Short communication

Enzymatic hydrolysis of rice straw in a tubular

reactor coupled with UF membrane

Sen Yang *, Wenyong Ding, Hongzhang Chen *

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China

Received 24 April 2005; received in revised form 1 August 2005; accepted 3 August 2005

Abstract

A tubular reactor coupled with UF membrane was used to investigate the enzymatic hydrolysis of rice straw. The results showed that the

high substrate concentration could be attained in the tubular reactor, and the rate of hydrolysis, as well as the yield of reducing sugar (RS), was

markedly enhanced in the membrane bioreactor due to the continuous removal of inhibitory products. When the substrate concentration was

175 g/l, the production of RS was 436 mg/g (dry weight of rice straw), while the final RS concentration in the product stream still remained a

high level, 27.2 g/l.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Membrane reactor; Cellulase; Enzymatic hydrolysis; Rice straw

www.elsevier.com/locate/procbio

Process Biochemistry 41 (2006) 721–725

1. Introduction

Lignocellulosic biomasses are widely considered as an

important source for the production of sugar streams that can

be fermented to ethanol and other organic chemicals [1].

And enzymatic hydrolysis of cellulosic material has been

extensively studied in the last decades to get soluble sugars

[2–6]. However, practical application of the enzymatic

hydrolysis has been deterred by the high cost of enzymes,

slow reaction rate, and lack of an ideal reactor system [7–9].

The use of a membrane reactor system is a promising

method in enzymatic hydrolysis. Cellulolytic enzymes and

unhydrolyzed cellulosic materials are retained within a

membrane reactor system, whereas hydrolyzed products

permeate the membrane. The membrane reactor offers

potentials in recovering and reusing of cellulolytic enzymes,

further improving yield and kinetics, reducing inhibition of

enzymes. Since the reactor coupled with the stirred-tank

type membrane module to retain the cellulase was employed

* Corresponding authors. Tel.: +86 10 82627067; fax: +86 10 82627071.

E-mail addresses: yangsen105@yahoo.com.cn (S. Yang),

hzchen@home.ipe.ac.cn (H. Chen).

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2005.08.002

by Ghose and Kostic [10], several types of membrane

bioreactor have been tested. Ohlson et al. [11] reported that

the enzymatic cellulose hydrolysis rate increased four times

in a membrane bioreactor compared with that obtained in a

conventional batch reactor. However, a present limitation in

the membrane bioreactor system is the low concentration of

RS, which has to be concentrated to get the optimal

conditions for the following fermentation process [12]. It is

known that this problem is partially caused by the glucose

inhibition on enzyme activity that lowers the productivity,

the high water content of the wet biomass and the water

required for assuring good rheological properties of the

reaction medium [1]. Moreover, continuous separation of

the sugars from the hydrolysis stage leads to a more diluted

product stream.

The aim of this study is to investigate the possibility of

enzymatic hydrolysis of cellulose in a tubular reactor

coupled with a hollow fibre UF membrane module. Rice

straw was used as model because of its abundance as

agricultural waste all around the world. The biomass was

pretreated in a steam-explosion plant being this pretreatment

one of the less expensive and most effective for perfor-

ming the enzymatic hydrolysis [13]. The advantages of

S. Yang et al. / Process Biochemistry 41 (2006) 721–725722

steam-explosion pretreatment arise from several factors:

hemicellulose degradation, lignin transformation, redistri-

bution within the cell wall, swelling of cell walls and

increase in functional specific gravity and water holding

capacity [14].

