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A conceptual model describing macromolecule degradation by suspended cultures and biofilms
8)
Pergamon
War. Sci. Tech. Vol. 37. No. 4-5. pp. 23/-234. 1998. ~ 1998 IAWQ. Published by Elsevier Science Ltd Printed in Greal Britam. 0273- I 223/98 S I 9'00 + 0'00
PH: 50273-1223(98)00112-7
A CONCEPTUAL MODEL DESCRIBING
MACROMOLECULE DEGRADATION BY SUSPENDED CULTURES AND BIOFILMS David R. Confer* and Bruce E. Logan** ... Department of Chemical and Environmental Engineering, University ofArizona. Tucson. AZ 85721. USA .... Department ofCivil and Environmental Engineering, The Pennsylvania State University. University Park, PA 16802 USA
ABSTRACf Macromolecular (> 1.000 daltons) compounds such as proteins and polysaccharides can constitute a significant portion of dissolved organic carbon (DOC) in wastewater. but limited information is available on how these compounds are degraded in suspended and fixed-film biological wastewater treatment systems. Bacteria cannot assimilate intact macromolecules but must first hydrolyze them to monomers or small oligomers. Here. we summarize experiments performed in our laboratory which indicate that the enzymes responsible for hydrolysis are primarily those that remain auached to the cell. In biofilm cultures fed macromolecular substrates. for example. no more than 8% of total hydrolytic activity was found to be located In the cell-free bulk solution. These and other experiments support a generalized mechanism for macromolecule degradation by biofilms that features cell-associated hydrolysis. followed by the release of hydrolytic fragments back into bulk solution. The extent of fragment release is larger for proteins (bovine serum albumin) than for carbohydrates (dextrans). @ 1998 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Biodegradation; biofilm; extracellular; hydrolysis; macromolecule; wastewater. INTRODUCTION A large percentage of dissolved organic matter (DOM) in wastewater can be composed of macromolecular substrates. Although size distributions of DOM in wastewaters can vary, 50 to 60% of dissolved organic carbon (DOC) has been reported to be > 1,000 daltons (I K) for domestic wastewater separated using Ultrafiltration membranes (Grady et al., 1984; Logan and Jiang, 1990). Dissolved proteinaceous material, defined as amino acids present as molecules larger than -2 K (but able to pass through a 0.2 ~m polycarbonate filter), has been reported to constitute more than 75% of the total amino acids in wastewater (Confer et at., 1995). The efficiency and kinetics of macromolecule degradation in wastewater treatment plants has not been extensively studied, despite predictions that the size distribution of DOM is an important factor in the efficiency of fixed-film bioreactors such as trickling filters (Logan et al., 1987). In this study we analyze and summarize recent studies we conducted in order to understand the fate of macromolecules in wastewater treatment systems. Because bacteria cannot directly assimilate large macromolecules without first hydrolyzing them to smaller molecules (either monomers or small oligomers), 231
232
D. R. CONFER and E. LOGAN
we sought to answer two questions: where are the enzymes responsible for hydrolysis located (in solution or attached to the cell); and are hydrolyzed molecules released into solution or held at the surface of the cells (or within a biofilm matrix)? METHODS The release of smaller molecules during macromolecules, degradation could occur either by excreted or cell• bound enzymes. In order to determine the location of these enzymes we examined the location of two representative enzymes (leucine aminopeptidase and a-glucosidase) during the breakdown of proteins and macromolecular sugars by acclimated cultures. Biofilms and suspended cultures were grown on either protein (bovine serum albumin, or BSA; 65 K) or dextran (70 K) as reported in Confer and Logan (l997a,b). The relative enzyme activities of these wastewater-inoculated suspended and biofilm cultures was probed using the fluorescent model substrate analogs L-leucine-7-amido-4-methylcoumarin e HCI (Leu-MCA) and 4• methylumbelliferyl-a-glucoside (MUF-a-glu). These molecules exhibit very little fluorescence until they are hydrolyzed by cell enzymes. Analogs were either added directly to the reactor (rates of hydrolysis in biofilm), or cultures (or the suspensions above the biofilm) were removed from reactors and incubated with these analogs; the rate of hydrolysis was measured directly as increases in fluorescence (Confer and Logan, 1998). Table I. Comparison of hydrolysis activities (measured in terms of moles of analog fluorescing) in the presence of a biofilm compared to those of suspended cells or the cell-free liquid. The high activities of a biofilm, relative to those of cell-free solutions, indicates that hydrolytic enzymes are primarily cell associated (Data from Confer and Logan, 1998) System
Analog
Conditions
Incubation time,min
Activity in Fraction, Jlmoles (% of Biofilm) Biofilm
Suspended
Cell-free
Biofilm (batch)
Leu-MCA
0.97 So 0.63 So 0.51 So 0.12 So
40 40 40 40
7.0 8.8 8.5 [0.1
1.1 (15) 0.93 (II) 1.8(21) 0.5 (5)
0.54 (8) 0.64 (7) 0.69 (8) 0.33 (3)
Biofilm (batch)
MUF-cx-glu
0.90 So 0.33 So
80 40
14.5 [4.
