Isolation and Selection of Appropriate Cellulolytic Mixed Microbial
Cultures for Cellulases Production from Oil Palm Empty Fruit Bunch
N.K. Abu Bakar,
In order to construct cellulolytic fungal mixed cultures,
screening and isolation of cellulolytic fungi was done using rotten oil
palm fruit bunches as microorganism source. Three isolated fungi had shown
the ability to degrade cellulose based on decolorization of CMC selective
agar using Grams iodine as color indicator. However, only two strains;
KS1 and KS5 were selected for construction fungal mixed culture. Based
on fungal interaction evaluation test done on PDA agar, both strains showed
contact deadlock inhibition interaction with each other. In correlation
to cellulase enzymes production, mixed cultures of strains KS1 and KS5
showed low enzymes activity compared to pure culture system. Although,
the cellulase enzymes production is low, total cellulase enzymes composition
was better than in pure culture system individually.
Cellulase enzymes complex is a multi-domain protein that consists of three
major enzymes components which are endo-β-(1-4)-D-glucanase, exo-β-(1-4)-D-glucanase
and β-glucosidase that works synergically in complex cellulose degradation
(Duff et al., 1987). In cellulose degradation
process, endo-β-(1-4)-D-glucanase or known as carboxymethyl cellulase act
by randomly cleave β-(1-4) linkages of glucose chain in the amorphous region
of cellulose to open up cellulose structure for subsequent attack from exo-β-(1-4)-D-glucanase
(Esteghlalian et al., 2002). On the other hand,
exo-β-(1-4)-D-glucanase (cellobiohydrolase) act to release cellobiose moiety
from end of glucose chain. Finally, β-glucosidase releases glucose from
cellobiose and short chain cellooligosaccharides (Krishna
et al., 1998; Rajoka et al., 2004;
Ikram-ul-Haq et al., 2005). For more than four
decades, many researches have been done on cellulase enzymes either in screening
and isolation of new strains, optimization processes involved or application
of enzymes in industrially. Yet, by far, application cellulases industrially
faced difficulties especially in total operational cost which mostly contributed
from the enzymes itself compared to the raw material used. Even though various
microorganisms have been reported to have the ability to produced cellulase
enzymes extracellularly, most studies suggested fungi have better enzymes production
compared to bacteria and yeast (Bakri et al., 2003).
Most reported cellulase enzymes producer are from Trichoderma species
and Aspergillus species (Bhat, 2000).
Trichoderma sp., was widely studied and used industrially especially
in production of β-(1-4) exoglucanase and β-(1-4) endoglucanase. Compared
to Trichoderma sp., Aspergillus sp., suffered low production of
β-(1-4) exogulacanase and β-(1-4) endoglucanse but high in β-glucosidase
enzymes (Madamwar and Patel, 1992). However, in order
to obtain high degradation of cellulose material, synergistic effect of all
three component of cellulase enzymes have to be achieved. Due to low production
of β-glucosidase by Trichoderma sp., many approaches have been suggested
to improve degradation of cellulosic material (Kovác
et al., 2009). Many suggested mixed culturing between two strains
and supplementation of β-glucosidase enzyme from Aspergillus improved
total cellulase enzymes activity of Trichoderma. Mixed cultures is a
cultivation system where two or more different microorganisms were introduce
in the same fermentation condition or environment (Yang
et al., 2003).
According to Correa et al. (1999), utilization
of fungi mixed culture resulted in higher product yield and growth rate especially
in poor nutritional agriculture residue and strengthens the protection of the
culture against contamination. Due to unique ability of individual strains of
microorganism in production of cellulase enzymes complex, it brings great interest
in understanding of its microbial ecology with correlation to cellulase enzymes
production. Hence, this study focus on the selection of appropriate mixed microbial
culture and microbial ecology interaction in correlation to cellulase enzymes
production using Oil Palm Empty Fruit Bunch (OPEFB) as substrate.
MATERIALS AND METHODS
The experiments were done from June 2008 until June 2009.
Screening and isolation of cellulolyic fungi: Sample of rotten oil palm empty fruit bunch was collected for screening and isolation of fungi-producing cellulases purposes. Serial dilution of sample were prepared in sterilized distilled water and 0.1 mL of diluted sample were spread on the surface of Potato Dextrose Agar (PDA) and incubated for 7 days at 30°C. Colonies with different morphological form were picked and sub-cultured to obtain pure culture. Stock cultures were maintained on PDA agar at 4°C for subsequent use as inoculum.
