10 Enzymatic Treatment of Waters and Wastes James A. Nicell McGill University, Montreal, Quebec, Canada I. INTRODUCTION The implementation of increasingly stringent standards for the discharge of wastes into the environment has motivated the development of alternative processes for the production of goods and for the treatment and disposal of wastes. Ultimately, these processes are developed to meet one or more of the following objectives: (1) to improve the efficiency of utilization of raw mate- rials, thereby conserving resources and reducing costs; (2) to recycle waste streams within a given facility to minimize the need for effluent disposal; (3) to reduce the quantity and maxi mize the quality of effluent waste streams that are created during production of goods; and (4) to transform wastes into marketable products. There are multitudes of ways in which the transfor- mation of wastes and pollutants can be carried out. Most of these methods may be classified as being chemical or biological in nature. Chemical transformations involve the application of reagents and reaction conditions to transform target species. Such processes involve a string of events that are usually well defined and can often be controlled to maximize efficiency. However, chemical processes often require the presence of excess quantities of reagents to accomplish the transformation to the desired extent. In addition, particularly harsh conditions (e.g., high temper- ature or extremes of pH) are sometimes required to facilitate the chemical transformations. This can present a problem once the desired transforma- tion has taken place because the resulting stream may be a low-quality mixture that cannot be disposed into the environment or reused without TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. subsequent treatment. Finally, many chemical treatment processes are not highly selective in terms of the types of pollutants that are transformed during treatment. Consequently, such processes are usually more econom- ical for the treatment of dilute wastewaters and are often used as a polishing step before waste discharge into the environment [1]. Biological processes have been used with much success for many decades. These processes are designed to take advantage of the biochemical reactions that are carried out in living cells. Such processes make use of the natural metabolism of cells to accomplish the transformation or production of chemical species. The metabolic processes occur as a result of a sequence of reactions conducted inside the cell that are catalyzed by proteins called enzymes. An important advantage of biologi cal systems is that they can be used to carry out processes for which no efficient chemical transformations have been devised. In addition, biological processes can often be conducted without the harsh conditions that are necessary during chemical transforma- tions. However, the use of microorganisms is beset with many rate-limiting factors. For example, costly and time-consuming methods may be necessary to produce microbial cultures that can degrade the targeted pollutant. Furthermore, severe conditions such as chemical shock, extremes of pH and temperature, toxins, predators, and high concentrations of the pollu- tants, intermediates, and products may irreversibly damage or metabolically inactivate microbial cells. Thus, the sensitivity of microorganisms to changes in their environment can make these processes difficult to control over the long term, and may subject them to frequent upsets. They also require a supply of macro- and micronutrients for the support of microbial growth, and often result in the formation of large quantities of biomass that ulti- mately must be discarded into the environment. In addition, the biochemical reactions occur at a rate that is limited by the metabolism of the micro- organism and, thus, are often slower than chemical processes. Moreover, whereas biological systems are commonly used to remove the bulk organic load in wastewaters, these systems often have difficulty in removing toxic pollutants to consistently low levels [1]. Therefore, conventional biological processes may not be able to improve water quality sufficiently to meet wastewater discharge criteria. In an attempt to overcome some of the problems associated with chemical and biological systems, recent research has focused on developing environmental applications of enzymes that have been isolated from their parent organisms. This concept of using enzymes in waste-treatment appli- cations is not new. In fact, applications were suggested as long ago as the 1930s [2]. However, recent interest in the development of enzymatic waste- treatment systems has grown for several reasons. Firstly, the rate of introduction of recalcitrant organic pollutants into the environment is on Nicell424 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. the rise, and it is becoming increasingly difficult to achieve an acceptable degree of removal of these pollutants