Proceedings of the
2016 Annual Conference on Sustainable Research and Innovation,
4 - 6 May 2016
Hydrolysis of Pretreated Prosopis juliflora
Stem to Simple Sugars using Immobilized
Enzymes
Wiseman T. Ngigi, David M. Menzwa, Ambrose Kiprop
1
I.
ABSTRACT- Prosopis juliflora is a noxious weed which can be put
into profitable use and help address the use of food based substrates
in the production of glucose which lead to food insecurity. In an
attempt to find profitable use, Prosopis juliflora stem was
investigated for its potential to produce glucose. In this research,
Prosopis juliflora stems were dried and milled into fine powder to
pass through a 0.5mm screen followed by alkali pretreatment then
hydrolysis using immobilized enzymes. The enzymes used were
cellulase, hemicellulase and cellobiase. Cellobiase was immobilized
using sodium alginate while cellulase and hemicellulase were
immobilized covalently using glutaraldehyde (GA). The factors
studied were pH, temperature, time and concentration of sodium
alginate (NaAl). The level of pH was varied at 4.5 - 7.5, temperature
varied at 40 - 70°C, time varied at 2- 72 hours while NaAl
concentration of was varied at 1.0% - 3.0% (w/v). The maximum
hydrolysis yield was established using a Central Composite Rotatable
Design (CCRD) in Response to Surface Methodology (RSM).
Mathematical models estimating the yield of simple sugars from
Prosopis juliflora stem were developed and analyzed for predicting
and enhancing the yield of glucose. Optimum glucose yield of 78.2%
(w/w) was obtained from Prosopis juliflora stem at 55°C hydrolysis
temperature, pH of hydrolysis medium of 6.0, 2.0 % (w/v)
concentration of sodium alginate (w/v) and 48 hours hydrolysis
period. Lowest glucose yield of 43.1% (w/w) was obtained at 55°C
hydrolysis temperature, pH of hydrolysis medium of 6.0, 3.7% (w/v)
concentration of sodium alginate and 48 hours hydrolysis period. The
immobilized enzyme was easily separated through simple filtration,
washed and recycled from the hydrolysis reaction mixture after each
hydrolysis reaction cycle of 48 hours. When reused in subsequent
hydrolysis reactions, the immobilized enzyme retained enzymatic
activity in subsequent substrate hydrolysis batches by upto 10 cycles
as demonstrated by the yield of glucose of 57.6% (w/w). The yield of
glucose obtained from the hydrolysis of Prosopis juliflora stem(
78.2%, w/w) using immobilized enzymes indicate that Prosopis
juliflora stem is suitable for the production of glucose which can be
used to produce bioethanol and marketable chemicals such as citric
and lactic acid. In addition the ability to recycle the immobilized
enzymes contributes significantly towards bringing down the cost of
enzymatic hydrolysis.
One of the major challenges facing the world in the 21st
century is to meet the energy demand for heating,
transportation, lighting and industrial processes. Recently
there has been an increased focus on bio-fuels due to the
impact of fossil fuel consumption on global warming and the
depletion of fossil fuel reserves [4].Several alternatives exist
which include biofuels such as bioethanol which is derived
from fermentation of simple sugars. In addition, the demand
for simple sugars is on the rise since they are used as raw
material for many industrial chemicals such as citric and lactic
acid. To address these challenges more research is required to
find out viable, renewable and sustainable sources of simple
sugars suitable for the production of bioethanol which is
renewable source of energy and industrial chemicals such as
citric acid and lactic acid. Subject substrates should not
compete with food sources. Substrates being considered
include agro-wastes (corn stover, wheat straws, sugar cane
bagasse, sorghum stalks), hardwood and softwood residues
from forest resources [17] which are generally referred to as
lignocellulosic biomass (LGB).The conversion of LGB to
biofuels and other chemicals involves the liberation of simple
sugars from biomass substrates which consist of three
fractions namely hemicellulose, cellulose and lignin.
Hemicellulose and cellulose contain sugars in polymeric form
that can be converted by enzymes, alkali or acids to simple
sugars. However, the lignin component, shields cellulose and
hemicellulose from microbial and enzymatic attack, hence the
use of LGB as a substrate requires some form of pretreatment
in order to make the cellulose and hemicellulose constituents
more accessible for enzymatic saccharification.
