EPA/600/A-92/174
RESPIROMETRIC METHODS FOR DETERMINATION OF BIODEGRADABILITY AND
BIODEGRADATION KINETICS FOR HAZARDOUS ORGANIC POLLUTANT COMPOUNDS
Henry H. Tabak
U.S. Environmental Protection Agency
Office of Research and Development
Risk Reduction Engineering Laboratory
Cincinnati, OH 45268
and
Sanjay Desai and Rakesh Govind
Department of Chemical and Nuclear Engineering
University of Cincinnati
Cincinnati, OH 45221
RISK REDUCTION ENGINEERING LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONHENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
Presented at the ACS Environmental Chemistry Division Symposium on
Chemical and Biochemical Detoxification of Hazardous Wastes II, Miami Beach,
Florida, September 10-15, 1989.
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RESPIROMETRIC METHODS FOR DETERMINATION OF BIODEGRADABILITY AND
BIODEGRADATION KINETICS FOR HAZARDOUS ORGANIC POLLUTANT COMPOUNDS
ABSTRACT
Electrolytic respirometry involving natural sewage, sludge and soil microbiota is
becoming prominent in fate studies of priority pollutant and RCRA toxic organics to
generate biodegradation/inhibition kinetic data. A developed multi-level protocol is
presented for determination of substrate biodegradability and toxicity, microbial
acclimation to toxic substrates and first order kinetic parameters of biodegradation
and for estimation of Monod kinetic parameters of toxic organic compounds, in order to
correlate the extent and rate of biodegradation with a predictive model based on
chemical properties and molecular structure of these compounds. Respirometric
biodegradation/inhibition and biokinetic data are provided for representative RCRA
alkyl, chloro- and nitro-benzenes, phenols, phthalates, ketones and selected CERCLA
leachate toxic organics. Data on the effects of the source of sludge biomass, temp-
erature and concentration of microbial inoculum and toxic substrate on the kinetics of
biodegradation are also included.
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INTRODUCTION
Electrolytic respirometry is attaining prominence in biodegradation
studies and is becoming one of the more suitable experimental methods for
measuring the biodegradability and the kinetics of biodegradation of toxic
organic compounds by the sewage, sludge and soil microbiota and for
determining substrate inhibitory effects to microorganisms in wastewater
treatment systems.
Biodegradation of toxic and hazardous organic compounds holds a great
promise as an important fate mechanism in wastewater treatment and in soil
detoxification. Information about the extent and rate of biodegradation is
a prerequisite for informed decision making on the applicability of the
biodegradation approach. Unfortunately, relatively little quantitative data
are available from which engineering judgement can be made, because of the
large effort required to assess biodegradation kinetics.
Current research in our laboratories has shown that it is possible to
assess biodegradation kinetic parameters from oxygen uptake data, obtained
through the use of electrolytic respirometry. This method greatly reduces
the work and expense involved in evaluation of biodegradation kinetics. The
ongoing biodegradation studies are concerned with the generation of
biokinetic database so that it can be ultimately used to establish a
possible correlation between molecular substrate configuration
(chemical/physical characteristics) and biomass activity (kinetic
parameters) as an index of biodegradation. The experimental respirometric
testing is also providing data on the concentration levels of toxic organics
inhibitory to microbial activity.
The ongoing biodegradation studies are concerned with the prediction of
the biological fate of toxic organic compounds using electrolytic
respirometry as an approach to measure the biodegradation of selected
organic compounds and generate biokinetic data so that these can be
ultimately used to establish a possible correlation between molecular
substrate configuration (physical/chemical characteristics) and biomass
activity (kinetic parameters) as an index of biodegradation. The
experimental respirometry testing is also providing data on the
concentration levels of toxic organics inhibitory to microbial activity.
Initially, the inter-laboratory, ring test, Organization of Economic
Cooperation and Development (OECD) studies at the EPA laboratory,
Cincinnati, Ohio, were undertaken to develop confirmatory respirometric
biodegradability testing procedure. Respirometric biodegradability and
biokinetic data were provided for the selected non-inhibitory and non-
adsorbing compounds, tetrahydrofuran, hexamine, pentaerythritol, 1-napthol,
sodium benzene sulphinate, thioglycolic acid and the biodegradable reference
compound, aniline.
Subsequently, similar electrolytic respirometry studies were initiated
to determine biodegradation kinetic parameters for selected representative
toxic compounds of varied classes of organics included in the Priority
Pollutant, RCRA and superfund CERCLA lists, and to demonstrate presence of
i
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any inhibitory effects of these organics of specified concentration levels
on the sludge biomass and on the metabolism of biogenic compounds.
The objectives of the present study were to utilize the electrolytic
respirometry oxygen uptake data to: (1) determine the biodegradability of
selected RCRA alkyl, chloro, and nitrobenzene*, phenols, phthalates, and
ketones and representative CERCLA leachate toxic organics; (2) generate
information on their acclimated times (t0) and the initiation and
termination time values for the declining growth phase (tj and t2); (3)
determine their first order kinetic parameters of biodegradation (specific
growth rate constants for the exponential growth phase (fi) and for the
declining growth phase (M'); (4) estimate the Monod kinetic parameters (jim,
Ks and Y) of these compounds without initial growth or growth yield
assumptions; (5) demonstrate presence of any inhibitory effects of these
compounds on the metabolism of the biodegradable reference compound,
aniline; and (6) to correlate the extent and rate of biodegradation of these
compounds with a predictive model based on chemical properties and structure
of these compounds.
-The purpose of this study was to obtain information on biological
treatability of the benzene, phenol, phthalate and ketone organics and of
the Superfund CERCLA organics bearing wastes in wastewater treatment systems
which will support development of an EPA technical guidance document on the
discharge of the above organics to POTWs. The study was to generate basic
information on the fate of CERCLA leachate organics during on-site treatment
and biodegradation and inhibition data for pollutants found in Superfund
site wastewater that could be discharged to POTWs. Respirometric
biodegradability, biokinetic and inhibition data were generated for the
selected RCRA benzene, phenolic, phthalate and ketone compounds.
BACKGROUND
Measurement of Oxygen Consumption
Measurement of oxygen consumption is one of the oldest means of
assessing biodegradability. Time consuming manual measurement of oxygen
uptake (dilution BOD measurements) was replaced gradually by a more direct
and continuous respirometric method for measurement of oxygen consumption in
biochemical reactions, for use in routine examination of sewage and in
control of sewage treatment process.
A rather comprehensive review of the use of respirometers for the study
of sewage and industrial waste and their application to water pollution
problems was published by Jenkins in 1960 (1). Montgomery's (2) review of
respirometric methods summarized the design and application of respirometers
for determination of BOD.
The application of respirometry was gradually directed to research
studies to assess the toxicity and biodegradation of specific wastes or
compounds, to evaluate factors affecting biological growth and to provide an
insight into nitrification reaction. Of the commercial respirometers which
have been developed for respirometric studies, the electrolytic
respirometers were shown to be most applicable for measurement and
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quantitation of biodegradation activity because they automatically produce
oxygen as needed, thereby eliminating some of the limitations of other
techniques and allowing output data to be collected automatically for direct
recording and processing (3-9). A recent detailed review of respirometric
techniques and their application to assess biodegradability and toxicity of
organic pollutants was published by King and Dutka (10).
Respirometric Biodegradability Testing
Most uses of electrolytic respirometry in biodegradability testing have
been for screening purposes to measure the extent of biodegradation as a
percentage of the theoretical oxygen demand exerted in some time period
(9, 11-17). A more recent study by Painter and King (18) concluded that a
procedure based on electrolytic respirometry was reliable for assessing
biodegradability, and could serve as an adequate Level I screening test for
biodegradability (19).
A considerable amount of studies using electrolytic respirometry to
determine the biodegradability of wastes and specific organics is available
in published literature and significant data on biodegradation of pollutants
based on oxygen uptake have been generated (2, 4, 5, 20-34).
There are many techniques that have been used to evaluate
biodegradation kinetics and these were reviewed in detail by Howard et al.
