APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1978, p. 51-53 Copyright X 1978 American Society for Microbiology
Vol. 35, No. 1
Printed in U.S.A.
Structural Factors Influencing the Biodegradation of Imidest D. M. ENNIS,`* A. KRAMER,2 C. W. JAMESON,3 P. H. MAZZOCCHI,3 AND W. J. BAILEY3 Department of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1,I and Food Science Program2 and Department of Chemistry,3 University of Maryland, College Park, Maryland 20742
Received for publication 28 February 1977
Comparative studies on the biodegradability of amides and imides are presented. Low-molecular-weight imides of varying chain lengths (4, 6, 7, 8, 18, and 20 carbons) were biodegradable. N-alkyl substitution ofamides and imides resulted in non-biodegradable derivatives when the amide portion was greater than two carbons in length. N-alkyl-substituted derivatives of acetamide or diacetamide, however, were biodegradable. Several soil isolates, including Aspergillus niger and species of Flavobacterium and Alcaligenes, were capable of growth with imides as sole N or C sources. From both the food industry and environmental viewpoints, an ideal plastic packaging material should be cheap and nontoxic, have desirable physical properties for specific food uses, and be degradable when discarded. Such a perishable package should be stable under conditions of storage (e.g., low light intensity, low moisture, and low temperature) and during transit in the channels of trade. Studies on the biodegradability of natural and synthetic polymers are now fairly numerous. In many cases, the presence of ester linkages in the main chain of the polymer has significantly increased biodegradability (2, 4, 5). Branching of polymers in some cases prevents biodegradation (5). As a first step in developing polymers that would be photo- and biodegradable, two studies were undertaken. The first of these was concerned with the mechanism of photolysis of simple imides. One of the principal photolysis mechanisms involves cleavage between a and ,f carbons adjacent to the imide functional group (C. W. Jameson, Ph.D. thesis, University of Maryland, College Park, 1975). In the case of polyimides, this type of cleavage should result in the formation of a series of N-acetyl imides and a mixture of high- and low-molecular-weight compounds terminating with a C = C. Photochemical cleavage on both sides of the imide moiety would result in the formation of diacetamide. In the present study, we investigated the biodegradability of low-molecular-weight imides, particularly those that would be expected to result from polyimide photolysis.
MATERIALS AND METHODS
Synthesis of imides. Imides were usually prepared by heating the appropriate primary or secondary amide with acetic anhydride containing 10 to 20% (by weight) acetyl chloride and refluxing for 24 to 48 h. The resultant dark-brown solutions were then vacuum distilled through a Vigreux column to remove excess reagent, and appropriate fractions were redistilled to collect product. In cases where mixtures of products resulted, the product was further purified by either preparative gas-liquid chromatography or recrystallization to yield compounds of 95 to 97% purity. The nuclear magnetic resonance and infrared data for all compounds were consistent with the structure assigned by previous workers (1, 7). Biodegradation studies. A modification of the rapid method of Ennis and Kramer (3) was used. In the modified method, 20 ml of nutrient medium containing test material was dispensed into 125-ml flasks, inoculated with 1 ml of nutrient broth which had been inoculated with a Maryland sandy loam (10%), and incubated at 35°C for 4 days. The nutrient medium consisted of 1 g of a pancreatic digest of casein (Trypticase; BBL, Cockeysville, Md.) and 0.67 g of dextrose per liter. For solid samples, 20 mg of test material was used and, for liquid samples, 20 pl. Due to the limited supply of N-propyl valeramide, N-methyl valeramide, N-acetyl-N-propyl valeramide, and N-acetylN-methyl valeramide, a further modification was found necessary. This modification involved using 1 of sample in 0.5 ml of nutrient medium, dispensed pilin 3-ml tubes. In these cases, the size of the inoculum was reduced to 10 pl. Headspace analysis for CO2 was performed after 10 to 34 days as described in the original method. Experiments were conducted under laboratory lighting conditions in the absence of ultraviolet irradiation. Isolate studies. Microorganisms capable of utilizing N-acetyl butyramide, N-acetyl pentanamide, Nacetyl palmitamide, and N-acetyl stearamide were isolated from soil by the enrichment culture technique during a study on hexamethylenediamine degradation.
t Scientific Article no. A2210, Contribution no. 5191 of the Maryland Agricultural Experiment Station, Food Science Program.
