Role of Microbial Extracellular Enzymes in the ... · amplified and sequenced. Sequence analysis...
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237
J Pharm Chem Biol Sci, September - November 2018; 6(3):237-249
Journal of Pharmaceutical, Chemical and Biological Sciences
ISSN: 2348-7658
CODEN: JPCBBG
September - November 2018; 6(3):237-249 Online available at https:/ /www.jpcbs.info
Role of Microbial Extracellular Enzymes in the Biodegradation of
Wastes
Monisha Khanna Kapur1*, Payal Das1, Prateek Kumar1, Munendra Kumar1, Renu
Solanki2
1Acharya Narendra Dev College, University of Delhi, Govindpuri, Kalkaji, New Delhi 110019, India 2Deen Dayal Upadhyaya College, University of Delhi, Sector 3, Dwarka, Opp NSIT, New Delhi,110078,
India
*CORRESPONDING AUTHOR
Dr. Monisha Khanna Kapur, Principal Investigator, Microbial
Technology Lab, Acharya Narendra Dev College, University of
Delhi , Govindpuri, Kalkaji, New Delhi 110 019
Email: [email protected],
ARTICLE INFORMATION
Received July 07, 2018
Revised September 02, 2018
Accepted September 12, 2018
Published November 30, 2018
INTRODUCTION
Actinomycetes are group of gram positive
filamentous soil bacteria [1, 2] well known as
producers of various types of extracellular
enzymes. These enzymes are produced within
the cell but are secreted and function outside the
cell [3]. Extracellular enzymes are of great
importance because they have large scale
applications in various industries such as paper,
textile, detergent, cosmetics, biosensors,
pharmaceuticals, agricultural, bioremediation
and biorefineries [5,6]. They can easily degrade
wastes into useful raw materials that can be
directly recycled in industries.
Research Article
The work is licensed under
ABSTRACT
Several soil bacteria produce extracellular enzymes having potential to degrade wastes into useful raw
materials that can be directly recycled in industries. Actinomycetes are well-known producers of
extracellular enzymes. In our study, during primary screening, isolate 196 was found to be efficient in
degrading sugarcane bagasse and corn stover and showed high cellulase activity. Similarly isolate 197
showed high xylanase activity, degrading rice straw and wheat bran; isolate 136 showed high chitinase
activity and degraded crustacean shells and isolate 244 showed maximum phosphatase activity,
degrading fruit pulp. Taxonomic characterization using 16S rDNA study revealed that these bacteria
isolated from soil belong to genus Streptomyces. Based on the results of primary screening, 196 and 197
were selected for secondary analysis. In secondary screening isolate 196 showed cellulase activity of
2.86 IU/ml/min at pH 6.80-7.0 and 2.92 IU/ml/min activity at 300C. Isolate 197 showed xylanase
activity at the rate of 3.86 IU/ml/min at pH 7.2 and 3.72 IU/ml/min at temperature range of 280C-300C.
Statistical analysis of fermentation conditions using one way ANOVA and Post Hoc test analyses
signified a notable pH and temperature effect on enzyme activity of isolates 196 and 197.
KEYWORDS: Extracellular enzymes; Actinomycetes; environmental wastes; fermentation conditions;
statistical analysis.
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Traditional chemical and physical treatments on
biodegradable waste materials are not efficient
methods for degradation because these processes
are costly, take longer time and involve use of
chemicals having hazardous properties.
Biodegradation using microbial extracellular
enzymes is an efficient and economical
alternative to the traditional methods [7].
Microorganisms can convert hazardous waste
materials into non-hazardous form by enzymatic
processes [8]. Microbes like bacteria, fungi and
yeast involved in biodegradation process use
organic waste material as the source of energy
resulting in the complete biodegradation of
wastes.
In the present study, biodegradable waste
samples were collected from agro-industrial,
fishery industries and household sites [9].
Samples were pretreated for converting them
into powdered substrates. Actinomycete cultures
from diverse habitats and producing
extracellular enzymes were subjected to primary
screening. Isolates showing high production of
enzymes were selected and subjected to
taxonomic characterization by polyphasic
approach. 16S rRNA genes of the strains were
amplified and sequenced. Sequence analysis was
done using “Sequencing analysis 5.1.1” software
and “EzTaxon” server, and it was found that
isolates 196, 197, 136 and 244 belong to the
genus Streptomyces. After analysis of sequence,
evolutionary trees based on 16S rDNA were
designed with the help of “Phylip-3.69” software
package. These isolates were then subjected to
secondary screening to determine range of
enzyme activity at different pH and temperature.
