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Water Environment Research Foundation

Field Validation of Biokinetic Coefficients for Predicting

Degradation of Organic Compounds
Project 98-CTS-3
Extant Respirometric Kinetic Parameter Estimation Procedure
Training Manual
Boris Eliosov and Timothy G. Ellis
March, 2002

Principal Investigator:
Timothy G. Ellis, Ph.D., P.E.
Department of Civil and Construction Engineering
Iowa State University
374 Town Engineering Building
Ames, Iowa 50011-3232
DISCLOSURE: This report has not been reviewed by THE FOUNDATION to determine
whether it contains patentable subject matter, nor has the accuracy of its information or
conclusions been evaluated. Accordingly, the report is not to be considered a published report
and is not available for general distribution, and its distribution is limited to employees and
advisors of the Foundation for the sole purpose of evaluating the progress and future course of
the project described in the report. Until the report has been reviewed and evaluated by THE
FOUNDATION, it should be neither disclosed to others nor reproduced, wholly or partially,
without written consent of THE FOUNDATION.

Extant Respirometric Kinetic Parameter Estimation Procedure

Training Manual

This manual is intended to provide the user with the information required to conduct extant
respirometric kinetic tests. The manual includes a complete list of recommended equipment and
chemicals, details on the experimental procedure, detailed instructions on data analysis, and an
estimate on staffing and training requirements. This manual was written as part of a research
project funded by the Water Environment Research Foundation.
The manual is divided into four sections. Section 1 outlines the rationale behind using
respirometry for kinetic measurements and basic mass balance equations. Section 2 includes a
description of the experimental setup for extant kinetic testing and guidance on collecting the
data. Section 3 provides step-by-step instructions for evaluation of extant kinetic parameter
values. Section 4 contains an estimate of manpower required for measuring extant kinetic
parameters. The manual is intended for use by wastewater treatment plant operations personnel,
engineers, scientists, and anyone else desiring to use this technique.
A copy of this Training Manual and templates for data analysis can be downloaded at:

Section 1. Background
Current research regarding the measurement of the rate of biodegradation of individual organic
compounds in activated sludge systems suggests that the use of extant kinetic tests is the most
appropriate. The term extant refers to the degradative ability that exists in the activated sludge
mixed liquor at the time of sampling. Thus extant kinetics refers to the biodegradation kinetic
parameters currently existing in the activated sludge system. An extant kinetic test is a test that
attempts to measure those kinetic parameters as closely to the original conditions as possible
(i.e., an extant kinetic test is a snapshot of the biodegradative ability of the biomass at the time of
What determines whether a kinetic test is extant or not is the test conditions imposed on the
biomass sample. Activated sludge systems are continuous flow systems (i.e., wastewater is
continuously flowing through the process). When we collect a sample of mixed liquor from the
aeration basin, we are taking it out of its flow through environment and are subjecting it to
conditions that are inherently different from the conditions in the aeration basin. The goal of the
extant test is to make the test conditions in the lab as similar as possible to the conditions in the
plant. An extant test seeks to maintain the characteristics of the biomass in the aeration basin, or
in other words, to cause as little change to the biomass as possible. A truly extant test would
have to be performed in situ (i.e., in the aeration basin itself) since as soon as we take a sample
out of the aeration basin, we are imposing a different environment on it. However, in situ
experiments are considerably more difficult to perform. Therefore, the extant test performed in
the laboratory ideally should be performed as quickly after sampling as possible.
In addition to performing the extant kinetic test quickly after sampling, it is important that the
conditions in the test itself do not alter the biomass. For example, it is important in an extant test

to expose biomass to as little substrate as possible to prevent significant growth. A common

recommendation is to limit the substrate concentration to approximately 0.02 times the biomass
concentration if both are measured in COD units (i.e., 0.02 mg substrate COD/ mg biomass
COD). A higher substrate to biomass concentration (SO:XO) ratio may allow the biomass to
grow significantly to alter the outcome of the test, and it would no longer be extant.
A low SO:XO ratio poses a number of challenges, however. For one, a low SO:XO ratio implies a
very rapid biodegradation response. A rapid test is good for preventing changes to the biomass.
It makes it difficult, however, to measure the rate of disappearance of the test compound,
especially if conventional analytical methods are employed. Consequently, surrogate
measurements of substrate disappearance are often used in place of conventional analytical
For instance, respirometry can be used to track the mineralization of a compound without having
to measure the compound analytically. Respirometry in an aerobic environment is the
measurement of the changes in the dissolved oxygen (DO) concentration or evolution of CO2 as
a result of biodegradation. It is often an attractive alternative in biodegradation studies since DO
(or CO2) concentrations can be monitored easily and continuously. In addition, output from a
DO meter can be connected to a personal computer for data storage and subsequent evaluation.
Respirometric techniques are based on the following relationship between substrate
biodegradation, biomass growth, and oxygen consumption:


OUR = mX



Where: q= specific rate of substrate degradation, h-1

OUR= oxygen uptake rate, mg (Lh)-1
X= competent biomass concentration, mg/L
m= specific rate growth of biomass, h-1
Y= yield coefficient, gCOD biomass formed/gCOD substrate utilized
Equations 1-1 and 1-2 yield the following expression for the DO concentration, substrate
concentration (S), and the competent biomass (portion of biomass involved in biodegradation of
the substrate) concentration (X) in a batch reactor:
(DO0 - DOt) = (S0 - St) (Xt-X0)


Where: DO0 and DOt = dissolved oxygen concentrations at time 0 and time t, respectively, mg/L
S0 and St = substrate concentrations at time 0 and time t, respectively, mgCOD/L
X0 and Xt = biomass concentrations at time 0 and time t, respectively, mgCOD/L
When biomass growth follows Monod kinetics (which it often does), the following expressions
can be written for substrate and biomass concentrations in a batch reactor:

= mX
- kd X
KS + S


Where: m = maximum specific growth rate, h-1

KS= half-saturation coefficient, mgCOD/L
kd= decay coefficient, h-1
Y KS + S


Equations 1-4 and 1-5 can be solved for various sets of kinetic parameters ( m , KS, Y, and kd),
and the solution can be used to calculate the DO profile from Equation1-3, which is then
compared with the measured one to find a set of kinetic parameters that provides the best
Often when an extant kinetic test is performed, the change in the biomass concentration can be
neglected. This is because the amount of substrate supplied to the biomass in an extant test is
small, and the resulting biomass growth is therefore negligible. As a result, the biomass
concentration can be eliminated from Equation 1-3 (Xt-X0=0) and only Equations 1-3 and 1-4
need to be solved, thus simplifying the calculation procedure for evaluating the values of m and
KS. The yield coefficient in this case can be calculated as follows:
Y= (1- oxygen consumed)/(substrate COD consumed)


It should also be noted that the boundary conditions are the initial and final concentrations of the
injected compound. Since the final concentration is not measured directly, it is important that
biodegradation is complete, and the final concentration can be assumed to be equal to zero. This
is typically verified in a respirometric test by the oxygen uptake rate returning to the endogenous
oxygen uptake rate.
When measuring biodegradation kinetics of specific compounds by activated sludge biomass, it
is important to recognize that only a fraction of the mixed liquor biomass has an ability to
degrade the test compound. This biomass is referred to as the competent biomass (X). An
independent estimate of the competent biomass concentration is required to evaluate the m from
the results of respirometric tests, because solution of Equations 1-3 and 1-4 can only provide an
estimates of the lumped parameter m X and KS (the system of two equations with three unknowns
[ m , X and KS] has an infinite number of solutions). The concentration of the competent biomass
can often be estimated from the fraction of COD contributed to the feed by the test compound
(i.e., the concentration of the competent biomass will be in direct proportion to the concentration
of the test compound in the feed). Research conducted in this study (WERF 98-CTS-3) suggests
that this approach is valid for some compounds. It was observed, however, that for some
compounds, especially those with extremely low concentrations in the influent, the COD fraction
significantly underestimates the concentration of the degrading population. In this case, an
alternate method to estimate the competent biomass fraction must be utilized. In order to

