Article

pubs.acs.org/IECR

Analysis of Hopf Points for a Zymomonas mobilis Continuous

Fermentation Process Producing Ethanol
Hangzhou Wang, Nan Zhang, Tong Qiu, Jinsong Zhao, Xiaorong He, and Bingzhen Chen*
Department of Chemical Engineering, Tsinghua University, 100084 Beijing, China

ABSTRACT: Zymomonas mobilis is one of the most promising industrial microorganisms for industrial ethanol production.
However, biomass, product and substrate oscillation behaviors have been observed under certain operating conditions in both
experiments and numerical simulations of continuous fermentations involving this organism. This article analyzed oscillation
behavior during fermentation based on Hopf bifurcation analysis, in which the Hopf singularity point and limit cycles were
introduced. All Hopf points in the operating parameter values panel were simultaneously located and identified for given sets of
operating conditions. Calculation of the distribution of Hopf points contributed to enhancing the stability of the fermentation
process, maintaining high product quality, and providing insights into system dynamics.

■ INTRODUCTION
Bioethanol is an important renewable and sustainable
alternative fuel source,1 both directly in the form of fuel
ethanol and as a blend with gasoline. Agricultural feed reactants
that are suitable for fermentation processes producing
bioethanol contain sugars, starches, and cellulose materials.
The yeast Saccharomyces cerevisiae2−5 and the bacterium
Zymomonas mobilis (Z. mobilis)3−6 are currently the most
important microorganisms for industrial production of ethanol.
Z. mobilis produces less biomass than Saccharomyces cerevisiae,
while more carbon is funneled to the ethanol fermentation. It
was reported that the ethanol yield of Z. mobilis could be as
high as 97% of the theoretical yield of ethanol to glucose,7
whereas only 90−93% can be achieved by Saccharomyces
cerevisiae.6 Thus, Z. mobilis has been promoted as a more
promising microorganism than yeast for the industrial
production of ethanol.8
Continuous fermentation using Z. mobilis is prone to
dynamic instability,2,3,6,9 in which sustained oscillations of
biomass, substrate, and product concentrations are routinely
observed, especially at low growth rate and high ethanol
concentrations.10 Oscillations are mainly caused by product
inhibition of enzymatic reactions, and experimental studies have
revealed that high temperatures may enhance such inhibitory
effects.11 Meanwhile, previous work has also showed that
oscillatory states are achieved at low dilution rates.10 These
oscillations increase the average residual glucose, which
correspondingly decreases the ethanol yield and declines
product quality.6 A considerable amount of work2,3,6,7,10−15
has demonstrated oscillatory behavior in fermentation
processes both experimentally and numerically. With regard
to bioethanol fermentation systems utilizing Z. mobilis, several
experimental and theoretical studies focused on dynamic
stability, which affects the industrial productivity of fuel
ethanol.3
In alcoholic fermentation, it is assumed that ethanol inhibits
the maximal specific growth rate indirectly by inhibiting the
synthesis of the main intermediate, which in turn determines
the overall growth rate.11 Therefore, the glucose conversion,
the ethanol yield, and the production rate will be favored by
minimizing ethanol inhibition.16 In addition, experimental and
theoretical studies also illustrated that variables such as dilution
rate and feed substrate concentration have an impact on static
and dynamic behavior in continuous stirred tank fermen-
tors.17,18
The existence of Hopf bifurcations were reported to cause
oscillatory behavior in fermentation processes with Z. mobilis.2
Optimal operating points and regions, calculated from
numerical analysis of oscillating biosystems, can be used to
avoid the oscillatory behavior that unfavorably decreases the
fermentation yield and declines product quality. Follow-up
design and control strategies can be developed to maintain
stability and guarantee high productivity and high product
quality.6,18 With properly selected operating conditions, these
Hopf points can be eliminated to avoid oscillations, directly
leading to a more stable and productive fermentation process.
The advantages of numerical analysis are listed as follows:
1. As the fermentation process is slow, its dynamic
simulation through experiment may be inefficient and
unable to locate points corresponding to certain dynamic
behaviors, such as bifurcation and oscillation.
2. Because only a limited number of experiments can be
performed, some dynamic behavior may be missed or
neglected.
Existing experimental work has shown the existence of
oscillatory behavior; researchers analyzed these dynamic
systems using single operation parameter analysis with all
other state variable changes monitored,1,2,14,16,18,19 detecting
frequent bifurcation, chaotic behavior, and even force
oscillations.1,14
However, all previous work has suffered limitations because
of the lack of ability to deal with two or three simultaneously
changed operation parameters. In this article, the effect of
operating condition values on product yield was analyzed, with
Received: May 17, 2012
Revised: December 12, 2012
Accepted: December 18, 2012
Published: December 18, 2012

