APPLiED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p. 1037-1041 Copyright C 1977 American Society for Microbiology
Vol. 33, No. 5 Printed in U.S.A.
Bacterial Metabolism of Quaternary Ammonium Compounds DEBORAH DEAN-RAYMOND'
AND
M. ALEXANDER*
Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853
Of 10 quaternary ammonium compounds tested for biodegradation by the biological oxygen demand technique, only decyl- and hexadecyltrimethylammonium bromides were decomposed by organisms derived from sewage and soil. A mixture consisting of individual strains of Pseudomonas and Xanthomonas grew in solutions containing decyltrimethylammonium bromide as sole carbon source. The xanthomonad metabolized this quaternary ammonium compound in the presence of other organic molecules. The products of this activity included 9carboxynonyl- and 7-carboxyheptyltrimethylammonium, suggesting that the terminal carbon of the decyl moiety is oxidized and the resulting carboxylic acid is subject to 8-oxidation.
Quaternary ammonium compounds are widely used in industry. Some are antifoam and antistatic agents and have surfactant properties. Among this class of chemicals are potent bactericides, and several of these quaternary ammonium compounds thus are included in antiseptic solutions, clinical preparations, and cosmetics. Almost half of the quaternary ammonium compounds used commercially are added to laundry preparations as water soften-
0.6 mg; ZnSO4 and MnSO4, 0.2 mg each. The solution was added to standard biological oxygen demand (BOD) bottles, and quaternaries were introduced to give a concentration of 10, 25, or 100 ,ug/ml. To measure the BOD, 10 ml of a 10% aqueous suspension of Williamson silt loam or 3.0 ml of a 10% solution of municipal sewage passed through Whatman no. 1 filter paper was used as inoculum. All treatments were done in duplicate. The rate of 02 depletion in the bottles was measured by means of a biological 02 monitor (model 53, Yellow Springs Instrument Co., Yellow Springs, Ohio). To isolate the ers (7). The biodegradation of members of this group active organisms, enrichments were prepared from BOD bottles exhibiting activity, the enrichments of substances has been the subject of study. For containing 100 to 500 ,mg of the quaternary per ml. that and Isaac reported (1) example, Barden The cultures were purified by streaking portions of cetylpyridinium bromide at low concentrations the enrichments on nutrient agar, and subsequently was completely oxidized by microorganisms the isolates were inoculated into basal salts medium from activated sludge, although somewhat containing the test compound to confirm the ability higher levels were toxic, and laurylpyridinium of the organism to use the chemical. Cultures were chloride at an initial level of 15 ,ug/ml was identified by the procedures outlined by Skerman found to be degraded by Pseudomonas picto- (8). To test the ability of the isolates to use quaternarrum (4). In addition, Desbordes and Jourdan (3) and related compounds, standard manometric ies and chloride cetylthat benzalkonium reported used (9). The bacteria were grown techniques trimethylammonium bromide were attacked by in 1 liter ofwere the basal salts solution containing the the other On hand, Pseudomonas. of species test substrate at a concentration of 0.05%. The culWinter (11) found no degradation of cetyltri- ture was incubated on a shaker for 3 days at 30°C, methylammonium bromide at a concentration and the cells were collected by centrifugation at 10,000 x g for 10 min, washed with 0.10 M phosphate of 10 ,ug/ml. The mechanism of bacterial degradation of buffer (pH 7.0), and suspended in fresh buffer. The quaternary ammonium compounds has yet to Warburg vessels contained 1.0 Amol of test sub7.0), and be defined, however. The present study was strate, 300 umol of phosphate bufferof(pH 60 ,ug. 40 to content a to cells protein equivalent designed to provide information on how these Oxygen utilization was measured until the rate widely used chemicals are metabolized by mi- uptake approached that of the endogenous control.of croorganisms. All treatments were performed in duplicate. To obtain products of metabolism, the bacteria MATERIALS AND METHODS were grown on a shaker at 30°C in 1.0 liter of a The basal salts solution contained, per liter: medium containing 0.2% yeast extract, 0.2% CasaKH2PO4, 5.5 g; Na2HPO4, 10 g; (NH4)2SO4, 1.0 g; mino Acids, and 0.05% decyltrimethylammonium MgSO4*7H20, 0.1 g; CaCl2 H20, 15 mg; Fe2 (SO4)3, bromide (DTM). After 2 to 3 days of growth, the cells were collected by centrifugation at 8,000 x g and I Present address: Ivorydale Technical Center, Proctor washed in 0.1 M phosphate buffer (pH 7.0). They were then incubated in a solution containing 0.05% and Gamble Co., Cincinnati, OH 45217. 1037 -
1038
APPL. ENVIRON. MICROBIOL.
