Final Report

College of Engineering - Department of Chemical Engineering
CHE 491: Senior Design 2

Spring 2015
EFFECT OF KINETIC INTERACTIONS ON BIOREACTOR PERFORMANCE

IN WASTE TREATMENT APPLICATIONS
(FINAL REPORT)
Group Members
Name
ID#
Mahren Masud
38975
Azka Vicar Mir
39786
Fahmida Anwar
39982
Reshma Sulthana Sherif
42501
Anisul Karim
44246
Sonam Ludhani
49634
Submission Date: 26th May 2015

Advisor: Dr. Zarook Shareefdeen
Coordinator: Dr. Hussain Ahmed
Executive Summary
An inlet stream to a waste treatment plant has various pollutants. In a biodegradation method of
treatment microorganisms use these pollutants as their source of carbon. This project was
designed to investigate the effects of these pollutants interacting with each other. Three systems
have been studied, namely benzene-toluene, toluene-phenol and benzene-phenol. Furthermore
the effect of these kinetic interactions on the overall bioreactor size and economics have also
been determined. For the benzene-toluene system the competitive interaction in the binary
mixture lead to an overall 80% increase in the reactor volume and costs increased by up to 30%
which are deemed as significant.
Table of Contents
Executive Summary ...................................................................................................................................... 1
List of Figures ............................................................................................................................................... 4
Introduction ................................................................................................................................................... 5
Background ................................................................................................................................................. 8
Wastewater Treatment .............................................................................................................................. 8
Bioreactors ................................................................................................................................................ 9
Bacterial Strains ...................................................................................................................................... 10
Bioreactor and Kinetics........................................................................................................................... 10
Pollutants ................................................................................................................................................ 10
Kinetics ................................................................................................................................................... 12
Monod Kinetics ................................................................................................................................... 12
Andrews Kinetics ................................................................................................................................ 13
SKIP Model ........................................................................................................................................ 14
Methodology ............................................................................................................................................... 15
MATLAB................................................................................................................................................ 15
MATLAB for modeling substrate and biomass behavior without interaction .................................... 15
MATLAB for modeling substrate interaction with biomass and each other for interaction ............... 16
PolyMath ................................................................................................................................................. 17
Literature Review.................................................................................................................................... 17
Results ......................................................................................................................................................... 20
Benzene and Toluene .............................................................................................................................. 20
PPO1 ................................................................................................................................................... 20
Consortium.......................................................................................................................................... 22
Toluene and Phenol................................................................................................................................. 24
Benzene and Phenol ................................................................................................................................ 26
Design ......................................................................................................................................................... 28
Equipment Sizing and Material of Construction..................................................................................... 28
Tank 1 (Holding/Storage Vessel) (T-101) ....................................................................................... 28
Tank 2 (Neutralization Tank) (T-102) ............................................................................................. 29
Reactor- (R-101) ................................................................................................................................. 30
Settling Tank- (T-103) ........................................................................................................................ 33
2
Air Stripper-(T-104)............................................................................................................................ 37
Benzene Data .......................................................................................................................................... 37
Adsorber-(T-105) ................................................................................................................................ 41
Pumps .................................................................................................................................................. 43
Piping .................................................................................................................................................. 44
Control ........................................................................................................................................................ 45
Costing ........................................................................................................................................................ 47
Reactor Size ................................................................................................................................................ 48
Data ............................................................................................................................................................ 48
Flow rate .................................................................................................................................................... 48
1000 gal/day ................................................................................................................................................ 48
Biomass ...................................................................................................................................................... 48
2000 mg/L ................................................................................................................................................... 48
Inlet Concentration of pollutants............................................................................................................. 48
500 mg/L ..................................................................................................................................................... 48
HSE- Health, Safety and Hazard................................................................................................................. 49
HAZOP ................................................................................................................................................... 49
UAE Regulations .................................................................................................................................... 53
Economic Analysis ................................................................................................................................. 54
Conclusion .................................................................................................................................................. 56
Future Work ............................................................................................................................................ 56
References ................................................................................................................................................... 58
Appendix ..................................................................................................................................................... 61
List of Figures
Figure 1: Plant Layout .................................................................................................................................. 6
Figure 2: Initial Substrate Conc. vs RE% (B-T) using PPO1 ..................................................................... 20
Figure 3: Graph of initial substrate conc. vs. removal efficiency using PPO1 interaction ...................... 21
Figure 4: Graph of initial substrate conc. vs. removal efficiency using consortium no interaction......... 22
Figure 5: Graph of initial substrate concentration vs. removal efficiency using consortium interaction 23
Figure 6: Graph of initial substrate concentration vs. removal efficiency interaction .............................. 25
Figure 7: Graph of initial substrate concentration vs. removal efficiency interaction .............................. 27
Figure 8: An ideal rectangular sedimentation tank illustrating the settling of discrete particles. [1] ......... 33
Figure 9 Pressure Drop Correlation Curve .............................................................................................. 41
Figure 10: Plant layout with Control and Instrumentation.......................................................................... 46
Figure 11: Bare Module Cost of Plant using CAPCOST ............................................................................ 47
Introduction
This project aims to study the effect of kinetic interaction on reactor sizing, economics
and overall performance of waste treatment. Feed streams with multiple pollutants are fed to bireactors for treatment. These bio-reactors contain microorganisms in order to feed on organic
matter, in this case the organic pollutants in industrial wastewater. When two pollutants enter a
bioreactor, bacteria may degrade one pollutant more over the other. These kinetic interactions
were studied in order to size the bioreactor. Monod, Andrews and SKIP models were employed
and parameters from literature [5] were obtained in order to design a continuous stirred tank
reactor (CSTR) model. A general plant layout was constructed and each equipment was sized to
estimate the overall plant cost. HAZOP was also performed on each equipment. Moreover,
comparisons of removal efficiencies and volume for interaction and non-interaction were
established. Initially, a benzene-toluene system was thoroughly examined where removal
efficiencies in interaction and non-interaction system was analyzed. Subsequently, different
pollutant systems were studied like toluenephenol and benzene-phenol. Figure 1 below
illustrates a simple plant layout which was used in preliminary equipment sizing using heuristics.
Figure 1: Plant Layout

Note: Not drawn to scale
Components
T101: Holding Tank
P101A/B: Centrifugal Pump
P102A/B: Dosing Pump
T102: Flow Equalization Tank/Neutralization Tank
R101: Bioreactor
T103: Settling Tank
P103A/B: Centrifugal Pump
T104: Air Stripper
T105: Adsorber
T106: Adsorber
6
In the schematic above the daily influx of wastewater is initially stored in the holding
tank (T-101). Then it is pumped (via P-101A/B) to an equalization tank (T-102) where the
composition of the water and other parameters such as pH and temperature are measured. If the
pollutant concentration is higher than what the bioreactor can process then the wastewater may
be diluted before being pumped to the bioreactor (R-101). The effluent from the CSTR is fed to a
settling tank (T-103) wherein the solid biomass is allowed to settle. The exit stream from the
tank is pumped (via P-103A/B) to a stripping tower which the feeds to the air stripper (T-104). It
has been determined that the post CSTR treatment should be performed using an adsorber (T105/T-106) [1] and not via liquid/liquid extraction or distillation. Liquid/liquid extraction will
require solvents which in turn will generate more chemical waste and distillation is not be
feasible since the outlet concentrations of the pollutants will be extremely low thereby making it
an exceptionally expensive method.
The adsorber will be packed with activated carbon. Recent research into high
performance activated carbon for benzene/toluene adsorption from industrial wastewater [1] has
shown promising results. Moreover, it has been cited by the US Environmental Protection
Agency as one of the best available environmental control technologies [1].
Background
Wastewater Treatment
Wastewater can be treated either physically, chemically or biologically. Physical means
of treatment is generally best applicable for wastewaters with high solid content and can be
usually carried out through sedimentation, filtration and screening. Examples of chemical
treatment are chemisorption and chemical oxidation.
Physical means such as filtration, sedimentation and screening aims to remove or separate solids
from a liquid stream. However, this project deals with studying industrial waste waters which is
high in organic pollutants not solids. [1]
Adsorption can be a physical or chemical surface phenomena where pollutants can be
removed using absorbers; in other words, physisorption and chemisorption. In physisorption, the
absorbate physically sticks on to the surface of absorbent molecules. The larger the surface area
of the absorbent, the stronger the absorption. In chemisorption, reaction takes place on the
absorbent surface during absorption. Adsorption using activated carbon as an absorbent is quite
popular due its high surface area. However, it can be very costly since activated carbon would
need to be replaced frequently. [1]
Chemical oxidation uses chemical oxidants in order to transform pollutants to less
harmful products. Common oxidizing agents are potassium dichromate, permanganate and
hydrogen peroxide. Although this method is effective in removing contaminants from waste
waters, it also results in toxic chemicals and more byproducts. Removing these chemicals would
be highly costly as additional separation units would be required. [2]
This project, on the other hand, focuses on biodegradation which is a type of biological
treatment. Basically, biodegradation uses micro-organisms to remove organic pollutants from
wastewaters and treatment can be either aerobic or anaerobic. Anaerobic treatment is carried out
in absence of oxygen whereas aerobic treatment uses oxygen to maximize the growth and
efficiency of biomass. Thus, this project was narrowed down to focusing on aerobic treatment.
Bioreactors
In general, bioreactors can simply be classified as either CSTR (continuous flow reactor),
PFR (plug flow reactor), batch or semi-batch. Based on the kinetics, the reactor can be sized.
Currently only a simple CSTR system has been sized and analyzed; however, in the future, more
reactors of this type will be looked at such as the ones mentioned below.
There are different types of reactors that can be used in this process:
1.
Fixed-film options bio-tower These towers consist of a layer of media and a rotary
distributor arm that sprays pretreated wastewater over the surface of the media. This
water moves downwards as air is circulated upwards. As the water moves downwards, a
biological slime of microbes [3] gets cultivated on the surface.
2.
Rotating biological contactors (RBCs) These consist of biomass coated media

arranged vertically on a horizontal rotating shaft. The media is 40% submerged in
wastewater and is exposed to atmospheric oxygen. As the surface area is high, biomass
population increases steadily and excess growth is continuously shed and removed.
3.
Submerged biological contactors (SBCs) This system is similar to RBCs except that
these contain 90% submerged wastewater and provide three times the surface area.
4.
Membrane bioreactors (MBR) These reactors combine both anoxic and anaerobic in
one system. They have submerged membranes that maintain an active biomass
production, thus reducing sludge-settling issues.
5.
Jet aeration This system works either by aerating wastewater mechanical surface
aeration or by injecting pure oxygen through submerged diffusers [4]
6.
Surface aeration This system uses a propeller that sprays wastewater into the air.
Bacterial Strains
As mentioned earlier, biodegradation requires microorganisms or bacterial strains to
remove pollutants. Such bacterial strains require a carbon source to grow which can degrade
hydrocarbons through bio-oxidation reactions. These microorganisms should also be able to
adapt to different pollutants.
So far, the bacterial strains which have been worked with are P. Putida, Rhodococcus,
Gliomastix indicus and consortium, where consortium is a mixture of different bacterial cultures.
Based on these bacterial strains, parameters for calculations were obtained from research.
Bioreactor and Kinetics
Bioreactor refers to an engineered device in which chemical processes are carried out
involving biological organisms. Within the bioreactor a reaction between the pollutants and
biological organism (i.e. bacteria) takes place. This biological reaction results in further growth
of the organism along with the production of one or many byproducts.
Pollutants
A pollutant is a substance presented into the environment that may have undesired effects
on the usefulness of a resource. There are two main groups of pollutants; Inorganic pollutants
10
and organic pollutants .This research focuses on organic pollutants since it deals with industrial
wastewater which is rich in organic pollutants.
11
Kinetics
The amount of bacteria present within the bioreactor is termed as biomass. The growth of
biomass (i.e. X) present within the bioreactor is proportional to the rate of change in biomass
(i.e. ).
The specific growth rate of biomass can be obtained by:
(1)
Equation 1: Growth rate of Biomass

Where rate of change of biomass is equal to proportionality (i.e. ) times the growth of biomass.
is also known as the specific growth rate of biomass. Several kinetic models can be used to
define or mathematically describe . The kinetic models used in this research project are as
follows.
Monod Kinetics
Monod model is a growth model that introduces the concept of limiting nutrients
(substrate). A nutrient is considered to be limiting when a relationship between its exhaustion
and end of its growth is established. This model describes the relation between the growth rate
and the concentration of the limiting nutrient:
= +
(2)
Equation 2: Growth rate using Monod model

The in the previous equation has been replaced another term. Here X is the concentration of
biomass at time t, S is the substrate concentration at t, m is the maximum specific growth rate and
K is the kinetic constant that supports half-maximum specific growth rate.
12
Andrews Kinetics
Andrews kinetic model evaluate the growth kinetics of inhibitory substrates, when the
cells grow on single substrate. This model defines as follows:

++
(3)
Equation 3: Growth rate using Andrews model

Where S is the substrate concentration at t, m is the maximum specific growth rate and K is the
kinetic constant that supports half-maximum specific growth rate and Ki is the inhibition
constant.
A binary mixture with competitive interaction containing (j, q) as pollutants can use the
following equations to define .

+ +
(4)
Equation 4: Maximum specific growth rate for pollutant j

=
, + +
(5)
Equation 5: Maximum specific growth rate for pollutant q
Where mj the maximum specific growth is rate of pollutant j and mq is the maximum specific
growth rate of pollutant q. Sj and Sq. is the substrate concentration of pollutants j and q, and Ks,j is
the kinetic constant for pollutant j and Ks,q is the kinetic constant for pollutant q and K qj is the
interaction parameter describing the effect of q on j and similarly Kjq is an interaction parameter
describing the effect of j on q .
13
SKIP Model
The SKIP model most closely determines the biodegradation of binary mixtures. It can be
used to fit unspecified inhibition types. Where , and / were determined from singlesubstrate experiments.
=
,1
,1 1
,2 2
+
+ 1 + (,1 ) 2 ,2 + 2 + (,2 ) 1
,2
,1
Equation 6: Growth rate using sum kinetics model
14
Methodology
In order to meet the objective of sizing a bioreactor that is acting with and without substrate
interactions, it was necessary to utilize intensive modeling and simulation programs. The results
of the model was evaluated in terms of removal efficiency. This was accomplished through the
use of MATLAB and PolyMath.
MATLAB
MATLAB codes were written for two purposes. MATLAB was first used for modeling substrate
behavior with biomass without the any other substrate interactions in the system. Then
MATLAB was used for modeling substrate interactions with biomass and each other when the
substrates are interacting with each other.
MATLAB for modeling substrate and biomass behavior without interaction
Please refer to Appendix for the specific MATLAB code.
In order to examine the change in substrate over time, it was assumed that biomass was constant
at 2000 mg/L and residence time of 30 minutes. The constants for the model such as specific
growth rate, yield and kinetic parameter were inputted into the program as fixed values. Based
on this, the kinetic model was programmed as a differential equation of change of substrate with
respect to time on MATLAB using the ode45 tool. A list of initial concentrations was developed
in intervals of 50 mg/L. The differential model was run for all values of initial concentration to
give the final concentration of the substrate. This value was used by the MATLAB program to
find the removal efficiency. Initial substrate concentration was plotted against final removal
efficiency as the output of the program.
15
The program required a guess for the final substrate concentration. However, considering that the
final value was a function of initial value and time, it was impossible to provide a constant value
for the guess that would be applicable for points of all concentration. Thus, the guess was
provided as a percentage of the initial concentration. For example, if the initial substrate
concentration is S0, the guess was stated as RS0 where R was usually 0.05 representing a guess
that the final concentration of the substrate would be approximately 5% of the initial substrate
concentration. This was used as a starting point by the MATLAB program to find the final
substrate concentration.
MATLAB for modeling substrate interaction with biomass and each other for interaction
Please refer to Appendix for the specific MATLAB code.
Similar to the first MATLAB model, it was assumed that biomass was constant at 2000 mg/L
and residence time of 30 minutes and a list of initial substrate concentrations were provided as an
input in steps of 50 mg/L. The constants for the model such as specific growth rates, yields,
kinetic parameters and interaction parameters were inputted into the program as fixed values.
This was used to develop the kinetic models for both substrates as differential equations using
the ode45 function.
However, in this case, the final substrate concentration was a function of time, initial
concentration and another substrates degradation as well. The interaction of one substrate upon
another was accounted for in the differential model by developing two equations each
representing the degradation of a substrate respectively while accounting for the interaction of
the substrates. The two differential equations were solved simultaneously to yield the final
concentrations for both substrates. The program used the final concentrations to calculate
16
removal efficiencies for both substrates. These values were plotted against the initial
concentrations to examine the effect of interaction on the removal efficiency.
The graphs for the two MATLAB programs were compared to see the difference that substrate
interaction made on the system.
PolyMath
Please refer to Appendix for PolyMath program.
PolyMath was used to confirm the results from the MATLAB simulations. The same data was
tested on PolyMath. A similar program was developed on PolyMath and the results were
analyzed. The analysis showed the same results as the MATLAB simulation and thus confirmed
the values.
Literature Review
Paper 1[6]
The paper published by Reardon, Mosteller & Bull Rogers, (2000) focuses on the bioremediation
of three aromatic compounds namely, benzene, toluene and phenol which are common pollutants
in the industrial and municipal wastewaters. The biodegradation was performed by a culture of
Pseudomonas putida F1 in batch cultivations separately on each component and then their
mixtures. The sum kinetics with interaction parameters (SKIP) model helped describe the binary
substrate results. This model, coupled with various parameters obtained from single and binary
substrate experiments aided in the prediction of the kinetics of the tertiary substrate mixture.
These kinetic interaction parameters have been extracted in order to be used as data for this
projects MATLAB modelling.
17
Paper 2[7]
This journal by Reardon, Mosteller, Bull Rogers, DuTeau & Kim (2002) deals with the same set
of aromatic compounds, benzene, toluene and phenol. The microorganisms involved are
Pseudomonas putida F1 and Burkholderia sp. strain JS150. The interaction parameters obtained
from this paper can be used to confirm the data obtained from the previous source and thereby be
used in modelling.
Paper 3[11]
This paper by Oh, Shareefdeen, Baltzis, & Bartha (1994) was actually the one that was used to
lay the groundwork for the research. The cultures used in this experiment was Pseudomonas
putida O1 and Consortium and the parameters have been obtained as documented below.
Paper 4 [12]
The paper by Zarook, Shaikh and Ansar (1996) helps lay some groundwork for conducting the
shock loading effects. It includes data for pseudo steady state and random variations to the feed.
A transient bio filtration model developed in this paper can be used for solving transient models
in a CSTR. It also depicts various transient responses of exit concentrations to the random
perturbations.
Paper 5 [13]
The paper published by Rafiei, Naeimpoor and Mohammadi (2014) deals with the performance
of hybrid bioreactors under inlet loading shocks. Conventional membrane bioreactors were
compared against hybrid reactors and it was found that the hybrid reactors had better resistance
18
to fouling effects and also were efficient. This article will be used to perform trials on membrane
bioreactors that are widely used in the chemical industry.
Paper 6 [14]
The paper published by Yao, Ren, Deng, & Wei (2010) discusses the substrate interactions of mcresol and pyridine as single and dual substrates in petrochemical and coking wastewater. The
phenol-biodegradation was done using the bacterium Lysinibacillus cresolivorans and the cell
growth and substrate biodegradation kinetics were studied. Results concluded that the Haldane
model worked well for the single substrate kinetics and dual substrates was found to exhibit
inhibitory effects. The kinetic parameters and results were extracted from the paper and included
into the pollutants database.
Safety book
This book provided a set of heuristics to follow in order to do sizing for most of the unit
operations illustrated in the plant layout.
Hazardous Waste Management
This book was used as a guide to size the air stripper and the adsorbers.
19
Results
Benzene and Toluene
The graphs obtained below are the results tabulated from the non-interaction and interaction models
between benzene and toluene using the bacterial strain PPO1 and Consortium.
PPO1
NO INTERACTION
Initial Substrate Conc. (S0) vs. Removal

Efficiency (RE%)
120
Removal Efficieny, RE (%)
100
80
60
Benzene
Toluene
40
20
0
0
200
400
600
800
1000
1200
Initial Substrate Conc., S0 (mg/L)
Figure 2: Initial Substrate Conc. vs RE% (B-T) using PPO1
Figure 1: Graph of initial substrate concentration vs. removal efficiency using PPO1 no
interaction
20
INTERACTION
Initial Substrate Conc. (S0) vs. Removal Efficiency

(RE%)
120
100
80
60
Benzene
Toluene
40
20
0
0
200
400
600
800
1000
1200
Figure 3: Graph of initial substrate concentration vs. removal efficiency using PPO1
interaction
Results for PPO1 (@ S0 = 500 mg/L)

Pollutant
Removal Efficiency (%)

No Interaction
Benzene
98.2
Toluene
96.9
Interaction
Benzene
15.5
Toluene
22.1
Table 4: Results for PPO1 @ S0 = 500 mg/L
21
Consortium
NO INTERACTION

(RE%)
120
100
80
60
Benzene
Toluene
40
20
0
0
200
400
600
800
1000
1200
Figure 4: Graph of initial substrate concentration vs. removal efficiency using consortium no
interaction
22
INTERACTION

(RE%)
120
100
80
60
Benzene
Toluene
40
20
0
0
200
400
600
800
1000
1200
Figure 5: Graph of initial substrate concentration vs. removal efficiency using consortium
interaction
23
Results for Consortium (@ S0 = 500 mg/L)

Pollutant
Removal Efficiency (%)

No Interaction
Benzene
97.5
Toluene
98.4
Interaction
Benzene
82.6
Toluene
95.5
Table 5: Results for Consortium @ S0 = 500 mg/L
Toluene and Phenol

1. Toluene non interaction was again simulated in MATLAB using parameters from a different
source [5]. Toluene was modelled using Monod kinetics in this study.
24
2. Phenol results illustrated using the Monod model.

