Zdravko Kravanja (Editor)

Proceedings of the 26th European Symposium on Computer Aided Process Engineering

June 12th - 15th, 2016, Portoroz, Slovenia.
c 2016 Elsevier B.V. All rights reserved.

Anaerobic Bio-reactor Modeling

Cansu Birgena,* , Heinz A. Preisiga , Alexander Wentzelb , Sidsel Markussenb , Bernd
Wittgensc , Ujjaini Sarkard , Anindita Gangulyd , Sudeshna Sahad and Sibashish
Baksid
a Norwegian University of Science and Technology, Chemical Engineering, Sem Slands vei 6,
Trondheim, Norway
b SINTEF Materials and Chemistry, Biotechnology and Nanomedicine, Richard Birkelands vei 3,

Trondheim, Norway
c SINTEF Materials and Chemistry, Industrial Process Technology, Sem Slands vei 2A, Trond-

heim, Norway
d Jadavpur University, Chemical Engineering, 188 Raja S. C. Mullick Road, Kolkata, India

*cansu.birgen@ntnu.no

Abstract
EcoLodge aims at providing a proof-of-concept for a new, integrated biotechnological production
process for C8 ester butyl butyrate, a promising supplement/substitute for diesel and jet engine
fuels, from lignocellulose derived C5 and C6 sugars. Butanol and butyric acid are the process
intermediates produced via anaerobic fermentation. This paper focuses on mathematical model
simulation for BuOH fermentation and integrated gas stripping unit. Inhibitory effects of butanol
and substrate are identified as main obstacles for high productivity butanol fermentation. There-
fore, in situ removal of inhibitory butanol from the bio-reactor by gas stripping is suggested taking
the substrate inhibition into consideration. Mathematical models from the literature are employed
together with experimentally estimated model parameters. Model simulations of the integrated
unit are performed in continuous mode for determination of optimum values for substrate con-
centration in feed and feed flow rate. The full domain of the operating ranges were explored for
three different inhibitory concentrations of butanol (5,8 and 10 g/l), which resulted in performing
14000+ simulation experiments. The results provide a clear insight into the feasible combinations
of the operating conditions. The optimal operating range for 5 g/l is obtained in this study will be
employed in experimental bio-reactor and gas stripping unit in the next phase of the project.

Keywords: Bio-reactor, Biobutanol, Fermentation, Modeling, Gas stripping

1. Introduction
EcoLodge aims at providing a proof-of-concept for a new, integrated biotechnological production
process for C8 ester butyl butyrate, a promising supplement/substitute for diesel and jet engine
fuels, from lignocellulose derived C5 and C6 sugars. Previous studies claim that BuB should have
excellent properties both as gasoline as well as diesel component (Lange et al., 2010). Therefore,
butyl butyrate is an interesting biofuel option of which butanol and butyric acid are the process
intermediates produced via anaerobic fermentation. This paper focuses on model simulation for
BuOH fermentation and integrated gas stripping unit to determine optimal operating points.
The process for production of butanol via anaerobic fermentation by Clostridia is known popularly
as acetonebutanolethanol (ABE) fermentation (Mayank et al., 2013). Clostridia are rod-shaped,
spore-forming Gram positive bacteria and typically strict anaerobes (Lee et al., 2008). Among
 2 C. Birgen et al.

many solventogenic clostridia, Clostridium acetobutylicum ATCC 824 remains the best studied
and manipulated strain (Durre, 2005). Therefore, it is chosen for butanol production in this paper.
Clostridial solvent production is biphasic; first phase is acidogenic phase during which acetate,
butyrate, hydrogen, and carbon dioxide are produced as major products. Acidogenic phase is ob-
served during exponential growth phase of cells (Andersch et al., 1983; Hartmanis and Gatenbeck,
1984). Second phase is solventogenic phase during which acids are re-assimilated and the solvent
is produced which consists of acetone, butanol and ethanol.
High cost of substrate, low solvent yield, low solvent tolerance, and culture degeneration are re-
garded as the main bottlenecks for BuOH fermentation (Durre, 2011). High cost of substrate poses
a problem when substrates such a sugar cane are used; however, our substrate is sugar derived from
lignocellulose. Its cost does not impose a major problem. The low solvent yield problem can be
approached by strain manipulation and optimization of the fermentation conditions.
High solvent yield would still create a problem due to solvent intolerance (Gu et al., 2011). There-
fore, researchers investigated various alternative techniques to recover butanol from the fermen-
tation broth. These techniques include adsorption, liquidliquid extraction, perstraction, pervapo-
ration, reverse osmosis, and gas stripping (Maddox, 1989). Gas stripping is widely applied as a
reliable option, since it is simple, does not require expensive equipment, and does not harm the
culture (Qureshi and Blaschek, 2001). During gas stripping, gas is sparged through the bio-reactor
and butanol is condensed and recovered. Therefore, the concentration of butanol in the broth is
kept below inhibitory level of 5g/l butanol as can be found in previous studies. However, gas strip-
ping would be more efficient when the butanol concentration in the broth is higher than 8 g/L, at
which the condensed vapor from gas stripping would have a butanol concentration higher than its
solubility, thus result in a highly concentrated organic phase (Xue et al., 2012).
Use of concentrated substrate solutions could enhance the solvent production if solvent intoler-
ance problem can be tackled. However, too high substrate concentration causes a lag phase due
to substrate inhibition (Ezeji et al., 2004). Hence, the inhibitory substrate concentration and min-
imum substrate concentration required for cell maintenance are 75 g/l and 20 g/l, respectively for
a continuous fermentation system. In a similar manner, substrate concentration in feed has to be
within a certain range to prevent drastic changes in the fermentation broth as the cells are sensitive
to great changes, therefore the range is defined as 250g/l - 500 g/l (Ezeji et al., 2005). Here we
illustrate how the operations improve when fulfilling the criteria for substrate concentration in the
feed, and meet the constraints in the substrate and product concentrations in the broth.

