(8) Berge, H., in “Phytotoxische Immissionen”, Paul Parey Verlag,
Berlin. 1963. (9) Thomas, M. D., Annu. Reu. Plant Physiol., 2,293-322 (1951). (10) Janone, G., Humus, 10,17-9 (1954). (11) Skelly, J. M., Moore, L. D., Stone, L. L., Plant Dis. Rep., 56,3-6 (1972). (12) Taylor. 0. C., Adu. Chem. Ser., No. 122 (1973). (13) Darley, E. F., Air Pollut., Proc. Eur. Congr., lst, 137-42 (1968).
(14) Code of Federal Regulations 40, Protection of Environment,
131-2, US. Government Printing Office, Washington, D.C., 1972. (15) Taylor, 0. C., Maclean, D. C., in “Recogqition of Air Pollution Injury to Vegetation: A Pictorial Atlas”, Jacobson, J. S., Hill, A. C., Eds., Air Pollution Control Association, Pittsburg, Pa., 1970.
Received for review July 28,1978. Accepted February 21,1979.
Microbial Degradation of Organic Compounds at Trace Levels Robert S. Boethling‘ and Martin Alexander* Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, N.Y. 14853
A sensitive method was developed for measuring the biodegradation of organic chemicals based on the formation of “TO2 from I4C-labeled compounds. It was shown that glucose at an initial concentration of 18 ng/L was degraded by microorganisms in culture a t rates well below those predicted by Michaelis-Menten kinetics from the degradation rates at higher glucose levels. T h e rate of glucose degradation a t 18 ng/L was affected by the density of the bacterial population. T h e maximum rate of biodegradation of dimethylamine, diethylamine, and diethanolamine added t o stream water was proportional to the initial amine concentration over a range of concentrations from several nanograms t o several milligrams per liter. In recent years, the production and use of synthetic organic chemicals have risen dramatically, and concomitantly the discharge of these chemicals into diverse ecosystems, both aquatic and terrestrial, has increased. T h e number and the identities of many of the substrates introduced into natural waters and soils are unknown, but knowledge of the activities o f society and environmental monitoring ( I ) suggest that the number and diversity of organic pollutants are large. Unfortunately, few generalizations exist to explain why some of these chemicals are biodegradable and disappear rapidly, whereas others are not subject to rapid microbial degradation or are not attacked at all. Concentration is one factor t h a t may govern the susceptibility of organic chemicals to microbial destruction in nature. Surprisingly, only a few studies on the effect of a wide range of concentrations on the persistence of organic chemicals have been reported ( 2 , 3). One reason for this scarcity of experimental data may be the lack of sensitivity of many techniques commonly used for the detection and quantification of organic compounds a t trace levels. The present study was designed t o assess the effect of a wide range of concentrations of biodegradable chemicals on the rate of their mineralization, Le., complete conversion to inorganic products. A sensitive method involving lT-labeled compounds was used for this purpose.
Materials and Methods Pseudomonas sp. was obtained from a n enrichment containing glucose, glycerol, and succinate as carbon sources ( 4 ) . Surface water samples were taken aseptically from Fall Creek in central New York and processed within 2 h of collection. All samples were analyzed for p H and total alkalinity by standard methods ( 5 ) .T h e water was amended with a n inorPres;;
address, Biocentrics, 480 Democrat Rd.. Gibbstown. N J .
