Use of Enzymes in Bioremediation - American Chemical Society

Chapter 10

Use of Enzymes in Bioremediation Jerzy Dec and Jean-Marc Bollag

Downloaded by NORTH CAROLINA STATE UNIV on September 17, 2012 | http://pubs.acs.org Publication Date: December 1, 2000 | doi: 10.1021/bk-2001-0777.ch010

Laboratory of Soil Biochemistry, Center for Bioremediation and Detoxification, The Pennsylvania State University, University Park, PA 16802

Bioremediation technologies rely on the activity of microbial or plant enzymes involved in the metabolic and cometabolic transformation of a variety of organic substrates. Many xenobiotic compounds can be degraded by intracellular enzymes and thereby undergo detoxification. To date, contaminants have been exposed to enzymatic degradation primarily by stimulating microbial growth in contaminated areas. As is well known, enzymes can be active outside the microbial cells. The extracellular activity of enzymes is expected to be increasingly exploited in future bioremediation technologies. Enzymes can be obtained i n large quantities from microbial populations grown under optimal conditions and without exposure to toxic chemicals. To prevent losses of enzymatic activity under severe field conditions, many investigations have been focused on developing methods to stabilize enzymes, preferably by immobilization on solid supports or by gel coating. The use of enzymes naturally stabilized in plant tissues also has been investigated. The feasibility of enzymatic treatment has been demonstrated at laboratory scale i n a number of studies. For example, hydrolases from Pseudomonas spp. and other bacteria have been shown to hydrolyze and detoxify organophosphate pesticides. Several fungal phenoloxidases effectively oxidized xenobiotic phenols and anilines to reactive intermediates that subsequently were detoxified through polymerization or binding to humus. Further studies are necessary to identify more enzymes that may be able to transform the increasing number of chemicals polluting the environment.

182

© 2001 American Chemical Society

In Pesticide Biotransformation in Plants and Microorganisms; Hall, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Introduction To date, enzymes have found many practical applications i n analytical chemistry and medicine (/, 2), and now attempts are underway to employ these biological catalysts for the protection of the environment (5, 4). Enzymatic processes are expected to replace many traditional industrial technologies to reduce emission of toxic by-products and to eliminate hazards associated with the use of dangerous chemicals, such as chlorine in paper bleaching or strong acids i n starch processing. Enzymes also have potential for bioremediation of polluted environments. Bioremediation is a process in which microorganisms or other biologically active agents are used to degrade environmental pollutants. The application of microorganisms to polluted areas is an established method of bioremediation (5, 6). Enzymatic treatment, however, is only at the stage of laboratory trials. The rationale for developing enzymatic cleanup methods is that enzymes are the ultimate cause of pollutant degradation during bioremediation procedures based on microbial activity. If applied extracellularly, enzymes may eliminate or at least considerably reduce the need to support specific microorganisms i n the treated areas. Enzymatic treatment is expected to have many advantages (7), such as the ease and simplicity of application, short treatment time, ability to target specific pollutants, low sludge volume, and the absence of ecological hazard associated with their use. Enzymatic treatment may have a number of advantages over microbial treatment. First, enzymes do not require an acclimatization phase as is often required by microorganisms. Enzymes can be used under a wider range of environmental conditions (e.g., pH, moisture, and temperature) than microorganisms. Unlike microorganisms, enzymes may be effective at both low and high pollutant concentrations. They can penetrate microporous sites in the soil matrix that cannot be entered by microorganisms. Enzymes are resistant to many inhibitors that may affect microbial metabolism. Furthermore, following isolation from genetically engineered microorganisms, enzymes can be released safely into the environment. One of the drawbacks of enzymatic treatment is the high cost of isolating enzymes from microbial cells, storing them and prolonging their stability under harsh environmental or industrial conditions. Another disadvantage is the need for cofactors, without which many enzymes do not show any activity. The most desired enzymatic action is complete mineralization of the toxic compounds. Unfortunately, as discussed in detail later, the majority of enzymes are not capable of mineralization when applied individually. Theoretically, mineralization may be possible i f several different enzymes are applied simultaneously, but all enzymes that are known to be involved i n the established mineralization pathways require very expensive cofactors. With individual enzymes, one can expect only a certain rate of pollutant degradation or transformation. However, as long as the transformation products are less toxic than the parent compounds, or, in other words, as long as there is a


184 detoxification effect, the enzymatic treatment may be considered successful, especially when the degradation products are susceptible to further transformation by microorganisms. This and other aspects of enzymatic treatment are the subject matter of this chapter.