2. Mathematical model for integrated fermentation and gas stripping unit

2.1. Process description
A schematic diagram of the integrated fermentation process is shown in Figure 1. The operation
objective of this continuous bio-reactor with gas stripping is to get constant and high concentration
cultivation of C. acetobutylicum ATCC 824 with high butanol production. The feed stream Fin
contains growth medium and substrate (Sin ) and the outlet stream, Fout contains stripped product
which is only butanol in this study. In the bio-reactor, anaerobic fermentation takes place and the
products are produced. Butanol is separated from the fermentation broth via gas stripping. The
stripping gas stream, Fgas , flows through the reactor and takes up the butanol from which then is
condensed and collected in condenser while the stripping gas is recycled back to the reactor.

2.2. Process model development

For the model we assume an ideally stirred tank reactor with constant volume of the liquid con-
tents. This implies that the intensive properties like the concentrations of the cell mass and sub-
strate are uniform throughout the liquid body. The Monod model is employed to describe the
Anaerobic Bio-reactor Modeling for Biofuel Production 3

Fgas

condenser
Fin, Sin
Fout

Vl, P, S, X

Feed tank Product tank

Bioreactor
Figure 1: Integrated fermentation unit

change in cell concentration over time. The model describes the cell growth rate, thus the cell
concentration, in terms of cell growth rate () and the decay term accounts for the natural death
of the cells (kd ). In this project we assume the latter to be negligible. Butanol inhibition is also
not considered due to the fact that the butanol concentration is always kept below the inhibitory
level as result of the in situ removal by gas stripping (Kovarova-Kovar and Egli, 1998). Thus, the
change in cell concentration in the whole unit over time [g/l/h] is described by:
dX
= cell growth - cell death = X kd X (1)
dt
The Monod model relates the cell growth rate to the concentration of a single growth limiting
substrate ( := f (S)). The two parameters are the maximum specific growth rate (max ), and the
substrate affinity constant (Ks ).
m S
:= (2)
Ks + S
The equation for change in substrate concentration links the specific substrate consumption rate
(qs ) and the cell concentration (X) with the inlet substrate concentration (Sin ), the inlet flow rate
(Fin ), and the active liquid volume (Vl ) [g/l/h]:
dS Sin Fin
= inflow - consumption = qs X (3)
dt Vl
The specific rate of substrate consumption (qs ) is defined in terms of the cell growth yield coeffi-
cient on the substrate (Yx/s ), cell growth rate (), and the maintenance term, which accounts for
the substrate consumption necessary for the cell survival (ms ) assumed negligible (Pirt, 1965):

qs := (4)
Yx/s + ms
dX
Yx/s := (5)
dS
 4 C. Birgen et al.

Butanol is the only product considered in this paper. Therefore, the model equation describes the
change in the butanol concentration over time and links the specific productivity rate (q p ) and cell
concentration (X) with the stripping rate (Rs ) [g/l/h]:

dP
= q p X Rs (6)
dt
The specific productivity rate (q p ) is defined in terms of production yield coefficient (Yp/x ), cell
growth rate () and maintenance factor for product (m p ,) which we assumed to be negligible as
did Luedeking and Piret (1959):

q p := Yp/x + m p (7)

The gas stripping rate of the product butanol, (Rs ), from the fermentation broth is modelled in
terms of the product concentration (P) and the gas stripping rate constant (Ks ) as reported by
Truong and Blackburn (Truong and Blackburn, 1984).

Rs := Ks P (8)

Considering the assumption that the liquid volume in the bio-reactor is constant, the product sep-
arated by gas stripping has the flow rate of Fout , [l/h]:

dVl
= Fin Fout = 0 (9)
dt
Rs Vl
Fout := (10)
BuOH

2.2.1. Experimental estimation of model parameters

Estimation of parameters by the fitting of experimental data to a model equation is a commonly ap-
plied practice (Mayank et al., 2013). Experiments were performed to determine and Ks and Yx/s
and Yp/x . Batch cultivation of Clostridium acetobutylicum ATCC 824 is performed on complex
growth medium containing glucose as growth limiting substrate. The cell concentration is mea-
sured using a spectrophotometer, which provides the optical density. Latter is plotted versus time
providing the cell growth rate. The data are used to estimate and Ks . The substrate and product
concentration in the fermentation broth is measured using high pressure liquid chromatography.
The resulting data are used to estimate Yx/s and Yp/x , respectively. All estimated and assigned
model parameters and experiment design considerations are shown in table below.