0.08 «I) 0.13 «I)
<0.06 «I) <0.07 «I)
Suspended cells
Leu-MCA
Batch (exponential growth phase)
160
23
0.95 (4)'
Suspended cells
MUF-cx-glu
CSTR (6=16 h)
75
2.4
0.21 (8)'
'Percent calculated relative to suspended rates for these data.
Wastewater samples were obtained from trickling filter effluent at the Roger Road Wastewater Treatment Plant in Tucson, AZ. These trickling filters are loaded with primary clarifier effluent, recycle, and secondary clarifier underflow providing relatively high concentrations of biofilm solids in the wastewater. RESULTS AND DISCUSSION Incubations were carried out over time periods assumed to be representative of hydraulic detention times in trickling filters (15 to 120 min; Tariq, 1975). When dextran and protein solutions were added to biofilm reactors, only 3 to 8% of the measured leucine aminopeptidase and <1% of a-glucosidase activity were attributed to enzymes in solution (i.e. was not cell associated) (Table I). A slightly larger fraction of the total hydrolysis of macromolecules was occurring in the liquid above the biofilm, ranging from 5 to 21 % of the total activity for Leu-MUF biofilm experiments, but < I % in MUF-a-glu experiments.
A conceptual model describing macromolecule degradation
233
The cell-associated nature of protein- and dextran-hydrolyzing enzymes is also evident in experiments using suspended cultures. When cells were grown in batch on protein. 96% of the Leu-MCA activity was cell associated (Table I). Similarly, 92% of a-glucosidase activity measured using MUF-a-glu was cell associated when cells were grown on dextran. Table 2. Comparison of hydrolysis rates in different biofilm systems. The trickling filter 'suspended biofilms'system is the effluent from a trickling filter containing a high solids concentration achieved by combining secondary clarifier underflow with recycled settling wastewater and primary clarifier effluent (a so-called activated trickling filter system). The biofilm reactors are biofilms grown on the inside of a 41 bottle filled with 0.41 of either Bovine Serum Albumin (Leu-MCA analog) or dextran (MUF-a-glu) analog. (Data from Confer and Logan, 1998) System
Analog
Incubation time (min)
Ratios of total hydrolysis after incubation
Biojilm
Susp. biojilm Cell-free
Set/I. or Cel1s
Cell-free
Cell-free
Suspended Biofilms: Trickling filter
Leu-MCA
94
_na
3.4
1.7
Suspended Biofilms: Trickling filter
MUF-aglu
70
_na
38
9.3
Biofilm Reactors (0.12 S.)
Leu-MCA
40
SO
_na
I.S
Biofilm Reactors (0.33 S.)