determine whether the COD fraction provides a reasonable estimate of the competent biomass
concentration, a model calibration can be performed.
During the model calibration, the m X and KS values should be measured in the mixed liquor
samples obtained from the activated sludge aeration basin. The concentration of the test
compound should also be measured analytically. Aeration basin effluent samples should be
obtained in a manner that minimizes loss of the test compound through volatilization or
continued biodegradation. Extant kinetic tests should be performed as described in Section 2, but
when analyzing the data, the value of the competent biomass concentration should be
temporarily assumed (e.g., 2 percent of the total biomass concentration in the mixed liquor). The
assumed value will have no effect on the outcome of the test and will be adjusted once the
calibration has been complete. After the set of kinetic parameters ( m , KS, and Y) is determined,
the value of m X is calculated, with X as the assumed value of the competent biomass
concentration. After the m X value is measured, the m is calculated based on the measured
concentration of the test compound as follows:
K (1 + k d q C ) 1 + k d q C
m = S
S eq C
The KS value from the extant kinetic test and the SRT at which the facility was operated at the
time of sampling should be used in Equation 1-7. The value of the decay coefficient can be
evaluated as described elsewhere (e.g. Grady et al., 1999) or assumed to be in the range of 0.0070.01 h-1 for municipal wastewater treatment plants. The competent biomass concentration, X,
can now be calculated using the value of m X from the results of extant kinetic tests and m
calculated from Equation 1-7:
X =
The competent biomass fraction, fC, can now be calculated as the ratio of the competent biomass
concentration to the total biomass concentration:
fC =
VSS 1.42
Where: VSS= volatile suspended solids concentration in the mixed liquor, mg/L
The average value of fC observed during the calibration period, is assumed to be constant and can
be used to calculate the competent biomass concentration after the model calibration is complete.
Several respirometric procedures have been used by various researchers to measure extant
kinetic parameters, and the experimental procedures are described in the literature. Ekama et al.
(1986) and Kappeler and Gujer (1992) used respirometers in which aeration was stopped to
measure the OUR, and the biomass was then reaerated for the next OUR measurement. Ubay et
al. (1998) and Szen et al. (1998) used an aerated 1 L reactor that was filled with wastewater and
seeded with acclimated biomass. Periodically samples of the mixed liquor were taken, and an
OUR was measured. Both techniques required a time period of 5-10 min for each OUR
measurement. A higher measuring frequency was achieved by the technique used by Brouwer et
al. (1998) by placing a biomass sample into a 3 L aerated batch reactor, and continuously
recycling the mixed liquor through the respirometer (HRT in the respirometer was 2 min), while

DO concentrations before and after the respirometer were measured every minute. The above
techniques do not account for the amount of oxygen provided to the biomass during the test, and
consequently, make it theoretically impossible to calculate Y from Equation 1-6. Kong et al.
(1996) performed respirometric tests in an aerated batch reactor. The DO concentration in the
reactor was continuously measured, and the rate of oxygen solubilization was incorporated into a
mathematical model. Theoretically, the procedure used by Kong et al. (1996) makes it possible
to uniquely identify Y, m , and KS (Dochain et al., 1995; Vanrolleghem et al., 1995). The authors
however, used an assumed value of Y in their calculations. One of the possible reasons for this
can be a more complex model used (due to accounting for oxygen solubilization) that reduced
practical identifiability of the model parameters.
The experimental procedure described in this Manual utilizes a sealed respirometer with no gas
phase where the DO concentration is continuously measured. This procedure has been
successfully used to measure biokinetic parameters for a variety of organic compounds (WERF
98-CTS-3) and combines the advantages of other techniques (i.e., simple mathematical model,
high measuring frequency, and identifiability of all the kinetic parameters). The general
procedure as described above, however, could be customized for use with a variety of
experimental equipment. A more detailed description of the extant test is provided for the
particular respirometry equipment and data acquisition and analysis software used in this

Section 2. Experimental setup and procedure

Recommended supplies

Respirometer Vessels
The respirometers should be water-jacketed in order to maintain the temperature during the tests
using a circulating constant temperature water bath. If not temperature controlled, the
temperature will rise during the experiments from the heat generated by the stir plate and friction
from the stir bar. Selection of the vessel volume is based on the amount of biomass available for
testing and the requirement to provide high mixing intensity during the test. When a magnetic
stirrer is used for mixing, a vessel volume of 100-250 mL can be recommended.
Dissolved Oxygen Monitor
Any dissolved oxygen meter with recorder output can be used (e.g., YSI Model 5300 Biological
Oxygen Monitor by YSI, Inc., Yellow Springs, OH). The use of a DO meter with recorder
output allows continuous data acquisition, thus greatly increasing the number of data points
collected during the test and assuring the maximum accuracy of kinetic parameter estimation.