© 2012 American Chemical Society 1645 dx.doi.org/10.1021/ie3013049 | Ind. Eng. Chem. Res. 2013, 52, 1645−1655
Industrial & Engineering Chemistry Research
rigorous Hopf points location in the operating region.
Moreover, the synergism between three multivariate operating
parameters was discussed.
As stated, numerical analysis methods can enable complete
and thorough analysis of dynamic systems. In this article, we
first introduce the numerical methodology for locating Hopf
point in steady state solutions as well as the limit cycles and
then apply the proposed methodology to the Z. mobilis
fermentation process.
■ INTRODUCTION TO HOPF POINTS AND
NUMERICAL ANALYSIS METHODS
In general, chemical processes are assumed to be described by
the following equation:
⎧ dx
⎪ = F (x , α )
⎨ dt
⎪
⎩ x(0) = x 0 (1)
where x represents state variables in the dynamic system such
as temperatures, pressures, components concentration, etc. (x
∈ Rn) and α is a parameter representing the operating
condition value or design variable, such as flow rate, initial
feedstock concentration, reactor volume, and so on. Equation 1
describes the dynamic behavior of a set of state variables under
specified operating conditions, providing access to better
understanding of system behaviors and optimization. The
solution satisfying eq 2 is the equilibrium solution representing
a set of practical state variable values under steady operating
conditions.
F (x , α ) = 0 (2)
As the main operating points, a steady state solution set
inevitably contains singular points that are worth looking into
to enable smooth operation. A thorough study of singularity
helps to screen and evade these distinct points, at which rich
dynamic behavior exists and stability is practically difficult to
maintain. Meanwhile, the accurate location of singular points,
including the clear distribution of Hopf points, will no doubt
contribute to safer decisions when a change in operating
conditions must be made, because a number of specific singular
points may possibly appear when switching across the operating
region. Here, the matrix Fx(x,α), which is the Jacobian matrix of
F(x,α), is introduced to assess stabilities of steady state
operation points. If x = x* is the solution of F(x,α) = 0, then
under the operating condition governed by α, the system will
maintain in the steady state when all state variables take a full
set values of x. In general, steady state points have different
stability characteristics and the stability of a system under
certain operating conditions and state variable values is
determined by the eigenvalues of the Jacobian matrix.
Specifically, the system is stable if all eigenvalues are located
on the left of the complex panel, otherwise the system is
unstable. In other words, when every real part of all the
Jacobian matrix eigenvalues is negative, re(λi) < 0, i = 1,2, ...,n,
then the system is stable when a minor disturbance is
encountered. On the contrary, if there is only one eigenvalue
that consists of a positive real part, the whole system would fall
into an unstable situation under which even a small disturbance
would cause extensive system changes. Stable steady state
operating conditions are desirable in both operation and design,
but some specific unstable steady situations also have
potentially practical and theoretical applications, among which
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the oscillatory phenomenon is one of the most significant. In
the oscillatory situation, state variables experience periodic
changes. Theoretically, oscillation corresponds to Hopf points
pertaining to singular points. A supercritical bifurcation occurs,
with orbital limit cycles, when the reaction system is situated
near this type of point. A pair of conjugate eigenvalues in
mirror symmetry, with at least one nonzero real part in the
other n−2 eigenvalues, can indicate this phenomenon. Periodic
systematic oscillations will be detected near these Hopf
singularity points, which clarifies the significance of locating
Hopf points. Currently, most research on Hopf points
emphasizes accurate search for and location of Hopf points
and limit cycles.
■ HOPF POINT DETECTION AND LOCATION
The unique feature of a Hopf point lies in its special pair of
conjugate pure image eigenvalues associated with the Jacobian
matrix at the steady state point. By the Hopf bifurcation
theorem, Hopf point detection can be achieved by calculating
eigenvalues. A more common way is to design a detection
function to identify Hopf points and the corresponding unique
stable steady state. Consider the function described in eq 3
ψH(x , α) = ∏ (λi(x, α) + λj(x, α))
i>j (3)
where λi are the eigenvalues of the Jacobian matrix at a specified
steady state point. According to theoretical analysis, this
function value will vanish when encountering a Hopf
bifurcation point, associated with a pair of conjugate pure
image eigenvalues, λ1,2 = ± iω0. Note that paired real
eigenvalues λ1 = κ, λ2 = −κ can be a special case of ψH = 0,
which corresponds to a neutral saddle situation, rather than the
Hopf point we are primarily concerned with. In that sense, the
Hopf point must be carefully identified from neutral saddle
points when the zero point of detection function is found. At a
generic Hopf bifurcation point, the function ψH is expected to
be real, smooth, and have a regular zero value, with a
nonsingular Jacobian of the proposed dynamic system assured
simultaneously at this specific point. Newton’s method can be
applied, and to avoid tedious explicit computation of all the
eigenvalues of A, the current work used a method based on the
bialternate product to compute ψH.
The procedure of Hopf point detection based on the
bialternate product is expressed in eq 4:
ψH(x , α) = det(2Fx(x , α) ⊙ In) (4)
Here, ⊙ represents the bialternate product of the matrix and
formulated as
apr aps δpr δps
(2A ⊙ In)(p , q),(r , s) = +
δqr δqs aqr aqs (5)
where
⎧1, i = j
⎪
δij = ⎨
⎩ 0, i ≠ j
⎪
In the matrix, rows are labeled by the multi-index (p, q) (p = 2,
3, ..., n; q = 1, 2, ..., p − 1), and columns are labeled by the
multi-index (r, s) (r = 2, 3, ..., n; s = 1, 2, ..., r − 1). The result of
the determinants is enumerated in eq 6:
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Industrial & Engineering Chemistry Research Article