08027. 0013-936X/79/0913-0989$01.00/0
ganic nutrient supplement (6)modified as follows: NaNO3 and NaHC0:j were omitted, Zn was added a t the same molarity as ZnSOCH20 rather than ZnClp, and NH4Cl was added a t 8.0 mg/L. Microbial populations were enumerated by plating appropriate dilutions in triplicate on nutrient agar containing 20 m M D-glUCOSe. T h e stream water contained 8-100 X lo3 bacterial cells/mL at the time of sampling. Microbial activity was monitored by measurement of I4CO2 produced from l4C-1abeled organic chemicals. AxeniC cultures of bacteria and stream water were incubated in 500-mL flasks that were modified to permit flushing with Nz subsequent to t h e incubation period. After specified periods of time, the contents of the flasks were acidified by adding 12 N HzS04 t o a final concentration of 0.12 N, the flasks were connected to a source of N2,and the COz in t h e flasks was flushed out and trapped in ethanolamine. If necessary, NaHC03 was added immediately before acidification to raise the concentration of bicarbonate plus carbonate t o at least 1.0 mM. T h e experimental flasks contained 100 or 200 mL of medium and were incubated a t 29 “C in the dark t o minimize photodecomposition of added organic chemicals and fixation by photosynthetic microorganisms of the I4CO2released from the degradation of the added 14C-labeled compound. Axenic cultures were incubated on a rotary shaker operating a t 140 rpm, but flasks containing stream water were incubated without shaking. Measurements of dissolved oxygen ( 5 ) showed t h a t the medium remained aerobic. Samples (1.0 g) of ethanolamine containing trapped 14C02were added to 9 m L of scintillation fluid ( 7 ) ,and the radioactivity was measured in a Beckman LS-1OOC liquid scintillation spectrometer (Beckman Instruments, Fullerton, Calif.). T h e data were corrected for quench by the external standard/channels ratio method and expressed as disintegrations per minute (dpm). T h e I4C-labeled chemicals used were not sufficiently volatile to be trapped in ethanolamine after acidification of the medium, they were not detectably sorbed at microgramhiter levels t o particulate matter in the media or to the incubation vessels, and they were not converted to 14C02 or other volatile products in autoclaved media. Pseudomonas sp. was grown in a mineral salts medium amended with 5 m M D-glucose to prepare inocula for biodegradation experiments or with the desired initial level of glucose in experiments assessing the biodegradability of low concentrations of this substrate. Growth of the cultures used as inocula was monitored spectrophotometrically a t 540 nm, and the cells were collected on 0.45-pm membrane filters when the cultures reached the late logarithmic phase of growth. The cells were washed with basal medium, resuspended in fresh medium without added glucose, and transferred to flasks
@ 1979 American Chemical Society
Volume 13, Number 8, August 1979
989
1.8 pg/L ( X I 0 1
Figure 2. Formation of C 0 2 from diethanolamine added to stream water at three initial concentrations
nolamine. The results of triplicate analyses showed that more than 95% of the initial I4C-labeled material was recovered as
CELLS/ML
1 4 ~ 0 ~ .
18ng/L 1 x 1 0 ~ 1 0
I
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3
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5
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Figure 1. Growth of Pseudomonas sp. and formation of C 0 2 from glucose. Upper panel: cell count. Lower panel: C02 formation. An inoculum of about 1.5 X IO4 cells/ml was used except where noted. The values for C o n formation at initial glucose levels of 18 and 1.8 pg/L are the averages of three determinations
containing basal medium and various concentrations of glucose. All media and glassware were brought to 29 "C, and the entire process from the collection of cells to the initiation of biodegradation experiments was carried out in 15 min or less. An inoculum of about 1.5 X lo4 cells/mL was used except where noted. Direct measurements of carbohydrate in the supernatant fluid immediately before collection of the cells were performed by the hexokinase, anthrone, and ferricyanide methods (8, 9). The results of measurements by these three procedures and of tests of the efficiency of removal of solutes by the filtration and washing process indicated that this potential contribution to the initial glucose level in experimental flasks was 2 ng/L or less. Scintillation grade ethanolamine and unlabeled dimethylamine and diethylamine hydrochlorides were obtained from Eastman Organic Chemicals (Rochester, N.Y.). Unlabeled diethanolamine was obtained from Fluka AG (Buchs, Switzerland). The sources and specific activities of the radiochemicals used were as follows: di[l4C]methylarnine-HC1 (Amersham, Arlington Heights, Ill.), 54 mCi/mmol; di[l'Tlethylamine (Amersham), 28 mCi/mmol; di[14C]ethanolamine (New England Nuclear, Boston, Mass.), 20.8 mCi/ mmol; [U-14C]glucose (New England Nuclear), 340 mCi/ mmol; and NaH14C03 (New England Nuclear), 59 mCi/ mmol.
