Treatment of Pulp and Paper Industry Process Waters with Oxalate

Chapter 5

Treatment of Pulp and Paper Industry Process Waters with Oxalate Oxidase: Compounds Interfering with the Activity 1

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Simona Larsson , Pierre Cassland , Leif J. Jönsson , and Nils-Olof Nilvebrant * Downloaded by PENNSYLVANIA STATE UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: August 7, 2003 | doi: 10.1021/bk-2003-0855.ch005

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STFI Swedish Pulp and Paper Research Institute, Box 5604, SE-114 86 Stockholm, Sweden Applied Microbiology, Lund University/Lund Institute of Technology, SE-221 00 Lund, Sweden Biochemistry, Division for Chemistry, Karlstad University, SE-651 88 Karlstad, Sweden 2

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Removal of oxalic acid from bleaching filtrates using the enzyme oxalate oxidase is a possibility to prevent problems with scaling in the pulp and paper industry. Bleaching filtrates contain compounds that interfere with the action of oxalate oxidase and a selection of cations, anions and organic acids, as well as hydrogen peroxide was investigated with regard to potential inhibitory effect on oxalate oxidase. While the effects of chloride and sulfate were rather limited in the concentration range studied (up to 20 mM), chlorate severely decreased the oxalate oxidase activity at 1.5 mM and sulfite at 0.10 mM. Under the conditions investigated, oxalate oxidase could stand inclusion of hydrogen peroxide very well up to 1.0 mM, while only 30 % activity remained in the presence of 20 mM hydrogen peroxide. The effect of organic acids was studied at pH 3.8 and formic acid showed more inhibitory effect than glycolic acid, which in turn displayed more effect than acetic acid that had no inhibitory effect at a concentration of 20 mM. Among the cations studied, the negative effect on oxalate oxidase activity increased in the order: calcium, magnesium < manganese (II) < iron (III) < copper (II), iron (II).

© 2003 American Chemical Society In Applications of Enzymes to Lignocellulosics; Mansfield, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Downloaded by PENNSYLVANIA STATE UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: August 7, 2003 | doi: 10.1021/bk-2003-0855.ch005

Introduction Precipitation of calcium oxalate, also known as scaling, is a current problem in the pulp and paper industry. Oxalic acid and calcium is present in bleaching filtrates due to occurrence in the raw material, but also due to formation of oxalic acid during bleaching (1-6). In order to decrease water usage, it is getting increasingly common to recirculate process water, which increases the problem with scaling. Selective removal of oxalic acid with the enzyme oxalate oxidase is a possibility to prevent scaling (7). Oxalate oxidasefrombarley is a secreted glycoprotein that is also known as germin (8-9). It catalyses the conversion of oxalic acid and molecular oxygen to carbon dioxide and hydrogen peroxide. It is a manganese-containing homohexamer that also possesses superoxide dismutase activity. The molecular mass of the subunits has been estimated to 26 kDa (10-11). A problem with the approach to eliminate oxalic acid by enzymatic treatment is the inhibition of oxalate oxidase that occurs in authentic process waters (7). In order to make the enzymatic treatment for removal of oxalic acid more efficient, it is therefore important to identify compounds in the bleaching filtrates that interfere with oxalate oxidase and with the help of this knowledge find the most appropriate method to remove or inactivate these interfering compounds or, alternatively, for enzymatic treatment select positions where the concentration of interfering compounds is low. Coupled assays are commonly used for determination of oxalic acid in medical samples and in studies of the properties of oxalate oxidase. In the coupled assays, hydrogen peroxide is first formed in the reaction catalyzed by oxalate oxidase. The hydrogen peroxide is then utilized in a second reaction, in which a peroxidase generally is employed to oxidize an aromatic substrate to a colored compound that is quantified spectrophotometrically. When coupled assays are used to study the effect of various compounds on oxalate oxidase activity, it is not clear whether the added compounds affect the oxalate oxidase or the peroxidase. In assays that rely on quantification of molecular oxygen (12) or on reactions coupled with hydrogen peroxide (11, 13-14), the presence of substances that directly interact with molecular oxygen and hydrogen peroxide may also lead to erroneous conclusions regarding the effect on the catalytic action of oxalate oxidase. Direct assays have also been employed to measure the effect of inhibitory compounds on barley oxalate oxidase activity (15-16). In these studies, measurements of substrate degradation were performed by using radiolabeled oxalic acid in combination with barley oxalate oxidase. However, due to that bleaching filtrates were not in the focus of attention in these investigations, a rather limited number of the studied compounds were the same as those that were examined in the current work.

