CONTINUOUS AND SEMICONTINUOUS REACTION SYSTEMS FOR HIGH-SOLIDS

ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSICS

By A Gonzlez Quiroga
ABSTRACT
The enzymatic hydrolysis of lignocellulosics is divided into three stages: a continuous enzyme adsorption
stage at low-solids loading (5% w/w) and with liquid phase recycling where enzyme adsorption is the primary
objective; a liquefying stage with high solids content (20% w/w) due to the thickening caused by pressing and
recycling liquid phase; and finally, a long-retention or long-reaction time stage where additional enzymes are
supplemented to the pumpable slurry and the final conversion is achieved. A detailed modeling and
simulation framework for the proposed reaction systems is presented. The micromixing limiting situations of
macrofluid and microfluid are used to predict conversion. The adsorption and liquefaction stages are modeled
as a continuous agitated tank reactor (CSTR) and a tower-type plug flow reactor, respectively. Two
alternatives for the long-retention or long-reaction time stage are studied: a train of 5 cascading CSTRs with
residence time by reactor ranging from 10 h to 50 h, and a battery of batch reactors in parallel with a
maximum reaction time of 180 h. Results show that glucose concentrations around 100 gl
-1
could be reached
with both of the reaction systems; however, a compromise between solids loading, conversion and volumetric
productivity has to be sought.
Keywords: Bioprocess design; enzymatic hydrolysis; lignocellulose; stirred tank reactor
INTRODUCTION
Modeling and simulation of continuous and semicontinuous reaction systems for chemicals and fuels
production from lignocellulosic materials is a useful approach for exploring process configurations. Previous
studies have highlighted the biochemical conversion of lignocellulosics as one of the most promising routes
[1-3]. In this conversion route, lignocellulosic materials are pretreated before being hydrolyzed in the
presence of enzymes to produce sugars, mainly glucose and xylose, which can be fermented to ethanol. This
paper focuses on the enzymatic hydrolysis stage and proposes a continuous and a semicontinuous reaction
system which address two current concerns: increasing solids loading while maintaining conversion and
reducing enzyme loading. These are fundamental issues to develop an economically feasible process.
Enzymatic hydrolysis of lignocellulosics has been typically carried out at 5-10% w/w solids to ensure a
proper contact with enzymes. However, solids loadings higher than 10% w/w are required to obtain cost-
effective concentrations of sugars at the outlet of the hydrolysis stage [4]. Due to the high water-holding
capacity of solids, the reaction medium cannot be efficiently sheared and mixed at solids loadings higher than
10% w/w [5]. In addition, conversion at increasing solids loadings exhibits a general decreasing trend [6]. On
the other hand, enzymes cost has been pointed out as one of the major cost of the biochemical conversion [7].
As a solid-liquid enzyme-catalyzed reaction, the reaction rate is directly related to adsorbed enzyme. And
again, a negative correlation between solids loading and adsorbed enzyme has been observed [6].
An alternative to increase solids loading has been the fed-batch operation where solids and enzymes are added
at different times. This strategy enables to operate at solids loadings higher than 10% w/w while overcoming
mixing constrains [8-10]. In fed-batch operations, two to four additions of solids have been reported, and
enzymes are added either all at the beginning of the reaction or added at each solids feeding event. Operating
in fed-batch mode offers additional advantages such as lower instantaneous solids concentration and lower
apparent viscosities, which could be benefit for enzyme adsorption.
Several studies have focused on recycling strategies of free (not adsorbed) and adsorbed enzyme to reduce
enzyme loading [11-13]. Results have showed that it is possible to recover and recycle a substantial amount of
enzyme provided that the lignin content of solids is low [11]. Authors also have suggested directly employing
the bound enzyme with residual substrate as a more effective method to recover enzymes [12]. However,
more research on enzyme recycling strategies needs to be undertaken, especially in larger trials, to elucidate
the real potential of these alternatives [13].
A recent study has demonstrated that adding enzyme at low-solids loading (around 5% w/w), followed by
filtering the mixture after short retention times (around 10 min), and supplementing the thickened pulp (to
around 20% w/w) with extra enzyme, results in conversions comparable to those with low-solids loading [14].
To our best knowledge, this operating strategy has been the most successful reported so far in terms of solids
loading handled, final conversion, volumetric productivity and enzyme utilization. When solids loading
increased from 5 to 20% w/w in a conventional operation, final conversions decreased markedly as had been
pointed out in previous research [6, 14].
Some proposals of continuous and semicontinuous reaction systems for enzymatic hydrolysis of
lignocellulosics are summarized in Table 1. As a common feature, the reaction systems is divided into two
subsystems with complementary goals: (1) liquefaction at high-solids loading and (2) long-time
retention/reaction where final conversion is achieved. During liquefaction, a significant drop in the apparent
viscosity of the reaction medium occurs (from 100,000 to 2 Pa.s Approx. [16-17]). The liquefaction stage has
been represented by a train of 2 or 3 cascading CSTRs with distributed feeding of substrate and enzyme [18-
20], and either a downward flow or an upward flow tower-type PFR [2, 15]. On the other hand, the long-time
retention/reaction stage has been represented by a battery of batch reactors in parallel [2], a train of cascading
CSTRs [15-23] and a PFR [17, 20]. A train of cascading CSTRs with distributed feeding of substrate and
enzyme seeks to translate the fed-batch operation into a continuous basis. It was pointed out that this last
operation strategy has the potential to increase solids loading beyond 10% w/w and significantly improve the
volumetric productivity of the reaction system [18].
Table 1 Summary of proposed continuous and semicontinuous reaction systems for enzymatic hydrolysis of
lignocellulosics
PROCESS REACTORS CONFIGURATION REF
SHF PFR followed by a train of 3 to 12 cascading CSTRs
PFR followed by two trains of 3 to 12 cascading CSTRs
Two PFR in parallel followed by a train of 3 to 12 cascading CSTRs
[15]
SHF A train of 3 cascading CSTRs
A train of 3 cascading CSTRs with liquid phase recycling from the third to
the first reactor, enzyme supplementation at the second reactor, and liquid
phase withdrawing at the outlet of the first reactor
A train of 3 cascading CSTRs with distributed feeding of enzyme and
distributed withdrawing of liquid phase
[16]
SHF A train of 10 cascading CSTRs or a PFR, both with and without recycle
A train of 10 cascading CSTRs with distributed feeding of substrate and
enzyme at the first two or three reactors
A train of 3 cascading CSTRs with distributed feeding of substrate and
enzyme at the first two or three reactors, followed by a PFR
[17-20]
SSF 3 trains of 6 cascading CSTRs [21]
SHF A train of 5 cascading CSTRs [22]
SHF A tower type PFR (downward flow) followed by 12 batch in parallel [2]
SSF A train of 4 STRs with intermittent feeding [23]