MUF-aglu
40
30
_na
1.9
na- not applicable
The experimental procedures used to analyze hydrolysis rates in biofilm reactors could obviously not be translated to full-scale trickling filters due to the cost of adding fluorescent analogs to large volumes of wa~tewater. However. we were still able to compare cell-associated and cell-free hydrolysis rates to determine whether cell-free hydrolysis rates significantly contributed to overall macromolecule hydrolysis rates in wastewater treatment systems. Samples taken from the trickling filter effluent were separated into three fractions: a suspension containing freshly sloughed biofilm and recycled biofilm solids from the secondary clarifier underflow (suspended biofilm); a settled suspension (settled); and a cell-free solution. We found that rates in the suspended biofilm-containing suspension were 4 to 38 times larger than rates measured in cell-free solutions (Table 2). When the samples were settled, the rates were 1.7 to 9.3 times larger than in cell-free solutions. These trickling filter results suggest that hydrolytic enzyme activity in fixed-film reactors occurs mostly in the biofilm matrix and not through the action of enzymes secreted into solution. Using our laboratory biofilm reactors, we found that biofilm hydrolysis rates were 30 to SO times as large as those that occurred in the supernatant. Just the presence of small concentrations of cells in the supernatant (produced in the biofilm reactors during the degradation of the macromolecules) was sufficient to produce cell-a~sociated rates that were several times larger than cell-free rates (Table 2). Although we were not able to directly measure hydrolysis rates in the trickling filter biofilms, the activity of biofilm-containing suspensions was many times larger than either that of settled suspensions (large biofilm pieces removed) or cell-free suspensions. The rates of settled suspensions from the trickling filter were 1.7 to 9.3 times larger than cell-free rates, and the rates of suspended cells in biofilm reactors were 1.5 to 1.9 times larger than cel/·free rates. Thus, we conclude from the biofilm experiments that macromolecule hydrolysis is primarily cell associated. Experiments using suspended cultures have shown that macromolecules are not completely consumed when they are hydrolyzed by the cells. although the fraction of hydrolytic product that accumulates varies depending on the type of macromolecule. Using suspended cultures, Haldane and Logan (1994) found that during the degradation of a model polysaccharide (dextran) hydrolytic fragments were released as shown by their accumulation in bulk solution. More extensive experiments using biofilm reactors were subsequently performed. Confer and Logan (1997a,b) measured the size distributions of dextran and bovine serum
234
D. R. CONFER and E. LOGAN
albumin using batch Ultrafiltration cells in biofilm reactors. Sugars were separated into size fractions of < I K, I-10 K and> I OK to <0.2 ~m based on measurements of carbohydrates using the anthrone method in dextran experiments. and proteins were measured as either -2 K to <10 K or >IOK to <0.2 ~m using a Coomassie protein assay that measures only those proteins larger than -ZK. They found that during protein degradation. a small. but measurable. fraction of protein accumulated in solution (maximum of 3 mg/l of a reactor fed 150 mg/I of protein). In dextran-degradation experiments, a larger proportion of the macromolecules (25%) accumulated in solution in a smaller «10 K) size fraction. Fluorescent analog results, and the size fractionation experiments indicating that hydrolytic fragments of proteins and polysaccharides accumulate in bulk solution during macromolecule degradation, support a generalized mechanism for macromolecule degradation that features cell-bound hydrolysis followed by the release of hydrolytic fragments back into bulk solution. These fragments are smaller molecules that will diffuse again to a cell's surface; this cell-bound hydrolysis and release sequence will be repeated until hydrolytic fragments are small enough to be assimilated by cells. Proteins are apparently hydrolyzed more efficiently than delltrans since proportionally Jess protein than dextran makes it out of the biofilm. The location and sequence of events necessary for macromolecule hydrolysis have important implications in how biofilm models for macromolecular substrates are constructed. The removal of DOM in trickling filters has been found to be primarily limited by mass transport from the bulk liquid