Figure D-1. Experimental setup

Test Compound Solutions

In extant kinetic tests, the initial concentration of the test compound should not typically exceed
2 % of the competent biomass concentration. The quantity of the tested compound that should
be injected depends therefore on the biomass concentration in the treatment plant sample. The
DO concentration in the test compound solution is often different than in the respirometer, and as
a result, injection of a large volume of the test compound solution can result in a significant drop
in DO concentration that is not related to biodegradation. For this reason, the injected volume
should be small compared to the respirometer volume. On the other hand, injecting a very small
volume (on the order of 1:L or less) can introduce a significant error related to measuring the
volume. A recommended concentration of the test compound solution can be estimated as
C =


Where: C=

concentration of the test compound solution, mgCOD/L
the ratio between the initial substrate and biomass concentration,
suggested range: 0.01-0.02


respirometer vessel volume, mL, suggested range: 100-250

competent biomass concentration, mgCOD/L
injection volume, mL, suggested range: 0.025-0.5

If solubility of the tested compound is lower than the concentration calculated from Equation 21, saturated solution should be used, and the injection volume is calculated as follows:
v = * 1000
Where: C*= saturation concentration of the tested compound, mgCOD/L
Buffer solution
To prevent pH from changing during the test, 5-10 mL of the buffer solution per L of the
biomass sample can be added to the aerated beaker. The value of pH of the buffer solution
should be same as in the biomass sample. For most practical applications, buffer with pH 6-8 is
required. To prepare the buffer solution (1.5 N), dissolve 207 g of sodium dihydrogen phosphate
(NaH2PO4H H2O) in 1L of distilled water, and adjust pH to the desired value by adding 6 N
KOH solution (336 g KOH in 1L). Add KOH to water slowly and use constant mixing to prevent
excessive heat buildup. Alternatively, commercial phosphate buffer solutions can be used.
A fresh biomass sample should be obtained. If testing cannot be performed immediately,
refrigerate sample at 4 0C.
PC with the Data Acquisition Hardware and Software
In order to be used for data acquisition, the PC should be equipped with data acquisition board
and connected to the DO monitor through the terminal board. Labtech Notebook for Windows
or similar software can be used. For specific PC requirements, please contact software


DO meter output should be connected to the data acquisition board through the screw terminal
box. The YSI 5300 DO meter has two recorder outputs (on the back panel of DO meter they are
called Channel one and Channel two), which allows measuring DO concentration in two
respirometers. Each output has two terminals on the back of the DO meter: negative (black), and
positive ground (red). Use shielded cable used to connect the DO meter output to the screw
board consists of three wires: black, red and green. Follow the instructions in Table 1 to connect
the DO meter to the screw board.

Table 1. Do meter output connection to the screw terminal board.

Screw # (corresponds to the number of
Shielded cable wire
pin on the computer board)

Red terminal on Channel 1
Black terminal on Channel 1
Red terminal on Channel 1
Red terminal on Channel 2
Black terminal on Channel 2
Red terminal on Channel 2


The following instructions are written for Labtech Notebook for Windows software (NB 19951100) to be installed on a personal computer equipped with a data acquisition board CIODAS08-PGL.
To install the Labtech software, follow the installation instructions. When prompted to select the
hardware, choose the manufacturer of the data acquisition card installed on your computer (e.g.,
Computer Boards). When the installation is complete, the data acquisition card has to be set up.
Run the Notebook program, click on the install command, and select Hardware and click on
Standard Installation. Choose the card model from the list (e.g., CIO-DAS08-PGL) and click
Add. Enter the board number (e.g., #0, to see it, run the board installation program, e.g.,
instacal). Click Done when finished.
After the hardware is installed, the data acquisition process should be customized according to
the experimental setup. To access the setup options, start the Labtech program, and click Setup
command on the tool bar and then click on the option you want to customize. The following
setup options are available:
Blocks: defines how many parallel respirometric tests are performed, and how often the
DO concentrations are measured.
Screens: defines how many windows will be open during the test, and the highest values
of time and DO to be displayed.
Traces: defines the color and line style of each of the DO vs. time curves displayed
during the test.
Logs: defines where and in what format the data collected is saved.
Click Setup command on the tool bar and then click Blocks
1. Number of blocks is equal to the number of DO probes used in parallel plus a time block.
Highlight Number of Blocks and type the number (e.g., 3 if two DO probes are used in
2. In order to customize block 2, highlight the box to the right of Current Block, type 2
and press Enter.
Double click on the box to the right of Block type and select Analog input

Double click on the box to the right of Interface device and select your data
acquisition card model
Double click on the box to the right of Input range and select 0-2.5 V. Note:
this range should be selected for DO meters with recorder output of 0-2 V (e.g.,
YSI Model 5300).
Highlight the box to the right of Scale factor and type DO saturation
concentration (mg/L) at test conditions (use values as given in Table 4).

Accept the default values for the rest of parameters.