(35, 36) and Grady (37). These techniques utilize continuous, fed-batch and
batch type reactors for providing data from which kinetic parameters can be
evaluated. The use of batch systems in biotechnology and biological
wastewater treatment represents a less labor intensive, less expensive and
much faster way to model biokinetics.
The kinetic parameters obtained by the above techniques should be
intrinsic, that is, dependent only on the nature of the compound and the
degrading microbial community and not on reactor system used for data
collection. If this condition is satisfied, then the parameters obtained
can be used for any reactor configuration and can be used in mathematical
models to estimate the fate of toxic organics.
Batch techniques are successful in obtaining intrinsic kinetic
parameters by applying non-linear curve fitting techniques to single batch
substrate removal curves, provided initial conditions are selected with
proper care (Simkins and Alexander (38, 39), Robinson and Tiedje (40), Cech
et al. (41), and Braha and Hafner (42). Batch systems can be used with
either acclimated or unacclimated biomass for providing kinetic data and
require that samples be taken at discrete time intervals during the course
of biodegradation [Tabak et al. (43), Larson and Perry (24) and Paris and
Rogers (44)].
Measurement of oxygen consumption through electrolytic respirometry is
a batch type technique which has been shown to be very promising for
automating data collection associated with biodegradation and intrinsic
kinetic parameters.
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measurements of oxygen consumption in respirometric batch reactors. With
the use of computer simulation techniques and non-linear curve fitting
methods, intrinsic kinetic parameters were obtained from oxygen consumption
data and were shown to be in agreement with those obtained from traditional
measurement of substrate removal (DOC, SCOD, 14C) or cell growth.
MATERIALS AND METHODS
Experimental Approach
The electrolytic respirometry approach to determine the biodegrad-
ability of the organic test compounds in this study was chosen because of
the specific advantages of the respirometric methods over that of manometric
procedures in tracking oxygen utilization during the exertion of biochemical
oxygen demand (BOD). These advantages are listed in Table 1. General
classification of respirometers based on principle of operation and on
techniques and applications is presented in Tables 2 and 3.
The electrolytic respirometry studies were conducted using an automated
continuous oxygen uptake and BOD measuring Voith Sapromat B-12 (12 unit
system) electrolytic respirometer-analyzer. The instrument consists of a
temperature controlled waterbath, containing measuring units, a recorder for
digital indication and direct plotting of the decomposition velocity curves
of organic compounds; and a cooling unit for the conditioning and continuous
recirculation of waterbath volume. The recorder shows the digital
indication of oxygen uptake and constructs a graph for these values of each
measuring unit. The cooling unit constantly recirculates water to maintain
constant temperature in the waterbath. Each measuring unit as shown in
Figure 1 is comprised of a reaction vessel with a carbon dioxide absorber
mounted in a glass joint flask stopper, an oxygen generator and a pressure
indicator. This measuring unit is interconnected by hoses, forming an air
sealed system, so that the atmospheric pressure fluctuations do not
adversely affect the results.
The activity of the microorganisms in the sample creates a vacuum which
is recorded by the pressure indicator, which triggers the oxygen generator.
The pressure conditions are balanced by electrolytic oxygen generation. The
quantity of the sample, the amperage for the electrolysis and the speed of
the synchronous motor are so adjusted that, with a sample of 250 mL, the
digital counter indicates the oxygen uptake directly in mg/L. The C02
generated is absorbed by soda lime. The nitrogen/oxygen ratio in the gas
phase above the sample is maintained throughout the experiment and there is
no depletion of oxygen. The recorder-plotter concomitantly constructs an
oxygen uptake graph for the selected values. The oxygen generators of the
individual measuring units are electrolytic cells which supply the required
amount of oxygen by electrolytic dissociation of a copper sulfate solution
combined with sulfuric acid.
The nutrient solution used in these studies was an OECD synthetic
medium (19, 56) consisting of measured amounts per liter of deionized
distilled water of (1) mineral salts solution; (2) trace salts solution, and
(3) a solution (150 mg/L) of yeast extract as a substitute for vitamin
solution.
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The microbial inoculum was an activated sludge from The Little Miami
wastewater treatment plant in Cincinnati, Ohio, receiving municipal
wastewater. The activated sludge sample was aerated for 24 hours before use
to bring it to an endogenous phase. The sludge biomass was added to the
medium at a concentration of 30 mg/L total solids. Total volumes of the
synthetic medium in the 500 ml capacity reactor vessels were brought up to a
final volume of 250 ml.
The test and control compound concentrations in the media were 100
mg/L, Aniline was used as the biodegradable reference compound, at a
concentration of 100 mg/L.
The typical experimental system consisted of duplicate flasks for the
reference substance, aniline, and the test compounds, a single flask for the
physical/chemical test (compound control), a single flask for toxicity
control (test compound plus aniline at 100 mg/L each) and an inoculum
control. The contents of the reaction vessels were preliminarily stirred
for an hour to ensure endogenous respiration state at the initiation of
oxygen uptake measurements. Then the test compounds and aniline were added
to it. The reaction vessels were then incubated at 25*C in the dark
(enclosed in the temperature controlled waterbath) and stirred continuously
throughout the run. The microbiota of the activated sludge used as an
inoculum were not pre-acclimated to the substrates. The incubation period
of the experimental run was between 28 to 50 days. A more comprehensive
description of the procedural steps of the respirometric tests is presented
elsewhere (33, 46).
For fully automatic data acquisition, frequent recording and storage of
large numbers of oxygen uptake data, the Sapromat B-12 recorders are
interfaced to an IBM-AT computer via Metrabyte interface system. The use of
Laboratory Handbook software package allows the collection of data at 15
minute intervals.
The oxygen utilization by the biomass based on the oxygen uptake
velocity (BOD) curves, consisting of the exponential and declining phases of
microbial growth was the basis for measurement of substrate utilization and
growth rate. Figure 2 provides a generalized plot of the substrate
concentration, biological solids concentration (biomass),•and oxygen
utilization during exertion of BOD, versus time in a respirometric
biodegradability treating vessel system. A typical relationship between the
BOD and substrate degradation curves as well as between biomass and
substrate concentrations and growth yield is illustrated in Figure 3.
Possible curves of oxygen uptake attributed to the organic test
substrate that can be generated in a respirometric run and which are
dependent on the acclimation time, the extent and rate of bio-oxidation of
the substrate, the presence of cometabolite(s) and the presence of
biocatalytic additive in the nutrient solution are demonstrated in Figure 4.
Factors affecting respirometric BOD determination have been taken into
consideration in the study, and are listed in Table 4.
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Chemical Analysis
Indirect analysis of culture samples from respirometric vessels during
the study included determination of chemical oxygen demand as soluble and
total COD and of dissolved oxidizable carbon (DOC) with the use of a Beckman
Model 915 B system.
Specific substrate analysis of culture samples for the residual parent
compound and the possible intermediate and final products of metabolism was
performed with the use of a Gas Chromatograph Varian 3700, equipped with FID
and EC detectors and with different detector columns (depending on the
substrate to be analyzed) and the Finnigan automated GC/EI-CI Mass
Spectrophotometer system. Samples were analyzed as dichloromethane extracts
of cultures (continuous liquid-liquid extraction) or by direct aqueous
injection.
Suspended solids and biomass were measured gravimetrically (solids
retained on a 0.45 pm filter) and by optical density determined
spectrophotometrically (percent transmission read at 540 nm).
Determination of Substrate Biodegradability from Oxygen Uptake Data
In this study, biodegradation was measured by three approaches: the
first, as the ratio of the measured BOD values in mg/L (oxygen uptake values
of test compound minus inoculum control - endogenous oxygen uptake values)
to the theoretical oxygen demand (ThOD) of substrate as a percent; the
second as a percentage of the test compound as measured by dissolved organic
carbon (DOC) changes [OECD Guidelines for Testing of Chemicals (Method DGXI
283/82, Revision 5) (56)]; and the third, as a percentage of the test
compound as measured by specific substrate analysis.
Graphical representation of percent biodegradation based on the
BOD/ThOD ratio were developed against time for each test compound. The
experimental DOC data for the initial samples and samples for reaction
flasks collected at the end of experimental run were used to calculate the
percent biodegradation based on the percent of DOC removal in the culture
system.