In Applications of Enzymes to Lignocellulosics; Mansfield, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

83 Rather than relying on any of the coupled assays, we have instead used an assay in which oxalic acid is directly quantified with ion chromatography to identify compounds that potentially could act as inhibitors of oxalate oxidase. A selection of compounds relevant for the pulp and paper industry was investigated including cations, anions and organic acids, as well as hydrogen peroxide.


Materials and Methods

Selected compounds The effect on the oxalate oxidase activity of selected inorganic cations, inorganic and organic anions, as well as hydrogen peroxide was tested. The pH of the stock solutions was adjusted to 3.8 using concentrated sodium hydroxide or hydrochloric acid. For comparing the effect of the anions chloride and sulfate, the sodium and potassium salts were used at the concentrations 100 uM, as well as 1.0, 10 and 20 mM. Sulfite (with sodium as the counter-ion) was studied at the concentrations 1.0, 10 and 100 uM, as well as 1.0, 10 and 20 mM. Chlorate was tested at the concentrations 1.0,1.5 and 2.0 mM. The effect of acetic, formic and glycolic acid at pH 3.8 was tested at the concentrations 100 uM, as well as 1.0,10 and 20 mM. Furthermore, the effect of formic acid at the concentrations 100 uM, as well as 1.0, 10 and 20 mM was tested at pH 2.2 and 4.5. The pH was adjusted with concentrated sodium hydroxide. The effects of cations, namely calcium, cupric, ferrous, ferric, magnesium and manganous ions, were studied using both the chloride and the sulfate salts. The cations were applied at the concentrations 100 uM, as well as 1.0,10 and 20 mM. The effect of hydrogen peroxide was also studied. The concentrations 100 uM, as well as 1.0,10 and 20 mM were used in the assays. All compounds were obtainedfromMerck (Darmstadt, Germany), with the exception of chlorate and oxalic acid solutions, which were obtained from Acculon Reference Standard solutions (AccuStandard Inc., New Haven, CT, USA).

Assay of oxalate oxidase activity The total volume of the assay was 2.00 mL. The assay mixture included the following components (in final concentrations): 250 mM succinate buffer (pH 3.8), 0.60 mM oxalic acid, and 100 ug/mL oxalate oxidasefrombarley seedlings (Sigma-Aldrich, St. Louis, MO, USA). The tested compounds were then added In Applications of Enzymes to Lignocellulosics; Mansfield, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

84 to the desired concentrations rangingfrom1.0 yM to 20 mM. The assay mixture was incubated at room temperature (23°C). Samples for ion chromatography analysis (200 were taken directly after the addition of oxalate oxidase (0 min) and at the end of the incubation (15 min). The samples were diluted 20 times and analysed immediately. All experiments were performed in duplicates and the mean values are presented.


