FINAL REPORT
on
EFFECT OF CONCENTRATION AND SOLUBILITY OF ORGANIC CHEMICALS
ON THEIR BIODEGRADATION
TECHNICAL DIRECTIVE 17
by
MARTIN ALEXANDER, HOWARD E. RUBIN, AND R.V. SUBBA-RAO
SUBCONTRACT NO. T6420(7197)-034
CONTRACT NO 68-01-5043
RONALD G. WILHELM, PROJECT OFFICER
ROBERT BRINK, TASK OFFICER
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
January 26, 1982
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TMs report is divided into three separate sections, each of which is
separately paginated. These sections represent three manuscripts, one of
which has been and two of which will be submitted for publication.
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Rates of Mineralization of Trace Concentrations of Aromatic Compounds
in Samples of Lake Waters and Sewage
HOWARD E. RUBIN, R. V. SUBBA-RAO, AND MARTIN ALEXANDER*
Laboratory of Soil Microbiology, Department of_ Agronomy,
Cornell University, Ithaca, New York 14853
The rates of mineralization of phenol, benzoate, benzylamine, pj-nitropbe-
nol, and di (2-ethylhexyl) phthalate added to lake water at concentrations
ranging from a few picograms to nanograms per milliliter were directly propor-
tional to chemical concentration. The rates were still linear at levels of
less than 1 pg of phenol or p-nitrophenoVml, but it was less than the pre-
dicted value at 1.53 pg of 2,4-dicMorophenoxyacetate (2,4-D)/ml. Mineraliza-
tion of 2,4-D was not detected in samples of lake water containing 200 ng/ml
of the chemical. The slope of a plot of the rate of phenol mineralization in
samples of three lakes as a function of its initial concentration was lower at
levels of 1 to 100 yg/ml than at higher concentrations. In lake water and sew-
age supplemented with less than 60 ng/ml of C-labelled benzoate or phenyl-
acetate, from 95 to 99% of the radioactivity disappeared from solution, indi-
cating that the microflora assimilated little or none of the carbon. The ex-
tent of mineralization of some compounds in samples of two lakes and sewage
was least in the water with the lowest nutrient levels. No mineralization of
2,4-D and. the phthalate ester was observed in samples of an oligotrophic lake.
These data suggest that mineralization of sore chemicals at concentrations of
less than 1 yg/rol is the result of activities of organisms different from those
functioning at higher concentrations or of organisms that metabolize the chem-
icals at low concentrations but .assimilate little or none of the substrate
carbon.
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2
Predicting the rate of decomposition of organic compounds in natural
waters is of considerable importance, especially for toxic chemicals. How-
ever, many of the routine tests used to assess rates of biodegradation in-
volve chemical concentrations far in excess of those likely to be found in
natural waters. Insufficient data exist to allow for extrapolation from the
high levels commonly used in laboratory studies to the far lower levels char-
acteristic of fresh or marine waters.
The rate of mineralization may be directly related to substrate con-
centration at all concentrations below those supporting the maximum rate
(Vmax) for a particular chemical. Conversely,, a threshold level may exist
below which the rate of deconposition is either less than that predicted
from a linear extrapolation of the rates at higher substrate levels or is
zero. Jannasch (5) demonstrated the existence of a threshold concentration
below which glucose decomposition did not take place in culture. Law and
Button (6) later found that the threshold concentration for glucose could be
lowered by the addition of amino acids. A subsequent study suggested that a
threshold level for certain compounds existed in natural waters (3).
This investigation was designed to determine the kinetics of mineraliza-
tion of several compounds in samples of freshwater and sewage. For this pur-
pose, a method was developed to test the rates at chemical concentrations
lower than those that have been studied heretofore:
MMERIALS AND METHODS
[U-14C]phenol (specific activity, 87 inCi/mmo!), 2,4-dichlorophenoxy[2-14C]-
acetic acid (28 nCi/mraol), and [U-C]aniline hydrogen sulfate (98.8 nCi/mmo!)
14
were obtained from Amersham Corp., Arlington Heights, 111. [Carboxyl-. CJ-
benzoic acid (25.6 mCi/mnol) was purchased from. New England Nuclear, Boston,
Mass. [M2tliylene-14C]benzylamine-HCl (6.9 nqi/mnol), phenyl [1-14C]acetic acid
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(1.0 nCi/mmol), and [carboxyl- C] di(2-ethylhexyl) phthalate (2.7 raCi/imol)
were obtained from California Bionuclear Corp., Sun Valley, Calif. pj-Nitro-
• 14
[2,6- Clphenol (26.6 nCL/nnol) was obtained from Tracerlab, Waltham, Mass.
Samples of fresh water were taken from Beebe Lake and Cayuga Lake in
Ithaca, New York and White Lake near Old Forge, N.Y. Ihese lakes are eutro-
phic, mesotrophic, and oligotrophic, respectively. Fresh sewage was from
the waste treatment facility of Ithaca, N.Y.
14
To study mineralization rates, which are taken to be the loss of C from
the test system, freshly sampled lake water was passed through a glass fiber
filter to remove the large particulates. She liquid was then amended with the
C-labelled compound to be tested and incubated without shaking in the dark
at 29 °C. All tests were conducted in triplicate with a single set of analyses
performed on the contents of each flask. The volume of lake water used varied
from 100 ml to 8 1 depending on the concentration and specific activity of the
14
chemical. The C-atnended waters were contained in 250-ml to 12-1 Erlemteyer
or flat bottomed Florence flasks. At regular intervals, 5.0- to 500-mL sam-
ples were removed and acidified with concentrated H-SO. to pH 2. Ihe amount
of radioactivity remaining was measured by liquid scintillation counting,
14
either after bubbling the sample for 60 min with air to remove the CO- or
after solvent extraction of the substrate from the water sample. Details of
the method will be published elsewhere (manuscript in preparation). Because
of its low water solubility, di (2-ethylhe3#l) phthalate was dissolved in acetone
before it was added to the reaction vessel. The acetone was allowed to evapo-
rate before the lake water was added. Abiotic and microbial mechanisms lead-
ing to the loss of each chemical were distinguished by adding the compound to
samples of lake water supplemented with 0.2% NaCN; no losses were detected under
these circumstances. A linear relationship was observed for the rate of disap-
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4
pearance of all chemicals as a function of tine, except for pj-nitrophenol,
for which the relationship was linear only at 605 fg/ml (manuscript in prepa-
ration) ; hence, the rates of disappearance of each chemical at each concentra-
tion were determined from the linear portion of a plot of the amount of chem-
ical that disappeared with time. •
RESULTS
lha effect of chemical concentration on the rates of mineralization of
several compounds added to Beebe Lake water is shown in Fig. 1. Ib present
all the data in a single figure, the rates and concentrations are expressed
on a logarithmic scale. Benzoate was the most rapidly mineralized compound,
and benzylamine and phenol were mineralized somewhat more slowly. The rates
of mineralization were slowest for 2,4-dLchlorophenoxyacetate (2,4-D), p-ni-
trophenol, and di(2-ethylhexyl) phthalate.
Hiese data show that a linear relationship existed between mineralization
rate and chemical concentration for all compounds over part of or the entire
range of concentrations studied. The correlation coefficient for the plot
of the logarithm of rate of carbon loss versus the logarithm of 2,4-D concen-
tration in the range between 7.2 and 500 pg/ml was 0.998. The observed rates
for 1.53 pg of 2,4-D/ml and 4.9 ng of 2,4-D/ml were 43 and 58% of the pre-
dicted rates and were outside the 95% confidence limits for the regression
line. Because no mineralization of 2,4-D occurred at 200 ng of 2,4-D/ml
(shown by the arrow pointing downward in Fig. 1), the finding of a slower
than predicted rate at 4.9 ng 2,4-D/ml is consistent with the apparent tox-
icity. However, the less than predicted rate at 1.5 pg/ml is noteworthy.
A similar statistical analysis indicated that the rate of mineralization
of di (2-ethylhexyl) phthalate at an initial concentration of 20.7 pg/ml was
4 times greater than the predicted value and was outside the 95% confidence
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5
limits for the regression line, which had a correlation coefficient of 0.996.
The data in Fig. 1 show that the rates of phenol and p_-nitrophenol min-
eralization were directly related to concentration from levels less than 1.0
pg/ml to greater than 100 ng/ml; i.e., by 5 or more orders of magnitude. The
rate at 100 yg phenol/ml was much lower than the rate predicted from the linear
plot derived from the lower concentrations; moreover, the plot at the still
higher concentration had a greater slope.
Phenol mineralization was studied further with samples of three differ-
ent natural waters (Fig. 2). Statistical analysis indicated that the three
plots.were different. The rates at every concentration were less in White
Lake water than in Beebe Lake water, except at 10 mg/ml, the highest level
tested. The decline in the slopes of the lines because of the rate at 100
yg/ml and the increase in slope at a concentration greater than 100 yg/ml are
noteworthy. The differences in rates between the two waters seem small on a
logarithmic plot, but the rates in White Lake water were 60% and 56% of those
in Beebe Lake water at 2 yg/ml and 200 fg/ml, respectively. A threshold was
not observed for phenol mineralization, even at 102 fg/ml' in White Lake and
at 191 fg/ml in Beebe Lake water.