Enzymes Tested for Bioremediation The idea of using extracellular enzymes for decontamination originated from research efforts to identify and characterize enzymes responsible for the transformation of xenobiotics in living organisms, mostly fungi and bacteria (8, 9, 10, 11, 12, 13, 14). As early as 1965, Kearney and Kaufman (9) reported that a soil bacterium identified as a species of Pseudomonas possessed an enzyme that could hydrolyze the herbicide isopropyl A^-chlorophenyl) carbamate and several other biologically active phenylcarbamates. The reaction pathway is presented i n Figure 1. The authors did not view their findings in terms of bioremediation or even detoxification; they were mostly concerned with losses in herbicidal activity and wanted to identify the responsible factors. Nevertheless, their discovery was noticed and remembered by those who later focused on the decontamination aspects. First reports that explicitly proposed the use of enzymes for cleanup appeared i n scientific journals shortly after the emergence of bioremediation technologies in the 1970s. One pioneering study in this area was on the enzymatic hydrolysis of parathion. carried out by Munnecke (15). The enzyme was isolated from a mixed culture of soil bacteria consisting mostly of Pseudomonas species. Barik and Munnecke (16) used a crude cell extract obtained by brief sonication of Pseudomonas bacteria at 150 W. The enzyme was immobilized on solid supports, such as ground Jena glass, or controlled pore glass beads. When applied to wastewater treatment, the enzyme removed 95% of parathion at initial concentrations ranging from 10 to 250 ppm. Soil treatment resulted in a complete, 100% hydrolysis of parathion that had been present at a concentration of 2500 ppm. The same enzyme showed considerable efficiency in the hydrolysis of several other organophosphate pesticides, such as triazophos, diazinon, and fenitrothion. The rate of enzymatic hydrolysis was from 11 to more than 2000 times faster than that of chemical hydrolysis (Table 1). Munnecke (17) prepared a list of enzymes that showed potential for pesticide hydrolysis. The list includes mainly esterases and a few acylamidases. Johnson and Talbot (18) reviewed microbial enzymes, mostly hydrolases, esterases, or amidases, that are capable of transforming a variety of pesticides, such as organophosphates, carbamates, phenylureas, acylanilides, or phenoxyacetates. Few of these enzymes, however, were used to demonstrate their ability to decontaminate water or soil. Recently, Karam and Nicell (4) presented a list of important enzymes that had been


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N—C—0—C—H CH

3

lsopropylN-(3-chlorophenyl)carbamate esterase CH

3

C0

+

2

HO—C—H CH

3

Figure I.

Hydrolysis of a phenyl carbamate by an esterase from Pseudomonas sp. (8).

Table 1. Enzymatic and Chemical Hydrolysis of Selected Insecticides (75) Pesticide

Parathion Triazophos Paraoxon EPN Diazinon Methyl parathion Dursban Fenitrothion Cyanophos

Enzymatic hydrolysis (nmol/min) 416 1360 500 12 200 354 36 217 58

Chemical hydrolysis (nmol/min) 0.17 1.35 0.95 1.05 1.40 2.90 0.89 1.05 0.79

Ratio (enzymatic/chemical) 2450 1005 525 11 143 122 40 205 73



186

Table 2. Enzymes Studied for Their Decontamination Potential (4) Enzyme Peroxidase

Source Horseradish root

(EC 1.11.1.7)