Figure 1. Schematic of Hopf analysis procedure.

⎧−aps ,
⎪
⎪ apr ,
r=q
r ≠ q, s = q
■ LOCATION OF LIMIT CYCLES
Bifurcation theory suggests that limit cycles are intrinsically of
⎪ concomitance with Hopf singularity points. The shooting
⎪ apr + aqs , r = p, s = q method, a widely accepted algorithm for calculating limit cycles,
(2A ⊙ In)(p , q),(r , s) =⎨ first converts the proposed question to a periodic boundary-
⎪ aqs , r = p, s = q
⎪ value problem for the cycle continuation in eq 7 as follows:
⎪−aqr , p=s
⎧ dx
⎪ T
⎩ 0, otherwise ⎪ = F (x , α )
(6) ⎨ dt 2π
⎪
Theoretically, the detection function equals zero when the ⎩ x(0) = η (7)
Hopf bifurcation occurs, but in practice, we first identify the
After parameter α is preset, we search for an initial value η
sign of the test function as positive or negative. If ψH(xi,αi)- and a period T satisfying eq 7 to obtain the periodic solution
ψH(xi+1,αi+1) < 0, this reveals a Hopf singularity point standing for the problem.
between (xi,αi) and (xi+1,αi+1); thus, the rigorous Hopf point Fortunately, software packages such as AUTO, 20−22
(xH,αH) location is subsequently found through a gradually CONTENT,23 MATCONT,24,25 DsTool,26 PyDSTool,27
reduced search space. XPPAUT,28 and some previous research by the authors’
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Industrial & Engineering Chemistry Research Article

Figure 2. Fermentation process.

group29 have implemented the method described and laid the theses. In this work, to decrease inhibition by ethanol, a
foundations for the current work. removal membrane was positioned at the bottom of the
The strategy for locating Hopf points in a dynamic system fermentor to remove ethanol produced during fermentation.
was summarized in Figure 1. Beginning with a model of the Continuous Ethanol Removal. A coupled membrane
fermentation process, this procedure was successively con- separation system for ethanol removal was proposed to prevent
ducted by calculating the steady state solution of the dynamic ethanol inhibition and increase the productivity and efficiency
system as parameters were changed, then identifying the steady of the fermentation process. The membrane has selectivity
state solutions to determine whether or not there are Hopf toward ethanol within the reaction environment, with a gas/
points. If there is a Hopf point and the first Lyapunov liquid sweep stream on the permeate side to remove product
coefficient is negative, orbital stable limit cycles in the system from the membrane surface.
and corresponding oscillatory behavior are sure to exist in the With the removal of ethanol, inhibition would be prevented
dynamic system. Finally, parameters were varied to find all and cell activity maintained at a high level, making it possible to
Hopf singularity points by this ergodic process, providing a use a higher glucose feed concentration, resulting in increased
fermentor cell density. The increased cell activity and cell
valuable reference for operating point optimization.
density contribute to higher volumetric productivity and a
Fermentation Process Flowchart. There are several
smaller fermentor volume. Additionally, the use of a
reported models10,11,13−15 describe the Z. mobilis fermentation
concentrated glucose feed will decrease the amount of process
process. Figure 2 shows a diagram of the continuous flow water.19
fermentor, while the data used in the numerical simulation is Model Assumptions. The model adopted in this paper was
listed in Table 1. The kinetics developed by Jobses et al. for based on the following simplified assumptions:30
production of bioethanol by Z. mobilis was adopted.1
Product Inhibition. Ethanol production by Z. mobilis is 1. The fermentor is considered to be a well-mixed system
subjected to end-product inhibition. The product alters the cell and is not controlled by diffusion.
2. The feed contains only nutrient (substrate).
membrane composition and inhibits enzymatic reactions such
3. The specific growth rate is defined by a Monod-type
as carrier-mediated transport processes and metabolic syn-
equation.
4. The maximum specific growth rate is proportional to the
Table 1. Parameters in the Fermentation Process Model concentration of the key component (e).
5. k1, k2, k3, Ks, ms, mp, Ysx, and Ypx are constants.
param. value unit
Model Development and Discussion. One of the most
Cs0 140 kg/m3
generally acknowledged models of fermentation processes is
DMin 0.5 h−1
AM 0.24 m2
the maintenance model, for which substrate consumption
Din 0.04 h−1
equation is as follows:
Ks 0.5 kg/m3 ⎛ μ ⎞
k1 16 h−1 rs = ⎜ + ms⎟Cx
k2 0.497 m3/kg·h
⎝ Ysx ⎠
k3 0.00383 m6/kg2·h where μ is the specific growth rate. The first term represents
ms 2.16 kg/kg·h the growth rate and the second term represents the
mp 1.1 kg/kg·h maintenance. Similarly, the product generation rate equation
Ysx 0.0244498 kg/kg is defined as
Ypx 0.0526315 kg/kg
Cp0 0 kg/m3 ⎛ μ ⎞
Ce0 0 kg/m3 rp = ⎜⎜ + mp⎟⎟Cx
Cx0 0 kg/m3 ⎝ Ypx ⎠
CpM0 0 kg/m3 The growth rate of biomass rx has its classical definition and
ρ 789 kg/m3 can be expressed in the form:
VM 0.0003 m3
VF 0.003 m3 rx = μCx
Pm 0.1283 m/h The rate of the key compound re is formulated as
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Industrial & Engineering Chemistry Research Article

