EFFECT OF SUBSTRATE CONCENTRATION, LENGTH OF INCUBATION AND pH IN

THE PROCESS OF ISOLATION MEDIA xylanase USING RICE STRAW

Albar Budiman, Sigit Setyawan
Advisor: Dr.Ir.Abdullah, MS
Department of Chemical Engineering, Faculty of Engineering of Diponegoro University
Jln.Prof.Soedarto, Tembalang, Semarang, 50239, Tel / Fax: (024) 7460058
Abstract
In a large number of straw deaerah in Indonesia is still considered as waste and in the end only be
burned away without any further use, while Indonesia as an agricultural country is producing a very
large straw with a total 230 million tons of straw per year.So far still limited use of straw as fodder and
fuel for household cooking, other than that no other uses that can optimally utilize the content of rice
straw.This study aims to utilize waste rice straw as raw material to produce xylanase enzyme.xylanase
enzyme in research using fungal strains of Aspergillus Niger.This research is divided into three stages,
namely preparation, enzyme production phase and testing phase.At this stage of preparation consisted
of cultivating strains and preparation of fermentation media consisted of mineral salt solution and
yeast extract as a source of organic nitrogen.In this fermentation medium substrate concentration
variation is 1%, 2%, 3% and 4% and the variation of incubation 2, 3, 4, 5 and 6 days.Then do the stage
production of the enzyme with the method of solid state Fermentation.And then conducted tests on the
substrate for the enzyme activity that has been produced.Now we know the best substrate
concentration of study followed by a variable different pH ie pH 5, 5.5, 6 and 6.5 with enzyme
production and poses the same test as sebelumnya.Berdasarkan research studies have been conducted,
it is known that each medium with a substrate concentrationdifferent incubation time have the same
optimum on day 4, while for the best substrate concentration to produce xylanase activity is at a
concentration of 1% substrate with the enzyme activity at 15:14 UI / ml at day 4 of incubation.As for
the pH optimum is obtained at pH 6 with 4-day incubation period with the enzyme activity at 15:33 UI
/ ml.
Key words: xylanase, Aspergillus Niger, rice straw, enzyme isolation.
Abstract
The purpose of this research is to Produce xylanase is degrading the which the agent of the component
of hemicelluloses.Hemicelluloses just like an adhesive Between lignin and cellulose.Then, due to
Degradation hemicelluloses, lignin cans be released Easily with small Addition of chlorine or even
without any Addition of it.Mold strains of Aspergillus Niger is Used to Produce xylanase.This research
is divided into three steps.First, preparation step consist of strains cultivation and preparation of
Fermentation medium.Fermentation medium contains of mineral salts and yeast extract as a source of
organic nitrogen.In this medium, Variations of substrate concentration Nowhere is 1%, 2%, 3% and
4%, and time of Incubations are conducted in 2,3,4,5 and 6 days.Second, enzyme production step by
Solid State Fermentation method.The last is a test step to calculate enzyme activities.After We knows
the optimum of substrate concentration continuing the research with different pH of the medium with
Same
variation of time of Incubation.This research for this second variation of pH is Same like the research
before.
Based on result of the research, it cans be concluded That EACH medium with different substrate
concentration have a different result in enzyme activity, the highest value of this variable is medium
with 1% substrate concentration and Incubation in 4 days, WHERE it's have 15:14 UI/ ml of enzyme
activity.For the variables of pH, pH optimum to Produce xylanase enzyme is pH 6 with 4 day
Incubation and the result of enzyme activity is 15:33 UI / ml.
Keywords: xylanase, Aspergillus Niger, Rice straw, immobilized enzyme.
INTRODUCTION
In a large number of straw deaerah in Indonesia is still considered as waste and in the end only be
burned away without any further use, while Indonesia as an agricultural country is producing a very
large straw with a total 230 million tons of straw per year.So far, the utilization of straw is still limited
as fodder and fuel for household cooking, other than that no other uses that can optimally utilize the
content of rice straw.Waste rice straw as the old plants, lignified cell wall has continued to form a
 complex bond, including cellulose and hemicellulose.Utilization of waste berlignoselulosa with
services mengguanakan microorganisms to produce enzymes that can degrade ekstrseluler
berligneselulosa material into its constituent fractions.And one of them is to use rice straw for
production of xylanase enzyme.
