Removal of Selected Pharmaceuticals and Personal Care Products from Wastewater using Soybean Peroxidase.

Personal care products and pharmaceuticals have been reported in various concentrations in the effluent of municipal sewage treatment plants (STP). Although they are generally found in the nanogram to microgram per liter range, many of them might have adverse health effects on humans at these concentrations. Conventional treatments applied at the STP are unable to effectively remove most of these recalcitrant compounds, thus there is a necessity for development of alternative treatment techniques. In this article, the efficiency of enzymatic treatment using soybean peroxidase in treating some commonly found micropollutants is discussed. The target compounds were, two phenolic surfactant breakdown products, nonylphenol and octylphenol, two antimicrobial agents, Triclosan and sulfamethoxazole and three phenolic steroids. The effects of the most important parameters pH, enzyme concentration and peroxide concentration have been evaluated for each compound. The treatment of synthetic wastewater was shown to be effective (≥95% removal), except for sulfamethoxazole, in concentration ranges of 10 s of µM at neutral pH with 2-5 mU/L of catalytic activity and 2-3 molar equivalents of hydrogen peroxide. The effectiveness of the treatment has also been determined for lower concentrations (6-9 nM) which approximate those in real wastewater. A matrix effect was found in the treatment of Triclosan in spiked real wastewater indicating that re-optimization of important parameters for STP treatment would be required to achieve high removal efficiency. A reverse-phase, solid-phase extraction technique was used to concentrate target analytes in real wastewater, enabling chromatographic detection by UV absorbance.

Soybean Peroxidase-Catalyzed Treatment of Azo Dyes with or without Fe° Pretreatment.

Representative azo dyes (Acid Blue 113 [AB113] and Direct Black 38 [DB38]) were treated in a single step with soybean peroxidase (SBP) and hydrogen peroxide (H2O2), or in two steps, zero-valent iron (Fe°) pretreatment followed SBP/H2O2. The purpose of this research was to compare both treatment processes and to determine which one was the optimal for degradation of each azo dye. For AB113, the preferred process was the single-step process, 1.0 mM AB113 required 2.5 mM H2O2, 1.5 U/mL SBP at pH 4.0 for ≥ 95% color and dye removal and 30% total organic carbon (TOC) removal. For DB38, due to the products formed after Fe° reduction, which are enzyme substrates (aniline and benzidine; two of four products) a two-step process was preferred, which allowed reduction in the required SBP and H2O2 concentrations by 5- and 2-fold, respectively, compared to a single-step treatment for ≥ 95% color, dye, and aniline/benzidine removal and 88% TOC removal.

Elimination of selected heterocyclic aromatic emerging contaminants from water using soybean peroxidase.

Widespread occurrence of various heterocyclic aromatic compounds is reported in concentrations from 1 to 20 µg/L in surface and groundwater as well as influents and effluents of wastewater treatment plants around the world. These so-called emerging contaminants and their metabolites can cause adverse effects on the environment and humans, even at very low concentration, hence raised environmental concerns. In this study, feasibility of soybean peroxidase-catalyzed removal of three selected heterocyclic aromatics from water was investigated, including sensitivity to the most important operational conditions, pH (range 3.6-9.0), H2O2 concentration (range 0.10-1.50 mM), and enzyme activity (range 0.001-5.0 U/mL). 3-Hydroxycoumarin and 2-aminobenzoxaozle were found to be substrates for the enzyme, having ≥95% and 45% removal efficiency with most effective pHs of 7.0 and 6.0, respectively. Time course study was also conducted to determine the initial first-order rate constants and half-lives; half-lives normalized for enzyme activity (0.0257 and 452 min for the respective substrates) are compared with those of 21 other compounds reactive with soybean peroxidase. High-resolution mass spectrometry was employed to characterize the plausible oligomerization products of enzymatic treatment, which revealed formation of dimers and trimers of the two substrates.

Kinetics and thermodynamics of thermal inactivation for recombinant Escherichia coli cellulases, cel12B, cel8C, and polygalacturonase, peh28; biocatalysts for biofuel precursor production.

Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes' thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.

Soybean Peroxidase Catalyzed Decoloration of Acid Azo Dyes.

