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Soybean peroxidase has been shown to be effective in removal of aromatic compounds from wastewater, while the use of additives effectively reduces enzyme concentration requirement, hence overall treatment cost. Enzymatic treatment, an oxidative polymerization, was successful in removal of over 95% of both aniline and o-anisidine. The originality of this study lies in the findings that the additives, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), Triton X-100, and sodium dodecanoate (SDOD), reduced enzyme concentration requirement, while polyethylene glycol (PEG, average molar mass of 3350 g/mol) had no effect on the required enzyme concentration. In addition, the presence of SDS also enhanced treatment by improving precipitation and color removal. These results are enabling advancement of soybean peroxidase-catalyzed treatment of anilines found in wastewaters as a new sustainable method.

Retention capacity values were measured in the laboratory for three non‐aqueous phase liquids (NAPLs), PCE, TCE and gasoline. The dry soils studied were sandy loam, clay, organic top soil and peat moss. For the conditions tested, it was determined that the retention capacity increased with an increase in the NAPL's density and the soil's porosity and a decrease in the soil bulk density. The largest observed retention capacity was for peat moss and PCE at approximately 7 gg‐1. A simple model was developed to correlate the findings and allow estimation of the retention capacity values for different soils and NAPLs. Applications of the retention capacity model are illustrated with typical examples.

The potential of oxidoreductases, such as laccases and peroxidases, to remove organic pollutants from industrial wastewater and process water is addressed in this short review, with an emphasis on the peroxidase work completed or in progress in the authors’ laboratory. The major drawback to this treatment is the cost of the enzyme. However, with new sources and recent advances in the biotechnology industry, it is becoming a feasible alternative. A niche where enzymatic treatment may be first applied is not as a primary treatment but as a secondary treatment (pretreatment or polishing) coupled to existing physico-chemical or biological processes to increase their overall efficiency and economy. Soybean seed coat peroxidase is well suited because of its stability, ease of extraction, widespread availability and potential for adding to the soy value chain. Since crude enzyme often works better than purified enzyme, the only additional cost may be in concentrating the extract. This review briefly covers aspects of the enzymatic treatment such as cost, use of additives for increased enzyme economy, enzyme recycling and studies already completed on industrial wastewaters.

The present study is an overview of the literature on the occurrence and potential risks of pharmaceutical substances in the wastewater treatment plants (WWTP), natural waters and drinking water treatment plants served by the Great Lakes Basin (Canada and the USA) between the years of 2007–2012. Large number of pharmaceutical substances, including anti-inflammatories, lipid regulators, antidepressants, antibiotics, beta blockers, anti-epileptics, anti-hypertensions and stimulants, in high ng/L concentrations, has been reported in the WWTP influents. Most of these compounds have also been detected in the WWTP effluents at comparable concentrations with the exception of caffeine, cotinine and salicylic acid suggesting the inefficiency of conventional treatment processes in the degradation of pharmaceutical compounds. Decreasing surface water concentrations have been observed with the distance downstream of the discharge point due to the dilution effect. Surface waters located around septic systems and agric...

Commercial production of soybean hull peroxidase is of interest because it is a renewable biocatalyst with many potential applications, it would not compete with animal feed, land or other agricultural resources and it could be easily incorporated with crushing operations, adding value to one of the fastest growing crops in the world. Further characterization of seed coat traits in combination with desirable seed characteristics will aid growers to select cultivars with higher potential end value. Therefore, we developed a lab-scale method in order to assess the soybean hull peroxidase levels in 65 different cultivars from four different seed suppliers, using 4–5 hulls from each. The simplicity of the method mimics a unit operation which could also be incorporated into a crushing plant. Herein we show that only 31 of the 65 samples would be classified as high activity hulls (the other 34 having little to no activity). Clearly, some varieties would have higher economic potential than others for the production of soybean hull peroxidase.

BACKGROUND: Studies with peroxidase (EC 1.11.1.7) have demonstrated that phenolic precipitate adsorbs free enzyme, inactivating it. The end-product inactivation model has been proposed to explain the effect. Additives, such as polyethylene glycol and Triton X-100, were reported to effectively extend enzyme lifetime by preventing the adsorption. RESULTS: It was found that soybean peroxidase (SBP, EC 1.11.1.7) trapped in precipitate during phenol polymerization retains activity. Contrary to the end-product inactivation model, recycling precipitate effectively utilized the active SBP. The minimum SBPconcentrationrequiredforthesubsequentbatchreactionremovalof1mmolL -1 phenolfromaqueoussolutionwasreduced from 1.2 to 0.5 U mL -1 . SBP adsorption on the precipitate was proven to be reversible by the addition of Triton X-100. Thus, a new explanation of SBP fate during the reaction is suggested: SBP is immobilized in situ in an active form with reduction of specific activity rather than inactivation. The adsorption is characterized by a Langmuir isotherm. CONCLUSIONS: The phenolic precipitate immobilizes SBP in an active form, consistent with the Langmuir isotherm model. Recycling the precipitate improves the enzyme economy in phenol removal. c � 2013 Society of Chemical Industry

The use of Triton X-100 (Triton) significantly improves enzyme economy for the removal of phenol from wastewater with crude soybean peroxidase (SBP, Enzyme Commission Number 1.11.1.7), a polymerization process that forms (poly) phenolic precipitates. The observation of SBP elution from phenolic precipitates in the presence of Triton was considered a competitive adsorption displacement phenomenon in order to explain the protection mechanism (Triton effect). Both SBP and Triton adsorption on the precipitates were characterized by Langmuir adsorption isotherms as single-adsorbate systems. As a binary-adsorbate system, the adsorption of Triton gradually reversed the adsorbed SBP back into the aqueous phase. The sigmoid elution curve was consistent with an orogenic displacement mechanism. A quantitative relationship between SBP elution as a function of Triton and precipitates concentrations was established by adapting the curves to a logistic function. The concept of using phenolic precipitates as an a...