As Rice Straw Waste Berlignoselulose
The development and progress in agriculture has led to an increase INDONESIA agricultural waste
which is largely a waste berlignoselulosa.Waste INDONESIA berlignoselulosa high potential such as
straw.In a large number of straw deaerah in Indonesia is still considered as waste and in the end only
be burned away without any further use, while Indonesia as an agricultural country is producing a very
large straw with a total 230 million tons of straw per year.So far, the utilization of straw is still limited
as fodder and fuel for household cooking, other than that no other uses that can optimally utilize the
content of rice straw.Waste rice straw as the old plants, lignified cell wall has continued to form a
complex bond, including cellulose and hemicellulose.Utilization of waste berlignoselulosa with
services mengguanakan microorganisms to produce enzymes that can degrade ekstrseluler
berligneselulosa material into its constituent fractions.
Substrates used in fermentation processes affect enzyme activity and productivity.The existence of a
specific substrate in the medium to stimulate the production of microorganisms for metabolites
secreting cells.The main nutrients for growth of microorganisms is a source of carbon, nitrogen, and
minerals, especially phosphate component.Formulation of the media in the growth and production of
fermentation is an important step in designing experiments in the scale of work (Stanbury and
Whitaker, 1984).
C5H8O4 + H2O C → 5H10O5
Xylan Xilose
OR
R
CH
CH
CH
OH
O
CH
CH
OH
OH
CH
CH
CH
OH
OH
O
CH
CH
+
H-OH
OH
Xylan
Xilose
Pictured above is the hydrolysis reaction of xylan carbon sources commonly used are molasses,
cereals, starch, glucose, sucrose and lactose.Production of xylanase enzymes are xylan as carbon
source.Xylan with xylanase activity produced by microorganisms will be hydrolyzed into
xylose.Xylan hemicellulose is xylose polymer which binds β-1, 4, with the number of monomer units
150-200 (Sunna and Antraniklan, 1997).Xylan chain branching and its structure is formed crystals so
that more solvent accessible compared with cellulose.Most of the xylan consists of 2-4
 heteroglikan.Heteroglikan a common is-Dxilan arabino, L-arabino-D-glukurono-Dxilan, 4-o-methyl-
D-glukorono-Dxilan, L-arabino-D-xylan, D-gluko-Dmannan, D-D-galaktogluko-Dmannan-, and L-
arabino-D-galaktan.Use of xylan in large-scale xylanase production was too expensive.Park et al.
(1992)
Production of xylanase enzyme from microorganisms.
Types of microorganisms that produce xylanase is already common fungus and bacteria.Examples of
some endoxilanase producing microorganisms are presented in Table 2.7.Several types of bacteria and
fungi
Known to be capable of producing extracellular xylanase.Xylanase from Clostridium acetobuty-licum
has been investigated by Lee et al.(1985), namely from 20 strains Clostri-dium sp.was
C.acetobutylicum NRRL B527 and ATCC 824 produced the highest xylanase.Strain NRRL B527
produced xylanase at pH 5.2, whereas strain ATCC 824 to produce xylanase, xilopiranosidase, and
arabinofuranosidase in the culture of anaerobic.Bacillus sp.xylanase-producing nature alkalofilik that
have been studied are Bacillus sp.YC 335 (Park et al., 1992), Bacillus sp.41m-1 (Nakamura et al.,
1993), and Bacillus sp.TAR-1, which also are thermophilic (Nakamura et al., 1994).Kubata et al.
(1992) have isolated Aeromonascaviae ME-1 producer of xylanase I from herbivorous insect gut,
while Dung et al.(1993) conducted a study β-1 ,4-xylanase 2 and 3 of A.caviae W-61.Irrawaddy (1992)
managed to produce cellulase and xylanase from Neurospora sitophila on substrates oil palm solid
waste.Richana et al.(2000) has conducted alkalofilik xylanase-producing bacterium isolated from
calcareous soil pH 7.9.
In producing enzymes from microorganisms, the important thing to do is start using the most active
strains of microorganisms are available.A strain selection program should be done by taking the
culture from nature or culture collections, and conduct tests of enzyme activity.The main requirements
for selection is the ease of methodology, so that a quick test for a large number of strains can be done.
