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Kinetics and Dynamics of Benzene Biodegradation with Pseudomonas putida

(ATCC 11172)

Conference Paper · October 2018

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 Kinetics and Dynamics of Benzene Biodegradation with Pseudomonas putida

Jun-Hsien Wang* and Jun-Chen Liao

Department of Chemical Engineering and Material Science

Minghsin University of Science and Technology
Hsinfong, Hsinchu, Taiwan 30401
*Email: bunny@must.edu.tw

Abstract non-perturbed node, either. All the theoretical predictions

An aerobe, Pseudomonas putida (ATCC 11172), was used
to study the benzene biodegradation kinetics first, in a
250mL shaking flask, at 25 ºC and pH 7.0  0.1. The
biomass concentrations were measured by the detection of
the optical density (OD600) by a UV/Vis- Spectrophotometer
(JASCO V-530) with a fixed wavelength at  = 600nm. The
benzene concentrations were measured by a Waters High
Performance Liquid Chromatography (HPLC). The aqueous
benzene concentration ranges of interest were 5 ~ 1800 mg
L-1. The net specific growth rates of benzene under these
concentration ranges were obtained by using the initial rate
method, and regressed nonlinearly by a modified Andrews’
model by the aid of a statistical package, SAS. It shows that
the benzene concentration effect on the biomass specific
growth rate can be well expressed by a modified Andrews’
model. The kinetic model parameters for benzene are as
follows: m = 1.798 h-1, KS = 63.74 mg L-1, and KI = 104.8
mg L-1. Besides, the true yield coefficient Y and the culture
self-maintenance coefficient c on benzene are derived by
analyzing the linear relationship between the apparent yield
coefficients, Yapp., and the net culture specific growth rates,
net. The result ends up with Y = 1.283 g g-1 and c =
0.05557 h-1.
The obtained kinetic data were furthermore used to
investigate the possible and meaningful multiple steady
states for the operation of benzene biodegradation in a
continuously-stirred tank reactor (CSTR). The outcomes
show that there exits three possible steady states under the
dilution rate D = 0.4 h-1 and benzene feed concentration s =
500 mg L-1 as follows: The steady state one (S.S.1) are
benzene concentration (s) 23.3 mg L-1 and biomass
concentration (b) 612 mg L-1 in the effluent stream, the S.S.2
are s = 285 mg L-1 and b = 276 mg L-1, and the S.S.3 are s =
500 mg L-1 and b = 0 mg L-1. Of these three steady states
mentioned above, the S.S.1 is a biomass survival stable
steady state, the S.S.2 is a biomass survival unstable steady
state, and the S.S.3 is a biomass washout stable steady state.
Meanwhile, from the mathematical point of view, the S.S.1
is a stable non-perturbed node, the S.S.2 is an unstable
saddle point, which contains two convergence directions and
two other divergence directions, and the S.S.3 is a stable
were carefully validated by a series of CSTR experiments.
1 Introduction
Benzene is widely used over the world [3]. It ranks in the top
20 chemicals for production volume. Some industries use
benzene to make other chemicals that are used to make
plastics, resins, and nylon and synthetic fibers. Benzene is
also used to make some types of lubricants, rubbers, dyes,
detergents, drugs, and pesticides [3]. According to medical
researches, benzene exposure affects the central nervous
system (CNS) and hematopoietic system and may affect the
immune system. Death due to acute benzene exposure has
been attributed to asphyxiation, respiratory arrest, CNS
depression, or cardiac dysrhythmia. Pathologic findings in
fatal cases have included respiratory tract inflammation,
lung hemorrhages, kidney congestion, and cerebral edema.
Aerobic and anoxic biodegradation of benzene have been
studied for many years up today to prevent from its lethal
toxicity to the public health [1,4,5,7,9,10,12-14]. Due to the
fact that the substrate inhibition effect on the net specific
growth rate of the species, Pseudomonas putida [2], a
reactor kinetic and dynamic analysis will provide detailed
knowledge of the possible and meaningful steady states in
the microbial degradation of benzene and enhance the
biodegradation rate, for a continuous operation
[5,11,15,17,18]. In this study, a widely utilized pure culture,
P. putida, was used to investigate the competitive inhibition
of benzene on the net specific growth rate of the species. The
kinetic data were analyzed to reveal the multiple steady
states, and the optimal operation strategy for benzene
removal or biodegradation.
2 Theory
2.1 Kinetic Model in a Batch Operation Mode
Material balance on substrate (benzene):
ds 1 1
    net   c  b     b
(1.1)
dt Y Y
Material balance on biomass:
db
    c  b   net b , (1.2)
dt
where the net specific growth rate of the biomass can be ^ s
described by a modified Andrew’s model [2] as follows: G s    sf  s    c  sf  s  (1.14)
s2
 s
^
K+s+
 net  2
 c , (1.3) KI
s
Ks 2.3 Dynamics Analysis in a CSTR Operation
KI The condition that steady states will be reached only holds
where c denotes the endogeneous maintenance coefficient, for the necessary and sufficient condition, F(s) = G(s). The
and Y the true yield coefficient. stability analysis can be done by means of the Jacob matrix
analysis [6,8,11,16] as follows:
The correlation between the apparent yield coefficient and ds 1
the true yield coefficient can be expressed as = D(s f  s )   net b = f ( s , b ) (1.15)
db Y net dt Y
Yapp   (1.4)
 ds   net   c 
db
=  Db +  net  s  b  g  s , b  (1.16)
dt
1   c  1 c  1 
 f f   D  b d  
  net
    (1.5) 
1
 net
Yapp Y net Y Y  net     f , g    s b   Y ds Y  (1.17)
s =    =  