DEAN-RAYMOND AND ALEXANDER
DTM in the phosphate buffer. After 24 or 48 h, the cells were removed by centrifugation, and the supernatant fluid was extracted with an equal volume of methylene chloride. The solvent was treated with HCl gas (prepared by dropping concentrated H2SO4 onto NaCl) until saturated, and it was then concentrated to about 2 ml in a Kuderna-Danish evaporator kept at 70°C. These extracts were analyzed by
chromatography. Analysis of the quaternary ammonium compounds was performed by the colorimetric procedure of van Steveninck and Maas (10). Bacterial growth was measured by either determination of optical density at 500 nm or determination of cell protein by the method of Lowry et al. (6). A Varian gas chromatograph, model 1700 (Varian Aerograph, Walnut Creek, Calif.), equipped with a flame ionization detector was used. The chromatograph was fitted with a stainless-steel column (1.8 by 3 mm) packed with 5% FFAP on Chromosorb W (80/100 mesh, acid washed). The flow rate of the carrier gas, N., was 20 ml/min. The temperatures of the detector and injector were 250 and 230°C, respectively. The column was programmed from 85 to 200°C at a rate of 4°C/min. Combined gas chromatography-mass spectrometry was performed with a Finnigan gas chromatograph-mass spectrometer, model 3300, equipped with a Systems Industries System 150 data processing system. The quaternaries were obtained from Eastman Kodak Co., Rochester, N.Y., and Pfaltz and Bauer, Flusing, N.Y. Amines were obtained from Pfaltz and Bauer and Lachat Chemicals, Mequon, Wis. Gas chromatographic packing materials were supplied by Applied Science Laboratories, State College, Pa., and Rohm and Haas, Philadelphia, Pa., supplied samples of Hyamine 10-X, a commercial quaternary preparation. gas
RESULTS
BOD. Using standard BOD techniques, the biodegradability of 10 quaternary ammonium compounds was tested at three concentrations. Oxygen measurements were made at 5- to 8-day intervals for a period of 60 days. Benzyltrimethylammonium chloride, phenyltrimethylammonium bromide, benzyltriethylammonium
bromide, didodecyldimethylammonium bromide, dioctadecyldimethylammonium bromide, hexadecyldimethylbenzylammonium chloride, phenyldimethylbenzylammonium chloride, and Hyamine 10-X were found to be resistant to attack by microorganisms in soil and sewage inocula. However, microorganisms derived from both soil and sewage metabolized DTM and hexadecyltrimethylammonium bromide (Table 1). DTM was extensively degraded within 8 to 10 days at all three concentrations tested. The low values for 02 consumption at 25 and 10 gg/ml with the soil inoculum resulted from the fact that the contents of the bottles were removed as an inoculum for enrichment cultures before complete 02 consumption was attained. Biodegradation of hexadecyltrimethylammonium bromide was evident at levels of 10 and 25 ,ug/ml, although the time to achieve detectable 02 uptake was longer than with DTM. The lack of activity on the hexadecyl compound at levels of 100 ,ug/ml probably is attributable to its toxicity. Characterization of active bacterium. DTM was chosen for further study because sewage and soil organisms used it rapidly at levels of 100 ,g/ml. After repeated serial transfer of the DTM enrichment culture at successively higher levels of the test compound, a mixed culture was obtained that was capable of growth at 500 ,ug/ml. This mixed culture, when plated on nutrient agar, showed the presence of at least five distinct colony types. The mixture was plated on 0.05% DTM-basal salts agar as well as nutrient agar, and individual colonies were selected. Isolates from nutrient agar and DTM-basal salts agar failed to grow, however, when inoculated into 0.05% DTM-basal salts broth. The enrichment culture was then diluted, and the dilutions were inoculated into DTMsalts medium and incubated for 10 days. The highest dilutions to give visible turbidity were plated on nutrient agar, but these dilutions
TABLE 1. Biodegradation of quaternary ammonium compounds by sewage and soil microorganisms Soil inoculum
Quaternary ammonium compound
DTM
Hexadecyltrimethylammonium bromide a
Sewage inoculum
Initial concn
(Ag/ml)
Days Das
02 consumeda
(jLg/ml)
Days Das 10 10 10
6.15 6.23 6.15
60 17
0.0 5.67
17
5.75
100
8
5.99
25 10
8
2.27 1.05
8
100 60 0.0 25 43 3.72 10 15 3.72 Corrected for 02 consumption in the absence of the quaternary.