2. Experimental methods and materials

2.1. Substrate and enzymes

Steam-exploded rice straw was prepared by treating

chopped rice straw (3–4 cm, containing 15% of water) in a

steam exploded vessel (1 m3) at 1.5 MPa for 10 min and then

discharge promptly. After the pretreatment, the solid residue

was dried without washed and used in the hydrolysis

experiments directly. Dry solids content (cellulose, hemi-

cellulose and lignin) was estimated according to the

procedures of Goering and Van Soest [15], after drying at

105 8C for 24 h. The composition of the rice straw after

pretreatment is 40.3 wt.% cellulose, 13.6 wt.% hemicellu-

lose and 3.1 wt.% lignin. The cellulase extracted from

Trichoderma reesei used in this work was obtained from

Ningxia xiasheng Co. (China); the filter-paper activity (FPA)

and the b-glucosidase activity were 110 FPU ml�1 and

37 IU ml�1, respectively [16,17].

2.2. Membrane bioreactor

A schematic diagram of the UFmembrane bioreactor was

shown in Fig. 1. The system consisted of a tubular reactor

with a length of 0.4 m and a diameter of 36 mm (Fig. 1) and a

hollow-fibre module made in Research Center for Eco-

Environmental Sciences (China). The module consisted of

10 fibres with an internal diameter of 1.0 mm, a wall

thickness of 0.1 mm, and a length of 10 cm. The total

membrane area was 0.00314 m2. The fibres were made of

polysulfone (PS) with a narrow molecular weight cut-off

(MWCO) of 10,000 Da. As the UF membrane used in the

Fig. 1. Experime

system has no separation selectivity towards RS permeation,

it is assumed that the measured RS concentration in the

permeate also represented the sugar concentration in the

bioreactor [7].

In the membrane bioreactor, Substrate was kept in the

tubular reactor with a porous filter, the buffer entered the top

of the tube through a distributor containing 20 capillary

tubes (0.5 mm inner diameter) and the flux was kept at a

constant speed of 200 ml/min by means of a recycling pump.

The highest water (buffer) content of the wet pretreated rice

straw was 5 ml/g straw (dry weight), so pretreated rice straw

with highest buffer content was used as substrate to keep the

volume of buffer flowing in the membrane bioreactor at a

constant. The tubular reactor was done under atmospheric

pressure and the RS produced in hydrolysis was dissolved by

buffer and separated from the system by the membrane. A

reservoir was used to continuously feed fresh buffer to

the reactor to keep the volume of buffer unchanged. The

pressure applied to the membrane module was conveniently

modified to obtain the desired permeation flow. The pressure

required was typically about 0–0.3 MPa. The permeate flux

was set at 0.4–0.7 ml/min and permeate was collected in a

measuring cylinder for flow control. An automatic fraction

collector was used for permeate collection in long-term

experiments.

2.3. Enzymatic hydrolysis

A weighed amount of pretreated rice straw with highest

buffer content was placed and an enzyme solution was added

to a final volume. The flowing buffer in the membrane

bioreactor was kept at a constant of 60 ml in all experiments.

The concentration of rice strawwas calculated as the amount

of dry substance per volume of the buffer. Pretreated rice

straw wasn’t additionally washed. The reactor was kept at

50 8C, the optimal catalytic temperature for the cellulases.

The reactions were conducted at a constant pH 4.8,

maintained by a sodium acetate buffer. An initial hydrolysis

period was conducted in the tubular reactor without flowing

ntal device.

S. Yang et al. / Process Biochemistry 41 (2006) 721–725 723

buffer. After 30 min, the recycling pump and the feeding

pump were turned on. At this point, the flowing buffer

(60 ml) entered the top of the tube through a distributor, the

pressure was applied, and a continuous permeate collection

started.

The RS concentration in the permeate flow was measured

using the dinitrosalicylicacid (DNS) method [18].

Fig. 3. Production of RS as a function of time for different substrate

concentration: (a) 125, (b) 155, (c) 175 and (d) 185 g/l.

3. Results and discussion

3.1. Effect of substrate concentration

Enzymatic hydrolysis under different substrate concen-

trations was carried out in the UF membrane bioreactor. The

traditional batch hydrolysis was also performed in 250-ml

glass flasks on a shaker for comparison. The substrate

concentration was varied from 125 to 185 g/l (w/v); the

process was lasted for 24 h with an enzyme loading of

20 FPU/g (straw). The reaction volume was maintained by

replenishing the reactor with buffer but without fresh

substrate.

