEG was not observed nor did it promote a reduction in the amount of LP-SBP required. As a consequence, the slope of the linear relationship previously found remained unchanged. Kinsley and Nicell14 evaluated the ef®ciency of PEG of different molecular weights as an MP-SBP activity protector over phenol (1, 2, 5 and 10 mmol dm 3) catalysis removal. Batch experiments of 3 h duration were performed at pH 7.0 using a 2:1 molar ratio of initial H2O2 to phenol. These authors established a linear relationship between MP-SBP minimum required dosages and initial phenol concentrations in order to achieve 95% phenol removal. In the absence of PEG, the relationship had a slope of 1.35 U cm 3 mmol dm 3, whereas in the presence of 320 mg dm 3 PEG-35000 the slope reduced to 0.32 U cm 3 mmol dm 3. A linear relationship was also shown between minimum PEG-35000 dosages and initial phenol concentrations with a slope of 35.2 mg dm 3 mmol dm 3.14 Caza et al 12 found optimum parameters to achieve 95% phenol (1 mmol dm 3) removal after 4 h 30 min retention time using the following conditions: pH 6.0, H2O2 concentration of 1.2 mmol dm 3, MP-SBP dosage of 0.6 U cm 3 in the presence of PEG-3350 and MP-SBP dosage of 0.9 U cm 3 in the absence of PEG and a minimum effective PEG-3350 dosage of 50 mg dm 3.12 Peroxidase activity was measured using an analytical technique equivalent to that previously described,12,14, enabling minimum SBP dosages to be compared. In this work, the minimum SBP dosage to catalyse removal of 1 mmol dm 3 phenol in the absence of PEG was similar to that reported by Caza et al 12 using different H2O2 concentrations and retention times. However under the reaction conditions used by Caza et al 12 we did not achieved removal ef®ciencies of 95% (data not shown). This may have been due to impurities in the seed hulls extract utilising H2O2 in side reactions before phenol removal was completed. Therefore, an extra dosage of H2O2 is necessary to account for these impurities. It should be noted that peroxidases are partially inactivated under the presence of excess H2O2 and thus its concentration should be limited.25 Kinsley and Nicell14 reported that in the absence of PEG, minimum SBP dosages were 1.7 times greater than those found in this study and 2.5 times less in the presence of PEG. The presence of other substances in the soybean seed hulls may be acting as SBP natural protectors. This phenomenon was also observed by Cooper and Nicell2 using horseradish crude extracts as J Chem Technol Biotechnol 77:851±857 (online: 2002)

the source of HRP. This presumption could explain why less LP-SBP than MP-SBP is required to catalyse the same amount of phenol removal, and the inef®ciency of PEG in the presence of natural enzyme protectants. Among the factors affecting the costs of an enzymebased process, reaction residence time is of particular concern. A low enzyme concentration reduces material costs but increases retention time to achieve a given phenol removal level; this leads to the use of bigger reactors to treat the same ef¯uent ¯ow rate. Therefore, there is a compromise between the reduction in variable costs achieved by using less enzyme and the increase in the capital investment in building a treatment facility. Synthetic solution samples with phenol concentrations of 1, 2, 5 and 10 mmol dm 3 were treated under optimised conditions, and the course of the phenol removal was monitored during the reactor retention time. Extracts consisting of an LP-SBP were prepared using hulls from sample C. Figure 3 shows the evolution of phenol conversion over a period of 3 h. Figure 3 shows that a period of 100 min was suf®cient to achieve maximum conversion and that longer retention times did not markedly increase reaction ef®ciency. The higher the phenol concentration, the shorter the retention time required to achieve the same conversion. Whilst a 1 mmol dm 3 solution of phenol needed a retention time of 100 min, 95% conversion of the phenol in the 10 mmol dm 3 sample took only 20 min. Using data from Fig 3, initial rates of phenol conversion were calculated for the ®rst 20 min of reaction. Figure 4 shows that the reaction rates increased as the

Figure 3. Percent of phenol removal as a function of reaction retention time. Reactions were performed using minimum LP-SBP dosages which are required to accomplish 95% phenol, a 2:1 molar ratio of initial H2O2 to phenol, a 3 h retention time and initial phenol concentrations of 1 mmol dm 3 (&), 2 mmol dm 3 (^), 5 mmol dm 3 () and 10 mmol dm 3 (~).

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Figure 4. Initial rate of phenol removal as a function of initial LP-SBP dosages calculated using the data from Fig 3 for the first 20min of reaction.

initial dosages of SBP-LP increased by a linear factor of 0.06 mmol min 1 U 1. Enzyme-based conversion might best be used as a pre-treatment in conjunction with conventional treatment processes. Process feasibility will depend on the quality of the ef¯uent. An observed drawback of the technique is the residual COD that results from the high organic content of extracts used as an LP-SBP. The COD of soybean seed hulls extracts measured was about 10 000 mg dm 3. To catalyse the removal of a 1 mmol dm 3 phenol solution (224 mg dm 3 COD) a 750 mg dm 3 LP-SBP COD was added using the minimum dosage required and consequently ten times this value for a 10 mmol dm 3 phenol solution (Table 2). However, the LP-SBP COD, unlike the phenol COD, is easily biodegradable and could be readily removed in stages, such as in maturation ponds. Moreover, the LP-SBP COD can also be decreased by partial puri®cation of the enzyme. According to Srinivas et al,26 an aqueous two-phase extraction technique (ATPE) may be used to remove nucleic acids and protein contaminants with low puri®cation costs. They also suggest addition of PEG and ammonium sulfate to SBP extracts to form a system which separates into two aqueous phases, and then removing the salt content from the SBP-rich phase by dialysis. Other techniques to remove impuri-