Screening of cellulase-producing fungi was done on CMC selective agar containing
0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4,
0.05% KCl, 0.2% carboxymethylcellulose (CMC) sodium salt, 0.02% peptone and
1.7% agar. Plates were spot plated with spores suspension of pure culture and
incubated at 30°C. After 3 days incubation, plates were flooded with Grams
Iodine solution for 3 to 5 min according to Kasana et
al. (2008) in order to observed and measured zone of clearance around
Fungi interaction evaluation: Fungal possible interaction was studied in nutrient-rich medium PDA agar. A loopful of each fungus strain spores suspension was cultured at 4.0 cm apart from each other in the same PDA agar to observe the consequences of different fungal interactions. Control were single cultured strains at one sided of equally divided PDA agar. Interacting fungi were incubated at 30°C for 7 days and observed at third and seventh day of incubation.
Production of cellulase enzymes: Locally isolated fungi of strain KS1 and KS5 were chosen for construction of fungal mixed culture. Each fungal culture were grown on PDA agar and incubated at 30°C for 7 day before harvested with sterile distilled water for subsequent use in inoculum preparation.
The basal medium described by Mandel and Weber (1969)
was used in this experiment. Its composition was (g L-1) KH2PO4
(2.0), MgSO4.7H2O (0.3), CaCl.2H2O (0.3),
CoCl2 (2.0), MnSO4.H2O (1.6), ZnSO4.H20
(5.0), 2 ml L-1 Tween 80 and 1 ml L-1 trace element. The
OPEFB obtained from mill was first ground to fibre with average length of 10
mm. It was delignified by soaking in 2% (w/v) NaOH for 4 h followed by autoclaving
at 121°C for 5 min according to Umikalsom et al.
(1997). The pretreated OPEFB was filtered, washed with distilled water until
no traces of alkaline could be detected and dried in an oven at 95°C for
In all fermentation, 100 mL medium (pH 5.0) was dispensed in 250 mL shake flask and inoculated with fungi spores suspension containing 1x106 spores mL-1. The flasks were incubated at 30°C with agitation speed of 150 rpm on rotary orbital shaker. Each experiment was performed in duplicates. Samples were withdrawn at regular time interval for analysis.
Analytical methods: The cellulase enzymes activity was measured according
to Wood and Bhat (1988) and Ariffin
et al. (2006). One unit activity of CMCase and FPase was defined
as 1 μmol reducing sugar released mL-1 enzyme/min. Meanwhile,
for one unit activity of β-glucosidase was defined as 1 μmol of p-nitrophenol
All experiments were duplicates and repeated twice for confirmation.
Samples withdrawn from fermentation broth were assayed for cellulase enzymes activity. Cellulase enzymes activity assay was done in order to determine the cellulase enzyme production for both pure and mixed cultures of locally isolated fungi.
Screening and isolation of celluloytic fungi: During screening process,
7 fungi were obtained from rotten palm oil fruit bunch. Only four fungi were
selected for isolation of cellulase producer based on they rapid growth on PDA
agar within seven days of incubation. All four selected fungi were subjected
to CMC agar (selective agar) for isolation of cellulase producer. Growth of
each fungus was observed after 3 days of incubation before each inoculated agar
were flooded with Grams Iodine solution for decolorization zone observation
and measurement. From Fig. 1, only three fungi showed decolorization
zone around mycelial growth on CMC agar. Decolorization zone made by fungi showed
secretion of cellulase enzymes by fungi in order to degrade cellulose structure
of CMC. Fungus strain SK3 showed the largest decolorization zones as compared
to strain SK1 and SK5 (Table 1).
strain KS1, KS3 and KS5 showed degradation of CMC agar with appearance
of decolorization of Grams Iodine
zone of clearance produced by four isolates on CMC agar stained with Grams
Iodine after 3 day incubation
However, due to its non sporulated fungi morphological form, strain SK1 and
SK5 were selected for the construction fungi cellulase mixed cultures.
Fungi Interaction on PDA agar: Visible fungi interaction evaluation
was tested on both SK1 and SK5 fungi strain on PDA agar within seven days of
incubation according to Iluyemi and Hanafi (2009). For
mixed culture interaction observation, PDA agar was divided into equal area
and each strain of fungi was inoculated 4 cm apart on PDA agar. As for control,
each strain were inoculated on one sided of equally divided agar. After 3 days
of incubation, both fungi grow well in the PDA agar yet did not perform any
direct contact to each growing mycelium as shown in Fig. 2.
Meanwhile, in PDA agar that contains only pure culture strain showed initial
growth of mycelium especially for strain KS1. Strain KS1 grown as greenish white
mycelium initially after 3 days incubation. Contradictly, growth of KS5 greenish
mycelium spread unevenly due to it morphological form that is highly sporulated
than KS1 strain. After 7 days incubation, full growth of mycelium from both
pure cultures can be observed. In mixed cultures agar, there was deadlock interaction
at touching point between two strains of fungus. However, strain KS1 mycelium
showed early stage of invasion toward strain KS5 mycelium according to Molla
et al. (2001). Mixed culture agar also showed slight inhibition where
visible demarcation line 1-2 mm between two strain fungi can be observed.
