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Use of Enzymes to Remove Organic Micropollutants from Potable Water
655
USE OF ENZYMES TO REMOVE ORGANIC MICROPOLLUTANTS FROM POTABLE WATER
B. DUSSERT, J. P. DUGUET, J. MANEM, J. MALLEVIALLE Centre de Recherche Lyonnaise cles Eaux - Degr6mont
38 rure du Pre‘sident Il’ilson, 78230 Le Pecq, FRANCE
ABSTRACT Enzymatic methods have shown promise for removing aromatic compounds from industrial wastewater (high strength). The removal of these compounds was studied at low levels which might be encountered in surface-waters. The results indicate that enzymatic oxidative coupling may be useful in eliminating aromatics which are not well removed in biological or physical water treatment.
1. INTRODUCTION Application of biotechnology to various fields have received a great deal of public and scientific attention recently. T w o broad areas which may be useful in (kinking-water treatment are the use of specialized and/or genetically engineered bacteria which degrade objectionable compounds to innocuous end-products, and enzymatic alteration of cornpounds to aid downstream unit. processes in their removal. Examples of existing biological unit processes are ammonia removal [ 1 1, carbonremoval [2], antl manganese removal [ 31 These examples fall into the category of specialized bacteria. A recent review [ 4 ] lists antl discusses many other examples of biological degradation of hazardous organic compounds. Enzymatic methods for the elimination of aromatic compounds from synthetic wastewaters have shown promise on the laboratory scale using a system of
656
hydrogen peroxide and horse-radish peroxidase [ 5,6, 7 1 . In this system, aromatic compounds are first converted to aromatic radicals, which then polymerize to form products which are less water soluble than the reactants [ 5 1 . The products thus can be more easily separated from water than their presursors. Klibanov et al. [ 5, 6, 7 ] have reported on the removal efficiencies for over 30 different aromatic amines and phenols from synthetic waters under laboratory conditions. Removal efficiencies ranged from 60% to greater than 99%. Furthermore, they have demonstrated that the presence of an easily removed substrate can enhance the removal efficiency for a more poorly removed compound. This fact is extremely important in drinking-water treatment, where a broad spectrum of organic compounds are usually present at low concentrations [ 8 , 9, 10 ] . Any such synergism would be very beneficial in potable water production. The studies on horse-radish peroxidase were conducted at high concentrations in synthetic wastewaters. Most of the aforementioned studies were conducted at 100 mg/L of aromatic and 100 to 1000 units/L of peroxidase. One aromatic amine has been studied as low as 0.5 mg/L, showing no effect of substrate concentration [ 7 1 . Thus, the peroxidase-peroxide system may have potential in drinking-water treatment for removal of problem aromatic compounds at low levels. Chlorinated phenolic compounds are examples of aromatics which may cause taste and odor problems in finished drinking-water 111 1 . These compounds may result from natural and domestic sources [12 1 , from wood preservatives [ 13 ] , or as by-products of chlorination in the treatment process [ l l ] . A common treatment process for the occurrence of chlorinated taste and odor problems is super chlorination, which converts the odor causing mono- and di-chlorophenols to trichlorophenols [ l l 1 which (lo not cause odor, but are suspected carcinogens [ 5 1. This paper will consider the efficacy or horse-radish peroxidase (HPR) for the removal of 2chlorophenol -- a taste and odor compound, and pentachlorophenol a wood preservative sometimes found as a contaminant in drinking-water. These compounds were evaluated at low as well as high concentrations, and in the presenc.e of potential competing compounds. Competing compountk may be innocuoua aromatic compounds or other specific micropollutants. Humic substances are organic compounds composed mainly of aromatic structures [ 14 ] which account for up to 90% of the background total organic carbon (TOC) in natural waters [ 15 ] . Recent studies have indicated that the presence of humic acids may deactivate horse-radish peroxidase [16 1.