(RE%)
100
Removal Efficiency, RE (%)
90
80
70
60
50
Toluene
40
Phenol
30
20
10
0
0
2000
4000
6000
8000
10000
12000
Initial Substrate Concentration, S0 (mg/L)
Figure 6: Graph of initial substrate concentration vs. removal efficiency interaction
25
Benzene and Phenol

1. Benzene results illustrated using the Monod model.
1. Phenol results illustrated using the Monod model.
26

(RE%)
100
Removal Efficiency,RE (%)
90
80
70
60
50
Benzene
40
Phenol
30
20
10
0
0
2000
4000
6000
8000
10000
12000
Initial Substrate Concentraton,S0 (mg/L)
Figure 7: Graph of initial substrate concentration vs. removal efficiency interaction
27
Design
The sizing of the plant (Figure 1) has been performed as follows:
Equipment Sizing and Material of Construction

Tank 1 (Holding/Storage Vessel) (T-101)
Purpose of the equipment
Storage of the entering waste water before any processing begins.
Assumed parameters
= 30
3
= 0.9
= 342367
Calculations for Sizing

Using the flow rate and time constant, volume can be calculated:
=
30
1
1 342367
= 7132.65
60 24
7132.65 = 27.00 3
Safety Factor of 1.5 to be added to volume estimation
= 27.00 1.5 = 40.50 3
Heuristic: For less than 3.8 m3 use vertical tank on legs

Heuristic: Liquids subject to breathing losses may be stored in tanks with floating or expansion roofs for
conservation
Horizontal tank with floating roof will be designed
Heuristic: L/D=2-5 with 3.0 as the optimal ratio
Assume L/D=3.0
=
= 3
=
2
3 = 40.50
4
= .
2
3
4
= = .
Therefore, Volume=40.50 m3, Diameter=2.580 m, Length=7.742 m

28
Tank 2 (Neutralization Tank) (T-102)

To hold the waste water while it is being brought to operating conditions. In this tank, parameters such as
concentration, pH and temperature will be adjusted before it enters the bioreactor.
Assumed parameters
= 30
3
= 0.9
= 342367

Using the flow rate and time constant, volume can be calculated. This holding tank follows the same
heuristics as the first one. However, for the second holding tank, the volume is assumed to be 50% more
than the first one to account for the addition of diluting fluid and pH neutralizers.
= 1 1.50 = 60.75 3
Assume L/D=3.0
=
= 3
=
2
3
4
2
3 = 60.75
4
= 2.954
= 3 = 8.863
Horizontal tank with floating roof, Volume=60.75 m3, Diameter=2.954 m, Length=8.863 m
Therefore, Volume=40.50 m3, Diameter=2.580 m, Length=7.742 m
29
Material of Construction
Considering all the available options and their associated pros and cons, three options for
material of construction have been finalized. Ideally, hasteloy, monel-nickel or stainless steel
would be used to construct the tank due to their high resistance to corrosion. However, they are
extremely costly to implement. This poses a major issue considering that petroleum industry
waste water typically has a high concentration of suspended particles which leads to severe
corrosion.
Considering this, mild steel will be used. To account for its low resistance to corrosion, a strong
polyphoshate inhibitor will be added. This protects against corrosion and prevents scaling from
occurring if the wastewater is left standing for a long period of time.
Reactor- (R-101)
Heuristics:
L/D = 1
Power input in homogeneous reaction stirred tank = 0.5 0.15 HP per 1000 gallons
Ideal CSTR is when 500 2000 revolutions of a well- designed stirrer
Benzene (no interaction)
0 = = 0.9
3
1000
0 = 0.9
=
900
1 3
0 = 400 /
= S = 20
@ 95% conversion
30
= 0.44 1
= 3.36
1
0.44 20
1
=
=
= 0.3767
+
3.36 + 20
=
1
0.65
0.3767
2000
= 1159.1
= 19.32 .
0 0 (0 )
=
900 (400 20)

=
= 17701.9 = .
19.32 .
4
= .
1
= .
= 20
0
Toluene (no interaction)
0 = = 0.9
0 = 0.9
3
1000
= 900
3
0 = 400 /
= S = 20
@ 95% conversion
31
= 0.72 1
= 15.07
0.72 1 20

1
=
=
= 0.4106
+
15.07 + 20
1
= =
1
0.64
0.4106
2000
= 1283.1
= 21.385
0 0 (0 )
=
900 (400 20)

=
= 15992.52 = .
21.385 .
4
= .
1
= .
= 17.7
0
Material of Construction for CSTR
Options:
1. Carbon steel inexpensive, it is capable of resisting abrasion and alkali. Moreover, it is
readily available. However, not resistant to acids and strong alkali and can cause
contamination.
2. Monel-Nickel Minimal discoloration and contamination. It is also resistant to chlorides.
However, can corrode in oxidizing environments and can be costly.
32
Settling Tank- (T-103)

Purpose of the equipment:
Continuously removing the biomass that is being deposited from the reactor using the process of
sedimentation.
Assumed parameter
Volumetric flow rate (Q) = 0.9 m3/min
Calculations for sizing of the settling tank:
Assume a Detention Time (DT) of 1 hour:
DT =
= = 1
. 9 3 60
= 54 3
Figure 8: An ideal rectangular sedimentation tank illustrating the settling of discrete particles. [1]
33
Table 1 Typical design values for a sedimentation basin. [1]
Using Table 1 the typical values are:

Depth of the settling zone (H) = 3.5m; and the ranges are:
Length of the settling zone (L) = 15-25 m
Width of the settling zone (W) = 3 -24 m
The lowest value of the length is too large for the purposes of this project. Therefore, a typical value
for depth is assumed at 3.5 m and a length to width ratio of 1:4 is also assumed [1]. These assumptions
are then utilized to find the length of the clarifier.
Volume = L x W x H
= 4
Volume = 4 2 x H
2
2
543
=
=
= 1.96
4
4 3.5
= 4 = 4 1.963 = 7.86
Therefore, using this length area can be calculate:

=
= 7.86 1.96 = 15.42 2
34
Therefore, the over flow rate is [1]:
Where:
vo is the overflow rate (m/min)
Q is the volumetric flowrate (m3/min)
A is the area (m2)
15.422
0.9
= 0.058 0.06
Assuming an efficiency () of 95 %:
Using the formula:
Where:
is the efficiency.
H is the depth of the settling zone.
h is the vertical fall over length.
= = 0.95 3.5 = 3.33
Using the h, the settling velocity (vs) can be calculate by:
35
3
3.33 0.9
= 0.055
0.06
2
3.5
15.42
To assure a good sedimentation tank design settling velocity (vs) must be greater than or equal to the
overflow rate (vo) [1].
It can be seen above that vs is equal to vo, hence it can be concluded that the design of the sedimentation
tank is reliable.
The Weir overflow rate (WOR):
=
= 2
= 2 1.96 = 3.92
Therefore,
3
0.9
3
=
= 0.229
3.92

Taking all available options into consideration and weighing their pros and cons, two material such as
stainless steel and carbon steel have been finalized. They are extremely costly however, they have high
resistance against corrosion and prove to be cost efficient in the long run.
36
Air Stripper-(T-104)
An air stripper was selected to remove the pollutants from the outlet of the settling tank as it is costeffective for removing volatile organic compounds and is very effective for low concentrations.
Sizing
Benzene Data
Benzene Data
Benzene
Concentration (mg/L)
1.4
Flowrate (gal/day)
Viscosity of Liquid (kg/m.s) (Engineering
Toolbox, n.d.)
Viscosity of Gas (kg/m.s) (Engineering Toolbox,
n.d.)
Density of liquid (kg/m3) (Engineering Toolbox,
n.d.)
Density of gas (kg/m3) (Engineering Toolbox,
n.d.)
Density of benzene (kg/m3) (Engineering
Toolbox, n.d.)
Critical surface tension (N/m) (Engineering
Toolbox, n.d.)
Surface Tension (N/m) (Engineering Toolbox,
n.d.)
Temperature (K)
Diffusivity of Benzene in water, DL (cm2/s)
(HyperTextBookShop, n.d.)
Molar Mass of Benzene (g/mol)
10,000
7.98E-04
1.90E-05
995.7
1.165
Packing (Tripack (PVC))

Size (m)
2.50E-02
F
28
at (m-1)
279
868
0.04
0.0712
303
1.02E0-5
Henrys Constant (LaGrega,

Buckingham, & Evans, 2001)
A
-3.19*103
B
5.53
78.11
3

, (
)=
78.11 1003
3
= 89.99
868 1000
3.19 103
3
, = exp ( + ) = exp (
+ 5.53) = 6.75 103 .
303
() =
6.75 103
= 0.270
8.25 105 303
37
( ) = 20
= 0.5,
= 0.202
Onda Correlations (LaGrega, Buckingham, & Evans, 2001)
8.31
, =
= 2.18 3
3
0.20 24 3600 2.204
.
10,000
, = 0.09234
(LaGrega, Buckingham, & Evans, 2001)
35.3147 3
3
=
= 0.015
264.172 24 3600
10,000
3
= 0.015 20 = 0.30
0.30
, =
, =
,
3
1.165 3 0.02832 3
= 0.049 2
2
0.20
.
2.18
=
= 9.79
279 0.798 103
2
2.182 279
=
= 1.36 104
2 995.72 9.81
2
2.182
=
= 2.40 104
995.7 0.0172 279
Equation 6- aw equation [1]
0.75 0.1 2
= 1 exp[1.45 ( )
(
) ( 2 )
0.05
0.2
2
(
)

= 279 [1 (1.45 0.560.75 9.790.1 (1.36 104 )0.05 (2.40 104 )0.2 )] = 81.901
Equation 7-kL equation [1]
1
3
3 0.5
0.4
(
) = 0.0051 (
) (
)
( )

38
3
995.7
[
]
3
(0.798 10 ) 9.81
2
0.5
3
2.18
0.798 103
= 0.0051 [
[
]
]
81.90 (0.798 103 )
995.7 (1.02 109 )
= 8.15 105
[279 0.025]0.4
Equation 8 - kG equation [1]

1
0.7 3
2
= 5.23 (
) (
) ( )

0.7
3
0.049
1.9 105
= 279 9.234 106 [5.23 (
)
(279 0.025)2
(
)
279 1.9 105
1.165 9.234 106
= 1.59 103
Equation 9 - Kla equation [1]
1
1
1
=
+

1
1
1
=
+
0.270 1.59 103 81.90 8.15 105 81.90
= 0.0056 1
Preliminary Design
, = 2.18
103
= 121.11 2
2
. 18
.
, = ( ) = 0.270 20 = 5.4
, =
= 1400
121.11
= 0.40
55,600 0.0056
(99.5% ) = 0.005 = 7
39
Equation 10- NTU equation [1]
( 1) + 1

=
ln [
]
1
1400
(5.4 1) + 1
5.4
=
ln [ 70
] = 6.3
5.4 1
5.4
, = = 6.3 0.4 = 2.5 = 8.3
20%,
= 8.3 1.20 = 10
Pressure Drop Calculations
= 2.18
0.2048 = 0.450 2
2
.
.
= 0.3
3
3
3
=
0.0085
35.3147 3
= 0.049
0.2048 = 0.010 3
2
.
.
0.5 0.450 0.073 0.5

( ) =
(
) = 1.5

0.010
62.2
2
0.012 28
=
= 1.9 105
0.073 62.2 32.17
Using the following chart, we can find the pressure drop in the tower. However, the calculated value for
the Ordinate is lower than the starting point in the chart. Hence, the lowest curve was assumed to be the
pressure drop.
40
Figure 9 Pressure Drop Correlation Curve [http://www.tankonyvtar.hu]
= 10 0.0015
= 0.015 2 = 3.7
Adsorber-(T-105)
Powdered activated carbon was used for adsorption process as it is widely used in biological treatment
processes.
= 70
= 0.0085
= 3.5
(95% )
= 0.0085
3
3
1000
= 0.25
1.165 29
41
, =
= 47.9 , = 0.533 [4]

= 47.9 0.00350.533 = 2.4
= 0.0024
= 0.0085
(0.07 0.0035)
103 3 3600 24
103
= 48.8
= 48.8
6 = 20,333
= 20,333
0.0024
1
30 6
= 3,660
1000
According to Hutchins [3],

Height of one column, d = 2.3m
Assuming,
Height of adsorption zone, AZ = 2m
, = (
2
) + 1 = ( ) + 1 = 1.9 = 2
2.3
Assuming L/D ratio of 2,

Diameter of adsorption unit = 1.15m
42
Pumps
Pump the waste from the holding tank to the bioreactor
Pump the water from the settling tank to the air stripper
Assumptions
= 0.9 3 /
= = 1000 /3
A centrifugal single stage pump will be used
Based on Heuristics:
Power for pumping liquids: kW = (1.67)*[Flow (m3/min)]*[P (bar)]/

Efficiency is selected as 60%
Pump 1-(P-101A/B)
P (bar) = = 1000 3 9.81 2 8 = 74.48 = 0.7848

1.670.9
Power =
3
0.7848
0.6
= 2.0
P1 (Pressure at suction) = Vapor Pressure inside tank (using Raoults law)

1 = + = 94.6 0.5 + 29.1 0.5 = 61.8 = 0.082
Assuming Benzene and Toluene are present in equal proportion
Pressure at discharge (P2) = P1 + P = 0.082 + 0.7848 = 0.8668
43
Pump 2-(P-103A/B)
P (bar) = = 1000
1.670.9
Power =
3
0.530
0.6
9.81
5.4 = 530 = 0.530
= 1.33
P1 (Pressure at suction) = Pressure inside previous unit operation

Pressure at discharge (P2) = P1 + P = 0.8668 + 0.530 = 1.40
Steel and stainless steel alloys provide protection against chemical and rust corrosion and have higher tensile
strengths than plastics, corresponding to higher pressure ratings.
Piping
Typical fluid velocities and allowable pressure drops, which can be used to estimate pipe sizes, are as
follows
Table 2:Typical pipe properties (Towler & Sinnott, 2008)
Total Pipe length= 43.8 m

Q=0.9 m3/min=0.015m3/s
44
From the above table taking the typical properties of non-viscous pumped liquid
4
=
0.0153
0.0153
4

3
1

= 0.0798 0.1382
However for economical pipe the following equation is given
, = 0.5 (Towler & Sinnott, 2008)
Q in 3 /s
= 0.90.5
= 0.122
Control
The control and instrumentation implemented in this design have been outlined in the plant
layout below.
The level gauge monitors the amount of liquid in the systems they are implemeted in.
Furthermore the TI (temperature indicator), TT (temperature transmitter) and TC (temperature
controller) work to control and maintain the temperature in the system since microorganisms are
very sensitive to any change to temperature and pH (pH indicators and controllers are also part of
the system).
45
The fluid flow is a very important factor in the adsorber and hence there is a FT (flow
transmitter) and FC (flow controller) in place to monitor this.
Finally a pressure relief valve is placed on the reactor in case of an excessive pressure build-up.
Figure 10: Plant layout with Control and Instrumentation
46
Costing
Figure 11: Bare Module Cost of Plant using CAPCOST
47
Reactor Size
The results illustrated below are the difference in reactor volume and thereby prices due to
kinetic interaction. This was performed using MATLAB. As tabulated below the same
parameters were used. The only variable was residence time (), which was manipulated to
obtain the same removal efficiency when there is no interaction.
Data
Flow rate
1000 gal/day
Biomass
2000 mg/L
Inlet Concentration of pollutants
500 mg/L
Pollutant
Sinlet
Soutlet
RE%
Benzene
500
12.6599
97.46802
Toluene
500
8.1119
98.37762
(h)
Pollutants
SB0
ST0
SB
ST
RE%Benzene
RE%Toluene
0.5
Benzene+Toluene
500
500
86.8235
22.7142
82.6353
95.45716
0.9
Benzene+Toluene
500
500
11.9554
4.0123
97.60892
99.19754
Volume(m3)
0.07885
0.1420
Cost ($)
11,600
15,000
Due to the effect of interaction the volume and the cost of the reactor increase by 80% and 30%
respectively.
48
HSE- Health, Safety and Hazard

HAZOP
Tank 1
Deviation
No Flow
Possible Cause
Line Fracture
Consequence
Action Required
Loss of feed to tank and

reactor. Reduced output
Regular maintenance and

inspection of lines. Set up
maintenance schedule
Wastewater discharged to
adjacent area
Estimate quantity
released and develop
management plan
Damage to pump due to

air flow
LSL to turn pump off if

level not high on enough
More Flow
Error in FCV
Tank level too high.

Reactor overfill
HSL to turn on pump and

turn off inlet flow.
More Pressure
Buildup of vapor
VOC release into air
Install floating roof
More acidic or basic

compounds present
Inflow conditions
Increased rate of
corrosion. Damage to
equipment
Add corrosion inhibitor to

protect tank
More hardness
Inflow conditions
Increased scaling. Loss of

efficiency and equipment
damage
Add resin to entering

flow to soften water
Tank 2
Deviation
No Dosage
Possible Cause
Consequence
Action Required
Damage of dosing pump
Equalization will not

occur. Biomass threat.
Switch to standby dosing

pump
Empty equalization supply

tanks
Control will not be

accomplished
Low level alarm on the

supply tanks
Error in indicators and

transmitters
Control will not be

accomplished.
Regular maintenance and

re-calibration of
transmitters.
49
No mixing
More Temperature
Uneven mixture.
Corrosion of mixer
paddles
Inhibitor added to tank

Control not accurate.
Industrial discharge at
high temp
Threat to biomass
Jacketed vessel cascade

control system
Pump malfunction
Delay to process
Switch to standby pump
Line Blockage
Fouling and pressure

build-up in pipes
Regular checking of pipes

and maintenance.
Inflow conditions
Threat to biomass
pH control in equalization
tank
Less Flow
Acidic or basic
compounds
Reactor
Deviation
Causes
Consequences
Action
High pressure
High volatile compounds

and increased CO2
production in the reactor
Temperature increase in
reactor
Install high temperature

alarm (TAH)
No/ very less degradation

of pollutants
Excessive organic
loading
Inefficient removal of
pollutants
Reduce inlet flow or add

additional aeration basins
or increase biomass in
reactor.
Low dissolved oxygen
Inadequate air supply
Removal efficiency
decreases
Increase air supply
More death rate than

growth rate
Improper environmental
conditions
No degradation of
pollutants
Use appropriate control

in order to improve
conditions.
High pH
Incoming effluent with a

larger pH
Decrease in biomass
growth
Adjust pH using a pH
indicator controller and
dosing pumps.
50
Settling Tank
Deviation
Causes
Consequences
Action
High flow
Issues with the FCV

(flow control valve)
The settling tank

washouts
HSL (high level switch)

to close FCV at the inlet.
No flow
Accumulation of
activated sludge
Overflow of untreated
water
HSL to open the FCV at

outlet
Low flow
Issues with the FCV
The detention time

increases
The FCV to be opened at

the inlet
Air Stripper
Deviation
Causes
Consequences
Action
More liquid flow in the

inlet
Increased pumping power

due to blockage in the
pipe
Flooding in the column
Flow control with Air to

Water set point
More pressure drop
Blockage in pipe
Flooding in the column
Regular maintenance
Less liquid flow
Less liquid pumped from

the settling tank
Liquid entering the

adsorber
Increase pump flowrate
Less liquid pressure
Pipe ruptured
Flooding
Regular maintenance
No liquid flow
pump failure
No stripping
Activate backup pump;

regular maintenance
51
Adsorber
Deviation
Causes
Consequences
Action
No flow
Pipe leak
Leaking of contaminated air
Regular maintenance
No adsorption
Saturated Activated Carbon
No adsorption
Regular maintenance,
Activate backup column
More concentration of
pollutants
Malfunction of Air stripper
Saturation of carbon
Activate backup adsorption

column
Other contaminants in the

inlet
Improper settling in the

settling tank
clogging of activated carbon
Maintenance of the settling

tank
Pumps
Deviation
Causes
Consequences
Action
Impingement of water on
the impeller causes
erosion corrosion
Activate standby pump
High Flow
High switch valve not

functioning
No flow
Gas Locking
No fluid enters or leaves

the pump; the pumping
system is useless
Activate the standby

pump and deviate flow
Less flow
Cavitation
Reduction in pump
efficiency; damage to
pump components
Low switch level (LSL)

on the preceding tank
should go off
Pump Failure
Pipe rupture due to liquid

build up
Activate standby pump
Reverse Flow
Regular maintenance
Regular maintenance
52
Piping
Deviation
Causes
Consequences
Action
No liquid flow
Blockage in pipe and

pipe Leak
Pump cavitation
If it occurs right before the primary pump, shut

primary pump and activate backup pump. If it
occurs in the common pipeline connecting both
the pump, activate alarm to shut down plant for
maintenance.
Less liquid flow
Partial pipe blocks and