In this paper, a previously developed and validated kinetic model [24] and the limiting micromixing situations
of macrofluid and microfluid [18, 19] are used to explore the capabilities of a continuous and a
semicontinuous reaction system. The following section presents the modeling framework which includes a
summary of the kinetic model, a detailed explanation of the operation strategies and the mathematical models.
MODELING FRAMEWORK
Kinetic model
The model is made up of two heterogeneous reactions for the breakdown of cellulose (C) into cellobiose (G
2
)
and glucose (G), and a homogeneous reaction for the breakdown of G
2
into G. The multi-enzyme system is
quantitatively represented by two enzymes concentrations, endoglucanase/cellobiohydrolase (EG/CBH) which
catalyze the heterogeneous reactions and -glucosidase (BG) which catalyze the homogeneous reaction. The
model incorporates EG/CBH adsorption on C and lignin (L), and BG adsorption on L. In addition, it takes into
account competitive inhibition of EG/CBH and BG by G
2
and G, and substrate reactivity [24].
The substrate, creeping wild ryegrass pretreated with sulfuric acid, was composed of 53% w/w C and 38%
w/w L on a dry basis. The model was fitted and validated under solids loadings from 4 to 12% w/w, EG/CBH
loadings from 15 to 150 FPU(g C)
-1
, BG loadings from 15 to 150 CBU(g C)
-1
, background G
2
of 10 gl
-1
and
background G of 30 and 60 gl
-1
.
Mass balances on C, G
2
, G, EG/CBH and BG were established as follow:

(1)

(2)

(3)

(4)