re = f (Cs)f (Cp)Ce
The formation rate of the key component is a function of the
substrate concentration, inhibited by ethanol, while its activity
is expressed in terms of concentration only.11 Subscript s
represents the substrate, while subscript p refers to the product.
f(Cs) is given by the Monod equation:
where the subscripts Din, Dout, DMin, and DMout represent their
respective dilution rates, and Ysx and Ypx refer to the yield
factors of biomass on substrate and product; k1, k2, k3 are
empirical constants; V stands for volume; mS and mP are
maintenance factors based on substrate requirements and
product formation. The membrane side and the fermentation
side are distinguished by subscripts M and F.

f (Cs) =
Cs
K s + Cs
■ HOPF POINTS IN THE SYSTEM AND THE LIMIT
CYCLES
The function f(Cp) is defined in polynomial form: The set of steady state points in the investigated process of
fermentation is shown in Figure 3. In the case of Cs0 = 150.3
f (Cp) = k1 − k 2Cp + k 3Cp2

Thus, the equation for re can be substituted into the

following expression:
k1 − k 2Cp + k 3Cp2
re = CsCe
K s + Cs
The base set of parameters in the current investigation is
listed in Table 1.19
The dynamic model for the continuous fermentation process
for substrate (s), microorganism (x), and the key compound
(e) are given by the following set of ordinary differential
equations:
dCx
= μCx + DinCx0 − Dout Cx
dt
dCs ⎛ μ ⎞
= −⎜ + ms⎟Cx + DinCs0 − Dout Cs
dt ⎝ Ysx ⎠
k1 − k 2Cp + k 3Cp2 Figure 3. Changes in product concentration with dilution rate.
dCe
= CsCe + DinCe0 − Dout Ce
dt K s + Cs
kg/m3, a Hopf point was detected at Din = 0.052 h−1, which
The product (ethanol) on the fermentor side (p), the coincides exactly with reported literature values.2,16 At the Hopf
product (ethanol) on the membrane permeate side (pM), and point, we have a product concentration Cp = 58.050 kg/m3,
the mass balance for the product (ethanol) over the fermentor which approaches the maximum point of product yield of Din =
are given by 0.046 h−1, Cp = 58.103 kg/m3. This fact highlights the
⎛ μ ⎞ importance of Hopf point investigation in the present dynamic
dCp
= ⎜⎜ + mp⎟⎟Cx + DinCp0 − Dout Cp system, because it is so close to the maximum conversion point
dt ⎝ Ypx ⎠ that we seek. In Figure 3 the Hopf singularity point is marked
as a red star, with the first Lyapunov coefficient l1 value of
A P
− M m (Cp − CpM) −0.00153. The negative sign means the point corresponds to a
VF supercritical Hopf bifurcation and the limit cycle is orbitally
stable. In Figure 3, the abscissa denotes the feedstock dilution
The mass balance for the product (ethanol) over the rate, ranging from zero to a reasonable maximum. As the
membrane is given by variable indicating the degree of feedstock volume diffusing into
dCpM AM Pm the reactor volume, the dilution rate has a relatively small value
= (Cp − CpM) + DMinCpM0 − DMout CpM in practice. Therefore, only a range of the dilution rate worthy
dt VF of consideration is discussed. As mentioned, the Hopf points
where the AM is the permeation area and Pm is the permeability are theoretically very close to a risky but high conversion point,
of the membrane for ethanol. and associated limit cycles are depicted in Figure 4. Each limit
The exit fermentor dilution rate can be obtained as follows: cycle is generated by a very small disturbance from the initial
operating parameter value at the Hopf point.
AM Pm According to bifurcation theory, experimentally observed or
Dout = Din − (Cp − CpM)
VFρ mathematically simulated oscillations in a dynamic system must
start and end at certain critical points in the system. It has been
The corresponding exit membrane dilution rate becomes proven that static and dynamic bifurcation behaviors, over a
AM Pm wide range of operating parameters, abound in the Z. mobilis
DMout = DMin + (Cp − CpM) fermentation process. In the current work, the fermentor
VMρ dilution rate, the membrane dilution rate, the initial substrate
1649 dx.doi.org/10.1021/ie3013049 | Ind. Eng. Chem. Res. 2013, 52, 1645−1655
Industrial & Engineering Chemistry Research
Figure 4. Homology limit cycles adjoining the Hopf point.
(glucose) concentration of feedstock concentration, and
permeation area were chosen as bifurcation parameters, not
only for their decisive role as operating variables in this system
but also because of their ease of manipulation and control
during practical fermentations. To avoid the oscillatory
behaviors caused by the existence of the Hopf bifurcation,
which as stated decrease the fermentation yield, system
stability, and controllability, decline product quality, we put
forward a rational method to position the entire set of Hopf
points in the operation parameter panel. In this proposed
method, all Hopf singularity points were located and assembled
in the operation parameter panel as a “risky curve”. Thus,
operating parameters can be located away from the undesirable
