APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1976, Copyright ©) 1976 American Society for Microbiology
p.
Vol. 32, No. 4
557-560
Printed in U.S.A.
Biodegradability of [14C]Methylcellulose by Activated Sludge F. A. BLANCHARD,* I. T. TAKAHASHI, AND H. C. ALEXANDER Dow Chemical Co., Environmental Sciences Research, Midland, Michigan 48640
Received for publication 21 May 1976
Three Methocel methylcellulose ethers of 1.9 degree of substitution with
[14C]methyl labels were shown to be biodegradable using batch-type activated
sludge tests. The maximum rate for conversion to 14CO2, attained after 1 week, was only 0.62 mg of methylcellulose/g of mixed liquor volatile solids per day. In 20 days, 55 to 73% of the radioactivity had been removed from solution as 14CO2, and the suspended solids contained 12 to 15% of the original radioactivity. Only 4% of the original methylcellulose appeared to be polymeric after the 20-day period. Thin-layer chromatography of supernatant liquid indicated at least two degradation products. The purpose of this study was to determine, using a sensitive radiotracer method, the biodegradability of several methylcelluloses by activated sludge. The biochemical oxygen demand (20-day) test (1) failed to show biodegradation of the Methocel (The Dow Chemical Co., Midland, Mich.) brand products. Small amounts of biological degradation of methylcellulose and similar compounds have been reported. Freeman et al. (2) observed a 93% decrease in viscosity of solutions of methylethylcellulose due to the growth of bacteria normally present in the manufactured product. Siu et al. (9) found minor (8%) weight losses in 9 to 10 days after inoculating 5,000 gg of methylcellulose solutions per ml with a cellulose-degrading fungus. Reese et al. (6) noted a slight hydrolysis of methylcellulose solutions by incubation with certain organisms for 30 days, but concluded that methylcellulose, with the high degree of substitution (DS) required for water solubility, is usually resistant to microbial attack. Reese (7), Savage (8), and Wirick (11) obtained small amounts of reducing sugars from methylcelluloses by enzyme action. MATERIALS AND METHODS The biodegradation of "4C-labeled methylcellulose by activated sludge was evaluated in batch-type cylinder biodegradation experiments according to procedures described previously (H. C. Alexander and F. A. Blanchard, Natl. Meet. Am. Chem. Soc., 168th, Atlantic City, N.J., Abstr. 35 Fuel, 1974; complete paper as Div. of Fuel Chem. Preprints 19:104-112, 1974). The 14C-labeled methylcelluloses were prepared to correspond as closely as possible to Methocel products MC 25, MC 100, and MC 4000 (designation in use before 6/1/74. MC 25 and MC 4000 are now named A 25 and A 4M. The MC 100 type is no longer available). Alkalicellulose was first prepared from cellulose and sodium hydroxide,
and then reacted with [14C]methyl chloride. The product was purified by degassing under vacuum, then by exhaustive washing with acetone, followed by exhaustive washing with boiling water and then drying under vacuum. The principal radioactive impurities expected were: ['4C]methanol, [14C]methyl ether, and unreacted ['4C]methyl chloride. Thin-layer chromatography (TLC), as given below, indicated that 99.4% of the product was polymeric. The properties of the methylcelluloses are given in Table 1. This table and later discussions in this paper use two definitions familiar to cellulose chemists. The DS is the average number of substituent groups attached to the ring hydroxyls of the anhydroglucose units of the cellulose. The maximum DS possible is three. The degree of polymerization (DP) represents the average number of anhydroglucose units per polymer chain. Settled sludge (at about 2% solids) was obtained from the Dow Chemical Co., Midland, Mich., General Wastewater Treatment Plant. A 500-ml portion was placed in a 2-liter cylinder. The volume was brought to 1.7 liters with the simultaneous addition of the 14C-labeled compound (to give 16.2, 28.3, and 25.7 mg/liter for MC 4000, MC 100, and MC 25, respectively) and dechlorinated water containing 1 ml of each of the standard biochemical oxygen demand nutrients per liter (1). The cylinder was aerated with C02-free air at 50 to 100 ml/min through a fritted glass bottom. Air from the cylinder, containing respired 14CO2, was passed through two traps in series. Each trap was filled with 15 ml of 40% monoethanolamine in 2-methoxyethanol (vol/vol). The contents were collected, assayed, and replaced periodically. Samples were prepared for liquid scintillation counting by mixing 3 ml with 6 ml of Econofluor scintillator (New England Nuclear Corp., Boston, Mass.) and 9 ml of methanol. Mixed liquor samples were withdrawn by syringe. To each 10-ml sample was added 0.5 ml of Formalin (37% formaldehyde by weight). Mixed liquor samples (0.3 ml) were combusted with a Harvey biological material oxidizer (R. J. Harvey Instrument Co., Hillsdale, N.J.) and then assayed by liquid scintillation counting. The mixed liquor volatile solids (MLVS) was 557
558
BLANCHARD, TAKAHASHI, AND ALEXANDER
APPL. ENVIRON. MICROBIOL.