into the biofilm, and it has been assumed that molecular size distributions are conserved (Logan et al., 1987). For macromolecules that are removed in a manner similar to the model protein ellamined here, diffusion of molecules into the biofilm could be considered a one-way process with negligible back-diffuion of hydrolytic products. However, if wastewaters contain larger fractions of dextran-like molecules, back-diffusion of hydrolysis products probably needs to be included in these models. For wastewaters in Tucson AZ examined by the authors. carbohydrates (measured using the anthrone test) are a negligible « I mgll) component of the DOC pool. while proteins (measured using total combined amino acids) are a larger fraction of the organic pool, suggesting that models that neglect back-diffusion of hydrolytic by-products are reasonable for domestic wastewaters. Whether this conclusion can be extended to other macromolecules and other wastewaters, however, is not known. ACKNOWLEDGEMENT This research was supported by the National Science Foundation. We thank Don Armstrong and other Pima County wastewater treatment plant personnel for their assistance in obtaining wastewater samples. REFERENCES Confer. D. R., Logan, B. E., Aiken. B. S. and Kirchman, D. L. (1995). Measurement of dissolved free and combined amino acids in unconcentrated wastewaters using HPLC. War. Environ. Res., 67. 118-125. Confer, D. R. and Logan. B. E. (1997a). Molecular weight distribution of hydrolysis products during the biodegradation of model macromolecules in suspended culture and wastewater biofilms: I. bovine serum albumin. War. Res., 31. 2127-2136. Confer. D. R. lind Logan. B. E. (1997b). Molecular weight distribution of hydrolysis products during the biodegradation of model macromolecules in suspended culture and wastewater biofilms: 2. dextran and dextrin. War. Res., 31. 2137-2146. Confer, D. R. and Logan, B. E. (1997). Location of protein and polysaccharide hydrolytic activity in suspended and biofilm wastewater cullUres. War. Res.• 32. 31-38. Grady, C. P. L. Jr.. Kirsh. E. J., Koczwara. M. K.• Trogovcich. B. and Watts. R. D. (1984). Molecular weight distributions in uctivuted sludge eflluents. War. Res., 18,239-246. Haldane. G. M. and Logan, B. E. (1994). Molecular size distributions of a macromolecular polysaccharide (dextran) during its biodegradution in batch and continuous culture. War. Res., 28. 1873-1878. Logan, B. E.. Hermanowicz, S. W. and Parker. D. S. (1987). A fundamental model for trickling filter process design. J. Waf. Pollur. Conr. Fed.• 59,1029-1042. Logan. B. E. and Jiang. Q. (1990). A Model for determining molecular size distributions of DOM. J. Envir. Engin.• 116. 1046• 1062. Tariq. M. N. (197S). Retention time in trickling filters. Prog. War. Technol.• 7(2). 225-234.
Pergamon
War. Sci. Tech. Vol. 37. No. 4-5. pp. 23/-234. 1998. ~ 1998 IAWQ. Published by Elsevier Science Ltd Printed in Greal Britam. 0273- I 223/98 S I 9'00 + 0'00
PH: 50273-1223(98)00112-7
A CONCEPTUAL MODEL DESCRIBING
MACROMOLECULE DEGRADATION BY SUSPENDED CULTURES AND BIOFILMS David R. Confer* and Bruce E. Logan** ... Department of Chemical and Environmental Engineering, University ofArizona. Tucson. AZ 85721. USA .... Department ofCivil and Environmental Engineering, The Pennsylvania State University. University Park, PA 16802 USA
ABSTRACf Macromolecular (> 1.000 daltons) compounds such as proteins and polysaccharides can constitute a significant portion of dissolved organic carbon (DOC) in wastewater. but limited information is available on how these compounds are degraded in suspended and fixed-film biological wastewater treatment systems. Bacteria cannot assimilate intact macromolecules but must first hydrolyze them to monomers or small oligomers. Here. we summarize experiments performed in our laboratory which indicate that the enzymes responsible for hydrolysis are primarily those that remain auached to the cell. In biofilm cultures fed macromolecular substrates. for example. no more than 8% of total hydrolytic activity was found to be located In the cell-free bulk solution. These and other experiments support a generalized mechanism for macromolecule degradation by biofilms that features cell-associated hydrolysis. followed by the release of hydrolytic fragments back into bulk solution. The extent of fragment release is larger for proteins (bovine serum albumin) than for carbohydrates (dextrans). @ 1998 IAWQ. Published by Elsevier Science Ltd