Type of microorganism that is commonly produced xylanase is from a class of fungi and
bacteria.Although the enzyme produced by a group of bacteria have resistance at higher temperatures
than the mold, but the activity of xylanase from a class of fungi is much higher than bacteria.In
addition, high production levels and the ease of making mushrooms cultivikasi more widely used in
industrial scale enzyme production (Bergquist et al, 2002).
The type of fungi that have the potential to produce xylanase enzyme that is fungus Aspergillus niger
and Trichoderma ressei.
Aspergillus niger is a mold of the class fungi imperfecti, scattered everywhere on a variety of
substrates, among others, present in fruits, vegetables and other foods that have been rotten.These
fungi play a role in the decomposition of polysaccharides in the wood, has a growth temperature of
300C - 370C, pH: 4-6 and aerobic.
According to an overview A.niger classified as follows:
Division: Fungi imperfecti
Sub-class: Hyphomyces
Order: Monoliales
Family: Monoleaceae
Genus: Aspergillus
Species: Niger
(Dwijoseputro, 1984)
Utilization lanase On Paper Making Process
In papermaking, the xylanase is used to remove the hemicellulose in the bleaching process.This
enzyme as a substitute way of chemicals so toxic chemical waste contamination will be avoided and
less expensive
(Ruiz Arribas et al., 1995).
Paper-making raw materials after the digester and the washing process, actually still in a dirty (low
degree of whiteness).To produce high-quality paper bleaching process needs to be done.Bleaching
process aims to remove lignin, hemicellulose causes brown color and contained extractive substances
from the washing and filtering.Bleaching process is usually done gradually, because it has the
advantage of which is the value of a high degree of whiteness.This gradual process consists of
chlorination stage, extraction, and the addition of chlorine dioxide.Chlorine is a toxic material, so that
the chlorine residual is discharged into waters of the river will make the biggest pollution
tinggi.Ternyata pollution in our country is the pollution from paper mills.Replacement of the use of
chlorine for bleaching paper has provided opportunities for biotechnology applications.Xylanase is an
enzyme that was first reported to the bleaching of paper and now has been used in several paper mills
(Bourbonnais et al., 1997; Viikari et al., 1991; 1994; Coughlan and Hazlewood, 1993).
The number of paper mills are already operating in Indonesia is currently more than 14 companies and
has not a single one using enzymatic processes in the bleaching process.Thus, to support the
preservation of the environment it needs to be applied to environmentally friendly process (clean
processing) in Indonesia.For the paper making process is expected xylanase used was a thermostable
and resistant to alkaline pH (Nakamura et al., 1993)
Type enzyme is endoxilanase
(Kantelinen et al., 1988; Paice et al., 1988; Viikari et al., 1994).
However, the combination of other xylanolitic and hemiselulolitik with endoxilanase has been shown
effective in improving the quality of the paper.The use of enzymes xylanase and the like in the paper
bleaching process helps reduce the amount of kappa and meningkatkanderajat white paper.A number
of studies the effect of xylanase on bleaching of paper carried out by enzymes derived from
Trichoderma sp.and found reduced use of chlorine tencapai 20-30%
(Viikari et al., 1991; 1994).
Utilization of Xylanase As Sugar xylose
Xylanase can also be used to hydrolyze xylan (hemicellulose) into sugar xylose.Xylan many derived
from agricultural waste and food industry.The development process of hydrolysis
enzymatically is a new prospect for the handling of waste hemicellulose (Biely, 1985; Rani and Nand,
1996; Beg et al., 2001).
Xylose sugar consumption is widely used for diabetics.In Malaysia, a lot of sugar xylose is used to
mix because toothpaste can serve to strengthen the gums.With the diversity of sugar xylose keguna
there needs
xylose production innovation toward them when they appear tersebut.Inovasi penghidro-lysis
lignocellulosic enzymes are already available.Sometimes to process the sugar xylose is not desirable
because it is less economical to remember the content of xylan is very low compared with
cellulose.However, consideration should be to make the process so the results are not only
multienzyme xylose only (from xylan), but also glucose (from cellulose and other oligo
saccharide).While these new technologies such as membrane technology, which can separate the
components according to molecular size and molecular weight fractionation, it can be done easily
glucose and xylose.