per h. The amounts consumed, in milligrams of compound per gram of MLVS per 4 h, during Property the initial 4 h of contact were: 0.0021 (MC 4000), Equiva2% SoluMeth0.0065 (MC 100), 0.0007 (MC 25), and 2.2 lent Meth- Sp act oxyl tion vis(phenol). ocel prod(,CiI conDSa cosity, DPb Non-methylcellulose compounds were presuct (centitent, g) ent in the MC 4000 supernatant liquid as deterpoise) wt (%) mined by TLC (Table 3). Radioactive materials 1.9 430 30.3 3,400 126 MC 4000 at R's of about 0.2 (x) and 0.5 (y) were already 31.3 502 1.9 82.5 128 MC 100 present in the 1-day sample. They were the 31.4 1.9 33.1 100 77 MC 25 predominate (80%) form of radioactive material a Degree of substitution. in solution by 12 days. By 20 days there was b Degree of polymerization. only 4% of the original radioactivity remaining in solution as polymer (the supernatant liquid initially 3 to 4 g/liter. After 20 days, it was 1.5 to 2.5 had 17% of the original radioactivity of which g/liter. Supernatant liquid was obtained by either 23% was polymer by TLC). TABLE 1. Properties of ['4C]methylcelluloses
settling or centrifuging the mixed liquor. Samples were added directly to 10 ml of Aquasol (New England Nuclear Corp.) scintillator solution. The radioactivity of the solids samples were obtained as the difference between that of mixed liquor and that of the supernatant liquid. One direct measurement on solids was made. The solids from a 20-day sample in the test of MC 4000 were separated by centrifugation and then washed, combusted, and counted. Selected supernatant liquids from the MC 4000 experiment (1, 12, and 20 days) were examined by TLC (n-propanol/ethyl acetate/water (7:2:1, vol/vol/ vol) with Quanta/gram QlF silica gel plates (Quantum Industries, Fairfield, N.J.). Reference compounds were: ['4C]methylcellulose, glucose, sucrose, and Dextran 20 (a polysaccharide with molecular weight = 14,500, from Pharmacia, Uppsala, Sweden). Plates were prepared, developed, exposed to I2 vapor, examined under ultraviolet light and marked off into 9 or 10 horizontal zones. Each zone was scraped into a counting vial, covered with liquid scintillator, and assayed. The results were converted into percentage recovery on the plate and a percentage distribution by zone.