Statistical analyses of fermentation conditions
including temperature and pH was done using
Post Hoc tests (Turkey HSD) and one way
ANOVA by IBM SPSS Statistics 19 software.
MATERIALS & METHODS
Collection of waste samples from selected
sites and their conversion into substrates
Collection of waste samples
Waste samples were collected from diverse
ecological habitats and were transferred to the
laboratory in zip-lock bags (Table 1).
Table 1: Collection sites for waste materials
Pre-treatment of waste samples to be used
as substrate
A multitude of different pre-treatment methods
have been suggested in literature. These are
placed under (a.) physical (grinding, irradiation,
milling), (b.) chemical methods (alkali/dilute acid
treatment, effects of organic solvents, oxidizing
acids), (c.) physicochemical (steam pre-
treatment/auto-hydrolysis, wet oxidation, hydro-
thermolysis) (d.) biological (combined use of
lignin degrading enzymes like peroxidases and
laccases) [10-12].
Screening of isolates at primary level for
checking production of Xylanase, Cellulase,
Chitinase, Phosphatase
Reviving of actinomycete cultures
Actinomycete cultures representing different
ecological habitats and known to be producing
extracellular enzymes were selected (Table 2).
Waste material used as
substrate
Collection site Waste degraded by
enzyme
Sugarcane bagasse Govindpuri Local Juice Corner
Cellulase
Corn Stover Okhla PhaseI, New Delhi
Wheat Bran Local atta chakki, Govindpuri, New
Delhi
Xylanase
Rice straw Matloda, Hisar, Haryana
Crustacean shells Ghazipur, New Delhi, Fish Market
Chitinase
Fruit pulp Jawahar Colony, Faridabad Local
Juice
Phosphatase
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The cultures were revived and restreaked on
Yeast extract – Malt extract (YM agar) [13, 14]
for getting purified culture plates.
Table 2: List of actinomycete isolates selected for Screening
Primary Screening
Media were prepared and supplemented with
specific pre-treated waste samples or with
commercial substrates.
1. Basal agar medium (pH 6.8-7.0) for
cellulase (composition (gL-1) - (NH4)2SO4
S.No. Properties Habitats Number assigned to
isolates/Colonies
1.
Cellulose
degrading
isolates
Agricultural Soil- Dhanaura, UP,
India
84
2. Agricultural Soil- Yamuna River,
Delhi, India
196
3. Dumping Site- Sarai Kale Khan,
Delhi, India
191
4. Purana Quila- Delhi, India 106
5. Diversity Park- Sarai Kale Khan,
Delhi, India
186
6. Dumping Site- Sarai Kale Khan,
Delhi, India
Isolate 194, Positive Control
(Previous study)
1.
Xylan degrading
isolates
Agricultural Soil- Dhanaura, UP,
India
43
2. Agricultural Soil- Yamuna River,
Delhi, India
88
3. Sarai kale Khan, Dumping Site,
Delhi, India
197
4. Sugar Plant- Dhanaura, UP, India 61
5.
Sugar Plant- Dhanaura, UP, India Isolate 169, Positive Control
(Previous study)
1.
Chitin
degrading
isolates
Agricultural Soil- Dhanaura, UP,
India
4
2. Agricultural Soil- Yamuna River,
Delhi, India
222
3. Sarai Kale Khan, Dumping Site
Delhi, India
88
4. Sugar Plant- Dhanaura, UP, India 66
5. Chemical Plant- Faridabad, HR,
India
136
6. Chemical Plant- Faridabad HR,
India
Isolate 130, Positive Control
(Previous study)
1. Phosphate
degrading
isolates
Agricultural Soil- Nainital, UK,
India
116
2. Agricultural Soil- Kashipur, UK,
India
161
3. Yamuna Bank- Delhi, India 79
4. Great Himalayan National Park-
Teerthan Valley, HP, India
244
5. Agricultural soil- Kashipur, UK,
India
Isolate 165, Positive Control
(Previous study)
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2.64, KH2PO4 2.38, K2HPO4.3H2O 5.65,
MgSO4.7H2O, trace salt solution
(composition gL-1) 1ml. CuSO4.5H2O
6.43, FeSO4.7H2O 1.1, MnCl2.4H2O 7.9,
ZnSO4.7H2O 1.5, agar 15 [13]. The
medium was supplemented with
sugarcane bagasse, corn stover and
cellulose (commercial substrate)
respectively.