Determination of Kinetic Parameters of Biodegradation
Monod equation, relating cell growth to biomass and substrate
concentration and the linear law, relating cell growth to substrate removal
are the most popular kinetic expressions which can provide adequate
description of growth behavior during biodegradation of substrate. The
Monod relation states that cell growth is first order with respect to
biomass concentration (X) and mixed order with respect to substrate
concentration (S) by tbe equation
dX/dt - (SumX)/(Ks + S) [I]
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Cell growth is related to substrate removal by the linear law by the
equation
dX/dt - -Y(dS/dt [II]
The kinetics of biodegradation were evaluated by quantifying is. , Ks and
Y kinetic parameters expressed in the equation for the rate of substrate
removal , rs:
(1)
where X is the concentration of biomass capable of utilizing the organic
substrate and Y is the biomass yield coefficient for the compound and in the
Honod equation:
It - /imSs/Ks + ss (2)
(if the compound is not inhibitory to its own biodegradation} or by the
Haldan euatin
Haldane equation:
H - MA/KS 4 Ss + (Sf/K,) (3)
if the compound is inhibitory. In these equations, pm is the maximum
specific growth rate, Ks is the half saturation coefficient, Kj is the
inhibition coefficient and S is the concentration of substrate.
A graphical presentation of the Honod substrate utilization equation is
illustrated in Figure 5 which provides a relationship between rate of
substrate utilization and substrate concentration.
Determination of Rates of Exponential and Declining Growth
The first order kinetic rate constants (specific growth rate
parameters) were determined by the linearization of the BOD curves or
transforming the typical BOD curve to the linear function of time t, by the
relationship of log dOM/dt to t, which gives straight lines expressing the
exponential and declining endogenous phases of the BOD curve as shown in
Figures 6 and 7. The slope of the Ln(d oxygen uptake/dt) versus t give
specific rate constants of the exponential growth phase (p values) and the
declining growth phase (/i1 values) of the BOD curve as described by Dojlido
(11), Tabak et al . (45), Oshima et al . (46), and Tabak et al . (54, 55).
Acclimation time values (t0) and the time values for the initiation and
termination of the declining growth phase (tj and t2) for each test compound
were determined from linearized expressions of BOD curves.
Estimation of Honod Kinetic Parameters
The estimations of the Honod Kinetic parameters, maximum specific
growth rate constant, ^ half saturation constant, K and growth yield
constant, Y were determined directly from experimental oxygen uptake curves
without the consideration of initial growth and growth yield assumption
[Jobbagy, Grady and Tabak (57) and Tabak et al . (54)].
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If the concentrations of the substrate, the products and the biomass
are all expressed in BOD units, then the oxygen uptake (Qu at any time in
the batch reactor may be calculated from
Ou ' (Sso - S.) - (X-X0) - (S-So) (4)
where Sso, Spo and X0 are the concentrations of substrate, products and
cells, respectively, at time zero.
To apply equation (4) for the determination of kinetic coefficients,
equations must be available which express the concentrations of soluble
substrate (Ss), soluble product (Sp) and biomass (X) as functions of time.
For batch reactors, those equations are:
dSs/dt - -(4/Y}SsX/Ks + S,) (5)
dS/dt - (Y|A/Y)S,X/(Ki + S.) (6)
dX/dt - ^S,X/(K, + S,) - KsbX/(Ks + S.) (7)
where Ss = soluble substrate concentration; Sp = soluble product
concentration; Y = product yield; and b = decay coefficient.
To calculate oxygen uptake in a batch reactor, equations (5), (6) and
(7) must be solved simultaneously, and the resulting values of Ss, S and X
over time are substituted into equation (4),
Determination of Y Constant
Y - the true yield parameter or the ratio of growth of biomass to
substrate utilization, can be obtained from the experimental oxygen uptake
curve at the initiation of the plateau of the curve as shown in Figure 8,
with the use of equation:
Y = U-Oupt/S0)-Yp (8)
A vertical line is drawn at the point of intersection of the tangents of the
exponential and plateau phases of the curve. The oxygen .uptake value
obtained at the point of intersection of the vertical line (drawn through
intersection of tangents) and oxygen uptake curve is the Oupt value -
[cumulative oxygen uptake value at the initiation of plateau].
The 0 t value is then plugged into the equation (8), Y - ($p~Qupt/S0)-
Y , where S0 is initial concentration of substrate and Yp is soluble product
concentration formed divided by initial substrate concentration, for Y
determination. In this study, product yield (Y ) was negligible.
Dete-nni nation of jim Constant
The initial estimate of nm is obtained by the technique of Gaudy et al .
(47, 48). If Y is assumed to be zero, equation (6) is eliminated and if b
(decay constant) is assumed to be zero, equation (7) is simplified to
dX/dt - (MmSsX)/(Ks + Ss) (9)
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Combining equation (5) and (9) and integrating from Sso to Ss and from X0 to
X gives:
X - X0 4 Y(SSO - S,, (10)
If the assumption that S » Ks, the term SS/KS + S? in equations (5) and (9)
or [I] approaches one, and these systems can be simplified through the use
of equation (10) to give equation
dX/dt = MmX (11)
Integrating equation (11) and combining with equation [II] and than
substituting in equation (4) with P and P0 both equal to zero gives
Ln[X0 4 0U/(1/Y)-1) - Ln(X0) 4 ^(tst) (12)
The plot of Ln[(X0 4 0U)/(1/Y) - 1)] versus time will give a straight line
with slope /im- The accuracy of /im(est) will depend upon the size of Ks, but
this value is good enough as an estimate.
/i_ - the maximum specific growth rate can be determined from
experimental oxygen uptake curve plot in the following manner:
(1) Values of the change of Ou with time (dOu/dt) or slopes are determined
along the entire experimental oxygen uptake curve as shown in Figure 9.
(2) These dOu/dt (slope) values are then plotted against the cumulative Ou
values for each time interval, as shown in Figure 10.
(3) The slope of the developed linearized form of oxygen uptake curve is
the estimated ^m value.
Determination of K. Constant
K - the half saturation constant or the substrate concentration at
which the specific growth rate is 1/2 the maximum specific growth rate can
be obtained from the experimental oxygen uptake curve in the following
manner:
(1) Value of 0 t can be calculated from the plot of (dOu/dt) versus Ou
provided the Ks value is 1 or less (insignificant in comparison to S0
value) and the plot contains a linear section with the slope jim, as
shown in Figure 11.
(2) Other (dO /dt) versus 0 plot in which the slope deviates from iim
because of larger Ks values (more significant in comparison to S0) is
illustrated in Figure 12.
(3) The value of dO /dt is determined at the intercept of the straight line
developed from Ihe plot of dOu/dt versus Qu (Figure 11) which contains
a linear section with slope nm.
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(4) Beginning with the value of 1/2 the intercept value, another straight
line (b) is constructed with the slope 1/2 that of the slope of
original line (a) whose slope is /im.
(5) At the point where line (b) intercepts the declining experimental curve
of the plot, a vertical line from that point of interception can
provide the value of Out on the x axis.
(6) This Out value is then used in the determination of Ks with the use of
the equation
St - So - (Out/(l-Yp-Y) - Ks
where S0 « initial substrate concentration and St •= substrate
concentration at time t.
(7) When the Out, Y, YpJ and S0 values are plugged into the equation, the
value of St can be calculated - which is the value of Ks (in systems
where Ks value is 1 or less).
Thus the oxygen uptake value Out associated with 1/2 of the estimate of
um is used in Ks = S0 - Out/l(l - Y) to get the estimate of K . The major
impact of Ks is upon the shape of the oxygen uptake curve in the region of
the plateau. Comparison of the experimental curves to a family of
standardized curves as an initial estimate provides an initial estimate of
Ks that is sufficient for non-linear curve fitting techniques for
quantitation of the kinetic parameters.
Quantitation of Honod Kinetic Parameters
The methodology for quantitation of the Monod kinetic parameters
requires the use of the above specific methods for estimating them initially
and subsequently followed by computer simulation methods coupled with non-
linear curve fitting techniques and is based on the use of measured values
of initial growth and growth yield. The method requires the use of the
kinetic equation relating growth rate of biomass in presence of substrate,
the substrate utilization rate, product formation rate and rate of oxygen
consumption from 02 uptake (BOD) curves to calculate and use the theoretical
oxygen consumption data to quantitate the biokinetic parameters.