Results T o measure the efficiency of' the COz-collection system, flasks containing either 100 or 200 mL of distilled water were amended with 3.0 yg/L of Hl4CO:3- and incubated in the dark a t 29 "C for 2 days. The 14C-labeledvolatile material was then collected in traps containing either 2.0 or 10.0 mL of etha990
Environmental Science & Technology
The method was first applied to study the effect of low substrate concentration on the biodegradation of organic chemicals by Pseudomonas sp. The results in Figure 1 are expressed both as the percent of the initial l4C-labeled sugar recovered as 14C02and as the amount of COz formed per liter. T o assess the relative rates of activity based on the amount of COz evolved, the slopes of the lines in Figure 1 should be compared after taking into account the division factors given in parentheses, these factors being included so that all the data could be presented in a single figure. The results thus show t h a t the rate of degradation of glucose at an initial concentration of 18 ng/L was much lower than the rates a t higher concentrations, and little degradation at this level occurred in the time of study. The effect of low substrate concentration was not the result of a net loss in viability because the results presented demonstrate an increase in population size. The data indicate that the bacteria grew rapidly in the basal medium even in the absence of added glucose to a population of more than 105 cells/mL and continued to grow more slowly for a t least 5 days. In the presence of glucose a t an initial level of 18 pg/L, the bacterial population was never more than threefold higher than in the absence of glucose. Glucose was degraded by Pseudomonas sp. at initial levels as low as 18 ng/L if the size of the inoculum was about 1.5 X lo5rather than 1.5 X lo4 cells/mL, however, and the rate of decomposition was more than tenfold greater for the larger inoculum incubated with the same substrate level. Control experiments indicated that the percent of the initial 14C-labeled sugar apparently assimilated by the cells was always approximately equal to the percent recovered as I4CO2. The effect of low substrate concentration on the biodegradation of organic chemicals by natural communities was examined by adding individual compounds to stream water. Glucose was rapidly metabolized at initial levels from 1.8 ng/L to 18pg/L, and 50-60% of the starting material was converted to C o n in 20 h. The production of 14CO2 from dimethylamine, diethylamine, and diethanolamine also was measured a t various initial concentrations. Secondary amines were studied because they have been shown to be precursors for the formation of carcinogenic nitrosamines in model ecosystems (10). The production of 'TO2 from di[14C]ethanolamineadded to stream water is shown in Figure 2, the data being expressed both as the percent of the initial 14C recovered as " T O 2 and as nanomoles of COJ formed per liter. At the lowest initial
9
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-
0 2
5-
3
r
s o
s
3-
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IO
4
4 6 LOG INITIAL AMINE CONCENTRATION l n q / ~ l
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Figure 3. Effect of initial concentration of diethanolamine, diethylamine, and dimethylamine added to stream water on the rate of degradation of these secondary amines
concentration (21 ng/L), degradation continued a t an approximately constant rate for a t least 4 days; at this time, only about 30% of the original compound had been converted to COZ. In contrast, a t an initial level of 210 pg/L, an accelerating curve for degradation of diethanolamine was obtained, and more than 50% was converted to CO2 in 4 days. At 2 1 mg/L, less than 5% of the original chemical appeared as C 0 2 in the period of the study. The results obtained with dimethylamine, diethylamine, and diethanolamine are summarized in Figure 3. T h e logarithm of the maximum rate of C 0 2 formation from each compound is plotted against the logarithm of the initial substrate concentration in order to present all the rates in a single figure. T h e data show that the maximum rate of CO2 formation was strongly influenced by the initial concentration of the parent compound. Thus, a decrease of approximately one order of magnitude in the maximum rate of degradation was observed for each successive decrease of one order of magnitude in the initial amine concentration.
