PaperVEA BBE0

8/11/2019 PaperVEA BBE0

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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 -1could 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


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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


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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 aPFR[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 1Summary of proposed continuous and semicontinuous reaction systems for enzymatic hydrolysis of

lignocellulosics

PROCESS REACTORS CONFIGURATION REF

SHF PFRfollowed by a train of 3 to 12 cascading CSTRs

PFR followed by two trains of 3 to 12 cascading CSTRs

TwoPFRin 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]


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SHF A train of 10 cascading CSTRs or aPFR, 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 typePFR(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 (G2)

and glucose (G), and a homogeneous reaction for the breakdown of G2into 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 incorporatesEG/CBHadsorption on Cand lignin (L), and BG adsorption onL. In addition, it takes into

account competitive inhibition ofEG/CBHandBGby G2and G, and substrate reactivity [24].

The substrate, creeping wild ryegrass pretreated with sulfuric acid, was composed of 53% w/w Cand 38%

w/wLon a dry basis. The model was fitted and validated under solids loadings from 4 to 12% w/w, EG/CBH

loadings from 15 to 150FPU(g C)-1,BGloadings from 15 to 150 CBU(g C)-1, background G2of 10 gl-1and

background Gof 30 and 60 gl-1.


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Mass balances on C, G2, G,EG/CBHandBGwere established as follow:

(1)

(2)

(3)

(4)

(5)

where:

r1: Heterogeneous reaction rate (Cto G2)

r2: Heterogeneous reaction rate (Cto G)

r3: Homogeneous reaction rate (G2to G)

t: Elapsed time

E1:EG/CBH

E2:BG

f: Free enzyme in solution

b: Bound enzyme

The kinetic rate equations r1, r2and r3have 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 (mainlyEG/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 andBG, with and without supplementary cellulase, were added into the thinned mixture and


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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 2were obtained.

Figure 1Simplified 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 ofEG/CBHenzymes at low solids loading, thicken to high-solids content and allow the

adsorbedEG/CBHenzymes to perform the necessary liquefaction which enables a thorough mixing of

supplementaryEG/CBH,BGand 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 2Sugar yields and sugar concentrations for the schemes evaluated by Xue et al. [14]. Cellulase loading

20FPU(g-substrate*)-1and 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 andBGsupplemented 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 andBGsupplemented 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


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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/CBHorEG/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 ARis to provide an environment with abundant free

aqueous phase beneficial for enzyme adsorption. The stream leaving theARcontains water-swollen partially

depolymerized solids with adsorbed enzyme and liquid phase with dissolved G2, Gand free enzymes. After

pressing in a Mechanical Separator (MS) liquid phase with dissolved G2, Gand enzymes is recycled to theAR

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 ARand 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/CBHorEG/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 lastNREL(National renewable energy laboratory) technical

report [2].

a)


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b)

Figure 2Schemes of the reaction systems proposed. Enzyme adsorptionThickening and liquid phase

recycling, followed by a tower typePFRand 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 theAR. To study the effect of thickening after enzyme adsorption, solids loading after the ARwere 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


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biomass, a micromixing behavior close to macrofluid was found in a CSTRfor 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 anEG/CBHloading of 15FPU(g-substrate)-1and aBGloading of 15 CBU(g-substrate)-1. It

was modeled and simulated the split addition ofEG/CBHwith and withoutBGaddition in theAR. The bound

concentration ofEG/CBHand 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) functionEis given by [26]:

( )(6)

where tis the reaction time, nrthe number of reactors and the residence time by reactor. TheRTDfunction

at the outlet of theAR(EAR) is given by:

(

)(7)

TheRTDfunction at the outlet of CSTRiof the cascade (Ei) is given by:

()(8)

where nriis the CSTRiof the cascade.

C, G2and Gconcentrations at the outlet of reactor i(Ci, G2iand Gi, respectively) are expressed in terms of the

kinetic model and the correspondentRTDfunction as follow:

(9)

(10)

(11)


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EachRTDfunction was numerically evaluated, and the maximal value of t(tdt) that guarantees a minimal

value of 0.9999 for eachRTDtime integral was used for the numerical evaluation of Equations 9 to 11. The

numerical values of C, G2and Gfor 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 iin the cascade, mass balances on C, G2and G, are expressed respectively as follow:

(12)

(13)

(14)

Batch model

For a batch reactor, the mass balances of C, G2and Gare expressed in the integral form as follow:

(15)

(16)

(17)

PFR model

It was assumed constant density of the slurry in thePFRso the reactor is described by the same set of

equations of the batch model, where tin the batch is equivalent to Pin thePFR.

RESULTS AND DISCUSSION

Results show that for a given residence or reaction time, cellulose conversion (XC) and Gconcentrations

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


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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

realRTDof 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 thePFRwhich implies that it is feasible to achieve well mixed conditions along the

cascade of CSTRor 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 Pafter 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 PFRcan be upward or downward.

Other options for thePFRare 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 PFRis expected to

be narrow. However, some channeling or laminar flow of the liquid phase could occur as the slurry moves

through thePFR.