consumeda
(Ag/ml)
VOL. 33, 1977
QUATERNARY AMMONIUM COMPOUND BIODEGRADATION
1039
were found to contain three organisms distin- Xanthomonas sp. did not grow or use DTM in guishable by colonial morphology. None of the unsupplemented medium or in solutions these organisms grew when inoculated individ- with either yeast extract or Casamino Acids, ually into DTM-basal salts broth. However, there was a visible increase in turbidity and when two of the organisms were inoculated about 70% of the DTM was metabolized when together into DTM-basal salts medium, growth this organism was provided with both yeast and metabolism of DTM occurred (Fig. 1). One extract and Casamino Acids. These observaof these organisms produces white and the tions suggest that Xanthomonas sp. was using other produces yellow colonies. Both bacterial a component of the yeast extract-Casamino types are gram-negative rods, possess polar fla- Acids mixture while acting on the DTM. gella, and give a positive Kovac oxidase test. To test further the metabolism of DTM, each Neither produces a fluorescent pigment. Conse- of the two bacteria was inoculated separately quently, they were identified as strains of Pseu- into a medium containing 0.05% DTM, 0.01% domonas and Xanthomonas. The third orga- yeast extract, 0.01% Casamino Acids, inorganic nism found in the dilutions was discarded be- salts, and an additional organic substrate in 1 cause it failed to grow in DTM-salts broth alone liter of water. For Pseudomonas sp., the addior in combination with either of the other two tional substrate was glucose (0.2%) or trimeorganisms. thylamine (0.2%). For Xanthomonas sp., the To determine the role of each of the bacterial additional nutrient was glucose (0.2%) or yeast populations in the mixture, the two axenic cul- extract plus Casamino Acids (0.2% of each). tures were separately inoculated into four me- The organisms were incubated on a shaker at dia, each of which contained the basal salts and 30°C. The pseudomonad grew well in media 0.05% DTM. One contained no growth factors, a with either glucose or trimethylamine but second contained 0.01% yeast extract, a third failed to metaboze DTM. The xanthomonad was supplemented with 0.01% Casamino Acids, multiplied in the media containing glucose but and the fourth contained both yeast extract and also did not metabolize DTM; however, when Casamino Acids (0.01% of each). The organisms grown in media supplemented with both yeast were incubated on a shaker at 30°C, and growth extract and Casamino Acids, it used all of the and metabolism of DTM were determined after intact quaternary compound provided. Some a 2-week period. Pseudomonas sp. did not grow growth occurred in the first 2 days, but then no or use DTM in any of the media. Although further increase in turbidity was evident (Fig. 2). Growth commenced again. after a few days and proceeded until day 8. DTM utilization began after day 1, leveled off from 2 to 4 days, and then proceeded readily again until the DTM was lost from the medium at day 8. Oxygen uptake. The extent of utilization of DTM was determined by manometric assessment of O., uptake. Cells from two member cultures grown on 0.05% DTM were incubated with DTM and compounds thought to be possible intermediates in its degradation. DTM was destroyed totally, 19.0 ,umol of O. being consumed per Amol of substrate (Fig. 3); the theoretical value is 18.5. Trimethylamine was also almost completely degraded. Decanoic acid and methyldecylamine were metabolized without a lag, but only about half of the theoretical quantity of 02 was consumed. Decane was not used. Since Xanthomonas sp. was capable of degrading DTM by itself in the presence of growth factors, it was tested in a similar experiment. The culture was grown in 1 liter of a medium containing 0.2% yeast extract, 0.2% Casamino Acids, and 0.05% DTM. After 2 days on a shaker at 30°C, the cells were collected by cenFIG. 1. Growth of a mixture of two bacterial popu- trifugation, washed three times in 0.10 M phoslations and DTM disappearance in solutions con- phate buffer (pH 7.0), and suspended in fresh buffer. DTM was used without a lag, but the 02 taining 0.05% DTM. HOURS
1040
DEAN-RAYMOND AND ALEXANDER
7E 2
a0. =k
FIG. 2. Bacterial development and DTM metabolism in a medium containing yeast extract and Casamino Acids.
IU)
o
U)
cz E
0
0
E
HOURS
FIG. 3. Oxidation of various substrates by resting cells derived from a two-member culture.
consumption was only about 60% of the theoretical value (Fig. 4). Decanoic acid was also used without a lag by the xanthomonad and to about the same extent as the two-member culture,
APPL. ENVIRON. MICROBIOL.