using conventional chemical and biological processes. Secondly, there is a need for the development of alter- native treatment methods that are faster, cheaper, more reliable, and simpler to implement than current processes. Thirdly, there is a growing recognition that enzymes can be used to target specific pollutants for treatment. And, finally, recent biotechnological advances foreshadow the production of cheaper and more readily available enzymes through genetic manipulation of microbial and plant cells and improved efficiency of isolation and puri- fication procedures. Much of this work remains very new and substantial hurdles must be overcome before full-scale industrial application of enzymes can become a reality. II. BACKGROUND AND FUNDAMENTALS OF ENZYMATIC PROCESSES A. Introduction to Enzymes Enzymatic systems fall between the two traditional categories of chemical and biological processes, since they involve chemical reactions based on the action of biological catalysts. Specifically, enzymes are catalysts that regu- late the multitude of chemical reactions that occur in living cells whether they are plant, animal or microbial. They carry out such cellular processes as energy conversion, food digestion, and biosynthesis. They have an orderly structure and the catalytic action occurs within a particular region of the enzyme protein. The active site usually consists of several amino acids with a specific conformation. Thus, they exhibit specificity and are characterized by showing an optimum tempe rature and pH for their actions. Similar to other catalysts, they accelerate the chemical reaction rate by lowering the activa- tion energy for a particular reaction. As shown in Fig. 1, the activation energy for the enzyme-catalyzed reaction is much smaller than that for the nonenzymatic reaction. This results in a much faster reaction rate. For example, the hydrolysis of sucrose by yeast invertase at 37jC is a trillion times faster than that caused by hydrogen ions alone [3]. The reactants of enzyme-catalyzed reactions are termed ‘‘substrates’’ and each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular product or products. The names of enzymes usually indicate the substrate involved. For example, hydrogen peroxide oxidoreductase is an enzyme that uses hydrogen per- oxide as its substrate to carry out the oxidation of organic substrates. Such formal names are often abbreviated such as in the contraction of hydrogen peroxide oxidoreductase to peroxidase. However, in the interest of avo iding Enzymatic Treatment of Waters and Wastes 425 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. confusion between enzymes with similar names, a numerical classification system is used to specifically identify the nature of the enzyme. This EC classification arose out of the work of the Enzyme Commission of the In- ternational Union of Biochemistry, which classifies enzymes into six general groups: (1) oxidoreductases, catalyzing oxidation–reduction reactions; (2) transferases, catalyzing the transfer of functional groups; (3) hydrolases, catalyzing hydrolysis reactions; (4) lyases, catalyzing the addition of groups to double bonds; (5) isomerases, catalyzing intramolecular rearrangements; and (6) ligases, catalyzing the condensation of two molecules coupled with the cleavage of a pyrophosphate bond of ATP or a similar triphosphate. The code consists of a sequence of four punctuated numbers: EC x.x.x.x. The first digit identifies the main class of the enzyme (as numbered above). The second and third digits further describe the kind of reaction being catalyzed. Enzymes catalyzing very similar but nonidentical reactions, e.g., the hydrolysis of different carboxylic esters, will have the same first three digits in their code. The fourth digit distinguishes them by defining the actual substrate, e.g., the actual carboxylic acid ester being hydrolyzed. Figure 1 The activation energy of an enzyme-catalyzed reaction compared to a noncatalyzed reaction. Nicell426 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. However, it should be noted that isoenzymes, i.e., different enzymes catalyzing identical reactions, would have the same four-digit classification. The classification system provides only the basis for a unique identification of an enzyme; the particular isoenzyme and its source still have to be specified. For example, peroxidases isolated from soybeans and horserad- ishes have the same classification, i.e., EC 1.11.1.7. Currently, there are approximately 3200 enzymes that have been listed and assigned classifica- tion numbers. The kinetics of many enzymatic reactions are often described using a simple model of enzyme catalysis, known as Michaelis–Menten kinetics. This model is based on an assumed string of events in which the enzyme E combines with the substrate S to form a complex ES (see also Fig. 1) according to E þ S ! k 1 k À1 ES ð1Þ which then dissociates into product P and free enzyme E according to ES ! k 2 P þ E ð2Þ where k 1 and