Figure 3 shows recycle ratioRR, and G2and Gconcentrations at the outlet of theARas a function of the

solids concentration after thickening (SAT). At this stage, it is expected a peak of adsorption of BG/CBH

enzymes on cellulose and lignin. WithoutBGaddition there is a fast release of G2and a slow release of G

(Figure 3a). IfBGis simultaneously added, it is expected a peak of adsorption ofBGon lignin and a

significant breakdown of G2into G(Figure 3b). No significant differences were found between recycle ratios

(RRs) with and withoutBGaddition in theAR. The adsorption model assumes thatEG/CBHenzymes adsorb

not only on cellulose but also on lignin andBGadsorbs 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 preventBGlosses by unproductive adsorption on

lignin, it should be added at a later stage. It has also been reported that BGadsorption on lignin depends on


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the type of enzyme and that this is not necessary unproductive [30]. Following this last concept, it is also

considered the addition ofBGin theAR.

a)


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b)

Figure 3G2, GandRRat the outlet of theARas a function of SAT. a)EG/CBH7.5FPU/g-substrate +BG15

CBU/g-substrate. b)EG/CBH7.5FPU/g-substrate

After leaving theARthere is a mechanical separation. From the mechanical separation, part of the non-

adsorbed enzyme in the liquid phase is recycled to theAR. As the main residence time in the ARwas fixed for

anyRR, 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 ( G2and G) are

recirculated. The inhibitory effect of G2and Gcould 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 theAR. Further research should be done to

set the mean residence time in theARbecause mixing times are strongly dependent on solids loading and the

ARvolume.


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Figure 4illustrates the concentration of G2with and withoutBGaddition in theARfor the continuous

(Figure 4a) and the semicontinuous system (Figure 4b). WhenBGis not added in theAR, G2exhibits a peak

at the outlet of thePFR. This peak is a strong function of SAT, and residence time.BGcannot be added

immediately after thickening because of the high apparent viscosity of the material which makes difficult the

homogenization. G2is a strong inhibitor ofEG/CBHactivity and concentrations of 16 gl-1could be considered

too high. This fact provides further support for operating with BGaddition in theAR, which resulted in a G2

peak of 3.3 gl-1. By operating with split addition of BG(as was set the addition ofEG/CBH) a G2peak

between 3.3 and 16 gl-1would be expected, so the G2peak could be a good indicator for adjusting both,BG

feeding strategy andBGloading. For a train of cascading CSTRs with a mean residence time of 30 h there

were not significant differences in G2concentrations between the cases (with and withoutBG) from the

second reactor of the cascade, and G2concentrations below 1 gl-1were obtained at the outlet of the third

reactor. For the battery ofBATCHsimilar results are observed after 10 and 60 h of reaction, respectively.

a)


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Figure 4G2profiles with and withoutBGaddition in theARand SAT=20% w/w. Results at the left or the

vertical line corresponds toARandPFRand 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 Rof 10 h is

insufficient to reach a conversion higher than 0.7. Values of Rbetween 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 lastNRELtechnical report [2] assumes aPFRwith 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


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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 Gconcentration at the outlet of the enzymatic hydrolysis stage

has to be guaranteed.

a)


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b)

Figure 5Cellulose conversion profiles withBGaddition theARand SAT=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 SATon cellulose conversion for the continuous system at outlet of the third and fifth reactors of

the cascade. The operation with SAT=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 SAT=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 SAT, although the

differences tend to be less significant at higher retention/reaction times (> 150 h).


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a)


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b)

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

Figure 7Effect 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 20FPU/g-substrate whereas the one set in the

current study was 15FPU/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


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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-1to 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-1would be required. Gconcentrations 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 Gconcentration 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 Gconcentration (Figure 8 b).

a)


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b)

Figure 8Glucose concentration for SAT=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 BGin theAR; however Xue et al. [14]

proposed to addBGafter liquefaction. The question of unproductive adsorption ofBGshould be clarified

experimentally. The split addition of enzymes implies enzyme adsorption in a partially hydrolyzed substrate

and in the presence of background Gand G2. Adsorption isotherms under these conditions should be obtained

to improve the calculation of adsorbed enzyme after thePFR. 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


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interesting procedure for quantifying the enzyme adsorbed onLand 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. Gconcentrations 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.


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Experimental work is essential to elucidate relevant aspects such as reutilization of recycle enzyme by

readsorption on fresh substrate andBGaddition 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

RTDof the reaction system, especially thePFR, is required to take advantage of the micromixing models and

improve predictions.

NOMENCLATURE

AR Adsorber-Reactor

C Cellulose [gl- ]

CBH Cellobiohydrolase enzyme

CBU Cellobiase Unit

CSTR Continuous Stirred Tank Reactor

E Residence time distribution function

E1 EG/CBH[g-protein.l-1]

E2 BG[g-protein.l-1]

EG Endoglucanase enzyme

FPU Filter Paper Unit

G Glucoe [gl-1]

G2 Cellobiose [gl-1]

L Lignin [gl- ]

MS Mechanical Separator

nr Number of reactors

NREL National Renewable Energy Laboratory

PFR Plug Flow Reactor

r1 Heterogeneous reaction rate (Cto G2) [gh-1]

r2 Heterogeneous reaction rate (Cto G) [gh-1]


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r3 Homogeneous reaction rate (G2to G) [gh- ]

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 CSTRReactor

P ReactorPFR

i CSTRi in the cascade

T Total

f Free (in solution)

b Bound

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