3. Highlight the box right to Current Block, type 3, press Enter, and follow the
instructions for step 2. When all the required blocks are customized, click on Done.
Click Set up command on the tool bar and then click Screens
1. Highlight the box to the right of Number of Windows and type 1.
2. Highlight the box to the right of Length of time and type 3600 (3600 seconds will
allow sufficient time for the complete mineralization of the compound, adjust if
3. Highlight the box to the right of Maximum X tic value and type 3600.
4. Highlight the box to the right of Maximum Y tic value and type 25.
5. Click on Done.
Click Setup command on the tool bar and then click Screens
1. Highlight the box to the right of Number of traces and type 2 if 2 DO probes are used.
(Number of traces is equal to number of blocks minus 1).
2. For each trace beginning from block 2 select color (make sure the selected colors are
different from the background color on your screen).
3. Set Y maximum displayed value at 25, and X maximum displayed value at 3600.
4. Click Done.
Click Setup command on the tool bar and then click Logs. Number of files depends on the
number of DO probes operated in parallel, and the desired file format. For convenience of data
analysis, each file should consist of three columns: time, and DO concentrations from two
probes. Thus, if two tests are performed in parallel, the number of files should be 1, for four
probes 2, for six probes 3, etc.
1. Highlight the box to the right of Number of files, type the number (e.g., 2) and press
2. Highlight the box to the right of Current file, type 1 and press Enter.
3. Highlight the box to the right of Data file name, and type the path C:\\directory

When the run is stopped, the file will be automatically saved under a name consisting of the
date and sequential number of the test (e.g., if the test was performed on January 12, the file
name will be 01120 for the first run, 01121 for the second run, etc.).
4. Highlight the box to the right of Number of columns in file and type 3.
5. Highlight block number and type 1 under column 1, 2 under column 2, and 3 under
column 3. Table 1 shows the Table you will see on the computer screen if the logs
setup is completed properly. Click Done if you have 3 blocks (2 DO probes).
6. If you operate 4 DO probes in parallel (5 blocks), highlight Current file, type 2 and
press Enter.
7. Repeat steps 3 and 4, but save the files in different directory (e.g., C:\\directory name
8. Highlight block number and type 1 under column 1, 4 under column 2, and 5 under
column 3. Click Done.
Table 2. Logs setup table.

Number of files [0..12]

Current file [1..2]
Data file name
Data Storage Mode
Number of header lines [0..4]
Header Line 1
Header Line 2
Header Line 3
Header Line 4
Data File Opening Mode
Data File Closing Mode
Number of Records to Close File
Number of Hours to Close File
No. of Columns in File
File Column Number
Block Number
Block Name
Block Units
Field Width (ASCII Files)
Decimal Places (ASCII Real Files)

[ASCII Real]

[Delete Existing File]

[End of Run]








Table 3. Extant respirometry equipment and supplies

YSI DO monitor

Model number



Catalog cost per


Total cost


Good sensitivity, 0-2 V output

YSI Standard Oxygen Probes


Use with high sensitivity membranes for better




Glass respirometer vessels

250 ml, flat bottom,

with two holes in
top for #2 stopper

Custom fabricated by Tudor Scientific Glass

(800) 336-4666



Computer Boards data acquisition



Available at



Screw Terminal Board


For connecting data acquisition board

to DO meter





For connecting screw terminal board to data

acquisition board



Shielded cable

M4 326 E-120910

For connecting screw terminal board to DO

meter output



Data acquisition software (available at












Labtech Notebook for Windows
Other supplies
Polyethylene tubing

For connecting water bath to respirometer

For aerating the respirometer vessels with

Capillary polyethylene tubing

Circulating constant temperature
water bath
Magnetic stir plates
Teflon Stir bars
Oxygen cylinder
1.5 N Phosphate buffer
10 mL and 50 mL Teflon syringes
250 :L and 500 :L syringes (fixed

S18500 Nuova II

11/2 H 5/16

Gas tight


Experimental procedure

1. Preparation of the DO probes. When high sensitivity membranes are used, it is

recommended to change them a day before performing respirometric tests, and to leave
overnight in aerated water. Then the DO probes should be calibrated using aerated tap
water (i.e., saturated with atmospheric oxygen and at the temperature of the test).
Equilibrium oxygen concentrations at various temperatures are shown in Table 4.
Table 4. Solubility of oxygen in water exposed to water saturated air at atmospheric
Temperature, OC