The determination of the kinetic parameters associated with
biodegradation requires a series of steps. The initial substrate (S ) and
biomass (X ) concentration must be carefully measured in COD units, the
ratio of trie two values must lie in a certain range in order to allow
independent evaluation of /Jm, K and Y [Simkins and Alexander (38, 39)].
Grady's studies (49, 50) have snown that a SsyX0 ratio of around 20 works
well. The value of Y may be estimated by determining the residual stable
SCOD concentration after substrate depletion (plateau area). It is
numerically equal to the residual SCOD divided by the initial SCOD. The
value of the decay coefficient, b, may be determined by fitting to the
oxygen consumption curve after the plateau when the only activity
contributing to oxygen consumption is endogenous metabolism and cell decay.
Once X0, Sso, Yp and b are known, /zm, Ks and Y may be determined by non-
linear curve fitting techniques [Grady (49, 50)].
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The technique involves the calculation of a theoretical oxygen
consumption using oxygen uptake equation (4) and equations for substrate,
product and biomass concentrations (5) - (7) with assumed Monod parameters.
The residual sum of squared errors (RSSE) associated with the difference in
calculated and experimental oxygen uptake values is used to obtain new
estimates. The above procedure is repeated until a minimum RSSE is found.
The Grid Search technique was selected as a most suitable non-linear
curve fitting technique for application in the determination of the kinetic
parameters from oxygen uptake data, because it can allow easy discrimination
between local minima and the global minimum RSSE. This technique enables a
comparison between the calculated and experimental oxygen uptake data.
Value of Y is fixed. For this value of Y, a pair of (im and Ks which give
RSSE is found on a Mro:K plane. The above procedure is repeated with other
values of Y. Values of nm, Ks and Y which give minimum RSSE associated with
the difference in calculated and experimental oxygen uptake data constitute
the best values of the kinetic parameters.
The values of jim> Ks and Y developed from grid search technique, which
when substituted into equations 5-7, will provide X and S values, which
(when substituted into oxygen uptake equation 4) will in turn provide
calculated oxygen uptake values at the region of the plateau, closest to the
experimental oxygen uptake values, with a minimum RSSE, will constitute the
best quantitative kinetic parameter values.
Development of Multi-Level Respirometric Biodegradation Testing Protocol
The oxygen consumption data generated with the use of electrolytic
respirometry have been adequately utilized for assessing the biodegradative
activity of sludge microbiota, the biodegradability/toxicity of toxic
organic compounds, as well as for the determination of the intrinisic
kinetic parameters of biodegradation. Methodologies have been developed for
quantitating biodegradability and biodegradation kinetics of representative
classes of RCRA toxic organics, with the resultant development of a
comprehensive multi-level respirometric biodegradation testing protocol
based on oxygen consumption data.
The methods of each successive testing level of the respirometric
protocol are characterized by increased complexity and a consequent higher
cost for performing the tests pertaining to each testing level.
Accordingly, the testing levels can be selected as appropriate to the
research needs.
Studies to assess the biodegradability and biodegradation rates of
toxic organics by sludge microbiota with the use of respirometric oxygen
uptake data can involve the use of one of more levels of the protocol,
depending on the amount of information needed for assessing the
biodegradability of the organic toxic pollutant or the toxicant bearing
waste for determination of its fate and rate of biotreatment in the
municipal or industrial waste treatment systems. A logic flow diagram of
the respirometric biodegradation testing protocol, providing a brief
description of each of the testing levels, is shown in Figure 13.
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RESULTS AND DISCUSSION
Respirometric biodegradability, biokinetic and Monod kinetic data for
selected RCRA alky! benzenes, phenols, phthalates and ketones are reported
in this paper. The electrolytic respirometry oxygen uptake data for the
test compounds, the control reference compound aniline, the inhibition and
endogenous control systems were generated revealing the lag phase
(acclimation phase), the biodegradation (exponential) phase, the different
bio-reaction rate slopes (characteristic of the test compound) as well as
the plateau region at which the biooxidation rate reaches that of the
endogenous rate of microbial activity. Figure 14 illustrates a
representative oxygen uptake curve for aniline and the endogenous controls.
Figure 15 shows the replicate pentaerythritol oxygen uptake curves and the
toxicity control (pentaerythritol plus aniline) curve, Figure 16 illustrates
a representative graphical treatment of the percent biodegradation of
pentaerythritol with time, which was developed for each test compound (OECD
studies).
Based on the biokinetic equations relating growth rate of microbiota in
presence of above compounds, the substrate utilization rate, and rate of
oxygen uptake (BOD) curves, specific growth rate kinetic parameters
(biodegradation rate constants) were derived as slope values of the
linearized plots (plots of the log of DO /dt) of exponential and declining
growth phases of the BOD curve. The acclimation time values (t0), and time
values for the initiation and the termination of the declining growth phases
(tj and t2) for the test compounds and aniline were also generated.
The estimations of the Monod kinetic parameters for benzene, phenol,
phthalate, and ketone compounds reported here, were determined directly from
experimental oxygen uptake curves without the consideration of initial
growth and growth yield assumption.
Respirometric Studies with Selected RCRA Alky! Benzene Compounds
The biodegradation of benzene, toluene, ethyl benzene, m- and
p-xylenes, tert-butyl benzene, sec-butyl benzene, butyl benzene, cumene, 1-
phenyl benzene and the reference compound, aniline at 100 mg/L concentration
by 30 mg/L sludge biomass (as measured by oxygen consumption by sludge
microbiota in mg 02/L) was followed over a period of 20 days. The
electrolytic respirometry oxygen uptake and BOD curves were generated and
graphical treatment of the percent biodegradation was established for each
compound. Figure 17 demonstrates typical oxygen uptake and BOD curves for
p-xylene and p-xylene + aniline and Figure 18 illustrates graphically the %
biodegradation of p-xylene with time.
The percent biodegradation data based on the BOD/ThOD ratios for
benzene, toluene, ethyl benzene, m- and p-xylene and the reference compound,
aniline, are summarized in Table 5. All of the above alky! benzene
compounds were shown to be biodegradable substrates at concentration levels
of 100 mg/L when exposed to 30 mg/L of activated sludge biomass under the
environmental conditions of the respirometric testing procedure, and within
the period of 20 days of incubation.
-------
The toxicity test control flask respirometric data revealed no
inhibitory effects by these test compounds at the 100 mg/L concentration
levels on the bio-oxidation of aniline by sludge microbiota.
Table 6 summarizes the bio-kinetic data for the benzenes studied,
showing the specific growth rate constants for the exponential growth phase
(p values) and for the declining growth phase (^' values) of the linearized
form of the BOD curves of these compounds, as well as the t«, t, and t2
kinetic parameters. Figure 19 shows a typical plot of 1^(50^/61) vs. time
for toluene, from which the kinetic parameters were determined.
Table 7 summarizes the Monod kinetic parameter {/im, Ks, Y ) data for
these benzene compounds.
Respirometric Studies with Selected RCRA Phenolic Compounds
The biodegradation of phenol, resorcinol, o-, m- and p-cresols,
catechol, 2,4-dimethyl phenol and the reference compound aniline at 100 mg/L
concentration levels and exposed to 30 mg/L biomass was followed over a
period of 20 days.
All of the phenols were shown to be biodegradable substrates under the
conditions of the respirometric testing procedure. The toxicity test
control flask respirometric data revealed no inhibitory effects by these
compounds at the 100 mg/L levels on the biodegradation of aniline by the
sludge biomass.
Table 8 summarizes the bio-kinetic data for the phenols studied,
showing the specific growth rate constants as well as the t0, tp and t2
kinetic parameters. Table 9 provides the Monod kinetic parameter data for
these phenolic compounds.
Respirometric Studies with Selected RCRA Phthalate Ester Compounds
Evaluation of the biodegradability and determining of bio-kinetics of
degradation of phthalate compounds, dimethyl phthalate, diethyl phthalate,
dipropyl phthalate and butyl benzyl phthalate was achieved with use of
respirometric oxygen uptake data.