Pipe leakage
Less flow in the

reactor and air
stripper
Regular maintenance, Increase pumping power

to raise flow, activate back up pump
Liquid flow in the

reverse direction
Pump cavitation or
pipeline block
No liquid flow to
the equipments
and Pipe rupture
Use one-way valve and regular maintenance
UAE Regulations
Organic compounds such as Benzene and Toluene are classified as carcinogens, and exposure to
high concentration is hazardous. According to Regulation & Supervision Bureau, the water
leaving any waste treatment can only have a maximum concentration of 10 g/L (Regulation,
2013). Therefore the calculated value of 7 g/L at a conversion of 99.5 % is with in this limit of
regulation and can be deemed safe. In addition, the Air quality standard by the UAE EHS
(Environment, Health and Safety) requires that the maximum amount of Benzene leaving any
plant cannot exceed the limit of 5 g/L (Department, 2013). This plant releases Benzene with a
concentration of 3.5 g/L at 95% conversion, which is lower than the allowable limit. Therefore
is it safe to release it into the atmosphere.
53
Economic Analysis
Wastewater treatment plants are categorized as public projects. The criteria that defines a public
project are: large investment, long life span of 30-50 years, no profit, funding from taxes and
subsidiaries, provides an essential service to the public on behalf of the government and is
politically inclined. Thus the bioreactor waste treatment facility can be considered a public
project.
While profitability analysis is generally adopted for a private sector analysis, public sector
projects do not have profitability as the main goal. In these cases, cost-benefit analysis and ratio
are used to evaluate the feasibility of completing a project. The cost-benefit ratio can be
described by the following equation:
( )
Benefits are the advantages to the public quantified in monetary terms. In this treatment plant,
the benefit would be the savings of the water treatment plant that would have otherwise occurred
using more expensive treatment methods.
Disbenefits are the disadvantages to the public quantified in monetary terms. For the case of this
project, the disbenefit would be the loss of income by using the land that could have otherwise
been relegated to another purpose in an industrial district.
The costs are the expenditures made by the government and include the daily operating costs and
maintenance expenditures. For example, transport and pump and pipe maintenance would be a
cost to this project.
54
Capital Investment is the investment in building the plant and the process. This includes land
costs and all installation costs of the equipment.
If the B/C analysis results in a value greater than 1, the project is considered economically
justified for the specified lifetime.
55
Conclusion
From the results obtained in this project one may conclude that the effect of kinetic interactions on the
bioreactor sizing is quite significant. As illustrated earlier the kinetic interactions cause an 80% increase
in reactor volume and a 30% increase in the overall cost.
Future Work
The system has been costed for two systems: a benzene and toluene system without interaction
and a benzene and toluene system with interaction. In the future, this work will be expanded for
different substrate systems such as toluene-phenol and benzene-phenol substrate interaction
systems.
Following this, the existing results and sizing parameters will be simulated on SuperPro where
the substrate systems will be tested in the sized equipment. This will give a good indication of
the overall efficiency of the process and its practical applicability.
To improve the general model and simulation process, the biomass mass balance will be taken
into account. The biomass is currently treated as a constant at 2000 mg/L. However, the biomass
has its own mass balance because it has its own growth and death rate while producing carbon
dioxide and water after the oxidation occurs. Therefore, for future work, the biomass mass
balance will be incorporated into the model and the product carbon dioxide will be accounted
for.
Furthermore, current costing does not take into account anything except for equipment purchase
and installation costs. In the future, costs for compressed air, corrosion inhibitor, hardness
reducing resin, utilities such as steam, biomass, acid and base for the neutralization process and
water for concentration equalization will be accounted for. In addition to this, because it is a
governmental project, there is no profitability analysis. However, an economic analysis can be
56
conducted based on a cost benefit ratio. This requires information such as benefits and
disbenefits to the public. In the future, this will be incorporated into the economic analysis to
provide a clear picture of how sustainable this project is in the long run and if the benefit of the
project is more than its various associated costs.
57
References
[1] T. E. Schultz, "Biological Wastewater Treatment," Chemical Engineering, vol. 112, no. 10,
pp. 44-46, 48-51, October 2005.
[2] S. A. Bolles, "Modeling wastewater aeration systems to discover energy savings
opportunities," n.d..
[3] ICON, "Pollutants in urban wastewater and sewage sludge," Office for Official Publications
of the European Communities, Luxembourg, 2001.
[4] Z. Shareefdeen, Effects of Kinetic Interactions on Bioreactor Performance in Wastewater
Treatment Applications, 2014.
[5] Zerajic, S., & Stevanovic, J. (2007). The Kinetic Models of the Bioprocess with Free and
Immobilized Cells.CI&CEQ, 13(4), 216-226. Retrieved October 20, 2014, from
http://www.doiserbia.nb.rs/img/doi/1451-9372/2007/1451-93720704216Z.pdf
[6] Reardon, K. F., Mosteller, D. C., & Bull Rogers, J. D. (2000). Biodegradation Kinetics of
Benzene, Toluene, and Phenol as Single and Mixed Substrates for Pseudomonas putida F1.
Biotechnology and Bioengineering, vol. 69, no. 4, pp. 387-400. [Online]. Available:
http://www.chemeng.queensu.ca/courses/CHEE884/documents/Reardonetal2000Biotechnol
Bioeng69-385.pdf. [Accessed: Sept. 29, 2014].
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Biodegradation Kinetics of Aromatic Hydrocarbon Mixtures by Pure and Mixed Bacterial
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58
[8] Chang, Y., Cheng, H., Lai, S., & Ning, H., Biodegradation of naphthalene in the oil refinery
wastewater by enriched activated sludge, International Biodeterioration & Biodegradation,
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wastewater treatment, Journal of Membrane Science, Volume 284, Issues 12, 1 November
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[14] Yao, H., Ren, Y., Deng, X., & Wei, C. (2011). Dual substrates biodegradation kinetics of mcresol and pyridine by lysinibacillus cresolivorans. Journal of Hazardous Materials, 186(2),
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60
Appendix
Meeting Minutes Sample
Senior Design 2 (CHE 491)

Meeting Minutes
Date: 23rd May 2015
Location: Library, IC1
Objective: Final Report and Presentation
Start
Time
Item
Responsible
17:30
0:02
Calls Meeting to order

Opening Remarks to welcome participants
Reviews Agenda
Time Keeper
Coordinator
17:32
0:01
Greetings and Welcome
Leader
17:33
0:03
Reviews Last Meetings Minutes
Leader
17:36
0:24
Discussion about Report

MATLAB
Sizing
Interaction sets
Plant layout
All Members
18:00
0:25
Discussion about Presentation

Prepare plant layout with instrumentation
Individual sizing slides
Economic analysis
All Members
18:25
0:03
Finalize what should be done by next meeting

Recorder reviews assignments
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61
Gantt Chart
62
MATLAB
Sample Code (Toluene Non-interaction)
%tau=1800 and X=2000 mg/L for all
%columns: umax(1/s), Ks, Ki, Kbt, Ktb, Y, S0, S, RE (all other units in
%mg/L or mg/mg or %)
tau=1800; %s
x=2000; %mg/L
load parametersoftoluene.txt %load file with all the parameters

umax=parametersoftoluene(1,1); %identify the parameter according to matrix position
ks=parametersoftoluene(1,2);
ki=parametersoftoluene(1,3);
Y=parametersoftoluene(1,6);
r=size(parametersoftoluene,1);%find number of rows

c=size(parametersoftoluene,2); %find number of columns
for m=1:r
B=@(s)parametersoftoluene(m,7)-s-(((1/Y)*umax*s*tau*x)/(ks+s));
if parametersoftoluene(m,7)>296
x0=0.3*parametersoftoluene(m,7);
else x0=0;
end
parametersoftoluene(m,c+1) = fzero(B,x0);
parametersoftoluene(m,c+2) = ((parametersoftoluene(m,7)parametersoftoluene(m,c+1))/parametersoftoluene(m,7))*100;
parametersoftoluene(m,c+3)=x0;
end
63
d=size(parametersoftoluene,2);
plot(parametersoftoluene(:,7),parametersoftoluene(:,d-1))
title('Initial Substrate Conc. (S0) vs. Removal Efficiency (RE%) toluene')
xlabel('Initial Substrate Conc., S0 (mg/L)')
ylabel('Removal Efficieny, RE (%)')
grid on
% fid = fopen('toluenesolvedputida.txt','wt');
% for ii = 1:size(parametersoftoluene,1)
%
fprintf(fid,'%g\t',parametersoftoluene(ii,:));
fprintf(fid,'\n');
% end
% fclose(fid);
64
Polymath
Non- Interaction
p.putida 01 for Benzene (monode):
tau=30 #mins
#Fo= 3785.41 lit/day
Fo=157.7255 #L/hr
V=(tau/60)*Fo #L
F=.95*Fo
mu=.44 #1/hr
ks=3.36 #mg/L
y=0.65 #g/g
X=2000 #mg/L
f(So)=Fo*So+F*S-(1/y)*((mu*S)/(ks+S))*X*V
So(0)=500 #mg/L
f(S)= Fo*So+F*S-(1/y)*((mu*S)/(ks+S))*X*V
S(0)=8.8812 #mg/L
Re= (So-S)/(So) *100
Results :
p.putida 01 for toluene (andrews):

tau=30 #mins
#Fo= 3785.41 L/day
Fo=157.7255 #L/hr
V=(tau/60)*Fo #L
F=.95*Fo
mu=.72 #1/hr
65
ks=15.07 #mg/L
ki= 44.43 #mg/L
y=0.64 #g/g
X=2000 #mg/L
f(So)=Fo*So+F*S-(1/y)*((mu*S)/(ks+S+ (S^2)/ki))*X*V
So(0)=500 #mg/L
f(S)= Fo*So+F*S-(1/y)*((mu*S)/(ks+S+(S^2)/ki))*X*V
S(0)=15.4809#mg/L
Re= (So-S)/(So) *100
Results :
#consortium for toluene (andrews):

tau=30 #mins
#Fo= 3785.41 L/day
Fo=157.7255 #L/hr
V=(tau/60)*Fo #L
F=.95*Fo
66
mu=.86 #1/hr
ks=11.03 #mg/L
ki= 78.94 #mg/L
y=0.71 #g/g
X=2000 #mg/L
f(So)=Fo*So+F*S-(1/y)*((mu*S)/(ks+S+ (S^2)/ki))*X*V
So(0)=500#mg/L
f(S)= Fo*So+F*S-(1/y)*((mu*S)/(ks+S+(S^2)/ki))*X*V
S(0)=8.1119#mg/L
Re= (So-S)/(So) *100
Results :
consortium for benzene (monode):

tau=30 #mins
#Fo= 3785.41 lit/day
Fo=157.7255 #L/hr
V=(tau/60)*Fo #L
F=.95*Fo
mu=.68 #1/hr
ks=12.22 #mg/L
y=0.71 #g/g
X=2000 #mg/L
f(So)=Fo*So+F*S-(1/y)*((mu*S)/(ks+S))*X*V
67
So(0)=500 #mg/L
f(S)= Fo*So+F*S-(1/y)*((mu*S)/(ks+S))*X*V
S(0)=8.8812 #mg/L
Re= (So-S)/(So) *100
Results:
68
Chemical Mixtures
Biodegradation Kinetics of Aromatic Hydrocarbon Mixtures by Pure

and Mixed Bacterial Cultures
Kenneth F. Reardon,1,2 Douglas C. Mosteller,1 Julia Bull Rogers,1 Nancy M. DuTeau,2 and Kee-Hong Kim1
1Department of Chemical Engineering and 2Department of Microbiology, Immunology, and Pathology, Colorado State University,
Fort Collins, Colorado, USA
Microbial growth on pollutant mixtures is an important aspect of bioremediation and wastewater

treatment. However, efforts to develop mathematical models for mixed substrate kinetics have
been limited. Nearly all models group either the microbial population (as biomass) or the chemical species (e.g., as biological oxygen demand). When individual chemical species are considered,
most models assume either no interaction or that the nature of the interaction is competition for
the same rate-limiting enzyme. And when individual microbial species are considered, simple competition for the growth substrate is the only interaction included. Here, we present results using
Pseudomonas putida F1 and Burkholderia sp. strain JS150 growing individually and together on
benzene, toluene, phenol, and their mixtures and compare mathematical models to describe these
results. We demonstrate that the simple models do not accurately predict the outcome of these
biodegradation experiments, and we describe the development of a new model for substrate mixtures, the sum kinetics with interaction parameters (SKIP) model. In mixed-culture experiments,
the interactions between species were substrate dependent and could not be predicted by simple
competition models. Together, this set of experimental and modeling results presents our current
state of work in this area and identifies challenges for future modeling efforts. Key words: benzene,
biodegradation kinetics, mixed growth substrates, phenol, Pseudomonas putida F1, toluene.
Environ Health Perspect 110(suppl 6):10051011 (2002).
http://ehpnet1.niehs.nih.gov/docs/2002/suppl-6/1005-1011reardon/abstract.html
Organic chemical mixtures are prevalent in

wastewater from industrial and municipal
sources as well as in contaminated groundwater. Common examples of chemical mixtures
that often become pollutants include gasoline
and other petroleum fuels, pesticides, and
wood-treating substances. Landfill leachates are
complex mixtures that contaminate groundwater supplies around the world. Pollutant mixtures may contain only organic chemicals or
may also include inorganics, heavy metals, or
radionuclides. The occurrence of contaminants
in mixtures is an important problem because
the removal or degradation of one component
can be inhibited by other compounds in the
mixture and because different conditions may
be required to treat different compounds
within the mixture. The work reported here
was motivated by the first of these issues as it
applies to pollutant biodegradation.
Researchers have noted that microbial
degradation (metabolism) of a compound in
a mixture can be strongly affected by other
substituents of the mixture (14). This has
been observed not only for mixtures of toxic
chemicals (bioremediation) but also for mixtures of pollutants and readily degraded compounds (wastewater treatment) and mixtures
of sugars (fermentation). To understand mixture effects, one must consider the metabolic
role each compound plays for the microorganisms. The terms homologous and heterologous have been proposed by Harder
and Dijkhuizen (5) for compounds that serve
the same or different roles, respectively.
Environmental Health Perspectives
The effects of other compounds in a

mixture of homologous carbon and energy
substrates on the biodegradation of a chemical can be positive, as in the case of increased
growth at low substrate concentrations (6,7)
or induction of required degradative
enzymes (8). More commonly, negative
interactions are reported. Reasons for
decreased biodegradation rates include competitive inhibition (911), toxicity (12), and
the formation of toxic intermediates by
nonspecific enzymes (13,14).
Although mathematical models of mixed
homologous substrate consumption and
microbial growth have been proposed [e.g.,
(2,11,1519)], this body of literature is
much smaller than that for the modeling of
single-substrate growth kinetics. Most models have been tested with only two substrates, and their applicability to larger
mixtures has been assumed without validation. More recently, models have been proposed and tested for larger mixtures.
Examples include the growth of Escherichia
coli on six sugars (16), the growth of a mixed
culture on benzene, toluene, ethylbenzene,
and o- and p-xylene (BTEX compounds)
(11), and the biodegradation of three
polycyclic aromatic hydrocarbons (20).
In addition to the interactions among
chemical components of a mixture undergoing
biodegradation, the interactions among microbial species in a mixed culture may be important. For example, Lewandowski and
co-workers (21) studied the biodegradation of
VOLUME 110 | SUPPLEMENT 6 | DECEMBER 2002
phenol by several two-species mixed cultures.

Excellent agreement between pure-and-simple
competition theory and experimental data
occurred when the two species in an experiment were both isolated from the same environment. However, when a mixture was
composed of two organisms from different
environments, there was no agreement with
the pure-and-simple competition model.
Conversely, in research by Murakami and
Alexander (22), interspecies interactions
beyond pure-and-simple competition, including interactions harmful to one species while
the other was unaffected, occurred between
members of a binary culture isolated from the
same sewage treatment plant.
At Colorado State University, our group
has been studying the biodegradation kinetics
of chemical mixtures for several years. The
long-term goal of this research is to understand (and model mathematically) the
biodegradation of complex chemical mixtures
by microbial communities. Our strategy is to
first learn from simpler (but representative)
systems: pure cultures degrading mixtures
and mixed cultures degrading single chemicals (Figure 1). In this report, we review our
results at this intermediate level and then
describe the results from a simple chemical
mixturemicrobial mixture experiment.
Finally, we discuss some preliminary results
that may provide insights on the observations
made previously.
This work has focused on the biodegradation of benzene, phenol, and toluene. These
monoaromatic compounds are ideal representatives of chemicals found in pollutant mixtures. They are produced in very large
quantities for use as fuels, solvents, and starting
This article is part of the monograph Application of
Technology to Chemical Mixture Research.
Address correspondence to K.F. Reardon, Dept. of
Chemical Engineering, 200 W. Lake St., Colorado
State University, Ft. Collins, CO 80523-1370 USA.
Telephone: (970) 491-6505. Fax: (970) 491-7369.
E-mail: reardon@engr.colostate.edu
This work was supported by grant 5 P42
ES05949-05 from the National Institute of
Environmental Health Sciences and by the Colorado
Advanced Technology Institute through a fellowship
for D.C.M. received from the Colorado Institute for
Research in Biotechnology. P. putida F1 was provided by D. Gibson, and Burkholderia sp. strain
JS150 was provided by J. Spain. T. Keefe provided
statistical analysis and assistance.
Received 18 December 2001; accepted 13 August
2002.
1005
Chemical Mixtures
Reardon et al.
materials for chemical syntheses (23). As an

outcome of this prevalent use, monoaromatics
are widespread environmental contaminants,
usually in mixtures. Thirty monoaromatics are
listed in the U.S. Environmental Protection
Agencys Priority Pollutants (24), and 11 of
these compounds are in the top 100 chemicals
on the Agency for Toxic Substances and
Disease Registrys Priority List of Hazardous
Substances (25). Two bacterial strains were
used in the work presented here: Pseudomonas
putida F1 and Burkholderia sp. JS150.
P. putida F1 uses toluene dioxygenase (TDO)
to initiate the metabolism of toluene, benzene,
phenol, and other aromatics (26). In contrast,
Burkholderia sp. JS150 can express at least
three initial dioxygenases (12). These strains
are thus interesting and distinct model systems
for the study of mixture biodegradation
kinetics.
Materials and Methods

Microorganisms. P. putida F1 is a wellcharacterized aromatic hydrocarbondegrading bacterium that can use toluene, benzene,
ethylbenzene, phenol, and other aromatics as
sole carbon and energy sources (26). The
biodegradation of toluene by P. putida F1
begins with the oxidation of the aromatic ring
by TDO to form cis-toluene dihydrodiol
(2628), which is then dehydrogenated to
form 3-methylcatechol. This molecule is then
cleaved at the meta position and then converted in three steps to acetaldehyde and pyruvate (2931). TDO also catalyzes the
oxidation of benzene (28,32,33) and phenol
(34,35). In both cases, catechol is formed after
dehydrogenation and is then further degraded
by meta ring cleavage and other reactions to
tricarboxylic acid cycle intermediates. Thus,
P. putida F1 uses the same metabolic pathway
to metabolize toluene, benzene, and phenol.
Burkholderia sp. JS150 is a nonencapsulated mutant of Burkholderia sp. JS1
obtained after ethyl methane sulfonate mutagenesis of strain JS1 (12). In addition to
toluene, benzene, and phenol, this species is
able to degrade a wide range of substituted
aromatic compounds. Strain JS150 has a
much greater metabolic capability than
P. putida F1, with the ability to synthesize at
Single chemical
Single microbial
species
Chemical mixture
Single chemical
Single microbial
species
Multiple microbial
species
Chemical mixture
Multiple microbial
species
Figure 1. Levels of complexity in biodegradation

kinetics research.
1006
least four ring-fission pathways and use three

separate initial dioxygenases (including a
nonspecific TDO) when grown on various
substrates (12).
Media. For all experiments, a modified
Hutners mineral base was used as the carbonfree medium (36), and toluene, benzene,
and/or phenol was added. Phenol was added
before autoclaving, but toluene and benzene
were added after autoclaving to minimize
losses from volatilization (37). For strain
maintenance, cultures of both bacteria were
grown on toluene vapors and stored at 70C
in 10% glycerol.
Chemicals. Benzene (Sigma, St. Louis,
MO, USA; HPLC grade), toluene (Baker,
Phillipsburg, NJ, USA; HPLC grade), and
phenol (Sigma, >99.5% pure) were used as the
carbon sources. Chloroform and p-xylene
(both from Baker; GC grade) were used to
prepare samples for gas chromatography (GC).
All chemicals used for media preparation were
reagent grade.
Analytical methods. Cell concentrations
were measured as optical density at 600 nm
(OD600) with a Bausch & Lomb Spectronic
21 spectrophotometer (Bausch & Lomb,
Rochester, NY, USA) and correlated to biomass concentration (37,38). To quantify the
two cell populations in the mixed culture
experiments, a fluorescence in situ hybridization (FISH) method was developed (39). In
this procedure, 30 L of a culture sample was
applied to slides, which were then dried,
fixed, and dehydrated. The samples were then
exposed to species-specific oligonucleotide
probes that were 5-end labeled with fluorescein isothiocyanate (40), rinsed, and prepared
for counting under a Leitz epifluorescence
microscope (Leica Microsystems AG, Wetzlar,
Germany) equipped with a BioQuant image
analysis system (R&M Biometrics Inc.,
Nashville, TN, USA)(39). For species
Burkholderia sp. JS150, correlations were also
developed between cell concentration
(cells/mL or cfu/mL) and biomass concentration (mg/L): 3.5 106 cells/mL equivalent to
1.0 mg/L biomass for cells grown on toluene,
and 2.4 10 6 cells/mL equivalent to
1.0 mg/L biomass for cells grown on phenol.
Benzene, toluene, and phenol concentrations were measured by GC. Aqueous samples
were extracted with chloroform, and p-xylene
was used as an internal standard (37). Samples
were stored at 4C in 2-mL screw-cap vials
with Teflon-lined rubber septa, until analysis.
Benzene, toluene, and phenol standards were
prepared as aqueous solutions and extracted
with chloroform/p-xylene. The detection limit
of this method for each of the three
compounds was 5 M.
Protocol for batch biodegradation
experiments. All data for biodegradation
kinetics modeling were obtained from batch
VOLUME
bioreactor cultivations inoculated from shake

flask cultures grown on the same carbon
source(s) used in the bioreactor. Two 3-L
Applikon batch bioreactors (Applikon,
Foster City, CA, USA) were used for the
biodegradation kinetic experiments. The
total initial substrate concentration was
approximately 0.5 mM in the liquid phase,
regardless of the number of substrates
involved. In mixture experiments, the substrates were added in approximately equimolar amounts. Henrys law was used to
calculate the amount of toluene or benzene
to be added. All experiments were run at the
operating and initial conditions found to
provide intrinsic biodegradation kinetics,
including a low inoculum size (expressed as
the ratio of substrate to cell mass; a value of
300 was used) (37). The bioreactor was run
as a closed system with no air sparging to
eliminate the substrate loss due to volatility.
The system operated aerobically (dissolved
oxygen levels remained above 5 mg/L),
30C, and without pH control (although the
pH remained in the range of 6.76.9). Less
than 1% of toluene and phenol was lost in
sterile control experiments. Biodegradation
experiments were performed in duplicate,
and replicates were not performed simultaneously. Additional experimental details can be
found in Reardon et al. (37).
Determination of biodegradation kinetics
model parameters. Several mathematical
models were compared for their ability to fit
or predict the experimental biodegradation
kinetics data. The values of all required
model parameters were determined by performing nonlinear curve fitting to the experimental data using SimuSolv, a modeling
and simulation package (Dow Chemical
Company, Midland, MI, USA). Simusolv
employed a Gear method to solve the differential equations and maximized the log of the
likelihood function (LLF) to optimize the
unknown parameters and discriminate
between models (37). The model with the
maximum LLF value and most homogeneous
errors residual plots was chosen. For each
final model, the percent variation explained
(PVE; similar to r 2 value for linear regression)
was calculated using the LLF. The average
value for each of the parameters was found by
separately determining the values for each of
the duplicate experiments and then averaging
these two values.
The tested models were those for cell
growth kinetics (as a function of growth substrate consumption). An equation was also
needed to model substrate depletion. For the
relatively nonvolatile substrate phenol, the
rate of consumption was described as
X
dS
=
,
dt
YX/S
[1]
110 | SUPPLEMENT 6 | DECEMBER 2002 Environmental Health Perspectives
m TOT = mL + mG
H
= mL 1 +
RT
VG
= mL . [2]
VL
Here, m refers to the mass of toluene in the