Analyses Oxalic acid was determined by ion-exchange chromatography (IC) with a Dionex 2020i-series ion chromatography system and using an IonPac AS4A-SC anion-exchange column (250 mm X 4 mm) and an AG4A-SC guard column (all from Dionex, Sunnyvale, CA, USA). The mobile phase consisted of 1.7 mM NaHC0 and 1.8 mM Na C0 and was applied at aflowrate of 2.0 mL/min. The concentration of oxalic acid was calculated by comparing the area of the oxalic acid peak with that of the external standard. The EZchrom software system version 2.31 (Scientific Software Inc., Pleasanton, CA, USA) was used for the quantification procedure. 3

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Results Inorganic anions Chloride and sulfate were tested in concentrations up to 20 mM. Inclusion of chloride resulted in almost no inhibition even at the highest concentration (Table I). Sulfate was more inhibitory than chloride. Slight inhibition (10 %) was observed for sulfate already at the concentration 1.0 mM (Table I). Sodium and potassium gave the same result when used as counter ions (data not shown). Chlorate and, in particular, sulfite were much more inhibitory than chloride and sulfate (Table I). Chlorate and sulfite caused almost complete inhibition at the concentrations 1.5 and 0.10 mM, respectively.

Hydrogen peroxide The potential inhibitory effect of hydrogen peroxide was also tested since it is commonly found in the process watersfromthe pulp and paper industry, where it is used as a bleaching agent. Hydrogen peroxide did not display any inhibition at the concentrations 0.10 and 1.0 mM (Table I). However, the activity of oxalate oxidase decreased with 15 % and 70 % at hydrogen peroxide concentrations of 10 mM and 20 mM, respectively.


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Organic acids Among different carboxylic acids that are present in bleaching filtrates, acetic, formic and glycolic acids are the most prominent (17-18). The potential inhibitory effect of these three acids on oxalate oxidase was therefore tested. Acetic acid was not inhibitory in the concentration range examined. Formic acid was the most inhibitory among the three organic acids. About one fourth of the activity was left when the concentration of formic acid was 20 mM at pH 3.8 (Table II). Glycolic acid showed an intermediate effect. Oxalate oxidase displayed 45 % activity in the presence of 20 mM glycolic acid (Table II). To test whether the difference between the three carboxylic acids was associated with the different pK values, additional experiments were conducted with formic acid. Reactions with formic acid were therefore also performed at pH 2.2 and 4.5. Formic acid should be mostly undissociated at pH 2.2 and mostly dissociated at pH 4.5. Performance of the reactions at either pH 2.2 or 4.5 led to a decrease in the rate of degradation of oxalic acid compared with the standard reaction at pH 3.8 (Table II). From the data in Table II, it can be calculated that the inclusion of 10 mM formic acid gave 7 % of the initial activity at pH 4.5,42 % of the initial activity at pH 3.8, and 63 % of the initial activity at pH 2.2. The inhibitory effect of formic acid was therefore more apparent at pH 4.5 than at pH 3.8 or 2.2. a

Cations Six different cations, including calcium, cupric, ferrous, ferric, magnesium and manganous ions, were tested. Both chloride and sulfate were employed as counter ions. For calcium, inclusion of the sulfate salt resulted in more degradation than when chloride was the counter ion (Table III). Addition of 20 mM calcium chloride gave a very slow reaction rate. The ferrous and the cupric ion followed by the ferric ion were the most inhibitory cations, displaying inhibition already at the lowest concentration tested (0.10 mM) (Table III). Calcium (chloride salt), magnesium and manganese displayed rather similar effect at the concentrations 10 and 20 mM (Table III). Calcium with sulfate as counter ion caused least interference (Table III). However, the low solubility of calcium sulfate should be considered when these results are interpreted.

Discussion In an attempt to clarify what kind of compounds in bleaching filtrates that are interfering with the catalytic action of barley oxalate oxidase, the effect of different inorganic anions, organic acids, metal ions and hydrogen peroxide were investigated. These groups of compounds are relevant with regard to process waters in the pulp and paper industry. There are several previous reports



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Table I. The effect of selected inorganic anions (the sodium forms of the salts were used) and hydrogen peroxide on the activity of oxalate oxidase at pH 3.8.*

Compound

Concentration (mM)

Remaining activity

Treatment of Pulp and Paper Industry Process Waters with Oxalate

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