(5)
where:
r
1
: Heterogeneous reaction rate (C to G
2
)
r
2
: Heterogeneous reaction rate (C to G)
r
3
: Homogeneous reaction rate (G
2
to G)
t: Elapsed time
E
1
: EG/CBH
E
2
: BG
f: Free enzyme in solution
b: Bound enzyme
The kinetic rate equations r
1
, r
2
and r
3
have been reported by [24].
Operation strategies
The operation strategies are based, in part, on the experimental work of Xue et al. [14]. In their study,
cellulase enzymes (mainly EG/CBH) were added to 5% w/w solids and mixed. After 10 minutes of retention,
the pulp was thickened to 20% w/w solids by vacuum filtration. After various time intervals supplementary
xylanase and BG, with and without supplementary cellulase, were added into the thinned mixture and
incubated for 48 h (Figure 1). When compared with a conventional hydrolysis where all solids and enzymes
are added at the beginning of the reaction, the results summarized in Table 2 were obtained.

Figure 1 Simplified scheme of the operation procedure tested at laboratory scale by Xue et al. [14]
According to the aforementioned results, the key for increasing solids content while maintaining conversion is
to mix a fraction of EG/CBH enzymes at low solids loading, thicken to high-solids content and allow the
adsorbed EG/CBH enzymes to perform the necessary liquefaction which enables a thorough mixing of
supplementary EG/CBH, BG and xylanase enzymes. Instead of operating under the two subsystems concept
summarized in Table 1, the results of Xue et al. [14] suggest a three subsystems approach: adsorption at low
solids loading, thickening and liquefaction, and long-time retention/reaction.
Table 2 Sugar yields and sugar concentrations for the schemes evaluated by Xue et al. [14]. Cellulase loading
20 FPU(g-substrate
*
)
-1
and total reaction time 50 h
Operation procedure Yield %
Concentra
tion [gl
-1
]
1) 5% w/w solids and all the enzymes added at the beginning 64 26
2) 20% w/w solids and all the enzymes added at the beginning 44 84
3) All substrate and cellulase added at the beginning. Slurry thickened to 20%
w/w after 10 min. Xylanase and BG supplemented after 8 h
59 114
4) All substrate and part of the cellulase added at the beginning. Slurry thickened
to 20% w/w after 10 min. Cellulase, xylanase and BG supplemented after 2 h
63 121

*
Glucan 61.1%; Xylan 15.0%; Acid insoluble L 20.0% and Acid soluble L 2.9%. % w/w on a dry basis
The current operation strategies seek to translate the Xue et al. [14] procedure into a continuous or
semicontinuous basis (Figure 2). Pretreated solids and enzymes (EG/CBH or EG/CBH+BG) are continuously
mixed at low solids loading (5% w/w) in a well stirred tank called Adsorber-Reactor (AR) with a mean
retention time (
AR
) of 0.2 h (12 min). The purpose of the AR is to provide an environment with abundant free
aqueous phase beneficial for enzyme adsorption. The stream leaving the AR contains water-swollen partially
depolymerized solids with adsorbed enzyme and liquid phase with dissolved G
2
, G and free enzymes. After
pressing in a Mechanical Separator (MS) liquid phase with dissolved G
2
, G and enzymes is recycled to the AR
and the thickened pulp is liquefied in a tower type plug flow reactor (PFR) with a mean retention time (
P
) of
2 h. The recycle ratio (RR) relates the volumetric flow sent back to the AR and the volumetric flow sent to the
PFR. Finally, the liquefied slurry is conveyed to a train of cascading continuous stirred tank reactors (CSTRs)
where additional enzymes (EG/CBH or EG/CBH+BG) are supplemented in the first reactor (Figure 2a), or a
battery of batch reactors in parallel where additional enzymes are supplemented in each reactor (Figure 2b).
The battery of batch has been proposed by the last NREL (National renewable energy laboratory) technical
report [2].

a)