gas phase (subscript G), liquid phase (L), or
the entire system (TOT). H is the Henrys
law constant, R is the gas constant, T is the
temperature, and VG and VL are the gas and
liquid phase volumes. Henrys law constants
of 8.08 103 atmm3/mol for toluene and
7.31 103 atmm3/mol for benzene at 30C
were used (41). The temperature and volume
of liquid remained essentially constant
during an experiment, and therefore the rate
of substrate consumption can be written as
(S L )X
dS
L =
.
dt
YX/S
[3]
In most experiments, a certain amount of

lag time was observed before any measurable
depletion of substrate or growth of organisms
occurred. Because the models do not account
for this lag time, time zero for modeling was
defined as the time when 2% of the substrate
had been consumed.
Results
Biodegradation of chemical mixtures by
pure cultures of P. putida F1. The first set
of experiments involved the use of P. putida
F1 to biodegrade benzene, toluene, phenol,
and their binary and tertiary mixtures. In
the single-substrate experiments, the growth
kinetics were well fit by the Monod model,
=
1 dX
S
= max L ,
X dt
K S + SL
[4]
in which max is the maximum specific

growth rate and KS is the Monod half-saturation constant. Equation (3) was used to
model the consumption (biodegradation) of
toluene and benzene. The Monod model
parameter values for each of the three substrates are listed in Table 1. The Monod
model provided the best fit for the biodegradation of toluene and benzene by P. putida
F1, although substrate inhibition has been
reported for growth on toluene by other
microorganisms (10). However, in the case of
growth on phenol, well known as an
inhibitory substrate, the fit to the experimental data was slightly improved by use of the
Andrews model (37):
=
1 dX
max S L
=
,
X dt
K S + S L + S L2 / K i
[5]
where K i is an inhibition parameter. In

addition, the growth pattern with phenol as a
substrate was different than that for toluene
in that biomass production continued for
approximately 10 hr after phenol was
depleted. This is indicative of the transient
production of an intermediate that was than
consumed for growth, and we therefore tested
various models that included such an intermediate. However, none of these yielded an
improved fit to the data (37). We chose to
use the Monod model rather than the
Andrews model because the differences
between the model fits were small and
because use of the Andrews model with its
additional parameter did not improve the
prediction of mixture experiments.
The results of a biodegradation experiment with toluene and phenol are shown in
Figure 2. Toluene was consumed before
phenol, and phenol biodegradation did not
begin until toluene was nearly depleted.
Although this sequential substrate consumption is reminiscent of diauxic growth, the
classic definition of that phenomenon (induction or derepression of catabolic enzymes)
does not apply here because P. putida F1 uses
the same enzymes to metabolize both substrates (35). Similarly, when this species was
grown on a 50:50 mixture of benzene and
phenol, benzene was degraded first, and phenol consumption did not begin until benzene
concentrations were near zero (Figure 3). In
the case of the toluenebenzene mixture, P.
putida F1 consumed both of these substrates
simultaneously during most of the cultivation, but toluene biodegradation began before
that of benzene, and toluene was depleted
first (Figure 4).
A common model for cell growth on
homologous substrate mixtures is a no-interaction sum kinetics model, in which the specific growth rate is the sum of the specific
growth rates on each substrate i (i). The rate
of consumption for substrate i can be
30
25

20
15
10
0
0
5
10
15
60
50
Phenol
Toluene
Biomass
Sum
Competitive
SKIP
40
30
20
10
20
25
30
35
Cell concentration (mg/L)
where S is the substrate concentration, t is

time, is specific growth rate, Y X/S is the
biomass yield, and X is biomass concentration.
To determine the yield, YX/S, the concentration
of cells produced (cells/mL) was divided by the
concentration of substrate consumed (mM).
Because toluene and benzene are volatile,
Equation 1 required modification to account
for the presence of toluene in both the gas
and liquid phases in the bioreactor. Microbial
growth rates depend on the liquid-phase substrate concentration only, whereas the biomass yield is a function of the change in total
mass of substrate. Because the cultivation
conditions were chosen to ensure that mass
transfer rates (from gas to liquid phase) were
always faster than biodegradation rates (37),
the masses of toluene in the liquid and gas
phases could be related using Henrys law,
yielding
Bacterial biodegradation kinetics of mixtures
Substrate concentration (mg/L)
Chemical Mixtures
0
40
Time (hr)
Figure 2. Experimental data and model output for

batch biodegradation of a toluenephenol mixture
by P. putida F1. Symbols indicate measurements of
liquid-phase toluene (), phenol (), and biomass
concentrations (
). Lines are predictions from the
sum kinetics, no-interaction model (dotted lines),
competitive inhibition model (dashed lines), and
SKIP model (solid lines). Adapted from Reardon
et al. (37).
Table 1. Parameters for Monod and SKIP models of biodegradation of mixtures.a

Microorganism
P. putida F1
Burkholderia sp. JS150
Growth
substrate
Toluene
Benzene
Phenol
Toluenephenol
Toluenebenzene
Benzenephenol
Toluenebenzenephenol
Toluene
Phenol
Toluenephenol
m
(per hour)
KS
(mg/L)
YX/S
(g/g)
I1,2
(-)
I2,1
(-)
PVE
0.86 0.01
0.73 0.03
0.11 0.01
*
*
*
*
0.39 0.01
0.31 0.03
*
13.8 0.9
0.12 0.02
32.0 2.4
*
*
*
*
1.01 0.28
0.51 0.38
*
1.28 0.13
1.20 0.05
0.80 0.07
*
*
*
*
1.03 0.09
0.88 0.005
*
N/A
N/A
N/A
55 5
5 0.3
18.5 1.5
*
N/A
N/A
80.6 6
N/A
N/A
N/A
0.01 0.002
0.01 0.003
0.01 0.002
*
N/A
N/A
0.6 0.03
98.4
86.6
93.9
98.1
95.7
94.2
96.7
96.3
99.1
97.3
aFor the parameters I and I , subscript 1 refers to the first chemical in the pair. The notation N/A is shown when a parameter was not used to model growth on the substrate indicated;
1,2
2,1
*indicates that previously determined values of that parameter (from single-substrate experiments) were used.
1007
Reardon et al.
modeled using Equation 1 or Equation 3, as

appropriate. Because the Monod model was
found to be suitable for biodegradation of
each of the three monoaromatics individually,
the no-interaction sum kinetics model is
[6]
where the subscripts 1 and 2 refer to each of

the two substrates. The predictions of this
model for the toluenephenol mixture are
shown in Figure 2. Comparison of these predictions with the toluenephenol data clearly
reveals mixture effects because phenol
biodegradation occurred later and at a lower
specific (per cell) rate than predicted by the
model. Thus, the presence of toluene inhibited phenol biodegradation. However, phenol
had little effect on toluene consumption.
Benzene also inhibited phenol biodegradation, although phenol did not have a significant impact on the rate of benzene
metabolism (37). Finally, when the model
was applied to toluenebenzene mixtures, the
biodegradation of benzene was predicted to
be earlier and faster than was actually measured, suggesting that the presence of toluene
inhibited the degradation of benzene. In contrast, the presence of benzene had little effect
on toluene consumption (37). Thus, mixture
effects (i.e., nonadditivity) were found with
all three pairwise combinations of these three
monoaromatics.
Because P. putida F1 uses TDO to
initiate catabolism of all three chemicals, one
might expect that these mixture effects are
due to competitive inhibition of this enzyme.
A sum kinetics model incorporating purely
competitive substrate kinetics (18) is
max,1S1
K S ,1 + S1 + I 2,1S 2
max,2S 2
K S ,2 + S 2 + I 1,2S1
[8]
Here, Ii,j indicates the degree to which

substrate i affects the biodegradation of substrate j, with larger values corresponding to
stronger inhibition. Yoon et al. (18) were the
first to propose a model of this type, which
25
30
25
20

10
Phenol
Benzene
Biomass
Competitive
SKIP
50
30
20
10
0
0
10
40
20

30
40
0
50
60
[7]
Predictions from this model are shown in

Figures 24 for each of the binary mixtures.
In the case of the toluenephenol mixture,
the model prediction for phenol degradation
represented the data better than did the nointeraction model, but the agreement with
the toluene data was worse. Thus, the onesidedness of the mixture effect was not well
predicted. Similar phenomena occurred
when Equation 7 was used to predict the
biodegradation of benzenephenol and
toluenebenzene mixtures. Models incorporating noncompetitive and uncompetitive
interactions have also been tested, but none
gave satisfactory results (37).
To account for these mixture effects, an
alternative model was formulated by incorporating an interaction parameter Ii,j into the
sum kinetics framework (37):
35
15
max,2S 2
.
K
K S,2 + S 2 + S,2 S1
K S,1
20
70
15
10
40
30
20
0
0
10
12
14
10
0
16
Time (hr)

batch biodegradation of a benzenephenol mixture
by P. putida F1. Symbols indicate measurements of
liquid-phase benzene (), phenol (), and biomass
concentrations (
). Dashed lines are predictions
from the competitive inhibition model, and solid
lines are curve fits for the SKIP model. Reprinted
from Reardon et al. (37) with permission from John
Wiley & Sons, Inc.

batch biodegradation of a toluenebenzene mixture by P. putida F1. Symbols indicate measurements of liquid-phase toluene (), benzene (),
and biomass concentrations (
). Dashed lines are
predictions from the competitive inhibition model,
and solid lines are curve fits for the SKIP model.
Reprinted from Reardon et al. (37) with permission
from John Wiley & Sons, Inc.
VOLUME
70
25
60
50
Toluene
Benzene
Biomass
Competitive
SKIP
Time (hr)
1008
we call sum kinetics with interaction parameters (SKIP). To obtain the values of the interaction parameters (Table 1), the SKIP model
was fitted to each set of binary mixture data
sets using values of m, KS, and YX/S determined from the single-substrate experiments.
The fitted SKIP model accurately describes
the biodegradation data for all three binary
mixtures (Figures 24), demonstrating that
the SKIP model can be used to fit unspecified
types of inhibition between two substrates.
The ability of the SKIP model to predict
the outcome of the 3-substrate mixture was
also examined. As shown in Figure 5, the consumption of toluene began first, followed by
benzene, and these two chemicals were then
degraded simultaneously. Significant phenol
consumption did not begin until the toluene
concentration was nearly zero and the benzene
concentration was low. A three-term version
of Equation 8 successfully predicted this pattern using parameters determined independently from the one- and two-substrate
mixture experiments (37).
Biodegradation of chemical mixtures by
pure cultures of Burkholderia sp. strain
JS150. A second study, using Burkholderia sp.
strain JS150, was performed to investigate
mixed-substrate biodegradation by a bacterium that employs different catabolic pathways to degrade the mixture components. The
Monod model was found to fit the biodegradation data well for both toluene and phenol
(Table 1) (38). During growth of strain JS150
on phenol, the release of several metabolites
into the medium was noted, and one was
identified as 2-hydroxymuconic semialdehyde
(38). However, these metabolites did not
inhibit phenol consumption.
When strain JS150 was grown on an
equimolar solution of toluene and phenol,
60

15
20
10

0
0
5
10
15
50
Phenol
Toluene
Benzene
Biomass
40
30
20
S
= max,1 1 + max,2 2 ,
K S ,1 + S1 K S ,2 + S 2
max,1S1
K
K S,1 + S1 + S,1 S 2
K S,2
Chemical Mixtures
10

0
20
25
30
Time (hr)
Figure 5. Experimental data and SKIP model

predictions for batch biodegradation of a
toluenebenzenephenol mixture by P. putida F1.
Symbols indicate measurements of liquid-phase
toluene (), benzene (), phenol (), and biomass
concentrations (
); lines are model predictions.
Reprinted from Reardon et al. (37) with permission
from John Wiley & Sons, Inc.
JS150
0.6
0.5
0.4
0.3
P. putida F1

0
0
15
10
0.2
0.1
20

0
10
15
20
25
30
35
0.35
Figure 6. Experimental data and model predictions

for batch biodegradation of phenol inoculated with
a 1:1 mixture of strains JS150:P. putida F1. Symbols
represent the experimental data values for phenol
(), strain JS150 (
), and strain F1 (
). Lines depict
the pure-and-simple competition model output.
Error bars represent one standard deviation based
on replicate analyses of each sample. Reprinted
from Bull Rogers et al. (39) with permission from
John Wiley & Sons, Inc.
12
Toluene
10
0.30
0.25
JS150
0.20
P. putida F1
0.10
0.05
0.15
0
0
Time (hr)
0.40
0
8
10 12 14 16
Time (hr)

for batch biodegradation of toluene inoculated
with a 1:1 mixture of strains JS150:P. putida F1.
Symbols represent the experimental data values
for toluene (), strain JS150 (
), and strain F1 (
).
Lines are the pure-and-simple competition model
output. Reprinted from Bull Rogers et al. (39) with
permission from John Wiley & Sons, Inc.
P. putida F1 grew faster and to a greater extent

than predicted by the model, and the growth of
strain JS150 was less than predicted. Given the
conflicting impacts on strain F1 in the phenolonly and toluene-only cultivations noted above,
it is interesting to note that the phenol pattern
dominated in this mixed substrate experiment.
In addition the concentrations of both substrates reached nondetect levels sooner than
predicted by the model, indicating that the
mixed culture is able to degrade the mixture
faster than either pure culture alone.
Discussion
The results presented here clearly illustrate
that the biodegradation kinetics of chemical
mixtures can be complex and difficult to
describe mathematically, even when the
chemicals serve as homologous substrates for
pure cultures of microorganisms. Although
these kinetics can in some cases be described
by relatively simple no-interaction (16) or
competitive inhibition (9,18,20) models, we
have demonstrated that such models are
inadequate for P. putida F1 growing on mixtures of toluene, benzene, and phenol and for
Burkholderia sp. JS150 growing on mixtures
of toluene and phenol. Furthermore, the
biodegradation kinetics of a mixed culture
growing on 1-butanol, 2-butoxyethanol, and
N,N-dimethylethanolamine also were not
well predicted by competitive inhibition
(42). These findings led us to develop the
SKIP model, in which a fitting parameter, Ii,j
was introduced to describe the influence of
chemical i on the rate of biodegradation of
chemical j. Using Ii,j values obtained from
the two-chemical experiments, we demonstrated the ability of the model to predict the
outcome of the three-chemical biodegradation experiments. The SKIP framework has
also been used as the basis of a model in
0.30
0.25
Toluene
Phenol
0.20
P. putida F1
10
JS150
0.15
0.10
0.05
0
0
15
10
15
20
Cell concentration (107 cells/mL)
Phenol
0.7
Phenol concentration (mM)
0.8
competition model. Further investigation

demonstrated that strain JS150 released a
metabolite, probably 2-hydroxymuconic semialdehyde, which strain F1 was able to use as a
growth substrate, and thus the interaction
between the two species included commensalism in addition to competition (39). Further
complexity was added by including Bacillus
subtilis American Type Culture Collection
7003, a species unable to grow on phenol, in
the mixed culture. When medium containing
phenol was inoculated with a 1:1:1 ratio of the
three microorganisms, B. subtilis grew to a
greater extent than did species JS150,
presumably by competing for metabolic
intermediates (40).
Purely competitive interactions were also
insufficient to describe the dynamics between
strains JS150 and F1 when similar experiments
were conducted with toluene (Figure 7). In
this case, P. putida F1 grew more slowly and to
a lesser extent than predicted by the pure-andsimple model. Using spent medium tests, this
was determined to be the result of inhibition
by an unidentified chemical released by species
JS150 (39). Thus, amensalism occurred along
with competition when these species grew
together on toluene.
Biodegradation of a chemical mixture by a
mixed culture. Finally, the biodegradation and
growth kinetics of the 1:1 mixed culture of
strains JS150 and F1 were examined for an
equimolar mixture of toluene and phenol. The
experimental results for the aromatic hydrocarbons and both microbial populations are
shown in Figure 8, along with the predictions
of a model based on the SKIP representation of
substrate consumption (Equation 8) and the
pure-and-simple kinetics representation of
microbial growth. As was the case when this
mixed culture was grown on either toluene or
phenol alone, the model predictions were poor.
Toluene concentration (mM)
toluene consumption began first. However,

phenol was degraded while toluene was
present in the medium, in contrast to the
experiments with P. putida F1. The use of the
no-interaction mixtures model (Equation 6)
revealed that the presence of each substrate
had an inhibitory effect on the biodegradation of the other. Competitive (Equation 7),
noncompetitive, and uncompetitive inhibition models were also tested, although not
mechanistically supported because strain
JS150 uses multiple biodegradation pathways
(12). The predictions from all three models
were poor. Finally, the SKIP model was
applied, with IT,P and IP,T values obtained by
fitting Equation 8 to the data. The model fits
were very good (PVE = 97.3%), and the
values of the interaction parameters indicate
that toluene inhibited phenol degradation
much more than the reverse.
Biodegradation of single chemicals by
mixed cultures. Biodegradation models for
mixed cultures often treat the microorganisms
as a single lumped quantity (e.g., total biomass). However, this was shown to be inadequate in the case of a 1:10 mixture of strain
JS150 and P. putida F1 growing on phenol
(39), suggesting that interactions between
these two species were important. Further evidence for complex interactions was obtained
by cultivating 1:1 mixtures of these species on
phenol and measuring the sizes of the two
populations using the FISH protocol (Figure
6). The resulting kinetics did not follow a
model derived from the concept of pure-andsimple competition, in which the only interaction is competition for a growth-limiting
substrate. Instead, P. putida F1 grew much
more than the model predicted, and strain
JS150 grew less than predicted by this simple
Substrate concentration (mM)
Chemical Mixtures
25
Time (hr)

for batch biodegradation of a toluenephenol mixture by the 1:1 binary mixture of strains JS150:P.
putida F1. Symbols represent the experimental data
values for toluene (), phenol (), strain JS150
(
), and strain F1 (
). Lines are predictions from a
pure-and-simple competition model between the
species and the SKIP model for substrate interactions. Reprinted from Bull Rogers et al. (39) with
permission from John Wiley & Sons, Inc.
1009
Chemical Mixtures
Reardon et al.
which a 13-chemical mixture was divided

into four groups and Ii,j values determined
for interactions between groups. In cases
without substrate inhibition, this modified
SKIP model accurately predicted the
experimental outcomes (43).
Although the differences between the
predictions of the SKIP and other models are
highly significant in a statistical sense, their
impacts do not necessarily appear large in the
batch experiments presented here. The novelty of the SKIP model is the inclusion of
inhibition terms that are different than those
in purely competitive, uncompetitive, or noncompetitive inhibition. In the case of the
toluenephenol mixture, toluene inhibits
phenol consumption to a much greater extent
than predicted by the other models, and
phenol inhibition of toluene degradation is
much less. In a batch experiment, the main
outcomes of this inhibition are a prolonged
lag phase before phenol consumption begins
and a faster toluene degradation rate. Because
toluene is rapidly consumed in this batch cultivation, the impacts on phenol degradation
are relatively small. However, in a continuous-flow bioreactor, the differences among
these various models would be much more
noticeable. Because phenol is not consumed
until toluene concentrations fall below some
low level (only accurately represented by the
SKIP model), the hydraulic residence times
and sizes of continuous bioreactors treating
toluenephenol mixtures would be substantially underpredicted unless the SKIP model
were used. Because aquifers can also be represented as continuous-flow bioreactors, the
result of using the SKIP versus another model
would be similar but expressed in terms of the
size of the contaminant plume and the length
of time required for remediation.
Despite the success of the SKIP model in
the cases presented here, the need to include
the fitting parameter Ii,j with no clear mechanistic basis is unsatisfying. This is particularly
true in the case of P. putida F1, where the
same set of enzymes appears to be involved in
the biodegradation of toluene, benzene, and
phenol. We have investigated this phenomenon further using two-dimensional polyacrylamide electrophoresis of soluble proteins (44).
Although this proteomic study has not been
completed, our current evidence points to differences in the cell membrane composition as
one of factors involved in these unexpected
kinetics. Based on the identification of acyl
carrier protein as one of the proteins with transient synthesis during biodegradation of
toluenephenol mixtures, we performed analyses of the phospholipid fatty acid content of
P. putida F1 cells. The predominant phospholipid fatty acid of cells growing on toluene was
cis-7-hexadecenoic acid (16:1w7c), whereas
cells growing on phenol had high levels of
1010
cyclopropylheptadecanoic acid (cy17:0) in

their membranes. The membranes of cells
growing on toluenephenol mixtures shifted
from 16:1w7c to cy17:0 after degradation of
toluene in the medium was complete (45).
Based on these findings, we have developed the
hypothesis that the inhibition of phenol
biodegradation in the presence of toluene is
caused by very slow transport of phenol into
the cell when the membrane has adapted to
the more hydrophobic environment. Then,
when toluene is depleted from the medium,
the membrane composition shifts to a form
through which phenol can more readily diffuse. A model based on this hypothesis has
been shown to predict toluenephenol mixture
results very well using only data from singlesubstrate experiments. We are continuing our
investigations into this hypothesis and will also
consider the implications of other proteins that
are differentially expressed by cells growing on
toluene versus phenol.
We have also shown here that the
interactions between microbial species in a
mixed culture are both significant for the
biodegradation kinetics and difficult to predict. In particular, we noted a large effect of
the carbon source on the nature of the microbial interactions, with commensalism occurring when the cells grew on phenol and
amensalism observed when toluene was the
growth substrate. We also noted that the
presence of a secondary degrader (B. subtilis)
had an additional impact on the biodegradations by introducing a new type of interaction. Although the mechanism of these
interactions could be determined after they
were observed, it seems unlikely that they
could be predicted from pure culture experiments without prior knowledge of all possible
metabolites produced by each species. Finally,
it is interesting to consider the question of
whether microbial species interactions
become less important as the mixed cultures
become more diverse. For example, although
the SKIP model alone did not describe the
kinetics of the binary P. putida F1/
Burkholderia sp. JS150 culture when total
biomass was used in the model, it was very
accurate in describing the biodegradation
kinetics of a larger (estimated 1020 species)
mixed culture growing on a mixture of 13
organic chemicals (43).
Conclusions. Although the biodegradation
kinetics of mixed microbial cultures growing
on mixtures of organic contaminants are
often assumed to be simple extensions of
pure-culture/single-substrate kinetics, we have
demonstrated that they are not. In the case of
pure cultures growing on aromatic chemical
mixtures, neither a no-interaction nor a competitive inhibition model accurately predicted
the mixture kinetics. To overcome this difficulty, we developed the SKIP model, which
VOLUME
used model parameters from single- and dualsubstrate mixture experiments to accurately
predict the outcome of the 3-substrate mixture experiment. When we conducted similar
experiments with a binary mixed culture
rather than pure cultures, we found that
interactions between the species had a significant impact on the biodegradation kinetics,
and that the nature of these interactions
depended on the growth substrate(s).
These findings reveal the significant
challenges that face efforts to model realworld biodegradation kinetics, in which
mixed substrates and mixed cultures are the
rule. Predictive modeling of these systems will
be difficult and time-consuming if one must
determine all pairwise chemical interactions
(e.g., as required by the SKIP model) and all
species interactions (with corresponding concentrations of inhibitors and metabolic intermediates). Options to these traditional
approaches may be developed through a fundamental understanding of the effects
involved [e.g., as hinted at by the proteomic
results in (44,45)] and by alternative modeling approaches such as that presented by Liao
et al. in this volume (46).
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1011
Biochemical Engineering Journal 67 (2012) 156166
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej
Regular article
Substrate interactions and kinetics study of phenolic compounds biodegradation