KEYWORDS Biodegradation; biofilm; extracellular; hydrolysis; macromolecule; wastewater. INTRODUCTION A large percentage of dissolved organic matter (DOM) in wastewater can be composed of macromolecular substrates. Although size distributions of DOM in wastewaters can vary, 50 to 60% of dissolved organic carbon (DOC) has been reported to be > 1,000 daltons (I K) for domestic wastewater separated using Ultrafiltration membranes (Grady et al., 1984; Logan and Jiang, 1990). Dissolved proteinaceous material, defined as amino acids present as molecules larger than -2 K (but able to pass through a 0.2 ~m polycarbonate filter), has been reported to constitute more than 75% of the total amino acids in wastewater (Confer et at., 1995). The efficiency and kinetics of macromolecule degradation in wastewater treatment plants has not been extensively studied, despite predictions that the size distribution of DOM is an important factor in the efficiency of fixed-film bioreactors such as trickling filters (Logan et al., 1987). In this study we analyze and summarize recent studies we conducted in order to understand the fate of macromolecules in wastewater treatment systems. Because bacteria cannot directly assimilate large macromolecules without first hydrolyzing them to smaller molecules (either monomers or small oligomers), 231
232
D. R. CONFER and E. LOGAN
we sought to answer two questions: where are the enzymes responsible for hydrolysis located (in solution or attached to the cell); and are hydrolyzed molecules released into solution or held at the surface of the cells (or within a biofilm matrix)? METHODS The release of smaller molecules during macromolecules, degradation could occur either by excreted or cell• bound enzymes. In order to determine the location of these enzymes we examined the location of two representative enzymes (leucine aminopeptidase and a-glucosidase) during the breakdown of proteins and macromolecular sugars by acclimated cultures. Biofilms and suspended cultures were grown on either protein (bovine serum albumin, or BSA; 65 K) or dextran (70 K) as reported in Confer and Logan (l997a,b). The relative enzyme activities of these wastewater-inoculated suspended and biofilm cultures was probed using the fluorescent model substrate analogs L-leucine-7-amido-4-methylcoumarin e HCI (Leu-MCA) and 4• methylumbelliferyl-a-glucoside (MUF-a-glu). These molecules exhibit very little fluorescence until they are hydrolyzed by cell enzymes. Analogs were either added directly to the reactor (rates of hydrolysis in biofilm), or cultures (or the suspensions above the biofilm) were removed from reactors and incubated with these analogs; the rate of hydrolysis was measured directly as increases in fluorescence (Confer and Logan, 1998). Table I. Comparison of hydrolysis activities (measured in terms of moles of analog fluorescing) in the presence of a biofilm compared to those of suspended cells or the cell-free liquid. The high activities of a biofilm, relative to those of cell-free solutions, indicates that hydrolytic enzymes are primarily cell associated (Data from Confer and Logan, 1998) System
Analog
Conditions
Incubation time,min
Activity in Fraction, Jlmoles (% of Biofilm) Biofilm
Suspended
Cell-free
Biofilm (batch)
Leu-MCA
0.97 So 0.63 So 0.51 So 0.12 So
40 40 40 40
7.0 8.8 8.5 [0.1
1.1 (15) 0.93 (II) 1.8(21) 0.5 (5)
0.54 (8) 0.64 (7) 0.69 (8) 0.33 (3)
Biofilm (batch)
MUF-cx-glu
0.90 So 0.33 So
80 40
14.5 [4.
0.08 «I) 0.13 «I)
<0.06 «I) <0.07 «I)
Suspended cells
Leu-MCA
Batch (exponential growth phase)
160
23
0.95 (4)'
Suspended cells
MUF-cx-glu
CSTR (6=16 h)
75
2.4
0.21 (8)'
'Percent calculated relative to suspended rates for these data.
Wastewater samples were obtained from trickling filter effluent at the Roger Road Wastewater Treatment Plant in Tucson, AZ. These trickling filters are loaded with primary clarifier effluent, recycle, and secondary clarifier underflow providing relatively high concentrations of biofilm solids in the wastewater. RESULTS AND DISCUSSION Incubations were carried out over time periods assumed to be representative of hydraulic detention times in trickling filters (15 to 120 min; Tariq, 1975). When dextran and protein solutions were added to biofilm reactors, only 3 to 8% of the measured leucine aminopeptidase and <1% of a-glucosidase activity were attributed to enzymes in solution (i.e. was not cell associated) (Table I). A slightly larger fraction of the total hydrolysis of macromolecules was occurring in the liquid above the biofilm, ranging from 5 to 21 % of the total activity for Leu-MUF biofilm experiments, but < I % in MUF-a-glu experiments.