b)
Figure 2 Schemes of the reaction systems proposed. Enzyme adsorption Thickening and liquid phase
recycling, followed by a tower type PFR and a) a train of cascading CSTRs, or b) a battery of batch in parallel
Reactor modeling
The following general assumptions were necessaries for modeling: isothermal reactors, steady state, well
mixed tanks in the macroscopic sense and plug flow. It was considered simultaneous adsorption and reaction
in the AR. To study the effect of thickening after enzyme adsorption, solids loading after the AR were ranged
from 5 to 20% w/w. A train of 5 cascading CSTRs was considered to explore the performance of the
continuous system. Residence times by reactor (
R
) along the cascade of equal size CSTRs were ranged from
10 h to 50 h. Incubation times of 180 h were simulated for the battery of batch.
The micromixing limiting situations of microfluid and macrofluid were used for predicting conversion [18-
20]. The state of microfluid may prevail if the incoming material immediately comes into intimate contact
with other fluid elements of all ages at molecular level as in an ideal CSTR. Conversely, the state of
macrofluid may be kept if the incoming material is broken up into discrete clumps in which elements of
different ages do not intermix at all while in the reaction system, and reaction proceed independently in each
fluid element. Real fluids in continuous flow do not exhibit the microfluid or macrofluid extremes in its
micromixing behavior and are termed partially segregated. For the enzymatic hydrolysis of lignocellulosic
biomass, a micromixing behavior close to macrofluid was found in a CSTR for residence times required to
achieve cellulose conversions from 0.50 to 0.85 [25]. For higher conversions, a gradual evolution to
microfluid may be expected as the solids structure collapse and the apparent viscosity of the slurry decreases.
It was considered an EG/CBH loading of 15 FPU(g-substrate)
-1
and a BG loading of 15 CBU(g-substrate)
-1
. It
was modeled and simulated the split addition of EG/CBH with and without BG addition in the AR. The bound
concentration of EG/CBH and the free (in solution) concentration of BG were calculated by means of the
Langmuir adsorption isotherms reported as part of the kinetic model [24].
Macrofluid model
For a well-mixed train of cascading CSTRs, the residence time distribution (RTD) function E is given by [26]:

) (6)
where t is the reaction time, nr the number of reactors and the residence time by reactor. The RTD function
at the outlet of the AR (E
AR
) is given by:

) (7)
The RTD function at the outlet of CSTR i of the cascade (E
i
) is given by:

) (8)
where nr
i
is the CSTR i of the cascade.
C, G
2
and G concentrations at the outlet of reactor i (C
i
, G
2i
and G
i
, respectively) are expressed in terms of the
kinetic model and the correspondent RTD function as follow:

(9)

(10)

(11)
Each RTD function was numerically evaluated, and the maximal value of t (tdt) that guarantees a minimal
value of 0.9999 for each RTD time integral was used for the numerical evaluation of Equations 9 to 11. The
numerical values of C, G
2
and G for evaluating Equations 9 to11 were obtained by numerical integration of
the linear differential equation system represented by Equations 1 to 3.
Microfluid model
For CSTR i in the cascade, mass balances on C, G
2
and G, are expressed respectively as follow:

(12)

(13)

(14)
Batch model
For a batch reactor, the mass balances of C, G
2
and G are expressed in the integral form as follow:

(15)

(16)

(17)
PFR model
It was assumed constant density of the slurry in the PFR so the reactor is described by the same set of
equations of the batch model, where t in the batch is equivalent to
P
in the PFR.
RESULTS AND DISCUSSION
Results show that for a given residence or reaction time, cellulose conversion (X
C
) and G concentrations
predicted by the macrofluid model are greater than those predicted by the microfluid model. As expected, the
predictions of both models get closer to each other as residence time increases. The differences between the
predictions of the micromixing models are significant along the train of cascading CSTRs. In the first reactor
of the cascade, experimental evidence suggests a micromixing behavior close to macrofluid [25]. For
intermediate reactors, a gradual micromixing evolution from macrofluid to microfluid may be expected as the
solids structure collapse releasing continuous phase and the apparent viscosity of the slurry decreases. The
current study assumes ideal flow patterns, however to take further advantage of the micromixing models, the
real RTD of the reaction systems have to be obtained.
An important issue is the implications of assuming ideal flow patterns. Solids can be efficiently mixed at an
initial loading of 5% w/w. Also, a significant drop in the apparent viscosity of the reaction medium is
expected at the outlet of the PFR which implies that it is feasible to achieve well mixed conditions along the
cascade of CSTR or the battery of batch. Due to the effect of solids type, pretreatment process, level of
thickening and enzymes loading on the liquefaction process, further experimental studies should be done to
set the residence time
P
after which the material can be pumped to and mixed in conventional agitated tanks.
On the other hand, the flow of the thickened pulp through the tower type PFR can be upward or downward.
Other options for the PFR are a baffled tubular reactor [27, 28] and a screw conveyor reactor [29]. Due to the
relative high consistency of the slurry, the residence time distribution at the outlet of the PFR is expected to
be narrow. However, some channeling or laminar flow of the liquid phase could occur as the slurry moves
through the PFR.
Figure 3 shows recycle ratio RR, and G
2
and G concentrations at the outlet of the AR as a function of the
solids concentration after thickening (S
AT
). At this stage, it is expected a peak of adsorption of BG/CBH
enzymes on cellulose and lignin. Without BG addition there is a fast release of G
2
and a slow release of G
(Figure 3a). If BG is simultaneously added, it is expected a peak of adsorption of BG on lignin and a
significant breakdown of G
2
into G (Figure 3b). No significant differences were found between recycle ratios
(RRs) with and without BG addition in the AR. The adsorption model assumes that EG/CBH enzymes adsorb
not only on cellulose but also on lignin and BG adsorbs on lignin [24]. Enzyme adsorbs rapidly on the
external surface occupying some of the sites available and only adsorbed enzyme is able to catalyze the
heterogeneous reactions. According to Xue et al. [14], to prevent BG losses by unproductive adsorption on
lignin, it should be added at a later stage. It has also been reported that BG adsorption on lignin depends on
the type of enzyme and that this is not necessary unproductive [30]. Following this last concept, it is also
considered the addition of BG in the AR.