   s , b    g g   
 D   net 
d
2.2 Kinetic Model in a CSTR Operation Mode b
Material balance on substrate:  s b   ds 
The characteristic equation or the corresponding determinant
ds 1 becomes
= D(s f  s )   net ( s )b (1.6)
dt Y b d 1
D     net
Material balance on biomass: Y ds Y
db =0 (1.18)
= -Db +  net b (1.7) d
dt b  D   net  
ds
For steady states, it implies
ds 1 Case 1. Biomass survival ( D =  net , 0 < s < sf )
= D(s f  s )   net ( s )b = 0 (1.8)
dt Y
b d 1
db D    D
=  Db +  net b = 0 , (1.9) Y ds Y
dt =0 (1.19)
where D denotes the dilution rate, D = F/V. d
b 
There exist two possible steady states, ds
Case 1: D =  net , 0 < s < sf (survival), or  b d   D b d = 0
 D 
2
(1.10) (1.20)
Case 2: b = 0, s = sf (washout).  Y ds  Y ds
From equations (1.8) and (1.9), we get for biomass survival, D d
12 
b = Y  sf  s 
b
(1.11) Y ds
Hence, equation (1.6) can be rewritten as (1.21)
 b d 
ds 1  2    D  
= D(s f  s )   net ( s )  sf  s   Y ds 
dt d
^ s If > 0 , then
= D(s f  s )    sf  s    c  sf  s  ds
s2 D d
K+s+ 12  b  0,
KI Y ds
(1.22)
(1.12)
 b d 
Define the substrate feed rate, F(s), and the substrate 1  2    D  0
consumption rate, G(s) as follows:  Y ds 
F  s   D(s f  s ) (1.13)  1  0, 2  0 .
and The criterion equation is
    