BACKGROUND: Studies with peroxidases (EC 1.11.1.7) have demonstrated extended lifetimes for conversion of phenolics into insoluble polymers in the presence of polyethylene glycol (PEG). Yet the mechanism of this ‘PEG effect’ has eluded investigators. The effectiveness of a laccase (EC 1.10.3.2) with PEGs of various average molar masses on the conversion of phenol, o-, m-, p-cresols, aniline, o-, m-, and p-toluidines was investigated. This structure-activity comparison, with and without the additive, could provide further insight into the ‘PEG effect’. RESULTS: The ‘PEG effect’ was observed only with the cresols and only with PEGs of average molar mass ≥ 1450. Other additives capable of H-bonding, such as polyvinylpyrrolidone, had similar results. When a ‘PEG effect’ was observed there was a linear relationship between the amount of additive used and the amount incorporated into the precipitate, suggesting a strong interaction between products and PEG, which was confirmed by nuclear magnetic resonance spectroscopy. Optimum pH was not affected by the quantity or molar mass of the PEG added. CONCLUSION: PEG only enhances the laccase-catalyzed oxidative coupling of certain substrates implying a product-additive relationship that may be related to the functionality and substituent positioning of the substrate. Copyright © 2011 Society of Chemical Industry

A two-step process for the removal of phenol and benzenediols, namely catechol, resorcinol, and hydroquinone, from buffered synthetic wastewater was investigated. The proposed process comprised laccase catalyzed conversion of these substrates using laccase from Trametes villosa followed by the removal of products generated using alum as coagulant. The effects of pH, laccase concentration, substrate concentration, and the presence of a hydrophilic polymer additive [polyethylene glycol (PEG)] to achieve ⩾95% removal of the substrate in a 3-h reaction period at room temperature were determined. The parent compound, phenol, required the most enzyme, followed by resorcinol, catechol, and hydroquinone. PEG showed no effect on any of the substrate conversions. Substrate conversion and enzyme inactivation were monitored over the reaction period. As phenol and benzenediols might coexist in industrial effluents, enzymatic treatment of an equimolar mixture of these four substrates was examined. Except for hydroquino...

This paper demonstrates, for the first time, the capability of soybean peroxidase (SBP), an enzyme, for catalyzing the removal of an aromatic thiol, 2-mercaptobenzothiazole (MBT), from aqueous solution. The optimum pH for enzymatic conversion of MBT in aqueous buffer was found to be in the range 6.0 to 9.0. The optimum hydrogen peroxide (H 2 O 2 ):MBT stoichiometry was 0.6. In terms of standard units (U) of catalytic activity, the minimum SBP concentration required for 95% conversion of 1.0 mM MBT in 3 hours was found to be 0.9 U/mL. The presence of polyethylene glycol at 50 mg/L can reduce the enzyme concentration required for the same conversion by 3-fold. It is proposed that these findings should be the basis for viable and cost-effective treatment of MBT in industrial wastewater and/or process water.

Soybean peroxidase (SBP), as a crude extract from soybean seed coats, was applied to remove 15 mM of phenols from coal-tar wastewater. The enzyme required for the conversion of phenols in coal-tar wastewater was less than that predicted by studies with synthetic wastewater. Step additions of both SBP and hydrogen peroxide reduced the SBP concentration requirement for >95% conversion of phenol. Polyethylene glycol (PEG) showed no improvement on the conversion efficiency, whereas sodium dodecyl sulfate (SDS) showed significant improvement that was better than step addition. Corroborative studies with synthetic wastewater have shown that Triton X-100 enabled the lowest SBP concentration for 95% conversion of 1.0 mM phenol followed by SDS, sodium dodecylbenzenesulfonate, and then PEG. Most significantly, evidence suggests that the anionic surfactants, SDS and sodium dodecylbenzenesulfonate, do not work in the same way as nonionic Triton X-100 or as PEG. Aluminum hydroxide gel (alum) was investigated ...

Crude soybean peroxidase (SBP), isolated from soybean seed coats (hulls), catalyzes the oxidative polymerization of hazardous aqueous phenylenediamines and benzenediols in the presence of hydrogen peroxide. Experiments were conducted to investigate the optimum operating conditions including pH, hydrogen peroxide-to-substrate concentration ratio and the minimum SBP concentration required to achieve at least 95% conversion of these pollutants in synthetic wastewaters. The substrate conversion and hydrogen peroxide consumption were monitored over the period of the reactions. Polyethylene glycol (PEG) was ineffective as an additive in enhancing the conversion efficiency. The enzymatically generated polymeric products from phenylenediamines could be removed with the aid of a surfactant, sodium dodecyl sulfate (SDS), whereas the polyvalent metal cation salt, aluminum sulfate (alum), was able to remove the products from benzenediols, except hydroquinone. Enzyme-catalyzed polymerization with SBP and subsequent removal of the polymeric products generated can provide an alternative means to the conventional methods for treating many aromatic wastewater pollutants, including the title compounds.