2. Mineral Salt agar medium (pH 7.2) for
xylanase (composition (gL-1) NaNO3 2.0,
KCl 0.5, Fe2 (SO4)3 0.01, MgSO4.7H2O
0.5, KH2PO4 0.14, K2HPO4.3H2O 1.2,
Yeast extract 0.02, agar 15 [15]. The
medium was supplemented with rice
straw, wheat bran and birchwood xylan
(commercial substrate) respectively.
3. Chitin medium (pH 8.0) for chitinase
(composition gL-1) KH2PO4 0.3,
ZnSO4.7H2O 0.001, MgSO4.5H2O 0.5,
K2HPO4 0.7, FeSO4.7H2O 0.01, agar 20
[16, 17]. The medium was supplemented
with crustacean shells and colloidal chitin
(commercial substrate) respectively.
4. Pikovskaya’s medium (pH 7.2) for
phosphatase (composition gL-1)
ZnSO4.7H2O 0.001, K2HPO4 0.7,
MgSO4.5H2O 0.5, KH2PO40.5,
FeSO4.7H2O 0.01, Bromophenol blue
(composition- 0.4g in ethanol, pH 6.5
using NaOH, working concentration=
0.08%, 0.16%, 0.24%, 0.32%, 0.40%), agar
20 [18, 19]. The medium was
supplemented with fruit pulp and
tricalcium phosphate (commercial
substrate) respectively.
The strains were subjected to plate assay
(primary screening) by spot inoculating
them on different media plates.
Inoculated plates were incubated for 7-21
days at 280C for secretion of extracellular
enzymes. Clear zone surrounding the
bacterial colonies indicates production of
enzyme. Zones of hydrolysis were
calculated by subtracting the size of
inoculum from the total diameter of zone.
Each experiment was carried out
independently with three replicates.
Study of taxonomic status of the strains
by Polyphasic approach using
Bioinformatics tools:
The highest enzyme producers (isolates
196,197,136 and 244) were selected for
determining their taxonomic status.
Genomic DNA isolation of highest enzyme
producers by Phenol-Chloroform method:
Genomic DNA was isolated by methods
standardized for actinomycetes [13, 20].
Polymerase chain reaction (PCR) of 16s
rRNA gene of isolates 196, 197, 136, 244:
Genomic DNA of the selected isolates was
subjected to 16s rRNA gene amplification
using specific primers and under defined
conditions (Table 3 and 4). The final product
was analyzed on agarose gel [1, 13, 20, 21].
Table 3: 16s rRNA gene specific Primers
Table 4: The PCR conditions used for 16s rRNA gene amplification
S.No. Primer Sequence of Primer
1. 8F 5’-AAGGAGGTGATCCAGCCGCA-3’
2. 1492R 5’-AGAGGTGATCCAGCCGCA-3’
3. 27F 5’-AGAGTTTGATCCTGGCTCA-3’
4. 1542R 5’-AAGGAGGTGATCCAGCCGCA-3’
PCR reaction mix (100µl) PCR conditions
MQ= 74.2µl 35 cycles
gDNA(template)= 3µl Initial denaturation: 5min. at 95°C
10X PCR Buffer= 10µl Denaturation: 30sec. at 95°C
dNTP(s)= 1.5µl Primer annealing: 30sec. at 55°C
Primer 8F „OR‟ 27F= 5µl Primer extension: 1min. 30sec. at 72°C
Primer 1492R „OR‟ 1542R= 5µl Final extension: 10min. at 72°C
Taq DNA polymerase= 1.3µl
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Elution and sequencing of amplified PCR
product(s) of isolates 196, 197, 136, 244:
The amplified PCR fragments were processed
using the QIAGEN gel extraction kit and were
submitted for sequencing. The sequences
obtained were analyzed and assembled using
Sequence analysis 5.1.1 software. The resultant
assembled sequence was then searched using EZ
Taxon and NCBI database and the FASTA
format sequences of its homologies (20 in total)
were copied and pasted in a notepad file [13,20
,22].
Phylogenetic tree construction using
“Phylip-3.69” for Taxonomical analysis of
Isolates 196,197,136,244:
The multiple FASTA sequence file was aligned
using ClustalX software. The resultant output
sequences were then used to construct
phylogenetic tree using Phylip 3.69. The tree was
viewed using Treeview software [1, 21, 23].