1-nitriloacetic acid 0.2g. Benzene and biomass
b d d b d
2 2
D
  D 4 b  D 0 (1.23) concentrations were measured with a reverse phase column
Y ds Y ds Y ds (SUPELCOTM C18 Hypersil ODS, 5m particle size, length ×
Two negative real Eigen values imply that the biomass I.D.: 250mm × 4.6mm) by a HPLC chromatography (Waters
survival outcome is a non-damping stable node, i.e., it is a 2487) at 254nm UV detector, and a UV/Vis-
stable steady state (S.S.1). spectrophotometer (JASCO V-530) at 600nm of constant
d wavelength, respectively.
If < 0 , then eqn. (1.20) implies
ds The CSTR experiments were performed in a well-stirred
D d glass vessel which was immersed in a thermostat (FIRSTEK
1 2  b  0, B402L, ROC), and the feed rate was controlled by a
Y ds peristaltic pump (Monastat 72-560-100, USA). The benzene

 
(1.24)
b d b d concentration range of interest was 5~1800 mg L-1, which is
1  2   D   0, if D >
Y ds Y ds corresponding to the upper limit of the saturation solubility
 1  0, 2  0 . of benzene in water, 1800 mg L-1. Besides, the biomass
concentrations in the CSTR experiments were measured by
The criterion equation is the dry cell weight (DCW) method.

 b d
 d
 b d
2 2
D
  D 4 b  D
Y ds Y ds
One of the two Eigen values is positive, and the other one is
negative; both are real roots, these indicate that the steady
state (S.S.2) for biomass survival is a meta-stable saddle
point, two directions are stable, and the other two directions
are unstable.
Case 2. Biomass wash-out ( b = 0, s = sf )
1
D     net  sf 
Y =0
 D   net  s f   
0
 D +  D   net +   = 0
1   D < 0
2   net  sf   D < 0, if D >  net  sf 
Two negative Eigen values imply that the biomass wash-out
outcome is a stable node (S.S.3).
3 Materials and Method
The pure culture, Pseudomonas putida (ATCC 11172), was
used to study the benzene biodegradation kinetics [5,12-14].
The cultivation media (per liter) was composed of beef
extract (3 g), peptone (5 g), KH2PO4 0.5g, K2HPO4 0.5g,
MgSO4·7H2O 0.5g, CaCl2·2H2O 0.08g, MnSO4·7H2O 0.1g,
ZnSO4·7H2O 0.02g, (NH4)2SO4 0.5g, Fe(NH4)2(SO4)2·6H2O
0.08g, and 1-nitriloacetic acid 0.2g, in a 250 mL
Erlenmeyer flask and adjusted pH to be 7.0  0.1. The
culture was inoculated to the sterilized media and incubated
at 25 °C for seven days. The culture was then injected into a
100 mL of sterilized serum bottle for kinetic study, which
contains benzene as the carbon source and trace elements
(per liter) of KH2PO4 0.5g, K2HPO4 0.5g, MgSO4·7H2O 0.5g,
CaCl2·2H2O 0.08g, MnSO4·7H2O 0.1g, ZnSO4·7H2O 0.02g,
(NH4)2SO4 0.5g, Fe(NH4)2(SO4)2·6H2O 0.08g, and

