ENZYMATIC HYDROLYSIS OF CELLULOSE IN MEMBRANE

BIORECTOR
Yik Wan Yap1 and Nazlee F.Ghazali1*
1
School of Chemical and Energy Engineering
Faculty of Engineering,
Universiti Teknologi Malaysia,
81200 Skudai, Johor Bahru,
Malaysia.

ABSTRACT

Enzymatic hydrolysis of cellulose of ultrafiltration in a membrane reactor using polyethersulfone

(PES) membrane was studied. Different enzyme loadings (1v/v%-4v/v%) were investigated on its
effect on flux, product yield, rejection and fouling mechanism at constant microcrystalline
cellulose concentration of 10g/L, TMP of 2 bar, cross flow velocity (CFV) of 120rpm. The
rejection of cellulase was above 94 % at all cases and high glucose permeation (above 93%) was
achieved as PES membrane with molecular weight cut off (MCWO) of 10kDa was feasible to
retain the cellulase with molecular weight (MW) which was larger than MWCO of the membrane
at the same time allow glucose to pass through. Reducing sugar concentration increased up to
6.13g/L at 72nd hour as showed proportional relationship with enzyme concentration. At 72nd
hour, as the enzyme concentration increased, the permeate flux showed declination 42.22 L/m2.h
to 26.15 L/m2.h. The cake layer model was determined to be the predominant factor contributed
to fouling as according to Hermia’s pore blocking model with highest R2of 0.99.

Keywords : cellulose, enzymatic hydrolysis, ultrafiltration, membrane reactor

1.0 INTRODUCTION

Cellulose can be found abundantly in natural residual lignocellulosic material has been widely
used in lignocelluloses biorefinery as these low-cost lignocellulosic materials can be converted
into fermentable sugars, which reduces the waste disposal costs and concomitantly meets the
growing demand for energy [4,7]. Enzymatic hydrolysis is often preferable as it is carried out in a
milder condition and has less impact on the environment. The enzymatic hydrolysis is carried out
using an enzyme known as cellulase, where it consists of a mixture of endoglucanases,
exoglucanases as well as β-glucosidases[3]. These three enzymes work synergistically to convert
cellulose into glucose. The endoglucanases and exoglucanases reduce cellulose into cellobiose,
then the cellobiose will be hydrolyzed into glucose by β-glucosidases. EMR has caught
researchers’ interest for its potential ability to prevent product inhibition and to increase the
product yield [3,8]. For the cellulose hydrolysis in a membrane reactor, ultrafiltration (UF) is
used as the separation process to retain cellulase as well as removing the reducing sugar produced
from the enzymatic reaction. The membrane used in UF is chosen based on the enzyme molecular
weight. UF membranes can retain large molecules with a molecular weight ranging from 10 to
100 kDa [6].

* Corresponding author : nazlee@ utm.my

2.0 MATERIALS AND METHODS

2.1 Membrane Reactor

Cellulase enzyme from Trichoderma reesei and the substrate of microcrystalline cellulose was used in
this study. TMP of 2 bar and crossflow velocity of 120pm ware set for the apparatus setup. The
hydrolysis reactor consisted of a 100 ml Scott bottle was connected to a crossflow ultrafiltration
cell. As this is an external loop membrane reactor, a peristaltic pump was used to transfer the
hydrolysate inside the reactor to the crossflow cell. Measuring cylinder was used to collect
permeate filtered from the hydrolysate.
Temperature Probe

Pressure Gauge Cross Flow

P Module

PES
membrane

Hydrolysate P
Stirrer Permeate

Hot Plate Magnetic Stirrer Peristaltic Pump Measuring Cylinder

Figure 1 Schematic diagram for enzymatic membrane reactor.

2.2 Enzymatic hydrolysis

In this study, different enzyme concentrations were investigated for their membrane performance
on reducing sugars production. Four different enzyme concentrations were studied (1, 2, 3 and 4
v/v% substrate). Cellulose will be used as the substrate and the concentration is fixed at 10 g/L.
The reaction temperature and pH were maintained at 50 °C and 5.0 respectively in sodium citrate
buffer (50Mm). The hydrolysis will be conducted for 72 hours continuously. Ultrafiltration will
be conducted at every 4 hours and 50% of hydrolysate will be collected as permeate. The volume
in the reactor was maintained at 100ml by adding fresh buffer. At the 24th hour, instead of only
fresh buffer, 10g/L of fresh cellulose substrate was also added. The replenishment of fresh buffer
was done every 4 hour after ultrafiltration while addition of fresh cellulose substrate was
conducted at 24th hour, 48th hour and 72nd hour. Some studies concluded that higher conversion of
cellulose into glucose can be achieved by removing glucose using membrane reactors [1]. For
cellulose hydrolysis, the membrane reactor consists of a reactor for enzymatic reaction and a
membrane separation unit [2]. After the hydrolysis is completed, the enzyme will be retained in
the membrane while the reducing sugar will permeate through the membrane. The main purpose
in enzymatic membrane reactor is to make sure that more than 90% of the enzymes are being
rejected while carrying out separation process, and at the same time, maintaining full enzymatic
activity inside the hydrolysis reactor [3].

