You are on page 1of 9

EFFECT OF SUBSTRATE CONCENTRATIONS AND pH ON THE

XYLOSE AND GLUCOSE CONCENTRATIONS ON HYDROLYSIS OF


OIL PALM (Elaeis guineensis) BUNCHES
Asti Arya, Mahfud Ainun Najib, Prima Ardini Pratiwi, Putri Nabila Adinda Adriansyah *
Department of Food Industrial Technology, Faculty of Agriculture Industrial Technology,
Padjadjaran University, Jatinangor. Email putrinabila_adriansyah@yahoo.co.id
Abstract
Oil palm contains cellulose, hemicellulose, and lignin. Based on the composition of oil
palm bunch, hemicellulose is a material with huge potential to be used in a very large
amount. Before utilized, hemicellulose must first be broken down into monomers with the
process of hydrolysis using enyzme. Enzyme has a very high specificity towards their
substrates. The action of the enzyme is influenced by pH. Not only at the enzyme, pH levels
can also affect the nature of the charge and shape of the substrate. Result of maximum
concentration of xylose from this study contained up to 29.823 g/L obtained from enzymatic
hydrolysis of substrate concentration of palm bunches at 15 g/L with a pH = 6. The optimum
conditions for obtaining the highest xylose concentration of research enzymatic hydrolysis of
the amount of substrate concentration and pH is the substrate concentration of 13,875 g / L
and pH value of 5,625, resulting xylose concentration up to 30,1934 g/L. Maximum cellulose
yield (72, 2062 g/L) was reached at pH of 6 and substrate concentration of 15 g/L. From the
solutions of optimization of this model, The best pH values obtained at 5 and best substrate
concentration at 10 g / L. This is consistent with the data that the enzyme cellic actual CTEC
has the optimum pH 5-5.5.
Keywords : Xylose, Glucose, pH, substrate, concentration, oil palm bunches
1. Introductions
Oil Palm is a plant that is easy to
cultivate in tropical areas such as
Indonesia. Palm oil waste consists of oil
palm bunches, wet solids, shells, fibers,
liquid waste and water condensate with
the percentage of each 23%, 4%, 6.5%,
13%, 50% and 3.5 % (Direktorat PHP.,
2006). Oil palm bunches is an organic
material containing; 42.8% C, 2.90% K 2O,
0.80% N, 0.22% P2O5, 0.30% MgO and
microelements include B 10 ppm, 23 ppm
Cu and 51 ppm Zn. In every 1 ton of oil
palm bunch contain nutrients equivalent to
3 kg of urea, 0.6 kg of RP, 12 kg and 2 kg
MOP kiserit. The content of oil palm bunch
include cellulose, hemicellulose, lignin,
ash, and water content
Cellulose
Cellulose is a polysaccharide
consisting of one type of monomer (homo
polysaccharides). Monomers of cellulose
are units of D-glucopyranose which binds
-1,4 glycosidic (Sjostrom, 1995). This

material can be found on the microfibril


plants. Properties owned by cellulose is
insoluble in water, but dissolved in a
solution of copper hydroxide ammonia and
ZnCl2 solution. Cellulose can be divided
into three parts, -cellulose, --cellulose
and cellulose. -cellulose is insoluble in
strong alkaline solution (NaOH). cellulose can be dissolved in an alkaline
medium and precipitate if the solution is
neutralized, while the -cellulose is a part
of cellulose which is soluble in alkaline
solution and in a fixed form when
neutralized (Fengel and Wegener, 1995).
Hemicellulose
Hemicellulose
consists
of
heterogeneous polysaccharides monomer.
Hemicellulose constituent monomers are
D-glucose, D-mannose, L-arabinose and
D-xylose (Sukarta, 2008). Hemicellulose is
one of the constituent components oil
palm bunches are also abundant in the
cell walls of plants. Xylan is hemicellulose
composed of -Dxilopiranosa. In general,