All of the above phthalates were shown to biodegradable under the
conditions of the respirometric tests and were shown not to exhibit any
inhibitory effects at the 100 mg/L levels on aniline biodegradation by the
sludge microbiota.
Tables 10 and 11 summarize respectively the biokinetic (first order)
and Monod kinetic parameter data for the selected phthalate esters under
study.
Respirometric Studies with Selected RCRA Ketone Compounds
Respirometric oxygen uptake data from the studies with the selected
ketone compounds, acetone, 2-butanone, 4-methyl-3-pentanone and a cyclic
ketone, isophorone were utilized to determine their biodegradability and
biodegradation kinetic parameters.
-------
All the ketones were shown to be biodegradable at 100 mg/L
concentration levels in media containing 30 mg/L biomass and did not exhibit
any toxicity to aniline biodegradation at these concentrations.
Tables 12 and 13 summarize respectively the first order and Monod
kinetic parameter data for these ketones.
CONCLUSIONS
The experimental data of respirometric studies with several classes of
organic compounds definitely demonstrate that it is possible to measure the
biodegradability (percent biodegradation - as a ratio of BOD to ThOD) and to
determine the kinetics of degradation of single organic compounds by using
only measurements of oxygen consumption in respirometric batch reactors.
The values of the kinetic parameters determined from oxygen consumption data
were demonstrated to be similar to those based on the measurements of
substrate removal and those made with cell growth data.
The generated data on biodegradation, biodegradation rates and
substrate inhibition kinetics through the use of electrolytic respirometry,
will enable the classification of biodegradability of toxic priority
pollutant and RCRA toxic organic compounds and ultimate projection of the
fate of organic compounds of similar molecular structure to those
experimentally studied by way of the established predictive treatability
models based on structure-activity relationships.
With the electrolytic respirometry approach, data base on the removal
of the above compounds by biodegradation fate mechanism can be adequately
generated to support the development of predictive models on fate and
removal of toxics in industrial and municipal waste treatment systems. A
possible relationship between the kinetic parameters and the effect of
different factors on these parameters, as determined through electrolytic
respirometry and the structural properties of the organic pollutant, can
eventually facilitate prediction of the extent and the rate of
biodegradation of organic chemicals in the field of wastewater treatment
systems from the knowledge of the structural properties of the pollutant
organics.
A preliminary predictive biodegradation - structure/activity model
based on the group contribution approach was developed from the generated
biodegradation kinetic data (first order kinetic parameters) with the use of
electrolytic respirometry. It is expected that the model will closely
predict the results found experimentally. In this way, the fate of other
organic compounds may be anticipated without the time and expense of
experimental work.
The electrolytic respirometry biodegradation studies will provide basic
pilot scale treatability information and data which will be used to confirm
methods to predict treatability and the need for pretreatment of
structurally related pollutants (e.g., by structure, anticipated
treatability properties, etc.). This study will thus provide a more
-------
extensive list of pollutants than was covered by experimental data, for
consideration in guiding the Agency to predict the fate of such compounds
without costly experimental testing.
Studies are currently in progress to determine the effect of
temperature, and different sources of sludge biomass (domestic and
industrial wastewater treatment) on the biodegradation kinetics derived from
electrolytic respirometry.
ACKNOWLEDGEMENTS
The authors wish to thank Mrs. Rena M. Howard and Mrs. Diana L.
Redmond, secretaries in the U.S. Environmental Protection Agency's Risk
Reduction Engineering Laboratory, Cincinnati, Ohio, for their excellent and
timely wordprocessing skills in preparing this manuscript for presentation
at the 12th U.S./Japan Conference on Sewage Treatment Technology, October
12-13, 1989 in Cincinnati, Ohio.
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-------
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-------
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TABLE 1
ADVANTAGES OF RESPROMETWC NCTHODS
1. Oxygen uptake ean b* monitored eontinuously and constantly »«a moim
precisely.
X. automation provides mumerieal er binary output «ata for direct
recording or processing (electrolytic) .
3. faapla mmy sot require iilution and therefore it» oxygen uptake
characteristics are measured IB « more natural atata.
Larger aaiapla volumes can ba v«*d so that »or» r«pra»antativ«
ar* obtained and »auBpling «rror» *r* »ini»i»«d.
I. Tb« ««jipl«» ar* «lx«d eontlnuouslf to provid* uuifona contact of
•icroorgaaiisa, cubttrata and oxygen.
«. He cheaical titr*tiou» are r*;uir*d.
7. 3k continuoui record of O2 uptake ie provided with BOB* units bavin;
automatic recording d*vic*».
•. >*rmit treatment plant* and in-streaa oonditions to be simulated
•ore clo»*ly than in dilution t**t.
9. Convenient for aeasuring the affect of various factors on ozygeo
uptake, *ueb as dilution, sut»trat* type and ooncentration, t*cp-
•rature and pretence of toxics, affect of pn, nutrient addition,
Tolua* and source of savage aeed and seed adaptation.
10. Can be used to determine bacterial growth and aubstrate raaoval
coefficients.
21. Much lover coefficient of variability can be obtained with
reipireaetry than with the dilution test because of the larger and
•ore representative aajsples used and the lack of dilution factor.
12. Conditions in nature and in a treatment plant Bay be simulate a
•ore closely in respiroaetar than in a BOD bottle.
13. Dsefull information ia of tan available In very much lass than ia S
days.
14. Zt ia possible to atop the test at a recognisable point en tee
oxygen curve (such as the beginning of the "plateau" which corre-
sponds to tba exhaustion of readily oxidiied aubstrate).
IS. Becoaaendad for stse in? *) routine examination of sewage and trade
vastes and in 1) control of aevage treatment processes, as well as
for C) research atudies because of the ease and precision vith
which variables can be controlled.
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TABLE 2 GENERAL CLASSIFICATION OF RESP1ROMETERS
Jjrpes Bisic Principle of Op-efjtk>n
]. Manometrk Determination of Oj weight changes in * closed system
by measuring o* responding to 02 pressure changes at
constant temperature and volume or volume changes at
constant pressure.
2, Electrolytic Same.
3. Dissolved 02 Us* of dissolved oxyfen probe lo make direct
depletion measurements of depletion of dissolved 02 from
solution.
TABLE 3 TYPES OF RESPIROMETERS BASED ON TECHNIQUES AND APPLICATIONS
Respirometers measuring us exchange
A. Small constant pressure respirometer? -
Measurements are made by observing the change in volume of
{as phase In contact with the respiring liquid, as gaseous 02 is
absorbed.
B. Small constant volume, respiromelers -
The changes in pressure due to 02 uptake is observed.
Calibration is necessary,
C. {.arge respirometers measuring gas eichange —
Recommended especially for studies of treatability.
respirome|ers —
Oxygen pressure is automatically maintained at a constant value
by an electrolysis cell.
Rpipiforneters in which gas tichange is not measured
Oxygen uptake (BOD) is measured by means of a solid O2 electrode.
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TABLE 4 FACTORS AFFECTING RESPIROMETRIC BOD DETERMINATION
Population dynamic* — growth characteristics
Substrate concentration - nutrient deficiency
Rate of oxygen transfer or CC>2 evolution
Type of inoculum
A. General - Indigenous microbial
populations
B. Addition of specific microbiota
to sample
C. Prouuoa
Concentration of inoculum
Dissolved oxygen concentration
Toxicily
Nitrification
Inhibition of
protein synthesis
Affect both
microoganism growth
and substrate
utilization rates
Temperature
pH value and buffering capacity
E/fect of light
Air bubble effect
Mixing
Turbulence
Nutrient concentration Mineral nutrients
Storage of sample
before analysis
Physico - chemical
(actors
R ef t
unt
A ruction Ycssel
> ery;er. senentcr
C pressure indicitor
stirrtr
2. »«=pl* (250 «J)
3' CC2 »tiorbtr
4 pressure iziicitcr
5 •lecticlrte
( •lictrodis
7 recorder
SCHEMATIC DIAGRAM OF A MEASURING UNIT
FIGURE 1
-------
§
I
1
I
!-
Generalized ptot of aubstrate concentration, btotogica!
tolds concentration, and oxygen utilization Airing
exertion of biochemtea! oxygen demand. Circles mark
Inflection points.