DISCUSSION The generally slow degradation rates observed for these methyl ethers are consistent with observations on the effects of DS on enzyme functioning. Reese et al. (6) found that the DS has a decided effect on enzymatic hydrolysis of water-soluble cellulose derivatives as measured by the production of reducing compounds, and that methylcellulose has the resistance anticipated for compounds of DS over one. The curves of Fig. 2 for MC 25 (DP 100) and MC 4000 (DP 430) are quite similar in general shape, location of the peak, and height of the peak. This agrees with the observations of Reese et al. (6) on carboxymethylcellulose that the DP does not affect the rate of enzymic hydrolysis. The several days needed to reach a peak rate of '4CO2 release from the methylcellulose might be due to an induction time for exoenzymes which are required for breaking the polymer into small enough fragments for cell entry. Organisms have been shown to produce enzymes catalyzing the hydrolysis of cellulose ethers and esters (3, 6). Induction of additional enzymes by the sludge organisms would probably be slow in response to the low concentration of methylcel-
RESULTS These radiotracer studies with Methocel MC 4000, MC 100, and MC 25 methylcellulose demonstrated, respectively, 73, 54, and 24% conversion of the radioactivity into 14CO2 in 20 days. The supernatant liquids retained 17, 21, and 24%, respectively. Radioactivity in the suspended solids accounted for 14, 11, and 21%, respectively. The biodegradation of MC 4000 is shown in detail in Fig. 1 and Table 2. Total The degradation rates found were slow. . 100 There was a gradual rate increase as seen in Supernatant ~~~Trap, Cumulative the evolution rate of 14CO2 through 7 to 9 days unatant @50 (Fig. 2). The maximum rates in milligrams of methylcellulose per gram of MLVS per day were: 0.59 (MC 4000), 0.66 (MC 100), and 0.68 (MC 25). For comparison, ['4C]phenol in a simio, , lar system (Alexander and Blanchard, Natl. 15 20 10 5 Meet. Am. Chem. Soc., 168th, Atlantic City, Time, Days N.J., 1974) at a similar loading reached a peak FIG. 1. Distribution of 14C activity during sludge in 3 h, with a rate of 1 mg of phenol/g of MLVS biodegradation of ['4C]methylcellulose. 0U
VOL. 32, 1976
BIODEGRADABILITY OF [14C]METHYLCELLULOSE
559
TABLE 2. "4C-labeled MC 4000 methylcellulose or equivalent in activated sludge and traps
14C02 collected in trapsa
eycmg of ['4C ['Cmethylcellulose/liter
__________________
As equivalent concn change in mixed liquor
Sludge vol
Day
Total in traps (Ag)
Mixed liquor Supematant
Incremen-
tal (mg/liter)
0 1.49 15.2 13.5 1 1.45 13.2 2 1.40 14.4 12.3 3 1.39 11.1 4 1.38 14.6 9.9 5 1.37 11.1 8.9 6 1.36 7.3 7 1.35 9.0 4.7 8 1.30 3.4 9 1.29 6.2 2.9 10 1.28 5.7 2.1 11 1.27 1.8 12 1.22 4.4 1.8 13 1.21 2.4 14 1.20 2.6 15 1.19 5.6 2.7 17 1.18 5.7 3.1 20 1.17 5.0 2.7 a Calculated as equivalent 14C-labeled methylcellulose.
0.6 _
8>
0.5 _-
2 T
226 383 540 821 1,483 2,291 3,418 2,881 1,620 980 440 130 74 72 44 26 24
Cumulative
0
0
2
4
6
8
10 12 Time, Days
14
16
18
20
FIG. 2. Comparison of biodegradation of three ['4C]methylcelluloses. The initial concentrations in Mg/ml were: 16.2 (MC 4000), 28.3 (MC 100), and 25.7 (MC 25). These represent loadings in mg/g of MLVS of 3.7 (MC 4000), 8.82 (MC 100), and 6.25 (MC 25).
