Green Polymer Chemistry: Biobased Materials and Biocatalysis

Chapter 5

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Emerging Enzyme-Based Technologies for Wastewater Treatment Andrew J. Maloney,1 Chenbo Dong,1,2 Alan S. Campbell,1,3 and Cerasela Zoica Dinu*,1 1Department

of Chemical Engineering, West Virginia University, 395 Evansdale Drive, Engineering Science Building, Room 445, Morgantown, West Virginia 26506 2Department of Civil and Environmental Engineering, Rice University, 6100 Main Street, Houston, Texas 77005 3Department of Biomedical Engineering, Carnegie Mellon University, 15B S 25th Street, Pittsburgh, Pennsylvania 15203 *E-mail: [email protected].

In an effort to reduce the need for increased treatment and because of its high importance, interest in “green-based technologies” for wastewater management has picked up in recent years. Green methods aim to be logistically feasible, reliable, efficient, less time and energy consuming and highly cost-effective. This review provides an overview of the state-of-the-art technologies currently used for wastewater treatment and proposes developing novel solutions using enzymes and enzyme-based conjugates to remediate active chemicals and their metabolic products thereby ensuring water reusability. Addressing the global challenges of water quality with biotechnological approaches will provide the optimum conditions for prolonged green decontamination and reduced logistical burdens.

© 2015 American Chemical Society In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


The Need for Wastewater Treatment Water is the foremost natural resource in the world; approximately 2.5% of the available source is in surface freshwater with only about 1/3 of this being readily accessible for human use (1). In the United States, 40% of the freshwater is used for agriculture, 42% for thermoelectric power generation, 5% for industry purposes, and only 9% is used by domestic households (2). However, many surface water resources are vulnerable to pollution; for instance, water can be contaminated relatively easily from fertilizer run-off (3), as a side effect of the industry and its usage of harsh organic solvents (4), or from everyday life (5) and the treatment of human waste and fecal matter (6). To address increasing water pollution concerns nationwide, investment policies and research strategies that increase water use efficiency and decrease water waste have been developed and implemented (7–9). For instance, the policies applied by the Environmental Protection Agency (EPA) and Natural Resources Defense Council (NRDC) have regulated the disposal of unused or expired drugs or of pharmaceutical discharges (10), and adopted strict metal ion guidelines (11) in order to reduce the impact of such pollutants or their metabolites if/when released accidentally. Complementary, research trends have looked at the development of industrial-grade products from reaction conditions that require milder solvent systems and reagents (12, 13), while research strategies considered both the contaminant characteristics and its persistence (14) in the environment (15). For instance, for a contaminant that does not mix with the ground water but only floats on its surface, remediation treatments were employed to either guide the contaminant to an “aspiratory” reservoir (16) or skim the contaminant from the water using material-based sponges, i.e., polypropylene fiber fabric (17) and woolen felt (18). Other treatments and decontamination technologies rely on chemical scrubbing (19), adsorption (20–22), precipitation via electrolysis (23), and chemical precipitation (24). While effective, such methods either need frequent replacement of the active decontaminating agent or are relatively inefficient, time consuming and expensive.

Engineered Nanoparticles-Based Strategies for Wastewater Treatment Recent studies applied nanotechnology and selected characteristics of nanomaterials to increase the efficiency, reliability and feasibility of wastewater treatment (Figure 1). When adsorbents made up of activated carbon (such as rice husks (22) and corn biochar (21)) or nanoparticles (such as modified silica or carbon nanotubes (20)) were used to remediate oil spills, considerably larger contaminant absorption capability was recorded (25). The super-hydrophobicity of carbon nanotubes for instance also prevented water adsorption, further confirming their increased potential for water clearance (25). While chemical adsorption tends to be successful for removal of contaminants, it can become inefficient in long term because the mechanism of removal relies on chemical or physical interaction of the absorbant with the substrate available surface area. 70 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


With the substrate becoming saturated, the efficiency of adsorption decreases overtime which will eventually lead either to replacement or active maintenance of the adsorption area to ensure sustainable wastewater treatment.

Figure 1. Diagram of a wastewater treatment platform of industrial products. The system is based on membranes containing nanotechnology products (i.e., carbon nanotubes, bucky balls, graphene or magnetic nanoparticles) capable to adsorb and degrade industrial contaminants.