2.3 Analysis
The samples were collected from permeate and hydrolysate and was mixed with mixed with DNS
reagent at 1:1 ratio and then heated in water bath of 90 ℃ for 5 minutes. The mixture was then
put in water bath to cool down to room temperature. The cooled down mixture was transferred to
a cuvette and measured its absorbance using Jenway 7305 Spectrophotometer at 540 nm.
Enzyme concentration will be determined based on the dye Brilliant Blue G and protein complex
formation in the solution using Bradford reagent. The protein complex causes a shift from 465 to
595 nm in the absorption maximum of the dye. The procedures are, protein sample will be mixed
with Bradford reagent in a ratio of 1:30 (e.g. 0.05 ml sample: 1.5 ml Bradford) and incubated at
room temperature for 5 minutes until the colour is developed. Then, the mixture will be recorded
at 595 nm using UV-VIS spectrophotometer.

2.4 Membrane Performance

The important parameters that measure the performance of a membrane process are flux and
rejection. Flux (J) is expressed as:
3600𝑠
𝐿 𝑉𝑝 (𝑚𝑙)×
ℎ
J (ℎ 𝑚2 ) = 𝑚𝑙 (1)
1000( )𝐴(𝑚2 )∆𝑡(𝑠)
𝐿

where J is the filtration flux, VP is the permeate volume, A is the effective membrane area,
Δt is the time elapsed since the first drop of permeate.
𝐶𝑃𝑖
𝑅𝑖 = (1 − ) × 100% (2)
𝐶𝐹

where CP is the concentration of solute i in permeate, CF is concentration in feed and Ri is the

rejection of enzyme/reducing sugar. In this study, a low rejection of reducing sugar (RS) and high
rejection of enzymes is desired. All experimental runs were carried out in triplicate and mean
values were reported.
2.5 Cellulose conversion
Cellulose conversion can be expressed as gram of reducing sugar (RS) produced over gram of
cellulose fed in the reactor shown below [3]:
𝑅𝑆 (𝑔)
𝐶𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑐𝑜𝑛𝑒𝑟𝑠𝑖𝑜𝑛 (%) = 𝐶𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑓𝑒𝑑 (𝑔) × 100 (3)

3.0 RESULTS AND DISCUSSION

3.1 Reducing Sugar Yield

As can be observed from Figure 2, the overall trend showed that the higher the enzyme
concentration the higher the yield of reducing sugar. As higher concentration of enzyme was used,
more enzymes have free active site for the binding of cellulose as substrate to catalyze the
hydrolysis process thus more cellulose can be converted to glucose. Based on Figure 2, there was
sharp decrease of the reducing sugar concentration at every 24 hour interval which was after
addition of fresh buffer. At 24th hour, the drastic drop of reducing sugar concentration at enzyme
concentration 1v/v% (2.369 g/L to 1.335 g/L), enzyme concentration 2v/v% (2.983g/L to
1.887g/L), enzyme concentration 3v/v% (5.274g/L to 2.097g/L) and enzyme concentration 4v/v%
(5.752g/L to 3.187g/L ). At 48th hour the drastic drop of reducing sugar concentration at enzyme
concentration 1v/v% (2.369 g/L to 1.335 g/L), enzyme concentration 2v/v% (2.983g/L to
1.887g/L), enzyme concentration 3v/v% (5.274g/L to 2.097g/L) and enzyme concentration 4v/v%
(5.752g/L to 3.187g/L ). The decrease in reducing sugar were due to the replenishment of citrate
buffer, causing the dilution of the reducing after the adding of fresh substrate.
Based on Figure 3, the conversion of microcrystalline cellulose increased from 25.97% to 61.32%
with the ascending enzyme concentration. Enzyme activity increased as the enzyme loading
increased. There was only slight increase of conversion of cellulose (59.34% to 61.32%) which
probably due to product inhibition and the effect of fouling that will greatly decrease the
concentration of reducing sugar production.
 8 Graph of Reducing Sugar Concentration Against Time

RS Concentration (g/L)
6
1 v/v%
4
2v/v%
2 3v/v%
4v/v%
0
0 20 40 60 80
Time (hr)
Figure 2 Reducing sugar concentration at different enzyme concentration
Reducing Sugar Yield Against Time
70
60
RS Yield (%)