2. MATERIALS AND METHODS The phenolic compounds, 2chloroptienol (2-CP) and pentachlorophenol (PCP) were taken from a phenol test kit (4--4570, Supelco, Inc., Bellefonte, PA). HRP
657
(Type VI, Sigma Chemical Co., St. Louis, MO, E.C. No. 1.11.1.7) was assayed for activity using the manufacturers’ instructions, yielding a value of 250 units per mg. Hydrogen peroxide (30%) was purchased from Prolabo (Paris, France). The various chlorophenols were mixed in water purified by the Milli-Q system (Millipore Corp., Bedford, Mass.) or filtered river water, as noted. Samples of the chlorophenols were placed in 250 mL flasks on a gyratory shaker (Model R-2, New Brunswick Scientific, Edison, NJ). The chlorophenol solution was adjusted to pH 6--7 by a 10 mM phosphate buffer. Peroxide and/or HRP were then added to the solution and the flasks were shaken at 175 rpm for 3 hours. Selected samples were centrifuged (Janetzki K24, Englesdorf, Germany) at 12,000 rpm to remove any suspended material. Afterwards, phenols were analyzed as described below. High pressure liquid chromatography (HPLC) was used to identify phenols and separate the products of the reaction. The system included a Dupont 870 pump module, 850 absorbance detector, and a 4.6 mm x 2 5 cm Zorbax ODS liquid chromatography column (all from DuPont and Co., Wilmington, DE). Analysis of 2 C P was achieved under the following conditions; mobile phase 33% MeOII in water at 1.5 mL/min; PCP analysis: mobile phase 50% MeOH in water at 1 5 mL/min; UV detection for 2 C P and PCP at 254 nm.
3. RESULTS AND DISCUSSION Table 1 shows the results for 2CP removal from synthetic solutions in MilliQ water, with and without centrifugation. Although the high removals previously described [ 6 ] were not attained at the 100 mg/L level, significant removal occurred. Almost complete removal occurred for the lower concentrations. At the higher concentrations, the solution turned a slight yellow-green color, and a precipitate was observed. Centrifugation did not produce a precipitate at the lowest concentration, thus it was eliminated from the experimental procedure for 2CP. The data in Table 1 indicate that lower substrate concentrations are best removed. This is an important finding for water treatment for 2 reasons. First, it indicates that the method may be applicable to trace organic removal in drinking-water. Second, it suggests that the enzyme and peroxide concentrations were in excess of requirements. Thus, applications of this technology may,, only need small additions of reactants for removal of trace aromatics. Table 2 presents the results for lower concentrations of 3CP in the presence and absence of natural humic substances. The sources of the humic materials were tap water in the laboratory and filtered river water from a pilot plant upstream of Paris on the Seine River (TOC = 2 mg/L in both cases). In all experiments, the
668
removal was greater than 95% for 2CP. The enzyme and peroxide concentrations were also varied with no effect on the results. It appears from these experiments that 100 units/L of horse-radish peroxidase, and 1 mM peroxide is sufficient for removal of low concentratioris of 2CP. Tab. 1. Removal and/or transformation of 2Chlorophenol by Enzymatic Oxidative Coupling of centrifugation 2CP (WlL)
Enzyme concentxation unitslL
Peroxide concentration
Centrifugation
- effect
%Removal
mM 75
100
1000
100
1000
75
100
1000
75-90
100
1000
7 5 -90
10
1000
95
10
1000
95
1
1000
1
1000
> 95 > 95
Conditions: all experiments conducted in MilliG water buffered (phosphate) at pH = 7 . 2 C P initially dissolved in methanol, resulting in 3-4 % v/v methanol in water.