 0 (1.25)
Y ds 4 Results and Discussion
4.1 Kinetic Model in Batch Operation Mode
The correlation between the apparent yield coefficient and
the true yield coefficient can be described by Equation (1.5).
The true yield coefficient and the endogeneous maintenance
coefficient can be obtained by the linear regression of the
reciprocals of both the apparent yield coefficient and the net
specific growth rate of the species, including both the
benzene concentrations greater and less than the
(1.26) breakthrough point, s*, at which the maximum specific
growth rate will be achieved, as shown in Figure 1. Both
approaching lines meet at a same intercept, 0.7792, which
(1.27) gives the true yield coefficient, Y = 1.283 g g-1 and the
endogeneous maintenance coefficients, c = 0.0556 h-1 or c
(1.28) = 0.241 h-1 for the benzene concentrations greater or less
than the breakthrough point, s* = 81.73 mg L-1, respectively.
This observation depicts that the substrate inhibition effect
on the net specific growth rate only happens at substrate
concentrations greater than the breakthrough point. This
coincides with the kinetic theory of the competitive
inhibition for an enzymatic reaction. On the other hand,
instead of depression, the net specific growth rate will be
enhanced at substrate concentrations less than the
breakthrough point. This phenomenon was already observed
by Wang [17]. These results imply that the modified
Andrews’ model including an endogeneous maintenance
coefficient term holds for high concentrations.
The specific growth rate of P. putida on benzene can be
illustrated by the modified Andrew’s model (Equation (1.3)),
as stated above. The non-linear regression done by the aid of
SAS package (SAS Institute Inc., Cary, NC, USA),
excluding the found value of c term which was determined
by Equation (1.5), the other three model parameters can be
established as follows:  = 1.7976 h-1, K = 63.74 mg L-1,
^
KI = 104.8 mg L-1. The substrate (benzene) dependence of
the net specific growth rate, net was shown in Figure 2. intersection points of both rates, which are b = 611.8 mg L-1
2 and s = 23.3 mg L-1 (S.S.1), b = 275.5 mg L-1 and s = 285.3
1.8 mg L-1 (S.S.2), and b = 0 mg L-1 and s = 500 mg L-1 (S.S.3),
1.6 respectively, lie together on a straight line, as shown by
1.4 y = 0.0438x + 0.7742 Equation (1.11), and pointed up in Figure 4.
R2 = 0.7098
1.2 300
Rate of Supply , F(s)
1/Yapp

1
250 Rate of Consumption, G(s)
0.8

0.6 200

F(s) or G(s)
0.4
y = -0.19334 x + 0.76756
R 2 = 0.99078 150
0.2

0 100
0 2 4 6 8 10 12 14 16 18
1/net
Experiment al dat a Upper bound
Lower bound Low subst rat e conc. dat a 50
線 性 (Experiment al dat a) 線 性 (Low subst rat e conc. dat a)

Figure 1: The correlation between the both reciprocals of 0

the apparent yield coefficient and the net specific growth 0 100 200 300 400 500
rate. Benzene Concentration, s (mg/L)
From Figure 2, we can easily realize the maximum specific Figure 3: The correlation between the substrate feed rate,
growth rate, max. = 0.702 h-1, which will occur at the F(s), and the substrate consumption rate, G(s) for D = 0.4h-1
breakthrough point, the latter can be derived by setting the and sf = 500 mg L-1.
first derivative of Equation (1.3) as zero,
700
1
s  K  K I  81.73mg L
*
. Besides, the specific growth S.S. 1 3 Steady States
600 Go to Biomass Washout S.S. 3
rate does not go through the zero point, which implies the Go to Stable Steady State S.S. 1
existence of the endogeneous metabolism coefficient, c, 500 b Return to S.S. 2
Return to S.S. 2
especially for the substrate concentrations greater than the
Biomass, b

400
breakthrough point.
300 S.S. 2
0.8
d
Specific growth rate,  (h )