DO, mg/L


DO, mg/L

Temperature, OC

DO, mg/L







Note: mixing intensity and temperature can affect performance of DO probes and the signal
response. Therefore, calibration should be performed at the same mixing intensity and
temperature as the kinetic test.
2. Add buffer to the biomass sample to maintain the desired pH.
3. Aerate biomass sample for 30 minutes prior to testing to oxidize residual substrate and
NH4 if it is a nitrifying culture.
4. Measure VSS concentration and pH in the biomass sample.
5. Place DO probe into a hole on top of the respirometer vessel. Fill the respirometer
vessels 70 to 80% full with biomass and bubble oxygen into the biomass solution until
DO concentration reaches approximately 20 mg/L. Close the second hole on top of the
vessel with a thermometer. Fill the vessels completely with the biomass through the
injection port and remove gas bubbles by turning the stir bar on and off frequently and
tapping the respirometer gently to move the air bubbles to the surface of the injection
port. Once the vessels are full and the air bubbles are removed, initiate data acquisition
program to start recording the DO concentration. Once the endogenous oxygen uptake
rate is established (typically 3-5 minutes after initializing data acquisition), inject the
sample with the desired substrate and write down the time of injection. The test is
terminated when the DO concentration drops below 1 mg/L.

6. Use the data to determine the extant kinetics with the Monod Curve Fitting procedure or
other appropriate model.

Section 3. Data Analysis

Normalizing a DO curve

The decrease in DO concentration in extant respirometric tests is a result of two processes:

oxidation of the injected compound by the competent biomass and endogenous respiration.
Since the goal of the test is to evaluate kinetic parameters describing biodegradation of the
injected compound, the DO curve must be normalized (i.e., the background oxygen uptake rate
should be subtracted from the measured OUR).
Procedure for Normalizing an Extant Respirometric Curve
1. The data file is first averaged with a Fortran program entitled ave.exe. This file was
developed to average two curves, and consequently, labtech notebook should be set-up to
list the time and two probe readings in each file. Initiate the averaging execution file and
follow the instructions given (typically a 2 or 4 second averaging interval will provide
enough data points to accurately average the profile).
2. The averaged file can easily be normalized in an MS Excel spreadsheet (MS Office 97
Professional Edition, Version 8.0 was used for this Manual). It can be imported by
simply opening excel and opening the averaged data file within excel. Excel will give
options for importing. Accept the excel defaults.
3. The data should now be listed in columns A, B, C. Data in column A is the time data in
seconds, B is the DO data from probe one, and C is the data from probe 2.
4. The endogenous respiration rate (OURE) must now be determined. It is equal to the OUR
measured prior to the injection of the test compound, and can be calculated as a slope of
the part of DO vs. time curve prior to the injection of the test compound (i.e., only data
measured before the test compound was injected can be used; if the compound was
injected at time that appears in the cell $A$35, the last data point to calculate the OURE is
$A$34). Use the slope function, and copy the slope from curve one into cell $D$1, and
the slope from curve two into cell $E$1. In order to do this, highlight cell $D$1, type in
the command prompt [=Slope(A4:A34,B4:B34)], and press Enter; then highlight cell E1,
type in the command prompt [=Slope(A4:A34,C4:C34)], and press Enter. The values in
cells D1 and E1 are equal to OURE for curve 1 and curve 2, respectively. Note: The
background slope should be the same at the beginning of the curve as the end of the
curve. For cases where the slope is different, an iteration must be performed as
described in step 7.
5. Now the normalized DO curves can be built. The data points for normalized curve
(DON) are calculated by subtracting the OURE multiplied by the time from the measured
DO concentration:
DO N = DO - OURE t
Data points begin in the row 4 (first three rows are occupied by the column name and
units). To normalize curve one, in cell D4 type:


[=B4-$D$1*A4] and use an auto fill function for the rest of the points. Then begin to
normalize curve two by typing in cell E4: [=C4-$E$1*A4] and calculate the rest of the
points using an auto fill function.
6. Now it is beneficial to plot the curves for observation. To plot the data curves and
normalized curve, select columns A, B, C, D, E beginning from the 4th row, and use chart
option in the insert menu. The beginnings and ends of the normalized curves should now
have no slope (see Figure D-2). If this is the case, skip step 7 (normalized data are in
columns A (time), D (curve 1), and E (curve 2).
DO, mg/L
Zero slope

Zero slope

Time, s.

Figure D-2. Normalized DO curve with zero slope (step 7 is required).

If the beginning of the normalized curve has no slope and the end has a slope (see Figure 2),
proceed as described in step 7.
DO, mg/L

Time, s.