8 7 6 5 4 3
0.90-1.00 0.73-0.90 0.60-0.90 0.48-0.60 0.37-0.48 Glucose 0. 26-0. 37 Dexranose
2 1 0
0.17-0.26 0.06-0.17 0-0.06 [14C]methylcellulosea
a
su
1-Day 12-Day 20-Day 0 0 0 0 0 0 0 12.2 8.6 0.5 8.8 6.9 0.9 1.5 4.6 2.6 34.1 32. 2 1.5
13.2 12.6
3.0 91.4
8.7 9.0) 20.2 23.2
The origin retained 99.4% of the radioactivity applied.
tached groups are reported to be resistant to attack (5). Some, perhaps most, of the methylcellulose was degraded through intermediates lulose supplied. In our experiments, the initial like x andy. x may be [14C]methyl oligosacchaenzyme concentration was probably very low rides such as cellobiose and y may be [14C]_ because the settled return sludge was diluted methyl glucoses. Levinson et al. (5) gave such an interpretation (substituted glucose and subwith fresh water. We believe that most of the large conversion stituted cellobiose) to enzyme derivatives of of radioactivity to "4CO2 was by total degrada- carboxymethylcellulose. Hagen et al (4) have tion of the polymer. Possibly, some of the degra- produced dye-substituted oligosaccharides and dation might be via detachment of the dye-substituted glucose by enzymic hydrolysis [14C]methyl groups from the polymer. Since of dye-substituted cellulose. Methyl-substituted this would lower the DS, it would be expected to oligosaccharides and methyl glucoses may be increase the biodegradability. However, at- similarly formed.
560
BLANCHARD, TAKAHASHI, AND ALEXANDER
With a 1.9 DS, the tracer methylcelluloses should have a composition (based on estimates of Savage [8] and Spurlin [10]) of 2% unsubstituted, 28% monomethyl (mainly 6-0-methyl), 49% dimethyl (mainly 2,6-0-methyl and 3,6-0methyl), and 21% trimethyl (2,3,6-0-methyl). The observed result of 73% converted to 14CO2 required considerable breaking of bonds between units with all degrees of substitution. It also required utilization of most of the released, substituted glucose molecules. Thus, methylcellulose is slowly biodegradable. In these experiments it was 96% degraded or otherwise removed from solution in 20 days by activated sludge. ACKNOWLEDGMENTS Wen Cheng of The Dow Chemical Co. Designed Polymers Research Laboratory developed the radiotracer synthesis method and prepared the tracer with some assistance from one of us (F.A.B.). We appreciate the assistance by Dave Gransden of our laboratory for much of the analytical work.
LITERATURE CITED 1. American Public Health Association. 1971. Standard methods for the examination of water and wastewater, 13th ed. American Public Health Association, New York.
APPL. ENVIRON. MICROBIOL.
2. Freeman, G. G., A. J. Baillie, and C. A. Macinnes. 1948. Bacterial degradation of sodium carboxymethylcellulose and methyl ethyl cellulose. Chem. Ind. (London), May 1, p. 279-282. 3. Gascoigne, J. A., and M. M. Gascoigne. 1960. Biological degradation of cellulose. Butterworths, London. 4. Hagen, P., E. T. Reese, and 0. A. Stamm. 1966. Reaction of reactive dyes with cellulose. IV. Enzymatic hydrolysis of reactive dyed cellulose. Helv. Chim. Acta 49:2278-2287. 5. Levinson, H. S., G. R. Mandels, and E. T. Reese. 1951. Products of enzymatic hydrolysis of cellulose and its derivatives. Arch. Biochem. Biophys. 31:351-365. 6. Reese, E. T., R. G. H. Siu, and H. S. Levinson. 1950. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J. Bacteriol. 59:485-497. 7. Reese, E. T. 1957. Biological degradation of cellulose derivatives. Ind. Eng. Chem. 49:89-93. 8. Savage, A. B. 1957. Temperature-viscosity relationships for water-soluble cellulose ethers. Ind. Eng. Chem. 49:99-103. 9. Siu, R. G. H., R. T. Darby, P. R. Burkholder, and E. S. Barghoorn. 1949. Specificity of microbiological attack on cellulose derivatives. Text. Res. J. 19:484488. 10. Spurlin, H. M. 1939. Arrangement of substituents in cellulose derivatives, J. Am. Chem. Soc. 61:22222227. 11. Wirick, M. G. 1968. A study ofthe enzymic degradation of CMC and other cellulose ethers. J. Polymer Sci. A-1, 6:1965-1974.
p.