Target pollutant Phenols Anilines PHA's, Phenols Phenols Anilines

Lignin peroxidase Phanerochaete (EC unknown) chrysosporium Manganese Phanerochaete peroxidase chrysosporium (EC unknown) Chloroperoxidase Caldariomyces Phenols fumago (EC 1.11.1.10) Laccase Rhizoctonia praticola Phenols (EC 1.10.3.2) Trametes versicolorAnilines Trametes villosa Tyrosinase Mushroom Phenols E C 1.14.18.1) Anilines Parathion Pseudomonas sp. Methyl parathion, hydrolase Flavobacterium sp. ethoxy-parathion, (EC unknown) diazinon, dursban, Streptomyces fensulfothion, coumaphos, potasan Alkylsulfatase Pseudomonas C12BAlkyl sulfates, alkyl (EC unknown) ethoxy sulfates, aryl sulfonates Cyanidase Alcaligenes Cyanide (EC unknown) denitrificans Cyanide hydratase Gloeocerospora Cyanide sorghi (EC 4.2.1.66) Stemphylium loti

Decontamination mechanism Ref. Polymerization Binding to humus Oxidation

32

Oxidation

51

Oxidation

50

Polymerization Binding to humic substances Polymerization

33

Hydrolysis

17

Complete surfactant degradation

52

50

Conversion to ammonia 53 and formate 54 Hydrolysis to formamide


187 tested for their decontamination potential (Table 2). The list contains several oxidoreductases, the parathion hydrolase, an alkylsulfatase, a cyanidase, and a cyanide hydratase. Many enzymes have been identified as responsible for cometabolic degradation of different xenobiotics in living organisms (see the next section), but currently, they appear too costly for application in bioremediation.


Enzymes Showing the Ability to Degrade Xenobiotics Some xenobiotics may be used by microorganisms as sources of energy and nutrients for growth. Another important mode of microbial transformation is cometabolism, in which microorganisms transform the xenobiotic molecules but are unable to proliferate on the resulting degradation products. According to literature data, oxidoreductases and hydrolases play a major role in both the metabolic and cometabolic transformation of xenobiotics (4, 18, 19). The decontamination potential of some enzymes is illustrated below by the transformation pathways of four common contaminants (2,4-D, atrazine, naphthalene, and 4-chlorobiphenyl). Based on various literature data, the herbicide 2,4-D is transformed by a dioxygenase from Alcaligenes eutrophus to 2,4-dichlorophenol with the release of glyoxylate (20, 21, 22). In turn, 2,4-dichlorophenol is hydroxylated by an oxygenase and the aromatic ring of the resulting dichlorocatechol is cleaved by a dioxygenase. Eventually, a series of further enzymatic reactions leads to mineralization. The intensely studied degradation pathway for atrazine involves the dehalogenation and hydroxylation of the herbicide by a hydrolase from Pseudomonas (23). Further steps, mediated by other hydrolases, can lead to mineralization (evolution of C 0 ) via gradual hydroxylation to cyanuric acid (24, 25) and aromatic ring cleavage (26). resulting in the release of biuret and then urea (Figure 2). Biodegradation of naphthalene may proceed through the oxidation of the compound to dihydroxynaphthalene by a dioxygenase from Pseudomonas sp. and then by a dehydrogenase (27, 28). The enzymatic hydroxylation gives rise to ring cleavage in the presence of an isomerase, and to mineralization of the resulting intermediates (salicylaldehyde and pyruvate) after they enter the general metabolic track. Similarly, dioxygenases, dehydrogenases, and hydrolases from Pseudomonas sp. (29, 30, 31) can mediate the transformation of 4-chlorobiphenyl to aromatic or aliphatic intermediates (2-hydroxypenta-2,4-dibenzoate and 4-chlorobenzoate) that are quickly metabolized to C 0 and water. Enzymes involved in the degradation pathways outlined above show great potential for bioremediation; unfortunately, most of them require expensive cofactors, such as NAD* N A P D or F A D . 2

2



188

N N. I H

OH

alrazine chlorohydrolase

I H

H Hydroxyalrazine

Atrazine

OH NA A H N-lsopropylammelide

x

N

NH (AA H 2

2

\

T

0

Biuret Figure 2.

