Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
Production, Optimization and Partial purification of Cellulase by
Aspergillus niger fermented with paper and timber sawmill
industrial wastes
*M. Charitha Devi and M. Sunil Kumar
Department of Virology, College of Sciences, Sri Venkateswara University, Tirupati, A.P, India
_____________________________________________________________________________________________
ABSTRACT
It was the goal to investigate the cellulase enzyme production ability of fungal strain Aspergillus
niger against the lignocellulosic bio wastes like saw dust, paper cellulose at varying
environmental parameters of pH (4-7), temperature (20-50°C) and incubation period (2-8
days). Production of cellulase was analysed by Dinitrosalicylic acid (DNS) and Filter paper
assay methods. In the DNS method, maximum enzyme production of 3.9 IU was achieved at
temperature of 45°C by Aspergillus niger in paper cellulose with pH of 5 on 7th day of growth.
The partial purification of the cellulase enzyme produced by Aspergillus niger in the waste
supplemented medium had two protein bands with the molecular weight of 33 and 24kDa
respectively.
Key words: Aspergillus niger, paper cellulose, DNS, FPA, Cellulase, Partial purification,
Fermentation.
_____________________________________________________________________________________________
INTRODUCTION
The cellulose constitutes the major form of stocking of glucose obtained through photosynthesis
and in the same time the major component of solar energy conversion to the biomass. The
cellulose is also major constituent of all the Plant materials and that is why it is the most
abundant organic material in nature, which is renewed every year. Because of its highly ordered
structure, the cellulose is very hard to be degraded and that is why it is unusable and stocked in
nature as waste. The capacity to degrade the natural cellulose implies the synthesis of the entire
cellulolytic system. Cellulose has been used by man for centuries, however, its enormous
potential as a renewable source of energy was recognized only after cellulose degrading enzymes
or “cellulases” had been identified (Bhat & Bhat, 1997). A cellulosic enzyme system consists of
three major components: endo-β-glucanase (EC 3.2.1.4), exo-β-glucanase (EC 3.2.1.91) and β120
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
glucosidase (EC 3.2.1.21) (Knowles et al., 1987). These components act synergistically in the
conversion of cellulose to glucose (Eveleigh, 1987). Ali and Akhand (1992) worked on cellulase
production by mesophilic Trichoderma isolate during growth on water Hyacinth under optimized
conditions. The cellulase complex of Aspergillus niger has been most thoroughly studied. It can
convert native cellulose as well as derived celluloses to glucose (King & Nessal 1969). Ahmad et
al., 2003 worked on Trichoderma harzianum for cellulase enzyme production by using different
carbon sources and reported that Carboxy methyl cellulose is the best for substantial amount of
enzyme production (Shazia shafique et al.,). Since growth of fungi as well as the enzyme
production depends on the composition of the growth media, pH, temperature. The effect of
environmental factors on the growth of fungi is generally less specific and restricted than the
effect on secondary metabolite production. For example, the ranges of water activity, growth
medium and pH within which formation of certain secondary metabolites occur, is narrower,
than the range of conidial growth (Northolt & Bullerman, 1982). Several studies were carried out
to produce cellulolytic enzymes from biowaste degradation process by many microorganisms
including fungi such as Trichoderma, Penicillium, Aspergillus spp. etc. by Mandels and Reese
(1985), Hoffman and Wood (1985), Brown et al. (1987), Lakshmikant and Mathur (1990) etc.
Similarly celluloytic property of bacterial species like Pseudomonas, Cellulomonas, Bacillus,
Micrococcus, Cellovibrio and Sporosphytophaga spp. were also reported (Nakamura and
Kappamura, 1982; Immanuel et al., 2006). The specific cellulolytic activity shown by the
bacterial species is found to be depending on the source of occurrence (Saxena et al., 1993). The
work on cellulase enzyme production by Aspergillus niger has been conducted all over the
world. But the physiological responses of same organism or species may vary with ecological
variations. Therefore, the present research work was aimed to evaluate the potential cellulase
production by native Aspergillus strains.
MATERIALS AND METHODS
Isolation and screening of Cellulase producing fungi:
The samples were aseptically collected from local industrial wastes like paper, timber- saw mills
etc., in and around Tirupati (Andhra Pradesh). Serially diluted sample prepared from the
industrial wastes were spread on surface of potato dextrose agar and incubated for 7 days at
30ºC. Colonies were picked and sub-cultured to obtain pure culture. Stock cultures were
maintained on potato dextrose agar at 4ºC. The isolated strains were carefully identified by
morphological characteristics include color of the colony and growth pattern studies, as well as
their vegetative and reproductive structures observed under the microscope.