Magnetic iron oxide nanoparticles were shown to adsorb organic compounds and inorganic metals from water while allowing for easy separation and recovery though an applied external magnetic field (26–28). Previous research strategies reported for instance that iron oxide submicron-wires could be successfully prepared by novel microwave heating method of graphite/Fe(CO)5 mixture; such wires showed enhanced performance for Cr(VI) removal from polluted water (29). Further, nanoengineered mesoporous organosilica synthesized using wet chemistry also demonstrated great ability for the removal of a wide range of organic and inorganic pollutants (30), from heavy metal species (31), to toxic anions (32), and radionuclides (30). Similarly, synthetic metallic catalysts obtained through organic synthesis reactions (33) demonstrated high efficiency and reliability when used for wastewater treatment (20–22); the solubility of such metal catalysts reduced the requirement for organic solvents usage and therefore decreased the amount of waste. 71 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Typically, remediation techniques based on metal catalysts rely on a chemical degradation reaction where the metal catalyst usage allows for its active recovery and thus reusability. Gil-Lorenzo et. al., for instance used pyrite nanoparticles as fenton-like reagents for the remediation of phtalocyanine (34), Han and Yan looked at deactivating chlorinated hydrocarbons through bimetallic nickel-iron nanoparticles for remediation (35), while others have looked at using BiOI-titanium dioxide (TiO2) for nanoparticle-based water treatment (36). Recently, it was also proposed that metal-oxide nanosupports immersed in water and under light irradiation generate hydrogen peroxide (H2O2) known to reduce biochemical oxygen demand (BOD) or chemical oxygen demand (COD) of industrial wastewater (37). H2O2 was shown to be a superior candidate for wastewater treatment because of its ability of oxidizing organic pollutants (34, 37, 38). Specifically, several research groups have looked at creating “green” fenton reagents in conjunction with a H2O2 source capable to oxidize the organic pollutants (34, 37, 39). TiO2-based photocatalysis for instance involves the generation of electron-hole pairs (e-/h+) (40) and formation of superoxide radical anions (O2-), hydroxyl radicals (•OH), and hydroperoxyl radicals (•OOH) (41–43). During TiO2-mediated Bis(2-chloroethoxy)methane photodegradation, the H2O2 was generated from a combination of 2(ClCH2CH2O)2CHO2• intermediate under UV light irradiation (44). In addition, the TiO2-based photocatalysis process was shown to eliminate sulforhodamine-B dye in aqueous environment (45) through the same mechanism of peroxide generation (46). The presence of H2O2 was confirmed during the dye degradation by measuring the amount of the generated H2O2 and determining both its rate of formation and the rate of dye decomposition. Other experiments have showed that both nitrogen-doped TiO2 under visible light and/or sunlight irradiation (47) and sintered nanocrystalline-TiO2 semiconductor electrode (NSE) used as anodes (48) at room temperature and in a photo-electrochemical reactors could generate H2O2 to be used for water purification (48). Additional studies have also shown that H2O2 could be generated upon irradiation of WO3 photoanodes or platinized WO3 nanoparticles, or TiO2 nanosupports synthesized by hydrothermal processing (49), with such particles showing increased wastewater treatment efficiencies (50). Despite this extensive body of research, the current production costs and recycling issues associated with the fabrication, characterization, and usages of such nano-based technologies inhibit their large-scale implementation (51). Further, even though the metallic nanoparticles have been promising in terms of reusability and decontamination efficiencies, they were also shown to possess environmental drawbacks. Specifically, heavier metals such as nickel have been shown to be carcinogenic if leakage occurs (52); further, any potential spills or leaks of the nanoparticle itself into the water supply could cause an even more detrimental effect (52) due to its accumulation and influence on the aquatic life (53). With accumulation depending on the metal concentration as well as the exposure time, water quality parameters such as pH, conductivity, and dissolved oxygen content also become affected (54) constituting a risk not only for the organisms that it contains but also for the predatory fishes, birds and mammals feeding on such contaminated stocks (55). 72 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Are Biological Means for Water Decontamination Feasible? The most effective means for surface water decontamination remain preventing further accidental or intentional release of contaminated sources and allowing for the natural biological, chemical, and physical life-regulated processes to break down the existing contaminant or its metabolic products over time. However, depending on the nature of the contaminant, this process is time consuming and at times, inefficient. Biologically “inclined” methods such as activated sludge (56), biotrickling filters (19), and vermicompost (57), or the use of biological species as the “workhorses” for synthesis (58) of decontamination species to replace the function of harsher chemicals (59), have been recently examined as a potential route for biological-based, i.e., green, decontamination. However, such methods face major drawbacks regarding their reliability, feasibility and efficiency. Specifically, a problem in conventional active sludge process for water treatment is the high sludge production (60); further, the conventional activated sludge process may not meet modern environmental protection requirements. Moreover, the biotrickling filter could not be successfully applied to highly concentrated volatile organic compounds (VOCs) because of its limited efficiency (61). It was also reported that when vermicompost for instance was used, the efficiency of the process was difficult to maintain at temperatures below 35°C (61). For biological means to be implemented for wastewater treatment, more complex processes that can increase methods efficiency, feasibility, reliability and shelf-life need to be implemented (62).