The influence of the substrate concentration on RS

concentration in permeate at different hydrolysis time was

shown in Fig. 2. The shape of the curves, with a peak in sugar

concentration after 2–4 h, is similar to those obtained for

hydrolysis of sallow in an UF membrane reactor [11]. This

means the RS concentration all amounts to the highest

after 2–4 h. Ohlson et al. [11] suggested that this shape

presumably depended not only on different dilution rates,

but also on the kind of pretreatment, enzyme/substrate as

well as cellobiase/cellulase ratio. The RS concentration in

the permeate increased with the increase in the substrate

concentration below 175 g/l. However, further increase in

the substrate concentration would lead to decrease in the RS

concentration. The RS concentration was the highest when

the substrate concentration is 175 g/l, in the enzymatic

hydrolysis step. The final concentration of RS (defined as

Fig. 2. RS concentration in permeate from the membrane bioreactor as a

function of time for various substrate concentration: (a) 125, (b) 155, (c) 175

and (d) 185 g/l.

the weight of RS per unit volume of the total permeate) after

enzymatic hydrolysis for 24 h was 4.6, 8.6, 27.2 and 18.7 g/l

when the substrate concentration was 125, 155, 175 and

185 g/l, respectively. While the RS concentration in

traditional batch reactor was 19.6, 26.9, 28.1 and 31.2 g/l,

respectively. Vlasenko et al. [2] examined the effect of

three distinct pretreatment procedures and six commercial

cellulases on enzymatic hydrolysis of rice straw. Hydro-

lyzates containing 27–30 g/l of redcing sugar were obtained

at 48 h using 100–150 g/l substrate concentrations and

1 FPU/ml enzyme concentration. However, only 33–38% of

carbohydrates were converted to soluble sugars under their

conditions.

Fig. 3 showed the effect of substrate concentration on RS

production. It could be seen that the production increased

with increasing substrate concentration and amounted to

the peak at 175 g/l substrata concentration. When the

operating time was fixed at 24 h, the RS production

increased from 276 to 436 mg/g (dry weight of rice straw)

as the substrate concentration increased from 125 to 175 g/l.

Similar results had also been observed for phenol-pretreated

wheat straw [19]. However, Wen et al. [20] believed that

when the ratio of enzyme to substrate was fixed, sufficient

enzyme could be supplied with increased substrate con-

centrations. And they reported that glucose yield remained

almost constant within the substrate concentration ranging

from 10 to 50 g/l. However, further increases in substrate

concentration (50–100 g/l) resulted in a lower glucose yield

due to the end-product inhibition caused by high concentra-

tion of glucose. In fact, when the ratio of enzyme to substrate

was fixed, enzyme concentration would increase with

increasing substrate concentration, and the higher the

enzyme concentration, the higher the productivity in terms

of g sugar/g substrate h. The reason that the concentration

and productivity of the RS doesn’t increase when the

substrate concentration beyond 175 g/l maybe due to the

end-product inhibition cannot be eliminated completely and

higher mass and heat transfer resistances in the tubular

reactor without stirring or vibration.

S. Yang et al. / Process Biochemistry 41 (2006) 721–725724

Fig. 4. Changes in the average hydrolysis rates in the UF membrane

bioreactor with different substrate concentration: (a) 125, (b) 155, (c)

175 and (d) 185 g/l.

Fig. 5. RS concentration in permeate from the membrane bioreactor as a

function of time for various dilution rates.

Fig. 4 showed the average reaction rate in different

hydrolysis time, and the reaction rate was defined as the

weight of RS produced per unit volume of substrate per unit

time, in the membrane bioreactor with different substrate

concentration. It had been generally observed that enzymatic

hydrolysis of cellulose in batch reactor had a rapid initial

rate followed by a continuous rate fall, which ultimately

tends to negligibly small, and the initial decline was

most drastic. The major expected benefit of operating a

continuous and selective in situ product removal during the

hydrolytic reaction is the reduction of product inhibitory

effect with consequential improvement of hydrolysis rate

[7]. An obvious increase in hydrolysis rate had been

observed, especially when the substrate concentration

beyond 155 g/l. However, the hydrolysis rate in membrane

bioreactor also declined drastically during the first several

hours (Fig. 4). This indicated that the hydrolysis rate was

limited not only by product inhibition, but also by other

factors [21], such as the decrease in the extent of adsorbed

enzyme, transformation of the structure of cellulose into a

less digestible form, and inhibition of the enzyme action by

the accumulated hydrolysis products.