Utilization of Xylanase for Animal Nutrition
Van Paridon et al.(1992) has researched the use of xylanase to a mixture of chicken feed the boiler, by
looking at the impact on the achieved weight and food conversion efficiency and its relationship with
intestinal viscosity.The same thing is done by Bedford and Classen (1992), who reported that a
mixture of chicken feed the boiler with a xylanase derived from T.longibrachiatum was able to reduce
the viscosity of digestion, thereby increasing the achievement of weight and food conversion
efficiency.
Utilization of Xylanase for Food and Beverage
Xylanase can also be used to clarify juice, coffee extraction, vegetable oil, and starch (Wong and
Saddler, 1993).The combination with cellulase and pektinase can for juice purification and
liquefaction of fruit and vegetables (Beg et al., 2001).
The efficiency of xylanase on bread quality improvements that have been done, namely xylanase
derived from Aspergillus niger var awamori is added to bread dough result in increase in specific
volume of bread and to further improve the quality of bread, therefore the combination of the addition
of amylase and xylanase (Maatet al., 1992). Although the potential use of xylanase enzyme to produce
quite diverse but also still faces several obstacles, including lack of superior strains of microorganisms
and the lack of knowledge about teknologiproduksi enzyme.On the other hand, experts from
developed countries acknowledged that countries rich in biodiversity, including Indonesia, is a source
 of microorganisms and the potential for bioprocess plants (Fox, 1994).
Seeing the potential berlignoselulosa abundant waste material, as well as wealth of biodiversity of
microorganisms in Indonesia, it is necessary to innovate in the direction of enzyme industry.A very
diverse user xylanase is produced in Indonesia if a xylanase-producing strains of microorganisms
superior and master the production technology.
RESEARCH METHOD
In the research on the influence of substrate concentration, pH and time of incubation and the variation
is in the process of isolation of xylanase Media Using Rice Straw.This research is divided into three
phases, namely, preparation phase, the enzyme production phase and testing phase.
The variables used are as follows
• The type of microorganism Aspergillus Niger
• The medium used rice straw
While the variable is Changing
• Concentration of substrate: 1%, 2%, 3% and 4%
• Long Incubation is 2, 3, 4, 5 and 6 days
• pH of fermentation medium: 5, 5.5, 6, 6.5
The tool used in this study are:
1.Beaker glass
2.Centrifuge
3.Erlenmeyer
4.Glass Measure
5.Autoclafe
6.Hot Plate and Stirer
7.Ose Wire
8.Measuring pipette
9.Eye dropper
10.Incubator Shaker
11.Spectrophotometer
12.Test tube
13.Scale
At this stage of preparation is done breeding fungus Aspergillus Niger on media Potato Dextrose Agar
(PDA) and then was processed substrate for fungal growth and production fermentation media.At the
stage poduksi enzymes, fungi that have been bred that would be inoculated on Aspergillus Niger
fermentation medium with substrate concentrations varying namely 1%, 2%, 3%, 4%, and then
incubated for 2, 3, 4, 5 and 6days.At this stage of the test, the enzyme that has been obtained from
enzyme production phase will be tested levels of protein and enzyme activity, after a known
concentration of the best substrate for xylanase enzyme production, the study continued with a
different medium pH of 5, 5.5, 6, and 6.5 with steps andthe same test as the previous research.
RESULTS AND DISCUSSION
Effect of Incubation Terahadap Old xylanase enzyme activity.
Xylanase activity expressed how much the ability of the enzyme xylanase in outline or convert xylan
into its products that is xylose..This activity is measured in International Units (IU), based on the
number micromol xylose liberated per minute in test conditions.The method used is a method of DNS
(Miller, 1959).
The test is performed on each enzyme is produced on a variety of long incubation time.From this test
to know the influence of incubation time on xylanase activity.Graph showing the relationship between
incubation time on xylanase activity is shown in graph 4.1.