singular points, unwitting steps across these points can be
avoided, and appropriate optimized values of the parameters
adopted.
■
PARAMETER INFLUENCES ON HOPF POINTS
Hopf points in Z. mobilis fermentation result in orbital stable
limit cycles and oscillation behaviors in the process, and it
follows from this, as mentioned, that the product conversion
rate, system stability, controllability, and product quality are all
adversely affected.
Here, four important operating parameters, including
dilution rate of feedstock, membrane side feedstock dilution
rate, the initial substrate concentration, and the permeation
area, were investigated. Each case study was undertaken in the
following way. First, the changes in product concentration were
observed and calculated under various operating conditions and
the results shown in a single figure to confirm the existence of
the Hopf points along with the steady state operation points in
the system. These two selected parameters were then changed
over a range wide enough to find all Hopf points in the
operating region formed orthogonally by these two parameters.
As can be seen, every point in the figure was obtained under
fixed corresponding operating parameters. Among all steady
state solutions of the system, a Hopf point intrinsically exists
with joint orbital stable limit cycles in practice, arising from the
oscillation. Furthermore, using the methodology proposed in
this paper, not only can these undesirable points be avoided but
the distances between current operating conditions and
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potential oscillatory operation points can also be measured to
better estimate the operating risk as a probability.
Case 1: Cs0 and Din. In this case, the dilution rate of the
feedstock and the initial substrate concentration were examined
because of their decisive roles as operating variables and their
ease of manipulation. The steady state solutions were
calculated; the Hopf points were marked in Figure 5, with
Figure 5. Steady state points and Hopf points.
the first Lyapunov coefficient labeled to imply that these Hopf
points are supercritical and will stimulate nearby periodic limit
cycles. To observe the membrane-side dilution influence on the
system by direct-viewing, results were combined within one
bifurcation map, as shown in Figure 5, under variable
membrane side dilution rates.
In Figure 5, it can be seen that the membrane side dilution
rate DMin changed from 0.1 to 0.9 h−1 at a step size of 0.2 h−1.
Under each given membrane side dilution rate, production
concentration is plotted against the feedstock dilution in the
steady state solution. Note that the clusters of production
concentration curves have generally delayed peaks with equal
changes in membrane side dilution. As may be expected, the
corresponding Hopf points were detected within the region of
low feedstock flow rate, where the negative first Lyapunov
coefficients indicate potential oscillation near the production
concentration peak. Another fact worth noticing is that the
product conversion rate peaks in the system decreased with
increasing membrane side dilution rate, which can be explained
by the conversion inhibition effect brought by increasing
membrane side dilution rate. Fortunately, with the decrease in
concentration of product, the Hopf point becomes increasingly
separated from the conversion peak, characterized by an
increasing first Lyapunov coefficient at the same time.
Details of this case study and its five Hopf points are listed in
Table 2. The same conclusion can be drawn after analyzing
homologous state and operating variables.
Figure 5 only describes the influence of a single parameter
(or two parameters to a limited extent) on the Hopf points.
However, it is more common for operating parameters to work
together and change simultaneously. Thus, we examined
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Industrial & Engineering Chemistry Research Article

Table 2. Hopf Points in the System Cs0 = 140 kg/m3, AM = results in Figure 7 indicate that the membrane permeation area
0.24 m2 has only a slight influence on product concentration as a
DMin Din Cp CpM first Lyapunov coefficient
function of feedstock dilution rate.
(h−1) (h−1) (kg/m3) (kg/m3) l1(10−4)
0.1 0.096 57.228 57.168 −8.968
0.3 0.395 53.154 52.988 −2.583
0.5 0.627 50.790 50.527 −1.825
0.7 0.832 48.995 48.641 −1.344
0.9 1.027 47.469 47.030 −1.073

another important parameter, the initial substrate concen-

tration. Specifically, while we fixed AM = 0.24 m2 and varied
DMin from 0.1 to 0.9 with a step-size of 0.2 h−1, we searched Din
and Cs0 to find the existence of Hopf points and then recorded
these latter two parameter values to plot the data in one figure.
Accordingly, all parameter values creating Hopf singularity
points over the range of fixed DMin and AM values are shown in
Figure 6.