OH HO^N^OH

N-isopropylammeJide isopropylaminohydrolase

0H

NH

hydroxyatrazine elhylaminohydrolase

cyanuric acid amidohydrolase

Cyanuric Acid

,,

,

^

u ^

.

biuret amidohydrolase^

O HN^NH jr

2

urease ^

2

Urea

Carbon dioxide

Degradation of atrazine by various hydrolasesfromPseudomonas sp. (23, 24, 26).


189


Decontamination of Water and Soil Using Various Oxidoreductases Klibanov et al. (32) discovered that horseradish peroxidase can transform toxic phenols and anilines to nontoxic polymers that can be removed from aqueous solution by filtration or sedimentation. Similar detoxification reactions were later observed with various phenoloxidases, such as laccase or tyrosinase (33, 34, 35). In a study of Dec and Bollag (36), the removal of various chlorophenols in the presence of horseradish peroxidase ranged from 40 to 100%, depending on the number and position of the chlorine atoms. Large removals also were found with the laccase of Trametes versicolor (91 to 100%). Tyrosinase was less effective, especially for pentachlorophenol; nevertheless, 100% of 4-chlorophenol was removed. Experiments using the electron spin resonance technique indicated that peroxidases and laccases oxidize substrates to free radicals that subsequently are involved in chemical coupling and oligomer formation (dimers i n the first stage) (37). Depending on the resonance form of the free radicals, some coupling reactions were accompanied by the release of one or two chloride ions (38). With tyrosinase, the oligomerization resulted from the formation of or/Ao-quinones and orthohydroxylated ions that may couple to each other through nucleophilic substitution. Two routes of coupling may exist, i.e., one with and the other without release of a chloride ion (39). Various phenoloxidases also may be involved in binding reactions between xenobiotics and soil organic matter. Just as with polymerization, binding presently is considered to be an efficient and safe method of detoxification (33). In the studies of Sarkar et al. (40\ C-labeled 2,4-dichlorophenol was bound to fulvic acid in the presence of various phenoloxidases (peroxidase, tyrosinase, a laccase of Trametes versicolor, and a laccase of Rhizoctonia praticola). The extent of enzymatic binding after 36 hours of incubation ranged from about 40% for tyrosinase to 70% for the laccase of Trametes versicolor. Experimental data indicated that, as with oligomerization, enzymatic binding may be controlled by a free radical mechanism, in which free radicals generated by the enzyme are involved in coupling with free radicals present in humic acid (38). Again, some coupling reactions may be accompanied by the release of chloride ions. 14

Stabilized Enzymes Enzymes must be stabilized to maximize their life-times under severe environmental or industrial conditions. Immobilization is one of the most efficient approaches for stabilizing enzymes. Several mechanisms may be used to immobilize



190 enzymes, such as covalent attachment to solid supports, adsorption on solid surfaces, entrapment in polymeric gels, encapsulation, or intermodular cross-linking (41). Solid supports used to immobilize enzymes can be divided into organic supports and inorganic supports. Organic supports may consist of synthetic matrices, such as acrylamide gels and ion-exchangers, or of natural matrices, such as lignin, humus, or cellulose. Among inorganic supports, one can use porous glass, metal oxides, and several other materials. As already mentioned, the parathion hydrolase used by Barik and Munnecke (16) was immobilized on ground glass or porous glass beads. In the studies of Leonowicz et al. (42) and Sarkar et al. (43), various enzymes including phenoloxidases that mediated polymerization and binding were immobilized on clay or soil. Prior to immobilization, these supports were treated with concentrated nitric acid, and then were activated with 3-aminopropyltriethoxysilane followed by glutaraldehyde. Enzymes immobilized by this method (glucose oxidase, p-Dglucosidase, laccase, and tyrosinase) showed increased resistance to elevated temperatures as compared to free enzymes. They were also considerably less susceptible to protease activity than the free enzyme. Moreover, losses of laccase activity during a 15-day exposure to a suspension of soil were remarkably reduced after immobilization as compared to losses sustained by the free enzyme. This finding constitutes great promise for using immobilized laccases for soil treatment. One of the most remarkable properties of immobilized enzymes is that they can be reused many times. As determined by Ruggiero et al. (44), laccase immobilized on different clays, such as kaolinite and montmorillonite, or on soil could be reused up to 24 times for the removal of C-labeled 2,4-dichlorophenol from polluted water. After each 2-hour cycle the treated water was replaced with a fresh portion of the polluted water. l4