Other investigators [ 16 ] have obperved a decrease in horse-radish peroxidase activity in the presence of natural humic acids. However, their experiments were conducted at up to 1600 mg/L of humic acid. A recent review [17] at water treatment for organic carbon removal included raw water TOC values in the range of 1--16 mg/L. The TOC value can be used as a rough estimate of humic substances as 90% of natural TOC is usually humic material [15 ] . Therefore, the experiments conducted here are for relatively low humic concentrations (2 mg/L). However, no effects was observed on 2CP even though the TOC was at a concentration of up to 200 greater than the 2CP. The humics did not appear to inhibit or compete for the peroxidase. Table 2 also shows that the greater than 95% removal persists for lower substrate concentration than have been previously reported, and that the enzyme and peroxide do not become limiting a t 1 mM hydrogen peroxide and 100 u n i t d l of enzyme activity. Previous cost estimates [5 ] have indicated that the enzyme application may be prohibitively expensive for potable water treatment. However, the actual
669
Tab. 2. Removal and/or transformation of 2Chlorophenol by Enzymatic Oxidative Coupling - with and without backgmund oxganics
2CP (mg/L)
Water type
0 -1
buffer pH 7
Enzyme concentration units/L
Peroxide concentntion mM
1000
5
0.01
1000
5
0.01
100
5
0.01
tap water
100
5
1
filtered river water
1000
5
0.3
1000
1
0.3
1000
5
0.3
1000
1
100
1
0.3
,
% Removal
> 95 > 95 > 95 > 95 > 95 > 95 > 95 > 95 > 95
Conditions: all experiments conducted without centrifuetion. 2 C P initially dissolved in methanol, resulting in 3 4 % v/v methanol in water.
requirements for the enzyme may be considerably less in drinking-water treatment. Therefore, future research is needed to determine the relationship between contaminant concentration and required enzyme doses. Table 3 shows a similar set of experiments for PCP. Initial experiments show much less removal for PCP than equivalent concentrations of 2 C P . For most experiments, the chlorophenol was initially dissolved in methanol and then added to water. Two experiments show that methanol may interfere with removal of PCP, because substantially better removal was obtained when methanol was eliminated. However, the removals for PCP remained less than those for2-CPelRnin the absence of methanol. PCP is more nonpolar than 2-CP and it may have remained associated with methanol after addition to water, even though there was no apparent emulsion. This may account for its lack of reactivity with the enzyme. compared to the 2CP. However, these data suggest that the enzyme may be specific for certain types of structures and that this may limit its applicability. Other compounds which have reported removal efficiencies less than 90% include 3chlorophenol, 2,6-dimethylphenol, aniline, 4chloroaniline, 4-bromoaniline, 4-fluoroaniline, and
660
Tab. 3.Removal and/or tmnsformation of pentachlorophenol by Enzymatic Oxidative Coupling ~~~
FCP (mg/L)
Enzyme concentration units/L
Peroxide concentration
Centrifugation
% Removal
mM
1000
-
5
20
1000
-
65
15
1000
-
62
lo*
"
Conditions:all experiments conducted in MilliQ water buffered (phosphate) at pH = 7. 'YPentachlorophenol (PCP) initially dissolved in methanol. Addition to water resulted in 3-4 % vlv methanol.
diphenylamine [ 5 ] . These compounds may be removable with greater concentrations of enzyme and peroxide, but the costs would likely be prohibitive. Many other questions need to be addressed before the potential use becomes a reality. Among these, some of the more important are: what are the products of the reaction, what is the fate of the products and the enzyme in the water treatment process, what is the potential for unwanted byproducts from other unit processes -- particularly chlorination, and what is the minimum level required for removal. This study indicated that precipitation was not visually obvious for the lowest concentration of 2CP. Thus the product of the reaction is assumed to remain water soluble. The use of enzymes in drinking-water treatment has several advantages over the use of whole bacteria. Enzymes often have high specificities for substrate, so there is better control over the compound removed and the product produced. The enzyme concentration in the water is controlled by the plant operator. Thus there is no dependence on the growth rate of the bacteria. This can be particularly important when the compound targeted for removal appean only sporadically in the raw water, such as taste and odor compounds. Bacteria require an incubation time to acclimate to the substrate and grow, but enzymes may be added as needs require.
4. CONCLUSION
The potential use of enzymes for trace aromatic compound removal has been shown on the laboratory scale. This type of examination can screen compounds for which the enzyme may be useful. 2CP, a cornpound of organoleptic concern, has
661 been shown to be effectively eliminated by the horse-radish peroxidase - peroxide system. This compound is a product o f prechlorination when phenol is in the water supply, and t h e enzyme system offers a n alternative to the usual practice of applying a n active carbon treatment (powdered o r granulated form) which requires investments o n t h e treatment site.