by Pseudomonas sp. cbp1-3
Jiao Liu, Xiaoqiang Jia , Jianping Wen, Zhengxi Zhou
Department of Biological Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University,
Tianjin 300072, PR China
a r t i c l e
i n f o
Article history:
Received 9 February 2012
Received in revised form 7 June 2012
Accepted 16 June 2012
Available online 26 June 2012
Keywords:
Biodegradation
Growth kinetics
Modeling
Substrate inhibition
Pseudomonas sp.
Phenolic compounds
a b s t r a c t
A new strain cbp1-3 was isolated from activated sludge of a coking plant in Tianjin and identied as
Pseudomonas sp. based on physiological and 16S rRNA gene sequence analysis, which could completely
degrade 1400 mg/L phenol, 800 mg/L m-cresol and 150 mg/L 4-chlorophenol (4-CP) as sole carbon and
energy source within 60 h, 40 h and 60 h, respectively. Investigation on substrate interactions in the
biodegradation of mixed phenols indicated that phenol and m-cresol exhibited a mutual inhibition to each
other, and they (only below 300 mg/L) both promoted the 4-CP biodegradation. However, 4-CP strongly
inhibited the biodegradation of other phenols in their mixtures despite of the low concentration. Further
analysis showed that the newly proposed kinetic model for cell growth on ternary substrates tted the
experimental data very well. And sensitivity analysis suggested that substrate interaction coefcients fi
(i = 12, 13 and 23) of the models were the most sensitive parameters. Finally, yield coefcient, calculated
for all conditions, taken as a whole, followed an allometric decline pattern with the increase of substrate
concentration.
2012 Elsevier B.V. All rights reserved.
1. Introduction
The widespread phenolic compounds are typical organic pollutants from pesticides, oil reneries, coking plants, pharmaceuticals
and so on. So far, it has still been difcult to dispose of these compounds safely owing to their chemical complexity [1]. Since the
biological treatment of phenolic compounds pollutants exhibits
many advantages such as the environmental friendly and cost effective, it has gained much interest as an attractive progress in this
eld recently [26]. In the past few decades, the related researches
primarily focused on the single-substrate biodegradation progress.
However, the biodegradation of multiple pollutants by different
microorganisms, which possessed diverse behaviors with complex
and unspecied interactions of the substrates, was considered to
be more applicable [7]. Moreover, the kinetics studies played great
important role in understanding the detailed behaviors of multiple pollutants biodegradation, in which all of these biodegradation
interactions studies had to be performed in model wastewater that
contained certain phenols with xed concentrations.
Several investigations on multiple pollutants biodegradation
have gained deep insight into mathematical models upon different interactions between substrates. Yoon et al. [8] developed a
Corresponding author. Tel.: +86 22 27890492; fax: +86 22 27403389.

E-mail address: xqjia@tju.edu.cn (X. Jia).
1369-703X/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bej.2012.06.008
generalized additive Monod model for cell growth on two substrates with pure competitive inhibition of them, whereas it failed
to predict the complex interactions of phenol, benzene and toluene
satisfactorily [9]. Reardon et al. [9] proposed a sum of kinetics with
interaction parameters model (SKIP model), similar to the additive
Monod model, to gure out the unspecied interactions of these
aromatic hydrocarbon mixtures. Additionally, the SKIP model was
also proved to be effective to describe the biodegradation of BTEX
compounds (benzene, toluene, ethylbenzene and xylene isomers)
successfully [10].
Substrate interactions have been considered in describing phenolic compounds biodegradation progresses in many researches
[1113] by using kinds of microorganisms such as Pseudomonas
species [6,1417]. In these researches, the additive specic growth
rate model, derived from the Andrew model and the additive
Monod model, was applied to describe the degradation of phenolic
mixtures with strong substrate inhibition. Especially, to account
for more complicated interactions between the corresponding
substrates, an alternative model was developed based on the
hypothesis that sorts of inhibitions had been taken into consideration in cell growth process to predict the microorganisms growth
on phenolic compounds mixtures at high concentrations [12,17].
Although most models stated above were capable of describing the
dual substrates biodegradation, they might not address the behaviors of the multiple substrates (more than two) biodegradation
appropriately.
J. Liu et al. / Biochemical Engineering Journal 67 (2012) 156166
Nomenclature
ai , ai , ai , a
stoichiometric coefcients (dimensionless) (i = 1,
i
2, 3)
fij
substrate interaction coefcient (L/mg)
substrate interaction coefcient (dimensionless)
Ii
ki , ki , ki reaction rate constants (i = +1, 1, +2, +3, 3, +4, 4,
, k , k , k ,
+5, 5) (unites: k+1 , k+3 , k+4 , k+5 , k+1
+5
+3
+4

k+1 , k+3 , k+4 , k+5 (L/(mg h)); k1 , k+2 , k3 , k4 , k5 ,
, k , k , k , k , k , k , k , k , k (h1 ))
k1
+2 3 4 5 1 +2 3 4 5
self-inhibition constant (mg/L) (i = 1, 2, 3)
Kii
Ksi
saturation constant (mg/L) (i = 1, 2, 3)
parameters in the cell growth model
pi
S
initial substrate concentration (mg/L)
S
global substrate concentration (mg/L)
SC
amount of carbon consumed (mg/L)
t
time (h)
X
biomass concentration (mg/L)
YX/C
observed overall yield coefcient (mg dry cell
formed /mg carbon consumed )
Greek symbols
mi
maximum specic growth rate on single substrate
(h1 ) (i = 1, 2, 3)
X
overall specic growth rate (h1 )
Xi
specic growth rate on single substrate (h1 ) (i = 1,
2, 3)
Subscripts
substrate, phenol
1
2
substrate, m-cresol
3
substrate, 4-CP
In the present work, we focused on the biodegradation processes

of three typical high toxic, carcinogenic, mutagenic and teratogenic
phenols (phenol, m-cresol and 4-chlorophenol (4-CP) [1820]) by
the newly isolated Pseudomonas sp. cbp1-3. New intrinsic kinetic
models of cell growth on ternary substrates were also proposed
based on the hypothesis of cell growth processes similar to enzymatic reactions and used to evaluate the complicated interactions
of the phenols.
2. Materials and methods
157
(Beijing, China). Gene alignment was implemented by the BLAST

program of NCBI (www.ncbi.nlm.nih.gov).
2.2. Culture media and cultivation conditions
The MM consists of 0.5 g/L (NH4 )2 SO4 , 0.8 g/L Na2 HPO4 , 0.2 g/L
KH2 PO4 , 0.1 g/L MgSO4 , 0.02 g/L yeast extract and 10 mL trace element solution [23]. LB medium containing 5.0 g/L beef extract,
10.0 g/L peptone, and 5.0 g/L NaCl was used as nutrient medium.
The initial pH of all of the mediums was adjusted to 7.2 with 2 mol/L
NaOH solution.
Strains were cultivated in 250 mL Erlenmeyer asks on a rotary
shaker at 180 rpm and 30 C. Phenolic compounds as carbon sources
were lter-sterilized through membranes (pore size of 0.22 m)
before adding to MM.
2.3. Phenolic compounds biodegradation
The strain was cultivated in 50 mL LB medium with 2% (v/v)
inoculation. After 24 h of incubation, cells were harvested at the late
exponential growth phase by centrifugation (10,000 g) for 10 min
at 4 C, then washed twice with 50 mL MM and resuspended in
MM as inocula at an appropriate concentration (OD600 5.0). In all
experiments, 2 mL inocula were inoculated into 100 mL MM with
varying initial phenol, m-cresol and 4-CP concentrations, respectively.
2.4. Analytical methods
Cell density was monitored spectrophotometrically by measuring the optical density at 600 nm (OD600 ). Dry cell weight
(DCW) was calculated from OD600 with linear correlation factor
(1 OD600 = 437.48 DCW mg/L). The dry cell weight was determined
by ltering a known volume of cell suspension then drying the
cells at 105 C [13]. The phenolic compounds concentration was
determined by the methods as described [16,20,21] with some
modications. The supernatant was prepared by centrifugation
at 6000 g for 10 min and then ltered through syringe lters (0.22 m). The residual substrate concentrations in prepared
supernatant were measured using high performance liquid chromatography (HPLC, 1200, Agilent, USA) equipped with a Zorbax
Eclipse XDB-C18 column (250 mm 4.6 mm, 5 m, Agilent, USA)
and a UV-detector (G1313B, Agilent, USA) at 280 nm. The mobile
phase of methanol/water (57/43, v/v) at a ow rate of 1.0 mL/min
at 30 C was adopted. The retention time for phenol, m-cresol and
4-CP was 3.87 min, 5.46 min and 6.75 min, respectively.
2.1. Isolation and identication of microorganism

2.5. Statistics
Strains were isolated as described by Jiang et al. [21] with some
modications. Activated sludge was collected from a coking plant
in Tianjin, and the coking wastewater contained kinds of phenols in
the range of 4002500 mg/L. Then the activated sludge was selectively enriched by ve steps for four weeks. The ve repeated
experiments were performed using phenol as the sole carbon
source in the mineral medium (MM) with the phenol concentration increasing from 200 to 1000 mg/L (interval of 200 mg/L). The
nal cultures were diluted and plated onto agar LuriaBertani (LB)
agar plates containing 1000 mg/L phenol, and then incubated afterwards at 30 C for 3648 h. Several microorganisms were obtained.
Following that, the degradation capacity (for phenol, m-cresol and
4-CP) of the obtained strains was further detected for the secondround selection of the expected strains.
The selected strain was identied by 16S rRNA gene sequence
analysis. The genome extraction and 16S rRNA gene amplication
of Pseudomonas sp. 13 were performed as described by Qiu et al.
[22]. DNA sequencing was served by the Beijing Genomics Institute
All experiments were repeated three times. Data shown in gures of Section 4 were the mean values of the experiments and the
error bars indicated standard deviation.
3. Modeling
3.1. Model development
Activation of the benzene ring by monooxygenase and ringssion by dioxygenase are two key steps in aromatic compounds
biodegradation pathways [3]. Co-metabolism usually plays an
important role in the metabolism of aromatic compounds such as
phenol, m-cresol and 4-CP ascribe to the two key steps catalyzed
by the same enzyme system of a microorganism. Therefore, when
mixed substrates are used as carbon sources for cell growth, the
complex interactions between them may usually be represented
as competitive, noncompetitive and uncompetitive inhibition,
158
analogous to enzymatic kinetics [17]. Then cell growth on three

phenolic compounds could be proposed based on enzymatic kinetics as the following reactions [8,12,17]:
k
+1 +2
X 2X
X + a1 S1
(1)
k1
k
(2)
k3
k+4
X + a1 S2 X S2
(3)
k4
k
+5
X + a
1 S3 X S3
(4)
k5
+1
k
+2
X + a2 S2 X 2X
(5)
k
k
+3
X + a2 S2 X S2
(6)
k
k
+4
X + a2 S1 X S1
(7)
k
k
+5

X + a
2 S3 X S3
(8)
k
k
+1
k
+2
X + a3 S3 X 2X
(9)
k
k
+3
X + a3 S3 X S3
(10)
k
3
k
+4
X + a3 S1 X S1
(11)
k
k
+5

X + a
3 S2 X S2
(12)
k
X , X and X are intermediate states of the microorganism, in

which substrates are consumed without cell growth. The param(i = 1, 2, 3) are stoichiometric coefcients,
eters ai , ai , ai and a
i
which represents the mean mass of each substrate consumed by
an organism to reach its intermediate state. The value of these
stoichiometric coefcients could be assumed to be 1 according to
Yoon et al. [8]. On the basis of pseudo-steady state assumption
for the intermediates: X , X , X , X S1 , X S2 , X S3 , X S1 , X S2 , X S3 ,
X S1 , X S2 and X S3 , the following equations can be easily derived
[12,17]:
Xi =
and
mi Si
S = I1
(13)
Ksi + Ii1 S
S1 + S12
Ki1
X =
3
Xi
(15)
i=1
+3
X + a1 S1
X S1
k
Consequently, the overall specied growth rate could be

described as follows:

+ I2
S2 + S22
Ki2
+ f12 S1 S2 + f13 S1 S3 + f23 S2 S3

+ I3
S3 + S32
The Andrew model [24] (sometimes referred as Haldane model),

which was widely used to evaluate the growth kinetics of inhibitory
substrates, could be obtained from Eqs. (13)(15) when the cells
grow on single substrate:
X =
m S
Ks + S + S 2 /Ki
(16)
So parameters mi , Ksi and Kii can be obtained separately from the
kinetics of individual cell growth on phenol, m-cresol and 4-CP,
respectively. Theoretically, the parameters Ii and fij both depend
on the ratios of the parameters Ksi , but they cannot strictly comply
with the relationships of the original reaction rate constants due
to the complexity of the system [17]. They could be determined
as independent parameters by tting the data of the mixedsubstrate biodegradation, and the parameter I1 is often set to
1 [12].
Based on the data of the exponential growth phase, the specic
growth rate was calculated as follows:
X =
d ln(X)
dt
(17)
where X is the cell concentration (mg/L).

Condence intervals (CI) of each kinetic parameter were calculated by the bootstrap method as described by Mishra et al. [25], by
using MATLAB R2008b. A ctional dataset was constructed by random sampling with replacement from the original dataset, which
was used to estimate the new values of parameters. Random sampling process was repeated 1000 times to obtain a set of values of
each estimated parameter. The condence interval (95%) of each
parameter was calculated based on the upper and lower 95 percentiles of the set of the parameter values.
To analyze the assimilation efciency of substrates in different
experiments, the observed overall yield coefcient YX/C (mg dry
cell formed /mg carbon consumed ) was dened as milligrams of biomass
(DCW) produced by the consumption of per milligram of carbon in
the substrates, and can be calculated as follows:
YX/C =
Xmax X0
SC
(18)
where Xmax and X0 are the maximum and initial biomass concentration, SC (mg/L) is the amount of carbon consumed by the
microorganism in the substrates when the biomass reached maximum.
3.2. Sensitivity analysis
Ki3
(14)
where Xi , mi , Ksi , Kii are the specic cell growth rate (h1 ), the
maximum specic cell growth rate (h1 ), the saturation constant
(mg/L) and the self-inhibition constant (L/mg) on one substrate,
respectively, while Ii (mg/L) and fij (L/mg) are the substrate interaction coefcients. These parameters can be obtained from the
original reaction rate constants ki , ki and ki . The global substrate
concentration S (mg/L), which means the integral effect of all substrates on the microorganism, is derived from individual impact
and cross-interactions of all substrates on cell growth with different coefcients. Its impact on the degradation of different substrate
could be characterized by the parameter Ii .
In order to nd the most inuential kinetic parameter on cell

growth, sensitive analysis is carried out, as described by Shashi and
Kumar [26] with some modications. Then the response variable
X is considered as a function of the fourteen parameters:
X = f (m1 , Ks1 , m2 , Ks2 , m3 , Ks3 , Ki1 , Ki2 , Ki3 , I2 , I3 , f12 , f13 f23 )
(19)
The rst order Taylors expansion of Eq. (19) was obtained as
follows:
X =
X
X
X
X
m1 +
Ks1 +
m2 + +
f23
m1
Ks1
m2
f23
(20)
159
Then sensitivity of X with respect to the parameter pi could

be indicated by the sensitive function X /pi (the slope of X
with respect to the parameter pi ). During the analysis only one
parameter was changed at a time with the others xed as constant.
All parameters varied from 90% to +100% of the tted values of
parameters (obtained in Section 3.1). Then a series of values of sensitive function X /pi is obtained. Moreover, normalization of the
series of values of X /pi was performed so that different slopes
could be plotted on the same plot:
Normalized slope =
(slope slopes)
SD
(21)
where slopes and SD indicates the mean value and standard deviation of the series of slope data, respectively.
4. Results and discussion
4.1. Characterization of the strain
Six strains were obtained and a further detection of biodegradation capability of strains showed that the strain cbp1-3 displayed
the best performance on the biodegradation of phenol, m-cresol
and 4-CP, especially of their mixture. Consequently, this strain
was selected for further investigations. The appearance for the
colony of cbp1-3 was a large, smooth, convex, light brown and
round on the nutrient agar. Microscopic observation showed the
strain was a short-rod gram-negative strain with mobility. Alignment of the partial 16S rRNA gene sequences of cbp1-3 (1440
nucleotides, Accession no. JN426990) demonstrated that, this strain
cbp1-3 was highly homologous (99%) to the type strains Pseudomonas putida GB-1 (Accession no. CP000712.1) and F1 (Accession
no. CP000926.1). Therefore, the strain cbp1-3 was identied as a
strain of Pseudomonas sp. temporarily. As one of the most popular
microorganisms, Pseudomonas species has shown great advantages
in biodegradation of phenolic compounds [6,1417]. Pseudomonas
sp. cbp1-3 may not hold the highest degradation rate compared
with some of the previously reported bacteria aiming at one specic
phenol, but it was the rst reported strain that could simultaneously degrade phenol, m-cresol and 4-CP as carbon and energy
sources. This advantage of cbp1-3 provided us a very good opportunity to study the interactions of these three phenols and to develop
new cell growth model based on complicated substrates.
4.2. Single-substrate biodegradation
The capabilities of cbp1-3 to degrade phenol, m-cresol and 4-CP
individually were investigated under different initial substrate concentrations. As shown in Fig. 1(a), the phenol degradation and cell
growth were examined with the initial concentration ranging from
800 to 1600 mg/L. Results showed that the time for complete degradation of phenol extended from 28 h to 60 h with the augmentation
of the initial phenol concentration. In addition, the nal biomass
also increased gradually and the lag phase prolonged noticeably
from 0 h to 32 h. However, cbp1-3 failed to degrade 1600 mg/L
phenol within 60 h.
The cell growth and biodegradation of m-cresol and 4-CP by
cbp1-3 were shown in Fig. 1(b) and (c), respectively. Similar to
the biodegradation of phenol, the time for the complete degradation of 4-CP and m-cresol was prolonged with the increase of the
initial substrate concentration. However, different from the phenol biodegradation, there was no obvious nal biomass increase in
the biodegradation of m-cresol and 4-CP. The maximum concentration of 4-CP that the strain cbp1-3 could completely degrade was
merely 150 mg/L, lower than that of m-cresol (800 mg/L) and phenol (1400 mg/L). These results indicated that 4-CP exhibited much
Fig. 1. Cell growth (open) and biodegradation (solid) of phenol (a), m-cresol (b) and
4-CP (c) in single-substrate systems.
stronger inhibitory on the substrates degradation by cbp1-3 than

phenol and m-cresol.
4.3. Dual-substrate biodegradation
A series of dual-substrate biodegradation experiments containing phenol, m-cresol and 4-CP under different initial concentrations
were performed. Fig. 2(a) showed the biodegradation of 600 mg/L
phenol and m-cresol varying from 100 to 500 mg/L. Although
phenol and m-cresol almost began to degrade simultaneously,
the complete biodegradation of phenol nished prior to that of
m-cresol. Moreover, higher initial concentration of m-cresol had
stronger inhibition on phenols biodegradation according to longer
time for the lag phase and complete biodegradation of phenol.
For instance, when the m-cresol concentration increased from 100
to 500 mg/L, the time for complete degradation of 600 mg/L phenol extended from 16 h to 26 h. In addition, the nal biomass
was generally enhanced by the increase of the initial substrate
concentration. In another situation, Fig. 2(b) represented the dualsubstrate biodegradation of 400 mg/L m-cresol and phenol from
200 to 600 mg/L. Just as the effect of m-cresol on phenol degradation, when the phenol concentration increased, the longer lag
phase and time for complete degradation of m-cresol as well as
160
Fig. 2. Cell growth and substrate biodegradation in phenol-m-cresol dual-substrate systems. (a) 600 mg/L phenol with m-cresol of 100500 mg/L and (b) 400 mg/L m-cresol
with phenol of 200600 mg/L.
the higher nal biomass were observed. For example, the time
for 400 mg/L m-cresol complete degradation increased from 22 h
to 32 h when the phenol concentration was enhanced from 200
to 600 mg/L. It can be concluded that mutual inhibition between
phenol and m-cresol occurred in these biodegradation processes,
and phenol was a preferential substrate for cbp1-3 compared with
m-cresol.
Experiments were conducted by varying initial 4-CP concentrations from 60 to 150 mg/L with phenol of constant concentrations
of 600 mg/L (Fig. 3(a)). Although the concentration of 4-CP was
much lower than that of phenol, the degradation of 4-CP could
only initialed after the almost fully depletion of phenol. Higher
concentrations of 4-CP could strongly inhibit phenol degradation
and cell growth. In these experiments, the time for complete
biodegradation of 600 mg/L phenol was prolonged from 20 h to
40 h when 4-CP concentration enhanced from 60 to 150 mg/L.
Fig. 3(b) showed the biodegradation of 100 mg/L 4-CP and phenol with the initial concentrations varying from 100 to 500 mg/L.
Phenol under low concentration from 100 to 300 mg/L gradually
accelerated the biodegradation of 4-CP. The time for 100 mg/L 4CP complete degradation were 26 h and 31 h with 300 mg/L phenol
or not, respectively. Although the lag phase of cbp1-3 growth on
100 and 300 mg/L phenol were almost vanished simultaneously,

the biomass accumulation of the latter was faster than that of the
former. In this case, phenol was easily utilized as carbon and energy
source by cbp1-3 to yield more biomass to promote the biodegradation of 4-CP. But cell growth and 4-CP biodegradation was strongly
inhibited by high concentration of phenol (from 300 to 500 mg/L),
where toxicity of phenol had stronger inuence on cell growth than
its supplies of carbon and energy.
The biodegradation behaviors of mixture of m-cresol and 4-CP
were very similar to that of phenol and 4-CP as mentioned above.
The effects of 4-CP varying from 60 to 150 mg/L on the biodegradation of 400 mg/L m-cresol were investigated (Fig. 4(a)). It was
observed that m-cresol was utilized for cells growth at the beginning of biodegradation, then 4-CP was degraded by cbp1-3. But
higher concentration of 4-CP exhibited stronger inhibition on cell
growth, and longer time was required for complete removal of all
substrates. In another case, Fig. 4(b) showed the biodegradation
of 100 mg/L 4-CP and m-cresol from 100 to 500 mg/L. The presence of m-cresol with low concentrations (from 100 to 300 mg/L)
could accelerate the biodegradation rate of 100 mg/L 4-CP gradually, whereas the opposite effect was observed with m-cresol over
300 mg/L.
161
Fig. 3. Cell growth and substrate biodegradation in phenol-4-CP dual-substrate systems. (a) 600 mg/L phenol with 4-CP of 60150 mg/L and (b) 100 mg/L 4-CP with phenol
of 100500 mg/L.
4.4. Ternary-substrate biodegradation