k À1 are the rate constants for the forward and reverse compo- nents of the first reaction and k 2 is the rate constant for the second reaction. It can be shown that in a well-mixed closed vessel, under conditions in which the ratio of the concentration of enzyme (E o ) to substrate (S) is very small, the enzyme remains fully active, and the forward and reverse components of the first reaction are in steady state, the rate of change of substrate and product concentrations ( P) can be written as [4]: dS dt ¼À dP dt ¼ ÀV max S K m þ S ð3Þ where t is time, V max is called the maximum or limiting velocity and is equal to k 2 E o ,andK m is known as the Michaelis constant and is equal to (k À1 +k 2 )/k 1 . The constants, V max and K m , are evaluated by fitting the Michaelis–Menten equation to experimentally determined initial rates. Initial-rate data are used since under initial conditions the influence of accu- mulating products on the enzyme is negligible, and since the reaction con- ditions including enzyme (E o ) and substrate (S o ) concentrations are known best at time zero. This equation is consistent with qualitative observations that hold true for many enzyme systems. That is, the rate of reaction is first order in sub- strate concentration at low values of concentration but approaches zero Enzymatic Treatment of Waters and Wastes 427 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. order as the substrate concentration increases. In addition, the rate of reac- tion is proportional to the total amount of enzyme present. This equation is useful in that it can be used, for instance, to compare the relative rates of various enzymes carrying out the same reaction. However, its application in engineering systems is somewhat limited. For example, the Michaelis– Menten equation can be integrated to give: V max t ¼ S o À S þ K m ln S o S  ð4Þ from which the substrate concentration, S, corresponding to any time, t, can be determined. While inactivation is not usually a significant problem in vivo (in the living organism) where enzyme synthesis compensates for any loss of previously active enzymes, enzyme deactivation in vitro (isolated from a living cell) cannot be overlooked in kinetic studies or reactor design. Thus, if the quantity of enzyme participating in the reaction declines through inactivation or inhibition processes, this equation cannot be used to ade- quately describe the performance of the reactor. In addition, if it is intended that the reactor system achieve a high degree of conversion of the substrate (which is often the case during the transformation of pollutants), as the reaction progresses the ratio of enzyme to substrate will become relatively large and the terms under which the Michaelis–Menten equation was derived will be violated. Of course, a major impediment to the application of this equation is that the enzyme reactions of interest may not follow the simple sequence of events described above. Therefore, studies of the mechanisms and kinetics of enzyme systems can be crucial for the design of efficient reactor systems. The importance of such studies will be demonstrated later in this chapter. B. Advantages and Limitations of Enzyme Applications for Pollutant Transformation When accomplishing the transformation of chemical species, cell-free en- zymes (i.e., those that have been isolated from their parent organisms) are often prefer red over intact organisms containing the enzyme because they act with greater specificity, their activity can be better standardized, they are easier to handle and store, and enzyme concentration is not dependent on bacterial growth rates [5]. This can lead to some important advantages of enzymatic processes over biological systems. For example, enzymes can be applied to transform targeted contaminants including many of those that may resist bio degradation. This catalytic action can be carried out on, or in the presence of, many substances that are toxic to microbes. In addition, Nicell428 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. some enzymes can operate over relatively wide temperature, pH, and salinity ranges compared to cultures of microorganisms. They can also be used to treat contaminants at high and low concentrations and are not susceptible to shock loading effects associated with changes in contaminant concentrations that can often irreversibly damage or metabolically inactivate microbial cells. Consequently, there are fewer delays associated with shutdown/startup periods that are normally required to acclimatize biomass to waste streams. Importantly, the catalytic action of enzymes enables the development of smaller systems of lower capital cost due to the high reaction rates associated with enzymatic reactions. In addition, because bacterial growth is not required to accomplish waste transformations, sludge production is reduced because no biomass is generated. Many of the advantages listed above allow for the development of stable enzymatic treatment systems