BACKGROUND: Several studies have demonstrated the feasibility of treating aqueous phenols and aromatic amines with oxidoreductases in synthetic wastewater samples. However, little work has been done on the effectiveness of enzymatic treatment on real wastewater. Here a comparison was made of the oxidative coupling of phenol catalyzed by laccase or soybean peroxidase (SBP) using synthetic and refinery wastewaters. RESULTS: Optimization of pH, enzyme concentration, effect of polyethylene glycol (PEG) addition, and reducing anions were examined for a 3 h reaction time. Laccase had an optimum pH of 5.6–6.0, while SBP had a broad optimum from 6.0 to 8.0. In synthetic samples in tap water to achieve ≥ 95% removal of 1.0 mmol L−1 phenol in 3 h required 0.12 and 1.5 U mL−1 of catalytic activity of laccase and SBP, respectively. In refinery samples comparable removals required 1.2- to 1.8-fold more enzyme than in synthetic tap water samples. Added PEG allowed for a small reduction in the SBP concentration for synthetic wastewater but was ineffective with either enzyme in treating refinery samples. Reducing ions increased the demand for oxidant but, with the exception of cyanide, phenol removal still occurred. CONCLUSION: Both laccase and SBP were effective in removing phenol from aqueous refinery samples, albeit at slightly higher concentrations than required for the corresponding synthetic samples. Copyright © 2008 Society of Chemical Industry

The priority pollutant lists of both the U.S. Environmental Protection Agency (U.S. EPA) and the European Union (EU) include diphenylamine (DPA), a contaminant found in wastewater of various industries. This work demonstrates the potential of using enzymatic treatment to remove DPA from buffered synthetic wastewater. This treatment method includes oxidative polymerization of DPA using laccase from Trametes villosa, followed by removal of those polymers via adsorptive micellar flocculation (AMF) using sodium lauryl sulfate (SDS) and alum. Researchers investigated the effects of pH, laccase concentration, molecular mass, and concentration of polyethylene glycol (PEG) in continuously stirred batch reactors to achieve 95% substrate conversion in three hours. Treatment of 0.19 mM DPA was best at pH 7 and an enzyme concentration from 0.0025 to 0.0075 standard activity unit/mL. Except for PEG 400, optimum enzyme and PEG concentrations decreased with an increase in PEG molecular mass. Optimum AMF conditions were pH 3.0 to 6.5, 200 mg/L of SDS, and 150 mg/L of alum.

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.

A two-step process for the removal of dinitrotoluene from water is presented: zero-valent iron reduction is coupled with peroxidase-catalyzed polymerization of the resulting diaminotoluenes (DAT). The effect of pH was examined in the reduction step: at pH 6 the reaction occurred much more rapidly than at pH 8. In the second step, optimal pH and substrate ratio, minimal enzyme concentration and effect of polyethylene glycol (PEG) as an additive for greater than 95% conversion of DAT, over a 3h reaction period were determined using high performance liquid chromatography. Two enzymes were investigated and compared: Arthromyces ramosus peroxidase (ARP) and soybean peroxidase (SBP). The optimal pH values were 5.4 and 5.2 for ARP and SBP, respectively, but SBP was more resistant to mild acid whereas ARP was more stable in neutral solutions. SBP was found to have a greater hydrogen peroxide demand (optimal peroxide/DAT molar ratio for SBP: 2.0 and 3.0 for 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT), respectively; for ARP: 1.5 and 2.75 for 2,4-DAT and 2,6-DAT, respectively) but required significantly less enzyme (0.01 and 0.1 U ml(-1) for 2,4-DAT and 2,6-DAT, respectively) to convert the DAT than ARP (0.4 and 1.5 U ml(-1) for 2,4-DAT and 2,6-DAT, respectively). PEG was shown to have no effect upon the degree of substrate conversion for either enzyme.

The effects of hydraulic retention time (HRT) and attachment media on the rate of microbial sulfate reduction with lactate as a carbon and energy source were investigated for sulfate reducing bacteria (SRB). Laboratory experiments were conducted in semi-continuous and continuous modes by using synthetic feed in three parallel 10-L PlexiglasTM upflow anaerobic reactors. The lactate:sulfate was maintained below 2 for proper SRB activity. Results indicated that 70% of the lactate fed was bio-oxidized and 30% was biosynthesized, which showed that the energy production reaction dominated over the synthesis reaction in non-packed reactors. Continuous mode operation of three upflow anaerobic suspended growth reactors yielded effluent sulfide concentrations as high as 190 mg/L at the optimum conditions of 20 h of HRT, 1.5 m/d of upflow velocity, and an organic loading rate (OLR) of 6 kg d–1 m–3, regardless of the presence of attachment media in the reactor. The critical HRT was 10 h. Lower retention times showed a significant decrease in the effluent sulfide concentration and washout of biomass. When the OLR was doubled, the rate of sulfide production increased proportionately. Key words: sulfate reducing bacteria (SRB), hydraulic retention time (HRT), support media, sulfide production.