Enzymes for Wastewater Treatment The efficiency of catalytic reactions as well as the high selectivity, make the enzyme-based water pollution monitoring and wastewater treatment system one of the preferable choices capable of reducing the logistical burdens associated with usage of harsh chemicals. Recent research has shown that the peroxidase family (EC:1.11.1.7) (63) is capable of degrading a wide spectra of aromatic compounds (64) from anilines (65) to phenols (66), and from aromatic dyes (67), to polyaromatic hydrocarbons (68), and polychlorinated biphenyls (69), all products of industrial contamination (70). For instance, Bayramoglu and peers used horseradish peroxidase (HRP) to remove phenol and p-chlorophenol from solution (66); phenolic compounds are known to be major pollutants of industrial wastewaters. The authors showed that HRP covalently immobilized onto magnetic beads retained a high activity and stability and performed higher phenol conversions than its free counterpart, thus indicating the potential of the immobilized HRP to be successfully used for large-scale continuous enzymatic degradation of such pollutants. Similarly, Bhunia et. al., used HRP and provided a systematic analysis of the efficiency of removal of azo dyes (i.e., Remazol blue); anaerobic transformations of such dyes could result in the formation and accumulation of highly toxic aromatic amines which are known for their carcinogenic effects (67). The authors showed that HRP has broad substrate specificity as well as as an optimum pH where dye degradation occurs with 73 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


high efficiency. Additionally, Koller et. al., used HRP and showed removal of about 90% of chlorinated dibenzodioxins (PCDD) and dibenzofurans (PCDF), two significant environmental pollutants (69). Specifically, the authors evaluated the efficiency of 2,5-Dichlorobiphenyl (PCB9) and 2,2′,5,5′-tetrachlorobiphenyl (PCB52) degradation when used at similar concentrations to what are currently found in the water contaminated sources and found that to be fast and efficient, in a reaction period of only 220 min. For a peroxidase-based reactor to be used for wastewater treatment, the enzyme needs to: (1) be immobilized to thus increase its reusability, lifetime, and improve its stability; and (2) allow efficient interaction with its substrate (i.e., H2O2 for the peroxidase family) to thus be capable of pollutant degradation. Immobilization onto nanosupports such as nanotubular aluminosilicate (71), graphene oxide (72), carbon nanotubes (73–75), graphene oxide sheets (74), nanodiamonds (76), and metal-oxide particles (77), was achieved either through physical or chemical means or through encapsulation into a gel (78) or membrane (79, 80). Optimization of the immobilization conditions at any of these nanosupport interfaces involved controlling the enzyme interactions with the nanosupport or encapsulator (with or without a linker) (74) to preserve its functionality and catalytic behavior. Typically, the immobilization interfaces could lead to non-specific interactions with the enzymes that could change its structure and conformation and decrease its catalytic activity (74). Previous studies have shown that additionally, protein-protein interactions (74, 81) at such nanosupport interfaces could also lead to enzyme loss of activity (82). For physical immobilization that relies primarily on nonspecific, hydrophobic interactions between enzyme and the nanosupport (77), it was observed that enzyme loadings (amount of enzyme/ amount of the support), activities and catalytic efficiencies (both relative to free enzyme in solution) are somewhat variable due to the inherent non-specificity of the interactions or the non-uniformity of enzyme loading (74). Complementary, for covalent immobilization that uses chemical reactions to form a covalent bond between the nanosupport and the enzyme (i.e., through a zero EDC/NHS chemistry between carboxylic acids and amines (73) or through the glutaraldehyde chemistry involving an interaction between amines and carbonyl groups) (83), it was shown that the chemical groups of the enzyme can be manipulated to interact with the functional groups of the nanosupport (83) with a variety of support functionalizations influencing the immobilization efficiency as well as its “initiator” (e.g., carboxylic acid (73), hydroxyl (76), amine (76), or carbonyl (76) respectively). Lastly, covalent immobilization though a linker molecule (e.g., polyethylene glycol, PEG) (74), was used to reduce direct contact between the nanosupport and the enzyme (74, 84). The potential benefits of using such polymeric linkers are associated with their ability to allow for controlling the distance between the enzyme and the nanosupport, thus minimizing nonspecific interaction and sterical conformation changes of the enzyme and thus increasing its catalytic efficiency. In addition, covalent association of enzymes via peptide-modified surfaces used as linkers were shown to maintain enzyme specific activity as well stability, and controlled its orientation at the support interface (85). While there was no single nanosupport recommended as the optimum one for 74 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