Mineralization was tested in samples of aquatic microbial habitats con-
taining dissimilar nutrient levels and communities. The samples were taken
in January. The data are presented as percent of the benzoate and phenyl-
acetate mineralized in 7 days and pj-nitrophenol and 2,4-D mineralized in 28
days. The extent of mineralization of one or both levels of phenylacetate
and pj-nitrophenol was greater in Beebe Lake water and sewage, and 2,4-D was
mineralized in this trial only in sewage (Table 1). Thus, the activity seems
to be related to the nutrient level, and/or possibly the species diversity, of
these environments. In two of the three environmental samples, the percent of
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6
g-nitrophenol mineralized was greater at the higher substrate concentration.
The almost coirplete -disappearance of radioactivity from solutions that initially
were amended with benzoate and phenylacetate indicates that little or none of
the substrate-carbon was assimilated by the tnicroflora. The previous finding
of 2,4-D breakdown in Beebe Lake water was made with samples taken in the spring
The mineralization of pj-nitrophenol in samples of sewage and lake water
is shown in Fig. 3. In several instances, an apparent lag phase was evident
before loss of the chemical was detected. This apparent lag phase was shorter
for comparable chemical concentrations in samples from sewage than from Cayuga
Lake. The reason for the decline in rate followed by a reinitiation of min-
eralization in Beebe Lake water is unknown. At the lower concentration, min-
eralization was slowest in Cayuga Lake water. The amount of substrate min-
eralized at any time period was different among the three environmental sam-
ples, the percent mineralization of the lower concentration increasing as the
nutrient status increased; i.e., in the order of Cayuga Lake, Beebe lake, and
sewage.
*
•
WVen White Lake water was amended with 2,4-D and di (2-ethylhexyl) phtha-
late at various concentrations, no mineralization was detected in 60 days.
In Beebe Lake water, in contrast, mineralization occurred immediately with
the phthalate ester and within 8 days for 2,4-D.
DISCUSSION
The rate of phenol mineralization is a linear function of concentration
at levels below 1 yg/ml, falls off between 1 and 100 yg/rol, and is again high
at levels above 100 pg/ml. These findings may reflect the activity of two
different kinds of organisms, oligotrophs (or oligocarbophiles) active at the
lower concentrations and eutrophs active at the higher concentrations. The
hypotheses that two such types of microorganisms exist is consistent with
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7
studies of axenic cultures. Ihe occurrence of eutrophs is shown by the stu-
dies of Jannasch (5) and Shehata and Marr (7), who found that stable microbial
populations could not be maintained in axenic continuous culture at low levels
of organic substrates, and of Boethling and Alexander (4), who noted that bac-
teria in axenic culture degraded 18 pg glucose/ml at rates far below those
predicted by Michaelis-Menten kinetics. Ihe existence of oligotrophs has been
the subject of considerable recent concern (8), and such organisms may grow
well on carbon sources added to media at concentrations of less than 10 yg
C/ml (1). Tha presence of sensitive oligotrophs in the water samples may
explain the total inhibition of 2,4-D mineralization at 200 ng/ml, a sensi-
tivity previously undescribed for heterotrophs.
Threshold concentrations below which there is little or no decomposition
of organic compounds have been reported for natural waters (3). A threshold
for a given compound may be evident in ecosystems containing eutrophs but not
oligotrophs capable of metabolizing a test chemical. Alternatively, in waters
containing organic compounds in addition to the substrate of interest, a eu-
troph may have a lower threshold for a given compound than might be predicted
from studies involving only a single substrate. Thus, Law and Button (6) re-
ported that the 210 ng/ml threshold for glucose of a marine bacterium was
reduced to less than 10 ng/ml by a mixture of amino acids. Effects of nutri-
ent levels in aquatic habitats on mineralization are suggested by the present
investigation. Nevertheless, it is not clear whether the absence of a detect-
able threshold noted here for most chemicals is a reflection of the existence
of an oligotrophic population or of diminished thresholds associated with
other nutrients. The lower than predicted rate at 1.53 pg of 2,4-D/ml sug-
gests that a threshold exists for this compound at still lower concentrations.
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8
Another possibility is raised by our observation that more than 95% of
the radioactivity in samples of lake water and/or sewage receiving behzoate
and phenylacetate and 93.7% of the radioactivity in sewage amended with 36.6
pg of 2,4-D/ml disappeared, Thus, little or none of the carbon had been as-
similated. It is thus possible that microorganisms carrying out such trans-
formations at these low concentrations have the previously unreported capacity
of mineralizing organic compounds without obtaining carbon, and presumably
energy, -from the substrate. This phenomenon is similar to conetabolism, the
cometabolizing populations deriving neither a nutrient nor energy from the
substrate they metabolize, but it is generally believed that mineralization
is not a consequence of cometabolism (2).
The finding of mineralization of 2,4-D and di (2-ethylhexyl) phthalate
in samples from Beebe Lake but not from White Lake points to a feature of
the environmental metabolism of synthetic chemicals that is often overlooked:
a mineralizable molecule may not be destroyed in a particular ecosystem, par-
ticularly in oligotrophic waters. The lack of decomposition may be attrib-
utable either to the absence of populations having the necessary catabolic
enzymes or to the existence of species that are potentially active but which
require growth factors that are not present in the nutrient-poor water. More-
over/ because 2,4-D decomposition was found in Beebe Lake water sampled at
one season but not another, time of year may determine the occurrence of
mineralizaton by means other than those linked with suppressed activity at
low temperature. The effect of season during which the sample was taken from
freshwaters also has been reported for the metabolism of N-nitrosodiethanol-
amine (9).
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9
ACKNOWLEDGMENT
this project was supported by the Environmental Protection Agency under
cooperative agreement CR806887. The statements do not necessarily reflect
the views and policies of the Environmental Protection Agency.
LITERATURE CITED
1. Akagi, Y., U. Simidu, and N. Taga. 1980. Growth responses of oligotrophic
and heterotrophic marine bacteria in various substrate concentrations,
and taxonomic studies on them. Can. J. Microbiol. 26:800-806.
2. Alexander, M. 1981. Biodegradation of chemicals of environmental concern.
Science 211:132-138.
3. Boethling, R. S., and M. Alexander. 1979. Effect of concentration of or-
ganic chemicals on their biodegradation by natural microbial communi-
ties. Appl. Environ. MLcrobiol. 37:1211-1216.
4. Boethling, R. S., and M. Alexander. 1979. Microbial degradation of organic
compounds at trace levels. Environ. ScL. Technol. 13:989-991.
5. Jannasch, H. W. 1967. Growth of marine bacteria at limiting concentrations
of organic carbon in seawater. Liitnol. Ooeanogr. 12:264-271.
6. Law, A. T., and D. K. Button. 1977. Multiple-carbon-source-limited
growth kinetics of a marine coryneform bacterium. J. Bacteriol.
129:115-123.
7. Shehata, T. E., and A. G. Marr. 1971. Effect of nutrient concentration on
the growth of Escherichia ooli. J. Bacteriol. 107:210-216.
8. Shilo, M. (ed.). 1979. Strategies of microbial life in extreme environ-
ments. Verlag Chemie, Weinheim, West Germany.
9. Yordy, J. R., and M. Alexander. 1980. Microbial metabolism of N-nitroso-
diethanolamine in lake water and sewage. Appl. Environ. Microbiol.
39:559-565.
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TABLE 1. Mineralization of aromatic compounds in
Test chemical
Benzoate
Phenylacetate
g-Nitrophenol
2,4-D
lake
Initial
concn
(pg/ml)
59
59,000
19
657
14.4
934
36.6
48,900
waters and sewage
Cayuga
Lake
94.5
98.8
77.8
70.9
9.5
58.4
0
0
% Mineralized
Beebe
Lake
96.3
98.6
98.3
96.3
27.9
56.3
0
0
Sewage
99.4
99.5
98.3
95.5
62.3
57.6
93.7
91.2
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. LEGENDS
FIG. 1. Rates of mineralization of_ several conpounds added a_t different
concentrations to Beebe-Lake water. DEHP is_ di (2-ethylhexyl) phthalate.
FIG. 2. Rate of mineraiization of_ several concentrations of phenol in
waters from three lakes.
FIG. 3. Mineralization of p-nitrophenol in sanples of sewage and lake
water.
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I mg
CHEMICAL CONCENTRATION (PER ML)
Figure 1
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a:
UJ
a
s
cc
Ul
K
O
<
isl
BEEBE LAKI
LAKE
LCAYUGA LAKE
Ing I pg Img
CHEMICAL CONCENTRATION'(PER ML)
Figure 2
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Figure 3
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Kinetics and Extent of Mineralization of Organic Chemicals
at Trace Levels in Freshwaters and Sewage
R. V. SUBBA-RAO, HOWARD E. RUBIN, AND MARTIN ALEXANDER*
Laboratory of_ Soil Microbiology, Department of Agronomy,
Cornell University, Ithaca, New York 14853
A sensitive and rapid method was developed to measure the mineralization
14
of C-labelled organic compounds at picogram-per-milliliter or lower levels
in samples of natural waters and sewage. From 93 to 98% of benzoate, benzyl-
amine, aniline, phenol, and 2,4-didilorophenoxyacetate (2,4-D) at one or more
concentrations below 300 ng/ml was mineralized in samples of lake waters and
sewage, indicating little or no incorporation of carbon into microbial cells.