it is classified as xylan, hemicellulose as it


is obtained through the extraction from
hemicellulose and xylan is the primary
component of the hemicellulose (Whistler,
1950). Xylan is found in the cell walls of
plants, which is about 30-35% of the total
dry weight (Joseleau et al, 1992).
According Sjostrom (1995), xylan is a
polymer of xylose, which binds -1,4xilopiranosa the number of monomer 150
to 200 units. Xylan has a branched chain
and not a crystalline structure that is more
easily accessible than the cellulose
solvent. The original structure of xylan is
very complex and can be substituted with
an acetyl group, L-arabinofuranosil and
glukoronosil as a side chain (Whistler,
1950).
Lignin
Lignin is a three-dimensional
polymer consisting of phenyl propane
units which are tied with ether bonds
(COC) and carbon bond (CC). Lignin is
resistant to hydrolysis because of its bond
with bond arilalkil ether (Judoamidjojo et
al, 1989), and does not dissolve in water,
acid solution and hydrocarbon solvent.
Lignin reactivity is influenced by functional
groups contained in the polymer lignin
itself. Methoxyl group-containing polymer
lignin, phenolic hydroxyl groups and some
aldehyde group on the side chain.
Functional groups which greatly affect the
reactivity of lignin is a phenolic hydroxyl
group and a carbonyl group.
Based on the composition of oil
palm bunch, hemicellulose is a material
with huge potential to be used in a very
large
amount.
Before
utilized,
hemicellulose must first be broken down
into monomers, and one way is to use the
process of hydrolysis. Hydrolysis is the
breakdown of complex molecules into
smaller molecules with the aid of acids or
enzymes as catalysts. An example of the
hydrolysis
process
is
breaking
hemicellulose into xylose and glucose
either by using acids or enzymes.
Enzyme is a protein that serves as
biocatalyst of biochemical reactions in
biological beings. Substances that are
described by the reaction are called
substrates, and the newly formed
subtance from the reaction are called

products. Enzyme has a very high


specificity towards their substrates, and
enzyme accelerates specific chemical
reactions without the formation of
byproducts. Enzyme plays a very
important in metabolic reactions. The
reaction of complex chemical reactions in
the body will be very slow without
enzymes. These enzymes work in an
appropriate environment.
The action of the enzyme is
influenced by pH. The optimal pH of
enzyme is around pH 7 (neutral), and if
the medium becomes highly acidic or
highly alkaline enzyme will become
inactivated (Gaman & Sherrington, 1994).
When the pH changes in certain media, it
leads to a change in the form of enzyme.
The structure of enzymes and amino acid
changes in the enzyme active site, there
by blocking the active site in combination
with a substrate.
Not only at the enzyme, pH levels
can also affect the nature of the charge
and shape of the substrate. In a narrow
pH range, structural changes in the form
of the enzyme and substrate may be
reversible. But for a significant change in
the levels of pH, enzymes and substrates
can undergo denaturation. In such cases,
they cannot identify with one another. As a
result, there will be no such reaction. This
is the reason why, pH affects enzyme
activity. An increase or decrease in pH
changes the ion concentration in the
solution. Therefore, the media should be
completely maintained by using a buffer to
prevent the change in ions.
2. Materials and Methods
The analysis begins by preparing a
number of materials such as oil palm
bunches, enzymes cellic CTEC, acetate
buffer solution, distilled water and a few
tools such as erlenmeyer, pipette volume.
After having prepared the tools
and materials to be used, oil palm
bunches were weighed at 3 g / L, 5 g / L,
10 g / L, 15 g / L and 17 g / L and added
buffer solution with pH 3.4, 4.0, 5.0, 6.0
and 6.2 respectively in accordance with
the order of the design program. The
solution is heated in an autoclave for
about 2 hours to hydrolyze the
hemicellulose components contained in

the oil palm bunches. The solution is


chilled down until it reaches room
temperature, so that when added to the
enzyme, the enzyme will not going to be
inactive. Enzymes are added at 1.5 mL
each erlenmeyer. Erlenmeyer contained
samples were kept in an incubator shaker
at 45oC for 45 hours.
The solution was poured on the
container for centrifuge. Centrifuge
separation performed at 6000 rpm for 5
minutes. Supernatant was separated from
the solids and 5 ml per erlenmeyer taken
for
analysis
using
absorbance
spectrophotometry at 540 nm. The
spectrophotometric absorbance obtained
is used to calculate the concentration of
xylose and glucose were obtained by
using a standard curve of each
compound. Concentration obtained in the
analysis using Design Expert program to
obtain
the
optimum
substrate
concentration and pH to get the desired
concentration xylose and glucose.
3. Results and Discussions
There are several factors that
affect the hydrolysis of cellulose, they are
the type of substrate, temperature, time,
pH, substrate concentration, enzyme
utilization and the type of reactants, many
of these factors are interdependent which
making the whole process become
complete
(Linko,
1975),
Cellulose
metabolic rate can be regulated by
multiple influences, namely environmental,
physical and chemical characteristics of
soil that have different cellulolytic capacity.
In this research, enzymatic hydrolysis with
independent variable parameters such as
substrate concentration of palm bunches
and pH values is measured.
Before testing the enzymatic
hydrolysis of the substrate and the
influence of pH, the research result should
be designed by design expert application
and obtained a wide range of pH values
as a factor 1 and a diverse concentration
of substrate which is used as a factor 2.
Response surface method is used to
determine the optimum value from
research conducted in addition to enhance
the value of diversity and the data are
arranged
randomly.
The
sampling
conducted
at
the
45
hour,