0.7
-1

200 c
0.6
100
0.5 a S.S. 3

0.4
0.3
0.2
0.1
0 and sf = 500 mg L-1.
-0.1 0 250
Benzene Concentration, s (mgL )
Figure 2: The substrate (benzene) dependence of the net
specific growth rate of Pseudomonas putida on benzene.
4.2 Kinetic Model in CSTR Operation Mode
From the analysis of Equations (1.6)-(1.9), we can get two
categories of possible steady states, as denoted by Equation
(1.10). Either case indicates that both biomass and substrate
concentrations have a linear relationship, b = Y  sf  s  .
From the results by analyzing the relation of the substrate
feed rate, F(s), and the substrate consumption rate, G(s),
there exits three possible steady states, as shown in Figure 3,
which stands for a dilution rate of 0.4 h-1, and a 500 mg L-1
of substrate in the feed stream. From Figure 3, the three
possible and meaningful steady states, obtained from the
0
0 100 200 300 400 500
Substrate, s
Figure 4: The phase portrait of benzene biodegradation by
Pseudomonas putida in a CSTR operation for D = 0.4h-1
500 750 1000 1250 1500 1750
-1 4.3 Dynamics Analysis in CSTR Operation Mode
The furthermore analysis of the dynamics in a CSTR
operation reveals the outcomes of three steady states which
were consisted of two stable nodes and one unstable saddle
point, as shown in Figure 4. At the S.S.1, a stable node
(biomass survival) can sustain any perturbation, as predicted
by Equations (1.22) and (1.23). At the S.S.3, the other stable
node (biomass wash-out), has the same tendency, as
predicted by Equation (1.28). But, at the S.S.2, a meta-stable
saddle point designates that some perturbations may
congregate back to the original point, but ironically, some
other perturbations may move away from this point, to either
one of the other two steady states, as predicted by Equations
(1.24) and (1.25). All these theoretical deductions had been
experimentally validated, as shown in Figures 5-10 and
Table 1.
 1200 1200
1200 1200 Benzene
Benzene
1000
Biomass 1000
Biomass
Benzene Concentration (mg/L)

Benzene Concentration (mg/L)

Biomass Concentration (mg/L)

1000 1000

Biomass Concentration (mg/L)

800 800
800 800

600 600 600 600

400 400 400 400

200 200 200 200

0 0 0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18
Time (hr) Time (hr)

Figure 5: Experimental validation of the stable node (S.S.1) Figure 7: Experimental validation of the instability of the
in a CSTR operation for D = 0.4h-1 and sf = 500 mg L-1. The saddle point (S.S.2) in a CSTR operation for D = 0.4h-1 and
process moves back to the original node after a perturbation sf = 500 mg L-1. The process moves away to a biomass
applied. wash-out outcome (S.S.3) after a perturbation applied.
When a biomass survival steady state (S.S.1) had reached, 1200 1200
an applied perturbation would end up with the same steady Benzene

Biomass Concentration (mg/L)

Benzene Concentration (mg/L)

Biomass
state, after three hours of continuous operation, as shown in 1000 1000

Figure 5. The steady state has very good coincidence with

800 800
the theoretical predicted values, as shown in Figures 3 and 4.
When another biomass wash-out steady state (S.S.3) had 600 600
reached, the system will go back to the same node from any
400 400
applied perturbation, as shown in Figure 6. The stable steady
state has excellent coincidence with the theoretical predicted 200 200
values as well, as shown in Figures 3 and 4.
0 0
1200 1200
0 2 4 6 8 10 12 14 16 18
Benzene
Biomass T ime (hr)
Benzene Concentration (mg/L)

1000 1000
Biomass Concentration (mg/L)

Figure 8: Experimental validation of the instability of the

800 800
saddle point (S.S.2) in a CSTR operation for D = 0.4h-1 and
600 600
sf = 500 mg L-1. The process moves away to a biomass
survival outcome (S.S.1) after a perturbation applied.
400 400