14
Assimilation of C by the cells mineralizing benzylamine in lake water was
not detected. Mineralization in lake waters was linear with time for aniline
at 5.7 pg to 500 ng/ml, benzylamine at 310 ng/ml, phenol at 102 fg to 10 mg/ml,
2,4-D at 1.5 pg/ml, and di-(2-ethylhexyl) phthalate at 21 pg to 200 ng/ml but
exponential at several p-nitrophenol concentrations. The rate of mineraliza-
tion of 50 and 500 ng of aniline/ml and 200 pg and 2.0 ng/ml of the phthalate
increased with time in lake waters. The phthalate and 2,4-D were mineralized
in samples from a eutrophic but not an oligotrophic lake. Addition to eutro-
phic lake water of a benzoate-utilizing bacterium did not increase the rate
of benzoate mineralization at 34 and 350 pg/ml but did so at 5 and 50 ng/ml.
Glucose and phenol reduced the percent of p-nitrophenol mineralized at p-
nitrophenol concentrations of 200 ng/ml but not at 22.6 pg/ml and inhibited
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the rates of mineralization at both concentrations. These results show that
the kinetics of mineralization, the capacity of the aquatic community to assim-
ilate carbon from the substrate or the extent of assimilation, and the sensi-
tivity of the mineralizing populations to organic compounds are different at
trace levels than at higher concentrations of organic compounds.
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3
Environmental problems may result from the slow mineralization of syn-
thetic organic chemicals introduced into soils and natural waters or from the
complete resistance of such compounds to microbial mineralization. Minerali-
zation refers to the conversion of organic substrates to inorganic products.
\
Cne of the reasons suggested for the lack of degradation of organic molecules
is their low concentrations (3). Concentrations of nutrients below which con-
tinuous cultures of axenic marine bacteria failed to grow have been observed
(6,8). Similarly, levels of 2,4-dichlorophenoxyacetate (2,4-D) and 1-naphthyl-
N-methylcarbamate below which mineralization by microbial coninunities of fresh-
waters did not occur have also been reported (3).
The mineralization of organic compounds at trace concentrations has been
observed to follow Michaelis-Msnten kinetics (3,4). First-order kinetics have
been reported for the degradation of chlorinated or nonchlorinated aromatic
molecules in estuarine river water and sediments (9). However, the microbial
mineralization of low levels of organic compounds was also reported to follow
a second-order rate equation (12). Thus, controversy exists on the type of
kinetic model needed to depict mineralization rates of the microbial communi-
ties of natural ecosystems. In addition to the concentration of the substrate,
the number and diversity of mineralizing microbial species may also play a
major role in the degradation of organic compounds, and the absence of or slow
mineralization of 2,4-D and 1-naphthyl-N-methylcarbamate has been ascribed to
the paucity of energy sources needed for high bacterial numbers (3).
This study was designed to assess the mineralization of low levels of
14
several C-labelled organic compounds in samples of natural waters. Attempts
were also made to enhance microbial activity by supplementing the waters with
readily available carbon and energy sources. Lake waters with different nat-
ural nutrient levels were used to assess the effect of nutrient status on the
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extent of mineralization. To achieve these goals, a sensitive and rapid meth-
14
od to detect mineralization of C-labelled compounds was developed.
MATERIALS AND METHODS
Natural waters. Water samples were collected from Cayuga Lake (Lansing,
N.Y.), Beebe Lake (Ithaca, N.Y.), and White Lake (Old Forge, N.Y.). The pH
values were 6.7 to 7.9, 7.4 to 8.7, and 6.8 and the dissolved organic carbon
contents were 1.3 to 2.7, 2.5 to 5.8, and 3.2 yg/ml for Cayuga, Beebe, and
White Lakes, respectively. The water samples were passed through a glass fi-
ber filter, no. 66085 (Gelman Sciences, Inc., Ann Arbor, Mich.), to remove
particulate matter. Sewage obtained from the settling tanks of the Ithaca,
N.Y. treatment plant was also filtered prior to use. White Lake water was
stored at 10 °C for 12 h, and all other water samples were processed within
2 to 4 h after collection to minimize the multiplication of bacteria during
prolonged storage prior to mineralization studies.
Measurement of mineralization. Glassware was soaked in dichromate-sul-
furic acid for 2 to 3 h and then rinsed several tines with distilled water
that had earlier been passed through Milli-Q Reagent-Grade Water System (Milli-
pore Corp., Bedford, Mass.). Labelled organic compounds with the highest avail-
able specific activity were added at various concentrations to particle-free
lake waters. At the higher substrate concentrations, most of the chemical
was added in the unlabelled form, and the labelled chemical was added to give
200 to 500 dpm/ml. The flasks containing the solutions were stoppered with
foam plugs and incubated in the dark at 29°C without shaking. The volume of
lake water added per flask varied from 100 ml to 8 liter depending on the con-
centration and specific activity of the chemical. The sizes of incubation
vessels were chosen to have at least one to four volumes of air for each
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5
volume of liquid to ensure aerobic conditions. At intervals, 5- to 500-ml
samples were taken from each flask, and the liquid was acidified with con-
centrated H?SO. to pH 2 and processed for measuring the radioactivity remain-
ing in the liquid. Mineralization of insoluble di-(2-ethylhexyl)phthalate
(DEHP) was measured by adding the chemical to the flask in an acetone solution,
and the solvent was evaporated prior to the addition of lake water; three
flasks were removed at intervals and the contents of each flask were adjusted
to pH 2 and extracted as described below.
14
The CO generated by the microbial mineralization of the test compounds
was removed from the solutions by bubbling air through the acidified samples.
The air was first passed through a manifold made of flexible Tygbn tubing and
10-yl sampling pipettes so that 200 samples could be aerated simultaneously.
A preliminary study in which a solution of NalT" CCL was acidified and bubbled
with air for various periods demonstrated that bubbling air for 60 min re-
14
leased >99% of the CO- in the solution, and this time period was thus used.
A 1- to 3-ml portion of the sample was added to 10 to 15 ml of Aqueous Count-
ing Scintillant (Amersham Corp., Arlington Heights, 111.) in 20-ml glass
scintillation vials, and 3- to 10-ml portions of the samples were added to
10 ml of Redi-solve MP (Beckman Instruments, Fullerton, Calif.) for samples
containing < 40 dpm/ml. Ihe radioactivity of the samples was measured for
10 min using a Beckman LS7500 scintillation system (Beckman). This proce-
dure was suitable for samples containing >10 dpm/ml.
Mineralization is expressed as percent of the added radioactivity which
disappeared from the solution, and the values are the averages of analyses
from three flasks. The variation among replicates was less than 5% of the
14
mean. Ihe C remained in solution when each of the test chemicals was in-
cubated in autoclaved or KCN-amended (200 yg/ml) waters under the test condi-
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6
tions, indicating that neither nonbiological mineralization nor sorption to
the walls of the incubation vessels had occurred.
To show that the procedure used for measuring mineralization was a valid
means for assessing the formation of volatile products, 220 pg/ml and 4.0 ng/ml
of both radioactive glucose and phenol were incubated in the dark for 18 h in
Beeba Lake water. At the end of the incubation period, more than 99% of the
radioactivity that disappeared from solution was recovered as labeled products
that were collected in an ethanolamine trap.
Mineralization at picogram concentrations or lower. Twenty to 1000 ml
of the samples acidified with concentrated H-SO. was used when the substrate
concentration was 20 pg/ml to 100 fg/ml, the sample size depending on the spe-
cific activity. For example, a 500-ml sample was initially chosen when the
phenol concentration was 100 fg/ml and the radioactivity was 0.2 dpm/ml. The
entire solution in the reaction flask was extracted for the insoluble substrate,
DEHP. A 1000-ml volume of lake water was incubated at a concentration of 20
pg/ml for DEHP having a low specific activity. The ionic strength of the
acidified solution was increased by the addition of NaCl to a concentration
of 2% prior to extraction of the sample. The solutions were extracted three
times either with ethyl acetate or, for DEHP, benzene in separatory funnels.
The solvent extracts were pooled and concentrated to 10 ml in a rotary flash
evaporator, and the remainder of the label was measured after adding the ex-
tract to 10 ml of Aqueous Counting Scintillant. Extraction efficiencies
greater than 98% were obtained for 2,4-D, p-nitrophenol, and phenol with
standard deviations of less than 3%. An extraction efficiency of 93 ± 2% was
obtained for DEHP. Unlabelled compound (10 yg/ml) was added prior to extrac-
tion of polar compounds at concentrations below 20 pg/ml to improve the ex-
traction efficiencies.