The feasibility of using the enzyme laccase to treat synthetic wastewater containing bisphenol-A (BPA) was examined. Optimization of pH, laccase concentration, polyethylene glycol (PEG) as an additive for >95% conversion and precipitation of BPA over 3 h of reaction period was determined through colorimetric assay and HPLC. PEG reduced enzyme inactivation, allowing a 5.2-fold reduction in the amount of laccase required for >95% removal of BPA in the range of 0.1–1 mM over 3 h. The fate of PEG after the reaction was also monitored. Linear relationships were found between the concentration of BPA (0.1–1 mM) and the optimum concentrations of laccase and PEG. Little PEG remained in the solution when up to 75 mg/L of PEG was used to treat 0.5 mM BPA. Beyond this level, PEG concentration increased linearly in the supernatant. It is inferred that an interaction between PEG and the polymeric products resulted in the protection of laccase.

A simple and accurate method has been developed for the field and laboratory analysis of four highly toxic substances found in certain industrial wastes in high concentrations. Trinitrobenzenesulfonic acid (TNBS) was used as a colorimetric reagent for 1,4-phenylenediamine (p-PD), 1,2-phenylenediamine (o-PD), 1,3-phenylenediamine (m-PD), and aniline in phosphate buffer of pH 7.4 to form relatively stable compounds that had high absorbance near 430 nm. With sodium sulfite present in the solution at 2 mmol·L–1 or above, higher extinction coefficients were attained and good linear relationships of absorbance with PDs and aniline concentrations were observed. Test times required to achieve maximum absorbance were 60 min for p-PD and m-PD, 30 min for aniline, and 200 min for o-PD. The reliable quantitation ranges were 2–20 µmol·L–1 for p-PD and m-PD and 2–40 µmol·L–1 for o-PD and aniline with the standard error of the estimates being less than 5% in these ranges. Key words: phenylenediamine, aniline, trinitrobenzenesulfonic acid, colorimetric test method.

Several different Microbial source tracking methods (MSTs) can be used to distinguish human from animal fecal contamination in water; In this study, experiments were carried out to test the effectiveness and reliability of three bacteria based approaches (the fecal coliforms to fecal streptococci ratio, antibiotic-resistant Clostridium perfringens, and human bifidobacteria) in a simulated groundwater micro-environment. The methods were evaluated in three phases: initially, the specificity of each indicator was validated on groundwater samples affected by known pollution source; then the variation of performance with time of each method was determined, and finally, the die-off coefficients for pure species of Clostridium perfringens and Bifidobacterium adolescentis were measured. The results indicate that only the determination of human bifidobacteria concentration can be considered reliable in distinguishing human from animal pollution in groundwater at the conditions applied. Nevertheless, human bifidobacteria were detectable only for two weeks after the contamination event. This study also shows that antibiotic resistant Clostridium perfringens detected using the Shahidi-Ferguson medium is not source specific, whereas it confirms that this species can be useful for timing general fecal contamination events.

The current research demonstrates the effects of certain parameters, usually present in municipal landfill leachate, on the growth of water hyacinth (Eichhornia crassipes), a floating aquatic plant, when used for treatment of leachate. Experiments were carried out to investigate the ability of water hyacinth to grow in leachate with different salinity ions concentrations, nutrients, pH, and heavy metals concentrations. The ability of water hyacinth to remove some parameters such as nitrogen, potassium, phosphorus, total solids, and chloride were also investigated. All experiments were conducted in batch reactors in a greenhouse environment. The leachate samples were collected from Essex–Windsor Regional Landfill, Windsor, Ontario. It was found that this treatment system required the presence of sufficient amount of nutrients (N, P, and K) to ensure plant growth. Optimum growth took place when the initial chloride and sodium concentrations were 560 mg L–1 and 330 mg L–1, respectively. The leachate pH for optimum water hyacinth growth was found to be in the range of 5.8 to 6.0. Total heavy metal concentrations below 0.10 mequiv L–1 supported the plant growth; concentrations above 0.91 mequiv L–1 inhibited the plant growth. Removal efficiencies of nitrogen, potassium, phosphorus, chloride, and total solids from leachate were found to be affected by the growth of water hyacinth. Higher removals were obtained with higher plant growth. Key words: water hyacinth, municipal landfill leachate, chloride, sodium, heavy metals, water hyacinth growth, leachate treatment, bioconcentration factor.

Batch experiments were conducted to investigate the effect of municipal landfill leachate pH on the growth of water hyacinth (Eichhornia crassipes). These experiments were carried out in a green house environment on leachate samples collected from Essex-Windsor Regional Landfill, Windsor, Ontario, Canada. It was found that water hyacinth plants survived in a pH range of 4.0 to 8.0. Both alkaline pH (above 8.0) and highly acidic pH (below 4.0) had inhibitory effect on the growth of plants. The pH range, for optimum growth of the water hyacinth plants was found to be 5.8 to 6.0. At optimum growth, water hyacinth had an average mean relative growth rate of 0.043 d-1. It was found that nitrogen compounds underwent different transformations depending on the pH of leachate. Plant uptake, nitrification and volatilization were among these transformations.

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.