REFERENCES 1. Y-Richard, “Biological Methods for the Treatment of Ground Water”, in Oxidation Techniqlps in Drinking Water Treatment, eds. H. Sontheimer and W.Kuhn, EPA-570/9-79-020,1979. 2. F. Fiessinger, J. Mallevialle, and A-Benedek, ”Interaction of Adsorption and Bioactivity in
Full Scale Activated Carbon Filters: The Mont Valerian Experiment”, in Treatment of Water by Granular Activated Carbon, eds. M. J. McGuire and I. H. Suffet, American Chemical Society Advances in Chemistry 202, ACS Books, Washington, D.C., 1983, p. 319. 3. R . Milliner, D.A. Bowles, and R. W. Brett, ”Biological Pretreatment at Tewkesbury”, Proceedings of the Society forwater Treatment Examination, v. 21, 1972, p. 318. 4. H. Kobayashi, and B.E. Rittmann, ”Microbial Removal of Hazardous OGanic Compounds”, Environmental Science and Technology, v. 16, n. 3, 1982, p- 170A. 5. B. N. Alberti. and A. M. Kh i n o v , ”Enzymatic Removal of Dissolved Aromatics from Industrial Aqueous Effluents”, in Biotechnology and Bioengineering Symposium No. 11, John Wiley and Sons, Inc. New York, NY, 1981, p. 373-9. 6. A.M. Klibanov, B.N. Alberti, E.D. Morris, and L.M. Felshin, ”Enzymatic Removal of Toxic Phenols and Anilines from Waste Waters”, Journal of Applied Biochemistry, v. 2, 1980, p. 414-21. 7. A.M. Klibanov, and E.D. Moms, ”Horseradish Peroxidase forthe Removal of Carcinogenic Aromatic Amines from Water”, Enzyme Microbiology and Technology, Y. 3, 1981, p. 11922.
8. I. H. Suffet, L. Brenner, and P.R. Cairo, ”GC/MS Identification of Trace Organics in Philadelphia Drinking Water During a 2-Year Period”, Water Research, v. 14, 1980, p. 853. 9. A. Bruchet, and J. Mallevialle, ”Etude de Cas #Elimination de Composes Organiques dans des Usine de Production d’Eau Potable: Realimentations a l e s d e N m T d m m t s d’Eaux Surfaceot d’Eaux Soutenaines”, Journal Francais D’Hydmlogie, v. 14, FASC 1 , n. 40. 1983, p. 31. 10. G.T. Coyle, et al, ”Broad Spectrum Analysis ofthe Removalof Trace Organics in an Ozone Granular Activated Carbon Potable Water Pilot Plant Study”, in Water Chlorination: Environmental Impact and Health Effects, Volume 4, ed. R. L. Jolley, A M Arbor Science, AnnArbor, MI, 1983, p. 421. 11. J.G. Smith, S. Lee, and A. Netzer, ”Model Studiesof Aqueous Ch1orination:TheChlorination of Phenols in Dilute Aqueous Solution”, Water Research, v. 10, 1976, p. 985-90. 12. R. A. Baker, ”F’henolic Analyses by Direct Aqueous Injection Gas Chromatography”, Journal of the American Water Works Association, v. 58, 1966, p. 751-60.
662
13. . L. Rudling, ”Determination of Pentachlophenol in Organic Tissues and Water”, Water Reseanfi, v.4, 1970, p-533-714. M. Schnitzer, and S . U.Khan, Humic Substances in the Enyironment, Marcel Dekker, New York, NY, 1972. 15. P. L. McCarthy, ’Organics in Water, an Engineering Challenge’’, Joumal of the Environmental Engineering Division, ASCE, V. 106, 1980, p. 1. 16. 2. Pflug, ”Effect of Humic Acids on the Activity of Two Peroxidases”, 2. Pflannernechr, Bodenkd., 1980, p.430-40. 17. P. V. Roberts, and R. S . Summers, ”Performance of GAC for TOC Removal”, Joumal of the American Water Works Association, v. 74, n. 2, 1982, p. 113.