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7
Isolation of a benzoate degrader. A bacterium using benzoate as sole
source of carbon was obtained from enrichments containing 0.2% benzoate in
inorganic salts solution, pH 7.0 (13). The solution was inoculated with
Beebe Lake water and incubated in the dark at 29 °C for 3 days, and the en-
richment was streaked on agar containing 0.2% benzoate and the inorganic
salts. Large numbers of cells were obtained by growing the organism in
500 ml of 0.2% benzoate-inorganic salts solution for 2 days at 29°C on a
rotary shaker operating at 100 rpm. The cells were washed twice with 0.1 M
phosphate buffer (pH 7.0) and resuspended in the buffer prior to inoculation
into lake water.
14
Chemicals. Aniline hydrogen sulfate [U- C] (98.9 mCi/mmol), 2,4-dichloro-
phenoxy[2-14C]acetic acid (21.7 mCi/mmol), nitrilotri[1- 4C]acetic acid (53
14
mCi/mmol), and [U- C] phenol (87 mCi/mmol) were from Amersham Corp. [Car-
14
boxyl- C] benzoic acid (25.6 irCi/imol) was from New England Nuclear, Boston,
Mass. [Methylene-14C] benzylamine HC1 (6.9 mCi/innol) and [carboxyl-14C]di(2-
ethylhexyl) phthalate (2.7 mCi/ititol) were from California Bionuclear Corp.,
14
Sun Valley, Calif. Nitro[2-6- C]phenol (26.6 mCi/mmol) was from Tracerlab,
Waltham, Mass. The radiocnemical purity of these compounds exceeded 99%.
Nonradioactive chemicals and solvents of highest purity were used without
purification.
RESULTS
Mineralization of test compounds. Benzoate at concentrations of 32 pg/ml
to 50 ng/ml was rapidly mineralized. More than 70% of the radioactivity in
the benzoate-amended lake water was lost, presumably because the chemical was
14
converted to CO within 18 h in samples from Beebe Lake (Fig. 1). From 63
to 83% of the compound was mineralized in 6 h. More than 94% of the benzoate
was mineralized in 58 h at the three higher concentrations. The extent of
-------
8
mineralization increased with increasing initial concentration of the chemical.
From 85 to 96% of the benzylamine added to Beebe Lake water at concen-
trations of 284 pg to 300 ng/ml was mineralized in 36 h (Fig. 2). From 93 to
97% of the benzylamine at all concentrations was mineralized within 3 days.
The mineralization of 5.7 pg to 500 ng of aniline/ml in samples of an
oligotrophic lake (White Lake) was measured for 21 days. Mineralization was
linear at several concentrations, but the rate increased after about one week
at the two highest concentrations, at which time mineralization had almost
ceased at the lower levels (Fig. 3). At the lowest substrate level, 5.7 pg/
ml, 98% of the parent molecule was mineralized in 6 days. At the other con-
centrations, 75 to 82% of the aniline was mineralized in 21 days, except that
14
only 41% of C was converted to volatile products at the highest concentra-
tion, 500 ng/ml.
Mineralization rates calculated from the slope of the curves in Fig. 3
were plotted against the initial aniline concentrations. Such a plot showed
a linear relationship betvreen mineralization rates and aniline concentration
from 5.7 pg/ml to 50 ng/ml (Fig. 4). Because mineralization at levels of 50
and 500 ng/ml was biphasic, the rates were calculated from the 1 to 3- and
9 to 21-day period; such calculations indicate that the initial rate at 500
ng of aniline/ml was less than that predicted from the linear relationship
suggested at the lower level.
Mineralization of phenol was measured in White Lake and Beebe Lake waters.
At concentrations of 1.0 yg/ml or less, 79 to 90% of the phenol was mineralized
in both the lake waters in 5 days (Fig. 5). Most of the chemical was min-
eralized in less than two days at the lower levels. Mineralization was evi-
dent without an apparent lag period at levels below 1 pg/ml in samples from
-------
9
both lakes. At 100 yg and 10 mg/ml concentrations, only 4 to 14% of the phe-
nol was mineralized in 5 days.
Mineralization of 2,4-D at levels of 20 pg to 200 ng/ml was not evident
in 30 days in water from mesotrophic Cayuga Lake and in 60 days in water
from oligotrophic White Lake. Mineralization occurred, however, in Beebe
Lake water and filtered sewage (Fig. 6). From 75 to 90% of the 2,4-D was
mineralized at concentrations of 500 pg/ml or less in Beebe Lake water, but
only 34% was mineralized at 4.9 ng/ml. More than 90% of this herbicide was
mineralized in sewage. Cn the other hand, no CO- was generated at the high-
est concentration tested, 203 ng/ml, in Beebe Lake water.
Mineralization of p-nitrophenol was slow in waters from Cayuga and Beebe
Lakes (Fig. 7). Mineralization was detected in the first day in Cayuga Lake
water, but an apparent lag of 10 to 15 days was observed at several concentra-
tions in Beebe Lake water. From 26 to 72% of the nitrophenol was mineralized
in 26 days in samples from Cayuga Lake and in 30 days in samples from Beebe
Lake.
DEHP was not mineralized in a 60-day incubation period in samples from
White Lake. In Beebe Lake water, in contrast, mineralization of the phthalate
proceeded with no detectable lag at all test concentrations (Fig. 8). Min-
eralization was linear at 21 pg and 200 ng/ml, but the rate increased with
time at the other concentrations. The extent of phthalate mineralization
was less than that of the other organic compounds tested, and 35 to 71% of
the chemical was mineralized in 40 days.
No mineralization of nitrilotriacetate added at 20 pg to 200 ng/ml was
noted in 60 days in White Lake water.
Extent of mineralization. Ihe extent of mineralization of the organic
compounds in water samples from lakes and sewage is indicated in Table 1.
-------
10
The percentages of 2,4-D and p-nitrophenol and of the higher concentrations
of phenol that were mineralized are not shown because mineralization was not
complete at the end of the incubation period. More than about 92% of the
benzoate and benzylamine was mineralized at all concentrations tested. The
extent of mineralization of phenol in Cayuga Lake water rose with increasing
initial concentrations of the test chemical. However, 91.3% of the phenol
added at levels below 1 pg/ml was mineralized in Beebe and White Lakes waters.
The percent of aniline mineralized in White Lake water was also highest at
an initial concentration of 5.7 pg/ml.
The possible incorporation of the radiolabel into microbial cells was
assessed by incubating Beebe Lake water for 8 days with concentrations of
benzylamine ranging from 24 pg to 250 ng/ml. The amended lake water was then
centrifuged at 27,000 X g_ for 10 min. No label was observed in the particu-
late fraction, and hence presumably the indigenous microflora did not convert
carbon from the substrate in detectable amounts into cell constituents.
Kinetics. Correlation coefficients obtained for the logarithm, the cube
root, and the actual amount of organic compound mineralized with time are pre-
sented in Table 2. The data are from tests conducted with Beebe Lake water
except as noted. The data for benzoate were omitted because the chemical was
mineralized within 6 h. Correlations were determined for the logarithm and cube
root of the amount mineralized during the period of incubation to assess the
possible importance of bacteria, fungi, and actinomycetes (11). Correlations
were obtained using the maximum number of data points from which the calcu-
lated coefficients were highest for all three types of data transformations.
All but a few correlation coefficients were statistically significant at or
below the 1% level of probability. Because the correlation coefficients were
highest for the linear expression of the rates for aniline, benzylamine, phe-
-------
11
nol, 2,4-D, and DEHP, the data indicate that the mineralization of these chem-
icals was indeed linear. Based on the values for the correlation coefficients,
the rate of mineralization of p-nitrophenol seemed to be exponential in Beebe
Lake water at all concentrations except at 605 fg, 110 pg, and 198 ng/ml; how-
ever, the differences in coefficients calculated from the logarithmic and cube-
root expressions of the rates were quite small. p-Nitrophenol mineralization
was linear at three of the test levels in samples of Cayuga Lake water, and
the differences in coefficients at the fourth concentration were quite small.
Correlation coefficients for cube-root transformations were higher than the
values obtained for the logarithmic expressions of the data at all concentra-
tions of aniline, benzylamine, phenol, 2,4-D, and DEHP.
Influence of supplemental energy sources. The effect of readily avail-
able carbon sources on the mineralization of phenol, p-nitrophenol, and 2,4-D
was tested by adding glucose or phenol (10 mg/ml) to samples of Cayuga Lake
water. From 78 to 96% of the phenol was mineralized in four days, and the
addition of glucose had no effect on the extent of phenol mineralization
(Table 3). The percent of the phenol that was mineralized rose with increas-
ing phenol concentrations, whether the sugar was present or not. The min-
eralization rates were similar in the presence or absence of glucose; the
slight apparent enhancement in rates by the sugar at the three lower phenol
levels was not statistically significant.