Phenols are present in petroleum refining wastewater. An enzymatic method for removing phenols from industrial aqueous effluent has been developed in the past several years. In this method, a peroxidase enzyme catalyzes the oxidation of phenol by hydrogen peroxide generation of phenoxyl radicals. These radicals diffuse from the active center of the enzyme into solution and react nonenzymatically to eventually form higher oligomers and polymers, which can be removed from wastewater by conventional coagulation and sedimentation or filtration. In this study, Arthromyces ramosus peroxidase (ARP) was applied to treat a petroleum refining wastewater containing 2 mM (188 mg/L) phenol in a batch and continuous-flow system. The latter consisted of a plug-flow reactor (PFR) where the reaction took place between phenol and hydrogen peroxide catalyzed by the enzyme in the presence of polyethylene glycol (PEG). A flocculation tank followed the PFR where alum and sodium hydroxide were added and then the polymers formed were settled in a sedimentation tank and removed from the system. Most (95 to 99%) of the phenol was removed by the same dose of ARP required for the treatment of synthetic wastewater containing an equal amount of phenol. Polyethylene glycol, as an additive, reduced enzyme inactivation and consequently reduced the enzyme dose and the cost of the treatment process. Step feeding of hydrogen peroxide was not effective in reducing the enzyme requirement. A significant removal of chemical oxygen demand was achieved when using PEG to reduce the enzyme dose.

Experiments were conducted to investigate the efficiency of using soybean peroxidase (SBP) to remove several different phenolic compounds from unbuffered synthetic wastewater. The phenol derivatives studied included parent phenol, chlorinated phenols, cresols, 2,4-dichlorophenol and 4,4′-isopropylidenediphenol (commonly known as bisphenol A). Optimum conditions to achieve at least 95% removal of these compounds were determined for the following parameters: pH, SBP dose in the absence and presence of polyethylene glycol (PEG), hydrogen peroxide to substrate ratio, and PEG dose. Experimental results showed that SBP efficiently removed aromatic compounds from synthetic wastewater in the presence of hydrogen peroxide. An increase in the hydrogen peroxide to substrate ratio beyond the optimum resulted in enzyme inactivation in all cases except for bisphenol A. The optimum pH for different phenolic compounds ranged from 5.5 to 8. For each substrate, the optimum enzyme dose in the presence of PEG varied significantly. The studies showed that PEG only slightly reduced the amount of SBP required for 95% removal of the substrate. For most of the substrates, an increase in PEG dose beyond the optimum dose did not significantly change the removal efficiency.

Sulfate-reducing bacteria, under anaerobic conditions, reduce sulfate, SO{sub 4}{sup {minus}2}, to sulfide, S{sup {minus}2}, which in turn can effectively precipitate heavy metals. In this research project, sulfate-reducing bacteria were grown in an upflow anaerobic fixed-film (UAFF) reactor using optimum growth conditions obtained in previous studies. These reactors were then fed with different heavy metals at increasing loading rates until complete failure occurred as metal removal reached zero and residual sulfide dropped to zero. The metal concentrations were measured as total, dissolved, and free ions both in the influent and in the effluent streams. The results of this research showed that 100% removal efficiencies could be obtained with individual concentrations up to 200 mg/L for Cu, 150 mg/L for Ni and Zn, 75 mg/L for Cr, 50 mg/L for Cd, and 40 mg/L for Pb. Also, the corresponding organic matter removal as total organic carbon was found to be about 50% of the influent total organic carbon. A set of mathematical equations were derived to express the mass balance inside the UAFF reactor, with respect to metal influent concentrations and sulfide production. These equations were corrected by incorporating a correction product, {alpha}{center_dot}{beta}, to represent the toxicity effect of the increasing metalmore » concentrations.« less

An overview of nitrification and denitrification in the natural environment is presented and the basic mechanisms of nitrification and denitrification in water/soil environments are described in this paper. Nitrification/denitrification is one of the most important techniques for removing inorganic nitrogenous compounds in water and soil environments. Nitrification and denitrification in aquatic and soil environments can be described by using First‐order kinetic and the methods for parameter estimation are discussed. The study indicated that nitrification and denitrification could take place simultaneously in aerobic and anaerobic zones in soil and water environment. The factors effecting nitrification and denitrification are discussed in this study. External carbon sources are the most important factors for completing denitrification and dissolved oxygen (DO) is very significant for nitrification.

A batch-mixed reactor and a plug-flow reactor were shown to have identical reaction kinetics for peroxidase-catalyzed removal of phenol. Semi-batch operation and step-addition operation of the plug-flow reactor with respect to hydrogen peroxide produced similar output. In the presence of polyethylene glycol, a continuous stirred tank reactor needed a long time to complete the reaction at the minimum enzyme dose. It was not necessary to stir the reaction mixture continuously during the reaction, hence polymerization and settling of products happened simultaneously. Recycling of the precipitates was not beneficial either for the removal efficiency or for the operation of a settling tank. A model previously developed to simulate the reaction in a batch reactor was used to predict the process in different types of reactor system. A plug-flow reactor system is recommended for the reaction in the presence of polyethylene glycol; the system consisting of a small mixing tank followed by a tank in which reaction and settling occur simultaneously. # 1999 Society of Chemical Industry

A kinetic model has been developed to simulate horseradish peroxidase-catalyzed polymerization of phenol in the presence of polyethylene glycol based on the following kinetics. The phenol conversion expression is a second-order Michaelis-Menten equation with respect to the concentrations of phenol and hydrogen peroxide. The enzyme inactivation is attributed to the polymer and hydrogen peroxide simultaneously. The inactivation by polymer is an apparently second-order reaction, first-order in each of enzyme and phenol, whereas the inactivation by peroxide is also an apparently second-order reaction, first-order in each of enzyme and hydrogen peroxide. The rates of consumption of hydrogen peroxide and polyethylene glycol are directly proportional to the rate of conversion of phenol. Experimental data show that the model output can predict the phenol removal and activity depletion realistically under a variety of reaction conditions. The model has been verified by predicting some independent experimental results on reaction stoichiometry, horseradish peroxidase dose effect, and semibatch operation with respect to hydrogen peroxide.