Secondary screening
Optimization of fermentation conditions
for cellulase and xylanase Based on the
results of primary screening, the colonies
showing maximum zone of clearance in case of
cellulase and xylanase were subjected to
submerged fermentation process (SmF). The
selected isolates were cultured in 25ml 148G
broth (g/L-1)- Beef extract 4g, Glucose 2.2g,
Bacto-peptone 5g, Tryptone 3g, NaCl 1.5g, Yeast
extract 0.5g (pH 7.5) incubated at 28 ֯C (5 to 6
days) at 200rpm on incubator shaker [6]. A
standard inoculum having average viable count
105 to 107 CFUs/ml was inoculated in respective
medium for enzyme production. Culture flasks
were kept for incubation at range of
temperature, pH values for determination of
optimum conditions for enzyme action.
Enzyme activity estimation for cellulase
and xylanase
Well grown, week old cultures were centrifuged
to obtain cell free supernatant which was taken
as crude enzyme source. Dinitrosalicyclic (DNS)
method was used to estimate enzyme activity for
both enzymes in crude extract. 0.5ml of crude
cell free extract from the culture flasks of
both processed wastes and commerc ial
substrates were added to 1.5ml volume of 2%
cellulose, prepared in 0.05M sodium citrate
buffer having pH value of 4.8 and incubated at
40°C for 30 minutes. To each tube, 3ml DNS
reagent was added, tubes were then incubated
for 5 minutes at 100°C [3, 13, 24, 25].
Absorbance was recorded at 575 nm using
spectrophotometer (UV Vis Elico SI-159).
Glucose standard curve was made and by
extrapolating the absorbance values on the
graph, amount of glucose in each sample was
determined. One unit of enzyme activity is the
amount of enzyme that releases 1μM of
glucose/ml/min. Each experiment was carried
out independently with three replicates.
Analyses of enzymatic activity by
univariate and multivariate methods using
statistical software
Experimental data was statistically evaluated
using Post-Hoc and ANOVA analyses at p < 0.05
(significance level) by using software IBM SPSS
19.
RESULTS
Collection of waste samples from selected
sites and their conversion into substrates.
In the present study, the waste samples collected
from various sites (Figure 1) were processed and
converted into powdered substrates by different
pre-treatment methods.
Utilization of enzymatic potential of bacterial
cultures for degradation of waste
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Figure 1: Collection sites of waste samples from varied habitats and their processing into
substrates
Reviving of actinomycete cultures
Actinomycete cultures representing different
ecological habitats and known to be producing
extracellular enzymes were selected and revived.
Primary Screening
During primary screening, isolate no. 196 was
found to be most efficient in degrading sugarcane
bagasse and corn stover and showed maximum
cellulase activity (32mm on bagasse and 24 mm
on corn stover substrates) followed by isolate no.
194 (positive control, 26mm and18mmm), isolate
no. 106 (12mm), isolate no. 84 (7mm and 5mm),
isolate 186 (3mm) and isolate 191 (no zone on
bagasse and corn stover substrates) (Figure 2).
Figure 2: Colonies with Cellulase enzyme activity showing degradation of processed waste
supplemented in growth medium plates in form of zones of clearance (denoted by arrows)
Colony 196 on BM +
bagasse plate (congo red
staining)
Colony 186 on BM +
bagasse
plate (congo red staining)
Colony194 (Positive
control) on BM +
bagasse plate (congo red
staining)
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Similarly isolate 197 showed maximum xylanase
activity (21mm on rice straw 15mm and wheat
bran substrates) followed by isolate no. 169
(positive control, 13mm and 18mm), isolate no.
61 (10mm and 7mm), isolate no. 85 (6mm and
4mm) and isolate no. 43 (3mm) (Figure 3).
Figure 3: Colonies with Xylanase enzyme activity showing degradation of processed waste
supplemented in growth medium plates in form of zones of clearance (denoted by arrows)
In case of chitinase, isolate no. 136 showed
maximum chitinase activity (30mm on
Crustacean shells substrate) followed by isolate
no. 130 (positive control, 22mm), isolate no. 222
(12mm), isolate no. 88 (9mm) and isolate no. 04
(5mm) ( (Figure 4). In phosphatase, isolate no.
244 showed maximum phosphatase activity
(20mm on Fruit pulp substrate) followed by
isolate no. 79 (15mm), isolate no. 165 (positive
control, 12mm), isolate no. 161 (9mm) and
isolate no. 116 (1mm) (Figure 5).