The percent of p-nitrophenol mineralized in 26 days also rose with in-
creasing initial substrate concentration (Table 3). On the other hand, the
amount of g-nitrophenol mineralized at one or both of the higher concentra-
tions decreased substantially when the water was amended with glucose or phe-
nol. Phenol amendment, however, increased the extent of mineralization of
22.6 pg of pj-nitrophenol/ml. The supplementary carbon sources also affected
-------
12
the mineralization rates. Phenol and glucose reduced the rates from 38 to 82%
and from 22 to 82%, respectively, as compared to waters with no supplemental
carbon source. The addition of glucose or phenol also caused a lag of 4 days
prior to the onset of mineralization of 23 pg/ml but not higher levels of
p-nitrophenol (data not shown).
Under these conditions, 20 pg to 200 ng/ml of 2,4-D was not mineralized
in 30 days in the presence of glucose or phenol as well as, as reported above,
in their absence.
Relative rates. A rate constant, which is defined as the mineralization
rate in pg/ml per day divided by the initial substrate concentration in pg/ml,
was calculated for each chemical concentration. Ihe data show a decline in
the rate constants at higher concentrations of phenol, aniline, 2,4-D, and
p-nitrophenol (Table 4). The rate constants for phenol also declined with
the diminished natural nutrient levels in sources of water in the order of
Beebe, Cayuga, and White Lakes, which are eutrophic, mesotrophic, and oligo-
trophic, respectively.
Influence of inoculation. A study was carried out to assess the effect
of addition of cells of a benzoate degrader to Beebe Lake water. Four concen-
4
trations of the aromatic compound were used. The lake water contained 1 X 10
bacterial cells/ml counted on trypticase soy agar and 30 cells/ml able to grow
on 0.2% benzoate-inorganic salts agar. The addition of 6,400 or 6.4 X 10
cells of a benzoate degrader isolated from water of the same lake did not in-
crease the rate constants at the lowest benzoate concentration, 34 pg/ml
(Table 5). However, such additions influenced the rate constants at 5.0 and
50 ng of benzoate/ml. It is noteworthy that the rate constant remained the
same at all benzoate levels when the water received 6.4 X 10 cells/ml.
-------
13
DISCUSSION
A sensitive and simple method was developed to detect the mineralization
14
of C-labelled organic chemicals at lower than picograms-per-milliliter levels,
—14
and as little as 10 fg (10 g) of phenol/ml was detected by the extraction-
liquid scintillation counting procedure. Detection of chemicals at lower
concentrations is possible by extracting a larger volume of water. Detection
at such low levels was not previously possible by conventional analytical
techniques. However, Button et al. (5) proposed a procedure similar to the
one described herein but not as sensitive. The present method, however, has
14
drawbacks similar to those of the CCL trapping technique used by Boethling
14
and Alexander (3) in that these procedures do not differentiate between CCL
and other volatile metabolites and between organic products of cometabolism
and the unmetabolized parent molecule. Msthods to assess mineralization that
14
involve the trapping of CCL liberated from the labelled compounds added to
soil or water (3,10) require leak-proof incubation and trapping systems, and
hence are more complex than the present procedure.
The mineralization of more than 98% of aniline, 94% of benzoate, 96% of
phenol, 97% of benzylamine, and 94% of 2,4-D at low concentrations suggests
i
that organic compounds may be mineralized without incorporation of carbon
into cellular components. This was confirmed by the absence of label in the
particulate fraction obtained at 27,000 X g_ from Beebe Lake water amended with
labeled benzylamine. Mineralization without assimilation suggests the exist-
ence of a mechanism of microbial transformation that is similar to cometabo-
lism in that carbon from the substrate is not incorporated into cell constit-
uents; the mechanism differs in that the mineralization at these low concentra-
;
tions is almost complete whereas cometabolism characteristically yields organic
products (1). Such complete mineralization of benzoate, phenylacetate, and
-------
14
2,4-D was also observed recently in sanples of aquatic habitats of varying
natural nutrient levels (H. E. Rubin, R. V. Subba-Rao, and M. Alexander, man-
uscript submitted). Incorporation of up to half of the carbon in the organic
substrate being metabolized is expected during growth-linked aerobic metabolic
14
processes (14). Boethling and Alexander (4) reported that 50 to 60% of C-
glucose added to stream water at low concentrations was mineralized. On the
other hand, it has been reported that only 0.92% of the glucose carbon being
metabolized during anaerobic growth was assimilated by Streptococcus faecalis,
although the medium contained 1% casein hydrolysate, several amino acids, vi-
tamins, and growth factors (2). Such anaerobic transformations did not occur
in the present studies because abundant oxygen was present in the gas phase
of the flasks.
The hypothesis that trace chemicals are mineralized without carbon assim-
ilation is supported by the observation that mineralization was linear with
time. The finding that the kinetics of mineralization of certain trace chem-
icals was cubic suggests a possible involvement of fungi and/or actinomycetes
in the process, although cubic kinetics would reflect growth and hence carbon
should be assimilated. A linear relationship between the cube root of oxygen
consumption and time was reported by Marshall and Alexander (11) for several
fungi and actinomycetes growing in liquid media. The possible importance of
fungi and actinomycetes in the transformation of organic pollutants in aquatic
environments is largely ignored. In contrast, the logarithmic kinetics for
p-nitrophenol metabolism indicate that bacteria may be implicated.
Paris et al. (12) observed that the rates of microbial metabolism of the
butoxyethyl ester of 2,4-D, malathion, and chlorpropham were proportional to
both initial bacterial and chemical concentrations. The rate constants for
benzoate mineralization shoved no such increase in rate with increasing num-
-------
15
bers of cells of a benzoate degrader. The addition to water samples of read-
ily available energy sources is reported to enhance the microbial transforma-
tion of synthetic compounds (7,13). In this study, glucose additions to water
from a mesotrophic lake (Cayuga Lake) did not enhance the mineralization of
phenol or p-nitrophenol. Cn the contrary, glucose or phenol markedly reduced
p_-nitrophenol mineralization.
The pattern of mineralization of aniline in samples of an oligotrophic
lake is noteworthy. The rate increased linearly with aniline concentrations
from 5 pg to 50 ng/ml, but the rate at higher concentrations was less than
that predicted from tests of lower concentrations. Thus, the rate of minerali-
zation of aniline may be reduced because it is effected by aniline-sensitive
oligotrophs. At concentrations of 50 and 500 ng/ml, mineralization was bi-
phasic. The first phase may be a result of the metabolism of oligotrophs
acting in the first few days, whereas the increase in mineralization after
about 7 days may reflect the replication of eutrophs active on this amine.
Similarly, the linear kinetics at lower aniline concentrations may be a con-
sequence of the metabolism of nongrowing oligotrophs. A biphasic mineraliza-
tion also was observed for DEHP at 202 pg and 2.0 ng/ml in samples of a eu-
trophic lake (Beebe).
The environmental significance of the extensive mineralization, the
absence or small amount of substrate-carbon assimilated, and the linear
kinetics of mineralization of synthetic compounds at low concentrations re-
quires further study. In addition, the practical importance of the finding
that certain transformations occur in eutrcphic but not oligotrophic waters
requires evaluation.
-------
16
This project was supported by the U. S. Environmental Protection Agency
under cooperative agreement CR806887. The statements do not necessarily re-
flect the views and policies of the Environmental Protection Agency. We
thank A. Baquero for his assistance.
-------
17
LITERATURE CITED
1. Alexander, M. 1981. Biodegradation of chemicals of environmental concern.
Science 211:132-138.
2. Bauchop, T., and S. R. Elsden. 1960. The growth of micro-organisms in
relation to their energy supply. J. Gen. Microbiol. 23:457-469.
3. Boethling, R. S., and M. Alexander. 1979. Effect of concentration of
organic chemicals on their bicdegradation by natural microbial com-
munities. Appl. Environ. Microbiol. 37:1211-1216.
4. Boethling, R. S., and M. Alexander. 1979. Microbial degradation of or-
ganic compounds at trace levels. Environ. Sci. Technol. 13:989-991.
5. Button, D. K., D. M. Schell, and B. R. Robertson. 1981. Sensitive and
accurate methodology for measuring the kinetics of concentration-
dependent hydrocarbon metabolism rates in seawater by microbial
communities. Appl. Environ. Microbiol. 41:936-941.
6. Jannasch, H. W. 1967. Growth of marine bacteria at limiting concentra-
tions of organic carbon in seawater. Limnol. Oceanogr. 12:264-271.
7. Juengst, F. W., Jr., and M. Alexander. 1976. Conversion of 1,1,1-tri-
chloro-2,2-bis(p-chlorophenyl)ethane (DDT) to water-soluble products
by microorganisms. J. Agric. Food Chem. 24:111-115.
8. Law, A. T., and D. K. Button. 1977. Multiple-carbon-source-limited
growth kinetics of a marine coryneform bacterium. J. Bacteriol.
129:115-123.
9. Lee, R. F., and C. Ryan. 1979. Microbial degradation of organochlorine
compounds in estuarine waters and sediments, p. 443-450. In A. W.
Bourquin and P. H. Pritchard (ed.), Microbial degradation of pol-
lutants in marine environments. EPA report 600/9-79-012. U. S.
Environmental Protection Agency, Gulf Breeze, Fla.
-------
18
10. Marinucci, A. C., and R. Bartha. 1979. Biodegradation of 1,2,3- and
1,2,4-trichlorobenzene in soil and in liquid enrichment culture.