Pretreatment of LGB is required in order to make the
recalcitrant LGB easily accessible to enzymatic
saccharification [23]. Pretreatment is normally done to disrupt
and disintegrate the lignin and break down the hemicellulose
portion of LGB in order for the enzymes to gain access to the
cellulose [3].
Secondly, even though several attempts have been made to
produce simple sugars from non-food substrates such as LGB,
the conversion of these substrates to simple sugars is often
faced with several draw backs. LGB is a renewable and
sustainable substrate ideal for the production of simple sugars.
This abundant substrate can be hydrolyzed to simple sugars by
enzymes. Whereas first generation bioethanol is commercially
Keywords: Central Composite Rotatable Design (CCRD), glucose,
hydrolysis, Prosopis juliflora
W.T. Ngigi, Department of Chemical & Process Engineering, Moi University,
P.O. Box 3900-30100, Eldoret-Kenya (Corresponding author – Email:
[email protected])
D. M. Menzwa, Department of Chemical & Process Engineering, Moi
University, P.O. Box 3900-30100, Eldoret-Kenya
A. Kiprop, School of Biological & Physical Sciences, Moi University, P.O.
Box 3900-30100, Eldoret-Kenya
INTRODUCTION
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Proceedings of the
2016 Annual Conference on Sustainable Research and Innovation,
4 - 6 May 2016
provide a cost effective method of hydrolysis as well as
confirming the potential of Prosopis juliflora stem as a viable
substrate for the production of simple sugars.
established and is produced from substrates such as corn and
sugar cane, it is not sustainable as food crops are used as
substrate [14]. On the other hand, second generation
bioethanol is not commercially established but can be
produced from sustainable (non-food crop). Enzymatic
hydrolysis is a major process step in the production of simple
sugars from LGB. This process uses enzymes to breakdown
LGB to simple sugars. Enzymatic hydrolysis of LGB to
simple sugars faces many challenges. One of the challenges is
the high quantity of enzymes required which increases the cost
of hydrolysis. One way of decreasing the cost of enzymes is to
recycle and reuse the enzymes by immobilizing them. Other
challenges include long hydrolysis time, irreversible
adsorption of enzymes to lignin, thermal denaturation of
enzymes, and separation of enzymes from product. In addition
the lack of sustainable non-food substrates presents a
challenge to the commercialization of processes for the
production of simple sugars from LGB. In an attempt to
address these challenges, this research study sought to develop
a process of enzymatic hydrolysis of pretreated Prosopis
juliflora stem to produce simple sugars using immobilized
enzymes with the aim of reducing the cost of enzymes due to
the ability to recycle/reuse the immobilized enzymes. In
addition, when used in immobilized state, the enzymes are
capable of withstanding high temperature operation thus
eliminating the challenge of thermal deactivation while at the
same time allowing the process to proceed faster at higher
temperatures thus reducing the time required to complete the
hydrolysis cycle. Finally, the problem of irreversible
adsorption of enzymes on lignin is addressed through
immobilization. Despite the wide interests in the production of
simple sugars from agricultural residues and woody substrates,
the hydrolysis of Prosopis juliflora stem fraction using
immobilized enzymes has not been reported in literature.
Furthermore, this LGB has never been explored as alternative
substrate for the production of simple sugars using
immobilized enzymes in Kenya. If successful, the study will
II.
MATERIALS AND METHODOLOGY
The research was carried out through literature review and
laboratory experimentation. The concentration of glucose was
analyzed using a spectrophotometer. Central Composite
Rotatable Design (CCRD) design approach were used to
determine the optimum level of the factors (temperature, pH,
and concentration of NaAl) affecting the hydrolysis of
pretreated Prosopis juliflora stem. All experiments were
carried out in triplicate and the mean values of the yield of
enzymatic hydrolysis were taken as the response. A total of 20
experimental runs were carried out based on CCRD. Statistical
software MATLAB (version R2010b) was used to generate
the CCRD matrix, plotting of graphs and subsequent analysis
of data. Three dimensional response surface and contour plots
were generated using MATLAB in order to evaluate the
effects of the variables on the response.
i. Reagents and standards
Glucose standard and reagents such as acetone, glucose
hexokinase (HK) reagent, sodium azide, calcium chloride,
sodium hydroxide, deionised water, sodium alginate,
glutaraldehyde, sulphuric acid, and sodium chloride were
purchased from Kobian Kenya limited. All reagents were
analytical grade. The enzymes used in this study were
cellulase, hemicellulase and cellobiase (β glucosidase). These
enzymes were purchased from Kobian Kenya limited. All
enzymes were ex Sigma Aldrich.
ii. Design of experiment
The CCRD matrix for factors under consideration is shown in
Table I. MATLAB was used in the design of the experiment
leading to the generation of the experimental design matrix
shown in Table III.
