Mathematics and Computers in Science and Industry
Study of enzymatic hydrolysis and liquid glucose
production by solid state fermentation from rice hull
using Trichoderma Species
(Study of rice hull using Trichoderma Species)
Azam Sinkakarmi
Arezou Ghadi
Department of Chemical Engineering, Islamic Azad
University, Ayatollah Amoli Branch,
Amol, Iran
[email protected]
Department of Chemical Engineering, Applied
Scientific University
Babol, Iran
[email protected]
Abstract— This study investigates enzymatic hydrolysis of rice
hull by Trichoderma Reesei to produce liquid glucose from
cellulose under solid state fermentation. Medium fortifying
effects on the rate of enzyme activity on (FPA, CMCASE) were
studied and the optimum condition of main culture medium and
optimization method for production of glucose were investigated.
Results showed that in terms of activity of filter paper and
activity of specific filter paper, medium containing pure rice husk
moistened with liquid basal is optimum and then medium
containing pure rice husk moistened with water has the highest
level of activity. Therefore if the aim is to achieve the highest
glucose concentration and highest concentration of filter paper,
basal liquid medium must be used in this fermentation, and if the
goal is to achieve the highest degree of carboxymethyl cellulase
activity, it is enough to moisten substrate medium containing
shell rice with tap water and moniter solid substrate fermentation
for 4 days at 30 °c.
Keywords— Rice husk, Trichoderma species, solid substrate
fermentation.
I.
mineral content such as sodium and potassium and it can
decrease the amount of crude fiber [6]. In Zaid et al.,
(2009) report, the amount of crude protein in the fermented
rice husk increased about 97% and the amount of crude
fiber has reduced about 45% [7]. In fermentation of rice
husk, solid state fermentation (S.S.F) and liquid state
fermentation (LSF) or submerged fermentation (SMF) can
be used; each of which, due to the type of method, has its
own advantages and disadvantages.
Solid state fermentation means control of growth of
microorganism on wet solid substrate in absence of free
water. This method is usually cheaper while equipment
used in liquid state fermentation (L.S.F) is more expensive.
Solid state fermentation (S.S.F) method does not need big
space, so that operations can be done in a pan bioreactor or
even in an Erlenmeyer. The greatest advantage of solid
state fermentation is that this method does not need soluble
substrate rather enough, just it is crucial to moisten the
substrate a little, in fact we do not need to free water.
The desired product is concentrated so its purification
is easier. Another advantage of SSF, due to the high
concentration of microorganisms and low humidity of
medium, is that it significantly reduces the microbial
contamination. Also, the amount of material in SSF is less
than SMF and enzymes show less susceptible to catabolic
inhibition or stimulation. However, the SSF method is
associated with some limitations, including limited use of
microorganisms capable of growth in semi-humid
environment and information about the increasing scale of
production in this way is negligible [8,9].
INTRODUCTION
In the last few decades, many efforts have been spent in
the study of enzymes with cellulolytic activity as potential
sources in obtaining energy from an abundant and
renewable cellulose especially the cellulase system of the
fungus Trichoderma sp. It has been known that cellulases
from Trichoderma sp. can be effectively hydrolyze
crystalline cellulose [1]. Lignocellulosic substrates, such as
rice husk are the agricultural wastes and by-products of
rice hulling plants. The major parts of them are cellulose
polymerase, hemi cellulose and lignin which can be used
to domesticate animals and birds as well as producing
some of other microbial metabolites such as cellulolytic
enzymes or ethanol [2-5].
In recent years, studies has been done to enhance the
nutritional value of rice husk in some countries.
Researches in Nigeria (2007) show that the fermentation of
rice husk using Trichoderma fungi for 40 days can cause a
significant increase in amount of crude protein, energy and
ISBN: 978-1-61804-247-7
II. MATERIAL AND METHODS
For this experimental research fungal strain
Trichoderma reesei (PTCC 5142) as lyophilized ampoule
was purchased from industrial scientific research
organization and transferred to physiologic serum under
367
Mathematics and Computers in Science and Industry
the sterile hood. Strains were cultured in steep medium of
Malt extract Agar (MEA), and incubated at 4°C for longterm maintenance. Husk was used in this study as substrate
and as carbon source.