and trimethylamine was not metabolized. Methyldecylamine and dimethyldecylamine inhibited endogenous respiration. Products of DTM metabolism. Because DTM was only partially metabolized by Xanthomonas sp., products must have accumulated in the reaction mixture. The products were extracted from a resting-cell suspension incubated with DTM in phosphate buffer. Prominent peaks appearing in gas chromatograms of the extract were selected for identification by combined gas chromatography-mass spectrometry. Electron impact mass spectra revealed that two of the components had similar spectra, showing prominent peaks at 45, 60, 73, 87, and 129 mass units, a fragmentation pattern characteristic of aliphatic acids. The peaks of highest mass number in the two spectra were 157 and 185, respectively. Although these peaks had the highest masses in the spectrum, they showed none of the other characteristics of molecular ion peaks. Chemical ionization spectra of the two gas chromatographic peaks were then obtained These spectra indicated that the molecular weights of the two compounds were 202 and 230, respectively. The molecular weights and fragmentation patterns are consistent with the structure being 7-carboxyheptyltrimethylammonium and 9-carboxynonyltrimethylammonium, respectively. These observations suggest that the bacterium oxidized the terminal carbon of the long alkyl chain of the quaternary and then cleaved acetyl units by 3-oxidation. Use of the data retrieval system with the chemical ionization spectra of the sample revealed the presence of compounds having molecular weights of 174, 146, and 118 mass units. These compounds were present in much smaller quantities than the other two. The products may be lower-molecular-weight members of a homologous series of carboxylic acids derived from the quaternary. DISCUSSION The results of BOD tests confirm earlier data that at least certain quaternary ammonium compounds are subject to biodegradation. In some instances, the failure to observe biodegradation may result from toxicity at the levels used. Thus, hexadecyltrimethylammonium bromide was only oxidized at the two lower concentrations. The toxicity of such compounds increases with increasing chain length of the alkyl moiety (2, 5). No isolate capable of growing on DTM as sole source of carbon was obtained, but a mixture of
VOL. 33, 1977
QUATERNARY AMMONIUM COMPOUND BIODEGRADATION
1041
and carboxymethyltrimethylammonium compounds. The substances with lower molecular weights were present in small quantities, perhaps because the rate of ,8-oxidation was affected by chain length. These acids and/or acetic acid might be released into the medium by the xanthomonad and thus be available to the pseudomonad as substrates for growth. When growing together on DTM, the two organisms may then act by protocooperation, the pseudomonad providing growth factors to its associate and the xanthomonad converting DTM to compounds used for energy by its part-
I-
co
ner.
z 0
E
ACKNOWLEDGMENTS The investigation was supported in part by a Public Health Service fellowship (5F22 ES01231) from the National Institute of Environmental Health Sciences. We thank T. Wachs and P. C. Wszolek for assistance in interpretation of the mass spectra.
HOURS
FIG. 4. Utilization of several compounds by DTMinduced resting cells of Xanthomonas sp. was able to proliferate on DTM without the addition of growth factors. Only one of the organisms, a strain of Xanthomonas, acted on DTM. Because this organism could not grow on DTM alone, it is probable that the second bacterium supplies one or more growth factors to the xanthomonad. Furthermore, Xanthomonas sp. cannot degrade DTM completely, and the products of partial degradation would be available to support replication of Pseudomonas sp. so that the two organisms together would be able to grow on DTM. The isolation of 9-carboxynonyl- and 7-carboxyheptyltrimethylammonium compounds suggests that the xanthomonad oxidized the terminal carbon of the longer alkyl chain and then cleaved acetyl units by 83-oxidation. Furthermore, the finding of products not only of 230 and 202 but also of 174, 146, and 118 mass units indicates the further operation of 13-oxidation since the difference between these molecules is 28 mass units, equivalent to CH2CH2. Thus, the sequence might then involve the sequential formation of the 9-carboxynonyl-, 7-carboxyheptyl-, 5-carboxypentyl-, 3-carboxypropyl-,
two bacteria
LITERATURE CITED 1. Barden, L., and P. C. G. Isaac. 1957. The effect of synthetic detergents on the biological stabilization of sewage. Proc. Inst. Civil Eng. 6:371-405. 2. Chuillon, J., and B. Baleux. 1968. Antibacterial action of certain quaternary ammonium compounds, p. 259268. In C. R. Congr. Int. Deterg., 5th, vol. 3. 3. Desbordes, J., and R. Jourdan. 1974. Influence of bacterial enzymes on molecular degradation of antiseptics utilized as preservatives in medical preparations. Dev. Biol. Stand. 24:77-90. 4. Janota-Bassalik, L., C. Olczyk, and M. Kaczorowska. 1969. Degradation of some cationic detergents by Pseudomonas pictorum. Acta Microbiol. Pol. Ser. B 1:31-34. 5. Korai, H., and K. Takeichi. 1970. Antimicrobial activity of quaternary ammonium compounds. (In Japanese) Hakko Kogaku Zasshi 48:635-640. 6. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. May, A., and A. Neufahrt. 1976. Ecological behaviour of cationic surfactants. Third report. The behaviour of distearyl dimethyl ammonium chloride in activated sludge units. Tenside 13:65-69. 8. Skerman, V. B. D. 1967. A guide to the identification of the genera of bacteria. The Williams & Wilkins Co., Baltimore. 9. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1972.
Manometric and biochemical techniques. Burgess Publishing Co., Minneapolis. 10. van Steveninck, J., and M. Maas. 1965. A colorimetric assay method for microquantities of quaternary ammonium detergents. Rec. Trav. Chim. Pays-Bas 84:1166-1168. 11. Winter, W. 1962. Der biochemische Abbau von Detergentien bei der Abwasserreinigung. Wasserwirtsch. Wassertech. 12:265-271.