BACKGROUND: Some industrial manufacturing processes generate and release dyes as water pollutants, many of which are toxic and hazardous materials. There is a need for milder, greener methods for dye treatment. OBJECTIVES: The objective of the present study was to investigate and optimize azo dye decoloration by a crude soybean peroxidase (SBP), based on two dyes that have widespread industrial use, but that differ greatly in structural complexity, Acid Black 2 and Acid Orange 7, and to investigate the effects of specific parameters on the removal process. METHODS: Batch reactors were used to remove 95% of the dyes' color and to produce substantial precipitates. RESULTS: The optimum pH for enzymatic decoloration of Acid Black 2 was in the acidic region, pH 4.4, and that of Acid Orange 7 occurred under neutral conditions, pH 6.9. The minimum enzyme activity needed for sufficient removal was 1.2 U/mL for both dyes at 0.5 mM. The minimum molar hydrogen peroxide/substrate ratio was 3 for Acid Orange 7 and 2.5 for Acid Black 2 to achieve approximately 95% removal. First-order fitting of progress curve data collected under the respective optimum conditions gave half-lives of 23.9 and 28.9 minutes for Acid Orange 7 and Acid Black 2, respectively. CONCLUSIONS: The feasibility of SBP-catalyzed treatment of industrial dyes Acid Black 2 and/or Acid Orange 7, or dyes that resemble them, as they might occur in industrial effluents, was successfully demonstrated. COMPETING INTERESTS: The authors declare no competing financial interests.

Enzymatic treatment for removal of hazardous aqueous arylamines, 4,4'-methylenedianiline and 4,4'-thiodianiline.

The search for an effective and sustainable treatment method to remove the recalcitrant atom-bridged bis-anilino compounds, 4,4'-methylenedianiline (MDA) and 4,4'-thiodianiline (TDA) from water is a major challenge and focus of this study. The escalating discharge of these two toxic and carcinogenic pollutants from industrial sources may pose a serious threat to the environment. Crude soybean peroxidase (SBP), isolated from soybean seed hulls (coats), catalyzes the oxidative polymerization of these aqueous pollutants in the presence of hydrogen peroxide. The effects of several process parameters, i.e., pH, hydrogen peroxide-to-substrate concentration ratio and SBP concentration, were investigated to optimize the performance of enzymatic treatment. The minimum effective SBP concentration required for removal of MDA was 0.70 U/mL, which was higher than that of TDA (0.15 U/mL). The reaction time course to achieve ≥95% removal of these compounds from water was determined under those optimum conditions. Identification of the transformed products was performed by means of high-resolution electrospray ionization mass spectrometry. The products generally observed were protonated oxidized oxidative dimers and higher oligomers (most commonly azo-coupled products). Michaelis constant, KM, and maximum reaction velocity, Vmax, obtained from the Michaelis-Menten (M-M) model revealed that TDA had a 65-fold lower KM than MDA (indicating TDA's higher affinity for SBP), and almost 5-fold higher Vmax than MDA. A pro-forma cost analysis is presented to assess the possibility of commercialization of enzymatic treatment as an alternative to conventional/traditional treatment methods.

Enzymatic treatment of sulfonated aromatic amines generated from reductive degradation of reactive azo dyes.

Anaerobic degradation, an effective treatment process of textile industry effluent, generates sulfonated aromatic amines, which are carcinogenic, mutagenic, and resistant to microbial degradation. These aromatic amines can be effectively removed by oxidative polymerization catalyzed by peroxidase enzyme. The amines, generated in this study from the anaerobic reduction by zero-valent iron of two reactive azo dyes (Reactive Red 2 [RR2] and Reactive Black 5 [RB5]), were successfully removed (90%) by Arthromyces ramosus peroxidase (ARP). For better understanding of the process, enzymatic treatment of two model compounds, diphenylamine (DPA) and 2-amino-8-naphthol-3,6-disulfonic acid (ANDSA), were also studied. Diphenylamine has a similar diarylamine bond as RR2. The ANDSA has a similar structure as the dye reduction products. The secondary amine bond in DPA and RR2 were oxidized by ARP. Enzymatic reaction of sulfonated aromatic amines generated soluble colored compounds, which were removed by coagulant. Optimum reaction parameters were also determined.

Molecular and biochemical characterization of recombinant cel12B, cel8C, and peh28 overexpressed in Escherichia coli and their potential in biofuel production.

