Cellulolytic Potential of Actinomycetes Isolated from Different Habitats

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Bioengineering and Bioscience 4(5): 88-94, 2016 http://www.hrpub.org
DOI: 10.13189/bb.2016.040503
Cellulolytic Potential of Actinomycetes Isolated
from Different Habitats
Anita Saini1, Neeraj K. Aggarwal1,*, Anita Yadav2
1Department of Microbiology, Kurukshetra University, India
2Department of Biotechnology, Kurukshetra University, India
Copyright©2016 by authors, all rights reserved. Authors agree that this article remains permanently open access under the
terms of the Creative Commons Attribution License 4.0 International License
Abstract Twenty actinomycete isolates capable of
utilizing lignocellulosic biomass were isolated from different
samples of agricultural field soil, compost and decaying
vegetation. Qualitative screening of all isolates during plate
assay method using Congo red showed 17 of them producing
a clear zone of hydrolysis around the colonies indicating
cellulose hydrolytic potential of the isolates. Quantitative
estimation of filter paper (FPase) and
carboxymethylcellulase (CMCase) activities exhibited by
the positive isolates showed that highest CMCase was
produced by NAA2 (0.366 IU/ml), while highest FPase
activity of 0.075 FPU/ml was observed in NAA14. All of the
isolates showed FPase activities comparable to that shown
by C. fimi (0.023 FPU/ml) taken as a standard, confirming
the cellulose degradation ability of the isolates.
Keywords Actinomycetes, Cellulolytic, CMCase, FPase,
Habitats
1. Introduction
The increasing demand for energy and rising cost of
rapidly depleting fossil fuels has necessitated the search for
renewable energy sources of energy [1]. Lignocellulosic
biomass, including industrial, forest, agricultural and
domestic wastes, is an attractive alternative to the current
fossil fuels based sources of energy [2]. Lignocellulose is
primarily composed of cellulose, hemicellulose and lignin
[3]. Cellulose is the most abundant biopolymer present on
the earth [4]. The end product of cellulose hydrolysis, i.e.,
glucose, a fermentable sugar, is the most desired chemical
for the synthesis of biofuel ethanol [5]. The degradation of
cellulose is a complex process, which can be carried out both
chemically as well as enzymatically [6]. Enzymatic
hydrolysis is, however, an environmentally benign process,
mediated through a complex of enzymes known as cellulases.
Cellulase enzyme system comprises three classes of enzymes:
(1) 1,4-β-endoglucanase, (2) 1,4-β-exoglucanase, and (3)
β-glucosidase (cellobiase), which act in synergism to
hydrolyze cellulose completely to the monomeric glucose
[7].
Cellulases enzymes are known to be produced by a wide
variety of microorganisms including bacteria, fungi and
nematodes [8]. Most of the research work on cellulose
degrading microbes has been focused chiefly on the fungi.
Actinomycetes are relatively less explored. Recently
research studies have gained momentum in search of bacteria,
including actinomycetes, for the production of potent
cellulose degrading enzymes. Actinomycetes are
filamentous, gram positive bacteria, found abundantly in soil
and other habitats rich in degrading organic matter. They are
known to actively take part in natural lignocellulose
degradation, as a result of their capability to synthesize
extracellular polymer hydrolases such as cellulases,
hemicellulases, xylanases, chitinases, pectinases, amylases,
peptidases, proteases and keratinases [9]. Reports have
shown cellulase production by different actinomycetes
belonging to Cellulomonas [10], Streptomyces [11],
Micromonospora [12], Actinopolyspora, Actinoplanes,
Microbiospora, Thermomonospora, Rhodococcus, Nocardia
and Thermoactinomyces genera [8]. The present study
focuses on isolation of cellulose degrading actinomycetes
from different habitats followed with evaluation of their
cellulolytic potential.
2. Material and Methods
2.1. Sample Collection
Samples of soil, compost and decaying vegetation, etc.
were collected from different regions of Haryana, Punjab &
Himachal Pradesh, India. The samples were stored in sterile
containers at 4oC till their processing.
2.2. Enrichment and Isolation of Cellulose Degrading
Actinomycetes
For the enrichment of cellulose hydrolyzing