Appl. Environ. Microbiol. 38:811-817
11. Marshall, K. C., and M. Alexander. 1960. Growth characteristics of
fungi and actinomycetes. J. Bacteriol. 80:412-416.
12. Paris, D. F., W. C. Steen, G. L. Baughman, and J. T. Barnett, Jr. 1981.
Second-order model to predict microbial degradation of organic com-
pounds in natural waters. Appl. Environ. Microbiol. 41:603-609.
13. Pfaender, F. K., and M. Alexander. 1973. Effect of nutrient additions
on the apparent cometabolism of DDT. J. Agric. Food Chem. 21:397-399.
14. Sokatch, J. R. 1969. Bacterial physiology and metabolism. Academic
Press, Inc., New York.
-------
TABLE 1. Extent of mineralization of organic compounds
in lake waters and
sewage
Mineralization of chemical (%)
Chemical
Benzoate
Phenol
Phenol
Phenol
Benzylamine
Aniline
2,4-D
DEHP
Environmental
source
Beebe Lake
Cayuga Lake
Beebe Lake
White Lake
Beebe Lake
White Lake
Sewage
Beebe Lake
^'a
10 pc£
M£
ND
91.3
91.3
ND
98.5
ND
ND
10-
100 pg
94.3
78.3
84.0
80.2
ND
75.1
94.5
71.2
0.1-
1 ng
97.6
83.5
ND
ND
96.1
77.5
ND
42.3-
1-
10 ng
97.8
92.3
84.1
85.2
96.6
82.3
ND
34.9
10-
100 ng
91.8
95.7
ND
ND
98.4
79.8
91.5
62.3
0.1-
i yg
ND
ND
85.0
85.2-
98.9
41.3^
ND
ND
concentration (amt/ml) that was tested fell within this range.
-Not determined.
Q
-Mineralization was not complete at the end of incubation.
-------
TABLE 2. Correlation coefficients for extent of mineralization of organic
chemicals with time
Correlation coefficients
Concentration Amount mineralized (M) Log M v/M"~vs
Chemical
Aniline^-
Benzylamine
Phenol-
Phenol
2,4-D
DEHP
p-Nitrophenol
pj-Nitrophenol—
(per ml)
5.7 pg
54 pg
505 pg
5.0 ng
50 ng
500 ng
310 pg
102 fg
975 fg
100 yg
10 mg
191 fg
933 fg
100 yg
10 mg
1.5 pg
21 pg
202 pg
2.0 ng
200 ng
605 fg
2.5 pg
5.3 pg
8.0 pg
14 pg
110 pg
1.9 ng
198 ng
27 pg
161 pg
1.4 ng
200 ng
vs time
0.989
0.989
0.995
0.991
0.991
0.977
0.993
0.975
0.998,
0.858g-
0.847-
0.967
0.996,
0.754-
0.997
0.995
0.998
0.943
0.992
0.994
0.990
0.971
0.941
0.915
0.979
0.985
0.892
0.967
0.994
0.997
0.991
0.989
vs time
0.926
0.890
0.898
0.917
0.915
0.821
0.963
0.939
0.964b
0.790-
0.892
0.950
0.62(£
0.973
0.955
0.984
0.813
0.918
0.964
0.985
0.995
0.987
0.934
0.993
0.958
0.961
0.994
0.983
0.980
0.994
0.946
time
0.950
0.944
0.943
0.964
0.961
0.895
0.989
0.953
0.97a
0.827^
0.809-
0.917
0.968
0.675
0.988
0.976
0.994
0.879
0.951
0.973
0.989
0.992
0.978
0.930
0.990
0.973
0.942
0.999
0.988
0.993
0.992
0.968
^Mineralization tested with White Lake water.
^tot statistically significant.
^Cayuga Lake samples.
-------
TABLE 3. Effect of unlabelled glucose or phenol on the mineralization of labelled
Labelled
chemical-
Phenol
p-Nitrophenol
phenol
Concentration
of labelled com-
pound (pg/ml)
52.7
379
19,200
194,700
22.6
13,900
200,000
and p-nitrophenol in Cayuga Lake water
Labelled compound mineralized (%)
No
amendment
78.3
83.3
92.3
95.7
45.5
59.0
72.2
Glucose
added
75.8
87.5
93.5
94.1
45.4
48.7
13.6
Phenol
added
«£
ND
ND
ND
64.6
18.3
18.4
Mineralization rate (pg/ml per h)
No
amendment
0.76
10.2
624
6850
0.056
2.59
144
Glucose
added
0.87
11.4
700
6330
0.044
1.17
101
Phenol
added
ND
ND
ND
ND
0.030
0.474
88.8
—Incubation period: 4 days for phenol and 26 days for p-nitrophenol.
K
done.
-------
TABLE 4. Mineralization rate constants for organic chemicals
Chemical
Benzoate
Benzylamine
Phenol
Aniline
2,4-D
p-Nitrophenol
DEHP
Lake
water
sample
Beebe
Beebe
Beebe
Beebe
Beebe
White
White
Cayuga
White
Beebe
Beebe
Beebe
Chemical
concentration
(per ml)
0.032- 50 ng
0.280-300 ng
0.190-100 pg
100 yg
10 mg
0.100-100 pg
0.100- 10 mg
0.053-195 ng
5.7 -5000 pg
50 - 500 ng
7 - 500 pg
5 ng
0.600- 14 pg
198 ng
0.021-200 ng
Rate
constant—
(per day)
3.14 ±
1.38 ±
1.32 ±
0.018
0.004.
0.579 ±
0.011 ±
0.648 ±
0.138 ±
0.027 ±
0.058 ±
0.026
0.051 ±
0.018
0.028 ±
0.51
0.20
0.39
0.098
0.001
0.213
0.034
0.019
0.006
0.009
0.013
The mean (± standard deviation) of the constants for all of the concen-
trations in the range given in the previous column.
-------
TABLE 5. Effect of inoculation of cells of a benzoate degrader on the rate
ol
No. of cells
added/ml
0
6,400
640,000
: mineralization of benzoate in I
Rate constant
345: 350
0.150 0.145
0.157 0.142
0.146 0.140
3eebe Lake wate
(per h)
5,000
0.124
0.143
0.143
r
50,000
0.104
0.114
0.145
HBenzoate concentration (pg/ml)
-------
LEGENDS
FIG. 1. Mineralization of_ benzoate in samples of Beebe Lake water.
FIG. 2. Mineralization of benzylamine added to samples of Beebe Lake
water.
FIG. 3. Mineralization of aniline added at various concentrations to
samples of White Lake water.
FIG. 4. Effect of concentration on the mineralization rates of_ aniline
incubated in samples of White Lake water.
FIG. 5. Mineralization of_ phenol added at different concentrations to
samples of White and Beebe lake waters.
FIG. 6. Mineralization of 2,4-D added at low concentrations to sanples
of_ Beebe Lake water and sewage. Curves not labeled "sewage" were conducted
with lake water.
FIG. 7. Mineralization of low concentrations of p-nitrophenol in samples
of Cayuqa and Beebe Lake waters.
FIG. 8. Mineralization of low concentrations of DEHP in samples of Beebe
Lake water.
-------
100
80
o
UJ
M
< 60
40
20
50ng/ml
A
o
/ ,
5ng/ml
,_ . .
369pg/ml
40
80
HOURS
120
Figure 1
-------
Figure 2
-------
Figure 3
-------
'"91
JOOpg
oe
£
lOpg
»-21 DAYS
J 1-3 DAYS
Z
o
<
N
<
£
1P9
lOOfgl
ipg
lOOpg Ing lOng lOOng
CONCENTRATION (PER ML)
Figure 4
-------
120
Figure 5
-------
0 60 -
N
DAYS
Figure 6
-------
K>
30
DAYS
Figure 7
-------
Figure 8
-------
Effect of Nutrients on the Rates of Mineralization of Trace
Concentrations of Organic Compounds in Natural Waters
H. E. RUBIN AND MARTIN ALEXANDER*
Laboratory of Soil Microbiology, Department of
Cornell University, Ithaca, New York 14853
A study was conducted of the effect of nutrients on the rates of min-
eralization of phenol and p-nitrophenol added to samples of natural waters
at concentrations of several pg/ml to 200 ng/ml. The water samples were
from Beebe Lake (eutrophic), Cayuga Lake (mesotrophic), White Lake (oligo-
trophic), and a stream with total organic carbon (TOC) concentrations of
38.5, 27.0, 5.7, and 7.9 yg/ml, respectively. The rate of phenol minerali-
zation was slowest in White Lake water, slightly more rapid in samples of
the stream, greater in Cayuga Lake water, and roost rapid in Beebe Lake water.