51
52
APPL. ENVIRON. MICROBIOL.
ENNIS ET AL.
RESULTS Effect of amide chain length. The effect of amide chain length on the biodegradability of imides is shown in Table 1. The StudentNewman-Keuls multiple comparison procedure (6) was used to separate means for which the analysis of variance had indicated a significant F ratio. All the N-acetyl imides tested, including N-acetyl stearamide, were biodegradable. The biodegradability of N-acetyl palmitamide and N-acetyl stearamide was demonstrated by isolating specific microorganisms that could utilize these materials as C or N sources. Effect of N-alkyl substitution. N-substitution of N-acetyl pentanamide with methyl and propyl groups prevented degradation as indicated by C02 analysis after 10 days. N-substitution of diacetamide with methyl, propyl, isobutyl, hexyl, and even phenethyl groups did not prevent degradation (Table 1). Alkyl substitution of amides. A study of Nalkyl substituted amides revealed that, in the case of valeramide, N-methyl and N-propyl substitution prevented degradation (Table 1). Nsubstitution of hexanamide with the hexyl group also prevented degradation. N-substitution (with propyl and hexyl groups) of acetamide did not interfere with biodegradability, as was the case with diacetamide (Table 1). Isolate studies. Aspergillus niger, a Flavobacterium species (I180), and Alcaligenes paradoxus were capable of utilizing N-acetyl butyramide and N-acetyl valeramide as sole carbon and nitrogen sources. Flavobacterium ferrugineum could use both substrates as sole nitrogen sources only, and a second Flavobacterium species (1222) could use both substrates as sole carbon sources only. A. niger and a species of Flavobacterium (I180) could utilize N-acetyl palmitamide and N-acetyl stearamide as sole carbon and nitrogen sources. F. ferrugineum and A. paradoxus could utilize both substrates as nitrogen sources only, and a second Flavobacterium species (1222) could not grow with either substrate as sole source of nitrogen or carbon. DISCUSSION Several N-acetyl imides of different chain lengths, including N-acetyl stearamide, are biodegradable, and soil isolate studies with four of these imides showed that they are readily metabolized by certain microorganisms, including A. niger. N-substituted imides with alkyl groups indicated that methyl and propyl substitution of N-acetyl pentanamide prevented rapid degradation. When the amide portion is shorter (e.g., in the case of N-alkyl-substituted diacetamide),
TABLE 1. Biodegradability of imide and amide substrates as determined by C02 production Substrate
Relative
CO2 produceda
N-alkyl substituted imides CH2-CO-NH-CO-(CH2)3CH3 CH3-CO-NH-CO-CH3 CH3-CO-N-CO-(CH2)3-CH3 CH3 CH3-CO-N-CO-(CH2)3-CH3
(OH2)2
201 151 100*
105*
CH3 CH3-CO-N--CO--CH3
(CH2)5
158
CH3
CH3-CO-N-CO-CH3 CH3
150
CH3-CO-N--CO-CH3 (CH2)2
144
CH3 CH3-CO-N-CO-CH3
CH3-C-CH3
153
CH3 CH3-CO-N-CO-CH3
CH2CH2Phenyl N-alkyl substituted amides CH3-(CH2)2--CO-NH2 CH3-(CH2)3-CO-NH2 CH3-(CH2)4-CO-NH2 CH3-(CH2)4CONH(CH2)5CH3 CH3(CH2)3CONH(CH2)2CH3 CH3-(CH2)3-C-NH-CH3 CH3CONH(CH2)2CH3 CH3CONH(CH2)5CH3
194 171 190 196 96* 89* 90* 143 143
Imides of varying chain lengths
CH3-CO-NH-CO-CH3 CH3-(CH2)2-CO-NH-CO-CH3 CH3(CH2)3-CO-NH-CO-CH3 CH3(CH2)4-C0-NH--C0-CH3
CH3(CH2)j4--C0-NH-C-CH3
151 174 201 204
_b
_b CH3(OH2)Ow 0-NH-CO-CH3 a (Yield of C02 from sample amended with test
substrate x 100)/yield of C02 from similar but unamended vessel. All means significantly higher than control (100 units) at >95% confidence as determined by Student-Newman-Keuls test, unless followed by an asterisk. 'Biodegradable, as determined by growth studies.