F. Measurement of glucose and protein concentration in
raw enzymatic solution
For measurement of glucose concentration in raw
enzymatic solution, kit of glucose oxidase (production of
Man Company), which is an enzymatic method for lump
sugar measurement was used. The produced glucose
concentration in crud enzymatic solution (mg/dl), was
calculated according to the existed direction in the kit. For
the measurement of amount solute protein, Loury method
has been used. Bovine Serum Albumin (BSA) solution is
as a standard solution and at the end the amount of samples
absorption read at 578 wave length and with using BSA
standard curve, protein concentration in the unknown
sample (mg/ml) was obtained.
A. Prepration of substrate
For preparation of substrate, husk has been incubated at
40C and then grinded, the resulting rice husk particles size
was less than 2 mm.
B. Prepration of pre culture
After elementary operation, 12.5 gr of rice husk powder
was mixed with 17 cc water in the Erlenmeyer (500 ml).
After preparation of solid bed, sterilization step 121°C and
15 psi in the autoclave is necessary. After that, the
temperature of the contents of the flasks should be brought
to room temperature.
G. Measurment of enzyme activity
This research is on the basis of calculation of filter
paper activity and after that calculation of endogluconase
activity. With respect to the references in this field,
experiments related to activity measurement done and at
last, the amount of activity (unit/ml) was calculated.
Statistical analysis: To analyze the data and comparison
the amount of produced glucose in cultural groups under
different incubation times and temperatures, the statistical
software SPSS and ANOVA were used and P<0.05 was
considered significant
C. Fermentation of culture
Spore suspension prepared from slants containing 3
strains that mentioned above with sterile distilled water,
and using sterile pipet at near the flame, impregnation
operation from slant to pre culture media took place and
then, pre culture flasks were incubated 30°C for 5 days.
D. Preparation of main culture
After the flasks were placed in the autoclave and
cooled at room temperature in the completely sterile
conditions, impregnation from preculture to main culture
took place. With sterile loop, a part of growth fungi colony
on a solid bed was transferred to flask that contained main
culture and was mixed and this process was repeated three
or four times till 5% growth fungi colony was transferred.
Then, after preparing fungi suspension with physiologic
serum, amount of 10 micro liter of this suspension was put
between lam and Lamel. Using optical microscope, the
number of fungal spores in each square was counted and
dilution factor that depends on the volume of physiological
serum was applied. Number of spores was calculated.
When the numbers of spores on a main culture reach to
8×105 wet weight1, again cultures were incubated at at
30°C for 5-7 days. After the mentioned time finished, the
culture was taken out from incubator.
H. Optimize the assay of enzyme activity
For optimization of enzyme activity during 4 days at
30°c, during which Trichoderma reesei microorganism
reaches its maximum growth [10], medium was considerd
the effects on various substrates by adding some
combination of mineral rich salts.
I. Calculation of filter paper
Since the sample is liquid enzyme and 0.5 ml of it is
diluted with 1ml citrate buffer after that allowed to react
for 1 hour in the oven and then according to the glucose
oxidase kit. to measure the glucose level 10 ml or 0.01 μl
of it was tested so calculation of filter paper is obtained
following equation:
E. Separation of enzymatic solution
For separation of raw enzymatic solution, first buffer
citrate at pH= 4.8 was prepared, and then, about 5 fold of
solid substrate weight, buffer citrate was added to flasks
(5×12.5=125 ml). Afterward, the flasks were put in the
shaker for 15 mins, until the contents are equal. Then the
solution was passed from Watman filter paper No.1. to
assure of no presence of fungi spores, the passed solution
from filter should be centrifuged at 40C and 4000 rpm for
20 min. This prevents turbidity in the experiment. 94By
this way, enzymatic solution that contains a lot of liquid
sugar (glucose), can be separated. Indeed, during
fermentation, oozed cellulose enzyme from Trichoderma
fungi, hydrolyze the existed cellulose in the rice husk and
convert it to glucose (lump sugar).
ISBN: 978-1-61804-247-7
(U/ml)FPA =
(1)
J. Calculate the specific activity of filter paper
Specific activity (SP.A) is the ratio of the concentration
of the enzyme on protein concentration and unit is defined
as units / mg prot.
(SP.A) =
368
(2)
Mathematics and Computers in Science and Industry
K. Calculate the molecular activity of filter paper
Molecular activity is defined as units/μmol. To
calculate the molecular activity of filter paper, it is
sufficient to divide the (u / ml) FP on (μ mol / ml) FP.
that standard curve with equation OD = 0.006 Cprot +0.03
is used. The protein concentration in the solution of 6
sample enzyme is shown in table 2.