USE OF ENZYMES TO REMOVE ORGANIC MICROPOLLUTANTS FROM POTABLE WATER
B. DUSSERT, J. P. DUGUET, J. MANEM, J. MALLEVIALLE Centre de Recherche Lyonnaise cles Eaux - Degr6mont
38 rure du Pre‘sident Il’ilson, 78230 Le Pecq, FRANCE
ABSTRACT Enzymatic methods have shown promise for removing aromatic compounds from industrial wastewater (high strength). The removal of these compounds was studied at low levels which might be encountered in surface-waters. The results indicate that enzymatic oxidative coupling may be useful in eliminating aromatics which are not well removed in biological or physical water treatment.
1. INTRODUCTION Application of biotechnology to various fields have received a great deal of public and scientific attention recently. T w o broad areas which may be useful in (kinking-water treatment are the use of specialized and/or genetically engineered bacteria which degrade objectionable compounds to innocuous end-products, and enzymatic alteration of cornpounds to aid downstream unit. processes in their removal. Examples of existing biological unit processes are ammonia removal [ 1 1, carbonremoval [2], antl manganese removal [ 31 These examples fall into the category of specialized bacteria. A recent review [ 4 ] lists antl discusses many other examples of biological degradation of hazardous organic compounds. Enzymatic methods for the elimination of aromatic compounds from synthetic wastewaters have shown promise on the laboratory scale using a system of
656
hydrogen peroxide and horse-radish peroxidase [ 5,6, 7 1 . In this system, aromatic compounds are first converted to aromatic radicals, which then polymerize to form products which are less water soluble than the reactants [ 5 1 . The products thus can be more easily separated from water than their presursors. Klibanov et al. [ 5, 6, 7 ] have reported on the removal efficiencies for over 30 different aromatic amines and phenols from synthetic waters under laboratory conditions. Removal efficiencies ranged from 60% to greater than 99%. Furthermore, they have demonstrated that the presence of an easily removed substrate can enhance the removal efficiency for a more poorly removed compound. This fact is extremely important in drinking-water treatment, where a broad spectrum of organic compounds are usually present at low concentrations [ 8 , 9, 10 ] . Any such synergism would be very beneficial in potable water production. The studies on horse-radish peroxidase were conducted at high concentrations in synthetic wastewaters. Most of the aforementioned studies were conducted at 100 mg/L of aromatic and 100 to 1000 units/L of peroxidase. One aromatic amine has been studied as low as 0.5 mg/L, showing no effect of substrate concentration [ 7 1 . Thus, the peroxidase-peroxide system may have potential in drinking-water treatment for removal of problem aromatic compounds at low levels. Chlorinated phenolic compounds are examples of aromatics which may cause taste and odor problems in finished drinking-water 111 1 . These compounds may result from natural and domestic sources [12 1 , from wood preservatives [ 13 ] , or as by-products of chlorination in the treatment process [ l l ] . A common treatment process for the occurrence of chlorinated taste and odor problems is super chlorination, which converts the odor causing mono- and di-chlorophenols to trichlorophenols [ l l 1 which (lo not cause odor, but are suspected carcinogens [ 5 1. This paper will consider the efficacy or horse-radish peroxidase (HPR) for the removal of 2chlorophenol -- a taste and odor compound, and pentachlorophenol a wood preservative sometimes found as a contaminant in drinking-water. These compounds were evaluated at low as well as high concentrations, and in the presenc.e of potential competing compounds. Competing compountk may be innocuoua aromatic compounds or other specific micropollutants. Humic substances are organic compounds composed mainly of aromatic structures [ 14 ] which account for up to 90% of the background total organic carbon (TOC) in natural waters [ 15 ] . Recent studies have indicated that the presence of humic acids may deactivate horse-radish peroxidase [16 1.