Vol. 33, No. 5 Printed in U.S.A.
Bacterial Metabolism of Quaternary Ammonium Compounds DEBORAH DEAN-RAYMOND'
AND
M. ALEXANDER*
Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853
Of 10 quaternary ammonium compounds tested for biodegradation by the biological oxygen demand technique, only decyl- and hexadecyltrimethylammonium bromides were decomposed by organisms derived from sewage and soil. A mixture consisting of individual strains of Pseudomonas and Xanthomonas grew in solutions containing decyltrimethylammonium bromide as sole carbon source. The xanthomonad metabolized this quaternary ammonium compound in the presence of other organic molecules. The products of this activity included 9carboxynonyl- and 7-carboxyheptyltrimethylammonium, suggesting that the terminal carbon of the decyl moiety is oxidized and the resulting carboxylic acid is subject to 8-oxidation.
Quaternary ammonium compounds are widely used in industry. Some are antifoam and antistatic agents and have surfactant properties. Among this class of chemicals are potent bactericides, and several of these quaternary ammonium compounds thus are included in antiseptic solutions, clinical preparations, and cosmetics. Almost half of the quaternary ammonium compounds used commercially are added to laundry preparations as water soften-
0.6 mg; ZnSO4 and MnSO4, 0.2 mg each. The solution was added to standard biological oxygen demand (BOD) bottles, and quaternaries were introduced to give a concentration of 10, 25, or 100 ,ug/ml. To measure the BOD, 10 ml of a 10% aqueous suspension of Williamson silt loam or 3.0 ml of a 10% solution of municipal sewage passed through Whatman no. 1 filter paper was used as inoculum. All treatments were done in duplicate. The rate of 02 depletion in the bottles was measured by means of a biological 02 monitor (model 53, Yellow Springs Instrument Co., Yellow Springs, Ohio). To isolate the ers (7). The biodegradation of members of this group active organisms, enrichments were prepared from BOD bottles exhibiting activity, the enrichments of substances has been the subject of study. For containing 100 to 500 ,mg of the quaternary per ml. that and Isaac reported (1) example, Barden The cultures were purified by streaking portions of cetylpyridinium bromide at low concentrations the enrichments on nutrient agar, and subsequently was completely oxidized by microorganisms the isolates were inoculated into basal salts medium from activated sludge, although somewhat containing the test compound to confirm the ability higher levels were toxic, and laurylpyridinium of the organism to use the chemical. Cultures were chloride at an initial level of 15 ,ug/ml was identified by the procedures outlined by Skerman found to be degraded by Pseudomonas picto- (8). To test the ability of the isolates to use quaternarrum (4). In addition, Desbordes and Jourdan (3) and related compounds, standard manometric ies and chloride cetylthat benzalkonium reported used (9). The bacteria were grown techniques trimethylammonium bromide were attacked by in 1 liter ofwere the basal salts solution containing the the other On hand, Pseudomonas. of species test substrate at a concentration of 0.05%. The culWinter (11) found no degradation of cetyltri- ture was incubated on a shaker for 3 days at 30°C, methylammonium bromide at a concentration and the cells were collected by centrifugation at 10,000 x g for 10 min, washed with 0.10 M phosphate of 10 ,ug/ml. The mechanism of bacterial degradation of buffer (pH 7.0), and suspended in fresh buffer. The quaternary ammonium compounds has yet to Warburg vessels contained 1.0 Amol of test sub7.0), and be defined, however. The present study was strate, 300 umol of phosphate bufferof(pH 60 ,ug. 40 to content a to cells protein equivalent designed to provide information on how these Oxygen utilization was measured until the rate widely used chemicals are metabolized by mi- uptake approached that of the endogenous control.of croorganisms. All treatments were performed in duplicate. To obtain products of metabolism, the bacteria MATERIALS AND METHODS were grown on a shaker at 30°C in 1.0 liter of a The basal salts solution contained, per liter: medium containing 0.2% yeast extract, 0.2% CasaKH2PO4, 5.5 g; Na2HPO4, 10 g; (NH4)2SO4, 1.0 g; mino Acids, and 0.05% decyltrimethylammonium MgSO4*7H20, 0.1 g; CaCl2 H20, 15 mg; Fe2 (SO4)3, bromide (DTM). After 2 to 3 days of growth, the cells were collected by centrifugation at 8,000 x g and I Present address: Ivorydale Technical Center, Proctor washed in 0.1 M phosphate buffer (pH 7.0). They were then incubated in a solution containing 0.05% and Gamble Co., Cincinnati, OH 45217. 1037 -
1038
APPL. ENVIRON. MICROBIOL.