Vol. 32, No. 4
557-560
Printed in U.S.A.
Biodegradability of [14C]Methylcellulose by Activated Sludge F. A. BLANCHARD,* I. T. TAKAHASHI, AND H. C. ALEXANDER Dow Chemical Co., Environmental Sciences Research, Midland, Michigan 48640
Received for publication 21 May 1976
Three Methocel methylcellulose ethers of 1.9 degree of substitution with
[14C]methyl labels were shown to be biodegradable using batch-type activated
sludge tests. The maximum rate for conversion to 14CO2, attained after 1 week, was only 0.62 mg of methylcellulose/g of mixed liquor volatile solids per day. In 20 days, 55 to 73% of the radioactivity had been removed from solution as 14CO2, and the suspended solids contained 12 to 15% of the original radioactivity. Only 4% of the original methylcellulose appeared to be polymeric after the 20-day period. Thin-layer chromatography of supernatant liquid indicated at least two degradation products. The purpose of this study was to determine, using a sensitive radiotracer method, the biodegradability of several methylcelluloses by activated sludge. The biochemical oxygen demand (20-day) test (1) failed to show biodegradation of the Methocel (The Dow Chemical Co., Midland, Mich.) brand products. Small amounts of biological degradation of methylcellulose and similar compounds have been reported. Freeman et al. (2) observed a 93% decrease in viscosity of solutions of methylethylcellulose due to the growth of bacteria normally present in the manufactured product. Siu et al. (9) found minor (8%) weight losses in 9 to 10 days after inoculating 5,000 gg of methylcellulose solutions per ml with a cellulose-degrading fungus. Reese et al. (6) noted a slight hydrolysis of methylcellulose solutions by incubation with certain organisms for 30 days, but concluded that methylcellulose, with the high degree of substitution (DS) required for water solubility, is usually resistant to microbial attack. Reese (7), Savage (8), and Wirick (11) obtained small amounts of reducing sugars from methylcelluloses by enzyme action. MATERIALS AND METHODS The biodegradation of "4C-labeled methylcellulose by activated sludge was evaluated in batch-type cylinder biodegradation experiments according to procedures described previously (H. C. Alexander and F. A. Blanchard, Natl. Meet. Am. Chem. Soc., 168th, Atlantic City, N.J., Abstr. 35 Fuel, 1974; complete paper as Div. of Fuel Chem. Preprints 19:104-112, 1974). The 14C-labeled methylcelluloses were prepared to correspond as closely as possible to Methocel products MC 25, MC 100, and MC 4000 (designation in use before 6/1/74. MC 25 and MC 4000 are now named A 25 and A 4M. The MC 100 type is no longer available). Alkalicellulose was first prepared from cellulose and sodium hydroxide,
and then reacted with [14C]methyl chloride. The product was purified by degassing under vacuum, then by exhaustive washing with acetone, followed by exhaustive washing with boiling water and then drying under vacuum. The principal radioactive impurities expected were: ['4C]methanol, [14C]methyl ether, and unreacted ['4C]methyl chloride. Thin-layer chromatography (TLC), as given below, indicated that 99.4% of the product was polymeric. The properties of the methylcelluloses are given in Table 1. This table and later discussions in this paper use two definitions familiar to cellulose chemists. The DS is the average number of substituent groups attached to the ring hydroxyls of the anhydroglucose units of the cellulose. The maximum DS possible is three. The degree of polymerization (DP) represents the average number of anhydroglucose units per polymer chain. Settled sludge (at about 2% solids) was obtained from the Dow Chemical Co., Midland, Mich., General Wastewater Treatment Plant. A 500-ml portion was placed in a 2-liter cylinder. The volume was brought to 1.7 liters with the simultaneous addition of the 14C-labeled compound (to give 16.2, 28.3, and 25.7 mg/liter for MC 4000, MC 100, and MC 25, respectively) and dechlorinated water containing 1 ml of each of the standard biochemical oxygen demand nutrients per liter (1). The cylinder was aerated with C02-free air at 50 to 100 ml/min through a fritted glass bottom. Air from the cylinder, containing respired 14CO2, was passed through two traps in series. Each trap was filled with 15 ml of 40% monoethanolamine in 2-methoxyethanol (vol/vol). The contents were collected, assayed, and replaced periodically. Samples were prepared for liquid scintillation counting by mixing 3 ml with 6 ml of Econofluor scintillator (New England Nuclear Corp., Boston, Mass.) and 9 ml of methanol. Mixed liquor samples were withdrawn by syringe. To each 10-ml sample was added 0.5 ml of Formalin (37% formaldehyde by weight). Mixed liquor samples (0.3 ml) were combusted with a Harvey biological material oxidizer (R. J. Harvey Instrument Co., Hillsdale, N.J.) and then assayed by liquid scintillation counting. The mixed liquor volatile solids (MLVS) was 557
558
BLANCHARD, TAKAHASHI, AND ALEXANDER
APPL. ENVIRON. MICROBIOL.