Table I: CCRD matrix for factors under consideration
Independent variables
o
Symbol
Range and levels
(- α)
(-1)
(0)
(+1)
(+ α)
Temperature, ( C)
X1
29.8
40
55
70
80.2
pH
X2
3.5
4.5
6
7.5
8.5
Conc. of NaAl (%, w/w)
X3
0.32
1
2
3
3.7
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2016 Annual Conference on Sustainable Research and Innovation,
4 - 6 May 2016
syringe without a needle at room temperature. The beads were
left in CaCl2 solution to cure for 30 minutes and then
thoroughly washed three times with distilled water and finally
stored at 4oC for use in further experiments. To immobilize
cellulase and hemicellulase, the two enzymes were mixed in
the ratio of 7:3 to constitute an enzyme loading of 10% (w/w)
then dissolved in 10 ml of 0.1 M citric acid buffer. Chilled
acetone was added into a 50 ml glass vial with a magnetic
stirrer. There after the enzyme-buffer mixture was added to
the chilled acetone. GA was then added drop wise to give a
solution with a concentration of 0.005 M which was
maintained at 10oC for 2.5 hours with gentle stirring. The
mixture was finally centrifuged for 10 min and the
immobilized cellulase/hemicellulase enzyme pellets obtained
upon decanting and washing them three times using buffer
solution. The washed, carrier-free immobilized cellulase:
hemicellulase enzyme system was stored in buffer solution at
4oC.
iii. Collection of samples
Prosopis juliflora stem samples were collected from Baringo
County and stored in sealed plastic bags. The samples were
then dispatched to the Chemical and Process Engineering
laboratory within 24 hours after harvesting for further studies.
iv. Drying and milling
The Prosopis juliflora stem was sun dried for two weeks then
cut into small sized pieces and dried using a hot air oven set at
105°C for 8 hours. The oven dried Prosopis juliflora stem
samples were then milled through a 0.5 mm screen into fine
powder and stored in sealed plastic containers. The chemical
composition of biomass under investigation was analyzed in
triplicate, as described by NREL-LAP procedures, with minor
modifications to suit the prevailing conditions. The procedures
which reference was made to include: [2], [13], [18], [19],
[20], [21] and [22].
v. Pretreatment of substrate
60 g of sample milled to an average particle size of 0.5 mm
was soaked in a dilute sodium hydroxide (1% NaOH) solution
at 10% solid loading for 24 hours at room temperature under
constant agitation using a magnetic stirrer. The slurry was then
filtered through a sieve to separate solid and liquid fractions.
The solids were thoroughly washed with distilled water to
remove the residual NaOH. The washed solid was then dried
at 60oC for 6 hours and stored in a sealed container for use in
the hydrolysis experiment.
vi. Enzyme immobilization
This procedure was adopted from enzyme immobilization
protocol [9]. 10% (w/w) enzyme loading of cellobiase was
mixed with sodium alginate (NaAl) solution to form separate
immobilizing mixture. The concentration of NaAl was set
depending on the experiment as determined by the CCRD.
The beads were formed by dripping the enzyme-polymer
solution from a height of 20 cm into an excess (100 ml) of
stirred 0.2 M calcium chloride (CaCl2) solution using a 20 ml
topped up with distilled water so that each vial consisted of a
working volume of 10 ml. Hydrolysis proceeded for 48 hours
and samples to be analyzed for glucose were stored at -4oC
prior to analysis. Each experimental treatment was performed
in triplicate and the average response calculated for each
experimental run. The level of factors in the experimental run
that gave the highest yield of glucose was considered to be the
optimum levels.