0.002.004.006.008.0010.0012.0014.0016.0001234567Lama Incubation (Days) Enzyme Activity (U /
ml) 1% Substrate constructive constructive constructive Substrate Substrate 2% 3% 4% Substrate
constructive
Graph 4.1 Effect of incubation duration on xylanase enzyme activity
From the graph 4.1 is known that for each substrate concentration proved to have an optimal
incubation time is almost equal or even the same can be said that on the fourth day, on substrate
 concentration of 1% indicates that there tersebesar enzyme activity on the fourth day was also on
substrate concentration of 2%, 3% and 4%.Substrate concentration on the medium with 1% for the
second day of incubation until the fourth day of incubation an increase in enzyme activity is very
significant, on day 2 enzyme activity menapai value of 10.93 UI / ml and an increase on the third day
at 12:50 UI / ml.Then on the fourth day reached the maximum point of the enzyme activity that is
equal to 15:14 UI / ml, after passing through the fourth day and into the fifth and sixth days of
incubation, xylanase activity decreased but not significant decrease in enzyme activity occurred at
13:29 UI / ml become 12:45UI / ml.And the same thing also happened on the medium with substrate
concentration of 2%, 3% and 4%.
As for the effect of substrate concentration in the media against xylanase enzyme activity can also be
seen in graph 4.1 where in the media with 1% substrate concentration xylanase enzyme activity
reached the highest point and then declined in the substrate concentration of 2% and has decreased in
substrate concentration of 3% and 4%.
In previous research conducted by Diah-Kristin, Gideon-Jang, (2005) using the fungus Trichoderma
reesei strain with incubation temperature of 30oC, and the incubation carried out for 7 days for
T.reesei.The selection condition is based on earlier studies which reported that in these conditions
produced xylanase activity can be maximized.Meanwhile, as a research variable is the acidity of the
fermentation medium at pH 4.5, 5, 5.5, 6; 6,5, 7, 7.5, 8, 8.5, and 9.
Microorganisms have varying growth period in which the metabolic activity of the microorganisms
have several phases in pertumbuhnnya.In the early growth phase is the phase through which growth
and metabolic activity will decrease after the peak growth of microorganisms passing phase,
penururnan phase is called the death phase.Growth phases are very influential on the enzymes
produced by microorganisms to aid digestion of food.
From the research that has been made known that the optimum time for incubation is four days,
because on the fourth day of incubation can be seen that the enzyme activity had the highest activity
compared with other days ie day 2, 3, 5 and 6.this occurs because the optimum growth period for
Aspergillus Niger is 3 and 4 days, it is marked with
increased viscosity and reduced viscosity broth medium as it passes through the optimal growth phase
(Prosetsa and Oi, 1997).
While the variation of substrate concentration, enzyme activity decreased along with increasing
concentrations of substrate.At a concentration of 1% substrate enzyme activity reached the highest
value then the enzyme activity began to decline in substrate with concentration of 2% and has
decreased in substrate concentration of 3% and 4%.Substrate concentration of 1% is an ideal condition
for Aspergillus Niger to produce xylanase enzyme because of substrate 1% represents ideal conditions
for submerged fermentation at a concentration of the adsorption of enzyme to substrate goes
well.Substrate that is not too high is the optimum state of submerged fermentation, this happens
because at these concentrations of oxygen diffusion and enzyme adsorption on the substrate will run
optimally (Stewart and Parry, 1981).
Effect of pH Terahadap xylanase enzyme activity.
Testing the effect of pH was done after the first test done is test the influence of substrate
concentration on enzyme activity of xylanase, in the first test showed that the concentration of
substrate is best for the growth media is a substrate of 1%, so the test for the effect of pH performed on
media with 1% substrate withlong incubation 2, 3, 4, 5 and 6 days indicated on the graph
4.2.0.002.004.006.008.0010.0012.0014.0016.0018.0055.566.5pHAktivitas Enzyme (
U / ml) Incubation 2 hariInkubasi 3 hariInkubasi 4 hariInkubasi 5 hariInkubasi 6 days
Graph 4.2 Effect of pH on xylanase enzyme activity
From the graph 4.2 show the relationship between pH and enzyme activity with incubation time
variation of 4.2 on the graph can be seen that the enzyme activity teringgi occurred at pH 6, at pH 5
enzyme activity is not too high even the smallest, then at pH 5.5 there is an increase of peak enzyme
activityoccurred at pH 6 and then decreased at pH 6.5.Aktitas highest at pH 6 and the 4th day of
incubation time of 15:33 to produce the enzyme activity UI / ml at pH 6.5 and then decreased to 15:19
UI / ml.whereas for pH 5 and 5.5 respectively enzyme activity of 12.90 UI / ml and 15:14 UI / ml.