The sulfate reducing bacteria (SRB) have the capability of reducing sulfate (SO4-2) under anaerobic conditions into sulfide (S-2) which can precipitate metals as metal sulfides. The optimum conditions for sulfide production by SRB utilizing lactate, in an upflow anaerobic fixed film reactor (UAFFR) were not previously established. The main objective of this research was to investigate these conditions for the growth of SRB to ensure the highest sulfide production under consistent behaviour of the system. Substrate containing lactate as the organic carbon source along with sulfate, nitrogen and phosphorus as the required nutrients was used as a feed to the UAFER which was seeded with SRB. It was found that an optimum sulfide production occurred with an organic loading rate (OLR) of 6 kg d-1 m-3, while the theoretical oxygen demand to sulfate ratio (ThOD/SO4) ranged from 1.5 to 2.25. Also, the optimum total nitrogen and phosphorus demands were determined to be about 250 and 50 mg L-1 respectively. A total nitrogen concentration above 600 mg L-1 started showing toxicity and lowered the sulfide production. The optimum ThOD:N:P for sulfide production and growth of SRB in the UAFFR was 100:5:1 under optimum conditions.

A biological process employing green sulphur bacteria to remove sulphide (S 2− ) from industrial wastewaters and convert it to elemental sulphur was investigated. This research was unique in that dissolved sulphide was present in the liquid influent fed into a continuous-flow photosynthetic bioreactor. A suspended-growth once-through continuous-flow stirred-tank bioreactor was successfully operated under five different experimental conditions. For the first three experiments, concentrated nutrient solution and sulphide stock solution were pumped separately into a 13.7 litre reactor at a hydraulic retention time of 45 h and S 2− loading rates of 2.1, 4.4, and 5.6 mg/h·l. At the lowest loading rate, nearly all influent S 2− was oxidized to sulphate. The middle loading rate resulted in complete conversion of S 2− to elemental sulphur. Steady state conditions were not achieved at the highest loading rate, and S 2− accumulated in the bioreactor. In two more experiments, nutrient medium and S 2− stock solution were separately fed into a 12.0 litre bioreactor at S 2− loading rates of 3.2 and 2.7 mg/h·l and hydraulic retention times of 173 and 99 h respectively. In these trials, the loading rates were adjusted to maintain a residual of 20 to 30 mg S 2− /h·l in the bioreactor, and consequently, there was nearly complete conversion of the consumed S 2− to elemental sulphur. A parameter was developed to relate the results from these experiments to those reported in the literature, where smaller reactors and higher bacterial concentrations were used in batch reactors fed with H 2 S (g) . This parameter described the capacity of the bioreactor to consume S 2− , and was calculated as the product of the radiant flux per unit reactor volume and the bacteriochlorophyll concentration.

Horseradish peroxidase has been proven effective in removing phenolic compounds in wastewater and additives such as polyethylene glycol have been found very effective in reducing the minimum enzyme dose required. The effect of additives on horseradish peroxidase-catalyzed removal of phenol was investigated in this study. In the absence of additive, active enzyme is predominantly inactivated by the polymer product formed during the reaction. The specific activity of horseradish peroxidase is higher due to the presence of additive. Experiments suggest that additives combine with the polymerization products formed during the reaction, because additives have a higher partition affinity with the polymer products than peroxidases. Most of the polymer product is coupled with additive so that less enzyme interacts with the polymer product. Horseradish peroxidase still combines with polymer products and becomes inactivated but at a much slower rate when additives are present. Consequently, the enzyme activity is protected by the additives.

Several additives, polyethylene glycol, gelatin and some polyelectrolytes, were studied to compare their behaviour in the removal of phenolic compounds from aqueous solution by horseradish-peroxidase-catalyzed polymerization. The effects of additives on optimum pH range, horseradish-peroxidase saving, reaction stoichiometry and minimum additive requirements were investigated. The fate of additive after reaction was also studied. The experimental results showed that all tested additives significantly reduced the horseradish-peroxidase requirement. Comparison among them revealed that polyethylene glycol was the best choice from all perspectives. It saved more peroxidase and had no negative overdose effect shown by gelatin and polyelectrolytes. At minimum polyethylene glycol dose, there was little polyethylene glycol remaining in solution after completion of reaction. However, a considerable amount of gelatin remained in solution even at the minimum gelatin dose. Also, gelatin produced more precipitate than polyethylene glycol.

Sulfate reducing bacteria biomass was examined for the biosorption of lead using a batch equilibrium method. The biosorption capacity, as a ratio of the lead removed to the sulfate reducing bacteria biomass used, was found to be equal to 0.3. This ratio was found to be constant under different conditions of the batch reactor and the biomass used. This information can be used in determining both the overall metal removal rate by the active sulfate reducing bacteria biomass due to sulfides precipitation and biosorption as well as the metal removal rate due to biosorption by inactive biomass.