vii. Optimal level of factors affecting hydrolysis
The method used was based on NREL LAP-009 standard
method [7]. 20 separate pretreated substrate samples equal to
the equivalent of 0.1 g of cellulose were weighed out and
added to 20 ml scintillation vials. 5.0 ml sodium acetate buffer
at the pH level determined by the experimental run (Table III)
was then added to each sample to form separate experiment
samples. Each vial was placed in a rotary shaker which was
then operated at 150 rpm for 30 min after which the vial was
placed in a water bath set at the temperature level determined
by the experimental run (Table III). To initiate the reaction,
the immobilized enzyme was added to the substrate samples
after reaching the desired reaction temperature as per the
experimental design. 0.1 ml sodium azide was added to
prevent microbial contamination. The volume of each vial was
then
hydrolysis mixture, collected and washed with distilled water
followed by enzyme assay buffer after which it was used in a
subsequent hydrolysis cycle. The resulting hydrolysis mixture
was analysed for glucose. Recycling was done until the yield
of glucose leveled out (the yield remained constant) which
was an indication of the decline in the activity of immobilized
enzymes. Each recycling was done for 48 hours. The number
of times the immobilized enzyme was recycled until the yield
started to level out was counted to stand for the total number
of cycles of reuse.
x. Analysis of glucose
This procedure was based on SIGMA glucose (HK) assay kit
technical bulletin [1].
xi. Statistical analysis
CCRD and RSM were applied in this study during
experimental design. The experimental data was matched with
second order polynomial equation. R2 and adjusted R2 were
used to determine the significance of developed models.
Fischer test at 95% confidence level was used to test the
statistical significance of the developed polynomial equations
viii. Effect of hydrolysis time on glucose yield
The optimum level of factors that gave the highest yield of
glucose was further used to investigate the effect of hydrolysis
time on the yield of glucose. Hydrolysis time was designed for
2 – 72 h. This time was chosen based on existing past
researches. Samples were analysed for glucose after 2, 6, 12,
18, 24, 36, 48, 60 and 72 hour.
ix. Recycling of immobilized enzymes
Using the optimal level of factors, hydrolysis was carried out
for 48 hours which was the complete hydrolysis cycle. After
each cycle, the immobilized enzymes were filtered from the
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III.
RESULTS AND DISCUSSION
i. Materials Characterization
Characterization of LGB was done to determine the
composition of Prosopis juliflora stem. The summary of the
chemical composition of Prosopis juliflora stem are as shown
in Table II
while the double tailed student’s t-test at 95% confidence level
was applied to test the significance of the various polynomial
coefficients. The statistical analyses were done on MATLAB
version R2010b. MATLAB was also used to plot response
surface and contour graphs which were analysed to observe
the hydrolysis trends and optimum hydrolysis results.
Table II: Chemical composition of Prosopis juliflora stem
Component
Cellulose
Hemicellulose
Lignin
Extractives
Ash
Moisture
TOTAL
Percentage
39.06
19.36
28.18
2.59
1.0
9.81
100
The chemical composition of Prosopis juliflora stem
compares favorably with that reported in [15]: 45-50%
cellulose, 25-35% hemicellulose and 25-35% lignin for
softwoods stems. The presence of high cellulose content in
Prosopis juliflora stem (39.06%) indicates its potential for use
as a feedstock for the production of glucose. The moisture
content of 9.45% found in Prosopis juliflora stem is below
10% and therefore there was no effect of moisture content on
the compositional analysis [12].
ii. Glucose yield from Prosopis Juliflora stem
The yield of glucose from Prosopis juliflora stem under
various experimental conditions is shown in Table III.
Table III: Glucose yield from Prosopis juliflora
Run
1
X1(OC)
-1
X2
-1
X3 (%)
-1
Glucose yield (%, w/w) (Actual)
57.4
Glucose yield (%, w/w) (Predicted)
58.4
2
1
-1
-1
66.4
66.8
3
-1
1
-1
58.3
58.8
4
1
1
-1
66.1
67.1
5
-1
-1
1
48.8
49.3
6
1
-1
1
49.2
51.5
7
-1
1
1
47.6
49.7
8
1
1
1
51.6
51.9
9
-1.682
0
0
54.3
52.9
10
1.682
0
0
63.2
61.8
11
0
-1.682
0
66.3
64.7
12
0
1.682
0
66.6
65.3
13
0
0
-1.682
62.3
61.5
14
0
0
1.682
43.1
41.0
15
0
0
0
78.2
77.5
16
0
0
0
77.2
77.5
17
0
0
0
76.9
77.5
18
0
0
0
77.8
77.5
19
0
0
0
77.3
77.5
20
0
0
0
77.4
77.5
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The coded values used were generated using MATLAB from Prosopis juliflora stem, the following 10 terms 2nd order
(R2010b). The actual and coded levels of factors are shown in regression polynomials (equation 1) was fitted to predict the
Table I. From the factors settings and actual yield of glucose yield of glucose from the hydrolysis of Prosopis juliflora stem.