Cellulase producing fungi were screened on selective carboxymetyl cellulose agar containing
(1%): NaNO3 2.0, KH2 PO4 1.0, MgSO4 .7H2O 0.5, KCl 0.5, carboxymethyl cellulose sodium
salt 10.0, peptone 0.2, agar 17.0.Plates were spot inoculated with spore suspension of pure
culture and incubated at 30ºC.After 3 days, plates were flooded with 1% Congo red solution for
15 minutes then de-stained with 1M NaCl solution for 15 minutes. The diameter of zone of decolorization around each colony was measured. The fungal colony showing largest zone of decolorization was selected for cellulase production.
121
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
Carbon source:
Fresh industrial wastes i.e., saw dust, paper cellulose were collected where the degraded samples
were collected for isolation of fungi, they were washed and blended by a mixer and air dried.
The blended materials were sieved and used as a carbon source for the production of cellulase.
Cellulase production:
The isolated Aspergillus niger pure culture harvested with sterile distilled water for inoculum
preparation was used for cellulase production. The basal mediums supplemented with known
volume of cellulosic substrates were used for production. 100ml of production medium (pH 5) in
250ml shake flask was inoculated with 2 ml of fungal spore suspension containing 105 spores per
ml. The flask was incubated at 30oC with agitation speed of 200 rpm in rotary shaker incubator.
After 7 days, culture filtrate was collected centrifuged at 6000 rpm for 15 min and supernatant
was used as crude cellulase source.
Cellulase assay:
Total cellulase activity in the culture filtrate was determined according to the method of Mandels
et al (1976).The reducing sugar released from filter paper per ml per min at 540nm was
determined by Dinitro salicylic acid (DNS) method. One unit of total cellulase activity was
defined as the amount of enzyme releasing 1µmole of reducing sugar per minute.
Optimization of production of cellulase enzyme:
Cellulase production depends upon the composition of the fermentation medium. Medium
optimization for over production of the enzyme is an important step and involves a number of
physico-chemical parameters such as the incubation period, pH, temperature and supplemented
Substrate in submerged fermentation. For the initial optimization of the medium, the traditional
method of “one variable at a time” approach was used by changing one component at a time
while keeping the others at their original level. The selected cellulolytic strains were grown in
selected media consisting of selected substrates for enzyme production. Studies were performed
in shake flasks to optimize different fermentation conditions for hyper cellulase production.
Carbon source:
The basal medium supplemented with different volumes of Cellulose substrates as carbon source
like paper cellulose and saw dust. The measured volume of Cellulose substrates was selected as
optimum amount of carbon source required for cellulase production.
Effect of Incubation period, temperature and pH:
To select the suitable temperature, pH, incubation period for fermentative production of the
enzyme the selected fungal strains were cultivated with varying temperatures of 30˚C-50˚C, pH
range 3-8, incubation period range of 2-8 days, by keeping all other parameters constant for
hours
Partial purification of cellulose
◦
All procedures of the cellulase purification were carried out at 4 C. The culture supernatant was
separated by centrifugation process, by using buffers like buffer A, 50mM Tris–HCl (pH 8.0);
buffer B, buffer A containing 80% saturated ammonium sulphate (Wood 1988). Followed by
Fractional ammonium sulphate precipitation by adding solid ammonium sulphate to the culture
122
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
filtrate to 80% saturation. After 12 h the resulting precipitate was collected by centrifugation at
10,000 × g for 30 min and dissolved in buffer A and dialysed overnight against three changes of
the same buffer. Insoluble material was removed by centrifugation at 10,000 × g for 10 min. The
clear supernatant was filter sterilized and stored at 0oC. Partially purified Enzyme was confirmed
by SDS- PAGE performed using the method of Laemmli (1970), with the stacking and
separating gel concentrations were 4% and 12% of polyacrylamide, respectively. After the
electrophoresis, the gels were stained with Coomassie brilliant blue R-250 (Sigma) for
visualization of protein bands.
Statistical analysis:
Analysis of variance (ANOVA) was performed on all data using the SAS (1985) statistical
package. The mean values were compared by the least significant difference (LSD) test at 5%
level of confidence.