A bioreactor is being developed that produces elemental sulfur suspended in aqueous bioreactor contents. The concentration of elemental sulfur must be measured explicitly in order to study the efficiency of the conversion of sulfide to elemental sulfur. Extracting the sample with ethanol gave erroneous results when sulfide was present in solution. The extraction of aqueous elemental sulfur into petroleum ether prior to colorimetric determination was tested. When the aqueous matrix was simply deionized water, the extraction was poor. The development of a method of extraction of the sulfur into chloroform prior to quantification by high-performance liquid chromatography is described. The efficiency of the extraction was found to be greater than 90% in all matrixes tested and linear for aqueous elemental sulfur concentrations up to 200 mg/L.

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.

Laboratory studies were conducted on the biodegradation of some selected lubricating oils. A standard method for determining the biodegradability of selected recalcitrant organic compounds was developed. Test results show that the species of bacteria present in the sample is an important factor affecting the biodegradation of selected lubricating oils. Results also indicate that it is difficult to degrade lubricating oils in water by using indigenous microorganisms in natural river or lake water. The study shows that up to 23% removal can be achieved biologically after 67 days of operation.

Horseradish peroxidase, once activated by hydrogen peroxide, initiates the oxidation of a wide variety of aromatic compounds. The products of this reaction undergo a non-enzymatic polymerization to form water insoluble aggregates which are easily separated from solution. This process shows great promise for removing aromatic compounds from industrial wastewaters which are not efficiently removed through conventional treatment methods. The capability of solubilized horseradish peroxidase in catalyzing the precipitation of phenols from solution in batch reactors is discussed. Studies have been conducted on the biological sulfate reduction process to remove metals from synthetic and actual industrial wastewaters. Under anaerobic conditions, bacteria of the genera Desulfovibrio and Desulfotomaculum produce hydrogen sulfide which reacts with metals to form metal sulfide precipitates. An anaerobic upflow fixed-film reactor with continuous flow was used to treat actual wastewater from a plating plant supplemented with lactate. The results indicate that soluble metals can be precipitated at efficiencies of up to 99%. The rates of organic conversion, sulfate reduction and metal removal have been found to be dependent on the hydraulic retention time.

The effect of batch and semi-batch additions of the stoichiometric amount of H2O2 on enzymatic removal of phenol has been studied over the phenol concentration range of 1-10 mmol/L. It is found that the ratio between the maximum H2O2 concentration during the reaction and the initial horseradish peroxidase concentration ((H2O2)max/(HRP)o) controls the rate of phenol removal by horseradish peroxidase in the presence of the additive, polyethylene glycol. An optimum range of this ratio is determined to be between 10 and 25 μmol/U. Empirical models are proposed to predict the values of (H2O2)max/(HRP)o required to treat given phenol concentrations under different modes for adding H2O2. On a etudie dans la gamme de concentration en phenol de 1 a 10 mmol/L l'effet de l'ajout en mode continu ou semi-continu de la quantite stochiometrique de H2O2 sur le retrait enzymatique du phenol. On constate que le rapport entre la concentration maximale en H2O2 lors de la reaction et la concentration initiale en peroxidase de raifort ((H2O2)max/(HRP)0) controle la vitesse de retrait du phenol par le peroxidase de raifort en presence de l'adjuvant, le polyethylene glycol. On a trouve que l'intervalle optimal pour ce rapport se situe entre 10 et 25 μmol/U. Des modeles empiriques sont proposes pour predire les valeurs de (H2O2)max/(HRP)o requises pour traiter des concentrations de phenol donnees dans des conditions d'ajout de H2O2 differentes.

The purpose of the present study was to establish a water quality model for a river network system in Beijing‐Tianjin, China and to develop a method for estimating parameters in the established model. The Network Grid method, which was used in the study, is based on the solution of optimal problems in the entire river network system. The parameters, the decay rate of total BOD (carbonaceous and nitrogenous), K1, the re‐aeration rate, K2, the decay rate of BOD for settleable solids, K3, the DO constant rate for photosynthesis, K4, and the nitrification rate of ammonia, K5, in network models are estimated simultaneously by the Network Grid method. The comparison of results indicates that the Network Grid method is suitable for the estimation of the parameters in river network model and the concentration distribution of organic pollutants calculated by the model is in accordance with that observed in situ.

Volatilization of selected organic compounds from water without agitation has been studied. Mathematial models, incorporating Fick’s diffusion law, are developed for volatilization under quiescent condition. A Theoretical model is obtained by solving a partial differential equation and is further approximated to a simplified model for practical applications. The volatilization-rate constant from this model is found to be inversely proportional to the square of the water depth instead of the thickness of liquid film multiplied by water depth used for turbulent condition. The experiments on volatilization of benzene, toluene, trichloroethylene (TCE), and tetrachloroethylene (PCE) were conducted in the laboratory. Both the theoretical model and the simplified model were applied to the experimental data, and correlation coefficients of better than 0.95 were obtained for all tested cases. However, for benzene, the dependency of volatilization-rate constant on water depth was found to be inversely proportional to the 1.81 power.

The apparent molecular mass concentrations of dissolved humic substances from the Detroit River and treated water from the Windsor Water Treatment Plant were determined by gel permeation chromatography over a 1-year period. The results of this research show significant seasonal variations in humic substance concentrations and these values are influenced by the turnover of water bodies. The gel permeation chromatograms of the samples indicate that there is a gradual reduction in the concentration of humic substances during the treatment process, the greatest change occurring after coagulation-sedimentation process. In this process, 76%, 40%, and 30% of the high, intermediate, and low molecular mass compounds, respectively, were either transformed in chemical composition or removed. Other parameters such as turbidity, chlorine demand (during prechlorination), and trihalomethanes concentration in the treated water were also analyzed and there appears to be a relationship between them. When the humic substance concentrations are high, generally the turbidity, chlorine demand, and total trihalomethanes concentrations in the drinking water are also high. Key words: water treatment, humic substances, molecular mass distribution.