77.5480 2.6482X
0.1687X
6.0985X
0.3000X X
1.5500X X
0.075X X
Y
4.4272X
9.2874X
ε
Where
Y
Predicted Glucose yield from Prosopis juliflora stem (%, w/w)
ε
Random error associated with Glucose yield prediction from Prosopis juliflora stem
7.1489X
(1)
Dimensionless values of the independent variables.
In order to test the significance of the various regression 95% confidence level was used. The analysis was done using
coefficients that describe the yield of glucose from Prosopis MATLAB and the results are as shown in Table IV.
juliflora stem in equation 1, a double – tailed student (t) test at
Table IV: t-TEST (tcritical = 2.262)
Term
β0
Coefficient
77.548
Se (βi )
0.65643
to
118.14
p- Value
4.6327E-017
Comment
Significant
β1
2.6482
0.4355
6.0809
0.00011866
Significant
β2
0.16873
0.4355
0.38745
0.70654
Insignificant
β3
-6.0985
0.4355
-14.003
6.7565E-08
Significant
β12
0.3
0.56903
0.52721
0.60955
Insignificant
β13
-1.55
0.56903
-2.7239
0.021416
Significant
β23
0.075
0.56903
0.1318
0.89775
Insignificant
β11
-7.1489
0.42389
-16.865
1.1283E-08
Significant
β22
-4.4272
0.42389
-10.444
1.0657E-06
Significant
β33
-9.2874
0.42389
-21.91
8.7831E-10
Significant
From Table IV, regression coefficients associated with cannot be dropped because it is part of model hierarchy. The
interaction terms X1 and X2, and X2 and X3 were insignificant reduced regression model equation 2 was plotted on a
and were hence collated with other errors and a reduced model response surface and contour graph as shown in fig. 1 for
for the yield of glucose from Prosopis juliflora stem was glucose yield.
obtained as equation 2. Although X2 is not significant, it
77.5480 2.6482X
0.1687X
6.0985X
1.5500X X
7.1489X
4.4272X
9.2874X
ε
Y
(2)
Where:
Y
Predicted Glucose yield from Prosopis juliflora stem (%)
ε
Random error associated with Glucose yield prediction from Prosopis juliflora stem
Dimensionless values of the independent variables.
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X1 & X3 vs Glucose (B) Surface Plot
Glucose Yield(B) (%)
100
80
60
40
40
20
0
35
2530
45
40 50
302535
55
65
70
80
60
55
75
1
55
0
-1
60
5045
40
35
1
0
-1
Concentration of NaAl. (Level)
Temperature. (Level)
Fig. 1. Glucose (B) vs X1, X3: Response surface and contour plots
55oC (0) and 2.0% (0) respectively that is both factors at the
central setting. This is illustrated by the smallest eclipse on the
contour plot. The results indicated by the plot are in agreement
with the experimental observations which showed that a
temperature of 55oC and a 2.0% (w/w) concentration of
sodium alginate led to optimum yield of glucose from
Prosopis juliflora stem.
Fig. 1 was plotted using MATLAB R2010b from the reduced
regression polynomial (equation 2) predicting the yield of
glucose from Prosopis juliflora stem. The plot shows the
response surface with contours depicting how both
temperature and concentration of sodium alginate influenced
the yield of glucose. The plot shows that the maximum yield
of glucose (78.2%) from Prosopis juliflora stem is obtained
when temperature and concentration of sodium alginate are
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X2 & X3 vs Glucose (B) Surface Plot
100
GlucoseYield(B) (%)
80
60
40
35
40
50
20
0
30 35
40
45
60
75
55
65
80
65
60
70
1
55
1
0
60
-1
0
55
-1
Concentration of NaAl. (Level)
pH. (Level)
Fig. 2. Glucose (B) vs X2, X3: Response surface and contour plots
Fig. 2 above shows the response surface and contour plot of
the interaction between concentration of NaAl and pH. The
smallest eclipse on the contour plot corresponds to the surface
peak and shows the maximum glucose yield. This
demonstrates that in order to achieve maximum yield of
glucose, both factors should be at the central settings.