VOL. 35, 1978
BIODEGRADABILITY OF IMIDES
53
methyl, propyl, isobutyl, hexyl, and phenethyl N-substituents do not affect rapid biodegradability. In the case of amides, a similar situation was found; alkyl substitution prevented degradation unless the amide was acetamide. The fact that all N-acetyl imides up to Nacetyl stearamide are biodegradable and the fact that N-hexyl diacetamide is also biodegradable suggest the possibility that existing polyamides, such as nylon 6,6[+NH-(CH2)6-NHCO(CH2)4-CO+J, might be modified by N-acetyl substitution to produce materials that are photoand then biodegradable. Degradation may be accelerated or retarded, depending on the availability of ultraviolet light. Perishable plastic packages, for example, may be preconditioned by natural or artificial ultraviolet light prior to land fill.
Center, University of Maryland. This study was supported by the Center for Materials Research under NSF project SK. We thank Eric Schmader and John Snyder for technical assistance.
ACKNOWLEDGMENS Statistical analysis was perfonned at the Computer Science
Ames. 7. Toth, G. 1970. Structure of diacylamines I. Acta Chim. Acad. Sci. Hung. 64:101-109.
LITERATURE
CITED
1. Cadwallader, D. E., and J. D. LaRocca. 1956. Synthesis
of some diacylimines. J. Am. Pharm. Assoc. 45:480 482. 2. Darby, R. T., and A. M. Kaplan. 1968. Fungal susceptibility of polyurethanes. Appl. Microbiol. 16:900-905. 3. Enmis, D. M., and A. Kramer. 1975. A rapid microtechnique for testing the biodegradability of nylons and related polyamides. J. Food Sci. 40:181-185. 4. Kaplan, A. L, R. T. Darby, ML Greenberger, and M. R. Rogers. 1968. Microbial deterioration of polyurethane systems Dev. Ind. Microbiol. 9:201-217. 5. Potts, J. E., R. A. Clendinning, W. B. Ackert, and W. D. Niegisch. 1971. The biodegradability of synthetic polymers. Union Carbide Corp., Bound Brook, N.J. 6. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods, p. 271-275. Iowa State University Press,
Vol. 35, No. 1
Printed in U.S.A.
Structural Factors Influencing the Biodegradation of Imidest D. M. ENNIS,`* A. KRAMER,2 C. W. JAMESON,3 P. H. MAZZOCCHI,3 AND W. J. BAILEY3 Department of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1,I and Food Science Program2 and Department of Chemistry,3 University of Maryland, College Park, Maryland 20742
Received for publication 28 February 1977
Comparative studies on the biodegradability of amides and imides are presented. Low-molecular-weight imides of varying chain lengths (4, 6, 7, 8, 18, and 20 carbons) were biodegradable. N-alkyl substitution ofamides and imides resulted in non-biodegradable derivatives when the amide portion was greater than two carbons in length. N-alkyl-substituted derivatives of acetamide or diacetamide, however, were biodegradable. Several soil isolates, including Aspergillus niger and species of Flavobacterium and Alcaligenes, were capable of growth with imides as sole N or C sources. From both the food industry and environmental viewpoints, an ideal plastic packaging material should be cheap and nontoxic, have desirable physical properties for specific food uses, and be degradable when discarded. Such a perishable package should be stable under conditions of storage (e.g., low light intensity, low moisture, and low temperature) and during transit in the channels of trade. Studies on the biodegradability of natural and synthetic polymers are now fairly numerous. In many cases, the presence of ester linkages in the main chain of the polymer has significantly increased biodegradability (2, 4, 5). Branching of polymers in some cases prevents biodegradation (5). As a first step in developing polymers that would be photo- and biodegradable, two studies were undertaken. The first of these was concerned with the mechanism of photolysis of simple imides. One of the principal photolysis mechanisms involves cleavage between a and ,f carbons adjacent to the imide functional group (C. W. Jameson, Ph.D. thesis, University of Maryland, College Park, 1975). In the case of polyimides, this type of cleavage should result in the formation of a series of N-acetyl imides and a mixture of high- and low-molecular-weight compounds terminating with a C = C. Photochemical cleavage on both sides of the imide moiety would result in the formation of diacetamide. In the present study, we investigated the biodegradability of low-molecular-weight imides, particularly those that would be expected to result from polyimide photolysis.