BACKGROUND: The high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of celB-, celC-encoded cellulases (cel12B and cel8C) and peh-encoded polygalacturonase (peh28) from Pectobacterium carotovorum subsp. carotovorum (Pcc) was carried out in our previous study using Escherichia coli as a host vector. The current study partially characterizes the enzymes' molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion. RESULTS: ß-Jelly roll topology, (α/α)6 antiparallel helices and right-handed ß-helices were the folds identified for cel12B, cel8C, and peh28, respectively, in their corresponding protein model structures. Purifications of 17.4-, 6.2-, and 6.0-fold, compared to crude extract, were achieved for cel12B and cel8C, and peh28, respectively, using specific membrane ultrafiltrations and size-exclusion chromatography. Avicel and carboxymethyl cellulose (CMC) were substrates for cel12B, whereas for cel8C catalytic activity was only shown on CMC. The enzymes displayed significant synergy on CMC but not on Avicel when tested for 3 h at 45 °C. No observed ß-glucosidase activities were identified for cel8C and cel12B when tested on p-nitrophenyl-ß-d-glucopyranoside. Activity stimulation of 130% was observed when a recombinant ß-glucosidase from Pcc was added to cel8C and cel12B as tested for 3 h at 45 °C. Optimum temperature and pH of 45 °C and 5.4, respectively, were identified for all three enzymes using various substrates. Catalytic efficiencies (kcat/Km) were calculated for cel12B and cel8C on CMC as 0.141 and 2.45 ml/mg/s respectively, at 45 °C and pH 5.0 and for peh28 on polygalacturonic acid as 4.87 ml/mg/s, at 40 °C and pH 5.0. Glucose and cellobiose were the end-products identified for cel8C, cel12B, and ß-glucosidase acting together on Avicel or CMC, while galacturonic acid and other minor co-products were identified for peh28 action on pectin. CONCLUSIONS: This study provides some insight into which parameters should be optimized when application of cel8C, cel12B, and peh28 to biomass conversion is the goal.

Removal of nitroaromatics from synthetic wastewater using two-step zero-valent iron reduction and peroxidase-catalyzed oxidative polymerization.

Degradation of nitroaromatics, which are significant environmental pollutants, is difficult to achieve. Zero-valent iron reduction of nitroaromatics coupled with peroxidase-catalyzed capture of the resulting anilines as a two-step strategy for removing nitroaromatics from wastewater and process water is investigated here. The concentration range of nitroaromatics studied was that which would be present in industrial wastewater streams. Studies were done in continuous-flow columns. The enzymatic treatment following zero-valent iron reduction was carried out in a plug-flow reactor using a crude preparation of the enzyme soybean peroxidase extracted from soybean hulls. The complete reaction time for the two steps was 5 to 5.5 hours. Operating parameters including pH, peroxide/substrate ratio, enzyme concentration, and alum concentration were optimized. Optimum conditions obtained were approximately neutral pH with a hydrogen peroxide/substrate molar ratio of 1.5 for all of the nitroaromatics tested. Alum concentrations between 50 and 100 mg/L were useful in removing the apparent color from the treated water.

Crude soybean hull peroxidase treatment of phenol in synthetic and real wastewater: enzyme economy enhanced by Triton X-100.

Soybean peroxidase (SBP)-catalyzed removal of phenol from wastewater has been demonstrated as a feasible wastewater treatment strategy and a non-ionic surfactant, Triton X-100, has the potential for increasing the enzyme economy of the process. Systematic studies on the enzyme-surfactant system have been lacking as well as demonstration of its applicability to industrial wastewater. This paper addresses those two gaps, the latter based on real wastewater from alkyd resin manufacture. The minimum effective Triton X-100 concentrations for crude SBP-catalyzed phenol conversion (≥95%) over 1-10 mM showed a linear trend. To illustrate translation of such lab results to real-world samples, this data were used to optimize crude SBP needed for phenol conversion over that concentration range. Triton X-100 increases enzyme economy by 10- to 13-fold. This treatment protocol was directly applied to tote-scale (700-1000 L) treatment of alkyd resin wastewater, with phenol ranging from 7 to 28 mM and total organic carbon content of >40 g/L, using a crude SBP extract derived from dry soybean hulls by simple aqueous elution. This extract can be used to remove phenol from a complex industrial wastewater and the process is markedly more efficient in the presence of Triton X-100. The water is thus rendered amenable to conventional biological treatment whilst the hulls could still be used in feed, thus adding further value to the crop.