Bioengineering and Bioscience 4(5): 88-94, 2016 89
actinomycetes, 1gm of the sample was inoculated in a liquid
enrichment medium consisting of minimal salt medium
(0.2% KH2PO4, 0.2% K2HPO4, 0.2% NH4NO3, 0.05% NaCl,
0.05% MgSO4.7H20, 0.001% Fe2SO4.7H2O, 0.001%
MnSO4.H20 (pH 7.0) supplemented with 1% cellulose
powder and 1% pretreated lignocellulosic biomass. This was
followed with the incubation at 30oC, at 120rpm for 4 days.
Before inoculation, the samples were dried at 60oC for 3 days
[13].
The actinomycete enriched samples were serially diluted
and appropriate dilutions were spread plated on minimal salt
medium containing 1% lignocellulosic biomass (pretreated
by soaking in O.5M NaOH solution at room temperature for
2h.). The plates were incubated at 30oC for 7-8 days till
appearance of actinomycete colonies.
2.3. Primary Screening of Cellulose Degrading
Actinomycetes
The isolates obtained were screened qualitatively by
Congo red assay method. The cellulose hydrolysis ability of
the colonies grown on the minimal agar medium
supplemented with 1% CMC (carboxymethyl cellulose) was
tested by flooding the plates with 0.1% Congo red dye
solution followed with destaining using 1M NaCl solution
for 15-20min. The appearance of a zone of hydrolysis around
the colonies indicates synthesis of extracellular cellulases by
the microbes. The cellulolytic potential of the positive
isolates was evaluated by measuring cellulolytic index (CI),
i.e., ratio of diameter of zone of hydrolysis to the diameter of
the colony.
2.4. Secondary Screening of Isolates Positive in Primary
Screening
The isolates positive in the primary screening were
activated in starch casein agar (SCA) medium (1% starch,
0.2% KNO3, 0.2% NaCl, 0.2% K2HPO4, 0.005 %
MgSO4.7H20, 0.002 % CaCO3, 0.001% FeSO4.7H2O and
1.5% agar agar, pH 7.0) till sporulation. For enzyme
production, 1 disc (10mm) of activated culture was
inoculated in 25ml of modified Mandel’s Medium
containing 0.1% peptone, 0.14% (NH4)2SO4, 0.2% KH2PO4,
0.03% urea, 0.03% MgSO4.7H2O and 1ml of trace element
solution containing 5mg/L FeSO4.7H2O; 1.6 mg/L
MnSO4,7H2O; 1.4 mg/L ZnSO4.7H2O and 2mg/L CoCl2.
The incubation was done at 30oC at 180rpm for 7 days. The
enzyme was extracted by centrifugation of the flask contents
at 10,000rpm at 4oC for 15-20min followed with filtration
through Whatman filter paper no.1. The filtrate obtained was
used as the crude enzyme for further assays.
2.5. Enzyme Assays
The crude enzyme extracts from all isolates were assayed
for CMCase and FPase activities using method by Ghose et
al [14]. The reaction mixture for determining CMCase
activity consisted of 0.5ml of appropriately diluted crude
enzyme extract and 0.5 ml of substrate (1% CMC prepared in
100mM sodium acetate buffer, pH 4.8). The reaction mixture
for FPase assay consisted of 0.5ml of enzyme and 0.5ml of
100mM sodium acetate buffer (pH 4.8) containing 50mg
(1x6cm) of Whatman filter paper no.1. The reaction was
carried out by incubating contents at 50oC for 10 min during
CMCase and for 30 min in FPase assay. The reactions were
terminated by addition of 3ml of dinitrosalicylic acid
followed with boiling for 5-10 min for the development of
colour [15]. Finally the amount of reducing sugars released
was determined spectrophotometrically at 540nm, by
comparing the results with a glucose standard. The activity
was expressed in U/ml. One unit (IU) of enzyme activity is
defined as the amount of enzyme required to release 1µmol
of glucose in the reaction mixture per minute under specified
conditions.
3. Results and Discussion
3.1. Isolation & Primary Screening of Actinomycete
Isolates
Total 20 isolates were obtained from different samples,
i.e., soil samples from waste land rich in decaying litter,
gardens, wetland; decaying leaves and vegetation; termite
infested wood and soil and compost. Qualitative screening
by Congo red plate assay method showed 17 of them
showing zone of hydrolysis indicating production of
extracellular cellulases by them. The diameter of zone of
hydrolysis as well as the CI values (ranging from 2.5 to 9.0)
observed for various isolates were different. The results were
compared with that found in case of the standard isolate,
Cellulomonas fimi NCIM 5015. Table 1 shows the results of
qualitative screening of the isolates along with the CI values
estimated for them. Colony morphologies of all positive
isolates and zone of hydrolysis shown by them have been
depicted in Figure 1 and 2 respectively.