with simpler process control [6]. Enzymes can also offer several advantages over conventional chemical processes. In particular, the significance of enzymes lies in their ability to carry out processes that are impractical or impossible through nonbiological chemistry. Their high degree of specificity allows enzymes to remove target pollutants selectively, which precludes undesirable or unnecessary reactions that would otherwise increase reactant consumption and, correspondingly, increase the cost of treatment. They also make efficient use of chemical re- agents and typically are characterized by a high reaction velocity. Their selectivity and high reaction rates make them ideal for the treatment of com- pounds that are present in trace quantities (i.e., micropollutants) or that cannot be removed by traditional physicochemical processes. Additionally, they operate at low temperature conditions, thereby reducing energy require- ments for processes normally conducted at elevated temperatures (e.g., ther- mal oxidation). They also operate under mild pH conditions, thereby reducing the impact of corrosion on reaction vessels and avoiding the need for waste neutralization (e.g., Fenton’s oxidation). Finally, an inherent ad- vantage of enzymes is their compatibility with the environment and their nonhazardous nature. Thus, enzyme residues that may be present following waste treatment are of low pollution potential. Whereas the above advantages are indeed significant, it should be noted that the majority of chemical and biological processes are not can- didates for replacement by enzymatic processes. That is, biological processes and some chemical processes (e.g., chemical or thermal oxidation) have a fundamental advantage over enzymatic systems, i.e., their ability to simul- taneously transform a broad range of compounds. For example, many mu- nicipal, agricultural, and industrial wastes consist of a mixture of organic compounds usually classified under the broad categories of biological oxy- gen demand (BOD) or chemical oxygen demand (COD). Once released into Enzymatic Treatment of Waters and Wastes 429 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. receiving water bodies, these collections of organic compounds result in the depletion of dissolved oxygen in the water column as a result of natural microbial processes. In many instances, the majority of these compounds can be efficiently degraded through the combined action of mixed cultures of microorganisms. In contrast, enzymes are biological catalysts whose actions are tailored to exclusively act upon specific chemical species. Thus, enzy- matic treatment will not result in the removal of a broad range of com- pounds from a waste stream, but will only accomplish the transformation of an individual compound or class of compounds. If the product of the reaction is less toxic, more readily degraded than the reactant, or has a commercial value, then an enzyme-based strategy could be effective. This limits the application of enzymatic processes to accomplish the transforma- tion of target species that are either problematic due to their toxicity or that have been identified as the raw materials from which enzymes can produce value-added products. In addition, because enzymatic reactions often require cosubstrates, these cosubstrates must be readily available. Water- or oxygen-requiring enzymes (e.g., hydrolases and oxygenases, respectively) are the most obvious candidates for waste applications, whereas enzymes that require cell-generated cosubstrates (e.g., ATP or NAD(P)H) would be less practical. Finally, it must be emphasized that some enzymes are quite fragile and their catalytic activities are sensitive to changes in ionic strength, the presence of metal ions, solvents, and other inactivating or inhibiting species. Therefore, wastewater characteristics will play a significant role in determining the feasibility of enzymatic treatment. III. POTENTIAL APPLICATIONS OF ENZYMES Recent research has focused on the development of enzymatic processes for the treatment of wastewaters, solid wastes, hazardous wastes, and soils. The environmental applications may be classified according to their objectives. For example, some processes are specifically designed to accomplish the transformation of target pollutants in wastewater streams to reduce toxicity. Alternatively, the conversion of waste materials can sometimes be achieved in a manner that produces a product with commercial value. Some applications that have recently been identified will be outlined below. A. Enzymatic Treatment to Improve Waste Quality Because of their high specificity to individual species or classes of com- pounds, enzymatic processes can be designed to specifically target selected compounds that are detrimental to the environment. Compounds that are Nicell430 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. candidates for this type of treatment are usually those that cannot be treated effectively or reliably using