MATERIALS AND METHODS
Synthesis of imides. Imides were usually prepared by heating the appropriate primary or secondary amide with acetic anhydride containing 10 to 20% (by weight) acetyl chloride and refluxing for 24 to 48 h. The resultant dark-brown solutions were then vacuum distilled through a Vigreux column to remove excess reagent, and appropriate fractions were redistilled to collect product. In cases where mixtures of products resulted, the product was further purified by either preparative gas-liquid chromatography or recrystallization to yield compounds of 95 to 97% purity. The nuclear magnetic resonance and infrared data for all compounds were consistent with the structure assigned by previous workers (1, 7). Biodegradation studies. A modification of the rapid method of Ennis and Kramer (3) was used. In the modified method, 20 ml of nutrient medium containing test material was dispensed into 125-ml flasks, inoculated with 1 ml of nutrient broth which had been inoculated with a Maryland sandy loam (10%), and incubated at 35°C for 4 days. The nutrient medium consisted of 1 g of a pancreatic digest of casein (Trypticase; BBL, Cockeysville, Md.) and 0.67 g of dextrose per liter. For solid samples, 20 mg of test material was used and, for liquid samples, 20 pl. Due to the limited supply of N-propyl valeramide, N-methyl valeramide, N-acetyl-N-propyl valeramide, and N-acetylN-methyl valeramide, a further modification was found necessary. This modification involved using 1 of sample in 0.5 ml of nutrient medium, dispensed pilin 3-ml tubes. In these cases, the size of the inoculum was reduced to 10 pl. Headspace analysis for CO2 was performed after 10 to 34 days as described in the original method. Experiments were conducted under laboratory lighting conditions in the absence of ultraviolet irradiation. Isolate studies. Microorganisms capable of utilizing N-acetyl butyramide, N-acetyl pentanamide, Nacetyl palmitamide, and N-acetyl stearamide were isolated from soil by the enrichment culture technique during a study on hexamethylenediamine degradation.
t Scientific Article no. A2210, Contribution no. 5191 of the Maryland Agricultural Experiment Station, Food Science Program.
51
52
APPL. ENVIRON. MICROBIOL.
ENNIS ET AL.
RESULTS Effect of amide chain length. The effect of amide chain length on the biodegradability of imides is shown in Table 1. The StudentNewman-Keuls multiple comparison procedure (6) was used to separate means for which the analysis of variance had indicated a significant F ratio. All the N-acetyl imides tested, including N-acetyl stearamide, were biodegradable. The biodegradability of N-acetyl palmitamide and N-acetyl stearamide was demonstrated by isolating specific microorganisms that could utilize these materials as C or N sources. Effect of N-alkyl substitution. N-substitution of N-acetyl pentanamide with methyl and propyl groups prevented degradation as indicated by C02 analysis after 10 days. N-substitution of diacetamide with methyl, propyl, isobutyl, hexyl, and even phenethyl groups did not prevent degradation (Table 1). Alkyl substitution of amides. A study of Nalkyl substituted amides revealed that, in the case of valeramide, N-methyl and N-propyl substitution prevented degradation (Table 1). Nsubstitution of hexanamide with the hexyl group also prevented degradation. N-substitution (with propyl and hexyl groups) of acetamide did not interfere with biodegradability, as was the case with diacetamide (Table 1). Isolate studies. Aspergillus niger, a Flavobacterium species (I180), and Alcaligenes paradoxus were capable of utilizing N-acetyl butyramide and N-acetyl valeramide as sole carbon and nitrogen sources. Flavobacterium ferrugineum could use both substrates as sole nitrogen sources only, and a second Flavobacterium species (1222) could use both substrates as sole carbon sources only. A. niger and a species of