Figure D-3. Normalized DO curve with different endogenous rates in the beginning and
in the end of the test (step 7 is required).

7. To calculate the remaining slope for the first curve, highlight cell F1 and type in the
command line [=Slope(An:Am,Bn:Bm)], and press Enter (m is the row number with the
last data point collected in the test, and n is the row number with data point measured 3-5
minutes before the test was terminated). To calculate the slope for the second curve,
highlight cell G1 and type in the command line [=Slope(An:Am,Cn:Cm)], and press
Enter. Copy the content of the cells B4 through Bx to the cells F4 through Fx, and the
content of the cells $C4$ through $Cx$ to the cells G4 through Gx (x is the number of the
last cell before the injection). In the cell under Fx (e.g., F35) type [=$B$35$F$1*($A$35-Ti)], where Ti is the time of injection. The value of Ti is the cell under Ax
(e.g., A35). Use an autofill function to fill the rest of the cells in column F. In the cell
under Gx (e.g., G35) type [=$C$35-$G$1*($A$35-Ti)]. Use an autofill function to fill
the rest of the cells in column G. The normalized curves data is now in columns A(time),
F (curve 1), and G (curve 2).
Evaluation of the competent biomass concentration.

Extant kinetic tests during the calibration period shall be performed at the same pH and
temperature as monitored at the wastewater treatment facility at the time of sampling.
In order to obtain a reliable estimate of fC, the duration of the calibration period should be 3
months and at least six estimates of fC should be obtained over this time period (i.e., one estimate
of fC every two weeks).
Evaluation of extant kinetic parameters.

The set of kinetic parameters that provides the best fit to the normalized DO curve can be found
using a variety of mathematical methods and software. The procedure described in this Manual
utilizes a spreadsheet template created in Microsoft Excel 2000. The template contains an
algorithm using numerical integration (fourth-order Runge-Kutta method) to calculate a
predicted DO profile as a function of the values of m , KS and Y. The defined objective function
is the sum of squared error between the observed and predicted DO concentrations. The set of
kinetic parameters at which the objective function is minimized is estimated using the Excel
Solver tool with the constraint that all the parameters have positive values.
Extant kinetic parameters are evaluated in an iteration process aimed at receiving a set of
parameters that provides the best fit of the normalized DO curve to that predicted by the selected
kinetic model.
Procedures of fitting a normalized DO curve to Monod kinetics.

Open the file named Monod.xls.

Enter the sample name in cell B5 followed by the date in cell B6.
Enter the substrate injection concentration in mg COD / L in cell B9.
Enter the competent biomass concentration in mg COD / L in cell B10. Estimation of the
competent biomass concentration is described below.
5. Enter the time interval between Dissolved Oxygen (DO) values in seconds in cell B11.

6. Copy the DO data from the normalized curve into the J column beginning at cell J32.
Use the paste special function and choose values to transfer the normalized curve.
7. Input the cell values for the initial DO concentration in cell B15. This cell averages the
normalized curve data points of the initial DO concentration. Select cell B15 and input
the final cell of the initial concentration in the average function.
8. Input the cell values for the final DO concentration in cell B16. This cell averages the
normalized curve data points of the final DO concentration. Select cell B16 and input the
initial and final cells of the final concentration in the average function. The row number
for the final DO is listed in cell N30
9. Highlight cell B26. Edit formula: it should read SUM(Km:Kn), where m is the raw
number from the cell M30, and n is the row number from the cell N30 (see step 4).
10. Enter the time value that the injection began (TR) in cell B22.
11. Highlight the SSE cell B26. Select the solver function under the tools heading. If not
already available, choose the Ad-ins and check the solver ad-in. When the solver options
menu comes up, click on solve.
12. The TR value should then be adjusted and the solver function initiated again until the
SSE is minimized. Iterate through by adjusting cell B22 incrementally up or down until a
minimum value for SSE in cell B26 is found.