2. MATERIALS AND METHODS The phenolic compounds, 2chloroptienol (2-CP) and pentachlorophenol (PCP) were taken from a phenol test kit (4--4570, Supelco, Inc., Bellefonte, PA). HRP
657
(Type VI, Sigma Chemical Co., St. Louis, MO, E.C. No. 1.11.1.7) was assayed for activity using the manufacturers’ instructions, yielding a value of 250 units per mg. Hydrogen peroxide (30%) was purchased from Prolabo (Paris, France). The various chlorophenols were mixed in water purified by the Milli-Q system (Millipore Corp., Bedford, Mass.) or filtered river water, as noted. Samples of the chlorophenols were placed in 250 mL flasks on a gyratory shaker (Model R-2, New Brunswick Scientific, Edison, NJ). The chlorophenol solution was adjusted to pH 6--7 by a 10 mM phosphate buffer. Peroxide and/or HRP were then added to the solution and the flasks were shaken at 175 rpm for 3 hours. Selected samples were centrifuged (Janetzki K24, Englesdorf, Germany) at 12,000 rpm to remove any suspended material. Afterwards, phenols were analyzed as described below. High pressure liquid chromatography (HPLC) was used to identify phenols and separate the products of the reaction. The system included a Dupont 870 pump module, 850 absorbance detector, and a 4.6 mm x 2 5 cm Zorbax ODS liquid chromatography column (all from DuPont and Co., Wilmington, DE). Analysis of 2 C P was achieved under the following conditions; mobile phase 33% MeOII in water at 1.5 mL/min; PCP analysis: mobile phase 50% MeOH in water at 1 5 mL/min; UV detection for 2 C P and PCP at 254 nm.
3. RESULTS AND DISCUSSION Table 1 shows the results for 2CP removal from synthetic solutions in MilliQ water, with and without centrifugation. Although the high removals previously described [ 6 ] were not attained at the 100 mg/L level, significant removal occurred. Almost complete removal occurred for the lower concentrations. At the higher concentrations, the solution turned a slight yellow-green color, and a precipitate was observed. Centrifugation did not produce a precipitate at the lowest concentration, thus it was eliminated from the experimental procedure for 2CP. The data in Table 1 indicate that lower substrate concentrations are best removed. This is an important finding for water treatment for 2 reasons. First, it indicates that the method may be applicable to trace organic removal in drinking-water. Second, it suggests that the enzyme and peroxide concentrations were in excess of requirements. Thus, applications of this technology may,, only need small additions of reactants for removal of trace aromatics. Table 2 presents the results for lower concentrations of 3CP in the presence and absence of natural humic substances. The sources of the humic materials were tap water in the laboratory and filtered river water from a pilot plant upstream of Paris on the Seine River (TOC = 2 mg/L in both cases). In all experiments, the
668
removal was greater than 95% for 2CP. The enzyme and peroxide concentrations were also varied with no effect on the results. It appears from these experiments that 100 units/L of horse-radish peroxidase, and 1 mM peroxide is sufficient for removal of low concentratioris of 2CP. Tab. 1. Removal and/or transformation of 2Chlorophenol by Enzymatic Oxidative Coupling of centrifugation 2CP (WlL)
Enzyme concentxation unitslL
Peroxide concentration
Centrifugation
- effect
%Removal
mM 75
100
1000
100
1000
75
100
1000
75-90
100
1000
7 5 -90
10
1000
95
10
1000
95
1
1000
1
1000
> 95 > 95
Conditions: all experiments conducted in MilliG water buffered (phosphate) at pH = 7 . 2 C P initially dissolved in methanol, resulting in 3-4 % v/v methanol in water.