Flavobacterium (I180) could utilize N-acetyl palmitamide and N-acetyl stearamide as sole carbon and nitrogen sources. F. ferrugineum and A. paradoxus could utilize both substrates as nitrogen sources only, and a second Flavobacterium species (1222) could not grow with either substrate as sole source of nitrogen or carbon. DISCUSSION Several N-acetyl imides of different chain lengths, including N-acetyl stearamide, are biodegradable, and soil isolate studies with four of these imides showed that they are readily metabolized by certain microorganisms, including A. niger. N-substituted imides with alkyl groups indicated that methyl and propyl substitution of N-acetyl pentanamide prevented rapid degradation. When the amide portion is shorter (e.g., in the case of N-alkyl-substituted diacetamide),
TABLE 1. Biodegradability of imide and amide substrates as determined by C02 production Substrate
Relative
CO2 produceda
N-alkyl substituted imides CH2-CO-NH-CO-(CH2)3CH3 CH3-CO-NH-CO-CH3 CH3-CO-N-CO-(CH2)3-CH3 CH3 CH3-CO-N-CO-(CH2)3-CH3
(OH2)2
201 151 100*
105*
CH3 CH3-CO-N--CO--CH3
(CH2)5
158
CH3
CH3-CO-N-CO-CH3 CH3
150
CH3-CO-N--CO-CH3 (CH2)2
144
CH3 CH3-CO-N-CO-CH3
CH3-C-CH3
153
CH3 CH3-CO-N-CO-CH3
CH2CH2Phenyl N-alkyl substituted amides CH3-(CH2)2--CO-NH2 CH3-(CH2)3-CO-NH2 CH3-(CH2)4-CO-NH2 CH3-(CH2)4CONH(CH2)5CH3 CH3(CH2)3CONH(CH2)2CH3 CH3-(CH2)3-C-NH-CH3 CH3CONH(CH2)2CH3 CH3CONH(CH2)5CH3
194 171 190 196 96* 89* 90* 143 143
Imides of varying chain lengths
CH3-CO-NH-CO-CH3 CH3-(CH2)2-CO-NH-CO-CH3 CH3(CH2)3-CO-NH-CO-CH3 CH3(CH2)4-C0-NH--C0-CH3
CH3(CH2)j4--C0-NH-C-CH3
151 174 201 204
_b
_b CH3(OH2)Ow 0-NH-CO-CH3 a (Yield of C02 from sample amended with test
substrate x 100)/yield of C02 from similar but unamended vessel. All means significantly higher than control (100 units) at >95% confidence as determined by Student-Newman-Keuls test, unless followed by an asterisk. 'Biodegradable, as determined by growth studies.
VOL. 35, 1978
BIODEGRADABILITY OF IMIDES
53
methyl, propyl, isobutyl, hexyl, and phenethyl N-substituents do not affect rapid biodegradability. In the case of amides, a similar situation was found; alkyl substitution prevented degradation unless the amide was acetamide. The fact that all N-acetyl imides up to Nacetyl stearamide are biodegradable and the fact that N-hexyl diacetamide is also biodegradable suggest the possibility that existing polyamides, such as nylon 6,6[+NH-(CH2)6-NHCO(CH2)4-CO+J, might be modified by N-acetyl substitution to produce materials that are photoand then biodegradable. Degradation may be accelerated or retarded, depending on the availability of ultraviolet light. Perishable plastic packages, for example, may be preconditioned by natural or artificial ultraviolet light prior to land fill.
Center, University of Maryland. This study was supported by the Center for Materials Research under NSF project SK. We thank Eric Schmader and John Snyder for technical assistance.
ACKNOWLEDGMENS Statistical analysis was perfonned at the Computer Science
Ames. 7. Toth, G. 1970. Structure of diacylamines I. Acta Chim. Acad. Sci. Hung. 64:101-109.
LITERATURE
CITED
1. Cadwallader, D. E., and J. D. LaRocca. 1956. Synthesis
of some diacylimines. J. Am. Pharm. Assoc. 45:480 482. 2. Darby, R. T., and A. M. Kaplan. 1968. Fungal susceptibility of polyurethanes. Appl. Microbiol. 16:900-905. 3. Enmis, D. M., and A. Kramer. 1975. A rapid microtechnique for testing the biodegradability of nylons and related polyamides. J. Food Sci. 40:181-185. 4. Kaplan, A. L, R. T. Darby, ML Greenberger, and M. R. Rogers. 1968. Microbial deterioration of polyurethane systems Dev. Ind. Microbiol. 9:201-217. 5. Potts, J. E., R. A. Clendinning, W. B. Ackert, and W. D. Niegisch. 1971. The biodegradability of synthetic polymers. Union Carbide Corp., Bound Brook, N.J. 6. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods, p. 271-275. Iowa State University Press,