DEAN-RAYMOND AND ALEXANDER
DTM in the phosphate buffer. After 24 or 48 h, the cells were removed by centrifugation, and the supernatant fluid was extracted with an equal volume of methylene chloride. The solvent was treated with HCl gas (prepared by dropping concentrated H2SO4 onto NaCl) until saturated, and it was then concentrated to about 2 ml in a Kuderna-Danish evaporator kept at 70°C. These extracts were analyzed by
chromatography. Analysis of the quaternary ammonium compounds was performed by the colorimetric procedure of van Steveninck and Maas (10). Bacterial growth was measured by either determination of optical density at 500 nm or determination of cell protein by the method of Lowry et al. (6). A Varian gas chromatograph, model 1700 (Varian Aerograph, Walnut Creek, Calif.), equipped with a flame ionization detector was used. The chromatograph was fitted with a stainless-steel column (1.8 by 3 mm) packed with 5% FFAP on Chromosorb W (80/100 mesh, acid washed). The flow rate of the carrier gas, N., was 20 ml/min. The temperatures of the detector and injector were 250 and 230°C, respectively. The column was programmed from 85 to 200°C at a rate of 4°C/min. Combined gas chromatography-mass spectrometry was performed with a Finnigan gas chromatograph-mass spectrometer, model 3300, equipped with a Systems Industries System 150 data processing system. The quaternaries were obtained from Eastman Kodak Co., Rochester, N.Y., and Pfaltz and Bauer, Flusing, N.Y. Amines were obtained from Pfaltz and Bauer and Lachat Chemicals, Mequon, Wis. Gas chromatographic packing materials were supplied by Applied Science Laboratories, State College, Pa., and Rohm and Haas, Philadelphia, Pa., supplied samples of Hyamine 10-X, a commercial quaternary preparation. gas
RESULTS
BOD. Using standard BOD techniques, the biodegradability of 10 quaternary ammonium compounds was tested at three concentrations. Oxygen measurements were made at 5- to 8-day intervals for a period of 60 days. Benzyltrimethylammonium chloride, phenyltrimethylammonium bromide, benzyltriethylammonium
bromide, didodecyldimethylammonium bromide, dioctadecyldimethylammonium bromide, hexadecyldimethylbenzylammonium chloride, phenyldimethylbenzylammonium chloride, and Hyamine 10-X were found to be resistant to attack by microorganisms in soil and sewage inocula. However, microorganisms derived from both soil and sewage metabolized DTM and hexadecyltrimethylammonium bromide (Table 1). DTM was extensively degraded within 8 to 10 days at all three concentrations tested. The low values for 02 consumption at 25 and 10 gg/ml with the soil inoculum resulted from the fact that the contents of the bottles were removed as an inoculum for enrichment cultures before complete 02 consumption was attained. Biodegradation of hexadecyltrimethylammonium bromide was evident at levels of 10 and 25 ,ug/ml, although the time to achieve detectable 02 uptake was longer than with DTM. The lack of activity on the hexadecyl compound at levels of 100 ,ug/ml probably is attributable to its toxicity. Characterization of active bacterium. DTM was chosen for further study because sewage and soil organisms used it rapidly at levels of 100 ,g/ml. After repeated serial transfer of the DTM enrichment culture at successively higher levels of the test compound, a mixed culture was obtained that was capable of growth at 500 ,ug/ml. This mixed culture, when plated on nutrient agar, showed the presence of at least five distinct colony types. The mixture was plated on 0.05% DTM-basal salts agar as well as nutrient agar, and individual colonies were selected. Isolates from nutrient agar and DTM-basal salts agar failed to grow, however, when inoculated into 0.05% DTM-basal salts broth. The enrichment culture was then diluted, and the dilutions were inoculated into DTMsalts medium and incubated for 10 days. The highest dilutions to give visible turbidity were plated on nutrient agar, but these dilutions
TABLE 1. Biodegradation of quaternary ammonium compounds by sewage and soil microorganisms Soil inoculum
Quaternary ammonium compound
DTM
Hexadecyltrimethylammonium bromide a
Sewage inoculum
Initial concn
(Ag/ml)
Days Das
02 consumeda
(jLg/ml)
Days Das 10 10 10
6.15 6.23 6.15
60 17
0.0 5.67
17
5.75
100
8
5.99
25 10
8
2.27 1.05
8
100 60 0.0 25 43 3.72 10 15 3.72 Corrected for 02 consumption in the absence of the quaternary.