Other investigators [ 16 ] have obperved a decrease in horse-radish peroxidase activity in the presence of natural humic acids. However, their experiments were conducted at up to 1600 mg/L of humic acid. A recent review [17] at water treatment for organic carbon removal included raw water TOC values in the range of 1--16 mg/L. The TOC value can be used as a rough estimate of humic substances as 90% of natural TOC is usually humic material [15 ] . Therefore, the experiments conducted here are for relatively low humic concentrations (2 mg/L). However, no effects was observed on 2CP even though the TOC was at a concentration of up to 200 greater than the 2CP. The humics did not appear to inhibit or compete for the peroxidase. Table 2 also shows that the greater than 95% removal persists for lower substrate concentration than have been previously reported, and that the enzyme and peroxide do not become limiting a t 1 mM hydrogen peroxide and 100 u n i t d l of enzyme activity. Previous cost estimates [5 ] have indicated that the enzyme application may be prohibitively expensive for potable water treatment. However, the actual
669
Tab. 2. Removal and/or transformation of 2Chlorophenol by Enzymatic Oxidative Coupling - with and without backgmund oxganics
2CP (mg/L)
Water type
0 -1
buffer pH 7
Enzyme concentration units/L
Peroxide concentntion mM
1000
5
0.01
1000
5
0.01
100
5
0.01
tap water
100
5
1
filtered river water
1000
5
0.3
1000
1
0.3
1000
5
0.3
1000
1
100
1
0.3
,
% Removal
> 95 > 95 > 95 > 95 > 95 > 95 > 95 > 95 > 95
Conditions: all experiments conducted without centrifuetion. 2 C P initially dissolved in methanol, resulting in 3 4 % v/v methanol in water.
requirements for the enzyme may be considerably less in drinking-water treatment. Therefore, future research is needed to determine the relationship between contaminant concentration and required enzyme doses. Table 3 shows a similar set of experiments for PCP. Initial experiments show much less removal for PCP than equivalent concentrations of 2 C P . For most experiments, the chlorophenol was initially dissolved in methanol and then added to water. Two experiments show that methanol may interfere with removal of PCP, because substantially better removal was obtained when methanol was eliminated. However, the removals for PCP remained less than those for2-CPelRnin the absence of methanol. PCP is more nonpolar than 2-CP and it may have remained associated with methanol after addition to water, even though there was no apparent emulsion. This may account for its lack of reactivity with the enzyme. compared to the 2CP. However, these data suggest that the enzyme may be specific for certain types of structures and that this may limit its applicability. Other compounds which have reported removal efficiencies less than 90% include 3chlorophenol, 2,6-dimethylphenol, aniline, 4chloroaniline, 4-bromoaniline, 4-fluoroaniline, and
660
Tab. 3.Removal and/or tmnsformation of pentachlorophenol by Enzymatic Oxidative Coupling ~~~
FCP (mg/L)
Enzyme concentration units/L
Peroxide concentration
Centrifugation
% Removal
mM
1000
-
5
20
1000
-
65
15
1000
-
62
lo*
"
Conditions:all experiments conducted in MilliQ water buffered (phosphate) at pH = 7. 'YPentachlorophenol (PCP) initially dissolved in methanol. Addition to water resulted in 3-4 % vlv methanol.
diphenylamine [ 5 ] . These compounds may be removable with greater concentrations of enzyme and peroxide, but the costs would likely be prohibitive. Many other questions need to be addressed before the potential use becomes a reality. Among these, some of the more important are: what are the products of the reaction, what is the fate of the products and the enzyme in the water treatment process, what is the potential for unwanted byproducts from other unit processes -- particularly chlorination, and what is the minimum level required for removal. This study indicated that precipitation was not visually obvious for the lowest concentration of 2CP. Thus the product of the reaction is assumed to remain water soluble. The use of enzymes in drinking-water treatment has several advantages over the use of whole bacteria. Enzymes often have high specificities for substrate, so there is better control over the compound removed and the product produced. The enzyme concentration in the water is controlled by the plant operator. Thus there is no dependence on the growth rate of the bacteria. This can be particularly important when the compound targeted for removal appean only sporadically in the raw water, such as taste and odor compounds. Bacteria require an incubation time to acclimate to the substrate and grow, but enzymes may be added as needs require.
4. CONCLUSION
The potential use of enzymes for trace aromatic compound removal has been shown on the laboratory scale. This type of examination can screen compounds for which the enzyme may be useful. 2CP, a cornpound of organoleptic concern, has
661 been shown to be effectively eliminated by the horse-radish peroxidase - peroxide system. This compound is a product o f prechlorination when phenol is in the water supply, and t h e enzyme system offers a n alternative to the usual practice of applying a n active carbon treatment (powdered o r granulated form) which requires investments o n t h e treatment site.