Figure 4: Colonies with Chitinase enzyme activity showing degradation of processed waste
supplemented in growth medium plates in form of zones of clearance (denoted by arrows)
Colony 197 on MSA + wheat
bran plate (congo red staining)
Colony 88 on MSA + wheat
bran plate (congo red
staining)
Colony 169 (Positive control)
on MSA + wheat bran plate
Colony 136 on CM+
crustacean shells plate Colony 61 on CM+
crustacean shells plate
Colony 136 on CM+
crustacean shells plate
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Figure 5: Colonies with phosphatase enzyme activity showing degradation of processed
waste supplemented in growth medium plates in form of zones of clearance (denoted by
arrows).
Study of taxonomic status of strains by
Polyphasic approach using Bioinformatics
tools
Genomic DNA isolation of highest enzyme
producers by Phenol-Chloroform method
Taxonomic characterization of highest enzyme
producers (isolates 196,197,136 & 244) was done
by polyphasic approach. Genomic DNA profile on
the gel showed a sharp single band for all the
strains.
Polymerase chain reaction (PCR) of 16s
rRNA gene of isolates 196, 197, 136, 244
Genomic DNA of the selected isolates were
subjected to 16s rRNA gene amplification using
specific primers. The final product was analyzed
on 0.8% agarose gel. Using Lambda DNA
EcoRI/HindIII double digested ladder as
reference it was found that the size of eluted
products of isolates 96, 197, 136, 244 was
≈1500bp which is equal to the size of 16s rRNA
gene.
Elution and sequencing of amplified PCR
product(s) of isolates 196, 197, 136, 244
The amplified product were then eluted and size
of the band obtained in each case was found to
be≈1500bp. The eluted product samples were
submitted for sequencing. The sequences
obtained were analyzed and assembled using
Sequence analysis 5.1.1 software. The resultant
assembled sequence was then searched using EZ
Taxon and the FASTA sequences of it homologs
(20 in total) were copied and pasted in a notepad
file which was used for construction of
phylogenetic trees.
Phylogenetic tree construction using
“Phylip-3.69” for taxonomical analysis of
isolates 196,197,136,244
After sequencing, the resultant assembled
sequences were compared with the sequences of
close Streptomyces species deposited in database.
The results showed that all the strains belong to
the genus Streptomyces. Actinomycete isolates
196, 197, 136, 244 were represented as distinct
clades in their respective rooted evolutionary
tress designed by neighbor-joining method.
Isolate 196: 16s rRNA gene sequence of 1409
nucleotides was generated for isolate 196.
Phylogenetic studies revealed that isolate 196
showed 100% similarity with Streptomyces
albolongus followed by 99.93%, 99.50%, 99.43%
with Streptomyces cavourensis, Streptomyces
puniceus, Streptomyces bacillaris respectively.
Isolate 197: 16s rRNA gene sequence of 1428
nucleotides was generated for isolate 197.
Phylogenetic studies revealed that isolate 197
has highest 16s rRNA similarity of 100% with
Streptomyces griseorubens followed by 99.79%,
99.79%, 99.79% with Streptomyces althioticus.
Isolate 136: 16s rRNA gene sequence of 1431
nucleotides was generated for isolate 136.
Phylogenetic studies revealed that isolate 136
has highest 16s rRNA similarity of 100% with
Streptomyces parvulus followed by 99.37%,
99.30%, 99.30% with Streptomyces
malachitospinus, Streptomyces olivaceus,
Colony 161 on PKV+ fruit
pulp plate (bromophenol
blue staining)
Colony 79 on PKV+ fruit
pulp plate (bromophenol
blue staining)
Colony 244 on PKV+ fruit
pulp plate (bromophenol
blue staining)
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Streptomyces pactum.
Isolate 244: 16s rRNA gene sequence of 1441
nucleotides was generated for isolate 244.
Phylogenetic studies revealed the isolate 244 has
highest 16s rRNA similarity of 100% with
Streptomyces parvulus followed by 99.37%,
99.30%, 99.30% with
Streptomycesmalachitospinus, Streptomyces
olivaceus, Streptomyces pactum.