A conceptual model describing macromolecule degradation
233
The cell-associated nature of protein- and dextran-hydrolyzing enzymes is also evident in experiments using suspended cultures. When cells were grown in batch on protein. 96% of the Leu-MCA activity was cell associated (Table I). Similarly, 92% of a-glucosidase activity measured using MUF-a-glu was cell associated when cells were grown on dextran. Table 2. Comparison of hydrolysis rates in different biofilm systems. The trickling filter 'suspended biofilms'system is the effluent from a trickling filter containing a high solids concentration achieved by combining secondary clarifier underflow with recycled settling wastewater and primary clarifier effluent (a so-called activated trickling filter system). The biofilm reactors are biofilms grown on the inside of a 41 bottle filled with 0.41 of either Bovine Serum Albumin (Leu-MCA analog) or dextran (MUF-a-glu) analog. (Data from Confer and Logan, 1998) System
Analog
Incubation time (min)
Ratios of total hydrolysis after incubation
Biojilm
Susp. biojilm Cell-free
Set/I. or Cel1s
Cell-free
Cell-free
Suspended Biofilms: Trickling filter
Leu-MCA
94
_na
3.4
1.7
Suspended Biofilms: Trickling filter
MUF-aglu
70
_na
38
9.3
Biofilm Reactors (0.12 S.)
Leu-MCA
40
SO
_na
I.S
Biofilm Reactors (0.33 S.)
MUF-aglu
40
30
_na
1.9
na- not applicable
The experimental procedures used to analyze hydrolysis rates in biofilm reactors could obviously not be translated to full-scale trickling filters due to the cost of adding fluorescent analogs to large volumes of wa~tewater. However. we were still able to compare cell-associated and cell-free hydrolysis rates to determine whether cell-free hydrolysis rates significantly contributed to overall macromolecule hydrolysis rates in wastewater treatment systems. Samples taken from the trickling filter effluent were separated into three fractions: a suspension containing freshly sloughed biofilm and recycled biofilm solids from the secondary clarifier underflow (suspended biofilm); a settled suspension (settled); and a cell-free solution. We found that rates in the suspended biofilm-containing suspension were 4 to 38 times larger than rates measured in cell-free solutions (Table 2). When the samples were settled, the rates were 1.7 to 9.3 times larger than in cell-free solutions. These trickling filter results suggest that hydrolytic enzyme activity in fixed-film reactors occurs mostly in the biofilm matrix and not through the action of enzymes secreted into solution. Using our laboratory biofilm reactors, we found that biofilm hydrolysis rates were 30 to SO times as large as those that occurred in the supernatant. Just the presence of small concentrations of cells in the supernatant (produced in the biofilm reactors during the degradation of the macromolecules) was sufficient to produce cell-a~sociated rates that were several times larger than cell-free rates (Table 2). Although we were not able to directly measure hydrolysis rates in the trickling filter biofilms, the activity of biofilm-containing suspensions was many times larger than either that of settled suspensions (large biofilm pieces removed) or cell-free suspensions. The rates of settled suspensions from the trickling filter were 1.7 to 9.3 times larger than cell-free rates, and the rates of suspended cells in biofilm reactors were 1.5 to 1.9 times larger than cel/·free rates. Thus, we conclude from the biofilm experiments that macromolecule hydrolysis is primarily cell associated. Experiments using suspended cultures have shown that macromolecules are not completely consumed when they are hydrolyzed by the cells. although the fraction of hydrolytic product that accumulates varies depending on the type of macromolecule. Using suspended cultures, Haldane and Logan (1994) found that during the degradation of a model polysaccharide (dextran) hydrolytic fragments were released as shown by their accumulation in bulk solution. More extensive experiments using biofilm reactors were subsequently performed. Confer and Logan (1997a,b) measured the size distributions of dextran and bovine serum
234
D. R. CONFER and E. LOGAN
