Professional Documents
Culture Documents
ID#
Mahren Masud
38975
39786
Fahmida Anwar
39982
42501
Anisul Karim
44246
Sonam Ludhani
49634
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 toluene- phenol and benzene-phenol. Figure 1 below
illustrates a simple plant layout which was used in preliminary equipment sizing using heuristics.
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.
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)
= +
(2)
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)
+ +
(4)
, + +
(5)
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
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
100
80
60
Benzene
Toluene
40
20
0
0
200
400
600
800
1000
1200
Figure 1: Graph of initial substrate concentration vs. removal efficiency using PPO1 no
interaction
20
INTERACTION
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
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
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
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
Benzene
97.5
Toluene
98.4
Interaction
Benzene
82.6
Toluene
95.5
24
90
80
70
60
50
Toluene
40
Phenol
30
20
10
0
0
2000
4000
6000
8000
10000
12000
25
26
90
80
70
60
50
Benzene
40
Phenol
30
20
10
0
0
2000
4000
6000
8000
10000
12000
27
Design
The sizing of the plant (Figure 1) has been performed as follows:
= 0.9
= 342367
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
= 3
=
2
3 = 40.50
4
= .
2
3
4
= = .
= 0.9
= 342367
= 3
=
2
3
4
2
3 = 60.75
4
= 2.954
= 3 = 8.863
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
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 )
=
19.32 .
4
= .
1
= .
= 20
0
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 )
=
21.385 .
4
= .
1
= .
= 17.7
0
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
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
2
2
543
=
=
= 1.96
4
4 3.5
= 4 = 4 1.963 = 7.86
34
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.
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
Material of Construction
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
279
868
0.04
0.0712
303
1.02E0-5
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
8.31
, =
= 2.18 3
3
0.20 24 3600 2.204
.
10,000
, = 0.09234
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
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
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
( 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
.
.
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
= 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
, =
= 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
2
) + 1 = ( ) + 1 = 1.9 = 2
2.3
42
Pumps
Purpose of the equipment
Pump the water from the settling tank to the air stripper
Assumptions
= 0.9 3 /
= = 1000 /3
Based on Heuristics:
Pump 1-(P-101A/B)
Power =
3
0.7848
0.6
= 2.0
43
Pump 2-(P-103A/B)
P (bar) = = 1000
1.670.9
Power =
3
0.530
0.6
9.81
= 1.33
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)
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.
46
Costing
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
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
No Flow
Possible Cause
Line Fracture
Consequence
Action Required
Wastewater discharged to
adjacent area
Estimate quantity
released and develop
management plan
More Flow
Error in FCV
More Pressure
Buildup of vapor
Inflow conditions
Increased rate of
corrosion. Damage to
equipment
More hardness
Inflow conditions
Tank 2
Deviation
No Dosage
Possible Cause
Consequence
Action Required
49
No mixing
More Temperature
Uneven mixture.
Corrosion of mixer
paddles
Industrial discharge at
high temp
Threat to biomass
Pump malfunction
Delay to process
Line Blockage
Inflow conditions
Threat to biomass
pH control in equalization
tank
Less Flow
Acidic or basic
compounds
Reactor
Deviation
Causes
Consequences
Action
High pressure
Temperature increase in
reactor
Excessive organic
loading
Inefficient removal of
pollutants
Removal efficiency
decreases
Improper environmental
conditions
No degradation of
pollutants
High pH
Decrease in biomass
growth
Adjust pH using a pH
indicator controller and
dosing pumps.
50
Settling Tank
Deviation
Causes
Consequences
Action
High flow
No flow
Accumulation of
activated sludge
Overflow of untreated
water
Low flow
Air Stripper
Deviation
Causes
Consequences
Action
Blockage in pipe
Regular maintenance
Pipe ruptured
Flooding
Regular maintenance
No liquid flow
pump failure
No stripping
51
Adsorber
Deviation
Causes
Consequences
Action
No flow
Pipe leak
Regular maintenance
No adsorption
No adsorption
Regular maintenance,
Activate backup column
More concentration of
pollutants
Saturation of carbon
Pumps
Deviation
Causes
Consequences
Action
Impingement of water on
the impeller causes
erosion corrosion
High Flow
No flow
Gas Locking
Less flow
Cavitation
Reduction in pump
efficiency; damage to
pump components
Pump Failure
Reverse Flow
Regular maintenance
Regular maintenance
52
Piping
Deviation
Causes
Consequences
Action
No liquid flow
Pump cavitation
Pump cavitation or
pipeline block
No liquid flow to
the equipments
and Pipe rupture
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].
[7] Reardon, K. F., Mosteller, D. C., Bull Rogers, J. D., DuTeau, N. M., & Kim, K. (2002).
Biodegradation Kinetics of Aromatic Hydrocarbon Mixtures by Pure and Mixed Bacterial
Cultures. Environmental Health Perspectives, vol. 110, no. 6, pp. 1005-1011. [Online].
Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1241285/ [Accessed: Sept. 29,
2014].
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,
Volume 86, Part C, January 2014, Pages 272-277, ISSN 0964-8305,
http://dx.doi.org/10.1016/j.ibiod.2013.09.018. [Accessed: Sept. 29, 2014]
[9] Yao, H., Ren, Y., Deng, X., & Wei, C., (2011) Dual substrates biodegradation kinetics of mcresol and pyridine by Lysinibacillus cresolivorans, Journal of Hazardous Materials,
Volume 186, Issues 23, 28 February 2011, Pages 1136-1140, ISSN 0304-3894,
http://dx.doi.org/10.1016/j.jhazmat.2010.11.118. [Accessed: Sept. 29, 2014]
[10] Pierre Le-Clech, Vicki Chen, Tony A.G. Fane, Fouling in membrane bioreactors used in
wastewater treatment, Journal of Membrane Science, Volume 284, Issues 12, 1 November
2006, Pages 17-53, ISSN 0376-7388, http://dx.doi.org/10.1016/j.memsci.2006.08.019.
[Accessed: Sept. 29, 2014]
[11] Oh, Y. S., Shareefdeen, Z., Baltzis, B. C., & Bartha, R. (1994). Interactions between
benzene, toluene, and p-xylene (BTX) during their biodegradation. Biotechnology and
Bioengineering, 44(4), 533-538. [Accessed: Sept. 29, 2014]
[12] Zarook, S. M., Shaikh, A. A., & Ansar, Z. (1997). Development, experimental validation
and dynamic analysis of a general transient biofilter model. Chemical Engineering Science,
52(5), 759-773. doi:10.1016/S0009-2509(96)00428-9 [Accessed: Dec. 16, 2014]
[13] Rafiei, B., Naeimpoor, F., & Mohammadi, T. (2014). Bio-film and bio-entrapped hybrid
membrane bioreactors in wastewater treatment: Comparison of membrane fouling and
removal efficiency. Desalination, 337, 16-22. doi:10.1016/j.desal.2013.12.025 [Accessed:
Nov. 22, 2014]
59
[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),
1136-1140. doi:10.1016/j.jhazmat.2010.11.118 [Accessed: Sept. 29, 2014]
[15] Department, E. (2013, 01). Trakhees. Retrieved 05 25, 2015, from
http://www.ehss.ae/forms/guidelineno.en-001airenvironmentjanuary2013.pdf
[16] Engineering Toolbox. (n.d.). The Engineering ToolBox. Retrieved from
http://www.engineeringtoolbox.com/
[17] Hutchins, R. (1974). New Method Simplifies Design of Activated Carbon Systems. Chem.
Engr. , 80, 133-138.
[18] HyperTextBookShop. (n.d.). Retrieved from
http://www.hypertextbookshop.com/biofilmbook/working_version/artifacts/tables/Modul
e_004/Table4-1_DiffCoeffH2O.htm
[19] LaGrega, M. D., Buckingham, P. L., & Evans, J. C. (2001). Hazardous Waste Management
and Environmental Resources Management. Thomas Casson.
[20] Regulation, W. Q. (2013, 04 10). Regulation & Supervision Bureau. Retrieved 05 25, 2015,
from http://www.rsb.gov.ae
[21] Speth, T., & Miltner, R. (1990). Benzene/GAC Isotherm. Retrieved from
http://iaspub.epa.gov/tdb/pages/contaminantProcess/contaminantProcessReferences.do
[22] Towler, G., & Sinnott, R. (2008). Chemical Engineering Design: Principles, Practice, and
Economics of Plant and Process Design. Oxford: Elsevier.
60
Appendix
Meeting Minutes Sample
Time
Item
Responsible
17:30
0:02
Time Keeper
Coordinator
17:32
0:01
Leader
17:33
0:03
Leader
17:36
0:24
All Members
18:00
0:25
All Members
18:25
0:03
Recorder
Meeting
Coordinator
18:28
18:30
0:02
Meeting Adjourned
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
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 :
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 :
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 :
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
1005
Chemical Mixtures
Reardon et al.
Single chemical
Single microbial
species
Multiple microbial
species
Chemical mixture
Multiple microbial
species
1006
[1]
m TOT = mL + mG
H
= mL 1 +
RT
VG
= mL . [2]
VL
[3]
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]
1 dX
max S L
=
,
X dt
K S + S L + S L2 / K i
[5]
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
Chemical Mixtures
0
40
Time (hr)
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.
[6]
max,1S1
K S ,1 + S1 + I 2,1S 2
max,2S 2
K S ,2 + S 2 + I 1,2S1
[8]
30
25
20
10
Phenol
Benzene
Biomass
Competitive
SKIP
50
30
20
10
0
0
10
40
20
30
40
0
50
60
[7]
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)
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)
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
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)
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
Phenol
0.7
0.8
Chemical Mixtures
25
Time (hr)
1009
Chemical Mixtures
Reardon et al.
1010
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).