consumeda
(Ag/ml)
VOL. 33, 1977
QUATERNARY AMMONIUM COMPOUND BIODEGRADATION
1039
were found to contain three organisms distin- Xanthomonas sp. did not grow or use DTM in guishable by colonial morphology. None of the unsupplemented medium or in solutions these organisms grew when inoculated individ- with either yeast extract or Casamino Acids, ually into DTM-basal salts broth. However, there was a visible increase in turbidity and when two of the organisms were inoculated about 70% of the DTM was metabolized when together into DTM-basal salts medium, growth this organism was provided with both yeast and metabolism of DTM occurred (Fig. 1). One extract and Casamino Acids. These observaof these organisms produces white and the tions suggest that Xanthomonas sp. was using other produces yellow colonies. Both bacterial a component of the yeast extract-Casamino types are gram-negative rods, possess polar fla- Acids mixture while acting on the DTM. gella, and give a positive Kovac oxidase test. To test further the metabolism of DTM, each Neither produces a fluorescent pigment. Conse- of the two bacteria was inoculated separately quently, they were identified as strains of Pseu- into a medium containing 0.05% DTM, 0.01% domonas and Xanthomonas. The third orga- yeast extract, 0.01% Casamino Acids, inorganic nism found in the dilutions was discarded be- salts, and an additional organic substrate in 1 cause it failed to grow in DTM-salts broth alone liter of water. For Pseudomonas sp., the addior in combination with either of the other two tional substrate was glucose (0.2%) or trimeorganisms. thylamine (0.2%). For Xanthomonas sp., the To determine the role of each of the bacterial additional nutrient was glucose (0.2%) or yeast populations in the mixture, the two axenic cul- extract plus Casamino Acids (0.2% of each). tures were separately inoculated into four me- The organisms were incubated on a shaker at dia, each of which contained the basal salts and 30°C. The pseudomonad grew well in media 0.05% DTM. One contained no growth factors, a with either glucose or trimethylamine but second contained 0.01% yeast extract, a third failed to metaboze DTM. The xanthomonad was supplemented with 0.01% Casamino Acids, multiplied in the media containing glucose but and the fourth contained both yeast extract and also did not metabolize DTM; however, when Casamino Acids (0.01% of each). The organisms grown in media supplemented with both yeast were incubated on a shaker at 30°C, and growth extract and Casamino Acids, it used all of the and metabolism of DTM were determined after intact quaternary compound provided. Some a 2-week period. Pseudomonas sp. did not grow growth occurred in the first 2 days, but then no or use DTM in any of the media. Although further increase in turbidity was evident (Fig. 2). Growth commenced again. after a few days and proceeded until day 8. DTM utilization began after day 1, leveled off from 2 to 4 days, and then proceeded readily again until the DTM was lost from the medium at day 8. Oxygen uptake. The extent of utilization of DTM was determined by manometric assessment of O., uptake. Cells from two member cultures grown on 0.05% DTM were incubated with DTM and compounds thought to be possible intermediates in its degradation. DTM was destroyed totally, 19.0 ,umol of O. being consumed per Amol of substrate (Fig. 3); the theoretical value is 18.5. Trimethylamine was also almost completely degraded. Decanoic acid and methyldecylamine were metabolized without a lag, but only about half of the theoretical quantity of 02 was consumed. Decane was not used. Since Xanthomonas sp. was capable of degrading DTM by itself in the presence of growth factors, it was tested in a similar experiment. The culture was grown in 1 liter of a medium containing 0.2% yeast extract, 0.2% Casamino Acids, and 0.05% DTM. After 2 days on a shaker at 30°C, the cells were collected by cenFIG. 1. Growth of a mixture of two bacterial popu- trifugation, washed three times in 0.10 M phoslations and DTM disappearance in solutions con- phate buffer (pH 7.0), and suspended in fresh buffer. DTM was used without a lag, but the 02 taining 0.05% DTM. HOURS
1040
DEAN-RAYMOND AND ALEXANDER
7E 2
a0. =k
FIG. 2. Bacterial development and DTM metabolism in a medium containing yeast extract and Casamino Acids.
IU)
o
U)
cz E
0
0
E
HOURS
FIG. 3. Oxidation of various substrates by resting cells derived from a two-member culture.
consumption was only about 60% of the theoretical value (Fig. 4). Decanoic acid was also used without a lag by the xanthomonad and to about the same extent as the two-member culture,
APPL. ENVIRON. MICROBIOL.