Figure 7. Steady state points and Hopf points.

As can be seen in Figure 7, although the permeation areas

differed markedly, their impact on product concentration as a
function of feedstock dilution was small and the steady state
solution curves almost coincided. In a similar manner, the
calculated Hopf points were located very close to one another
and were almost constant. Thus, it can be concluded that the
permeation area was not a sensitive parameter for this system.
From Table 3, it can be seen that AM contributes little to Cp
and CpM, as shown in Figure 7.

Table 3. Hopf Points in the System with Cs0 = 140 kg/m3,

DMin = 0.5 h−1
AM Din Cp CpM first Lyapunov coefficient
Figure 6. All Hopf points in the parameter panel. (m2) (h−1) (kg/m3) (kg/m3) l1(10−4)
0.1 0.650 50.578 49.954 −1.564
0.3 0.624 50.817 50.607 −1.770
In Figure 6, the Hopf points can be seen to change in
0.5 0.620 50.861 50.734 −1.825
response to the variations in feedstock dilution rate and initial
0.7 0.618 50.879 50.788 −1.847
substrate concentration. When the feedstock dilution rate or
0.9 0.617 50.889 50.818 −1.827
the initial substrate concentration was low, the system displayed
a higher probability of generating Hopf points. Notably, even if
one parameter was altered only over a certain narrow range,
while the other was varied more aggressively, Hopf points were Furthermore, at a fixed value of permeation area, Hopf points
located, which reveals that there is no universal safe operating were fixed within the operating panel with respect to initial
region for every parameter. The position of the Hopf points, as substrate concentration and feedstock dilution rate, as shown in
expected, changed with dilution rate on the membrane side. It both Figures 7 and 8. Thus, in the analysis that follows, the
is demonstrated in Figure 6 that a greater membrane side permeation area was not considered because it is not a sensitive
dilution rate causes a heavier density of Hopf points within the variable in this system. In contrast, the dilution rate on both the
operating range as the feedstock dilution rate increases. feedstock and the membrane sides, together with the initial
Meanwhile, locations across the Hopf points are shown in substrate concentration in the feed were discussed in more
this figure by a dashed line, which marks the Hopf points under detail.
a constant substrate concentration of 140 kg/m3. Case 2: DMin and Cs0. In case 2, we focused on membrane
After examining D Min , the final parameter A M was side dilution and the initial substrate concentration in the
investigated because the diversity of membranes used in feedstock.
fermentation processes may cause different permeation areas Simulation was carried out under five given feedstock
of the membrane. Here, we selected AM from 0.1 to 0.9 at dilution rates and one membrane-side dilution rate. The
intervals of 0.2 m2, and fixed DMin = 0.5 h−1, to examine the product concentration changed with the initial feedstock
influence of membrane area on product concentration. The substrate concentration, as shown in Figure 9.
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Table 4. Hopf Points in the System AM = 0.24 m2, DMin = 0.5

h−1
Din Cs0 Cp CpM first Lyapunov coefficient
(h−1) (kg/m3) (kg/m3) (kg/m3) l1(10−4)
0.02 418.671 58.833 58.526 −3.048
0.05 250.692 58.201 57.897 −1.375
0.1 195.376 57.267 56.968 −1.070
0.3 155.680 54.330 54.047 −1.222
0.5 144.557 52.050 51.780 −1.574

Figure 8. All Hopf points in the parameter panel.

Figure 10. All Hopf points in the parameter panel.