per h. The amounts consumed, in milligrams of compound per gram of MLVS per 4 h, during Property the initial 4 h of contact were: 0.0021 (MC 4000), Equiva2% SoluMeth0.0065 (MC 100), 0.0007 (MC 25), and 2.2 lent Meth- Sp act oxyl tion vis(phenol). ocel prod(,CiI conDSa cosity, DPb Non-methylcellulose compounds were presuct (centitent, g) ent in the MC 4000 supernatant liquid as deterpoise) wt (%) mined by TLC (Table 3). Radioactive materials 1.9 430 30.3 3,400 126 MC 4000 at R's of about 0.2 (x) and 0.5 (y) were already 31.3 502 1.9 82.5 128 MC 100 present in the 1-day sample. They were the 31.4 1.9 33.1 100 77 MC 25 predominate (80%) form of radioactive material a Degree of substitution. in solution by 12 days. By 20 days there was b Degree of polymerization. only 4% of the original radioactivity remaining in solution as polymer (the supernatant liquid initially 3 to 4 g/liter. After 20 days, it was 1.5 to 2.5 had 17% of the original radioactivity of which g/liter. Supernatant liquid was obtained by either 23% was polymer by TLC). TABLE 1. Properties of ['4C]methylcelluloses
settling or centrifuging the mixed liquor. Samples were added directly to 10 ml of Aquasol (New England Nuclear Corp.) scintillator solution. The radioactivity of the solids samples were obtained as the difference between that of mixed liquor and that of the supernatant liquid. One direct measurement on solids was made. The solids from a 20-day sample in the test of MC 4000 were separated by centrifugation and then washed, combusted, and counted. Selected supernatant liquids from the MC 4000 experiment (1, 12, and 20 days) were examined by TLC (n-propanol/ethyl acetate/water (7:2:1, vol/vol/ vol) with Quanta/gram QlF silica gel plates (Quantum Industries, Fairfield, N.J.). Reference compounds were: ['4C]methylcellulose, glucose, sucrose, and Dextran 20 (a polysaccharide with molecular weight = 14,500, from Pharmacia, Uppsala, Sweden). Plates were prepared, developed, exposed to I2 vapor, examined under ultraviolet light and marked off into 9 or 10 horizontal zones. Each zone was scraped into a counting vial, covered with liquid scintillator, and assayed. The results were converted into percentage recovery on the plate and a percentage distribution by zone.