Cellulase enzymes production by single and mixed cultures: Production
of cellulase enzymes by single and mixed cultures were carried out in shake
flask fermentation using Oil Palm Empty Fruit Bunch (OPEFB) as substrate. Figure
3a-c show the production of cellulase enzymes by single
and mixed cultures. Based on the results obtained, single cultures of strain
KS1 and KS5 gave highest activities activity in certain component of cellulase
enzymes. No mutual synergism was observed between strain KS1 and KS5 in mixed
cultures fermentation. However, according to maximum enzymes production shown
in Table 2 indicated that even though the production of respective
enzymes was low in mixed cultures fermentation compared to single culture fermentation,
improved total enzymes composition can be observed in mixed cultures.
of fungi single culture (KS1 and KS 5) and mixed culture fungi (KS1 and
KS 5) during 7 day cultivation on PDA agar. (a) 3rd day of observation
and (b) 7th day of observation
enzymes profiling by strain KS1, KS 5 and mixed cultures
enzymes production by locally isolated fungi
Production of cellulase enzymes can be done either as pure culture fermentation or mixed culture fermentation.
However, lack of deep understanding especially in fungi interaction during
mixed culture hindered the advantages of utilization mixed culture for production
of industrially important enzymes. Based on results obtained from the experiments,
2 potential cellulase producer fungi had been selected for the construction
of appropriate cellulolytic mixed microbial for cellulase enzymes production
(Fig. 1). Visible interaction evaluation between opposition
colonies revealed that KS1 and KS5 fungi strain possessed a deadlock with mutual
inhibition interaction between each other (Fig. 2). The appearance
of demarcation line of 1-2 mm according to Stahl and Christensen
(1992) confirmed the deadlock inhibition interaction between two strains.
Many researches also found that deadlock interaction is the most common fungi
interaction when grown on nutrient-rich medium (Iluyemi
and Hanafi, 2009; Stahl and Christensen, 1992; Myvan
and Shearer, 1988). Rapid growth rate of strain KS1 may caused invasion
of mycelium to strain KS5 territory after seven day incubation. As for correlation
of cellulase enzymes secretion to fungal interaction, the production of cellulase
enzymes was done in submerged fermentation for both culture systems (Fig.
3). In mixed cultures system, low activity of cellulase enzymes especially
for FPase and CMCase can be observed compared to strain KS5 pure culture fermentation.
In comparison to strain KS1, mixed culture resulted higher FPase and CMCase
activities up to 0.44 and 19.68 U mL-1, respectively. Similar results
also reported by Castillo et al. (1994), where
mixed culture of T. reesei and A. niger produced 4 FPA/g substrates
compared to single culture of T. reesei 5 FPA/g substrates. However,
if comparison were drawn between both fungi strains, KS1 strain showed better
β-glucosidase composition compared to KS5 strain which 0.26 U mL-1.
In contrast, KS5 strain produced higher activity of FPase and CMCase enzymes,
0.6 and 28.19 U mL-1, respectively. Deadlock inhibition interaction
observed in mixed culture of strains KS1 and KS5 on PDA agar might explain low
cellulase enzymes activities obtained in mixed culture system compared to pure
culture system. Other than that, production of protease from either fungus strains
as defensive mechanism resulted in low enzyme activity. Competition for available
carbon resource among the strains may also contribute to low cellulase enzymes
activity in mixed culture system. Even though mixed culture of both strain suffered
low enzymes activity, in term of total enzymes composition it showed better
enzymes composition compared to single strain cellulase enzymes production same
as agreed by Castillo et al. (1994) and Yusoff
et al. (2000). It appeared that the results showed no synergistic
effect between two fungi and doesnt support for cellulase enzymes production
using fungi mixed culture system using both isolated fungi strains. However,
further investigation on saccharification of cellulosic materials using cellulase
enzymes produced by both pure and mixed cultures system need to be done in order
to verify the effectiveness of using fungi mixed cultures for the production
of cellulase enzymes.
Fungal interaction during mixed culture is essential for construction of fungal mixed culture for the production of cellulase enzymes. Synergistic interaction between each fungus cultures need to be achieved in order to obtain higher enzymes production. The need for investigation especially in saccharification of cellulosic material using crude cellulase enzymes might further explain effectiveness of fungal mixed culturing for cellulase enzymes production.
1: Ariffin, H., N. Abdullah, M.S.U. Kalsom, Y. Shirai and M.A. Hassan, 2006. Production and characterisation of cellulase by Bacillus pumilus EB3. Int. J. Eng. Technol., 3: 47-53.
Direct Link |
2: Bakri, Y., P. Jacques and P. Thonart, 2003. Xylanase production by Penicillium canescens 10-10c in solid-state fermentation. Applied Biochem. Biotechnol., 108: 737-748.