traditional techniques. Alternatively, enzymatic treatment can be used as a pretreatment step to remove one or m ore compounds that can interfere with subsequent downstream treatment processes. For example, if inhibitory or toxic compounds can be removed selectively, the bulk of the organic material could be treated biologically, thereby minimizing the cost of treatment. Many enzymes are susceptible to inactivation in the presence of other chemicals. Therefore, it is likely that enzymatic treatment will be most effective in those streams that have the highest concentration of the target contaminant and the lowest concen- tration of other contaminants that may tend to interfere with enzymatic treatment. The following situations are those where the use of enzymes might be most appropriate [1]:  removal of specific chemicals from a complex industrial waste mix- ture before on-site or off-site biological treatment;  removal of specific chemicals from dilute mixtures, for which con- ventional mixed-culture biological treatment might not be feasible;  polishing of a treated wastewater or groundwater to meet limita- tions on specific pollutants or to meet whole effluent toxicity criteria;  treatment of wastes generated infr equently or in isolated locations, including spill sites and abandoned waste-disposal sites; and  treatment of low-volume, high-concentration wastewater at the point of generation in a manufacturing facility to allow reuse of the treated process wastewaters, to facilitate recovery of soluble prod- ucts, or to remove pollutants known to cause problems downstream when mixed with other wastes from the plant. Some potential applications of enzymes that have been identified for the improvement of waste quality include the transformation of toxic and color- causing aromatic compounds, cyanide, pesticides, surfactants, and heavy metals. In addition, some physical modifications in waste characteristics have been achieved through the mixing of solid wastes with enzymes. These applications are summarized in Table 1, and some promising applications are described in the following. 1. Aromatic Pollutants Aromatic compounds constitute one of the major classes of pollutants and are heavily regulated in many countries. They are found in the wastes of a variety of industries including coal conversion, petroleum refining, resins and plastics, wood preservation, metal coating, dyes and other chemicals, textiles, mining and dressing, and pulp and paper. Most aromatic compounds are Enzymatic Treatment of Waters and Wastes 431 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. Table 1 Cell-Free Enzymes Used to Accomplish the Transformation of Pollutants Enzymes a Proposed application Acylamidase Transformation of pesticides including acid analides (e.g., propanil, karsil) and phenoxyacetates (e.g., 2,4-D, MCPA) [53] Alcalase Hydrolysis of tannery waste for chrome recovery [110] Amylases (in the presence of proteases) (EC 3.2.1) Reduced treatment time for activated sludges from food processing wastes [63,111] Cellulase glucanglucanohydrolase (EC 3.2.1.4) glucan exo-cellobiohydrolase (EC 3.2.1.91) B-glucosidase (EC 3.2.1.21) Degradation of sludge from municipal wastewater treatment plants [61]; treatment of hazardous wastes [112] Chloroperoxidase (EC 1.11.1.10) Oxidation of phenolic compounds [29,85,102] Cyanidase b Cyanide decomposition [56,57] Cyanide hydratase (EC 4.2.1.66) Cyanide hydrolysis [57,58] Dehalogenases Degradation of agrochemicals, solvents, degreasers, flame retardants, and chemical intermediates used in the production of high volume chemicals [113]; detoxification of chlorophenols [61] Esterase Transformation of pesticides including phenylcarbamates (e.g., CIPC, IPC) and organophosphates (e.g., malathion, paraoxon, parathion) [53] Laccase (EC 1.10.3.2) Oxidation of phenols, dyes, and polycyclic aromatic hydrocarbons [48,49], decolorization of Kraft bleaching effluents, binding of phenols and aromatic amines with humus [47] Lignin peroxidase Transformation of phenols, aromatic amines, polyaromatic hydrocarbons, and other aromatic compounds, decolorization of Kraft bleaching effluents, treatment of dioxins, pyrene [86–89,114] Lipase (EC 3.1.1.3) (with carbohydrase and protease) Improved sludge dewatering [59] Lysozyme (EC 3.2.1.17) Cell lysis resulting in sludge degradation and improved dewatering [61] Mn peroxidase Oxidation of monoaromatic phenols and aromatic dyes [2] Muramidase Dewatering of pulp and paper sludges [61] Parathion hydrolase Hydrolyzation of organophosphate pesticides [53,55] Pectinesterase (EC 3.1.1.11) Degradation of pectin [64] Peroxidase (EC 1.11.1.7) Transformation of phenols and aromatic amines [7–24], decolorization of Kraft bleaching effluents [21], dewatering of phosphate slimes [60] Perma-Zyme b Improve the strength and stability of pure clayey soils and soil–fly ash mixtures [62] Proteases Degradation of proteins, improving sludge dewatering [61] Tyrosinase (EC 1.14.18.1) Transformation of phenols [42,44] Ureases Treatment of urinous waste waters [53] a EC classifications are provided wherever they were specified in literature sources. b Commercial enzyme preparation that may consist of more than one enzyme. Nicell432 TM Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. [...]