Little information was available for the biodegradation of
ternary mixtures of phenolic compounds utilized as sole carbon
and energy sources. Wang and Loh applied conventional carbon
sources such as glucose to co-metabolize phenol and chlorophenol that could not be degraded alone [7,17]. Another study found
that a mixture of 4-CP, 4-nitrophenol and phenol could be completely co-metabolized by Arthrobacter chlorophenolicus A6 [27]. In
this study, phenols biodegradation and cell growth on ternary substrates were further investigated. Typical proles were shown in
Fig. 5. The data presented in Fig. 5(a) was consistent with the results
of experiment T4 (Table 1). It was shown that phenol and m-cresol
almost began to degrade simultaneously after a 12 h lag phase.
Compared with the corresponding single-substrate biodegradation, time required for complete degradation of 4-CP shortened to
24 h, while time for complete degradation of phenol and m-cresol
was prolonged to 18 h and 22 h, respectively. Additionally, when
the concentration of phenol increased to 400 mg/L (corresponding
to experiment T22), the complete degradation time was prolonged
to 24 h and 40 h for complete transformation of m-cresol and 4-CP,
respectively (Fig. 5(b)). As shown in Fig. 5(c) and (d) (data

corresponding to experiment T6 and T7), the increase of the concentration of m-cresol and 4-CP resulted in deceleration of the other
phenols degradation. In addition, low concentration of phenol and
m-cresol promoted the 4-CP biodegradation, whereas high concentration of them exhibited the contrary effect. For example, time for
Table 1
Summary of experiments in ternary-substrate systems.
Experiment no.
T1-T3
T4-T6
T7-T9
T10-T12
T13-T15
T16-T18
T19-T21
T22-T24
T25-T27
4-CP, 4-chlorophenol
Initial concentration (mg/L)

Phenol
m-Cresol
4-CP
200
200
200
300
300
300
400
400
400
100, 250, 400

100, 250, 400
100, 250, 400
100, 250, 400
100, 250, 400
100, 250, 400
100, 250, 400
100, 250, 400
100, 250, 400
50
100
150
50
100
150
50
100
150
162
Fig. 4. Cell growth and substrate biodegradation in m-cresol-4-CP dual-substrate systems. (a) 400 mg/L m-cresol with 4-CP of 60150 mg/L and (b) 100 mg/L 4-CP with
phenol of 100500 mg/L.
100 mg/L 4-CP complete degradation with 200 mg/L phenol and
100 mg/L m-cresol was 24 h, which was shorter than that of 32 h
without phenol and m-cresol (Fig. 5(a)). However, a longer time of
40 h was required for that with 400 mg/L phenol and 100 mg/L mcresol as shown in Fig. 5(b). Overall, although the ternary-substrate
biodegradation processes were complicated, the interactions of the
phenols exhibited similar behaviors to that in the biodegradation
processes of dual-substrate systems.
Additionally, it should be pointed out that the symbols of
Figs. 15 reected the presupposed initial concentrations of these
three phenols. However, in the actual experiments, the measured
concentrations could be a little bit different from the prepared concentrations.
4.5. Interaction analysis in phenols biodegradation
Generally, in a bacterium, phenolic compounds were usually catalyzed by the same enzyme system. The key metabolites
with same structure of catechol were generated and cleaved by
monooxygenase and dioxygenase in their biodegradation [3,4]. The
substrate specicity and enzymatic activity of the two key enzymes
were dependent on the structures of enzymes and substrates.
The chlorine substituent and methyl group on benzene ring had

electron-attracting and steric hindrance effect to impede catalysis
of the two oxygenases. Besides, stronger substrate inhibition of 4CP and m-cresol decreased the activity of the two enzymes. Thus,
4-CP and m-cresol were more difcult to be degraded than phenol
as observed in this study. Other researchers also found the similar
phenomenon: only 375 mg/L 4-CP could be completely degraded
within 40 h by a good chlorophenols degrader, A. chlorophenolicus A6 [4,20], whereas the concentrations of phenol and cresol
mineralized could reach to 3000 and 1500 mg/L, respectively
[18,28].
Moreover, compared with 4-CP and m-cresol, phenol was lower
toxic to induce more easily the synthesis of oxygenases [29], and
more readily biodegradable with higher enzyme reactive activities. Therefore, when the three coexisted phenolic compounds were
catalyzed by the same enzymes system, phenol was usually rstly
utilized as primary substrate to promote the degradation of cresol
and 4-CP by co-metabolism [11,1517]. And then m-cresol began
to degrade, which followed by the 4-CP biodegradation. However,
further work for gaining insight into the interaction mechanisms is
necessary by investigating the global metabolism of cell growth on
phenolic mixtures, which is being undergoing in our lab.
163
Fig. 5. Typical cell growth and substrate biodegradation in ternary-substrate systems.
4.6. Kinetics study of phenolic compounds biodegradation

4.6.1. Intrinsic kinetics parameters estimation
Batch cultures were performed in the MM with different concentrations of phenol (01400 mg/L), m-cresol (0800 mg/L) and
4-CP (0150 mg/L), respectively. On the basis of the single substrate
experiments, the parameters mi , Ksi and Kii for the biodegradation of phenol, m-cresol and 4-CP as the sole carbon source were
determined, respectively. The tted values of parameters with condence intervals (95%) (Table 2) were obtained by a nonlinear
least-square regression analysis using MATLAB R2008b. Larger saturation constant (Ksi ) means lower substrate afnity, and smaller
value of self-inhibition constant (Ki) indicates stronger substrate
inhibition [30]. The values of saturation constant and self-inhibition
constant for the biodegradations of phenol, m-cresol and 4-CP were
in ascending and descending orders, respectively. It was indicated
that the strain cbp1-3 exhibited a substrate preference in the following order: phenol, m-cresol and 4-CP, which is in agreement
with the conclusion of the substrate toxicity order as mentioned in
Section 4.1.
The information derived from single- or dual-substrate
biodegradations could be applied to more complex systems [31].
Eqs. (13)(15) were used to describe the dual- and ternarysubstrate biodegradation. Then, coupled with the determined
parameters calculated based on single-substrate experiments as
mentioned above, the parameters I2 , I3 , f12 , f13 and f23 could be
obtained on the basis of the data of dual- and ternary-substrate
experiments. All of these parameters with condence interval (95%)
were listed in Table 2. Finally, four nonlinear curve ts were performed for the determination of all the parameters, the small
residual sum of squares (RSS, <5.65 103 ) and high correlation
coefcient (R2 > 0.979) of each nonlinear curve t indicated that
the model tted the experimental data very well.
The newly developed models for cell growth on three substrates

in this research have a similar part of Eq. (13) with models such as
Monod model, the additive Monod model [8], the skip model [9]
and other more complex models [12,17] deriving from cell growth
progresses reactions similar to enzymatic kinetics. The variable part
between these models can be integrated to a parameter of global
which reects the complex interaction
substrate concentration (S),
of substrates. Moreover, other appropriate and meaningful forms
of S may be selected or obtained for the biodegradation in different
systems.
4.6.2. Yield coefcients of cell growth
The yield coefcient of cell growth signicantly depended
on the medium composition and environment conditions. Three
Fig. 6. Overall yield coefcient of cell growth on single, dual and ternary substrates.
164
Fig. 7. Plots of normalized X1 /pi with normalized parameters pi over the range of 90% to +100% in four different experiments of T4 (a), T22 (b), T6 (c) and T7 (d).
patterns: the constant pattern [32], the linear decline pattern [33]
and the allometric decline pattern [19] were summarized by Xiao
et al. [34] to describe different trends of yield coefcient correlated
with substrate concentration. Pirt [35] proposed that yield coefcient had a direct relation with impact of substrate on specic
growth rate and maintenance energy. Lower yield coefcient may
be obtained ascribe to lower specic growth rate and higher maintenance energy which could be resulted in by stronger inhibition of
phenolic compounds with higher concentration. As shown in Fig. 6,
the observed overall yield coefcient YX/C indicated the assimilation efciency of substrates in single-, dual-, and ternary-substrate
systems (mentioned in Figs. 15). Relatively small changes in the
yield coefcient of cell growth on phenol (0.350.39) was observed
owing to low growth rates at high substrate concentration, while
the yield coefcient of cell growth on m-cresol (0.441.78) and 4-CP
(2.264.30) varied distinctly. The yield coefcient of cell growth on

phenolic mixtures distributed above that on phenol and below that
on 4-CP, with a mean value of 0.88. Moreover, complex interactions
of phenols in their biodegradation usually had indistinct effects on
the metabolism of cells besides substrate inhibition. Thus, irregular
uctuation of yield coefcient of cell growth on phenolic mixtures
(especially in the ternary-substrate systems listed in Table 1, but
not shown in gure) could be observed. However, in this work,
the yield coefcient, taken as a whole, performed the allometric
decline pattern accompanying with the increase of substrate concentration.
4.6.3. Sensitivity analysis of parameters
Sensitivity analysis was performed to identify sensitive parameters in the cell growth model. Slopes X1 /pi , X2 /pi , X3 /pi
Table 2
Kinetic parameters of Pseudomonas sp. cbp1-3 in ternary-substrate systems.
Parameters
Value
RSS
R2
CI (95%)
m1 (h1 )
Ks1 (mg/L)
Ki1 (mg/L)
m2 (h1 )
Ks2 (mg/L)
Ki2 (mg/L)
m3 (h1 )
Ks3 (mg/L)
Ki3 (mg/L)
I1
I2
I3
f12 (L/mg)
f13 (L/mg)
f23 (L/mg)
0.275
6.90
530.7
0.541
12.58
73.9
0.416
26.50
14.3
1
0.315
0.499
3.54 104
4.92 103
5.60 103
5.65 103
0.985
2.63 104
0.997
6.34 104
0.979
(0.253, 0.330)
(1.00, 20.00)
(389.2, 655.1)
(0.407, 0.696)
(1.00, 25.79)
(51.8, 109.2)
(0.376, 0.456)
(6.49, 38.27)
(12.51, 16.55)
4.29 104
0.989
Data sources
Phenol alone
biodegradation
m-Cresol alone
biodegradation
4-CP alone
biodegradation
Set value
4-CP, 4-chlorophenol; CI, condence intervals; RSS, residual sum of squares.
(0.273, 0.358)
(0.434, 0.561)
(1.78 104 , 6.00 104 )
(4.11 103 , 5.64 103 )
(4.32 103 , 6.75 103 )
Dual-and
ternary-substrate
biodegradation
165
Fig. 8. Effect of phenols concentration on X1 /pi (black, squared), X2 /pi (red, circle), X3 /pi (blue, regular triangle) and X /pi (olive green, inverse triangle). A series
of values of one slope, such as X1 /m1 , was obtained to calculate the mean value with the corresponding parameter pi varying from 90% to +100% in one experiment,
then four mean values of each parameter were obtained in different experiments of T4, T22, T6 and T7 (Table 1) corresponding to the four points from left to right, with error
bars indicating the variation range of the slopes. X /mi was equal to Xi /mi and X /Ksi was equal to Xi /Ksi (i = 1, 2 and 3). (For interpretation of the references
to color in this gure legend, the reader is referred to the web version of this article.)
and X /pi with respect to the same parameter showed a similar

trend ascribe to their similar mathematical structure (pi means a
parameter). Thus the change trends of normalized slopes in four
typical experiments (Table 1: T4, T22, T6 and T7) were shown by
taking X1 /pi for example (Fig. 7). And the real values of slopes
/pi in the four experiments were compared in Fig. 8 ( was
X1 , X2 , X3 and X , respectively).
Fig. 7 showed that the parameters could be divided into three
groups: the rst group of m1 and Ks1 was insensitive and had stable impact on X1 ; the second group of Kii (i = 1, 2 and 3) were
sensitive when normalized parameter less than about 40%; the
third group of Ii (i = 2 and 3) and fi (i = 12, 13 and 23) had a similar stable variation tendency. But Fig. 8 showed that the absolute
values of /fi were much larger than /Ii or other slopes,
which indicated that fi had the most important effects on cell
growth. Moreover, great changes of /fi were observed in different experiments with different concentrations of phenols in Fig. 8,
and it demonstrated that the interactions of the phenols had great
relationship with the phenols concentration and played much signicant role in cell growth. So the parameters fi were considered
as the most sensitive parameters.
5. Conclusions
Strain cbp1-3, newly isolated and identied as Pseudomonas
sp., could effectively degrade phenol, m-cresol, 4-CP and their
mixtures. Substrate interactions were investigated in mixed phenols biodegradation: Phenol and m-cresol had a mutual inhibition
to each other, both of which enhanced the biodegradation of 4CP when their concentrations were below 300 mg/L, while 4-CP
showed strong inhibition on the other two phenols biodegradation.
New kinetic models were developed to describe the cell growth
behavior on ternary substrates, which tted the experimental data
very well. The self-inhibition constants Kii indicated a substrate
preference order as follows: phenol, m-cresol and 4-CP. In sensitivity analysis, the most sensitive parameters of fi (i = 12, 13 and
23) indicated that the interactions between the phenols play the
most signicant role in the cell growth. Finally, the observed overall yield coefcient was calculated, which signicantly depended on
the composition and concentration of the substrates and generally

decreased as the initial substrate concentration increased.
Understanding of the mechanisms of substrate interaction was
important as well as the new kinetic models proposed in this
work, which help us better understand and utilize the complicated
phenolic compounds biodegradation processes, like progress optimization and scale-up. And a further insight into mechanisms of
substrate interaction from the cell physiology perspective is also
necessary, which would be facilitated by global metabolism analysis undergoing in our lab. It should also be pointed out that the
unique ability of Pseudomonas sp. cbp1-3, to degrade these three
typical phenolic compounds, also makes this strain very promising
to be applied in practical bioremediation of complicated phenols in
a wide range of concentrations.
Acknowledgements
The authors wish to acknowledge the nancial support provided by the National Natural Science Foundation of China (No.
20906070), the Seed Foundation of Tianjin University and the
Program of Introducing Talents of Discipline to Universities (No.
B06006).
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Journal of Environmental Chemical Engineering 1 (2013) 865874
Contents lists available at ScienceDirect
Journal of Environmental Chemical Engineering

journal homepage: www.elsevier.com/locate/jece
Biodegradation of dual phenolic substrates in simulated wastewater by

Gliomastix indicus MTCC 3869
Shashi Kumar, Deepika Arya, Abhinav Malhotra, Surendra Kumar *, Brajesh Kumar
Department of Chemical Engineering, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 3 June 2013
Received in revised form 24 July 2013
Accepted 28 July 2013
The biodegradation of phenol, resorcinol, and p-cresol by Gliomastix indicus was carried out by
conducting batch experiments in single and the dual substrate systems: phenolp-cresol, and phenol
resorcinol, at various initial concentrations in simulated aqueous solution. The growth kinetic model was
selected and interaction parameters were estimated. Competitive inhibition type of substrate
interaction was involved in this dual substrate systems. The interaction parameter values were found
as Ia,1 = 0.044, Ia,2 = 1.17 for phenolp-cresol, and Ia,1 = 1.09, Ia,2 = 0.052 for phenolresorcinol system.
The variation in maintenance energy expenditure with specic growth rate was incorporated to model
specic degradation rate. A linear model for specic degradation rate was developed for single substrate
system, while a non linear model was developed for dual substrate system. A set of model equations was
proposed and solved to describe the biodegradation dynamics of substrates in the two dual substrate
systems. The simulation results were found to be consistent with the experimental data.
2013 Elsevier Ltd. All rights reserved.
Keywords:
Biodegradation
Wastewater treatment
Interaction parameter
Maintenance energy
Introduction
Environmental pollution due to phenolic compounds is a major
problem, which is being faced by developing countries today.
Phenol and its derivatives such as resorcinol, and cresols are widely
found in the efuents of many industries which include oil
reneries, ceramic plants, steel plants, coal conversion process
plants, and textile, phenolic resin, paper and pulp, pharmaceutical,
fertilizers, pesticides, plastics, petrochemical, explosive production, rubber industries. The discharge range of phenolic compounds depends upon the type of industry. The discharge ranges of
concentration of phenol, resorcinol and p-cresol from a few
industries have been reported by Gonzalez-Munoz et al. [1],
Gonzalez et al. [2], Kira et al. [3], Phutdhawong et al. [4], Kumara
and Paruchuri [5], Babich and Davis [6]. Once the phenolic
compounds present in untreated industrial efuents, are released
into the environment, they persist in the water for a week or more
and adversely affect life forms. They react with metal ions and
other compounds present in the waste resulting into the formation
of more toxic complex compounds [7]. Phenol and resorcinol are
not potential carcinogens. But United States environment protection agency has classied p-cresol as pollutant of group C (possible
human carcinogens) and listed it as priority pollutant [8,9]. World
Health Organization (WHO) has set a limit level of 0.001 mg/L to
* Corresponding author. Tel.: +91 9897077460; fax: +911332273560.

E-mail addresses: skumar@iitr.ernet.in, skumar.iitroorkee@gmail.com
(S. Kumar).
2213-3437/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jece.2013.07.027
regulate the concentration of these phenolic compounds in

drinking water [10]. The European Union listed phenol among
the substance undesirable in excessive amounts and has decided
that its amount in water (lakes, streams) should be limited to
0.3 mg/L to protect human health from the possible harmful effects
on exposure to phenol by drinking water and eating contaminated
water plants [11].
Various treatment technologies have been studied for the
treatment of wastewater contaminated with phenols in order to
reduce the toxicity of wastewater and thereby to reduce
the environmental load of harmful phenolic compounds. In the
biodegradation technique a large number of reports on the
biodegradation of phenolic compounds by bacteria are available
in the literature [1221]. But the application of yeast and fungal
strain has not been studied in detail. However, the lamentous
fungi are being used in many biological processes like production
of organic acids, enzymes, hormones, antibiotics, steroids, and
treatment of environment problems [22,23]. In recent years of
research interest has been focused on the degradation of industrial
efuents by the fungi in order to solve the environmental
problems. In the present research programme fungal strain
Gliomastix indicus MTCC 3869 has been used for biodegradation
of phenol, resorcinol, and p-cresol in synthetic wastewaters.
During single substrate biodegradation of a toxic compound,
the biomass growth inhibition occurs due to the enhanced
substrate toxicity, beyond a certain initial concentration in the
culture medium. This substrate inhibition effect results into
the slow biomass growth rate, lower biomass yield, and increase
in the energy expenditure for maintenance of the cells. Usually the
866
S. Kumar et al. / Journal of Environmental Chemical Engineering 1 (2013) 865874
Nomenclature
Abbreviations
PSSH
pseudo-steady state hypothesis
SKIP
sum kinetics with interaction parameters
Notations
f
[] substrate interaction coefcient
[] inhibition to the degradation of substrate 1 in
Ia,1
the presence of substrate 2
[] inhibition to the degradation of substrate 2 in
Ia,2
the presence of substrate 1
[L/mg] inhibition to the degradation of substrate 1
Ib,1
in the presence of both the substrates 1 and 2
[L/mg] inhibition to the degradation of substrate 2
Ib,2
in the presence of both the substrates 1 and 2
K
[mg/L]2 substrate interaction coefcient for substrate used in Eqs. (6)(12)
[mg/L] inhibition constant
Ki
[mg/L] saturation constant of biomass growth for
KSi
substrate i
[h1] reaction rate constants
k2, k8
[h1] maintenance energy coefcient for substrate i
mSi
[h1] specic degradation rate of substrate i
qSi
[h1] maximum specic degradation rate
qSmax
qS0
[h1] initial specic degradation rate
[h1] maximum initial specic degradation rate
qS0max
[mg/L] concentration of substrate i
[Si]
[S0]
[mg/L] initial substrate concentration
t
[h] time
[X]
[mg/L] biomass concentration
[mg/L] initial biomass concentration
[X0]
[mg/L] total biomass concentration
[XT]
(Yx/s)oi [g/g] observed growth yield coefcient
[g/g] true growth yield coefcient
(Yx/s)T
(Yx/s)Ti [g/g] maximum growth yield coefcient with
respect to substrate i
Greek letters
mg
[h1] specic growth rate
mgmax [h1] maximum specic growth rate
mgi
[h1] specic growth rate for substrate i
industrial efuents contain a mixture of pollutants in wastewater