DISCUSSION The generally slow degradation rates observed for these methyl ethers are consistent with observations on the effects of DS on enzyme functioning. Reese et al. (6) found that the DS has a decided effect on enzymatic hydrolysis of water-soluble cellulose derivatives as measured by the production of reducing compounds, and that methylcellulose has the resistance anticipated for compounds of DS over one. The curves of Fig. 2 for MC 25 (DP 100) and MC 4000 (DP 430) are quite similar in general shape, location of the peak, and height of the peak. This agrees with the observations of Reese et al. (6) on carboxymethylcellulose that the DP does not affect the rate of enzymic hydrolysis. The several days needed to reach a peak rate of '4CO2 release from the methylcellulose might be due to an induction time for exoenzymes which are required for breaking the polymer into small enough fragments for cell entry. Organisms have been shown to produce enzymes catalyzing the hydrolysis of cellulose ethers and esters (3, 6). Induction of additional enzymes by the sludge organisms would probably be slow in response to the low concentration of methylcel-
RESULTS These radiotracer studies with Methocel MC 4000, MC 100, and MC 25 methylcellulose demonstrated, respectively, 73, 54, and 24% conversion of the radioactivity into 14CO2 in 20 days. The supernatant liquids retained 17, 21, and 24%, respectively. Radioactivity in the suspended solids accounted for 14, 11, and 21%, respectively. The biodegradation of MC 4000 is shown in detail in Fig. 1 and Table 2. Total The degradation rates found were slow. . 100 There was a gradual rate increase as seen in Supernatant ~~~Trap, Cumulative the evolution rate of 14CO2 through 7 to 9 days unatant @50 (Fig. 2). The maximum rates in milligrams of methylcellulose per gram of MLVS per day were: 0.59 (MC 4000), 0.66 (MC 100), and 0.68 (MC 25). For comparison, ['4C]phenol in a simio, , lar system (Alexander and Blanchard, Natl. 15 20 10 5 Meet. Am. Chem. Soc., 168th, Atlantic City, Time, Days N.J., 1974) at a similar loading reached a peak FIG. 1. Distribution of 14C activity during sludge in 3 h, with a rate of 1 mg of phenol/g of MLVS biodegradation of ['4C]methylcellulose. 0U
VOL. 32, 1976
BIODEGRADABILITY OF [14C]METHYLCELLULOSE
559
TABLE 2. "4C-labeled MC 4000 methylcellulose or equivalent in activated sludge and traps
14C02 collected in trapsa
eycmg of ['4C ['Cmethylcellulose/liter
__________________
As equivalent concn change in mixed liquor
Sludge vol
Day
Total in traps (Ag)
Mixed liquor Supematant
Incremen-
tal (mg/liter)
0 1.49 15.2 13.5 1 1.45 13.2 2 1.40 14.4 12.3 3 1.39 11.1 4 1.38 14.6 9.9 5 1.37 11.1 8.9 6 1.36 7.3 7 1.35 9.0 4.7 8 1.30 3.4 9 1.29 6.2 2.9 10 1.28 5.7 2.1 11 1.27 1.8 12 1.22 4.4 1.8 13 1.21 2.4 14 1.20 2.6 15 1.19 5.6 2.7 17 1.18 5.7 3.1 20 1.17 5.0 2.7 a Calculated as equivalent 14C-labeled methylcellulose.
0.6 _
8>
0.5 _-
2 T
226 383 540 821 1,483 2,291 3,418 2,881 1,620 980 440 130 74 72 44 26 24
Cumulative
0
0
2
4
6
8
10 12 Time, Days
14
16
18
20
FIG. 2. Comparison of biodegradation of three ['4C]methylcelluloses. The initial concentrations in Mg/ml were: 16.2 (MC 4000), 28.3 (MC 100), and 25.7 (MC 25). These represent loadings in mg/g of MLVS of 3.7 (MC 4000), 8.82 (MC 100), and 6.25 (MC 25).