Direct Link |
3: Castillo, M.R., M. Gutierrez-Correa, J.C. Linden and R.P. Tengerdy, 1994. Mixed culture solid substrate fermentation for cellulolytic enzyme production. Biotechnol. Lett., 16: 967-972.
CrossRef | Direct Link |
4: Duff, S.J.B., D.G. Cooper and O.M. Fuller, 1987. Effect of media composition and growth conditions on production of cellulase and β-glucosidase by a mixed fungal fermentation. Enzyme Microb. Technol., 9: 47-52.
5: Esteghlalian, A.R., S.D. Mansfield and J.N. Saddler, 2002. Cellulases: Agents for fiber modification or bioconversion? The effect of substrate accessibility on cellulose enzymatic hydrolyzability. Biotechnol. Pulp Paper Industry: Prog. Biotechnol., 21: 21-36.
6: Correa, M.G., L. Portal, P. Moreno and R.P. Tengerdy, 1999. Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugarcane bagasse. Bioresour. Technol., 68: 173-178.
7: Ikram-ul-Haq, M.M. Javed, T.S. Khan and Z. Siddiq, 2005. Cotton saccharifying activity of cellulases produced by co-culture of Aspergillus niger and Trichoderma viride. Res. J. Agric. Biol. Sci. 1: 241-245.
Direct Link |
8: Iluyemi, F.B. and M.M. Hanafi, 2009. Mycelial growth interactions and mannan-degrading enzyme activities from fungal mixed cultures grown o palm kernel cake. Afr. J. Biotechnol., 8: 2283-2288.
9: Kasana, R.C., R. Salwan, H. Dhar, S. Dutt and A. Gulati, 2008. A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine. Curr. Microbiol., 57: 503-507.
CrossRef | Direct Link |
10: Krishna, S.H., K. Prasanth, G.V. Chowdry and C. Ayyanna, 1998. Simultaneous saccharification and fermentation of pretreated sugar-cane leaves to ethanol. Proc. Biochem., 38: 825-830.
11: Kovac, K., G. Szakacs and G. Zacchi, 2009. Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride. Bioresour. Technol., 100: 1350-1357.
12: Madamwar, D. and S. Patel, 1992. Formation of cellulases by co-culturing of Trichoderma reesei and Aspergillus niger on cellulosic waste. World J. Microbiol. Biotech., 8: 183-186.
13: Mandel, M. and J. Weber, 1969. Exoglucanase activity by microorganisms. Adv. Chem., 95: 391-414.
14: Molla, A.H., A. Fakhru`l-Razi, S. Abd-Aziz, M.M. Hanafi and M.Z. Alam, 2001. In vitro compatibility evaluation of fungal mixed culture for bioconversion of domestic wastewater sludge. World J. Microbiol. Biotech., 17: 849-859.
15: Rajoka, M.I., I.S. Durani and A.M. Khalid, 2004. Kinetics of improved production and thermostability of an intracellular β-glucosidase from mutant-derivative of Cellulomonas biozotea. Biotechnol. Lett., 26: 281-285.
16: Myvan, H.Z. and C.A. Shearer, 1988. In vitro hyphal interaction among wood and leaf inhabiting Ascomycetes and fungi imperfect freshwater habitats. Mycologia, 80: 31-37.
Direct Link |
17: Stahl, P.D. and M. Christensen, 1992. In vitro mycelial interactions among members of a soil microfungal community. Soil Biol. Biochem., 24: 309-316.
18: Umikalsom, M.S., A.B. Ariff, H.S. Zulkifli, C.C. Tong, M.A. Hassan and M.I.A. Karim, 1997. The treatment of oil palm empty fruit bunch fibre for subsequent use as substrate for cellulase production by Chaetomium globosum Kunze. Bioresour. Technol., 62: 1-9.
19: Wood, T.M. and K.M. Bhat, 1988. Methods of measuring cellulase activities. Method Enzymol., 160: 87-112.
20: Yang, Y.H., B.C. Wang, L.J. Xiang, C.R. Duan, Q.H. Wang and J. Lian, 2003. Construction of a microbial consortium used for solid-state fermentation on rice chaff. Colloids Surfaces B: Biointerfaces. 32: 51-56.
21: Yusoff, W.M.W., M.I. Massadeh, O. Omar and J. Kader, 2000. Sugar cane bagasse degradation by mixed culture of T. reesei and A. terreus in solid substrate fermentation. Pak. J. Biol. Sci., 3: 1758-1761.
CrossRef | Direct Link |
22: Bhat, M.K., 2000. Cellulases and related enzymes in biotechnology. Biotechnol. Adv., 18: 355-383.
CrossRef | PubMed | Direct Link |