RESULTS
Isolation and screening of Cellulase producing fungi
The fungi were isolated and screened from the samples collected from paper and timber- saw
mills for cellulase production by congo red assay. The isolated strains were carefully identified
by morphological Characteristics include color of the colony and growth pattern studies. Some
of the microscopic characteristics examined under the microscope include spore formation and
color (Fig.1). Isolated fungal strains were subjected to screening, the colony forming units per
gram of sample (CFU/g) were calculated (Fig:2). The diameter of hydrolytic zone and colony
diameter were measured (Fig:3). Isolate PIW-1 and TSMW-1 showed highest zone of
hydrolysis as 12 mm out of 19 mm and 13mm out of 17mm of colony diameter.
Figure.1: Microscopic features of Aspergillus sp. stained with lactophenol cotton blue at magnifications of 40X
Table.1: Colony forming units of isolated fungi per gram of sample (CFU/g).
S. No.
Nature of sample
1.
Paper industrial waste
2.
Timber saw mill waste
Dilution
10-3
10-4
10-5
10-3
10-4
10-5
Number of Fungal population (CFU)
45
14
04
24
03
-
123
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
Table.2: The diameter of hydrolytic zone and colony diameter
S. No.
1.
2.
3.
4.
5.
6.
Colony Microscopic Colony diameter Hydrolytic Zone
colour
observation
(mm)
(mm)
Black Aspergillus sp.
PIW-1
19
12
Black Aspergillus sp.
12
5
PIW-2
Black Aspergillus sp.
10
PIW-2
Black Aspergillus sp.
TSMW-1
17
13
Black Aspergillus sp.
9
TSMW-2
8
4
Black Aspergillus sp.
TSMW-3
PIW, Paper industrial waste; TSMW, Timber saw mill waste.
(-) indicates diameter of hydrolytic zone is less than 2mm.
Strain No
Figure.3: Isolated fungal primary colonies (A, B, C and D) and CMC-Congo red plate assay of Aspgergillus sp.
Fig.4. Effect of incubation period on cellulase production
124
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
Fig.5. Effect of pH on cellulase production
Fig.6. Effect of temperature on cellulase production
Fig.7. SDS-PAGE analysis of partially purified endo and exo glucanase. Lane 1- Marker proteins lanes 2
partially purified enzyme stained with Comassie brilliant blue.
125
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
Optimization of production of cellulase enzyme:
Maximum activity was achieved at the cultural optimized conditions - recorded by Aspergillus
niger PIW-1 and TSMW-1 in Papercellulose with temperature of 45°C, pH of 5 at the growth of
7th day. The activity calculated was 3.9 IU/min/ml and 3.8 IU/min/ml respectively.
Partial purification of cellulose
Two protein bands with the molecular weight of 33 and 24kDa respectively were identified in
SDS-PAGE analysis of partially purified endo and exo glucanase.
DISCUSSION
This study could establish that paper industrial waste, timber saw mill waste which does not find
any significant commercial use especially in developing countries like India and is disposed of in
municipal bins for rotting could serve as an ideal substrate for production of cellulases. Hence,
the technology using these cheap and readily available substrates for production of cellulases in
optimum quantities in about 96 h holds promise for the future. This study could also demonstrate
that a single enzyme, if partially purified, could be used for saccharification of lignocellulosic
biomass rather than using a concoction of cellulases. The results are significant for the study on
cellulase production and provide a potential approach for the industry. Several fungal species
were isolated from diverse environments for the production of cellulase enzyme, the two fungal
isolates of Aspergillus sp. possessing the potential cellulolytic activity were selected for the
production of cellulase enzyme by carboxy methyl cellulose clearance plate assay by measuring
the clearing zone. Czapak dox broth was selected as viable media for the production of cellulase.
The production of cellulase in the medium containing papercellulose and saw dust as substrates
were suitable in case of Aspergillus sp. submerged fermentation and gave highest production of
total cellulase activity. The cultural conditions were optimized for higher yield of cellulase
enzyme. For the initial optimization of the medium, the traditional method of “one variable at a
time” approach was used by changing one component at a time while keeping the others at their
original level. The optimized cultural conditions resulted in increased total cellulase production
of 3.9 IU and 3.8 IU for Aspergillus sp. PIW-1, TSMW-1 respectively. The results obtained in
this study are better or comparable with the results obtained by Krishna (1999) or Ananda
Muniswaran et al. (1994) who used banana stalk and coconut coir for production of cellulases,
respectively. However, Kang et al. (2004) have reported higher enzyme yields using different
ratios of rice straw and wheat bran using Aspergillus sp. Thus, this study could reveal that if the
crude filtrate prepared using above treatments could be partially purified using ammonium
sulphate precipitation followed by membrane filtration, it could alone be sufficient for efficient
saccharification of pretreated lignocellulosic material. Two bands showing cellulolytic activity
were detected on gel during electrophoresis of the partially purified enzyme. The molecular
weights of these bands were estimated to be 24 and 33 kD. These bands (proteins) may be
isoenzymes or the different subunits of the same enzyme protein on electrophoresis gel (Coral et
al., 2002).