a)

b)
Figure 3 G
2
, G and RR at the outlet of the AR as a function of S
AT
. a) EG/CBH 7.5 FPU/g-substrate + BG 15
CBU/g-substrate. b) EG/CBH 7.5 FPU/g-substrate
After leaving the AR there is a mechanical separation. From the mechanical separation, part of the non-
adsorbed enzyme in the liquid phase is recycled to the AR. As the main residence time in the AR was fixed for
any RR, its volume is not constant but varies proportional to (1+RR). By recycling liquid hydrolysate from
reaction medium of higher reaction times, significant concentrations of enzyme inhibitors (G
2
and G) are
recirculated. The inhibitory effect of G
2
and G could be partially alleviated by recycling at early retention
times. In the batch laboratory experiments reported by Xue et al. [14] the retention time for enzyme
adsorption was set as 10 min (0.17 h) and the liquid stream with dissolved sugars and cellulase enzymes
leaving the mechanical separation was rejected. The current operation strategies propose to recycle this stream
avoiding sugars or enzyme losses even at higher retention times in the AR. Further research should be done to
set the mean residence time in the AR because mixing times are strongly dependent on solids loading and the
AR volume.
Figure 4 illustrates the concentration of G
2
with and without BG addition in the AR for the continuous
(Figure 4a) and the semicontinuous system (Figure 4b). When BG is not added in the AR, G
2
exhibits a peak
at the outlet of the PFR. This peak is a strong function of S
AT
, and residence time. BG cannot be added
immediately after thickening because of the high apparent viscosity of the material which makes difficult the
homogenization. G
2
is a strong inhibitor of EG/CBH activity and concentrations of 16 gl
-1
could be considered
too high. This fact provides further support for operating with BG addition in the AR, which resulted in a G
2

peak of 3.3 gl
-1
. By operating with split addition of BG (as was set the addition of EG/CBH) a G
2
peak
between 3.3 and 16 gl
-1
would be expected, so the G
2
peak could be a good indicator for adjusting both, BG
feeding strategy and BG loading. For a train of cascading CSTRs with a mean residence time of 30 h there
were not significant differences in G
2
concentrations between the cases (with and without BG) from the
second reactor of the cascade, and G
2
concentrations below 1 gl
-1
were obtained at the outlet of the third
reactor. For the battery of BATCH similar results are observed after 10 and 60 h of reaction, respectively.

a)

Figure 4 G
2
profiles with and without BG addition in the AR and S
AT
=20% w/w. Results at the left or the
vertical line corresponds to AR and PFR and are the same for the two reaction systems. a) Continuous reaction
system with
R
=30 h; b) Semicontinuous reaction system
Cellulose conversions for the reaction systems are depicted in Figure 5. It is important to bear in mind that
these results apply for the pretreated substrate used for fitting the kinetic model. Differences in solids type and
pretreatment process could lead to significant differences in both, final conversions and conversions profiles.
However the modeling and simulation framework presented here remains useful even if a more detailed
kinetic model were to be used. For the train of five cascading CSTRs it is manifest that a
R
of 10 h is
insufficient to reach a conversion higher than 0.7. Values of
R
between 20 and 30 h seems to offer a balance
between conversion and residence time. A cellulose conversion of 0.8 could be reached after 80 h in the
batch; similar conversions could be attained in a train of 4 cascading CSTRs with a mean residence time by
reactor of 30 h.
The last NREL technical report [2] assumes a PFR with a residence time of 24 h followed by a batch with a
retention time of 60 h to reach a final cellulose conversion of 0.9. From Figure 5 it is evident that for the
current substrate and enzyme loadings such a conversion could not be attained even in a batch with reaction
times higher than 150 h. Conversions higher than 0.8 could lead to prohibitive residence or reaction times but
from an economical point of view a minimal G concentration at the outlet of the enzymatic hydrolysis stage
has to be guaranteed.