Figure 9. Steady state points and Hopf points.
Product concentration increased gradually with the increase
of initial substrate concentration of feedstock. And the turning
points seem to serve as an upper limit of concentration no
matter how the substrate concentration increases. This baffle
effect can be interpreted since at the begin in the fermentation
process, the Z. mobilis grow with the increase of initial
concentration of feedstock, but when the growth reaches the
maximum, the produce capability has no potential to constantly
increase. However, the dilution rate of feedstock also has an
impact on product concentration. It is worth noting that the
greater feedstock dilution rate is, the earlier turning points will
appear, with low concentration at high dilution rate. Hopf
points, shown in Figure 9, tighten therewith the maximum
point. Corresponding values are listed in Table 4.
Similarly, in Table 4, we find all the first Lyapunov
coefficients are negative, indicating the existence of stable
limit cycles. The location of Hopf singularity points in the
parameter panel changed significantly with the initial substrate
concentration and the membrane side dilution rate, as shown in
Figure 10.
1652
From Figure 10, it can be seen that, once again, the entire
system has a turning point on each curve. The position of the
Hopf points, although always below the turning point, changes
markedly as the initial substrate concentration increases,
whereas the rates decreased above the turning points. The
dilution rate of feedstock influenced the curve shape to a large
extent, with larger dilution values giving lower initial substrate
concentrations at which Hopf points were encountered.
Case 3: Din and DMin. In this case, the dilution rate on both
sides of the membrane were considered. Calculation results are
depicted in Figure 11, showing a complete three-parameter
bifurcation diagram. When the initial substrate concentration
and the feedstock dilution rate were both fixed, the product
concentration decreased progressively with the membrane side
dilution rate because the feedstock dilution rate had only a low
value (0.02 h−1), which gave a sufficiently long resident time for
the product concentration to achieve its maximum. With an
elevated membrane-side dilution rate, the ethanol was gradually
removed through entrainment, resulting in a drop of the
product concentration. This effect would be intensified by
further increases in the membrane-side dilution rate. Mean-
while, when the initial substrate concentration in the feedstock
increased, the product could maintain maximum growth rate
for a longer time, even at the same membrane-side dilution rate.
All Hopf points are marked in Figure 11, and actual values are
listed in Table 5.
Contrary to to the preceding cases, Table 5 clearly indicates
that Hopf points are generated at the maximum growth rate,
adjacent to the value of product concentration, as for the
membrane-side product concentration. The Hopf points in the
dilution rate and membrane-side dilution rate panel are
dx.doi.org/10.1021/ie3013049 | Ind. Eng. Chem. Res. 2013, 52, 1645−1655
Industrial & Engineering Chemistry Research Article

feedstock and membrane side, together with the initial substrate

concentration, are the most sensitive parameters contributing
to the product yield and product quality, and they significantly
influence the location of Hopf points.