spectrophotometry is used to determine


absorbance values for each sample. From
the results of the absorbance values,
concentration of xylose and glucose can
be measured with the standard equation
of each component.
Xylose Concentration
Table 1. Result of Xylose Concentration
with independent variable concentration
substrat of oil palm bunches and pH
value.
Xylose
concentrati
Concentrati
Run pH
on
on
(g/L)
(g/L)
1
5
10
28,5680
2
5
10
29,1757
3
5
17
29,2946
4
6
15
29,8230
5
3,4
10
15,4373
6
5
17
29,6645
7
4
15
27,0885
8
6,2
10
29,2021
9
6
5
27,1678
10
3,4
10
19,0172
11
5
3
26,5601
12
4
5
16,3487
13
4
15
26,1638
14
5
10
29,1757
15
6
5
27,1678
16
6
15
28,8322
17
5
10
28,3170
18
5
10
27,9868
19
5
3
26,6658
20
6,2
10
28,8322
21
4
5
19,1229
Enzymatic hydrolysis observations
result with independent variables such as
the amount of substrate and pH were
analyzed using Design Expert application
and obtained results that a large number
of the substrate concentration affect the
xylose concentration, results obtained by
ANOVA which showed significant outcome
data. According to Salle (1974), the higher
the concentration of substrate may
increase or reduce the speed of an
enzymatic reaction, if the concentration of
the substrate is considerably less than the

amount of enzyme then an increase in the


content of substrates will increase the
reaction speed. The rate of enzyme
activity
increases
with
increasing
substrate concentration until a certain
point. Once the enzyme is saturated with
substrate, the addition of substrate level
will have no effect on the speed of the
reaction. The lower levels of the substrate
that will generate the maximum activity,
which is saturating the enzyme, the
greater the reaction between the enzyme
with its substrate (Volk and Wheeler,
1986).
Analysis of the pH showed that
different pH values affect the xylose
concentration, results obtained by ANOVA
which showed significant outcome data.
Enzymes are proteins composed of amino
acids, therefore, the effect of pH is closely
related to the acid-base properties owned
by the protein. Generally, the enzyme
showed activity optimum point at a
particular pH (Martoharsono, 1993).
Volk and Wheeler (1988) states
that the concentration of hydrogen ions, ie
the acidity or alkalinity of a solution greatly
affects the activity of an enzyme. This is
because the amino acids that constitute
the active center of the enzyme must be in
a proper state of ionization in order to
become active. Each enzyme has a pH
maximum, optimum and minimum. The pH
values vary with the temperature, the type
and concentration of substrate, type of
buffer solution (buffer), the presence or
absence of compound inhibitor or activator
and the enzyme needed time to work
(Salle, 1974).

A regression model exhibits lack-of-fit


when it fails to adequately describe the
functional relationship between the
experimental factors and the response
variable. Lack-of-fit can occur if important
terms from the model such as interactions
or quadratic terms are not included. It can
also occur if several, unusually large
residuals result from fitting the model.
Result of lack of fit in this research shows
that P-value < or the model does not fit
the data. The "Lack of Fit F-value" of 5.77
implies the Lack of Fit is significant. There
is only a 1.11% chance that a "Lack of Fit
F-value" this large could occur due to
noise. this is caused by a number of
disturbances in the research process.