all enzymes being tested, some general trends have been observed. For instance, research has shown that more curved nanosupports are more desirable than their flatter counterparts because they tend to limit the nonspecific interactions as well as enzyme-enzyme interactions (73, 86–88). Campbell et. al., for instance have demonstrated that soybean peroxidase, glucose oxidase, and chloroperoxidase have activities ranging from 3% to nearly 30% depending on the immobilization method and the charge and morphology of the nanosupport being used (74, 77). Immobilization was shown to increase the mechanical and thermal stability of the enzymes while decreasing the probability of enzyme leaching into solution (75); mechanical and thermal stabilities are needed when reusability and increased lifetime of the enzyme-based conjugates are considered. Particularly, Verma et al., showed that the thermal stability of lipase was greatly improved upon its immobilization onto carbon nanotubes (89), while Klein and coworkers showed a similar phenomenon for β-D-Galactosidase immobilized onto chitosan (90). Further, affinity immobilization was shown to exploit the specificity of the enzyme to the substrate, in different physiological conditions, to increase enzyme immobilization capacity and reusability (91). Moreover, entrapment of enzymes in gels or fibers was shown to result in reduced enzyme leaking and increase in its mechanical stability (80). However, even though analysis and improvements of efficiency, selectivity and stability of enzymes immobilized onto nanosupports have offered opportunities for enzyme implementation in wastewater treatment (92), the poor performance of an enzyme-based technology due to the limited reaction time or the limited available substrate required for enzyme functionality, have hindered its large scale, industrial implementation.

Proposed Means and Support Studies for the Next Generation of Self-Sustainable Wastewater Treatment Platforms We now propose that the next generation of green and efficient wastewater treatment platforms based on enzymes can be fabricated through direct immobilization of the biocatalyst onto a nanosupport which is capable to generate enzyme’s required substrate through a reaction at its interface. Such a strategy would not only allow for the immobilization and reusability of enzyme but would further permit for the substrate required by the enzyme to be generated on demand thus creating a self-sustainable wastewater treatment platform. To demonstrate the feasibility of this proposal, our preliminary studies showed that carbon nanotubes can be used as nanosupports for dual enzyme immobilization and natural catalytic chain reactions at their nanointerfaces (73). Specifically, the approach involved immobilizing two enzymes namely glucose oxidase (GOX) and chloroperoxidase (CPO) onto carbon nanotubes either via physical and chemical means. The two enzymes maintained their functionality and were able to generate their products with high efficiencies as well as allow for a chain reaction to occur at the carbon nanotube interface to lead to hypochlorous acid generation—a potent microbial decontaminant (Figure 2) (73). However, while carbon nanotubes allowed for high loadings and efficiencies did not allow 75 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


for creation of a self-sustainable system since the two enzymes both required the substrated to be added for the reaction to occur.