X1 & X2 vs Glucose (B) Surface Plot
Glucose Yield(B) (%)
100
80
60
40
55
6560
20
0
75
45 50 55 60 65
80
70
1
0
55
1
55
-1
60
50
45
0
-1
pH. (Level)
Temperature. (Level)
Fig. 3. Glucose (B) vs X1, X2: Response surface and contour plots
Fig. 3 above shows the response surface and contour plot of
the interaction between pH and temperature. The smallest
eclipse on the contour plot corresponds to the surface peak and
shows the maximum glucose yield. This demonstrates that in
order to achieve maximum yield of glucose, both factors
should be at the central settings. In order to test the
significance of the model depicting the yield of glucose from
Prosopis juliflora stem, ANOVA was performed using
MATLAB and the results are shown in Table V.
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Table V: Prosopis juliflora stem: ANOVA for the regression significance
P - value
Source
Sum of Squares
df
Mean of Squares
F- test (observed)
F- test (critical)
Prob > F
Model
2558.9759
9
284.3307
109.7630
3.02
0.0000
Error
25.9041
10
2.5904
Total
2584.8800
19
From Table V the model representing the yield of glucose from
Prosopis juliflora stem has an F- value of 109.7630 which is
higher than the critical F – value of 3.02 at 95% confidence
level. This shows that the developed model equation 1
significantly evaluates the experimental data involving glucose
yields from pretreated Prosopis juliflora stem. The model F –
value (109.7630) implied that the model was significant and
there was negligible (0.00%) chance that an F- value this large
could occur due to noise. In addition, regression analyses
indicate that coefficient of determination, R2 for the model
describing glucose yield is 0.9900 with an adjusted R2 of 0.9810
which indicates that the developed model is significant. This
implies that there is a better correlation between observed and
predicted values of responses.
iii. Effect of hydrolysis time on glucose yield
The results of the effect of time on the yield of glucose during
hydrolysis of Prosopis juliflora stem are shown in Table VI.
Table VI: Effect of hydrolysis time on glucose yield
Time (hour)
2
Glucose (mg/ml)
1.50
Yield (%, w/w)
13.5
6
2.99
26.9
12
3.69
33.2
18
5.03
45.3
24
6.72
60.5
36
7.93
71.4
48
8.70
78.3
60
8.73
78.6
72
8.76
78.8
It is necessary to determine the best level of time for the
hydrolysis reaction. Reference [16] reported that longer time
of hydrolysis can cause inhibition from substrate and product
which results into a lower hydrolysis yield. From Table VI,
the highest yield of glucose obtained was 78.8 % (w/w) at a
time period of 72 hours. Comparing this with the yield of
glucose at 48 hours (78.3%, w/w) leads to the conclusion that
the best time for carrying out hydrolysis is a period of 48
hours. After this time period, it is not economically feasible to
carry out further reactions because even a 24 hour (after 72
hours) increase in time results into a negligible (0.64%)
increase in the yield of glucose. The results in Table VI were
plotted as shown in fig. 4 which indicates that after 48 hours,
the yield of glucose started to level off.
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A graph of glucose yield against time
100
90
Glucose yield (%, w/w)
80
70
60
50
40
30
20
10
0
2
6
12
18
24
36
48
60
72
Time (hour)
Fig. 4.Effect of hydrolysis time on glucose yield
Time is an important factor during chemical reactions because
the rate of any chemical reaction varies with time. It is
important to find that optimum time to operate with since time
determines productivity so that a shorter reaction period
means higher productivity due to increased number of batches
within a given operating time frame. From the results, it can
be seen that at time 48 hours, the yield of glucose was
optimum. This time was considered to be the optimal level.
This can be explained as follows: the ability of the
immobilized enzyme system to operate at high temperature
resulted in an increase in the rate of reaction as chemical
reactions are known to proceed at a faster rate at higher
temperature which in turn reduced the time for each batch
during hydrolysis. In addition, the presence of immobilized
cellobiase ensured that the resulting cellobiose was quickly
converted to glucose.
iv. Recycling of immobilized enzymes
A batch process was used to study the effectiveness of
immobilized enzymes upon recycle. The results of this
experiment are shown in Table VII for Prosopis juliflora stem.