Figure 1. Contour graph of Xylose


Concentration

Glucose Concentration
Hydrolysis is the breakdown of
complex compounds into simpler ones
such as the breakdown of the

Figure 2. 3-D surface graph of Xylose


Concentration shows result the maximum
concentration of Xylose
Figure 1 and 2 show result of xylose
production with independent variable
substrate concentation and pH, figure 1
shows contour graph and figure 2 shows
3-D surface. Result of maximum
concentration of xylose from this study
contained up to 29.823 g/L obtained from
enzymatic
hydrolysis
of
substrate
concentration of palm bunches at 15 g/L
with a pH = 6. Based on the analysis of
response surface method in the Design
Expert, the optimum conditions for
obtaining the highest xylose concentration
of research enzymatic hydrolysis of the
amount of substrate concentration and pH
is the substrate concentration of 13,875
g / L and pH value of 5,625, resulting
xylose concentration up to 30,1934 g/L.

polysaccharide compound in the oil palm


bunches, cellulose and hemicellulose
hydrolysis to glucose and xylose . This
process can occur chemically or
enzymatically. This research hydrolysis
process is carried out with the help of
enzymes cellic ctec .
Glucose can be made as a product
of cellulose hydrolysis.
Hydrolyzed
cellulose will produce a simple sugar
called glucose accompanied by the
formation of water. The primary goal of
hydrolysis is to convert as much cellulose
as possible into monomeric sugars as
quickly as possible, which is achieved by
adding celluloses and hemicelluloses. A
high cellulose conversion can be achieved
by increasing either the enzyme dosage or
the hydrolysis time (Novozymes, A/S,
2010)

research model . Significant lack of fit


which means there are quite a lot of noise
on the results of this research
A regression model exhibits lackof-fit when it fails to adequately describe
the functional relationship between the
experimental factors and the response
variable. Lack-of-fit can occur if important
terms from the model such as interactions
or quadratic terms are not included. It can
also occur if several, unusually large
residuals result from fitting the model.
In the calculation of this study
ANOVA get the value of R - squared of
0.9203 (92 %) indicating that the study
results is quite good as r - squared has a
high value and has more than 75 % value.
After the results were compared
with models that have predicted , the
model still not in a very good result
because it has not approached the curve
line prediction . Predictions and actual
curve relationship can be seen in the
image below .

Figure 3. Cellulose Hydrolysis Process


(Held, 2012)
In the case of this glucose
analysis, quadratic model in were used to
study the effects of pH and substrate
concentration that applied to palm
bunches fermentation with cellic ctec
enzyme. Quadratic model were used
because on analysis using expert design
software 9.0.5 results it was concluded
that the quadratic form is the form most
appropriate for this study
After the hydrolysis process, the
data included the results of the
concentration of glucose inserted to
ANOVA calculation . In the ANOVA
calculation, we got the information that
this research model is significant as the
calculation results of the research, but
also there was found a significant lack of
fit . Lack of fit indicates the number of
interference (noise) contained in this

Figure. 4. Predicted VS Actual Curve


The red line is the predicted curve
while the colored dots are the result of
research. As we can see the results
showed that the predicted glucose
concentration incompatible with the
results. Results of the study did not follow
the predicted curve so that the results
have not been good.

Linear or not, the value of this


assay is in its ability to predict glucose
content with the desired accuracy. The
impact of the form of the standard curve
on accuracy become evident with
evaluation of predicted vs actual curve.
The standard curves were used to predict
the glucose concentrations of standards
using the measured absorbance values of
the standard that have been used to
generate the curve.
Results
of
treatment
were
analyzed by using response surface
method (RSM) . The yield of hemicellulose
and cellulose after treatment was used to
calculate the efficiency of treatment.
Quadratic models were developed to
study the effect of pH and substrate
concentration on fermentable sugar yield
on both cellulose and hemicellulose.

Figure 5. Respone surface Method for the


cellulose yield
Figure
5
shows
a
three
dimensional display of response surface
for the yield of cellulose (glucose) after
hydrolysis
treatment,
respectively.
Maximum cellulose yield (72, 2062 g/L)
was reached at pH of 6 and substrate
concentration of 15 g/L. From the
solutions of optimization of this model,
The best pH values obtained at 5 and best
substrate concentration at 10 g / L. This is
consistent with the data that the enzyme
cellic actual CTEC has the optimum pH 55.5 .
Results stated that the rise of pH ,
the concentration of glucose tends to