FIGURE 2
'-E C3J«VCS O*" TMf
TO THC ^ftOftUW 5JJ«STHAT£
FIGURE 4
Th«
BOD Cunrt
T**t Com pound
Dtf ndition Curvi
•i h
Tlm«
di»§rim for Ihi rri»tionihip of S, J •"
-------
o
"D
V
at
x
I
tr»
ELAPSED TIME
FIGURE 6 Log ( dy/dt ) vs t Plot
( PENTAERYTHRITOL-A }
10
10
10
10
10
10
•AAAAAAAAAAAAAA
10 20 30 40 SO
ELAPSED TIME Cdoy.)
FIGURE 7 L°9 ' dv/dt ' vs * Plot
( PENTAERYTHRITOL & ANILINE )
FIGURE 8 DETERMINATION OF THE START OF THE PLATEAU
IN OXYGEN CONSUMPTION FOR USE IN ESTIMATING Yf
* y "t
-------
Z
o
p
Q.
5
z
O
o
Z
ui
O
X
o
FIGURE 9 PLOT OF OXYGEN UPTAKE RATE
( Ou VERSUS TIME )
ui
** UI
Ou/Nt) varaim Out curva
tr*Mr »ocbon wfth a slope* mlo be unod In eettmatno
\h» vnJuos of KJTI can
h coftipn/lson to SQ .
I canoa whoc« K^volues me
UM-0.1. K5-5, TC-0 4,10-5. 50" 100
FIGURE 12 PWof('*Ou/A1) v«r»u»iOutlnwhlchM»i»lop«d9irW»«
from f (T^xx;nimn K n vnltioB ato moco monificnnt in
ownpnrlnon to tlQ.CK^i- 5)
-------
LOGIC FLOW DIAGRAM FOR THE RESPIROMETRIC BIODEGRADATION TESTING PROTOCOL
LEVEL I -- BIODEGRADATION SCREENING METHOD
DETERMINATION OF PRIMARY BIODEGRADATION FROM OXYGEN UPTAKE DATA
DETERMINATION OF ACCLIMATION
TIME
ACCLIMATION TIME STUDIES
DETERMINATION OF PERCENT
BIODEGRADATION
BIODEGRADABILITY TESTING STUDIES
DETERMINATION OF MICROBIAL
INHIBITION/TOXICITY
SUBSTRATE INHIBITION/TOXICITY
STUDIES
ACCLIMATION
OR
BIODEGRADATION
OR
INHIBITION
OR -
(1) DETERMINATION OF ACCLIMATION
(LAG) TIME OF MICROBIAL BIOMASS
TO TOXIC SUBSTRATE (T0 VALUES)
(2) DETERMINATION OF THE INITIATION
AND TERMINATION TIME VALUES FOR
DECLINING GROWTH PHASE OF BOD
CURVE (T, AND T, VALUES)
(1) MEASUREMENT OF THE RATIO OF
BOD TO THOD IN MG 02/L OF THE
TOXIC SUBSTRATE AS A PERCENT.
(2) MEASUREMENT OF THE PERCENTAGE
OF THE RESIDUAL TOXIC SUBSTRATE
BY DOC VALUE CHANGES.
(3) MEASUREMENT OF THE PERCENTAGE
OF THE RESIDUAL TOXIC SUBSTRATE
BY THE SPECIFIC SUBSTRATE ANALYSIS,
(1) DETERMINATION OF THE INHIBITORY
EFFECTS OF TOXIC SUBSTRATE OR THi
TOXICANT BEARING WASTE ON
MICROBIAL BIOMASS AS A FUNCTION
OF CONCENTRATION.
(2) DETERMINATION OF THE INHIBITORY
EFFECTS OF TOXIC SUBSTRATE OR
TOXICANT BEARING WASTE ON THE
BIODEGRADATIVE ACTIVITY OF
BIOMASS ON BIOGENIC SUBSTRATE(S)
(3) DETERMINATION OF SUBSTRATE
INHIBITORY LEVELS TO BIOMASS AND
TO NORMAL RATE OF BIODEGRADATION
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LOGIC FLOW DIAGRAM FOR THE RESPIROMETRIC BIODEGRADATION TESTING PROTOCOL
LEVEL II -- STUDIES OF BIOKINETICS OF BIODEGRADATION BASED ON RESPIROHETRIC OXYGEN UPTAKE DATA
DETERMINATION OF SPECIFIC GROWTH RATES AND THE
INITIAL ESTIMATES OF MONOD KINETICS OF BIODEGRADATION
DETERMINATION OF THE RATES OF EXPONENTIAL AND
DECLINING GROWTH PHASE OF BOD CURVE
DETERMINATION OF M AND M' CONSTANTS
DETERMINATION OF THE MONOD KINETIC PARAMETERS
FROM THE EXPERIMENTAL OXYGEN UPTAKE CURVES
DETERMINATION OF HM, Ks AND Y PARAMETERS
r:
APPLICATION OF 0, UPTAKE DATA AND BOD VALUES TO
THE DETERMINATION OF BOD KINETICS. UTILIZAITON
OF BIOKINETIC DATA (EXPONENTIAL AND DECLINING
GROWTH RATES) FOR BIODEGRADATION EFFICIENCY
DETERMINATION.
FIRST ORDER KINETIC RATE CONTSTANTS (SPECIFIC
GROWTH RATE PARAMETERS) DETERMINED BY
LINEARIZATION OF BOD CURVES THROUGH A RELATIONSHIP
OF LOG (DOU/DT) TO T WHICH GIVES STRAIGHT LINES
EXPRESSING EXPONENTIAL AND DECLINING PHASES OF
BOD CURVES. SLOPES OF THESE LINES REPRESENT M
AND M' PARAMETER VALUES RESPECTIVELY.
APPLICATION OF 02 UPTAKE DATA AND BOD VALUES FOR THE
DETERMINATION OF THE INITIAL ESTIMATES OF MONOD KINETIC
PARAMETERS DIRECTLY FROM EXPERIMENTAL 02 CONSUMPTION
CURVES WITHOUT THE CONSIDERATION OF INITIAL GROWTH AND
GROWTH YIELD ASSUMPTION.
THESE INITIAL KINETIC PARAMETERS ESTIMATE DATA CAN BE
SUCCESSFULLY APPLIED FOR BIODEGRADATION EFFICIENCY
DETERMINATION IN WASTEWATER TREATMENT SYSTEMS.
SPECIFIC METHODOLOGIES HAVE BEEN DEVELOPED FOR
OBTAINING THE INITIAL ESTIMATES OF THE MONOD KINETIC
PARAMETERS.
USE OF PRE-ACCCLIMATED BIOMASS OR BIOMASS ACCLIMATED IN
RESPIROMETRIC VESSELS.
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LOGIC FLOW DIAGRAM FOR THE RESPIROMETRIC BIODEGRADATION TESTING PROTOCOL
LEVEL III -- STUDIES TO QUANTITATE HONOD KINETIC PARAMETERS FROM RESPIROHETRIC OXYGEN UPTAKE DATA
USE OF INITIAL ESTIMATES OF KINETIC PARAMETERS, UH, Ks AND Y, TO QUANTITATE THESE BY
MEANS OF COMPUTER SIMULATION METHODS COUPLED WITH NON-LINEAR CURVE FITTING TECHNIQUES
METHODOLOGY BASED ON THE KINETIC RELATIONSHIP
BETWEEN GROWTH RATE, OXYGEN UPTAKE RATE AND
TOXIC SUBSTRATE CONCENTRATION IN A MASS BALANCE
EQUATION TO DEVELOP INTRINSIC KINETIC PARAMETER
DATA FOR PREDICTION OF THE FATE OF THE SUBSTRATE
IN FULL SCALE TREATMENT SYSTEMS
METHODOLOGY SUPPORTED BY PROOF-OF-CONCEPT DATA WHICH
SHOW AN AGREEMENT BETWEEN THE VALUES OF THE KINETIC
PARAMETERS OBTAINED FROM 02 CONSUMPTION DATA AND THOSE
OBTAINED FROM TRADITIONAL MEASUREMENT OF SUBSTRATE
REMOVAL (DOC, SCOD, 14C) OR CELL GROWTH.