a)

b)
Figure 5 Cellulose conversion profiles with BG addition the AR and S
AT
=20% w/w. a) Continuous reaction
system with; b) Semicontinuous reaction system
According to the results of Xue et al. [14], one of the main features of the current reaction systems would be a
significant increase in solids loading with final conversions similar to those achieved at low-solids loading.
As an initial solids loading of 20% w/w falls outside the interval of validation of the adsorption and kinetic
model, it is not possible to obtain results for an operation with such an initial solids loading. Figure 6 shows
the effect of S
AT
on cellulose conversion for the continuous system at outlet of the third and fifth reactors of
the cascade. The operation with S
AT
=20% w/w and
R
=30 h shows reductions in conversion of 19% and 13%
in the third and fifth reactor when compared to the operation with S
AT
=5% w/w (Figure 6a). Likewise, the
operation with
R
=50 h shows reductions in conversion of 14% and 9% in the third and fifth reactor. Contrary
to expectations, there are substantial differences between conversions at increasing S
AT
, although the
differences tend to be less significant at higher retention/reaction times (> 150 h).

a)

b)

Figure 6 Effect of thickening on cellulose conversion for the continuous system at a)
R
=30 h and b)
R
=50 h

Figure 7 Effect of thickening on cellulose conversion for the batch system
There are a number of reasons for the apparently unexpected results of Figures 6 and 7. First of all the
substrate type and pretreatment process of the study of Xue et al. [14] and those of the kinetic model [24] are
different. Also, the cellulase loading used for Xue et al. was 20 FPU/g-substrate whereas the one set in the
current study was 15 FPU/g-substrate. Enzyme loading has a significant and complex impact on process
economics and it was preferred a conservative value. Determining an economical enzyme loading requires
optimization of various parameters (such as temperature, solids loading, residence/retention time, etc.) which
is beyond the purposes of the current study. Higher enzyme loadings like the one of the study of Xue et al.
could significantly reduce retention/reaction times and keep conversions closed to those of 5% w/w. Finally,
Xue et al. reports the supplementation of xylanase enzyme after liquefaction but its action is not included in
the current model. It is important to highlight that when a solid loading of 20% w/w was added all at once
(procedures 2 of Table 1) a reduction of 43% in sugar yield was observed when compared with the operation
with split addition of enzyme (Procedure 4 of Table 1). In this sense, a continuous or semicontinuous
operation with split addition of enzyme as the proposed here offers clear advantages in terms of cellulose
conversion.
A critical issue for the enzymatic hydrolysis of lignocellulosic biomass is the volumetric productivity of the
reaction system. It has been stated that ethanol concentration in the broth entering distillation should be higher
than 40 gl
-1
to make an economically feasible process [4]. Assuming an ethanol yield of 0.48 g(g G)
-1
, a G
concentration of at least 83 gl
-1
would be required. G concentrations in Figure 8 were calculated as the
average between the macrofluid and the microfluid prediction. Figure 8a shows that depending on
R
, cascade
with different numbers of CSTRs fit the aforementioned cutoff. For instance, with
R
=10 h it is no possible to
attain a G concentration of 83 gl
-1
, whereas with
R
=30 h 3 CSTRs are required. On the other hand, reaction
times of 60 h are enough for attain the mentioned G concentration (Figure 8 b).