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Table VII: Recycling of immobilized enzymes
Cycle (number)
0 (initial batch)
Glucose (mg/ml)
8.66
Yield (%, w/w)
77.9
1st
8.53
76.8
nd
8.34
75.1
rd
8.24
74.2
4
th
8.13
73.2
5
th
7.94
71.5
6th
7.60
68.4
7th
6.86
61.7
8th
6.59
59.3
9th
6.49
58.4
10th
6.40
57.6
2
3
The results in Table VII were plotted as shown in fig. 5.
A graph of glucose yield against number of cycles
100
Glucose yield (%, w/w)
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
Number of cycles
Fig. 5. Effect of recycling immobilized enzymes on glucose yield
These results indicate that the immobilized enzymes can be
reused for 7 consecutive cycles without significant decline in
the yield of glucose (yield at over 60%, w/w). After 9 cycles,
the yield of simple sugars was still reasonably high. Reference
[10] reported that they were able to recycle immobilized
Trichoderma reesei for five times during their study on
hydrolysis of pure form of cellulose. The ability to recycle the
immobilized enzymes can be explained as follows:
immobilized enzyme was shielded from thermal destruction
by the immobilizing agent at high temperature. The increased
thermal stability of the immobilized enzyme was due to
attachment of the enzyme to the carrier protecting them from
autolysis [5]. In addition, a decline in the yield of glucose was
noted after the seventh cycle. This was due to a decline in the
activity of enzymes within the carrier system which was
probably caused by the leakage of cellobiase from the
247
Proceedings of the
2016 Annual Conference on Sustainable Research and Innovation,
4 - 6 May 2016
enzymes tend to become (partly) unfolded and/or inactivated,
implying that they are no longer able to perform the desired
tasks within the expected efficiency levels. The reason why
immobilized enzymes are able to function even at higher
temperature is because immobilization provides a thermal
shield to the enzymes, enabling them to withstand high
temperatures although up to a certain extent. The ability of
immobilized enzyme to withstand higher temperatures is
advantageous because it allows the hydrolysis reaction to be
carried out enzymatically at higher temperature. From
chemical reaction kinetics, heat speeds up the movement of
substrate and enzyme molecules in the solution, which
increases the number of collisions between the enzyme and
substrate. This speeds up the reaction thus minimizing time
spent in each batch. This in turn leads to overall cost
reductions which enhances the economic viability of
immobilized enzymatic hydrolysis. Another possible
explanation as to why the yield of glucose declined when
using free enzymes is because of the effect pH has on the
activity of enzymes. Comparing with free enzymes, the yield
obtained in the case of immobilized enzymes is higher at pH
value of 6.0. From literature, free enzymes show a decrease in
yield at pH of about 5.0. This is due to a decline in enzyme
activity as a result of enzyme inactivation which can be
explained as follows: Enzymes have ionic groups on their
active sites (regions within the enzyme where reactions are
believed to occur) and which must be in a suitable form (acid
or base) to function. Any variation in the pH of the reaction
medium leads to changes of the ionic form of the active site
which affects the enzyme activity. This results in a decrease in
the rate of enzyme catalysed reaction which in turn leads to a
decline in the yield of glucose. For these reasons, enzymes are
only active within a certain pH range. The stability of
immobilized enzymes at high pH levels can be due to the
charge on the immobilizing agent and the manner in which the
enzyme is bound [6]. The immobilized forms of enzymes are
more stable within a wide range of pH which makes them
suitable for commercial use [6]. In addition, the ability of
immobilized enzymes to operate within a wide range of pH
values provides a flexible opportunity of performing
hydrolysis process with minimum effect on yield of simple
sugars in situations where process variables may cause a
variation in pH.
immobilizing agent due to loss of mechanical strength of the
beads as a result of repeated separation and washing of the
beads after each cycle. The recycled immobilized enzymes
used in the hydrolysis of pretreated Prosopis juliflora stem
clearly established that recyclable application of the
immobilized enzyme system is attainable with high hydrolysis
efficiency. This leads to cost reduction which goes a step
further in making the production of glucose from Prosopis
juliflora stem economically viable. These results indicate that
calcium alginate and GA are excellent materials which can be
used for enzyme immobilization.
v. Model Validation
In order to validate the model obtained, the levels of factors
that were found to be the optimum values were used in
carrying out separate experiments using Prosopis juliflora
stem as substrate. The results of these experiments gave rise to
a maximum glucose yield of 77.3% which was similar to that
obtained in the model prediction shown in Table IV for
Prosopis juliflora stem.
vi. Comparing Immobilized and Free Enzymes
In order to determine the effectiveness of the immobilized
enzyme, pure form of cellulose was used as the substrate.