and trimethylamine was not metabolized. Methyldecylamine and dimethyldecylamine inhibited endogenous respiration. Products of DTM metabolism. Because DTM was only partially metabolized by Xanthomonas sp., products must have accumulated in the reaction mixture. The products were extracted from a resting-cell suspension incubated with DTM in phosphate buffer. Prominent peaks appearing in gas chromatograms of the extract were selected for identification by combined gas chromatography-mass spectrometry. Electron impact mass spectra revealed that two of the components had similar spectra, showing prominent peaks at 45, 60, 73, 87, and 129 mass units, a fragmentation pattern characteristic of aliphatic acids. The peaks of highest mass number in the two spectra were 157 and 185, respectively. Although these peaks had the highest masses in the spectrum, they showed none of the other characteristics of molecular ion peaks. Chemical ionization spectra of the two gas chromatographic peaks were then obtained These spectra indicated that the molecular weights of the two compounds were 202 and 230, respectively. The molecular weights and fragmentation patterns are consistent with the structure being 7-carboxyheptyltrimethylammonium and 9-carboxynonyltrimethylammonium, respectively. These observations suggest that the bacterium oxidized the terminal carbon of the long alkyl chain of the quaternary and then cleaved acetyl units by 3-oxidation. Use of the data retrieval system with the chemical ionization spectra of the sample revealed the presence of compounds having molecular weights of 174, 146, and 118 mass units. These compounds were present in much smaller quantities than the other two. The products may be lower-molecular-weight members of a homologous series of carboxylic acids derived from the quaternary. DISCUSSION The results of BOD tests confirm earlier data that at least certain quaternary ammonium compounds are subject to biodegradation. In some instances, the failure to observe biodegradation may result from toxicity at the levels used. Thus, hexadecyltrimethylammonium bromide was only oxidized at the two lower concentrations. The toxicity of such compounds increases with increasing chain length of the alkyl moiety (2, 5). No isolate capable of growing on DTM as sole source of carbon was obtained, but a mixture of
VOL. 33, 1977
QUATERNARY AMMONIUM COMPOUND BIODEGRADATION
1041
and carboxymethyltrimethylammonium compounds. The substances with lower molecular weights were present in small quantities, perhaps because the rate of ,8-oxidation was affected by chain length. These acids and/or acetic acid might be released into the medium by the xanthomonad and thus be available to the pseudomonad as substrates for growth. When growing together on DTM, the two organisms may then act by protocooperation, the pseudomonad providing growth factors to its associate and the xanthomonad converting DTM to compounds used for energy by its part-
I-
co
ner.
z 0
E
ACKNOWLEDGMENTS The investigation was supported in part by a Public Health Service fellowship (5F22 ES01231) from the National Institute of Environmental Health Sciences. We thank T. Wachs and P. C. Wszolek for assistance in interpretation of the mass spectra.
HOURS
FIG. 4. Utilization of several compounds by DTMinduced resting cells of Xanthomonas sp. was able to proliferate on DTM without the addition of growth factors. Only one of the organisms, a strain of Xanthomonas, acted on DTM. Because this organism could not grow on DTM alone, it is probable that the second bacterium supplies one or more growth factors to the xanthomonad. Furthermore, Xanthomonas sp. cannot degrade DTM completely, and the products of partial degradation would be available to support replication of Pseudomonas sp. so that the two organisms together would be able to grow on DTM. The isolation of 9-carboxynonyl- and 7-carboxyheptyltrimethylammonium compounds suggests that the xanthomonad oxidized the terminal carbon of the longer alkyl chain and then cleaved acetyl units by 83-oxidation. Furthermore, the finding of products not only of 230 and 202 but also of 174, 146, and 118 mass units indicates the further operation of 13-oxidation since the difference between these molecules is 28 mass units, equivalent to CH2CH2. Thus, the sequence might then involve the sequential formation of the 9-carboxynonyl-, 7-carboxyheptyl-, 5-carboxypentyl-, 3-carboxypropyl-,
two bacteria
LITERATURE CITED 1. Barden, L., and P. C. G. Isaac. 1957. The effect of synthetic detergents on the biological stabilization of sewage. Proc. Inst. Civil Eng. 6:371-405. 2. Chuillon, J., and B. Baleux. 1968. Antibacterial action of certain quaternary ammonium compounds, p. 259268. In C. R. Congr. Int. Deterg., 5th, vol. 3. 3. Desbordes, J., and R. Jourdan. 1974. Influence of bacterial enzymes on molecular degradation of antiseptics utilized as preservatives in medical preparations. Dev. Biol. Stand. 24:77-90. 4. Janota-Bassalik, L., C. Olczyk, and M. Kaczorowska. 1969. Degradation of some cationic detergents by Pseudomonas pictorum. Acta Microbiol. Pol. Ser. B 1:31-34. 5. Korai, H., and K. Takeichi. 1970. Antimicrobial activity of quaternary ammonium compounds. (In Japanese) Hakko Kogaku Zasshi 48:635-640. 6. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. May, A., and A. Neufahrt. 1976. Ecological behaviour of cationic surfactants. Third report. The behaviour of distearyl dimethyl ammonium chloride in activated sludge units. Tenside 13:65-69. 8. Skerman, V. B. D. 1967. A guide to the identification of the genera of bacteria. The Williams & Wilkins Co., Baltimore. 9. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1972.
Manometric and biochemical techniques. Burgess Publishing Co., Minneapolis. 10. van Steveninck, J., and M. Maas. 1965. A colorimetric assay method for microquantities of quaternary ammonium detergents. Rec. Trav. Chim. Pays-Bas 84:1166-1168. 11. Winter, W. 1962. Der biochemische Abbau von Detergentien bei der Abwasserreinigung. Wasserwirtsch. Wassertech. 12:265-271.