REFERENCES AND NOTES
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20. Guha S, Peters C, Jaff P. Multisubstrate biodegradation
kinetics of naphthalene, phenanthrene, and pyrene mixtures. Biotechnol Bioeng 65:491499 (1999).
21. Lewandowski GA, Baltzis BC, Kung C-M, Frank ME. An
approach to biocatalyst modeling of mixed populations
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22. Murakami Y, Alexander M. Destruction and formation of
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1997.
1011
Regular article
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
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.
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
157
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
+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
(10)
k
3
k
+4
(11)
k
k
+5
X + a
3 S2 X S2
(12)
k
mi Si
S = I1
(13)
Ksi + Ii1 S
S1 + S12
Ki1
X =
3
Xi
(15)
i=1
+3
X + a1 S1
X S1
k
+ I2
S2 + S22
Ki2
+ I3
S3 + S32
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)
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 .
X
X
X
X
m1 +
Ks1 +
m2 + +
f23
m1
Ks1
m2
f23
(20)
159
(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.
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
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.
T1-T3
T4-T6
T7-T9
T10-T12
T13-T15
T16-T18
T19-T21
T22-T24
T25-T27
4-CP, 4-chlorophenol
m-Cresol
4-CP
200
200
200
300
300
300
400
400
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.
163
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
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
(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.)
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
166
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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
866
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
867
(1)
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
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
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)
mg
k2 X 1 k8 X 2
X T
(4)
mg
mgmax1 S1
D1
mgmax2 S2
D2
(5)
[(Fig._1)TD$IG]
0.12
869
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
0.059
0.45
0.4
0.052
0.35
0.045
0.3
0.038
0.25
0.2
0.031
0.15
0.1
0.05
0.024
0.017
0.01
0
200
400
600
800
1000
1200
mg mg1 mg2
(16)
mg
mgmax1 S1
2
mgmax2 S2
K S2 S2
[(Fig._2)TD$IG]
1400
S2 2
K i2
(17)
870
Table 2
Estimated values of biomass growth kinetic model parameters for phenol.
Model
Mathematical expression
mg
Haldane
mg
Yano
mg
Webb
mg
mgmax S
S K S S2 =K i
m
S
gmax
S K S S2 =K i SK S =K i
mgmax S
2
S K S SK 1 KS
i S
mgmax S 1 K
2
S K S SK
0.98
2.10
(35)
53.59
0.97
2.75
(36)
0.99
2.16
(37)
0.95
6.42
(38)
78.29
0.485
53.56
0.262
36.08
128.5
0.240
29.88
114.9
Ki (mg/L)
784.6
9510
Dmg %
Equations
350
300
250
150
100
50
0
40
50
mg
0:462S1
2
S1
78:29 S1 44:29
0:044S2
60
0:512S2
2
S2
1:17S1
91:87 S2 21:99
(18)
To study the effect of the presence of resorcinol on biodegradation, phenol, and resorcinol have been taken in combinations of
100 mg/L phenol300 mg/L resorcinol, 200 mg/L phenol200 mg/L
resorcinol, and 300 mg/L phenol100 mg/L resorcinol. The results
of single substrate biodegradation study show that resorcinol is an
inhibitory substrate like phenol. it causes substrate inhibition
effect in the medium at an initial concentration of 90 mg/L. Hence,
the biodegradation of resorcinol in presence of phenol has been
studied at the concentrations higher than 90 mg/L.
The analysis of Eq. (17) for phenolresorcinol system by
LevenbergMarquardt nonlinear regression program applied on
the four inhibition patterns, shows that competitive cross
inhibition takes place during the biodegradation of phenol with
resorcinol. The values of interaction parameters have been
obtained as Ia,1 = 1.09, Ia,2 = 0.052 for phenolresorcinol degradation system, which indicate that the resorcinol has stronger
inhibition effect on phenol degradation in comparison to the
inhibition caused by the phenol to resorcinol degradation. During
the experimental study it has been observed that the fungus
consumed resorcinol in preference to phenol. The specic growth
rate of biomass in phenolresorcinol system is expressed as
0:462S1
2
S1
78:29 S1 44:29
1:09S2
200
30
mg
20
44.49
0.462
[(Fig._3)TD$IG]
10
R2
KS (mg/L)
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
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)
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
Competitive inhibition
Competitive inhibition
Competitive
Competitive
Competitive
Competitive
Competitive
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
mgi
Y X=S
m1i ki 1
Ti
mgi
mgmaxi
!
(23)
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)
mgi
(29)
(30)
R2 1
For phenol
(31)
R2 1
For p-cresol
(32)
R2 1
For phenol
(33)
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
Boundary conditions
Equations
At t = 0, XT = XTo,
S1 = S1o, S2 = S2o
(39)
(40)
(41)
2
g1
(42)
(43)
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)
[(Fig._4)TD$IG]
350
250
200
150
100
50
0
0
10
15
20
25
30
35
(44)
300
873
40
874
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