Model parameter/experiment design consideration Symbol Value

Maximum specific growth rate (1/h) max 0.18
Substrate affinity constant (g/l) Ks 1.2
Cell growth yield coefficient (g cells/g substrate) Yx/s 0.28
Product yield coefficient (g product/g cells) Yp/x 0.41
Minimum substrate concentration in fermentation broth (g/l) Smin 20
Maximum substrate concentration in fermentation broth (g/l) Smax 75
Minimum substrate concentration in in feed (g/l) Sinmin 250
Maximum substrate concentration in feed (g/l) Sinmax 500
Maximum butanol concentration in fermentation broth (g/l) Pmax 5, 8, 10
Gas stripping rate constant (1/h) Ks 0.059
Anaerobic Bio-reactor Modeling for Biofuel Production 5

3. Results and Discussion

A simple fermentation model is constructed for a continuous bio-reactor equipped with gas strip-
ping using the Monod kinetics. The model is used to determine the optimal substrate feed con-
centration and the optimal feed flow rate whilst considering the substrate and butanol inhibition
by simulating the process at all combinations of feed and inhibition constraints and meeting the
composition constraints imposed by the gas stripping. Inhibitory values for butanol concentra-
tion in fermentation broth are taken from literature as 5 g/l, 8 g/l, 10 g/l as well as the inhibitory
substrate concentration and minimum substrate concentration required for cell maintenance as 20
g/l and 75 g/l (Ezeji et al., 2005; Xue et al., 2012). The substrate concentration in feed has to be
within a certain range to prevent drastic changes in the fermentation broth because the cells are
sensitive to large changes. Literature Ezeji et al. (2005) suggest a range of 250g/l - 500 g/l. All
the design considerations (Smin , Smax , Sinmin , Sinmax , Pmax ) are implemented to compute the optimal
Sin and Fin so that fermentation process can operate continuously without inhibiting the growth
of cells. The model is simulated by Matlab at steady state. Figure 2 is plotted for pre-defined

Figure 2: Substrate concentration in broth (S) Figure 3: Substrate concentration in feed (Sin )
vs. Substrate flow rate in feed (Sin *Fin ). vs. Flow rate of feed stream (Fin ).

range of S 20 75 [g/l] and corresponding Sin *Fin values which are obtained by evaluating
Equation 3 at steady state. Figure 2 shows
clearly that S increases as Sin *Fin increases.
The relation is not linear: the rate of change
accelerates with higher values of Sin *Fin ,
which indicates that the system is more sen-
sitive to changes at higher values of Sin *Fin .
This trend would result in dramatic changes
in the fermentation broth, which is not desired
as it would strongly affect the cells. The data
are used used to determination the relation be-
tween Sin and Fin . As can be seen in Figure
3, different combinations of Sin and Fin are de-
termined for three different Pmax values. Each
set of Sin and Fin indicates an operating point Figure 4: Substrate concentration in broth (S) vs.
which fulfils the criteria. A total of 14,056 dif- Cell concentration in broth (X).
ferent combinations are obtained of which 941
points fulfil the criteria for Pmax =5 g/l, 10,157 points fulfil the criteria for Pmax =8 g/l, and 2958
points fulfil the criteria for Pmax =10 g/l.
A combination of Sin and Fin is chosen for each Pmax value. The change of cell concentration (X)
with changing substrate concentration in feed (S) are illustrated in Figure 4. X is increasing with
 6 C. Birgen et al.

decreasing S, which implies that the cells are growing by consuming substrate. For Pmax =5 g/l,
response of X to changing S is quicker compared to other two. Trends of the curves for Pmax =8 g/l
and Pmax =10 g/l closely match.

4. Conclusions
A simple reactor model combined with Monod kinetics for the anaerobic fermentation of sugar
to butanol using C.acetobytlicum ATCC 824 has been used to explore an optimal operation range
with respect to feed concentration, feed rate as well as the product concentration constraint main-
taining suitable conditions for the cells whilst enabling a continuous stripping of the butanol from
the reactor. The full domain of the operating ranges were explored, which resulted in performing
14000+ simulation experiments. The results provide a clear insight into the feasible combinations
of the operating conditions. Cells can still be productive under some toxic levels of butanol; how-
ever, it will decrease the overall productivity and life time of the bio-reactor. Therefore, inhibitory
butanol concentration is chosen Pmax as 5 g/l in this study so that no inhibiton occurs. The op-
timal operating range for Pmax =5 g/l is obtained in this study will be employed in experimental
bio-reactor and gas stripping unit in the next phase of the project.

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