50
40
30
20
10
0
1 2 3 4
Enzyme Cncnetration (v/v%)

Figure 3 RS yield at different enzyme concentration

3.2 Flux Declination

Based on Figure 4, the flux profile achieved the lowest value for enzyme concentration of 1v/v%
(20.47 L/m2.h), for the enzyme concentration of 2v/v% (19.76 L/m2.h), for enzyme concentration
of 3 v/v% (17.73 L/m2.h), and enzyme concentration of 4 v/v% (16.59 L/m2.h) at 72nd hour.
During the ultrafiltration at 72nd hour, the amount particles molecules accumulated on membrane
surface achieved the highest amount as compared to earlier time, thus forming cake layer that
brings to blockage of membrane pores.
Graph of Flux Against Time
50
45
40
35
Flux (L/m2.h)

30 1v/v%
25
20 2v/v%
15 3v/v%
10 4v/v%
5
0
0 20 40 60 80 100
Time(min)

Figure 4 Flux Profiles at 72nd hour

As the hydrolysis process was carried at constant substrate concentration 10g/L, the declination of
flux increased as the enzyme concentration increased. At the lowest enzyme concentration of
1v/v%, the flux were the highest at 72nd hour (42.224 L/m2.h), as compared enzyme concentration
of 2v/v%, 3v/v% and 4v/v%. As less enzyme was being used, more membrane pores were free
and left unoccupied to allow the hydrolysate solution to pass through thus higher flux value can
be obtained.

3.3 Rejection
As can be observed from Figure 5 that the rejection were almost constant at different enzyme
concentration which were very high percentage for enzyme rejection and very low percentage for
glucose rejection. Both permeation of glucose and rejection of enzyme does not affect by the
enzyme concentration but depends on the molecular weight cut off of membrane used. The
permeability of the solute feed depends on the pore size of the membrane rather than the
concentration of enzyme [5].

Graph of Rejection Against Enzyme Concentration

120
100
Rejection (%)

80 Reducing Sugar
60
40
Enzyme
20 Cellulase
0
1 2 3 4
Enzyme Concentration (v/v%)

Figure 5 Rejection of reducing sugar and enzyme

3.4 Fouling Mechanism

As the enzyme concentration increased, the fitting for the four types of blocking decreased as
indicated by decreasing R2 value. While the best fitting for all different enzyme concentration
was observed at the cake layer blocking model. Marshall explained that the formation of cake
layer as the large size enzymes particles accumulated on the membrane surface. While in
complete pore blocking model, the lowest R2 were recorded at 72nd hour for enzyme
concentration 3v/v% (0.8197) and enzyme concentration 4v/v% (0.7477). This indicates that the
flux was less likely decreased due to enzymes particles plugged individual pores and the flow
diverted to other pores that plugged successively [5].

Table 1 Regression coefficient (R2) for pore blocking model at different enzyme concentration
Complete Pore Blocking Standard Pore Blocking
T(h)
1v/v% 2v/v% 3v/v% 4v/v% 1v/v% 2v/v% 3v/v% 4v/v%
24 0.9663 0.9164 0.9112 0.7676 0.9752 0.9676 0.8286 0.7837
48 0.9701 0.9147 0.9464 0.7753 0.9783 0.9414 0.9621 0.7916
72 0.9263 0.9386 0.8197 0.7477 0.9476 0.9298 0.9384 0.7588
 Intermediate Pore Blocking Cake layer model
T(h)
1v/v% 2v/v% 3v/v% 4v/v% 1v/v% 2v/v% 3v/v% 4v/v%
24 0.9818 0.9866 0.9597 0.7973 0.9862 0.9953 0.9859 0.8177
48 0.9847 0.9628 0.9746 0.8059 0.9881 0.9888 0.9971 0.8291
72 0.965 0.9412 0.8481 0.7684 0.9873 0.9577 0.8694 0.7833

CONCLUSIONS

Enzymatic hydrolysis of cellulose using ultrafiltration membrane reactor was proved able to
extend enzyme reusability up to 72 hours. PES 10kDa was suitable to be embedded in cross flow
module for the above process as it provided high retention of enzyme more than 94% and the
same time allowed permeation of glucose more than 93%. Besides, the permeate flux decreased
from 42.22 L/m2.h to 26.15 L/m2.h as the enzyme concentration increased from 1v/v % to 4v/v%
as it was determined the cake layer model was the predominant fouling mechanism.

ACKNOWLEDGEMENTS

Support for this work by the University Technology Malaysia, assistance from University
Laboratory Management Centre (PPMU) is gratefully appreciated.

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