8 7 6 5 4 3
0.90-1.00 0.73-0.90 0.60-0.90 0.48-0.60 0.37-0.48 Glucose 0. 26-0. 37 Dexranose
2 1 0
0.17-0.26 0.06-0.17 0-0.06 [14C]methylcellulosea
a
su
1-Day 12-Day 20-Day 0 0 0 0 0 0 0 12.2 8.6 0.5 8.8 6.9 0.9 1.5 4.6 2.6 34.1 32. 2 1.5
13.2 12.6
3.0 91.4
8.7 9.0) 20.2 23.2
The origin retained 99.4% of the radioactivity applied.
tached groups are reported to be resistant to attack (5). Some, perhaps most, of the methylcellulose was degraded through intermediates lulose supplied. In our experiments, the initial like x andy. x may be [14C]methyl oligosacchaenzyme concentration was probably very low rides such as cellobiose and y may be [14C]_ because the settled return sludge was diluted methyl glucoses. Levinson et al. (5) gave such an interpretation (substituted glucose and subwith fresh water. We believe that most of the large conversion stituted cellobiose) to enzyme derivatives of of radioactivity to "4CO2 was by total degrada- carboxymethylcellulose. Hagen et al (4) have tion of the polymer. Possibly, some of the degra- produced dye-substituted oligosaccharides and dation might be via detachment of the dye-substituted glucose by enzymic hydrolysis [14C]methyl groups from the polymer. Since of dye-substituted cellulose. Methyl-substituted this would lower the DS, it would be expected to oligosaccharides and methyl glucoses may be increase the biodegradability. However, at- similarly formed.
560
BLANCHARD, TAKAHASHI, AND ALEXANDER
With a 1.9 DS, the tracer methylcelluloses should have a composition (based on estimates of Savage [8] and Spurlin [10]) of 2% unsubstituted, 28% monomethyl (mainly 6-0-methyl), 49% dimethyl (mainly 2,6-0-methyl and 3,6-0methyl), and 21% trimethyl (2,3,6-0-methyl). The observed result of 73% converted to 14CO2 required considerable breaking of bonds between units with all degrees of substitution. It also required utilization of most of the released, substituted glucose molecules. Thus, methylcellulose is slowly biodegradable. In these experiments it was 96% degraded or otherwise removed from solution in 20 days by activated sludge. ACKNOWLEDGMENTS Wen Cheng of The Dow Chemical Co. Designed Polymers Research Laboratory developed the radiotracer synthesis method and prepared the tracer with some assistance from one of us (F.A.B.). We appreciate the assistance by Dave Gransden of our laboratory for much of the analytical work.
LITERATURE CITED 1. American Public Health Association. 1971. Standard methods for the examination of water and wastewater, 13th ed. American Public Health Association, New York.
APPL. ENVIRON. MICROBIOL.
2. Freeman, G. G., A. J. Baillie, and C. A. Macinnes. 1948. Bacterial degradation of sodium carboxymethylcellulose and methyl ethyl cellulose. Chem. Ind. (London), May 1, p. 279-282. 3. Gascoigne, J. A., and M. M. Gascoigne. 1960. Biological degradation of cellulose. Butterworths, London. 4. Hagen, P., E. T. Reese, and 0. A. Stamm. 1966. Reaction of reactive dyes with cellulose. IV. Enzymatic hydrolysis of reactive dyed cellulose. Helv. Chim. Acta 49:2278-2287. 5. Levinson, H. S., G. R. Mandels, and E. T. Reese. 1951. Products of enzymatic hydrolysis of cellulose and its derivatives. Arch. Biochem. Biophys. 31:351-365. 6. Reese, E. T., R. G. H. Siu, and H. S. Levinson. 1950. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J. Bacteriol. 59:485-497. 7. Reese, E. T. 1957. Biological degradation of cellulose derivatives. Ind. Eng. Chem. 49:89-93. 8. Savage, A. B. 1957. Temperature-viscosity relationships for water-soluble cellulose ethers. Ind. Eng. Chem. 49:99-103. 9. Siu, R. G. H., R. T. Darby, P. R. Burkholder, and E. S. Barghoorn. 1949. Specificity of microbiological attack on cellulose derivatives. Text. Res. J. 19:484488. 10. Spurlin, H. M. 1939. Arrangement of substituents in cellulose derivatives, J. Am. Chem. Soc. 61:22222227. 11. Wirick, M. G. 1968. A study ofthe enzymic degradation of CMC and other cellulose ethers. J. Polymer Sci. A-1, 6:1965-1974.