90 Cellulolytic Potential of Actinomycetes Isolated from Different Habitats
Tab le 1. Qualitative Screening of Cellulolytic Actinomycetes & their CI values
S. No. Actinomycete
Isolate Source of Isolate CI value
Standard C. fimi NCIM 5015 1.4
1 NAA 1 Waste Land Soil, Ambala City, Haryana, India 5.0
2 NAA 2 Waste Land Soil, Ambala City, Haryana, India 6.0
3 NAA 3 Waste Land Soil, Ambala City, Haryana, India 6.3
4 NAA 4 Garden Soil, Ambala City, Haryana, India 5.0
5 NAA 5 Garden Soil, Ambala City, Haryana, India 6.6
6 NAA 6 Wetland Soil, Trilokpur, Himachal Pradesh, India 5.0
7 NAA 7 Termite Infested Soil, Kurukshetra, Haryana, India 4.0
8 NAA 8 Termite Infested Wood, Kurukshetra, Haryana, India 2.5
9 NAA 9 Garden Soil, CPPRI, Uttar Pradesh, India 5.0
10 NAA 10 Garden Soil, CPPRI, Uttar Pradesh, India 4.8
11 NAA 11 Decaying Leaves, Trilokpur, Himachal Pradesh, India 4.9
12 NAA 12 Kitchen garden Soil, Kurukshetra, Haryana, India 3.7
13 NAA 13 Compost, Kurukshetra, Haryana, India 6.8
14 NAA 14 Compost, Kurukshetra, Haryana, India 5.7
15 NAA 15 Compost, Kurukshetra, Haryana, India 9.0
16 NAA 16 Decaying Vegetation, Kurukshetra, Haryana, India 3.0
17 NAA 17 Decaying Vegetation, Kurukshetra, Haryana, India 3.2
All results were recorded in triplicates & CI values were calculated as average of the three results

Bioengineering and Bioscience 4(5): 88-94, 2016 91
Figure 1. Colony Morphology of Different Actinomycete Isolates (on SCA medium)

92 Cellulolytic Potential of Actinomycetes Isolated from Different Habitats
Figure 2. Zone of Hydrolysis of Different Actinomycete Isolates (on CMC agar medium)
Figure 3. CMCase activity shown by different actinomycete isolates

Bioengineering and Bioscience 4(5): 88-94, 2016 93
Figure 4. FPase activity shown by different actinomycete isolates
3.2. Secondary Screening of Actinomycete Isolates
Quantitative screening of isolates showed that the isolate
NAA2 produced largest amount of endoglucanase (CMCase),
i.e., 0.366 U/ml. The standard isolate of C. fimi NCIM 5015
produced 0.185 IU/ml of CMCase and 0.023 FPU/ml of
FPase. Four other isolates, i.e., NAA3 (0.186 IU/ml), NAA6
(0.228 IU/ml), NAA7 (0.197 IU/ml) and NAA15 (0.257
IU/ml), also showed CMCase values higher than or close to
that of the standard culture. Figure 3 represents the data of
the CMCase activities shown by various isolates. On the
other hand, all of the isolates showed FPase activities higher
than or near to that shown by C. fimi. Highest FPase activity
(0.075 FPU/ml) was observed in NAA14. NAA11, NAA5
and NAA10 produced 0.061, 0.058 and 0.056 FPU/ml of
FPase respectively. Figure 4 depicts the FPase activities
exhibited by all isolates. From the results, this can be
concluded that zone of hydrolysis is not proportional to the
synthesis of cellulolytic enzymes in the production medium.
Also there’s no correlation between FPase and CMCase
activities.
Many other reports have revealed the cellulolytic potential
of different actinomycetes isolated from various ecological
niches. Jeffrey and Azrizal [16] isolated cellulolytic
actinomycetes from different soil samples and screened them
for the production of halo around colonies indicating
cellulose hydrolysis. Identification studies revealed that all
isolates belonged to Streptomyces group. Bui [17]
successfully isolated Streptomyces and Actinomyces
actinomycetes from coffee exocarps showing high
cellulolytic potential. Das et al [9] isolated cellulose
degrading Streptomyces griseochromogenes, Streptomyces
rochei, Streptomyces plicatus and Streptomyces
enissocaesilis from diverse habitats. In another study by
Mohanta [6], cellulolytic actinomycetes were isolated from
mangrove forest sediments, which produced large zone of
hydrolysis and exhibited FPase and CMCase activites in the
range of 0.266±0.001 to 0.734±0.001 IU/mL and
0.501±0.014 to 1.381±0.024 IU/mL respectively.
4. Conclusions
85% of the actinomycetes isolated from different samples
showed cellulose degradation potential in the plate assay.
Comparison of the endoglucanase and FPase production by
the positive isolates with the standard isolate of C. fimi
confirmed the cellulose hydrolysing capability of the isolates.
Quantitative comparisons revealed high potency of the
isolates. The present work is, however, a preliminary study
which requires further elaboration. Future research can be
executed focused on optimization of physicochemical
parameters for enhancement of cellulases production by the
isolates. The cellulolytic potential of the isolates can
especially be beneficial in hydrolysis of the cellulosic
materials such as the saccharification of lignocellulosic
biomass for ethanol production.
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