Use of Plant Materials Containing Peroxidase Activity As is well known, many technological developments were inspired by nature. Immobilization of enzymes is no exception. Enzymatic activity that can be detected in all samples of fresh soil originates largely from naturally immobilized extracellular enzymes (45). Many plant tissues contain enzymes that by all appearances are immobilized (46). In an attempt to use these natural resources, Dec and Bollag (47) tested horseradish roots, which are known to contain large amounts of peroxidase. The experiments were carried out using horseradish cut into small pieces to decontaminate wastewater from a company that manufactured the herbicide 2,4-D. The wastewater contained up to 850 ppm of 2,4-dichlorophenol and other chlorinated phenols. The decontamination reaction was initiated by adding hydrogen peroxide as an electron acceptor. Peroxidases that were naturally immobilized in horseradish could remove up to 100% of the initial 2,4-dichlorophenol depending on the concentration



191 of hydrogen peroxide. High removals were also achieved using other plant materials containing peroxidases, such as potato or white radish. As shown i n the studies of Roper et al. (48% 55% of 2,4-dichlorophenol that had been incubated with cut horseradish precipitated from aqueous solution in the form of a polymer, and 24% was covalently bound to the plant tissue. The remainder consisted of oligomer products that either were dissolved in aqueous solution (12%) or physically sorbed to the plant tissue (8%). Horseradish application resulted i n 99% removal of 27 compounds among 50 compounds tested, including eight U.S. Environmental Protection Agency priority pollutants. A n experiment for the removal of 2,4-dichlorophenol at varying p H proved that cut horseradish tissue was at least as effective as purified peroxidase obtained from Sigma (St. Louis. MO). In fact, the pH range for nearly complete removal of 2,4dichlorophenol was greater in the presence of horseradish (pH 3 to 8) than i n the presence of purified enzyme (pH 4 to 7). Moreover, similar to enzymes immobilized on solid supports, cut horseradish could be recycled (47). Peroxidase naturally immobilized i n horseradish tissue removed 100% of 2,4-dichlorophenol from 15 fresh portions of polluted water. Each cycle was stopped after 30 minutes of incubation. The plant material retained significant amounts of peroxidase activity (about 50%) by cycle 30, when the experiment was terminated. Using cut horseradish, it was possible to detoxify chlorinated phenols through their binding to soil. In the study of Flanders et al. (49\ horseradish combined with different peroxides mediated irreversible binding of 2,4-dichlorophenol to soil, immobilizing up to 92% of the pollutant. In sharp contrast, the immobilization observed in the control samples (untreated soil, or soil treated only with horseradish or peroxide) did not exceed 12%, clearly indicating that peroxidase activity present in the plant tissue was instrumental in enhancing the binding. The detoxification process was completed in 30 minutes. The immobilization of 2,4-dichlorophenol increased with increasing soil moisture. Apparently, as more water was added, contact between the pollutant and the enzyme was improved. The hydrogen peroxide was quickly decomposed in soil; therefore only 43% of 2,4-dichlorophenol was bound. With calcium peroxide, however, binding increased to 92%, probably because slow release of H 0 from calcium peroxide allowed efficient utilization by the peroxidase present in horseradish. Although the effect of calcium peroxide requires further investigation, it is clear that plant materials containing peroxidases can mediate detoxification processes and, therefore, they constitute a promising remediation option. 2

2

Conclusions Extracellular enzymes show great promise for their exploitation in future bioremediation technologies. They can be used either individually to enhance microbial degradation or in mixtures to provide for a complete mineralization of


192 xenobiotics. The use of artificially or naturally immobilized enzymes may considerably improve the efficiency of enzymatic treatment.


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Use of Enzymes in Bioremediation - American Chemical Society

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