creates problems for their biodegradation because different
operating conditions such as pH and temperature may be required
for the biodegradation of different compounds. Besides, the
biodegradation of one substrate may be inhibited by the presence
of other substrate present in the wastewater. The interaction
among these multiple substrates is complex due to their toxicity
and competition for microbial enzymes and cofactors [24].
Therefore, it is necessary to study the way of interaction of
substrates with each other and its effect on their degradation. In
dual substrate system the biodegradation rate remains low,
usually due to substrate toxicity, competitive inhibition, and the
formation of toxic intermediates by nonspecic enzymes
[15,20,25]. Various types of substrate interaction patterns including competitive inhibition and non competitive inhibition have
been observed in different dual substrate biodegradation system
[2629]. The pollutants like phenol resorcinol, p-cresol are toxic to
growing cells. They damage the cells inhibiting their metabolism

and stop their growth when present in higher concentration.
In majority of biodegradation studies research workers have
frequently reported biomass growth kinetics and substrate
degradation kinetics without taking into account the changes in
maintenance energy requirement of the culture. A signicantly
higher amount of maintenance energy is required during the
biodegradation of toxic substrates, in comparison to other cultures
where energy providing substrate is nontoxic substance like
glucose, fructose, molasses etc. Experimentally it has been
observed that the biomass growth yield and requirement of
maintenance energy of microorganism vary with the different
concentrations of toxic substrate in the medium [3032]. The
energy requirement for the cell maintenance depends upon the
microorganism and the toxic substrate under biodegradation.
The operating condition such as temperature, concentration of
nutrients, and pH value in the medium also affect the maintenance
energy requirement of microorganisms. The cells under biodegradation studies require a minimum, constant and continuous
amount of maintenance energy to tolerate the toxicity of the
substrate and for their survival at each growth phase. This amount
of maintenance energy is consumed by the cells for maintenance
activities while the rest of the maintenance energy is produced for
the growth of microorganism. Further, when substrate inhibition
to biomass growth takes place in the medium, the degree of
toxicity of substrate including the production of various intermediates and extracellular products, affect the biomass growth
yield adversely and lead to the higher requirement of energy for
the maintenance of the cells [33,34]. Therefore, the biomass
growth yield and the substrate degradation are not directly
proportional to each other. Substrate degradation takes place even
though the biomass growth yield is low, because the consumed
substrate is utilized for more energy generation to be utilized for
higher maintenance of microbial cells at enhanced concentration
of the toxic substrates like phenol, resorcinol, and p-cresol, causing
the inhibition to biomass growth and to their own degradation.
Hence, the concept of energy expenditure for maintenance of cells
is needed to provide proper description of biodegradation
dynamics. The knowledge of biodegradation dynamics is signicant to design the biodegradation unit and to predict the changes
in the concentration of a component in the wastewater during its
removal by biodegradation technology.
The present study reports the quantication of maintenance
energy during the degradation of a mixture of phenolic substrates,
and an analysis of substrate interactions during biomass growth,
and the degradation dynamics. To the best of our knowledge, there
is no other biodegradation study available in the literature which
quanties the variation in the maintenance energy expenditure
when substrate concentration in the mixture is in inhibition range,
and biomass growth follows inhibition kinetics. The work
presented here, is a reasonable starting point for the development
and validation of mathematical model to describe the specic
degradation rate, growth rate, maintenance energy expenditure,
growth yield, and degradation dynamics for mixtures of two
homologous substrates. The results provide in depth knowledge of
the degradation of organic pollutants for phenolic wastewater
treatment, required to design a biodegradation facility.
Materials and method
Microorganism and cultivation condition
The fungus G. indicus MTCC 3869 was procured from the
Institute of Microbial Technology (IMTECH), Chandigarh, India and
maintained on the potato dextrose agar (PDA) medium containing:
potatoes (200 g/L), dextrose (20 g/L) and agar (15 g/L), at pH 6
using 1N NaOH by serial transfer on the PDA medium after every

two weeks duration and incubating at 28 8C. For biodegradation
studies, a mineral salt medium supplemented with phenol,
resorcinol, and p-cresol as single substrate and phenol in
combination with p-cresol and resorcinol as dual substrate along
with the ingredients of K2HPO4 (1 g/L), FeSO47H2O (0.01 g/L),
NH4NO3 (3 g/L), MgSO47H2O (0.5 g/L), KCl (0.5 g/L) was used [35].
Chemicals
All the chemicals used in the experimentation including phenol,
resorcinol and p-cresol were of AR grade with more than 99%
purity. These chemicals were procured from HiMedia Laboratories
Pvt. Ltd. Mumbai, Loba Chemie Pvt. Ltd. Mumbai, Ranbaxy Fine
Chemicals Ltd., New Delhi, Reidel Chemicals, Hapur.
867
phenol, resorcinol, and p-cresol respectively while in dual

substrate biodegradation system detection was realized at
275 nm. The retention time for phenol, resorcinol, and p-cresol
were 3.35, 2.53, and 4.54 min respectively. For the analysis of
biomass growth, oven dry method was used. The biomass was
found in the form of a pellet at the bottom of the centrifuge tube
after the centrifugation. The biomass pellet was washed with the
distilled water using centrifugation again. After washing, the
biomass pellet was taken out on lter paper in a petri plate and was
kept in the oven at 80 8C for 24 h. After evaporation of water from
the biomass pellet, dry biomass was weighed using a chemical
balance for the analysis of biomass growth. Each batch experimental run was repeated three times under identical conditions
and the values were averaged to get true experimental values.
Kinetic modeling
Inoculum development and experimentation

Acclimatization experiments were conducted for familiarization of fungal strain to phenolic environment. In the present
research work, the three phenolic compounds phenol, resorcinol
and p-cresol were degraded using fungal strain G. indicus MTCC
3869 at high concentration of phenol up to 1000 mg/L, resorcinol
up to 1300 mg/L, and p-cresol up to 700 mg/L. To acclimatize the
fungus, 2% glucose was added for appropriate growth of the fungus
in modied czapeck medium containing substrate (phenol/
resorcinol/p-cresol). Cultures were acclimatized, by exposing them
to the toxic substrate in a series of conical asks (250 mL) with
working volume of 100 mL. Glucose concentration was decreased
gradually along with increasing concentration of the toxic
substrate into the medium for a period of 2 months. The developed
inoculum was used for all batch culture experiments during the
exponential growth phase of the culture. pH 6 and temperature of
28 8C in the incubator were maintained during the whole
experimentation.
For each concentration of the substrates, fourteen asks of
250 mL capacity with 50 mL working volume were used and kept
in BOD incubator-cum-orbital shaker at 28 8C and 150 rpm.
Inoculation step was done in aseptic conditions of UV chamber
and 5% V/V inoculum was taken. The single substrate biodegradation experiments were conducted in the range of 70 to 1000 mg/L
for phenol, 50 to 700 mg/L for p-cresol, and 90 to 1300 mg/L for
resorcinol. The batch experiments of the interaction between
mixed substrates were performed in the three combinations of
phenol with p-cresol, and phenol with resorcinol. The combinations of phenol and p-cresol were as 100 mg/L phenol with 300 mg/
L p-cresol, 200 mg/L phenol with 200 mg/L p-cresol, and 300 mg/L
phenol with 100 mg/L p-cresol. Similarly, phenol and resorcinol
were used in the three combinations; 100 mg/L phenol with
300 mg/L resorcinol, 200 mg/L phenol with 200 mg/L resorcinol,
and 300 mg/L phenol with 100 mg/L resorcinol.
Determination of biomass and substrate concentrations in the sample
The experimental asks were taken out at different time
intervals for sampling, to study the biomass growth and substrate
degradation. The samples of 2 mL volume were taken from each of
the experimental asks and were subjected to centrifugation at
8000 g for 15 min at 25 8C, for the estimation of biomass and
residual substrate concentrations. Supernatant was separated out
for the analysis of substrate concentrations by high performance
liquid chromatography (HPLC, Waters system, USA) with a
Symmetry1 C18, 5 mm (250 mm 4.6 mm, Waters, Ireland)
column. Elution was performed with 400/300 (v/v) methanol/
water at a ow rate of 1.0 mL/min, and detection was realized with
a photodiode array detector (Waters 2998) at 271, 274, 277 nm for
During single or dual substrate batch biodegradation process,

the growth rate of biomass is expressed as
dX
mg X
dt
(1)
where mg is specic growth rate (h1) and [X] is the biomass

concentration at any time t. In order to model the growth kinetics
for mixed substrates, various sequences of reactions analogous to
enzymatic reactions have been proposed in the literature
[14,40,41,43]. In the present situation, one possible sequence of
reactions based on the enzymatic reactions for the mixed substrate
system consisting of substrates S1 and S2, is given in Table 1. In
these reactions X1 and X1 and X2 are different intermediate states of
the microorganisms in which substrates S1 and S2 are consumed
respectively but no biomass growth has occurred. Other intermediates are X 1 S1 , X 1 S2 , X 1 S1 S2 , X 2 S2 , X 2 S1 , and X 2 S1 S2 complexes.
In most of the dual substrate systems during two substrate
reactions, it appears that a ternary intermediate complex X 1 S1 S2
and X 2 S1 S2 may be formed with both substrates [36]. These facts
are incorporated in Eqs. (e), (f), (k), (l) of Table 1. Eqs. (b) and (h) are
Table 1
Reaction schemes and concentration of active intermediates.
Reaction
Equation
k1
X S1 !X 1
(a)
k1b
k2
X 1 S1 !2X
(b)
k3
X S1 !X 1 S2
(c)
k3b
k4
X 1 S2 !X 1 S2
(g)
k4b
k5
X 1 S1 S2 !X 1 S1 S2
k5b
k6
X 1 S2 S1 !X 1 S2 S1
k6b
X S 2 !X 2
X 1 !2X
(e)
k10
X 2 S1 !X 2 S2
X 2 S1 !X 2 S1
k11
(n)
(o)
k11b
k12
k12b
(l)
Equation
X S2
K S2
X S2 2
X 2 S2
K S2 K i2
(r)
X 2
(s)
X S2 S1
X 2 S1
K S2 K 12
(t)
(p)
X 2 S2 S1
X S1 S2
K S1 K 11 K 31
(k)
Concentration of
intermediates
X 1 S2 S1
(j)
k10b
X 2 S1 S2 !X 2 S1 S2
(m)
X S1 S2
K S1 K i1 K 21
(f)
k9b
k10
(i)
X S1 2
K S1
X S1 2
X 1 S1
K S1 K i1
X 1 S1 S2
(d)
k7b
k8
X 2 S2 S1 !X 2 S2 S1
Equation
X S1 S2
X 1 S2
K S1 K 11
Equations
k7
(h)
Concentration of
intermediates
X 1
Reaction
X S2 S1
K S2 K i2 K 22
(u)
(q)
X 2 S1 S2
X S2 S1
K S2 K 12 K 32
(v)
1
k1
1
k7
1
k3 1
k4 1
k5 1
k6
;
K S1 k1b k2 K S2 k7b k8 K i1 k3b K 11 k4b K 21 k5b K 31 k6b
1
k9 1
k10 1
k11 1
k12
K i2 k9b K 12 k10b K 22 k11b K 32 k12b
868
assumed to be irreversible rst-order reactions, whereas remaining Eqs. (a), (c)(g), (i)(l) are assumed to be reversible, and are of
rst order with respect to each of the reactants and products. In
Table 1, Eqs. (a)(c) represent substrate inhibition by S1 and Eqs.
(d)(f) represent substrate inhibition by substrate S2. Eqs. (g)(i)
and (j)(l) represent the cross inhibition between substrates S1 and
S 2.
The active intermediates may follow two reaction pathways. In
one pathway, the active intermediate may be deactivated which is
just the reverse reaction of their formation (reversible reaction). In
the alternative pathway, the active intermediate decomposes
spontaneously to form stable products (irreversible reaction) [37].
It is very difcult to measure the concentration of active
intermediates because they are highly reactive and very short
lived. Consequently, the evaluation of reaction rate laws in their
present form becomes quite difcult. Besides, in most of the
instances it is not possible to eliminate the concentration of active
intermediates in the differential form of the mass balance equation
to obtain the solution. However, an approximate solution may be
obtained, exploring the pseudo-steady state hypothesis (PSSH)
method. In pseudo-steady state approximation, the rate of
formation of active intermediates is assumed to be equal to its
rate of disappearance. As a result, the net rate of formation of active
intermediates is zero. Thus, the concentration of active intermediates can be expressed in terms of the concentrations of
biomass and substrate. The approximation by PSSH is applicable
due to two conditions of intermediates: they have very short life
time because of their high reactivity and are present in low
concentrations. Further, it is known that the net rate of formation
of any reaction species involved in many simultaneous reactions is
the sum of the rates of formation of that reaction species in each
reaction. On this basis, the net rate of formation of ith reaction
species occurring in N different reactions can be generalized as,
ri
N
X
r ij ;
j 1!N
(1a)
where
k2 mgmax1
D1 K S1 S1
S1 2 S1 S2 S1 2 S2
S2 2
f S2 f
K 51
K 41
K i1
K i2
f
S2 2 S1
K 42
D2 K S2 S2
k8 mgmax2
S2 2 S1 S2 S2 2 S1 S1 S1 2
K 52
K 42
f
K i2
fK i1
S1 2 S2
fK 41
K S1
;
K S2
K 41
1
K 42
(6)
(7)
(8)

1
1
;
K i1 K 21 K 11 K 31
(9)

1
1
;
K i2 K 22 K 12 K 32
(10)
1
f
1
;
K 51 K 12 K 11
(11)
1
1
1
K 52 K 12
fK 11
(12)
The values of mgmax1, KS1, Ki1 and mgmax2, KS2, and Ki2 can be
obtained separately from the kinetics of individual biomass growth
on phenol, resorcinol, and p-cresol as sole energy and carbon
source. The other parameters including f can be determined as
independent parameter by tting the experimental data.
j1
In above series of reaction step Eqs. (a)(l) of Table 1, each

reaction is elementary in which the reaction orders and
stoichiometric coefcients are identical. Thus, on applying
Eq. (1a) and PSSH on Eqs. (a)(l), the net rate of formation of
the active intermediates in Eqs. (a)(l) are formulated. These net
rate rates of formation of all intermediates are set to zero to get
concentrations of intermediates in terms of biomass and substrate
concentrations as represented by Eqs. [(m)(v)] in Table 1.
The total biomass growth rate can be represented in terms of
total biomass concentration [XT], as
dX T
d
dt
dt

X X 1 X 2 X 1 S1 X 1 S2 X 1 S1 S2 X 1 S2 S1

X 2 S1 X 2 S2 X 2 S1 S2 X 2 S2 S1
(2)
On applying PSSH in Eq. (2) and combining it with Eqs. (1), (b),
and (e) one gets
dX T dX
mg X T k2 X 1 k8 X 2
dt
dt
(3)
Eq. (3) yields
mg
k2 X 1 k8 X 2
X T
(4)
The substitution of values from Eqs. (m)(v) in Eq. (4), gives
mg
mgmax1 S1
D1
mgmax2 S2
D2
(5)
Results and discussion

Growth kinetics
Single substrate system
The biodegradation ability of fungus G. indicus has been
investigated up to the initial concentrations of 1000, 700, and
1300 mg/L for phenol, p-cresol, and resorcinol respectively.
Biodegradation of initial phenol concentration of 700 mg/L takes
place in 98 h. The biodegradation of p-cresol and resorcinol with
the same initial concentration of 700 mg/L takes place in 130 and
69 h respectively. Figs. 1 and 2 show the variation of specic
growth rate and observed biomass growth yield with initial
concentrations of the three substrates. The specic growth rate and
observed biomass growth yield increase up to the initial
concentration of 70 mg/L of phenol, 50 mg/L of p-cresol, and
90 mg/L for resorcinol. Beyond these inhibitory initial concentrations, the biomass growth yield and specic growth rate tend to
decrease because of increased substrate toxicity at higher initial
substrate concentrations. This decline trend shows that phenol, pcresol, and resorcinol are inhibitory substrates.
Specic growth rate of resorcinol is much higher than the
specic growth rate of phenol and p-cresol, because phenol and pcresol cause stronger substrate inhibition to the biomass growth
in comparison to resorcinol. Therefore, resorcinol is utilized more
than the phenol and p-cresol by the fungal cells in single
substrate system. To study the biomass growth kinetics of the
single substrates, four single substrate growth kinetic models
listed in Table 2 have been selected [38]. The kinetic parameters
of the models have been estimated by non linear least square
[(Fig._1)TD$IG]

0.14
0.12
Specific growth rate (h-1)
percent standard deviation
Specific growth rate phenol

Specific growth rate p-cresol
Specific growth rate resorcinol
869
Dmg % 2:1 (Table 2). The equa-
tions of the best t single substrate kinetic models for the three
substrates, with estimated values of kinetic parameters can be
restated as follows:
0.1
mg
0.08
mg
0.06
0.04
mg
0:462S
2
For phenol
(13)
For p-cresol
(14)
S
S 78:29 44:49
0:512S
S
S 91:87 21:99
0:185S

S2
S
1 1790
S 19:83 376
For resorcinol
(15)
0.02
0
0
200
400
600
800
1000
1200
1400
Initial substrate concentration (mg/L)

Fig. 1. Effect of initial substrate concentration on specic growth rate of phenol,
p-cresol and, resorcinol.
technique using MATLAB 7.2 based on Windows XP. The

estimated values of kinetic parameters are also given in
Table 2 for phenol. The growth kinetic parameters for resorcinol
and p-cresol have been estimated in previous study [38].
Goodness of the t of experimental data with the models under
study has been judged by measuring R2 and the percent standard

deviation Dmg % between experimental and predicted values of
specic growth rate for each model [39]. It is clear that lower the
value of percent standard deviation, the better is the t of

experimental data. Yano R2 0:974;
Dmg % 1:73 and

Dmg % 1:03 models
Andrews and Noack R2 0:993;
0.059
0.45
Observed biomass growth yield (g/g)
0.4
0.052
0.35
0.045
0.3
0.038
0.25
0.2
0.031
0.15
Observed growth yield phenol

Observed growth yield p-cresol
Observed growth yield resorcinol
Maintenance energy coefficient phenol
Maintenance energy coefficient resorcinol
Maintenance energy coefficient p-cresol
0.1
0.05
0.024
0.017
0.01
0
200
400
600
800
1000
1200
mg mg1 mg2
(16)
mg1 and mg2 represent specic growth rates of the biomass on

substrates S1 and S2 respectively. The mathematical expressions of
mg1 and mg2 indicate that mg1 and mg2 depend on both the
substrates due to inhibition and interaction effects of one substrate
on the other. Similar types of expressions have been proposed by
many authors [4042]. Furthermore, in the study by Yan et al. [50]
on phenol and m-cresol, the values of coefcients of S21 S2 , S22 S1 , S21
and S22 in Eqs. (6) and (7) are found to be negligibly small. Finally, an
alternative model analogous to Eq. (5) that takes into account the
unspecied type of substrate inhibition has been formulated. This
model consists of the interaction parameters that are treated as
unknown. According to this description, Juang and Tsai [15]
expressed specic growth rate in dual substrate system as
mg
mgmax1 S1
2
K S1 S1 SK1i1 Ia;1 S2 Ib;1 S1 S2
mgmax2 S2
K S2 S2
Maintenance energy coefficient (h-1)
[(Fig._2)TD$IG]
were found best t to the specic growth rate data of resorcinol

and p-cresol respectively [39]. For phenol, the predictions of
Andrews and Noack model are in the best agreement with the
experimental data of specic growth rate with R2 = 0.98 and
Dual substrate system

A series of batch biodegradation experiments were conducted
to study the biomass growth rate and interaction between the two
substrates in the two dual substrate degradation systems. Eq. (5)
indicates that the specic growth kinetic model for dual substrate
system can be expressed as
1400
Initial substrate concentration (mg/L)
Fig. 2. Effect of initial substrate concentration on observed biomass growth yield

and maintenance energy coefcient.
S2 2
K i2
(17)
Ia;2 S1 Ib;2 S1 S2
where Ia,1 represents inhibition to the degradation of S1 in the

presence of S2. Ia,2 represents inhibition to the degradation of S2 in
the presence of S1. While Ib,1 shows inhibition to the degradation of
S1 due to the presence of both S1 and S2. Similarly Ib,2 shows the
inhibition to the degradation of S2 due to the presence of both S1
and S2. These interaction parameters take into account all
interactions jointly as considered in Eq. (5). The values of kinetic
constants mgmax, KS, Ki are the same as in the single substrate
biodegradation system (1 and 2 on sufx refer to substrates S1 and
S2 respectively). There is a concept of four substrate interaction
patterns for dual substrate system applied to various studies
[40,44]. The conditions corresponding to these interaction patterns
are applied to Eq. (17) to get nal results to represent and discuss
the growth kinetics. The conditions Ib,1 = 0, Ib,2 = 0, Ia,1 6 0, Ia,2 6 0
represent the competitive cross inhibition between the two
substrates (pattern 1). The conditions Ib,1 6 0, Ib,2 6 0, Ia,1 = 0,
Ia,2 = 0 represent the uncompetitive cross inhibition (pattern 2).
The conditions Ib,1 = 0, Ib,2= 0, but either Ia,1 60 , Ia,2 = 0 or Ia,1 = 0,
Ia,2 6 0 indicate the competitive partial inhibition (pattern 3). The
conditions Ia,1 = 0, Ia,2 = 0, but either Ib,1 6 0, Ib,2 =0 or Ib,1 = 0,
Ib,2 6 0 show the uncompetitive partial inhibition between the two
substrates (pattern 4).
870
Table 2
Estimated values of biomass growth kinetic model parameters for phenol.
Model
Mathematical expression
Andrews and Noack
mg
Haldane
mg
Yano
mg
Webb
mg
mgmax S
S K S S2 =K i
m
S
gmax
S K S S2 =K i SK S =K i
mgmax S

2
S K S SK 1 KS
i S
mgmax S 1 K
2
S K S SK
Concentration of substrates, phenol/p-cresol (mg/L)
0.98
2.10
(35)
53.59
0.97
2.75
(36)
0.99
2.16
(37)
0.95
6.42
(38)
78.29
0.485
53.56
0.262
36.08
128.5
0.240
29.88
114.9
Ki (mg/L)
784.6
9510
Dmg %
Equations
350
300
250
150
100
50
0
40
50
mg
0:462S1
2
S1
78:29 S1 44:29
0:044S2
60
Degradation time (h)