Hydrolysis was carried out at optimum level of factors
obtained in objective one. From the results the yield of glucose
was 89.8%. This yield was higher than the glucose yield of
63% (w/w) that was obtained in [10] when they hydrolyzed
microcrystalline cellulose (pure form of cellulose) using
immobilized Trichoderma reesei. In addition, a separate
experiment was conducted using free enzymes at optimal level
of factors. A glucose yield of 61.4% (w/w) was obtained using
Prosopis juliflora stemas the substrate. This result indicates
that there was a decline in the yield of glucose when using free
enzymes. This can be explained as follows: The rate of
enzyme catalysed reactions increases with temperature up to a
certain level above which the effectiveness of the enzyme
decreases. The immobilized enzymes exhibited maximal yield
of glucose at 55°C. In reference [8] it is reported that the
optimal reaction temperature for immobilized xylanase at
between 52.5°C and 65°C while that of immobilized βxylosidase shifted from 45°C to between 50°C and 60°C. The
optimum level of temperature (55°C) seemed higher for free
enzymes to efficiently operate. Reference [11] reported a
maximum hydrolysis temperature of 50°C during their study
on production of fermentable sugars through hydrolysis using
free cellulases and cellobiase. They concluded that above
50°C the activity of the enzymes decreased as temperature
increased because of denaturation. The reason as to why
immobilized enzymes were able to operate at a higher
temperature is because the immobilizing agent (NaAl and GA)
retained the three dimensional shape of the enzymes at higher
temperature preventing the enzymes from folding. The free
enzyme underwent denaturation at 55°C, that is the enzymes
were denatured and lost their three dimensional structure,
making them inactive. At elevated temperatures, many
IV.
CONCLUSION
The aim of this research study was to produce glucose from
pretreated Prosopis juliflora stem through hydrolysis using
immobilized form of enzymes and establish the number of
times the immobilized enzymes can be reused in hydrolyzing a
fresh batch of pretreated Prosopis juliflora stem. The
following conclusions were drawn from the study:
 Optimum glucose yields of 78.2% (w/w) from Prosopis
juliflora stem were obtained at 55oC hydrolysis
temperature, 6.0 pH of the hydrolysis medium, 2.0%
(w/v) concentration of sodium alginate and 48 hours
hydrolysis time.
248
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Proceedings of the
2016 Annual Conference on Sustainable Research and Innovation,
4 - 6 May 2016
The recycled enzyme system, when used in multiple
hydrolysis batches, maintained high hydrolysis yields.
The results indicate that with up to ten times, the yield of
glucose was reasonably high at 57.6% (w/w) which was
an indication that the immobilized enzyme system was
capable of being recycled with high hydrolysis efficiency
for up to 10 times.
The ability to recycle the immobilized enzymes presents
an opportunity of reducing the cost of enzymes that are
used in hydrolysis of LGB.
The hydrolysis enzymes were successfully immobilized
on glutaraldehyde and calcium alginate and showed
better thermal stability and increased tolerance to pH of
up to 6.0 in addition to resisting mechanical degradation
during washing and reuse.
Prosopis juliflora stem is a suitable substrate for the
production of simple sugars through hydrolysis using
immobilized enzymes.
The quantity of glucose produced from Prosopis juliflora
stem is of sufficient quantity which can be used in the
production of bioethanol and marketable products such
as citric acid and lactic acid.
Since Prosopis juliflora stem is a cheap renewable
resource, this research study is useful in the conversion
and utilization of this renewable LGB into marketable
products such as citric acid and lactic acid which will
result into good economic, environmental and social
significance.
ACKNOWLEDGEMENT
The authors acknowledge the financial support provided by
Moi University, and the staff of Moi University, Chemical and
Process laboratory for their support and guidance during the
course of experimental work.
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