ties from peroxidase solutions were reported by Johnson et al 17 using a protein ®xative or a detergent to the extracts to precipitate dissolved proteins and non-proteic lipophilic impurities. This stage is followed by the addition of a non-solvent for peroxidases, such as acetone or isopropanol, forming an enzyme precipitate, which may be redissolved in water, recovering SBP as the supernatant solution. The economic signi®cance of the enzyme-based technique has been assessed by several authors and is based mainly on source cost of the enzyme (Table 1). In Brazil, Bunge SA sells one package of 25 kg of hulls at R$4.00 (US$1.00 = R$2.5 rate of 08/08/2001) for animal-feeding purposes. A rough cost estimation may be calculated for treatment of 1 m3 wastewater using the experimental data obtained. Considering the minimum amount of LP-SBP to achieve 95% removal of phenol (0.8 U cm 3 mmol dm 3) and using the value of LP-SBP activity of sample C of hulls (85 U g 1), costs were estimated in US$ 0.60 m 3 mmol dm 3 phenol. This value agrees with those published by Pokora and Johnson15 and Ibrahim et al 21 and is competitive with conventional treatment processes using the Fenton reaction, activated sludge or activated carbon16,21 (Table 1).

4 CONCLUSIONS

Soybean seed hulls extracts can be used as an LP-SBP source to catalyse phenol removal in aqueous solution. A linear relationship with a slope of 0.8 U cm 3 mmol dm 3 between minimum LP-SBP dosage and initial phenol concentration was found for 95% phenol removal ef®ciency. This relationship remained unaltered when 1000 mg dm 3 of PEG-6000 was added to the solution. Minimum LP-SBP dosages were 1.7 times lower than those published by Kinsley and Nicell14 using an MP-SBP. A retention time of 100 min was suf®cient to achieve 95% phenol removal ef®ciency under the conditions studied. This retention time decreased with increasing phenol concentration.

ACKNOWLEDGEMENTS

The authors thank CoordenacËaÄo de AperfeicËoamento de Pessoal de NõÂvel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientõ®co e TecnoloÂgico (CNPq) for ®nancial support. Thanks are

COD (mg O2 dm 3) Initial phenol (mmol dm 3) Table 2. Theoretical analysis of COD resulting from the phenol removal reactions performed using the minimum dosage of LP-SBP required to achieve 95% efficiency

856

1 2 5 10

Initial Phenol

LP-SBP

Total

Final (considering 95% phenol removal)

224 448 1120 2240

750 1500 3750 7500

974 1948 4870 9740

761 1522 3806 7612

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Soybean peroxidase removal of aqueous phenol

extended to Bunge Alimentos SA for providing the samples of soybean seed hulls. REFERENCES 1 Rio Grande do Sul, (SSMA) Secretaria de SauÂde e do Meio Ambiente, State law 05/89, Brazil, pp 14±15 (1989). 2 Cooper VA and Nicell JA, Removal of phenols from a foundry wastewater using horseradish peroxidase. Water Research 30(4):954±964 (1996). 3 Aitken MD, Waste treatment applications of enzymes: opportunities and obstacles. Chemical Engineering Journal 52:B49±B58 (1993). 4 Nicell JA, Saadi KW and Buchanan ID, Phenol polymerization and precipitation by horseradish peroxidase enzyme and an additive. Bioresource Technology 54:5±16 (1995). 5 Klibanov AM, Alberti BN, Morris ED and Felshin LM, Enzymatic removal of toxic phenols and anilines from waste waters. Journal of Applied Biochemistry 2:414±421 (1980). 6 Klibanov AM and Morris ED, Horseradish peroxidase for the removal of carcinogenic aromatic amines from water. Enzyme and Microbial Technology 3(2):119±122 (1981). 7 Nakamoto S and Machida N, Phenol removal from aqueous solutions by peroxidase-catalyzed reaction using additives. Water Research 26:49±54 (1992). 8 Wu J, Taylor KE, Bewtra JK and Biswas N, Optimization of the reaction conditions for enzymatic removal of phenol from wastewater in the presence of polyethylene glycol. Water Research 27(12):1701±1706 (1993). 9 Wu Y, Taylor KE, Biswas N and Bewtra JK, Comparison of additives in the removal of phenolic compounds by peroxidase catalyzed polymerization. Water Research 31(11):2699±2704 (1997). 10 Gillikin JW and Graham JS, Puri®cation and developmental analysis of the major anionic peroxidase from the seed coat of Glycine max. Plant Physiology. 96:214±220 (1991). 11 McEldoon JP, Pokora AR and Dordick JS, Lignin peroxidasetype activity of soybean peroxidase. Enzyme and Microbial Technology 17:359±365 (1995). 12 Caza N, Bewtra JK, Biswas N and Taylor KE, Removal of phenolic compounds from synthetic wastewater using soybean peroxidase. Water Research 33(13):3012±3018 (1999). 13 Wright H and Nicell JA, Characterization of soybean peroxidase

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