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(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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