Thus, a direct relationship between nutrient levels and the rate of phenol
mineralization was evident. Beebe Lake water was diluted from 38.5 yg to
3.85 ng TOC/ml with distilled water. The original microbial community was
removed from Beebe Lake water by centrifugation, and the cells were washed
twice with phosphate buffer and then added to the lake water dilutions. The
mineralization rate decreased as the nutrient level decreased. Because the
microbial community was the same at the different dilutions, species diver-
sity and numbers were not factors affecting the rate of phenol mineraliza-
\
tion. At a concentration of 38.5 yg carbon/ml, yeast extract and arginine
stimulated the rate of phenol mineralization in pH 7.2 distilled water inoc-
ulated with the washed cells from Beebe Lake. Inorganic salts were also stint-
-------
2
ulatory. The mineralization rate was not altered'in tris-buffered water.
Arginine did not affect the rate of phenol mineralization in sanples of the
stream water. Similar findings were obtained with pj-nitrophenol. These
data indicate that the presence of inorganic and organic nutrients in nat-
ural waters affects the rate of mineralization of organic compounds in trace
concentrations.
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3
Ihe destruction of organic pollutants in natural waters is of consider-
able importance. Environmental factors that may affect the rate of .destruc-
tion of pollutants include the size and diversity of the microbial community
(16,17), sorption (13), chemical structure (3), conetabolism (2,3), and nu-
trient supply (12,13).
i
Nutrient supply probably has a significant inpact on the mineralization
of organic compounds in natural waters. In studies by Vaccaro (17) and Tanaka
(16), it was shown that a direct relationship existed between nutrient levels
and microbial populations and diversity in eutrophic waters. Subsequently,
Nesbitt and Watson (12,13) demonstrated that the rate of mineralization of
high concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) was linked to
nutrient concentration. However, all of the studies of the effect of nutrient
supply on mineralization of organic compounds have used concentrations of
chemicals far higher than exist in nature. Furthermore, the rates of mineral-
ization of substrates at high concentrations are far different from those oc-
curring at natural substrate concentrations (3) and may follow different
kinetics (6).
This study was undertaken to determine the effect of nutrient supply
in natural waters on the mineralization of chemicals in trace concentrations.
The effect that different secondary carbon sources and trace elements have on
mineralization of organic compounds was also studied.
MATERIALS AND METHODS
14
[U-C]Phenol (specific activity, 87 mCL/mnol) was obtained from Amersham
14
Corp., Arlington Heights, 111., and pj-nitro[2,6- Clphenol (26.6 mCL/mtnol) was
purchased from Tracer lab, Waltham, Mass. When inorganic nutrients were added
to the solutions, the supplement contained (per liter of distilled water) MgSO4«7-
0.10 g; MgCl2, 0.066 g; (NH4)2SO4, 0.50 g; CaCl2«2H20, 0.02 g; FeCl-j, 1.0
-------
4
rag; Na2Mo04'2H20, 0.062 g; and MnSO.-F^O, 0.023 g. The waters were buffered
either with Tris (7) or phosphate buffers (5).
Total organic carbon (TOC) analysis was performed using a Beckman (Beck-
nan Instruments, Inc., Fullerton, Calif.) model 915A TOC analyzer fitted with
a Beckman model 865 infrared analyzer. Sodium1 oxalate at concentrations of
0 to 20 yg/ml was used as the standard. The flow rate of the conpressed air
was 150 inl/min, and the temperature of the column was 970 °C.
Samples of fresh water were taken from Beebe Lake, Cayuga Lake, and
Bifield Creek near Ithaca, New York and .from White Lake near Old Forge,
New York. Beebe Lake is eutrophic, Cayuga Lake is mesotrophic, and the
stream and White Lake are oligotrophic.
A natural aquatic microbial cotmtunity was added to distilled water in
the following manner. After passing fresh samples of Beebe Lake water through
a glass fiber filter no. 66085 (Gelman Sciences, Inc., Ann Arbor, Mich.) to
remove large particulates, the cells were removed by centrifugation at 41,000
X g at 4°C, washed twice with 0.3 mM phosphate buffer, pH 7.2 (5), and con-
centrated 100-fold. A suspension of these cells was added to distilled water
to give a cell count equal to that originally found in Beebe Lake water.
To study mineralization rates, the water samples were passed through
14
a glass fiber filter, and then the water was amended with the C-labelled
compound to be tested. The samples were incubated in the dark at 29 °C with-
out shaking. The method used to determine mineralization rates has been
previously described (14,15). At intervals, mineralization was determined
in distilled water adjusted to pH 7.2 with NaOH, 52.8 mM tris buffer, pH
7.2 (7), or distilled water augmented with inorganic salts, glucose, adenine,
arginine, propionate, or yeast extract. The organic compounds were added
either to distilled water or natural waters at the same concentration, 38.5
Vig/ml, of organic carbon that was present in Beebe Lake.
-------
RESULTS
The rates of mineralization of different concentrations of phenol ware
determined in samples of Beebe Lake, Cayuga Lake, stream water, and White
Lake. The test samples of these waters had TOC concentrations of 38.5, 27.0,
7.9, and 5.7 ug/ml, respectively. The data indicated that a direct relation-
ship existed between the rate of phenol mineralization and the concentration
of organic carbon present in these natural waters (Fig. 1). At phenol con-
centrations ranging from 2.0 pg/ml to 200 ng/ml, the rates of mineralization
were proportionately higher as the organic carbon level of the water increased.
It is also evident that mineralization rates in these waters were directly
proportional to phenol concentration over a range of 5 or more orders of mag-
nitude.
To aid in determining why the rates of phenol mineralization were dif-
ferent in the several waters, Beebe Lake water (initial TOC, 38.5 yg/ml) was
diluted with distilled water, and the dilutions were then amended with a sus-
pension of the normal microflora. The data indicated that the rate of min-
eralization decreased as the extent of dilution increased with the undiluted
lake water having the fastest rate and distilled water the slowest rate (Table
1). These data indicate that a dilutable factor is present that can affect
the rate of mineralization. Because species diversity and the size of the
microbial community were the same at each dilution, it is likely that some
other factor, such as nutrients, was the dilutable factor affecting the min-
eralization rate.
To determine if organic nutrients affected phenol mineralization, ar-
ginine, adenine, glucose, propionate, or yeast extract (in amounts equiva-
lent to 38.5 yg of carbon/ml) was added to distilled water containing 2.0 ng
-------
6
of phenol/ml. The liquid was inoculated with a portion of the microbial com-
munity from Beebe Lake. The data indicate that the apparent lag phase was
shortened by arginine, and that phenol mineralization was stimulated by yeast
extract (Fig. 2). Qi the other hand, adenine and glucose did not shorten the
lag phase and suppressed the subsequent rate of phenol mineralization. Pro-
pionate almost totally suppressed phenol mineralization in the test period.
The effect of various carbon sources at 38.5 yg of carbon/ml on the
rates of mineralization of phenol at various concentrations was then deter-
mined (Table 2). The microbial community from Beebe Lake was added to dis-
tilled water in the presence of phenol and the various organic materials.
Hie rate of phenol mineralization was often highest in the presence of yeast
extract. Arginine was also stimulatory as compared to the water without a
second carbon source. In contrast, glucose at low levels was inhibitory, and
phenol at a concentration of 2 yg/ml was not mineralized in the presence of
glucose. Therefore, organic compounds can either augment or decrease the
rate of phenol mineralization.
The rate of phenol mineralization was determined in a solution contain-
ing inorganic salts, tris buffer (pH 7.2), or arginine at a concentration of
38.5 yg C/ml. The solution was inoculated with cells from Beebe Lake water.
The addition of the buffer did not increase the rate in water alone (Table 3).
Addition of the inorganic salts increased the rate at which phenol was min-
eralized. The rate was also increased upon the addition of arginine. The
greatest rate was obtained in solutions amended with arginine and inorganic
salts. Thus, certain organic compounds and inorganic nutrients are stimula-
tory.
Arginine at 38.5 yg of carbon/ml also was added to samples of stream
'water to determine its effect on the mineralizaton of phenol in natural waters.
-------
7
No significant difference in rates of phenol mineralization was noted in the
presence or absence of arginine (Table 4). Hence, the arginine stimulation
was not evident in a natural water with low levels of carbon but was noted
when no other nutrients were present.
To determine if nutrients might affect the mineralization of a more
slowly mineralized compound, p-nitrophenol at various concentrations was
added to distilled water containing cells derived from Beebe Lake and to
stream water. Arginine (38.5 yg of carbon/ml) was added to half the samples.
The mineralization rate was slower in distilled water than in stream water
with no supplemental carbon (Table 5). Arginine stimulated the rate of pj-
nitrophenol mineralization in distilled water, but only occasionally had an
effect in samples of stream water. Thus, nutrients in natural waters appar-
ently have a similar stimulatory effect as added organic nutrients for both
of the phenols.
DISCUSSION
Nutrient content of natural waters has been shown to increase the popu-
lation density of eutrophs (16) and the rate of uptake of glycine, glucose,
malate, phenylacetate (17), and 2,4-D (12,13). Hoover, the effect of natu-
rally occurring nutrients on the rates of mineralization of trace concentra-
tions of compounds has not previously been investigated. The present data,
however, suggest that nutrient levels stimulate the mineralization rate of
the test compounds independent of the concentration of the two phenols.
t
Furthermore, nutrients in lake waters can increase not only the mineraliza-
tion rate but also the final percent of the pollutant mineralized (15). In
addition, with arginine as a second source of carbon, a decrease was noted
in the apparent lag phase before active phenol decomposition, indicating that
arginine was stimilating the growth of phenol-degrading microorganisms.