TABLE I.
TABLE II. THE PROTEIN CONCENTRATION IN THE SOLUTION OF 6
THE PROTEIN CON CENTRA TION IN THE
SAMPLE ENZYME
Methods of medium optimization
•
Medium tested
6 medium were numbered respectively and operated as
follows:
1-11gr nolignin rice crust moist within 7.48 ml basal
medium.
2- 11gr nolignin rice crust moist within 7.48 ml of tap
water.
3- 11gr rice crust (pure culture) 7.48 ml
4-11gr purified rice crust moist within 7.48 ml of tap
water.
5- 8.8 gr rice crust mixture with 2.2 gr wheat husk and
moist within 7.48 ml basal medium.
6- 8.8 gr wheat crust mixture with 2.2 gr wheat husk (ratio
of 8 to 2) and moist within 7.48 ml of tap water.
After preparing the cultures autoclave them for 20 min
in 121°C. After cooling the cultures, from T.reseei pure
culture under sterile conditions, fertilization operation is
done to mediums and then to autoclave flasks containing
medium cultures in optimum time and temperature (30°c
temperature for 4 days)[10] then the media is removed
from the incubator and 55 ml citrate buffer is given to them
and placed on shaker and then the same operation is
performed (filtration, centrifugation filter) finally what is
achieved is raw enzymatic solution from 6 cultures. First
optical absorption of sample obtained by glucose oxidase
method in λ=500 nm then with Lowry method
concentration of protein in the crude enzyme solution was
measured and ultimately FPA and CMCase activity was
obtained. Results of produced glucose concentration in the
5142 mediums to optimize fungal medium is shown in
table 1.
No lignin in liquid basal
medium containing rice crust
Medium containing no lignin
rice crust with water
Liquid basal medium containing
pure rice crust
Medium containing pure rice
crust with water
Shell liquid basal medium
containing wheat and rice husk
Mixed medium crust of wheat
and rice flakes with water
protein concentration
mg/ml
0.044
0.04
0.3
0.34
0.22
0.4
Rice husk is an organic source and has high percent of
protein, after that medium containing pure wheat crust
contains high protein concentration.
B. Calculation of filter paper activity, specific activity and
molecular activities of filter paper
In terms of activity of filter paper and specific activity
of filter paper, medium containing pure rice husk which
soaked in liquid basal is optimum medium and then
medium containing pure rice husk that has been moistened
with water has the highest level of activity. Activity,
specific activity, molecular activity and relative activity of
tested media cultures are shown in table3.
13B
C. Calculation of activity, specific activity and molecular
activity of carboxymethyl cellulase
The filter paper activity and specific activity of filter
paper medium containing pure rice husk which soaked in
liquid basal is optimum medium and then medium
containing pure rice husk that has been moistened with
water has the highest level of activity. Activity, specific
activity, molecular activity and relative activity of CMCase
in tested media culture is shown in table4.
14B
III. RESULT AND DISCUSSION
2B
A. Measuring of enzymatic soluble protein of 5142 species
by lowry's method for optimization of fungal growth
medium
Samples and dilutions is prepared according to the
Lowry method. Bovine serum albumin standard samples
adsorption standard is done in λ=578 nm wavelength so
12B
TABLE І . RESULTS OF PRODUCED GLUCOSE CONCENTRATION IN THE 5142 MEDIUMS TO OPTIMIZE FUNGAL MEDIUM
Culture
Glucose concentration in
the crude enzyme solution
mg/dI or mg/100cc
Absorbance of standard
solution
(OD) λ=500nm
Absorbance of sample (OD)
λ=500nm
No lignin wheat crust Basal medium
12.35
0.34
0.042
No lignin wheat crust Free basal medium
14.7
0.34
0.05
Pure wheat crust Basal medium
50
0.34
0.17
Pure wheat crust Free basal medium
412
0.34
0.14
48.5
0.34
0.165
23.5
0.34
0.08