METHODOLOGY REQUIRES THE USE OF KINETIC EQUATIONS
RELATING GROWTH RATE IN PRESENCE OF TOXIC SUBSTRATE,
SUBSTRATE UTILIZATION RATE, SOLUBLE PRODUCT
FORMATION RATE AND RATE OF OXYGEN UPTAKE IN ORDER
TO CALCULATE AND USE THE THEORETICAL 02 CONSUMPTION
DATA (0, UPTAKE CURVES) TO QUANTITATE THE
BIOKINETIC PARAMETERS.
METHODOLOGY IS BASED ON THE USE OF MEASURED VALUES OF
INITIAL GROWTH (X0) (BIOMASS CONCENTRATION), SUBSTRATE
CONCENTRATION (Sso) AND GROWTH YIELD (Y), PRODUCT YIELD
(Yp) (DETERMINED BY FITTING 02 UPTAKE CURVE AFTER THE
PLATEAU (02 CONSUMPTION DUE TO ENDOGENOUS METABOLISM AND
CELL DECAY).
r:
or
ONCE X0, Sso, AND Yp AND B (CELL DECAY) VALUES ARE KNOWN, THE KINETIC
PARAMETERS, UH, Ks AND Y ARE QUANTITATED BY THE NON-LINEAR CURVE FITTING
TECHNIQUES. INITIAL KINETIC PARAMETER ESTIMATES ARE USED IN THE METHOD.
THE NON-LINEAR CURVE FITTING TECHNIQUE (GRID SEARCH METHOD) WAS SHOWN TO BE
MOST APPLICABLE, SINCE IT ALLOWS EASY DISCRIMINATION BETWEEN THE CALCULATED
AND EXPERIMENTAL 02 UPTAKE DATA. RSSE ASSOCIATED WITH DIFFERENCES IN
CALCULATED AND EXPERIMENTAL 02 UPTAKE VALUES ARE USED TO OBTAIN NEW ESIMATES
OF KINETIC PARAMETERS. THIS PROCEDURE IS REPEATED TILL MINIMUM RSSE IS FORMED,
GRID SEARCH TECHNIQUE - VALUE OF Y IS FIXED. FOR THIS VALUE OF Y, A PAIR
OF UK AND Ks VALUES WHICH GIVE RSSE IS FOUND ON A UH:K, PLANE. THE ABOVE
PROCEDURE IS REPEATED WITH OTHER VALUES OF Y. VALUES OF IL, K, AND Y WHICH
-ATED
GIVE MINIMUM RSSE ASSOCIATED WITH THE DIFFERENCE IN CALCUL/
AND EXPERIMENTAL
0, UPTAKE DATA CONSTITUTE THE BEST VALUES FOR THE KINETIC PARAMETERS.
LEVEL III USES EITHER PRE-ACCLIMATED BIOMASS OR BIOMASS ACCLIMATED IN RESPIROMETRIC VESSELS.
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LOGIC FLOW DIAGRAM FOR THE RESPIROMETRIC BIODEGRADATION TESTING PROTOCOL
LEVEL IV -- STUDIES TO DETERMINE THE EXTENT OF BIODE6RADAT10N
TESTS FOR ASSESSMENT OF ULTIMATE BIODEGRADABILITY DURING RESPIROMETRIC EXPERIMENTS
RADIOISOTOPE RESPIROHETRY
ELECTROLYTIC RESPIROMETRY COUPLED
WITH RADIOISOTOPE TECHNOLOGY
RESPIROMETRY COUPLED WITH
SPECIFIC SUBSTRATE ANALYSIS
USE OF 14C LABELED TOXIC SUBSTRATES. TRAP HCO
AND MEASURE 14C INTERMEDIATE METABOLITES DURING
INCUBATION. MEASURE RADIOACTIVITY OF THE LIQUID
AND GASEOUS PHASES OF THE CONTENTS IN THE
RESPIROMETRIC VESSEL SYSTEM BY SCINTILLATION
COUNTER TECHNIQUES.
LIQUID AND GASEOUS SAMPLES FOR THE RADIOACTIVITY
ANALYSES CAN BE TAKEN INTERMITTENTLY DURING THE
RESPIROMETRIC RUN BY MEANS OF SPECIALLY ADAPTED
SYRINGES FROM SIDE ARMS OF THE RESPIROMETRIC
VESSEL.
ANALYSIS OF THE RESIDUAL PARENT TOXIC SUBSTRATE,
INTERMEDIATE METABOLITES AND END PRODUCTS OF
METABOLISM BY GC, GC/MS AND HPLC TECHNOLOGY.
LIQUID AND GASEOUS SAMPLES OF THE CULTURE SYSTEM
CAN BE MANUALLY TAKEN FROM SIDE ARMS OF THE
RESPIROMETRIC VESSEL WITH SPECIALLY ADAPTED
SYRINGES DURING THE RESPIROMETRIC RUN.
AUTOMATED ANALYSIS OF THE CULTURE SYSTEM CAN BE
PERFORMED BY WAY OF SPECIALLY DESIGNED SYSTEM
COUPLING THE RESPIROMETRIC VESSELS TO THE
ANALYTICAL INSTRUMENTS.
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500
en
i
300
ft.
x
3
I
x
o
200
100
0
50
10 20 30 40
ELAPSED TIME COoy*)
FIGURE 14 OXYGEN UPTAKE DATA ON ANILINE
500
50
10 20 30 <0
ELAPSED TIME COoys)
FIGURE 15 BIOLOGICAL OXYGEN UPTAKE CURVE
(Run 1 Sample No. 10-12)
100 •
o
o
ID
50-
ELAPSED TIME (doy«)
FIGURE 16 BIODEGRATION (x BOD REMOVAL) CURVE
( PENTAERYTHRITOL )
30
-------
J
^-» -
I 1 1 I I 1 1 1 I I I 1
FIGURE 17 BIOCHEMICAL OXYGEN UPTAKE AND BOD DATA FOR
P-XYLENE AND P-XYLENE • ANILINE
HI
g "
~ TO
I «
C
D
2 *
CD
X »
2V
II
I
5111
TIME. DAYS
p - XYLENE
10 II \2 I)
FIGURE 18 BIODEGRADATION DATA FOR P-XYLENE
O
c
9
m
x
MO
t.x
TIME
TOLUENE
l.w
FIGURE 19 PLOT OF LOG (AOu/At) FOR TOLUENE
31
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TABU 5. SUWttRY Of RCSPIKMCTIIIC SlOOEGRAaftTIQ* DA1A FOR SELECTED BENZENES
PERCENT 8IOOE5RA0AT1QN (BASED ON X 600 REMOVAL)
TIM
(S*j>i) Am line
0.0 0.0
1.0 2.58
2.0 3.«
3.0 3.94
4.0 14.95
5,0 102.0
6.0 102.0
7,0 102.0
8.0 102.0
9.0 102.0
10.0 102.0
11.0 102.0
12.0 102.0
13.0 102.0
TABLE 6.
COMPOUNDS
Aniline
(E»peri«ent 1)
(Enperiaent 2}
Benzene
Ethyl benzene
Toluene
p-Iylene
••Xyltne
tert-Butjrl benzene
sec -But? 1 benzene
Cuaene
Butyl benzene
1-Pheny) hexjne
Benzene
0.0
2.11
2.11
4,15
4.97
71.5
74.22
81.85
93.37
93.89
95. 45
95.45
96.13
»7.46
SUWMRY OF
ThOO
for 100
310
310
308
317
313
317
317
322
322
320
322
326
toluene
0.0
2.78
8.81
8S.11
89.04
94.34
97.92
100. 0
101.18
103.48
103.48
103.48
103.48
103.48
BIO-KlNniC DATA
't>
•g (
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TABU 7. SUMMIT Of MOWO K1KCTIC PARAMETEH DAI* FOR SELECTED KH2M CmPOUNDS
tWPQUNDS
Anil me
Benzene
Ethyl benzene
Tolouene
p-iylene
•-Xylene
tert-8utyl benzene
sec-Butyl benzene
Cuaene
lutyl btniene
I-Phenyl hexane
it - oaxiBua specific
K, • half saturation
Tf » growth Jf1t)d, •$
IK"
4.00
4. SO
4.00
2.00
3.90
2.00
4.40
3.50
2.44
3.30
4.00
growth rate.