-------
8
Both organic and inorganic compounds stimulated the rate of phenol min-
eralization, the inorganic salts having a greater stimulatory effect than ar-
ginine alone. Hie largest increase in the rate of mineralization occurred
with the inorganic salts and arginine together, which probably reflects what
occurs in many natural environments, which often have both inorganic and or-
ganic nutrients available for the microflora.
Mineralization of low concentrations of organic chemicals is probably a
result of activities of oligotrophic populations. These organisms are prob-
ably quite sensitive to organic chemicals at higher concentrations, a view
consistent with the finding that propionate at 38.5 pg of carbon/ml prevented
mineralization of phenol at all concentrations of the aromatic compound and
that glucose was completely inhibitory at one phenol level. It has been pre-
viously shown that mineralization of 2,4-D in freshwaters sometimes ceased at
concentrations as low as 200 ng of 2,4-D/ml, presumably because of the sensi-
tivity of the active organisms (14). The significance of the oligotrophs,
which have been reported in natural waters (1,8,11), thus requires further
inquiry.
Concentrations of substrates below which no mineralization occurs have
been demonstrated in pure culture (9) and in some natural waters (6). The
failure to observe such thresholds in some waters (14) may result from the
presence in these waters of nutrients that influence the indigenous community.
In support of this view is the finding of Law and Button (10) that the thresh-
old concentration for glucose in culture was lowered in the presence of argi-
nine and was lowered beyond the limits of detection in the presence of a mix-
ture of amino acids. A threshold may be expected in environments in which the
indigenous populations are obligate eutrophs that are not able to metabolize
the test chemicals at low levels regardless of the presence of other nutrients.
-------
ACKNOWLEDGMENT
This project was supported by the Environmental Protection Agency
under cooperative agreement CR806887. The statements do not necessarily
reflect the views and policies of the Environmental Protection Agency.
The technical assistance of Ms. Deirdre M. Brophy is greatly appreciated.
-------
10
LTTERAIURE CITED
1. Akagi, Y., N. Taga, and U. Simidu. 1977. Isolation and distribution of
oligotrophic marine bacteria. Can. J. Microbiol. 23:981-987.
2. Alexander, M. 1979. Role of oonetabolism, p. 67-75. In A. W. Bourquin
i
and P. H. Pritchard (ed.), Microbial degradation of pollutants in
marine environments. U. S. Environmental Protection Agency, Gulf
Breeze, Fla.
3. Alexander, M. 1980. Helpful, harmful, and fallible microorganisms: Im-
portance in transformation of chemical pollutants. In D. Schlessinger
(ed.), Microbiology 1980. American Society for Microbiology, Wash-
ington, D.C.
4. Alexander, M. 1981. Biodegradation of chemicals of environmental concern.
Science 211:132-138.
5. American Public Health Association. 1976. Standard methods for the exami-
nation of water and wastewater. American Public Health Association,
Washington, D.C.
6. Boethling, R. S., and M. Alexander. 1979. Effect of concentration of or-
ganic chemicals on their biodegradation by natural microbial communi-
ties. Appl. Environ. Microbiol. 37:1211-1216.
7. Qomori, G. 1955. In Colowick and Kaplan (eds.). Methods in enzymology,
vol. 5, p. 138. Academic Press, New York.
8. Kuznetsov, S. I., G. A. Dubinina, and N. A. Lapteva. 1979. Biology of
oligotrophic bacteria. Annu. Rev. Microbiol. 33:377-387.
•V**i*
9. Jannasch, H. W. 1967. Growth of marine bacteria at limiting concentrations
of organic carbon in seawater. Limnol. Oceanog. 12:264-271.
-------
11
10. Law, A. T., and D. K. Button. 1977. Multiple-carbon-source-liroited growth
kinetics of a marine coryneform bacterium. J. Bacteriol. 129:115-123.
11. Maledj, K., and J. Overbeck. 1980. Studies on uptake kinetics of oligo-
carbophilic bacteria. Arch. Hydrobiol. 89:303-312.
«ww
12. Nesbitt, H. J., and J. R. Watson. 1980. Degradation of the herbicide
2,4-D in river water. I. Description of study area and survey of
rate determining factors. Water Fes. 14:1683-1688.
13 Nesbitt, H. J., and J. R. Watson. 1980. Degradation of the herbicide
2,4-D in river water. II. The role of suspended sediment, nutrients
and water temperature. Water Res. 14:1689-1694.
14. Rubin, H. G., R. V. Subba-Rao, and M. Alexander. 1981. Rates of minerali-
zation of trace concentrations of aromatic compounds in samples of
lake waters and sewage. Appl. Environ. Microbiol., submitted for
publication.
15. Subba-Rao, R. V., H. E. Rubin, and M. Alexander. 1981. Kinetics and ex-
tent of mineralization of organic chemicals at trace levels in fresh-
waters and sewage. In preparation.
16. Tanaka, N., Y. Ueda, M. Onizawa, and H. Kadota. 1977. Bacterial popula-
tions in water masses of different organic matter concentrations in
Lake Biwa. Jap. J. Limnol. 38:41-47.
17. Vaccaro, R. F. 1969. The response of natural microbial populations in
seawater to organic enrichment. Liimol. Oceanog. 14:726-735.
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TABU! 1. Mineralization of phenol at different dilutions of Beebe Lake water with a constant natural
microbial population.
Initial phenol
ooncn/ml
(ng/ml)
0.020
0.20
2.0
20
200
Mineralization rate (pg/ml j?er h)
Beebe
Lake
1.4
13.9
144
1,600
16,800
10 X
diluted
1.1
14.3
134
1,200
14,000
100 X
diluted
1.2
14.4
134
1,300
12,300
1000 X
diluted
1.0
13.0
121
1,100
10,800
10,000 X
diluted
0.94
12.8
130
890
10,200
Distilled
H20
0.89
12.9
93
1,100
10,000
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TABLE 2. Rates of phenol mineralization in distilled water amended with
organic compounds and cells from Beebe Lake.
Initial
concn/ml
19.6
1.82
19.8
199.8
2.0
pg
ng
ng
ng
vg
Mineralization rate (amt/ml
Yeast
extract
1.
195.
1.
19.
233.
81
0
25
0
3
pg
pg
ng
ng
ng
Arginine
2
177
1
21
195
.55 pg
.5 pg
.5 ng
.25 ng
.7 ng
Adenine
220
76.4
1.23
20.0
220
fg
pg
ng
ng
ng
per h)
Glucose
191.7 fg
95 pg
1.0 ng
12.25 ng
oa
Distilled
water
1.38
185
746.7
21.7
171.4
pg
pg
pg
ng
ng
aNo mineralization.
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TABLE 3. Mineralization of phenol in the presence-of arginine in a salts
solution or in tris buffer.
Initial
phenol
concn/ml
(ng/ml)
0.020
2.0
200
Mineralization rate
Salts
467
74
7
fg
.5 pg
.75ng
Salts +
arginine
487
90.8
8.8
fg
pg
ng
Tris
314
45.8
5.1
(amt/rnl per
h)
Tris +
arginine H-O
fg
pg
ng
363
53.5
6.3
fg
pg
ng
333
46.7
5.6
fg
pg
ng
H2° +
arginine
363
57.5
6.8
fg
pg
ng
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TABLE 4. Mineralization of phenol in sanples of stream water in the
presence of arginine.
Initial phenol
ooncn
(ng/ml)
0.020
2.0
200.
Mineralization rate
Phenol
0.041
3.50
286
(pg/ml per h)
Phenol + arginine
0.042
3.68
307
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TABLE 5. Mineralization of p-nitrophenol in distilled water inoculated
with cells from Beebe Lake and in stream water.
Initial
p-nitrophenol
ooncn
(ng/ml)
0.020
0.20
2.0
20
200
Stream
water
0.25
3.04
48.0
438
5,600
Mineralization i
Stream
water +
arginine
0.20
2.50
51.8
577
5,800
rate (pg/ml per h)
Distilled
water
0.18
2.42
37.8
391
1,260
x
Distilled
water +
arginine
0.58
3.00
48.9
495
6,850
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FIG. 1. Rates of phenol mineral!zation in waters from Beebe Lake,
Cayuga .Lake, White Lake, and a_ stream.
FIG. 2. Effeet of organic compounds on phenol mineralization in
distilled water inoculated with cells fron Beebe Lake.
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FIG. 1. Rates of phenol mineralization in waters from Beebe Lake, Cayuga Lake,White Lake, and a stream
10 .
1 ng-
.c
jjj 100
a
10 -
Ipg-
100 .
10 fg
Beebe Lake
Cayuga Lake
Stream water
White Lake
1 pg
10
100
1 ng
PHENOL
10
100
1 yg
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FIG. 2. Effect of organic conpounds on phenol mineralization in distilled
water inoculated with cells from Beebe Lake
I
§
Yeast extract
addition
.ucose
Propionate
8
10
HOURS
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