Figure 2. Michaelis-Menten kinetics of enzyme-based MWNTs conjugates. a) CPO-based conjugates (physically immobilized-open circles; covalently immobilized- filled triangles) kinetics relative to free CPO in solution (filled squares). b) GOx-based conjugates (physically immobilized-open circles; covalently immobilized- filled triangles) kinetics relative to free GOx in solution (filled squares). Reproduced with permission from reference 78. Copyright 2012 Elsevier. 76 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


77 Figure 3. a) Scanning Electron Microscopy (SEM) images of pristine titanium dioxide revealing their morphology and aspect ratios; b) Functionalization of photocatalyst pristine titanium dioxide results in the formation of carboxyl functionalized nanobelts or TiO2-NBs. c) FTIR spectrum of TiO2-NBs reveals the presence of the carboxyl peak at 1731 cm-1, confirming -COOH functionalization. d) CPO enzyme immobilization onto TiO2-NBs with and without the use of a PEG linker. The PEG linker brings the enzyme away from the nanosupport. The concept of hypochlorus (HOCl) generation using the CPO-based conjugates. The UV irradiation of the TiO2-NBs photocatalysts will generate hydrogen peroxide (H2O2) to be used by CPO for in situ HOCl-based decontamination.

In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Considering for instance TiO2, a photocatalyst that produces reactive oxygen species from water when excited under UV light (93–95), one can envision using this support as a platform to produce the necessary H2O2 to be used as the substrate by immobilized peroxidases to thus ensure a viable self-sustainable platform for wastewater treatment as proposed above. Even though this photocatalyst has been extensively studied for the decontamination of a wide variety of water contaminants (94, 96) as well as contaminants in the air (97), to our knowledge there are no previous studies that attempted using this nanointerface to generate H2O2 for efficient enzyme kinetics. Our preliminary results showed that these nanosupports can be produced from pristine anatase TiO2 via hydrothermal processing (98). The nanosupports were found to be 60-300 nm wide and several micrometers in length (SEM; Figure 3a). The pristine anatase TiO2 nanosupports were further carboxyl functionalized using 3-triethoxysilylpropyl succinic anhydride to allow the formation of TiO2 nanobelts or TiO2-NBs (Figure 3b); no significant changes were identified in the TiO2-NBs morphology or length distribution upon such carboxyl functionalization. Carboxyl functionalities were confirmed using Fourier Transform Infrared Spectroscopy (FTIR; Figure 3c); specifically, the large peak identified at 1731 cm-1 confirmed the presence of the C=O bond present onto the TiO2-NBs (99).

Table 1. Characterization of CPO-TiO2 Hybrid System for in Situ Generation of HOCl Parameter

CPO-TiO2

CPO-PEG-TiO2

Loading

0.10 ± 0.03

0.04 ± 0.02

Loading function of enzyme offered (%)

20

8

Specific retained

0.30 ± 0.13

12.00 ± 1.63

Vmax (μM μg-1 s-1)

0.54 ± 0.11

4.64 ± 0.87

Km (μM H2O2)

270 ± 80

490 ± 70

Kcat (s-1)

22.68 ± 4.62

194.88 ± 36.54

η

0.02

0.17

(mg protein/mg nanosupport)

activity relative to free enzyme (%)

In order to generate enzyme-nanosupport hybrid systems, we used the TiO2-NBs as scaffolds for the covalent immobilization of model CPO through EDC/NHS chemistry (100, 101) or EDC/NHS chemistry with a PEG spacer (75) (Figure 3d). The PEG spacer was highly hydrophilic, with a length of 3.2 nm; previous studies have shown that such a spacer has little or no chemical effect on enzyme immobilization however it allows for its improved solubility of 78 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