... cellobiohydrolase, and cellobiase Another study involving the fungal enzyme preparation Econase was conducted to investigate the effect of cellulolytic enzymes on MSW degradation [73] Econase consists mainly of endo-1,4-h-D-glucanase, cellobiohydrolase, and exo-1,4-h-D-glucosidase in addition to a number of other enzymes The use of Econase seemed to enhance the degradation of MSW as well as cellulose degradation. .. materials from pulp and paper and municipal solid wastes to produce alcohol; degradation of grain, fruit and vegetable wastes to produce glucose syrups [64] Bioconversion of shellfish waste to N-acetyl glucosamine for yeast production [69] Conversion of galactose from whey hydrolysis to L-ascorbic acid [64] Improved juice yields and dissolved solids resulting in less waste [115] Degradation of apple... Treatment of Waters and Wastes 451 technologies for the treatment of aqueous phenols using peroxidase enzymes, particularly those isolated from horseradishes and soybeans Therefore, such enzymes will be used in the discussion below as an example of fundamental and ongoing research that is required when developing enzymatic processes for eventual full-scale application A Candidate Wastes and Enzymes Aromatic... applications Amylases: a-amylase (EC 3.2.1.1) glucoamylase (EC 3.2.1.3) Cellulolytic enzymes: cellulase (EC 3.2.1.4) cellobiohydrolase (EC 3.2.1.91) cellobiase (EC 3.2.1.21) exo-1,4-h-D-glucosidase (EC 3.2.1.74) Chitinase (EC 3.2.1.14) L-Galactono-lactone oxidase (EC 1.1.3.24) Pectinases Pectin lyase (EC 4.2.2 .10) Lactase (EC 3.2.1 .108 ) Proteases a Hydrolysis of starches found in rice and grain wash waters...Enzymatic Treatment of Waters and Wastes 433 toxic and must be removed from wastes before they are discharged into the environment Several methods of treatment based on the use of peroxidases and polyphenol oxidases have been proposed as potential alternatives to conventional methods for the treatment of such compounds Peroxidase enzymes are produced in the cells of many microorganisms and plants They catalyze... pesticides, detoxification of pesticide containers and spray tanks, and the pollution of surface and groundwater by pesticide runoff [19,53] Common treatment methods include incineration, chemical methods, and landfilling However, these systems have serious limitations including high cost, production of hazardous byproducts, disposal of chemical reagents, and the TM Copyright © 2003 by Marcel Dekker, Inc... writing-grade paper, and tissue IV BARRIERS TO FULL-SCALE APPLICATION Enzymes have undoubtedly stimulated the research community’s interest and have been investigated for their potential use in many types of applications Applications have been demonstrated that aid in the efficient use of raw materials, improve the quality and reduce the quantities of wastes for disposal, and catalyze the transformation... postsedimentation handling Cellulase and the bacterial enzyme lysozyme, or muramidase, were also used for sludge dewatering [61] Whereas cellulase was used with penicillin and gave rather poor results, lyzozyme was allegedly able to alter floc matrices and to cause a dramatic increase in dewatering rates Cellulase has also been used for the treatment of low-level radioactive, mixed, and hazardous /chemical wastes. .. reduction, deproteination, and demineralization to produce a chitin material that can be easily bioconverted by chitinase to the monomer N-acetyl glucosamine The latter serves as a substrate for the single-cell protein production An unspecified enzyme was also used to treat leather wastes [70] The degradation of leather wastes yielded a water-soluble hydrolysate that could be concentrated and dried to produce... peroxidases for the treatment of phenolic wastes As shown in Fig 3, tyrosinase catalyzes two distinct oxidation reactions of phenols Molecular oxygen binds to the initial state of tyrosinase (Edeoxy) bringing it to an oxygenated state (Eoxy) Thereafter, monophenols or o-diphenols are oxidized by tyrosinase to oquinones in cycle I (Eoxy, Eoxy-M, and Emet-D) or cycle II (Eoxy, Eoxy-D, Emet, and Emet-D), respectively . of goods; and (4) to transform wastes into marketable products. There are multitudes of ways in which the transfor- mation of wastes and pollutants can be carried out. Most of these methods may. specific chemicals from a complex industrial waste mix- ture before on-site or off-site biological treatment;  removal of specific chemicals from dilute mixtures, for which con- ventional mixed-culture. (E oxy ). Thereafter, monophenols or o-diphenols are oxidized by tyrosinase to o- quinones in cycle I (E oxy , E oxy-M , and E met-D ) or cycle II (E oxy , E oxy-D , E met , and E met-D ), respectively. Quinones