200
0 0
0 2
Figure 6: Experimental validation of the stable node (S.S.3)
in a CSTR operation for D = 0.4h-1 and sf = 500 mg L-1. The
process moves back to the original point after a perturbation
applied.
Besides, when a perturbation was applied to a system which
was located at a meta-stable saddle point (S.S.2), there are
two possible outcomes: one is the departure from the
original point, eventually the system will shift to either one
of the other two stable steady states, as validated and shown
in Figures 7 and 8. The other possible outcome is the
junction back to the original saddle point, as validated and
shown in Figures 9 and 10.
Figures 7 and 8 demonstrate the instabilities of the saddle
point (S.S.2) in Figure 4. When a perturbation applied to the
system at S.S.2, the outcomes depend on the values of
biomass and benzene concentrations. For the case of
200
benzene concentrations less than the value at S.S.2, and the
biomass concentration is greater than Y(sf – s), the system
will apart from S.S.2 and shifts to S.S.1, as shown in Figures
4 6 8 10 12 14 16 18
4 and 7. But for the case of benzene concentrations greater
Time (hr)
than the value at S.S.2, and the biomass concentration is less
than Y(sf – s), the system will apart from S.S.2 and shifts to
S.S.3, as shown in Figures 4 and 8.
Conversely, Figures 9 and 10 show the stabilities of the
saddle point (S.S.2) in Figure 4. When a perturbation applied
to the system at S.S.2, the outcomes for the case of benzene
concentrations less than the value at S.S.2, and the biomass
concentrations less than Y(sf – s), the system will return to
S.S.2, as shown in Figures 4 and 9. But for the case of
benzene concentrations greater than the value at S.S.2, and
the biomass concentrations greater than Y(sf – s), the system
will go back to S.S.2 as well, as shown in Figures 4 and 10.
All experimental data were listed in Table 1. The results
shown in Figures 7-10 were summarized in Figure 4, which
shows the detailed systematical phase portrait of benzene
biodegradation by P. putida.
 1200 1200 relationship in the phase portrait. A stable node can sustain
Benzene any perturbation; on the contrary, an unstable saddle point
Benzene Concentration (mg/L)

Biomass Concentration (mg/L)

1000 Biomass 1000
may depart from or return to the original point, depending on
800 800
the location of the initial operating condition in the phase
portrait. Furthermore, performing a saddle node bifurcation
600 600 analysis may provide an advanced information concerning
models of transitions and instabilities as some important
400 400
control parameter, such as the dilution rate, is varied [16].
200 200 Table 1: Operating conditions for experimental validation of
the dynamics behavior of benzene biodegradation.
0 0
0 2 4 6 8 10 12 14 16 18
Steady States SS 1 SS 2 SS3
Time (hr)
Figure 9: Experimental validation of the stability of the Outcome survival Survival Wash-out
saddle point (S.S.2) in a CSTR operation for D = 0.4h-1 and Stability Stable Unstable Stable
sf = 500 mg L-1. The process moves back to the original point Predicted
concentration 23.3 285.3 500
after a perturbation applied. of benzene
(mg L-1)
1200 1200 Predicted
Benzene concentration 611.8 275.5 0
of biomass
(mg L-1 DCW)
Benzene Concentration (mg/L)

1000 Biomass 1000

Perturbed
Biomass Conc. (mg/L)

800 800
benzene 119.1 417.4 107.7 152.4 387.6 398.6
concentration
(mg L-1)
600 600 Perturbed
biomass 538.5 93.8 516.7 230.1 200.5 0
concentration
400 400 (mg L-1 DCW)
Final
200 200
benzene 22.3 491.4 24.4 281.9 282.2 493.0
concentration
(mg L-1)
0 0 Final biomass
0 2 4 6 8 10 12 14 16 18 concentration 609.2 0 614.3 268.6 267.3 0
(mg L-1 DCW)
Time (hr)
Figure 10: Experimental validation of the stability of the
saddle point (S.S.2) in a CSTR operation for D = 0.4h-1 and Literature References
sf = 500 mg L-1. The process moves back to the original [1] Alvarez, P.J.J. and T. M. Vogel, “Degradation of BTEX and their aerobic metabolites by
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[2] Andrews, J.F., ”A mathematical model for the continuous culture of microorganisms utilizing
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[3] The Centers for Disease Control and Prevention (CDC). http://www.bt.cdc.gov/
[4] Collins, L.D. and A.J. Daugulis, “Biodegradation of phenol at high initial concentrations in
As displayed in the theory section, the S.S.1 always holds two-phase partitioning batch and fed-batch bioreactors,” Biotechnol. Bioeng. J., 55(1), pp.
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