the nanosupport interface (84). The CPO-TiO2-NBs and CPO-PEG-TiO2-NBs conjugates showed enzyme loadings of 0.10 ± 0.03 and 0.04 ± 0.02 mg enzyme/mg nanosupport respectively, which represented 20% and 8% of the amount of protein initially offered during the immobilization process (Table 1). The activity of the CPO immobilized at the TiO2-NBs interface was evaluated using the colorimetric reaction showing the conversion of monochlorodimedon to dichlorodimedon in the presence of H2O2 (74). The CPO-TiO2-NBs conjugates retained about 0.3%, while CPO-PEG-TiO2NBs conjugates retained about 12% activity when compared to the activity of the same amount of free CPO in solution (Table 1). The low activity observed for CPO-TiO2-NBs was attributed to the interface reactions between the enzyme and the TiO2-NBs nanosupport surface. Specifically, previous studies have shown that nanosupports with lower curvature favor enzyme-enzyme interactions and their non-specific attachment which in turn could lead to enzyme denaturation (75, 102, 103). Further, at the working pH of 4.8 that is slightly above the isoelectric point of CPO (pI = 4) (104), the enzyme has a net negative charge while the TiO2-NBs (isoelectric point, pI = 6.5) (105) has a net positive charge, thus favoring the adsorption or non-specific immobilization of the enzyme and leading to the observed reduced enzyme activity. Meanwhile, the higher activity observed for the CPO-PEG-TiO2-NBs was presumably due to the PEG spacer that brough the enzyme away from the nanosupport thus reducing its non-specific interaction with the nanosupport as well as its denaturation at the nanointerface (106). Kinetic constants were evaluated for the free CPO and compared to the kinetics of the CPO-based conjugates, i.e., CPO-TiO2-NBs and CPO-PEG-TiO2-NBs, by using non-linear regression plots (107). Namely, the Km (substrate concentration at which the initial reaction rate is half maximal) and Vmax (maximum initial rate of an enzyme catalyzed reaction) values of the CPO-TiO2-NBs and CPO-PEG-TiO2-NBs conjugates are shown in Table 1 and reflect comparison to the free CPO in solution. The Km values were on the same order of magnitude for all analyzed samples; specifically they were found to be 480, 270 and 490 μM, respectively, for the free, CPO-TiO2-NBs and CPO-PEG-TiO2-NBs conjugates indicating that there was no significant conformational change of the enzyme active site upon immobilization. The apparent Km for the directly covalently conjugated enzyme was decreased by about 40% when compared to that of the free enzyme or the PEG covalently conjugated enzyme that showed no significant change. Vmax values were on the same order of magnitude (i.e., 27, 0.5 and 4.6 µM mg-1 s-1, respectively for the free, CPO-TiO2-NBs and CPO-PEG-TiO2-NBs conjugates); the Vmax for the covalently immobilized enzyme decreased about 98% and about 81% for the PEG immobilized samples when compared to the free enzyme in solution. The smaller Vmax obtained for the covalently immobilized enzyme was presumably due to the enzyme coming into direct contact with the nanosupport, which lead to decreased substrate reaction and consequently slow reaction rate. Our reported Km and Vmax results are on the same order of magnitude with previous reports for carbon nanotubes (103), polymer coated magnetic nanoparticles interfaces (108) or mesoporous silicate (109). Any deviations are presumably resulted from the environmental differences in which the experiments were performed. 79 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


The efficiency factor η was also calculated from the maximum reaction rates of the immobilized CPO (both CPO-TiO2-NBs and CPO-PEG-TiO2-NBs) relative to the rate of the free enzyme in solution using:

where νimmobilized is the reaction rate of the immobilized enzyme (directly through covalent immobilization or through PEG and covalent immobilization respectively) and νfree is the reaction rate of the free enzyme (Table 1). The efficiency factor for the CPO-TiO2-NBs was 0.02 while the η for the CPO-PEG-TiO2-NBs was 0.17. The reduction in the efficiency factor of the immobilized enzyme further confirmed the non-specific interactions with the nanosupport interface and thus enzyme denaturation (75, 102, 103). Further tests of enzyme-nanosupport hybrid systems will evaluate the efficiency of both H2O2 and subsequently hypochlorus acid generation, their rate of production and decomposition, all upon enzyme-based conjugates irradiation under UV (i.e. UV-A (λ = 316-400 nm) or UV-C (λ = 235-280 nm). Upon demonstration of the hypothesis for this CPO model system, the approach could be extended to soybean peroxidase (SBP) for its applicability in green treatments of products resulted from industrial contamination of water (70) such as anilines (65), phenols (66), or aromatic dyes (67), and polyaromatic hydrocarbons (68).

Conclusions Because of enzyme’s versatility and high catalytic efficiency which allow decontamination in mild, green conditions, there is presently a wide variety of ongoing research for designing enzyme-based platforms that allow efficient wastewater treatment. The challenges that limit such platforms large-scale, industrial implementation are however associated with the high cost of synthesizing enzymes in situ and/or the use of non-standardized immobilization techniques to ensure improved enzyme reusability, stability and extended shel-f-life. Future research is undergoing to permit great control over the immobilization and reusability of enzymes while ensuring that the substrate to be used can be generated on demand to thus overcome the observed limitations and form the next generation of self-sustainable wastewater treatment platforms.

Acknowledgments National Science Foundation (CBET-1033266) supported this work. A.J. Maloney thanks the Barry Goldwater Scholarship and Excellence in Education Program. 80 In Green Polymer Chemistry: Biobased Materials and Biocatalysis; Smith, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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