The use of a peroxidase from the fungus Coprinus macrorhizus for the removal of toxic organics from synthetic wastewater is explored in this study. Removal of phenols demonstrated a dependence of enzyme lifetime on enzyme concentration in batch reactors. Maximum removal of phenol under optimal conditions in a batch reactor at enzyme activity above 0.3 U ml −1 and up to 1.2 U ml −1 was 53% in the present study. Continuous addition of C. macrorhizus peroxidase (CMP) to the reactor over periods varying from 0.5 to 3 h did not improve the removal. Similarly, continuous addition of H 2 O 2 did not improve the removal of phenol. A colorimetric assay for H 2 O 2 indicated its depletion in the batch reactors. H 2 O 2 : phenol stoichiometry was greater than one if all the H 2 O 2 was added at the beginning of the reaction. Instability of the H 2 O 2 and possible decomposition by contaminating catalase in the sample preparation are two possibilities for its depletion. To overcome this depletion in the reactors regardless of the enzyme concentration, discontinuous addition of either CMP or H 2 O 2 or both was adopted. Significant improvement in phenol removal (90%) was obtained when either H 2 O 2 or CMP was added in three discrete aliquots over 0.5 h. With discontinuous addition of both reactants, the amount of CMP could be reduced to 0.3 U ml −1 while achieving 91% removal of phenol at equimolar concentration of H 2 O 2 . Thus, discontinuous addition of the reactant(s) increased the turnovers obtained by CMP and conserved the 1 : 1 phenol to peroxide stoichiometry in the clearance reaction. Spectral observation of the CMP utilized in this study showed the presence of a contaminating cyanide-like complex of the enzyme, as was observed by another group using a similar microbial peroxidase.

This study investigated the feasibility of a two-step process for the removal of benzene from buffered synthetic wastewater. Benzene is outside the scope of enzymatic removal. In order to remove it from wastewater using enzyme, its pretreatment by modified Fenton reaction was employed to generate the corresponding phenolic compounds. In the first phase, the optimum pH, H2O2 and Fe2+ concentrations and reaction time for the Fenton reaction were determined to maximize the conversion of benzene to phenolic compounds without causing significant mineralization. The pretreatment process was followed by oxidative polymerization of the phenolic compounds catalyzed by a laccase from Trametes villosa. Factors of interest for the three-hour enzymatic treatment were pH and laccase concentration. Under optimum Fenton reaction conditions, 80% conversion of the initial benzene concentration was achieved, giving a mixture containing oxidative dimerization product (biphenyl) and hydroxylation products (phenol, catechol, resorcinol, benzoquinone and hydroquinone). Enzymatic removal of biphenyl and benzoquinone was not possible but 2.5 U/mL laccase was successful in removal of the rest of the phenolic products.

The effect of the additive, polyethylene glycol (PEG), on the horseradish peroxidase (HRP) catalysed removal of phenol from wastewater has been studied over the phenol concentration range of 1–10 mM (0.1–1.0 g/l). The optimum pH, HRP concentration, PEG concentration and the molar ratio of hydrogen peroxide and phenol have been investigated in the presence of PEG at room temperature in order to achieve the maximum phenol removal efficiency with the minimum cost. The effect of concentrations of HRP and PEG on reaction time was also investigated. Experimental results showed that the addition of PEG had significant protective effect on the activity of HRP. The amount of peroxidase required was reduced 40- and 75-fold less than that required without PEG for 1 and 10 mM phenol solutions, respectively. The higher the phenol concentration, the more effective was the addition of PEG. In the presence of PEG, the optimum pH is 8.0 and the optimum molar ratio of hydrogen peroxide and phenol is around 1.0. The minimum doses of HRP and PEG required for at least 95% removal were determined for several phenol concentrations and two empirical models are proposed to predict the minimum HRP and PEG doses required for 95% removal over the entire phenol concentration range of 1–10 mM. Under the optimum reaction conditions described above, the reaction times for at least 95% removal from 1 and 10 mM phenol solutions were 5 and 3 h, respectively. An increase in HRP concentration significantly reduced the reaction time; however, an increase in PEG concentration showed negligible influence.

Horseradish peroxidase enzyme catalyzes the oxidation of toxic aromatic compounds, especially phenols and aromatic amines, in the presence of hydrogen peroxide. The reaction products polymerize to form high molecular weight materials which readily precipitate from solution; hence, providing a means for the treatment of wastewaters which contain aromatic compounds. The catalytic lifetime of horseradish peroxidase enzyme can be extended by optimizing treatment conditions such as pH and temperature and by maintaining a low instantaneous enzyme concentration in the reaction mixture. The enzyme catalyzed polymerization process was implemented in a continuous stirred tank reactor (CSTR) configuration because reactant and enzyme concentrations are lowered immediately upon entering the reactor causing a reduction in inactivation of HRP through free radical bonding and compound III formation. Catalytic turnovers achieved in single and multiple CSTRs in series were significantly higher than those observed in batch reactors when sufficient retention time was provided.