increased while the higher concentration


of the substrate is not necessarily
increased the glucose concentration. This
was consistent with Michaelis - Menten
theory which states that the higher the
substrate is not necessarily the higher its
product. If the substrate is too high, it may
become the inhibition of the enzyme while
the pH relationship with enzyme is
dependent upon each of the pH optimum
of each enzyme. A change in pH can alter
the rates of enzyme catalyzed reactions,
with many enzymes exhibiting a bellshaped curved when enzyme activity is
plotted against pH. Changes in pH can
alter the following :
1 The ionization state of the
substrate or the enzyme binding
site for substrate
2 The ionization state at the catalytic
site on the enzyme
3 Protein molecules so that their
conformation and catalytic activity
change
Table 2. Relation Between pH, Substrate
Concentration,
and
Glucose
Concentration
pH
Substrate
Glucose
Concentrati concentratio
on (g/L)
n (g/L)
5
2.92893
64.4875
5
2.92893
64.7375
6
5
64.675
4
5
40.3312
6
5
65.925
4
5
46.8937
5
10
69.2375
5
10
68.675
3.58579
10
38.175
6.41421
10
70.7375
3.58579
10
46.6437
5
10
70.675
5
10
68.6437
5
10
67.8625
6.41421
10
69.8625
6
15
72.2062
4
15
65.7375
4
15
63.55
6
15
69.8625
5
17.0711
70.9562
5

17.0711

71.8312

4. Conclusion

Held, P. 2012. Optimization of Polymer


Digestion and Glucose Production in
Microplates. Available online at
http://www.biotek.pt.

Result of maximum concentration


of xylose from this study contained up to
29.823 g/L obtained from enzymatic
hydrolysis of substrate concentration of
palm bunches at 15 g/L with a pH = 6.
The optimum conditions for
obtaining the highest xylose concentration
of research enzymatic hydrolysis of the
amount of substrate concentration and pH
is the substrate concentration of 13,875
g / L and pH value of 5,625, resulting
xylose concentration up to 30,1934 g/L
Maximum cellulose yield (72, 2062
g/L) was reached at pH of 6 and substrate
concentration of 15 g/L. From the
solutions of optimization of this model,
The best pH values obtained at 5 and best
substrate concentration at 10 g / L. This is
consistent with the data that the enzyme
cellic actual CTEC has the optimum pH 55.5 .

Judoamidjojo, R.M, E.G. Said, L. Hartoto.


1989.
Biokonversi.
Depdikbud.
Dirjen Pendidikan Tinggi. Pusat
Antar Universitas Bioteknologi, IPB,
Bogor

5. References

Salle, A.J. 1974. Fundamental Principles


of Bacteriology. Tata Mc Graw Hill.
New Delhi.

Anonim. Michaelis-Menten kinetic theory


of enzyme action Available online
at attic.volgmed.ru
Corredor, D. Y., Bean, S., Wang, D.2007.
Pretreatment
and
Enzymatic
Hydrolysis
of
Soghum
Bran.
Proquests Agriculture
Journals.
84,1:61-66.
Direktorat Pengolahan Hasil Pertanian.
2006.
"Pedoman
Pengelolaan
Limbah Industri Kelapa Sawit".
Jakarta: Ditjen PPHP.
Fengel, D. dan Wegener. 1995. Wood:
Chemistry, Ultrastructure, Reactions.
Terjemahan S. Hardjono. UGM.
Press, Yogyakarta.
Gaman, p.m & K.B. Sherrington. (1994).
Food Science, Introduction to Food
Science, Nutrition and Microbiology.
Gadjah Mada University press.

Linko, M. 1975. An Evaluation of


Enzymatic Hydrolysis of Cellulosic
Materials.
Technical
Research
Centre of Finland. Biotechnical
Laboratory. Helsinki. Finland. p: 2745.
Martoharsono,
Soeharsono.
1993.
Biokimia Jilid II. Gajah Mada
University Press. Yogyakarta.
Novozymes A/S. 2010. Cellic CTec2 and
HTec2 - Enzymes for hydrolysis of
lignocellulosic materials. Available
online at www.scienceplease.com.

Sjostrom, E. 1995. Wood Chemistry. Jilid


II. Diterjemahkan oleh Hardjono S.
UGM Press, Yogyakarta.
Sjostrom, E. 1995. Wood Chemistry. Jilid
II. Diterjemahkan oleh Hardjono S.
UGM Press, Yogyakarta.
Sukarta, I Nyoman. 2008. ADSORPSI
ION
Cr3+
OLEH
SERBUK
GERGAJI KAYU ALBIZIA(Albizzia
falcata):
Studi
Pengembangan
Bahan Alternatif Penjerap Limbah
Logam
Berat.
http://damandiri.or.id/file/nyomansuk
artaipbbab2.pdf
Volk, W.A, : Wheeler, M.F, 1986, Basis
Microbiology, 6th edn., Harper &
Row, New York
Whistler, R.L. 1950. Xylan. Di dalam
Hudson, C.S. dan Sidney (eds).
Advances
in
Carbohydrate

Chemistry.

Volume

V.

General

Polysaccharides. Academic Press,


New York.