From production and concentration of RS data, a 175 g/l

substrate concentration seems to be optimal. In the present

study we therefore chose to work with 175 g/l substrate

concentration.

3.2. Effect of enzyme loading

The optimal ratio between enzyme and substrate is very

important for the efficient use of cellulase enzyme. Thus, the

effect of the enzyme loadingon theRSproductionwas studied

with 175 g/l substrate and a time of 24 h. The production of

RSwas 220, 398, 420 and 425 mg/gwhen the enzyme loading

was 10, 20, 30 and 40 FPU/g, respectively. The high cost of

enzymes, however, makes high dosage impractical for this

type of application [2]. For this reason, an enzyme loading of

20 FPU/g was selected for the remaining experiments.

3.3. Effect of dilution rate

The dilution rate [D (h�1) = permeate flow (ml/h)/

reaction volume (ml)] is thought to be limited by the

operating conditions such as the concentration of substrate

within the reactor and filtration module. Hydrolysis of

175 g/l substrate was performed with 20 FPU/g enzyme

loading at different dilution rates over 24 h time period.

Fig. 5 showed the RS concentration in permeate as a

function of time for various dilution rates. It has been

generally observed that an increased dilution rate means a

higher conversion rate and a lower concentration of RS in

permeate from the membrane reactor [11]. In this system, an

increased dilution rate (from 0.057 to 0.075 h�1) means a

higher production (from 266 to 439 mg/g) and concentration

of RS in permeate from the membrane reactor. It could be

explained by the end-product inhibition cannot be elimi-

nated completely with low dilution rate.

3.4. Deactivation of cellulase in the tubular reactor

The soluble enzyme activity during hydrolysis was

investigated. It has been reported that irreversible adsorption

to nondegradable cellulose, thermal inactivation and shear

stress cause an undesirable deactivation of cellulase in

hydrolysis [21–24]. In our system, deactivate cellulase due to

shear stress could be negligible since the tubular reactor

doesn’t stir or vibrate. The thermal stability of cellulase was

investigated in the membrane bioreactor without substrate

under specified conditions. The FPA and b-glucosidase

activityweremaintained stably during 24 h, so loss of activity

during hydrolysis was attributed to reversible and irreversible

adsorption. After 24 h, when hydrolysis was complete,

soluble FPA and b-glucosidase activity was 50 and 70% of

the initial added enzyme activity, showing that the non-

convertible fraction of the substrate adsorbed a considerable

fraction of the enzymes. These enzymes are partially released

when fresh substrate is available, and data with enzyme

S. Yang et al. / Process Biochemistry 41 (2006) 721–725 725

recovery and recycle in semi-continuous and continuous

feeding of rice straw is to be reported in the future.

4. Conclusions

In conclusion, high substrate concentration and high RS

concentrationwere successfully realized in a tubular reactor

coupled with UF membrane. The rate of hydrolysis, as well

as the yield of RS, was markedly enhanced in an UF

membrane bioreactor due to the continuous removal of

inhibitory products. When the substrate concentration was

175 g/l, the production of RS was 436 mg/g (dry weight of

rice straw), while the final RS concentration in the product

stream still remained a high level, 27.2 g/l. After 24 h, when

hydrolysis was complete, soluble FPA and b-glucosidase

activity was 50 and 70% of the initial added enzyme

activity.

Acknowledgements

This study was performed thanks to funding by National

Basic Research Program of China (2004CB719700),

Hi-Tech Research and Development Program of China

(2001AA514023) and Knowledge Innovation Programm of

Chinese Academy of Sciences (KJCXZ-SW-206-2).

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