albumin using batch Ultrafiltration cells in biofilm reactors. Sugars were separated into size fractions of < I K, I-10 K and> I OK to <0.2 ~m based on measurements of carbohydrates using the anthrone method in dextran experiments. and proteins were measured as either -2 K to <10 K or >IOK to <0.2 ~m using a Coomassie protein assay that measures only those proteins larger than -ZK. They found that during protein degradation. a small. but measurable. fraction of protein accumulated in solution (maximum of 3 mg/l of a reactor fed 150 mg/I of protein). In dextran-degradation experiments, a larger proportion of the macromolecules (25%) accumulated in solution in a smaller «10 K) size fraction. Fluorescent analog results, and the size fractionation experiments indicating that hydrolytic fragments of proteins and polysaccharides accumulate in bulk solution during macromolecule degradation, support a generalized mechanism for macromolecule degradation that features cell-bound hydrolysis followed by the release of hydrolytic fragments back into bulk solution. These fragments are smaller molecules that will diffuse again to a cell's surface; this cell-bound hydrolysis and release sequence will be repeated until hydrolytic fragments are small enough to be assimilated by cells. Proteins are apparently hydrolyzed more efficiently than delltrans since proportionally Jess protein than dextran makes it out of the biofilm. The location and sequence of events necessary for macromolecule hydrolysis have important implications in how biofilm models for macromolecular substrates are constructed. The removal of DOM in trickling filters has been found to be primarily limited by mass transport from the bulk liquid into the biofilm, and it has been assumed that molecular size distributions are conserved (Logan et al., 1987). For macromolecules that are removed in a manner similar to the model protein ellamined here, diffusion of molecules into the biofilm could be considered a one-way process with negligible back-diffuion of hydrolytic products. However, if wastewaters contain larger fractions of dextran-like molecules, back-diffusion of hydrolysis products probably needs to be included in these models. For wastewaters in Tucson AZ examined by the authors. carbohydrates (measured using the anthrone test) are a negligible « I mgll) component of the DOC pool. while proteins (measured using total combined amino acids) are a larger fraction of the organic pool, suggesting that models that neglect back-diffusion of hydrolytic by-products are reasonable for domestic wastewaters. Whether this conclusion can be extended to other macromolecules and other wastewaters, however, is not known. ACKNOWLEDGEMENT This research was supported by the National Science Foundation. We thank Don Armstrong and other Pima County wastewater treatment plant personnel for their assistance in obtaining wastewater samples. REFERENCES Confer. D. R., Logan, B. E., Aiken. B. S. and Kirchman, D. L. (1995). Measurement of dissolved free and combined amino acids in unconcentrated wastewaters using HPLC. War. Environ. Res., 67. 118-125. Confer, D. R. and Logan. B. E. (1997a). Molecular weight distribution of hydrolysis products during the biodegradation of model macromolecules in suspended culture and wastewater biofilms: I. bovine serum albumin. War. Res., 31. 2127-2136. Confer. D. R. lind Logan. B. E. (1997b). Molecular weight distribution of hydrolysis products during the biodegradation of model macromolecules in suspended culture and wastewater biofilms: 2. dextran and dextrin. War. Res., 31. 2137-2146. Confer, D. R. and Logan, B. E. (1997). Location of protein and polysaccharide hydrolytic activity in suspended and biofilm wastewater cullUres. War. Res.• 32. 31-38. Grady, C. P. L. Jr.. Kirsh. E. J., Koczwara. M. K.• Trogovcich. B. and Watts. R. D. (1984). Molecular weight distributions in uctivuted sludge eflluents. War. Res., 18,239-246. Haldane. G. M. and Logan, B. E. (1994). Molecular size distributions of a macromolecular polysaccharide (dextran) during its biodegradution in batch and continuous culture. War. Res., 28. 1873-1878. Logan, B. E.. Hermanowicz, S. W. and Parker. D. S. (1987). A fundamental model for trickling filter process design. J. Waf. Pollur. Conr. Fed.• 59,1029-1042. Logan. B. E. and Jiang. Q. (1990). A Model for determining molecular size distributions of DOM. J. Envir. Engin.• 116. 1046• 1062. Tariq. M. N. (197S). Retention time in trickling filters. Prog. War. Technol.• 7(2). 225-234.