■ RESULTS AND DISCUSSION

In our research work, four important operating parameters,
including dilution rate of feedstock, membrane side feedstock
dilution rate, the initial substrate concentration, and the
permeation area, were investigated, because of their decisive
roles as operating variables and their ease of manipulation. As
operating conditions changed, the steady state solution curve
and the location of Hopf point appear in very distinguishing
manners. Specifically, we found that
1. Hopf points, in the present fermentation dynamic
system, locate very close to the maximum conversion
point, and this fact highlights the importance of Hopf
point investigation.
Figure 11. Steady state points and Hopf points.
2. Increasing the membrane-side dilution rate will result in
delayed and decreased the product conversion rate peak
Table 5. Hopf Points in the System Din = 0.02 h−1, AM = 0.24 value, while the undesired Hopf point becomes
m2 increasingly separated from the desired conversion peak.
3. Hopf points have a high probability to appear in areas of
Cs0 DMin Cp CpM first Lyapunov coefficient either low feedstock dilution rate or low initial substrate
(kg/m3) (h−1) (kg/m3) (kg/m3) l1(10−3)
concentration; thus, there is no universal safe operating
150 0.049 58.701 58.671 −5.710 region for a single parameter.
200 0.133 58.731 58.648 −3.884 4. The membrane permeation area is not a sensitive
250 0.218 58.757 58.622 −2.598 parameter and only has a slight influence on product
300 0.302 58.781 58.594 −1.651 concentration. Almost coincident steady state solution
350 0.386 58.803 58.565 −0.949 curves and Hopf points were detected.
5. Baffle effect of product concentration and the maximum
illustrated in Figure 12. Hopf points exist at each given initial turning point were observed, while the position of Hopf
substrate concentration with various dilutions. points changes markedly as initial substrate concen-
tration increases.
6. The three-parameter bifurcation diagram showed that
when the initial substrate concentration is fixed and the
feedstock dilution rate is low, the product concentration
decreased progressively with the membrane side dilution
rate, but when the initial substrate concentration in the
feedstock increased, the product could maintain a
maximum growth rate for a longer time.
7. At low initial concentration, the Hopf points appear at
almost the same level of feedstock dilution rate, whereas
at higher initial concentrations, the Hopf points arise in a
region of relatively low dilution rate but high membrane
dilution.
8. Product conversion rate, system stability, controllability,
and product quality all depend heavily on these four
operating variables. The synergism between multivariate
operating parameters has been first discussed.
The emphasis of this article is on locating Hopf singularity
points in the process. As a result, with four major manipulate
Figure 12. All Hopf points in the parameter panel. parameters and their combinations, also within a large
operation condition parameters scopes, we have studied the
Hopf singularity points thoroughly in our research work. The
It can be observed from Figure 12 that at low initial results showed the universal existence of Hopf singularity
concentration, the Hopf points appear at almost the same level points in this process within a large operational space. Although
of feedstock dilution rate, whereas at higher initial concen- it seems there were many singularity points in the panel
trations, the Hopf points arise in a region of relatively low constructed by operational variable, suitable operation points
dilution rate but high membrane dilution. for designing process were even more in the panel. Because
From all of these analyses, we can now conclude that, among only the points located exactly in the critical curve were not
all operating parameters, the dilution rates on both the recommended, many more points away from the curve in the
1653 dx.doi.org/10.1021/ie3013049 | Ind. Eng. Chem. Res. 2013, 52, 1645−1655
Industrial & Engineering Chemistry Research
panel are feasible to the process design. Also, in practice, the
operation condition parameter space was limited into a smaller
operational space, which would meets complex constraints; the
operation variables and their combinations also will be reduced,
so there would be less Hopf singularity points, even no Hopf
singularity points would be encountered in some situations.
In general situations at process design phase, it is a better
method to determine the operation ranges first, after that, to
use the method in this article to detect whether there are Hopf
singularity points or not. Most of the time, there would be
seldom Hopf singularity points within the small feasible
operational range that meets all the complicated practical
constraints. Even in the worst situation, there was a Hopf
singularity curve in the operational space; these points are
feasible to process design except that these operation points
just locate in the critical curve; for insurance, these points very
close to the critical curve also can be excluded in process
design.
■ CONCLUSION
In this article, a typical fermentation process using Z. mobilis to
produce ethanol was investigated. Although it is a more
promising microorganism than yeast for the industrial
production of ethanol, continuous fermentation of Z. mobilis
undergoes oscillatory behavior in which biomass, product, and
substrate cycle under certain fermentation conditions. There-
fore, it is important to search for a reasonable approach that
helps to avoid the oscillatory behaviors caused by Hopf
bifurcations, which decrease fermentation yield, system stability
and controllability, and decline product quality. This article
rigorously analyzed the Hopf point variation under carefully
designed operating conditions and discussed its influence on
product yield and quality. The article primarily focused on the
operating conditions and product yield rather than complicated
theoretical bifurcations and chaotic behavior. A numerical
analysis method for locating the Hopf singularity point and
limit cycles was introduced and applied to the Z. mobilis
fermentation process for producing ethanol. In our research,
the entire set of Hopf points have been solved in the operating
parameter panel. Furthermore, to avoid tedious explicit
computation of all the eigenvalues of a matrix, the current
work used a method based on the bialternate product to
compute the test function to determine Hopf point. It is the
proposed method makes it possible for us to first locate and
assemble these Hopf points as a “risk curve”, with reduced
computational work and better accuracy. The calculated
distribution of Hopf points can enable all operating parameters
to be located away from undesirable singular points and
avoidance of unwitting steps across these points, with
appropriate and optimized values. Furthermore, the proposed
method can be applied to other fermentation processes and
improved models. Design and control strategies can be
developed at a very early stage, due to the a priori identification
of potential Hopf points, and this will contribute to the
achievement of high productivity, product quality, and stability
in dynamic processes.
■ AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 10 62784572. Fax: +86 10 62770304. E-mail:
dcecbz@tsinghua.edu.cn.
1654
Article
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors gratefully acknowledge support from China
Postdoctoral Science Foundation (No. 20100480282), China
Postdoctoral Science special Foundation (No. 2012T50099),
and the National Basic Research Programme (No.
2012CB720500).
■
NOMENCLATURE
Ci Concentration of component i (kg/m3)
ri Production rate of component i (kg/m3 h)
Cs0 Inlet substrate concentration (kg/m3)
DMin Inlet membrane dilution rate, inlet flow rate/membrane
volume (h−1)
DMout Output membrane dilution rate, output flow rate/
membrane volume (h−1)
Din Inlet fermentor dilution rate, inlet flow rate/for monitor
volume (h−1)
Dout Output fermentor dilution rate, output flow rate/
fermentor volume (h−1)
AM Area of membrane (m2)
XS Substrate conversion
KS Monod constant (kg/m3)
k1 Empirical constant (h−1)
k2 Empirical constant (m3/kg h)
k3 Empirical constant (m6/kg2 h)
ms Maintenance factor based on substrate (kg/kg·h)
mp Maintenance factor based on product (kg/kg·h)
Ysx Yield factor based on substrate (kg/kg)
Ypx Yield factor based on product (kg/kg)
VM Membrane volume (m3)
VF Fermentor volume (m3)
Pm Membrane permeability (m/h)
Greek Symbols
ρ Ethanol density (kg/m3)
μ Specific growth rate (h−1)
Subscripts i
s Substrate (glucose) inside the fermentor
s0 Influent glucose to the fermentor
p Product (ethanol) inside the fermentor
p0 Influent ethanol to the fermentor
e Internal key component inside the fermentor
e0 Influent internal key component to the fermentor
x Biomass (microorganisms) inside the fermentor
x0 Influent biomass to the fermentor
pM Product (ethanol) inside the membrane
pM0 Influent ethanol to the membrane
■
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