a)

b)
Figure 8 Glucose concentration for S
AT
=20% w/w for a) the continuous and b) the semicontinuous reaction
system
To strengthen the kinetic model there are suggestions related with adsorption, kinetic rates and long-time
enzyme substrate interactions. Following the proposed scheme, recycled enzyme would be reutilized by
readsorption on fresh substrate; however experimental evidence is necessary to elucidate the potential of this
alternative. Simulation results show that it is beneficial to add BG in the AR; however Xue et al. [14]
proposed to add BG after liquefaction. The question of unproductive adsorption of BG should be clarified
experimentally. The split addition of enzymes implies enzyme adsorption in a partially hydrolyzed substrate
and in the presence of background G and G
2
. Adsorption isotherms under these conditions should be obtained
to improve the calculation of adsorbed enzyme after the PFR. Also it would be desirable to include xylanase
adsorption and xylan hydrolysis in the kinetic model. The kinetic model used in this work accounts for
substrate reactivity, however enzyme deactivation is not taken into account [24]. The model presents an
interesting procedure for quantifying the enzyme adsorbed on L and a global adsorbed enzyme deactivation
rate could improve predictions.
A question that remains is whether to use the continuous or the semicontinuous operation. In general,
continuous processing is the preferred mode of operation for the production of commodity chemicals because
of reduced labor cost, improved process control and uniform product quality. On the other hand, a
semicontinuous operation provides for greater flexibility and lower investment costs, and could be preferred
at pilot plant research. As reaction systems are constrained by the huge required reaction volumes, a
compromise between solids loading, conversion and volumetric productivity of the system has to be sought.
This compromise could imply that the higher conversions (>0.85) assumed in economic evaluations of the
technology are not realistic.
CONCLUSIONS
Reaction systems for enzymatic hydrolysis of lignocellulosic biomass proposed so far divide the system in a
liquefaction stage at high-solids loading and a long-reaction/retention stage. A promising operation strategy
results from dividing the system into three stages: 1) a continuous enzyme adsorption stage at low-solids
loading (around 5% w/w) with short retention times followed by thickening and liquid phase recycling; 2) a
continuous liquefying stage at high-solids loading (around 20% w/w) were adsorbed enzyme performs the
thinning effect until obtaining a pumpable slurry and 3) a long-retention/reaction time stage were the final
conversion is achieved.
Differences in raw material and pretreatment process could lead to significant differences in the predicted
retention times and enzyme loadings. However, the modeling and simulation framework presented here
remains useful even if more detailed kinetic model is to be used; besides this approach can be extended form
SHF to SSF. The prediction of conversions for the limiting micromixing situations of macrofluid and
microfluid is a useful approach specially for cascading CSTRs. G concentrations around 100 g/L could be
attained with both of the proposed reaction systems. However, a compromise between solids loading,
conversion and volumetric productivity has to be sought because of the high required reaction volumes. 0.85.
Experimental work is essential to elucidate relevant aspects such as reutilization of recycle enzyme by
readsorption on fresh substrate and BG addition in the adsorption stage. Future kinetic models should
incorporate xylanase adsorption, xylan hydrolysis and a global enzyme deactivation rate. For detailed design
purposes, it is imperative to link kinetics and rheology. Semi-empirical relations to connect the progress of the
enzymatic hydrolysis with insoluble solids concentration and yield stress should be developed. Besides, the
RTD of the reaction system, especially the PFR, is required to take advantage of the micromixing models and
improve predictions.
NOMENCLATURE
AR Adsorber-Reactor
C Cellulose [gl
-1
]
CBH Cellobiohydrolase enzyme
CBU Cellobiase Unit
CSTR Continuous Stirred Tank Reactor
E Residence time distribution function
E
1
EG/CBH [g-protein.l
-1
]
E
2
BG [g-protein.l
-1
]
EG Endoglucanase enzyme
FPU Filter Paper Unit
G Glucoe [gl
-1
]
G
2
Cellobiose [gl
-1
]
L Lignin [gl
-1
]
MS Mechanical Separator
nr Number of reactors
NREL National Renewable Energy Laboratory
PFR Plug Flow Reactor
r
1
Heterogeneous reaction rate (C to G
2
) [gh
-1
]
r
2
Heterogeneous reaction rate (C to G) [gh
-1
]
r
3
Homogeneous reaction rate (G
2
to G) [gh
-1
]
RR Recycle ratio
RTD Residence Time Distribution
SAT Solids after thickening [% w/w]
SHF Separated Hydrolysis and Fermentation
SSF Simultaneous Saccharification and Fermentation
STR Stirred Tank Reactor
Greek Symbols
Residence time [h]
A variety of glucosidase enzyme
Change
Subscripts
R CSTR Reactor
P Reactor PFR
i CSTR i in the cascade
T Total
f Free (in solution)
b Bound

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