Wheat flakes mixed with rice husk
Basal medium
Wheat flakes mixed with rice husk
(Free basal medium
ISBN: 978-1-61804-247-7
369
Mathematics and Computers in Science and Industry
TABLE ІІІ. ACTIVITY, SPECIFIC ACTIVITY, MOLECULAR ACTIVITY AND RELATIVE ACTIVITY OF CMCASE IN TESTED MEDIA CULTURE
Standard solution ODs= 0.34
Number of tested
culture medium
OD sample
content CMCase
Activity
CMCase
(u/ml)
Activity of
filter paper
Specific activity
SP.A(units/mgpropt)
Molecular activity
(units/µmol)
Relative
activity
1
0.09
288
17
6545.4
200
0.55
2
0.04
134
13
3350
200
0.26
3
0.13
422
120
1407
200
0.8
4
0.16
520
49
1529
200
1
5
0.14
460
26
2091
200
0.9
6
0.11
360
26
900
200
0.7
TABLE ІV. ACTIVITY, SPECIFIC ACTIVITY, MOLECULAR ACTIVITY AND RELATIVE ACTIVITY OF CMCASE IN TESTED MEDIA CULTURE
Standard solution ODs= 0.34
Number of tested
culture medium
λ=500nm
λ=500nm
Activity of filter paper
Specific activity of filter
paper
Molecular activity
F.P
Relative
activity
1
0.055
0.05
17
386
205
0.14
2
0.042
0.038
13
325
200
0.11
3
0.105
0.068
120
400
200
1
4
0.095
0.08
49
144
200
0.41
5
0.08
0.072
26
118.2
200
0.22
6
0.06
0.052
26
65
200
0.22
REFERENCES
IV. CONCLUSION
15B
3B
[1]
Enzymatic hydrolysis of rice husk of production of
glucose (sugar liquid) has been studied. 5142 T.reseei
strain has maxium growth if incubated in optimum time
and tempreture (30°c in 4 days) on the pure rice husk
substrate which didn’t lignin formation and moist with
liquid basal medium and has the highest enzymatic activity
and glucose production. However highest glucose
concentration obtained in the crude enzyme solution which
substrate contains only pure rice husk that miosted with
tap water. If to the rice husk medium addamount of wheat
husk (ratio of 8 to 2) and the surface of substrate is wet
with tap water glucose produced approximately 2-fold
increase compared to before.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
ISBN: 978-1-61804-247-7
370
Gaden, E.L., Mandels, M.H., Reese,E.T., Spano,L.A.Eds, 1976,
“Enzymaticconversion of cellulosic maerials: Technology and
Applications”, Biothechnol. Bioeng. Symp.No.6, Jhon Wiley and
sience, Newyork P.319.J.
I RR. Singhania, PK. Sukumaran, A. Pilli, et al. “Solid state
fermentation of lignocellusic substrate for cellulase production by
Trichoderma reesei”, NRRL460. Indian J Biotech 2006, 5, 332-6.
LR. Lyud, PJ. Weimer, WH. Van Zyl, IS. Pretorious. Microbial
cellulose utilization: Fundamentals and biotechnology”, Microbial
Mol Biol Rev 2002, 66, 506-77.
Y. Sun, J Cheng. “Hydrolysis of
lignocellulosic materials for
ethanol production: A review”, Bioresour technol 2002, 83, 1-11.
J Zaldivar, J Nielson, L Olsson. “Fuel ethanol production from
lignocelluloses: A challenge for metabolic engineering and process
integration”, APPL Microbiol Biotechnol 2001, 56, 17-34.
A Z Aderolu, A Iyaya, AA Onilude. “Changes in nutritional value
of rice husk during Trichoderma viride degradation. Bulgarian J
Agricultural Science, 2007, 13, 583-589.
AA Zaid , O Ganiyat. “Comparative utilization of biodegraded and
undegraded rice husk in Clarias gariepinus diet”, African J
Biotechnol 2009, 8 (7): 1358-1362.
C Krishna. “Solid-state fermentation systems – An overview”,
Critical Rev Biotechnol, 2005, 25, 1-30.
Mathematics and Computers in Science and Industry
Method”, 2011 International Conference on Biotechnology and
Environment Management IPCBEE vol.18 (2011) © (2011)IACSIT
Press, Singapoore
[9]
CN Aguilar, G Gutiérrez-Sánchez, PA Rrado-Barragán, R
Rodríguez-Herrera, JL Martínez-Hernandez, JC Contreras-Esquivel.
“Perspectives of solid state fermentation for production of food
enzymes”. American J Biochem Biotechnol 2008, 4 (4): 354-366.
[10] A.Ghadi , S. Mahjoub, R. Mehravar, “Management of Glucose
Production Process from Rice Husk by Solid State Fermentation
ISBN: 978-1-61804-247-7
371