The enzyme is a protein that has a biochemical activity as a catalyst for a reaction.Because it is a
 protein, this enzyme is very susceptible to environmental conditions.The change of concentration
substrate or pH environment will result in the activity of enzymes involved changing although there
are many other things that can also affect enzyme activity such as temperature or medium
composition.Therefore, each enzyme has a specific pH and temperature which causes the activity
reaches an optimum state.Conditions of optimum pH and temperature will be supported enzyme
catalyst in a reaction by doing good.Whereas
temperature and pH are less fit will result in damage or inactive protein in an enzyme that causes the
function and activity of this enzyme is reduced.
In this study the optimum conditions found at a concentration of 1% substrate incubation time and to
obtain the optimum incubation time on day 4.PH test showed high enzyme activity obtained at pH 6.
Effect of Substrate Concentration on Protein Concentration
The purpose of testing this protein is to determine the amount of protein contained in crude xylanase
and calculate the enzyme specific activity.By knowing the specific activity of enzyme can know the
amount of enzyme activity in proteins.
Proteins are dissolved in the fermentation medium should be measured to determine the amount of
enzyme protein is synthesized by microbes and to calculate the specific activity of the
enzyme.However, soluble protein is measured not absolutely reflect that all enzymes measured are
synthesized by microorganisms, because in the medium also contains soluble protein in the form of
residual media (yeast extract) or the result of a secreted protein metabolism of microorganisms.In
addition, not all proteins are a group of xylanase enzyme.
Here is a graph of the relationship of protein concentration produced by Aspergillus Niger with
substrate concentration and incubation time are shown in graph 4.3
202530354023456Lama Incubation (Days) Concentration of Protein (mg / l) 1% Substrate
constructive constructive constructive substrate substrate 2% 3% 4% substrate constructive
Graph 4.3 Effect of Substrate Concentration on Protein Concentration
Determination of protein in this study carried out by using the method of Lowry.From Graph 4.3
explained with increasing time of incubation, protein levels will rise by about 4 days.After 4 days of
protein levels tend to be constant.The highest protein content was found in the substrate concentration
of 1% with a long incubation period of 4 days.
Effect of pH on Protein Concentration
Effect of pH on the concentration of proteins with different long incubation time is shown in graph
4.4.
202530354055.566.5pHKonsentrasi Protein (
mg / l) Incubate 2 hariInkubasi 3 hariInkubasi 4 hariInkubasi 5 hariInkubasi 6 days
Graph 4.4 Effect of pH and incubation time on Protein Concentration
In graph 4.4 explained that the effect of pH on protein content is not too significant.While the effect of
pH on the incubation time is significant.In this study pH 6 is the optimum pH to obtain the highest
protein content with a long incubation period of 4 days.
CONCLUSIONS AND SUGGESTIONS
Conclusion
1.The content of lignocellulose in rice straw can be utilized to the enzyme xylanase.
2.On variations of substrate concentration showed that the substrate concentration of 1% has the
highest enzyme activity value when the media was incubated for 4 days, with a value of enzyme
activity at 15:14 UI / ml.At pH variation of media, obtained optimum pH for enzyme activity were pH
6, with the incubation time for 4 days and the value of their enzymatic activity at 15:33 UI / ml.
3.The optimum condition for xylanase production can be achieved when the substrate concentration of
1% with medium pH 6 and incubation time for 4 days.
Suggestion
1.For further research, the application needs to be done xylanase enzyme in the degradation of
hemicellulose, and needs to be done well of a study regarding the conversion of xylose into ethanol
that can be used as an alternative fuel production.
2.Need to be the choice of other strains that are quite specific in the enzyme cellulase-free xylanase
ACKNOWLEDGMENTS
 On this occasion we thank Almighty God for guidance that has been given - his, Mr Dr.Ir.Abdullah,
MS as lecturers for their guidance during this that has been given and all those who have helped so
that this research can be resolved.
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