REFERENCES 1. Y-Richard, “Biological Methods for the Treatment of Ground Water”, in Oxidation Techniqlps in Drinking Water Treatment, eds. H. Sontheimer and W.Kuhn, EPA-570/9-79-020,1979. 2. F. Fiessinger, J. Mallevialle, and A-Benedek, ”Interaction of Adsorption and Bioactivity in
Full Scale Activated Carbon Filters: The Mont Valerian Experiment”, in Treatment of Water by Granular Activated Carbon, eds. M. J. McGuire and I. H. Suffet, American Chemical Society Advances in Chemistry 202, ACS Books, Washington, D.C., 1983, p. 319. 3. R . Milliner, D.A. Bowles, and R. W. Brett, ”Biological Pretreatment at Tewkesbury”, Proceedings of the Society forwater Treatment Examination, v. 21, 1972, p. 318. 4. H. Kobayashi, and B.E. Rittmann, ”Microbial Removal of Hazardous OGanic Compounds”, Environmental Science and Technology, v. 16, n. 3, 1982, p- 170A. 5. B. N. Alberti. and A. M. Kh i n o v , ”Enzymatic Removal of Dissolved Aromatics from Industrial Aqueous Effluents”, in Biotechnology and Bioengineering Symposium No. 11, John Wiley and Sons, Inc. New York, NY, 1981, p. 373-9. 6. A.M. Klibanov, B.N. Alberti, E.D. Morris, and L.M. Felshin, ”Enzymatic Removal of Toxic Phenols and Anilines from Waste Waters”, Journal of Applied Biochemistry, v. 2, 1980, p. 414-21. 7. A.M. Klibanov, and E.D. Moms, ”Horseradish Peroxidase forthe Removal of Carcinogenic Aromatic Amines from Water”, Enzyme Microbiology and Technology, Y. 3, 1981, p. 11922.
8. I. H. Suffet, L. Brenner, and P.R. Cairo, ”GC/MS Identification of Trace Organics in Philadelphia Drinking Water During a 2-Year Period”, Water Research, v. 14, 1980, p. 853. 9. A. Bruchet, and J. Mallevialle, ”Etude de Cas #Elimination de Composes Organiques dans des Usine de Production d’Eau Potable: Realimentations a l e s d e N m T d m m t s d’Eaux Surfaceot d’Eaux Soutenaines”, Journal Francais D’Hydmlogie, v. 14, FASC 1 , n. 40. 1983, p. 31. 10. G.T. Coyle, et al, ”Broad Spectrum Analysis ofthe Removalof Trace Organics in an Ozone Granular Activated Carbon Potable Water Pilot Plant Study”, in Water Chlorination: Environmental Impact and Health Effects, Volume 4, ed. R. L. Jolley, A M Arbor Science, AnnArbor, MI, 1983, p. 421. 11. J.G. Smith, S. Lee, and A. Netzer, ”Model Studiesof Aqueous Ch1orination:TheChlorination of Phenols in Dilute Aqueous Solution”, Water Research, v. 10, 1976, p. 985-90. 12. R. A. Baker, ”F’henolic Analyses by Direct Aqueous Injection Gas Chromatography”, Journal of the American Water Works Association, v. 58, 1966, p. 751-60.
662
13. . L. Rudling, ”Determination of Pentachlophenol in Organic Tissues and Water”, Water Reseanfi, v.4, 1970, p-533-714. M. Schnitzer, and S . U.Khan, Humic Substances in the Enyironment, Marcel Dekker, New York, NY, 1972. 15. P. L. McCarthy, ’Organics in Water, an Engineering Challenge’’, Joumal of the Environmental Engineering Division, ASCE, V. 106, 1980, p. 1. 16. 2. Pflug, ”Effect of Humic Acids on the Activity of Two Peroxidases”, 2. Pflannernechr, Bodenkd., 1980, p.430-40. 17. P. V. Roberts, and R. S . Summers, ”Performance of GAC for TOC Removal”, Joumal of the American Water Works Association, v. 74, n. 2, 1982, p. 113.