•0 biomass
•9 Substrate
0.38
0.23
0.23
0.20
0.26
0.14
0.66
0.64
0.66
0.80
0.67
K.
6.15
J.30
9.00
9.09
8.33
11.50
1.80
2.39
2.44
5.17
2.15
i.
6.10
22.16
11.81
54.74
67.00
35.61
19.04
22.00
17.64
34.50
35.09
constant; concentration af substrate it *Jl.
tit amass formed/*;
substrate consumed.
TABLE 8. SUWARY OF 8IO-K1NCTIC DATA FOR SELECTED PHENOLIC COMPOUNDS
COMPOUNDS
Aniline
Phenol
Rtsorcinol
p-Cruol
o-Crtsol
•-Crejol
Cattchol
2,4-OlMthj'l phenol
ThOO
for 100 «j
310
238
189
252
252
252
189
262
(days)
4.00
1.00
1.50
1.00
1.20
1.44
0,85
2.00
t,
(days)
4.70
1.56
1.83
1.34
1.78
2.04
0.94
2.64
(days)
4.83
1.67
2.14
1.52
1.90
2.40
1.12
2.84
(day-1)
2.78.
2.75
S.42
4.70
3.76
1.17
11. BO
3,21
«£l)
3.29
• .26
6.53
S.78
3.J2
C.15
11.15
5.02
ji " tpectflc growth rate constant for •xpontntial growth phast of 800 curvi,
f' - specific growth rttt constant for declining grtxrth phisi ef 100 curve.
38
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TABLE 9. SUNKARY OF NONOO KINETIC PARAMETER DATA FOR SELECTED PHENOLIC COMPOUNDS
COMPOUNDS
Aril me
Pttenol
Resorcinol
p-Cresol
o-Cresol
•-Cresol
Catechol
2,4-di»ethyl phenol
Lag Ti«e
(t,)
d»ys
4.00
1.00
1.50
1.00
1.20
1.44
0.8S
2.00
K blOmlSS
•q suostrjte
0.38
O.S8
0.48
0.33
0.41
0.46
0.49
0.39
(day-1)
6.15
9.82
12.22
6.11
4.10
7.97
12.80
5.62
Ai
6.10
9.43
6.31
27.78
16.41
17.62
43.87
14.07
p - Mximun specific growth rate.
K, - half saturation constant; concentration of substrate at
1t • grovth yield, m) btomiss formed/*) substrate consuaed.
TABLE 10. SlftWRY OF BIO-KINETIC DATA FOR SELECTED PtiTHALATE ESTER COMPOUNDS
COMPOUNDS
Aniline
DiMthyl phthalate
Oitthyl phthalate
Oipropyl phthalate
Butyl benzyl phthalate
ThOO
for 100 m)
310
168
195
211
226
(days)
4.00
3.46
2.00
2.40
2.00
t,
(days)
4.70
3.98
2.97
2.87
2.28
(days)
4.83
4.25
3.30
3.40
2.80
(daj-l)
2.78
2.76
2.16
2.04
4.12
*'
3.29
4.71
2.92
2.00
2.33
* • specific growth rate constant for exponential growth phase of BOO curve.
t' • specific growth rate constant for declining growth phase of BOO curve.
34
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TUU 11. SWMMY OF NONOO KINtTIC PARAMETER DATA Ft* SELECTED PHTMALATE ESTER COMPOUNDS
COMPOUNDS
Aniline
DiMthy) pnthalate
Diethyl phthtlate
Dipropyl pnthalate
lutyl benzyl phthalate
Lag Tiae
days
4.00
3.46
2.00
2.40
2,00
M b>0«i}>
•g substrate
0.38
0.43
0.46
0.48
0.61
•A,,
6. IS
7.07
3.00
5.78
7.80
i.
C.10
41.68
11.67
IS. 81
36. 25
ft • BixlKM specific grwth rite.
K, * htlf siturttion constant; concentration of substrate it
T( • grovth yield, m) biowis forBXt/Bg substrate consumed.
COMPOUNDS
iz. sumwr or BIO-KINCTK DATA FOR SELCCTED «TOHE CWPOUWK
ThOO t, t, t; |i „'
for 100 «g (days) {days) (days) (day-JJ (day-1)
AnlUne
Acetone
2-Buttnone
4-Nethyl-2-p«ntanone
Iiophorone
310
221
144
272
278
4.00
3.70
2.00
i.as
22.30
4.70
3.99
2.20
2.24
23.70
4.83
4.18
2.35
2.35
25.40
2.78
2.45
2.41
2.31
0.73
3.2S
3.98
4.98
4.80
0.38
- specific growth rate constant for exponential growth phase of BOO curve.
• specific growth rate constant for declining growth phase of MO curve.
TABU 13. SUMMARY OF HONDO KINETIC PARAMETER DATA fOfi SELECTED KCTONE COMPOUNDS
COMPOUNDS
Aniline
Acetone
2-Butanone
4-Ncthyl -2-pentanone
Isophorone
Lag Tiae
(t,)
days
4.00
3,70
2.00
l.BS
22.30
M bio«»ss
•g substrate
0.38
0.36
0.39
0.4S
0.43
,-ft-,,
6. IS
4.86
5.11
(.40
1.S7
A,
i.10
9.76
10.79
24.70
27.42
H - MIIBUB specific growth rate.
K, * half saturation constant; concentration of substrate at
T, * growth yield, •? bloaass foraed/ag substrate contused.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before complef
1. REPORT NO,
EPA/600/A-92/174
2,
4, TITLE AND SUBTITLE
RERSPIROMETRIC METHODS FOR DETERMINATION OF BIODEGRAD-
ABILITY AND BIODEGRADATION KINETICS FOR HAZARDOUS
ORGANIC POLLUTANT COMPOUNDS
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
. AUTHORCS)
1)HH Tabak;
2) S. Desai; 3) R. Govind
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
1)US EPA, ORD, RREL, WHWTRD, TAB, BTS, Cincinnati, OH
45268
2)Dept. of Chem. & Nuc. Engrg., Univ. of Cinti, Cinti,
OH 45221
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
CR-812939-010
12. SPONSORING AGENCY NAME AND ADDRESS
Risk Reduction Engineering Laboratory - Cincinnati, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPF <-«= RiPORT AND PERIOD COVERED
Publish^ Paper
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES project Of ficer i Henry H . Taba k 1513)569-7861 ...
CIiatn€«1 a«d Biochemical Detoxification of Hazardous Wastes II, Miami
Beach. FA , 9/10-15/89
16. ABSTRACT
Electrolytic respirometry involving natural sewage, sludge and soil microbiota is
becoming prominent in fate studies of priority pollutant and RCRA toxic organics
to generate biodegradation/inhibition kinetic data. A developed multi-level protocol
is presented for determination of substrate biodegradability and toxicity, microbial
acclimation to toxic substrates and first order kinetic parameters of biodegradation
and for estimation of Monod kinetic parameters of toxic organic compounds, in order to
correlate the extent and rate of biodegradation with a predictive model based on
chemical properties and molecular structure of these compounds. Respirometric
biodegradation/inhibition and biokinetic data are provided for representative RCRA
alkyl, chloro- and nitro-benzenes, phenols, phthalates, ketones, and selected CERCLA
leachate toxic organics. Data on the effects of the source of sludge biomass, temp-
erature and concentration of microbial inoculum and toxic substrate on the kinetics
of biodegradation are also included.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIEHS/OPEN ENDED TERMS C. COSAT1 Field/Group
kinetics
biodegradability,
protocol, toxic organic
compounds
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
38
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Farm 2220-1 (R**. 4-77) PREVIOUS EDITION ts OBSOLETE
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