Fig. 3. Effect of degradation time on concentration of substrates, phenol/p-cresol.
0:512S2
2
S2
1:17S1
91:87 S2 21:99
(18)
To study the effect of the presence of resorcinol on biodegradation, phenol, and resorcinol have been taken in combinations of
100 mg/L phenol300 mg/L resorcinol, 200 mg/L phenol200 mg/L
resorcinol, and 300 mg/L phenol100 mg/L resorcinol. The results
of single substrate biodegradation study show that resorcinol is an
inhibitory substrate like phenol. it causes substrate inhibition
effect in the medium at an initial concentration of 90 mg/L. Hence,
the biodegradation of resorcinol in presence of phenol has been
studied at the concentrations higher than 90 mg/L.
The analysis of Eq. (17) for phenolresorcinol system by
LevenbergMarquardt nonlinear regression program applied on
the four inhibition patterns, shows that competitive cross
inhibition takes place during the biodegradation of phenol with
resorcinol. The values of interaction parameters have been
obtained as Ia,1 = 1.09, Ia,2 = 0.052 for phenolresorcinol degradation system, which indicate that the resorcinol has stronger
inhibition effect on phenol degradation in comparison to the
inhibition caused by the phenol to resorcinol degradation. During
the experimental study it has been observed that the fungus
consumed resorcinol in preference to phenol. The specic growth
rate of biomass in phenolresorcinol system is expressed as
0:462S1
2
S1
78:29 S1 44:29
1:09S2
200
30
phenolp-cresol system can be expressed as
mg
Exp. phenol 100 mg/L

Exp p-cresol 300 mg/L
Exp p-cresol 200 mg/L
Exp. Phenol 300 mg/L
Exp. p-cresol 100 mg/L
Model
20
44.49
0.462
[(Fig._3)TD$IG]
10
R2
KS (mg/L)
The results of single substrate degradation study reveal that

phenol, p-cresol, and resorcinol start to cause substrate inhibition
in the medium at the initial concentration of 70, 50, and 90 mg/L
respectively [39]. Hence, the substrate interaction, in the dual
substrate biodegradation systems: phenolp-cresol, and phenol
resorcinol has been studied at the initial concentrations higher
than the inhibitory concentrations of these substrates. The
biodegradation of Phenol and p-cresol has been studied in three
combinations of concentrations; 100 mg/L phenol300 mg/L pcresol, 200 mg/L phenol200 mg/L p-cresol, and 300 mg/L phenol
100 mg/L p-cresol. Figs. 13 show the biodegradation trend of
phenol and p-cresol with biomass growth curve for the combinations of phenol and p-cresol. The values of interaction parameters
have been estimated by LevenbergMarquardt nonlinear regression program in MATLAB 7.2. Eq. (17) describes four type of
substrate inhibition during dual substrate degradation. The
equation was solved applying the four conditions given for the
four patterns of substrate interaction in the dual substrate system.
The conditions given for pattern 1 are found applicable to the
experimental data of phenolp-cresol system. The estimated
values of interaction parameters Ia,1 and Ia,2 show the involvement
of competitive cross inhibition in phenolp-cresol system as
the substrate interaction. Values of interaction parameters
(Ia,1 = 0.044, Ia,2 = 1.17) imply that the phenol inhibits p-cresol
degradation more than the p-cresol causes inhibition to phenol
degradation in the medium. Experimental study reveals that the
phenol is consumed preferentially by fungus G. indicus in phenol
p-cresol system. The specic growth rate of fungal biomass in
K (mg/L)
mgmax(h1)
0:185S2

2
S2
2
0:052S1
1 1790
19:83 S2 S376
(19)
The expressions of Eqs. (18) and (19) are similar to SKIP model
or Sum Kinetics with Interaction Parameters equation as
proposed and discussed by Yoon et al. [43]. This study on phenol,
resorcinol, and p-cresol in dual substrate system indicates the
ascending order of toxicity of these substrates for G. indicus as
resorcinol > phenol > p-cresol. Phenol, resorcinol, and p-cresol are
homologous substrates; therefore, competitive cross inhibition is
likely to take place [45]. The results of dual substrate degradation
study are supported by the reported ndings of Saravanan et al.
[38] and Wang et al. [46], where the less toxic substrate causes
stronger inhibition to the degradation of more toxic substrate in
the dual substrate degradation system. A few studies on different
dual substrate degradation systems with the estimated values of
their interaction parameters have been summarized in Table 3
along with the values of interaction parameters obtained for the
two dual substrate systems in the present study. All the model
parameters are corresponding to SKIP model.
871
Table 3
The estimated values of interaction parameters by SKIP model for different dual substrate systems.
Dual substrate system
Microorganism
Interaction parameter values

Ia,1
Ia,2
Ib,1
Ib,2
Phenolm-cresol
Phenolsodium salicylate
Alcaligenes faecalis
Pseudomonas putida
4.82
0.277
4.12
0.126
1.46
0.509
Phenolm-cresol
Benzene toluene
Benzene phenol
Toluene phenol
Phenolm-cresol
Phenolresorcinol
Phenolp-cresol
Phenolp-cresol
Phenolp-cresol
Phenolp-cresol
Phenolresorcinol
Mixed culture
3.9
5.16
1.08
1.03
2.91
9.9
0.49
0.27
0.14
1.79
4.72
1
8.6
0.044
1.09
7.46
Pseudomonas putida F1
Candida albicans PDY-07
Trichosporon Cutanium R57
Trametes versicolor
Trichosporon cutaneum
Aspergillus awamori
Gliomastix indicus MTCC 3869
0.3
1.17
0.052
Degradation kinetics
The specic degradation rates of substrates S1 qS1 and S2 qS2
can be represented by Eqs. (20) and (21) respectively as given
below
qS1
mg1
Y X=S
(20)
qS2
(20a)
mg2
Y X=S
(21)
T2
dS2
qs2 X T
dt
(21a)
The corresponding substrate degradation rate equations for

substrates S1 and S2 can be described

by using Eqs. (20a) and (21a)
respectively. Here Y X=S T1 and Y X=S T2 are the maximum growth
yield coefcients with respect to substrates S1 and S2. Eqs. (20) and
(21) hold true when the maintenance requirements are negligible.
In the biodegradation, a part of substrate is used by the biomass to
form new biomass cells and another part is used to perform
metabolic activities irrespective of growth. These non-growth
metabolic activities are performed by the consumption of energy,
termed as maintenance energy expenditure and for a substrate i
they are described by maintenance energy coefcient mSi. Pirt [44]
initially dened maintenance coefcient as the minimum substrate consumption to maintain the cell activity. Thus, apart from
biomass growth, some measure of maintenance energy is needed
to provide proper description of the substrate degradation
dynamics in dual substrate system. Maintenance energy was
considered as a constant quantity specic to substratemicroorganism system in various dual substrate degradation kinetic
modeling studies [14,43].
The maintenance energy coefcient msi can be incorporated in
dening specic degradation rate qSi for the given substrate i in
dual substrate system as follows:
qSi mSi
mgi
Y X=S
(22)
Ti
In this approach, maintenance denotes extra substrate consumption not used for growth purposes. Neijssel and Tempest [47]
and Hemping and Mainzer [48] reported the measurements of the
maintenance energy and found the variation in it. Pirt [44]
described the maintenance as growth rate dependent parameter
References
Competitive inhibition
Competitive + Uncompetitive
inhibition
Bai et al. [14]

Juang and Tsai [15]
Saravanan et al. [38]
Abuhamed et al. [42]
Competitive
Competitive
Competitive
Competitive
Competitive
Wang et al. [46]

Aleksieva et al. [51]
Alexieva et al. [52]
Alexieva et al. [53]
Yemendzhiev et al. [54]
This study
inhibition
inhibition
inhibition
inhibition
inhibition
and postulated a modication to this theory considering maintenance dependent on specic growth rate and by including a
portion that decreases with the increasing specic growth rate.
Accordingly, the Eq. (17) is rewritten as
qSi
T1
dS1
qs1 X T
dt
Type of substrate inhibition
mgi
Y X=S
m1i ki 1
Ti
mgi
mgmaxi
!
(23)
where mgi is specic growth rate, m1i denotes the constant

maintenance energy coefcient and the third term of the equation
is the growth rate dependent maintenance energy coefcient for
substrate i in the dual substrate system. ki is a constant.
The specic substrate degradation rate for substrate i can also
be dened in terms of observed growth yield coefcient Y X=S oi as
follows:
qSi
mgi
Y X=S
(24)
oi

Here it is important to distinguish Y X=S oi from Y X=S Ti . Y X=S Ti
considers consumption of substrate for biomass growth only,

while

Y X=S oi is the yield corrected for maintenance. It implies that
Y X=S Ti is supposed to be higher and less variable with substrate

concentration than Y X=S oi :. Experimentally it has been found that
observed biomass growth yield for phenol, resorcinol, and p-cresol
varies with the initial substrate concentration in the single
substrate systems. At each initial substrate concentration, the
observed growth yield has been determined by linearizing biomass
growth with substrate degradation. Fig. 2 shows observed growth
yield coefcient proles as a function of initial substrate
concentrations for phenol, resorcinol, and p-cresol. The maximum
observed growth yield value of 0.437 g/g has been estimated at the
concentration of 70 mg/L for phenol, 0.443 g/g at 90 mg/L for
resorcinol, and 0.31 g/g at 50 mg/L for p-cresol. For the estimation
of maintenance energy coefcient values at each initial concentration of phenol, resorcinol, and p-cresol the linear expression for
mSi used in Eq. (23) has been applied. The decreasing trend of

observed growth yield coefcient Y X=S oi and increasing maintenance energy coefcient mSi beyond the inhibitory initial substrate
concentration results in the reduction of observed growth yield.
This study concludes that the substrate inhibition reduces the
specic growth rate as well as biomass growth yield due to the
increase in the value of maintenance energy coefcient. Therefore,
relating
specic degradation rate with the growth rate in terms of
Y X=S Ti [Eqs. (20) and (21)] is incorrect [33]. On comparing Eq. (24)

with Eq. (23), it is clear that Y X=S oi incorporates the maintenance
energy expenditure. For phenol, resorcinol, and p-cresol Eq. (23)
872
can be restated as

mg
mg
0:020 0:0212 1
qS
0:437
0:129
For phenol
(25)
qS

mg
0:0135 0:054 1
0:437
0:132
For resorcinol
(26)
qS

mg
0:0229 0:011 1
0:437
0:102
For p-cresol
(27)
mg
mg
where the values of m1i and ki have been calculated by plotting the
values
of specic degradation rate qS against specic growth rate
mg values for each substrate in single substrate system.
Variation of maintenance energy expenditure as a function of
initial substrate concentration has been shown in the Fig. 2.
Pirt [44] dened maintenance energy coefcient and thereby
the specic degradation rate as a linear function of specic growth
rate as is clearly shown in Eq. (23). This type of linear relationship
between qSi and mgi is applicable to single substrate degradation
system as shown above. In dual substrate system, the linear
relationship between qSi and mgi has been used
in

empirical
formulation [14,33,43]. In these formulations Y X=S Ti has been
derived empirically as the maximum yield after correcting for
constant maintenance energy expenditure.
The sensitivity analysis of maintenance done by Bodegom [49]
indicates the importance of various non-growth components, and
emphasizes that the overall maintenance depends nonlinearly on
relative growth rate, relative death rate, growth yield, and
endogenous metabolism. The maintenance is a dynamic process
and ideally maintenance description should incorporate the
dynamics of each non-growth component. There is no constant
relation between these non-growth parameters. The simple
combinations of these parameters cannot be made due to partial
overlapping of these parameters. The conceptual analysis on
various non-growth components by Bodegom [49] shows strong
dependence of overall maintenance on growth rate. This overall
maintenance depends on the growth rate in a non linear way.
Further, the analysis on growth yield in case of dual substrate
system indicates that it is difcult to nd out the growth yield for
substrate i present in the mixture because the biomass only
represents the total growth, and its decomposition in two parts
corresponding to two substrates is extremely difcult. Therefore,
instead of Eq. (24), Eq. (22) is considered to describe specic

degradation rate for dual substrate system where Y X=S Ti may be
assumed to be a constant parameter, not measured experimentally
but estimated empirically by tting the experimental data to
empirical mathematical model.
In view of the above discussion, to generate a non-linear
function of mgi, it is convenient to express maintenance energy
coefcient mSi for substrate i in the following second degree
polynomial form
mSi A1i A2i mgi A3i m
2
gi
(28)
With the substitution of Eq. (28) in the model, Eq. (22) to

describe the specic degradation rate of substrate i in dual
substrate system, can be transformed in the following Eq. (29)
mgi
A1i A2i mgi A3i m2gi

qSi
Y X=S Ti
(29)
where A1i, A2i and A3i are constants of polynomial expression. On

keeping Y X=S Ti constant, Eq. (29) can be reduced to
qSi A1i A4i mgi A3i m2gi

where, A4i Y 1 A2i
X=S Ti
(30)
The experimental values of specic degradation rates qS1 and

qS2 are calculated using Eqs. (20) and (21) for phenol, resorcinol,
and p-cresol in the two dual substrate systems; phenolp-cresol,
and phenolresorcinol. To study the variation of specic degradation rates of the substrates with specic growth rate of biomass at
each combination of phenol with p-cresol and resorcinol, the
proposed model Eq. (30) has been applied. For each substrate in
dual substrate system, the constants involved in Eq. (30) are
estimated by using curve tting tool in MATLAB 7.2. Thus, the
specic degradation rates for each substrate can be expressed as
follows:
Phenol p-cresol system
qS1 0:038 1:071mg1 2:460m2g1
R2 1
For phenol
(31)
qS2 0:046 2:175mg2 62:56m2g2
R2 1
For p-cresol
(32)
qS1 3:369 108:2mg1 886:7m2g1
R2 1
For phenol
(33)
qS2 2:520 51:34mg2 274:9m2g2
R2 1
For resorcinol
Phenolresorcinol system
(34)
The values of regression coefcient (R2 = 1) in above expressions conclude that simulated model predictions are well
consistent with the experimental data. These results justify the
idea of inclusion of maintenance energy expenditure varying
nonlinearly with the growth rate, to quantify the specic
degradation rate in dual substrate system. Thus, it is suggested
that Eq. (30) may be very well adopted for the assessment of
specic degradation rate for the substrate in dual substrate system.
Computed substrate degradation proles in dual substrate system
In the present study, the substrate degradation proles with
time have been computed for the two dual substrate degradation
systems under study; phenolp-cresol and phenolresorcinol. For
this purpose a set of model equations mentioned in Table 4 along
with boundary conditions has been used. In the exponential
growth phase of batch culture, since the substrate consumption in
lag phase is negligibly small in comparison to high substrate
concentration in the mixture, the initial substrate concentration of
both the substrates in dual substrate system can be used as initial
boundary conditions required to solve the model equations. The
model equations have been solved simultaneously using ordinary
differential equation solver tool of MATLAB 7.2 for the both dual
substrate systems. The simulated results are discussed below.
Phenol p-cresol System
Fig. 3 shows the comparison of the model predictions and the
experimentally determined degradation data on phenol and pcresol in dual substrate system at the total initial substrate
concentration of 400 mg/L. The model corroborates the experimental data of phenol and p-cresol degradation well for each
combination of the two substrates. These trends conclude that the
model proposed for the specic degradation rate [Eqs. (31) and
(32)] by incorporating maintenance energy is quite correct. In a
combination of 100 mg/L phenol and 300 mg/L p-cresol the
observed biodegradation times of phenol and p-cresol are 33 h
and 50 h respectively. In the single substrate degradation system, it
takes 10 h for the 100 mg/L phenol and 38 h for 300 mg/L p-cresol.
Since observed rate of degradation of either substrate in dual
substrate system is slower than the degradation rate of either

Table 4
Model equations for substrate degradation dynamics.
Model equations

dX T
mg1 mg2 X T
dt
dS1
qS1 X T
dt
dS2
qS2 X T
dt
Boundary conditions
Equations
At t = 0, XT = XTo,
S1 = S1o, S2 = S2o
(39)
(40)
(41)
2
g1
(42)
qS2 A12 A42 mg2 A32 m2g2
(43)
qS1 A11 A41 mg1 A31 m
mgmax1 S1

K S1 S1 S21 =K i1 Ia;1 S2 Ib;1 S1 S2
mgmax2 S2

mg2
K S2 S2 S22 =K i2 Ia;2 S1 Ib;2 S1 S2
mg1
(45)
substrate alone in single substrate system, a competitive type of

cross inhibition between phenol and p-cresol is thus observed [27].
Therefore, the presence of p-cresol in phenolp-cresol system
decreases the phenol degradation rate. On increasing the phenol
concentration in combination with p-cresol the biodegradation
time of both the substrates is further increased due to the
competitive cross inhibition caused by both substrates on each
other. These observations support the ndings of growth kinetic
studies on phenolp-cresol system (Section 3.1.2) carried out to
test the four substrate interaction patterns (Eq. (18)).
Phenolresorcinol system
Fig. 4 shows the experimental and model predictions of
degradation proles for phenol and resorcinol in phenolresorcinol system, at the total initial substrate concentration of 400 mg/L.
The simulated values of the model show good agreement with the
experimental data of phenol and resorcinol degradation for each
combination of phenol and resorcinol. Fig. 4 shows that it takes less
time for resorcinol degradation in comparison to phenol. The
biodegradation time for 100 mg/L resorcinol in combination with
300 mg/L phenol has been observed 36 h while it took 38 h for the
degradation of 300 mg/L phenol. Biodegradation of 100 and
300 mg/L phenol occurs in 7 and 32 h respectively, in single
substrate degradation system. When resorcinol is present with
phenol in the medium it causes inhibition to phenol degradation.
Since the biodegradation time for both the substrates in dual
[(Fig._4)TD$IG]
Concentration of substrates, phenol/resorcinol (mg/L)
350

Exp. resorcinol 100 mg/L
Exp phenol 200 mg/L
Exp. resorcinol 200 mg/L
Exp. Phenol 100 mg/L
Exp. Resorcinol 300 mg/L
Model
250
200
150
100
50
0
0
10
15
20
25
30
35
substrate system is larger than the biodegradation time for these

substrates in single substrate system, the competitive cross
inhibition is expected to occur between phenol and resorcinol.
This fact is supported by the growth kinetic studies on phenol and
resorcinol described by Eq. (19) with condition Ib,1 = Ib,2 = 0. The
degradation dynamic proles clearly support to conclude that the
models represented by Eqs. (33) and (34) describe well the specic
degradation rates for phenolresorcinol system.
Conclusion
(44)
300
873
40
Degradation time (h)

Fig. 4. Effect of degradation time on concentration of substrate, phenol/resorcinol.
G. indicus degraded phenol, p-cresol, and resorcinol in the dual

substrate systems efciently. Competitive inhibition type of
substrate interaction was found between phenol and p-cresol,
phenol and resorcinol. Specic degradation rate model with the
variation of maintenance energy expenditure was proposed and
the model predictions were found consistent with the experimental data. Further, biodegradation dynamics of these substrates was
modeled. Simulated model predictions supported the ndings of
growth kinetic studies well. The substrate degradation kinetic
models proposed in this study can be applied to the design of
treatment unit for the biodegradation of mixture of phenolic
compounds on commercial scale.
Acknowledgments
This work was nancially supported by the University Grant
Commission (UGC), Bahadur Shah Zafar Marg, New Delhi: 110002,
India.
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College of Engineering - Department of Chemical Engineering

CHE 491: Senior Design 2

EFFECT OF KINETIC INTERACTIONS ON BIOREACTOR PERFORMANCE

Azka Vicar Mir

Reshma Sulthana Sherif

Submission Date: 26th May 2015

Figure 1: Plant Layout

Rotating biological contactors (RBCs) These consist of biomass coated media

Equation 1: Growth rate of Biomass

Equation 2: Growth rate using Monod model

Equation 3: Growth rate using Andrews model

Equation 4: Maximum specific growth rate for pollutant j

Equation 5: Maximum specific growth rate for pollutant q

Equation 6: Growth rate using sum kinetics model

Initial Substrate Conc. (S0) vs. Removal

Removal Efficieny, RE (%)

Initial Substrate Conc., S0 (mg/L)

Figure 2: Initial Substrate Conc. vs RE% (B-T) using PPO1

Initial Substrate Conc. (S0) vs. Removal Efficiency

Removal Efficieny, RE (%)

Initial Substrate Conc., S0 (mg/L)

Results for PPO1 (@ S0 = 500 mg/L)

Removal Efficiency (%)

Initial Substrate Conc. (S0) vs. Removal Efficiency

Removal Efficieny, RE (%)

Initial Substrate Conc., S0 (mg/L)

Initial Substrate Conc. (S0) vs. Removal Efficiency

Removal Efficieny, RE (%)

Initial Substrate Conc., S0 (mg/L)

Results for Consortium (@ S0 = 500 mg/L)

Removal Efficiency (%)

Table 5: Results for Consortium @ S0 = 500 mg/L

Toluene and Phenol

2. Phenol results illustrated using the Monod model.

Initial Substrate Conc. (S0) vs. Removal Efficiency

Removal Efficiency, RE (%)

Initial Substrate Concentration, S0 (mg/L)

Figure 6: Graph of initial substrate concentration vs. removal efficiency interaction

Benzene and Phenol

1. Phenol results illustrated using the Monod model.

Initial Substrate Conc. (S0) vs. Removal Efficiency

Removal Efficiency,RE (%)

Initial Substrate Concentraton,S0 (mg/L)

Figure 7: Graph of initial substrate concentration vs. removal efficiency interaction

Equipment Sizing and Material of Construction

Calculations for Sizing

Heuristic: For less than 3.8 m3 use vertical tank on legs

Therefore, Volume=40.50 m3, Diameter=2.580 m, Length=7.742 m

Tank 2 (Neutralization Tank) (T-102)

Calculations for Sizing

Horizontal tank with floating roof, Volume=60.75 m3, Diameter=2.954 m, Length=8.863 m

Therefore, Volume=40.50 m3, Diameter=2.580 m, Length=7.742 m

Ideal CSTR is when 500 2000 revolutions of a well- designed stirrer

Benzene (no interaction)

900 (400 20)

Toluene (no interaction)

900 (400 20)

Material of Construction for CSTR

Settling Tank- (T-103)

Table 1 Typical design values for a sedimentation basin. [1]

Using Table 1 the typical values are:

Therefore, using this length area can be calculate:

Therefore, the over flow rate is [1]:

= = 0.95 3.5 = 3.33

Power for pumping liquids: kW = (1.67)[Flow (m3/min)][P (bar)]/