Discussion In the method used, the recovery of respired l4C0:, was essentially quantitative over a wide range of initial concentrations of tKe labeled chemical. T h e procedure used for assay of biodegtadation did not distinguish between conversion of the test chemical to 14C02 and to other volatile metabolites t h a t are trapped in ethanolamine, and the absence of "TO2 formation d'id not preclude the possibility of cometabolic alteration of the parent compound. High affinity transpok-t systems for simple organic molecules are common in bacteria (11).Nevertheless, the rates of degradation of glucose a t the lowest initial concentration by Pseudomonas sp. were far below those predicted by Michaelis-Menten kinetics. Related effects of low substrate concentration have been reported by Jannasch (2) and by Shehata and Marr ( 3 ) ,who demonstrated that stable, axenic microbial populations could not be maintained in continuous culture a t low concentrations of simple organic molecules. Where an organic substrate is present a t levels too low to permit a significant increase in the population of active degraders, the rate of decomposition should depend in part upon the initial density of this population. Thus, with Pseudomonas sp. incubated with 18 ng of glucose/L, the very low rate of degradation with an inoculum of 1.5 X IO4 cells/mL was increased more than tenfold if the inoculum was tenfold larger,
and the rates of degradation were approximately constant for a t least 6 days with both inoculum levels. Therefore, the density of the active population may be an important factor governing the rate of decomposition a t low substrate concentration. The kinetics of I4CO2 production from di[14C]ethanolamine added to stream water support this argument because the rate of I4CO2 formation a t - a n initial diethanolamine level of 21 ng/L was approximately constant in'tkie period of study, wherea$ the rate a t 210 pg/L increased exponentially. The maximum rate of biodegradation of the three amines in stream water was proportional to their initial concentration over a wide concentration range. These data suggest that caution should be exercised in extrapolating from laboratory experiments to natural waters, in which concentrations of organic chemicals are usually much lower than those used in laboratory'assessments of biodegradation rates. Factors in addition to substrate concentration may influence the biodegradation of organic chemicals in natural waters. The role of solid surfaces in effecting a concentration of bacteria and/or nutrients is well documented (12, 13) but poorly understood. The presence of other utilizable carbon sources may lower the threshold concentration for bacterial breakdown of an individual carbon source (14). Moreover, a natural selection may occur in aquatic environments to favor bacterial species able to grow a t the prevailing low substrate concentration (15). Nevertheless, it is possible that the persistence of some chemicals even at levels as low as a few microgramsfliter might cause serious environmental problems following bioaccumulation and subsequent toxicity to species a t higher levels in food chains. The present findings have direct relevancy to laboratory tests designed to assess biodegradability in natural ecosystems because these tests are usually performed with chemicals in concentrations of several milligrams per liter, and it is clear from the data given here that the rates of chemical loss so obtained are far higher than those occurring a t lower chemical concentrations. This is evident from the results of the studies of the effect of concentration on the biodegradation of secondary amines in stream water, the compounds thus being able to persist for longer periods than would be expected from tests done with high amine levels.
Literature Cited (1) .Junqclaus, G . A., Lopez-Avila, V.,Hites, R. A., Enuiron. Sei. Technol., 12,88-96 (1978). ( 2 ) Jannasch, H. W., Limnol. Oceanogr., 12,264-71 (1967). ( 3 ) Shehata, T. E., Marr, A. G., J . Racteriol., 107, 210-6 (1971). ( 4 ) Cook, A. M., Daughton, C . G., Alexander, M., J . Racterio[., 133, 85-90 (1978). (,5) American Public Health Association, "Standard Methods for the Examination of Water and Wastewater", 14th ed., American Public Health Association, Washington, D.C., 1976. (6) United States Environmental Protection Agency, "Algal Assay Procedure-Bottle Test", United States Environmental Protection Agency, Corvallis, Ore., 1971. ( 7 ) Bray, G. A,, Anal. Riochem., 1,279-85 (1968). ( 8 ) Herbert, D., Phipps, P. J., Strange, R. E., Methods Microbiol., 5B,209-344 (1971). (9) Ashwell, G., Methods Enzyrnol., 3,73-105 (1957). (10) Ayanaha, A,, Alexander, M., J . Entiiron. Qual., 3, 83-9 (1974). ( 1 1 ) Midgley, M., Dawes, E. A,, Hiochem. J . , 132, 141-54 (1973). (12) ,Jannasch, H. W., Pritchard, P. H., Mem. I s f . I t a l . Idrobiol. Dott. Marco de Marchi, Suppl., 29,289-308 (1972). (13) ZoBell, C . E., J . Racteriol., 46, 39-56 (1943). (14) Law, A. T., Button, D. K., J . Racteriol., 129, 115-23 (1977). (15) Keunen, J . G., Boonstra, H. G., Schroeder, H. G., Veldkamp, H., Microb. Ecol., 3, 119-30 (1977). lieceiced for reL'iecc,October 5 , 1978. Accepted April 6 , 1979. This rc.search i i m ,\upported in part by Public Hrnlth Seri)ice Training ( ; r a n t No. ES00098 from the Diuision of Enoironrnental Health Sciences and by National Science Foundation Grant N O ENV7,519797.
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