Section 4. Manpower required for evaluation of extant biokinetic parameters

The manpower requirement was evaluated for an experimental set up consisting of 1 DO monitor
with two recorders outputs, 2 respirometers, and personal computer with the Windows operating
Prior to the beginning the kinetic tests, this manual must be studied, data acquisition card and
software should be installed, and the DO monitor recorder outputs should be connected to the
data acquisition card. The manpower required for studying the manual is approximately 20-22
manh. The manpower required for installing the data acquisition card is approximately 8
manh. Connecting the DO monitor recorder outputs to the data acquisition card will require 2
additional manh. Installation and setup of the data acquisition software requires up to 8 manh
(less time is required if the personnel is familiar with operating the data acquisition programs).
Total manpower required for completing the experimental set up is approximately 40 manh.
Manpower required for evaluation of one set of extant kinetic parameters.
The following operation should be performed to evaluate the extant kinetic parameters for one
test compound:
Changing the DO probes membranes (for 2 DO probes): 0.15 manh
Calibration of DO probes: 0.5 manh
Respirometric tests: 2 manh
Data analysis: 4 manh
Most wastewater treatment plants collect mixed liquor samples on a daily basis for measuring
biomass concentration. For this reason, no additional manpower will be usually required to
collect the biomass sample and measure the biomass concentration.
Manpower required for performing the respirometric tests is based on the experimental data
collected in this project that indicates that an average of four measurements provides a reliable
estimate of the extant kinetic parameters. An average duration of one respirometric test was

assumed to be one hour. This number can vary depending on the test compound type, and
biomass properties. Manpower estimate for data analysis is provided for a person that only has
a basic knowledge of using MS Excel. As the personnel gains experience, the manpower
requirement can be reduced to 2 manh. The total manpower required for evaluation of one set
of kinetic parameters is 5-7 manh depending on the personnel qualifications.
Manpower required for evaluation of the competent biomass fraction.
In order to obtain a reliable estimate of the competent biomass fraction, six sets of extant kinetic
parameters should be measured over a three month period on biweekly basis, and six
measurements of the of the test compound concentrations in the effluent should be performed. It
is not possible to evaluate the manpower required for measuring test compound concentrations
because it strongly depends on the type of the test compound, concentration level to be
measured, available equipment, and personnel qualifications. Note: once the three months
calibration period is complete, no additional estimates of the competent biomass fraction are
necessary unless plant operating conditions change.
Evaluation of the six sets of kinetic parameters: 30-42 manh (5-7 manh) biweekly
Calculation of the competent biomass fraction: 2 manh
The total manpower required for evaluation of the competent biomass fraction is 32-42 manh
plus manpower required to measure effluent concentrations of the test compound.
Once a competent biomass concentration is estimated, eight to ten sets of kinetic parameters
collected over the period of 3-4 months are required to predict the effluent concentration of the
test compound.
Based on the above considerations, the following estimate of manpower required can be
Completing the experimental set up 40 manh
Personnel training 80 manh
Evaluation of the competent biomass concentration 42 manh
Collecting the data for predicting the effluent concentration of the test compound 60
Total: 222 manh over a period of 7 months (approximately 10 manh/week).
References for more detailed information:
Brouwer H., A. Klapwijk, and K.J. Keesman. 1998. Identification of activated sludge and
wastewater characteristics using respirometric batch experiments. Wat. Res., 32, 4: 1240-1254.
Dochain, D., P.Vanrolleghem, and M.Van Daele. 1995. Structural identifiably of
biokinetic models of activated sludge respiration. Water Research, 29: 2571-2578.
Ellis, T.G., D. S. Barbeau, B.F. Smets, and C.P.L.Jr. Grady. 1996. Respirometric
technique for determination of extant kinetic parameters describing biodegradation. Water
Environment Research, 68: 917-926
Ellis, T. G. 1995. The use of batch and fed-batch respirometric techniques for
determining extant kinetic parameters describing single and multiple substrate biodegradation.
Ph.D. Dissertation, Department of Environmental Systems Engineering, Clemson University,
Clemson, SC.


Grady, C. P. L. Jr., G. T. Daigger, and H.S. Lim.1999. Biological Wastewater Treatment:

Theory and Applications, 2nd Edition, Marcel Dekker: New York.
Grady, C. P. L. Jr. and B. S. Jr. Magbanua. 1996. Evaluation of biodegradation rates of
toxic organic chemicals, Project 92-TFT-2, Final Report to the Water Environment Research
Foundation, Alexandria, Virginia.
Grady, C. P. L. Jr., B. F. Smets, and D. S. Barbeau .1996. Variability in kinetic parameter
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Kappeler J. and W. Gujer. 1992. Estimation of kinetic parameters of heterotrophic growth
under aerobic conditions and characterization of wastewater for activated sludge modeling. Wat.
Sci. Tech., 25: 125-139.
Vanrolleghem, P., M.Van Daele, and D. Dochain. 1995. Practical identifiably of
biokinetic model of activated sludge respiration. Water Research, 29: 2561-2570.