CONCLUSION
The fungi as enzyme sources have many advantages that, the enzymes produced are normally
extracellular, making easier for downstream process. The development of economically feasible
126
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
technologies for cellulase production and for the enzymatic hydrolysis of cellulosic materials
will enable to utilize the large quantities of biomass such as the residues of both food industries
and agriculture. Thus the present investigation was selected to conduct an extensive study on
cellulases from A. niger. Present study was aimed at isolation of promising cellulase producing
Aspergillus sp. and its identification, optimization of cultural conditions for production of
cellulolytic enzymes. Fungal cultures were initially identified as species of the genera of
Aspergillus based on cultural, morphological, microscopic characterstics. The cellulololytic
activity of these cultures was studied by standard CM-cellulose and congo red plate assay
method. The process development is the key step in fermentation processes. The study related to
process development involves optimization of different fermentation conditions (physical and
nutritional) towards enhancement of cellulolytic enzymes production. Shakeflask cultural
conditions (physical and nutritional factors) for cellulolytic enzymes production by the isolated
promising Aspergillus niger were optimized. Rice straw was selected as a best substrate for
cellulase production using Aspergillus niger. The cultural conditions were optimized for higher
yield of cellulase enzyme. One factor at a time (OFAT) strategy was used for the optimization of
medium components. The selected cellulolytic strains were grown in selected media for enzyme
production and the studies were performed in shake flasks. Cellulase production with Aspergillus
niger was highest at temperature 45 ˚C, pH-5.0, incubation time (7 days) and in presence of
substrates (rice straw). the partial purification of cellololytic enzymes from the culture filtrate
was performed by ammonium sulphate salt precipitation followed by desalting through dialysis.
The partial purification of cellololytic enzymes from the culture filtrate was performed by
ammonium sulphate salt precipitation followed by desalting through dialysis. Two protein bands
with the molecular weight of 33 and 24kDa respectively
Acknowledgement
This work was supported by DBT (Department of Biotechnology) Ministry of Sciences &
Technology, New Delhi, We are very thankful for providing the financial support and my
colleagues for their moral support.
REFERENCES
[1]. Ananda Muniswaran, P. K., Selvakumar, P., and Narasimha Charyulu, N. C. L. (1994).
Journal of Chemical Technology and Biotechnology, 60, 147–151.
[2]. Badal, C.S. (2004). Process Biochemistry. 39, 1871–1876.
[3]. Bhat, M. and Bhat, S. (1997) Biotechnology Advances 15, 583- 620.
[4]. Coral G, Arikan B, Unaldi MN, Guvenmes H (2002). Turk J. Biol. 26:209-213.
[5]. Ghose, T.K. (1969) Biotechnology and Bioengineering 11, 239-261.
[6]. Han, Y.W. and Callihan, C.D. (1974) Applied Microbiology 27, 159-165.
[7]. Jahangeer S, Khan N, Jahangeer S, Sohail M, Shahzad S, Ahmad A, Ahmed Khan S(2005).
Pak.J. Bot.
[8]. Kang, S. W., Park, Y. S., Lee, J. S., Hong, S. I., & Kim, S. W. (2004). Bioresource
Technology, 91, 153–156.
[9]. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951) J. Biology Chem. 48:
17-25.
[10]. Miller, G. L. (1959) Anal. Chem. 31: 426-428.
[11]. Nakamura K, Kppamura K (1982) J. Ferment. Technol. 60 (4): 343 - 348.
127
Available online at www.scholarsresearchlibrary.com
M. Charitha Devi et al
J. Microbiol. Biotech. Res., 2012, 2 (1):120-128
______________________________________________________________________________
[12]. Reese, E.T. and Mandels, M. (1984) Rolling with the time: production and applications of
Trichoderma reesei cellulase. Annual Report of Fermentation Processes. 7, 1–20.
[13]. Shamala, T. R. and Srikantaiah, K. R. (1986) Enzyme Microbial Technology 8, 178-182.
[14]. Wood, T.M. and Bhat, M.K. (1988), Methods in Enzymology, 160, 87-112.
128
Available online at www.scholarsresearchlibrary.com