Secondary screening
Based on the results of primary screening,
isolates 196 (for cellulase) and 197 (for xylanase)
were selected for quantitative analyses using
fermentation parameters of pH and temperature
so as to identify the conditions at which enzymes
show highest levels of activity. In case of isolate
196, maximum cellulase activity was 2.86
IU/ml/min at pH 6.8-7.0 and 2.92 IU/ml/min
activity at temperature 300C (Table 5, Figure 6
and 7). Isolate 197 showed maximum xylanase
activity, 3.86IU/ml/min at pH 7.2 and 3.72
IU/ml/min activity at temperature range of
280C-300C (Table 6, Figure 8 & 9).
Table 5: Optimization of fermentation conditions for Isolate 196 (Cellulase producer)
Table 6: Optimization of fermentation conditions for isolate 197 (Xylanase producer)
Analyses of enzymatic activity by
univariate and multivariate methods
using statistical software One way ANOVA was used for statistical
analyses of fermentation conditions including
temperature and pH, which showed a notable
temperature and pH effect on enzyme activity
of isolates 196 and 197. For isolate 196 it was
F (5, 12) = 111288.172, p = .000 for pH and F
(5, 12) = 111288.172, p = .000 for temperature.
For isolate 197 it was F (5, 12) = 66581.475, p
= .000 for pH and F (5, 12) = 137621.778, p =
.000 for temperature.
Results were re-checked using Post Hoc
analysis where a statistically significant
variation in activity was observed at various
values of temperature and pH. In conclusion,
with increasing temperature and pH, enzyme
activity increases initially, then attains a
maximum level and finally decreases gradually
Cultures pH Enzyme activity (IU/ml/min) Temp.
(°C)
Enzyme activity
(IU/ml/min)
Isolate 196
(Agricultural
soil,
Yamuna
river, Delhi)
Substrate-
Sugarcane
baggase
6.4 0.67 26 0.90
6.6 0.89 28 0.92
6.8 2.86 30 2.92
7.0 2.80 32 0.65
7.2 0.63 34 0.12
7.4 0.056 36 0.09
CULTURES pH Enzyme activity
(IU/ml/min)
Temp. Enzyme
activity
(IU/ml/min)
Isolate 197
(Dumping Site, Sarai kale
Khan, Delhi)
(Substrate-Rice straw)
6.6 0.72 240C 0.68
6.8 0.92 260C 0.78
7.0 1.12 280C 3.72
7.2 3.86 300C 3.70
7.4 0.96 320C 1.12
7.6 0.091 340C 0.61
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hence producing a bell shaped curve. For
isolate 196, maximum cellulase activity was at
pH range 6.80-7.0, temperature 300C (Figure
10(a, b)) and for isolate 197, maximum
xylanase activity was at pH 7.2, temperature
range 280C -300C (Figure 11 (a, b)).
DISCUSSION
Actinomycetes are well known producers of
extracellular enzymes [26, 27, 28, 29]. In the
current study actinomycetes were isolated from
diverse habitats including Agricultural Soil-
Yamuna River, Delhi; waste dumping site (Sarai
Kale Khan) Delhi; chemical plant (NTPC)-
Faridabad and Great Himalayan National
Park- Teerthan Valley and were screened for
their ability to produce extracellular enzymes.
Isolation of actinomycete strains from varied
habitats have been reported by researchers
earlier too for screening of extracellular enzyme
producers [30-33]. They have isolated bacterial
strains from diverse habitats and screened the
cultures for presence of cellulase, xylanase,
chitinase and phosphatase activity respectively.
During primary screening, isolate no. 196 was
found to be most efficient in degrading
sugarcane bagasse and corn stover and showed
maximum cellulase activity (32mm on bagasse
and 24mm on corn stover substrates) as
compared to other colonies and positive control.
Similarly isolate 197 showed maximum xylanase
activity (21mm on rice straw and 15mm on
wheat bran substrate), isolate 136 showed
maximum chitinase activity (30mm on
crustacean shells substrate) and isolate 244
showed maximum phosphatase activity (20mm
on fruit pulp substrate).
Nayaka and Vidyasagar [34], screened isolates
from different habitats and found them to yield
zones of clearance for degradation of sugarcane
bagasse in the range of 19-52mm. Porsuk et al.,
[35] obtained zones of clearance of 12-25mm for
rice straw degrading bacterial isolates. Similar
experiments with wheat bran, crustacean shell
and fruit pulp degrading bacteria have been
reported in literature [33, 36-38].
Taxonomic characterization of selected isolates
was done by polyphasic approach. 16SrRNA gene
sequence analyses showed that isolates 196, 197,
136 and 244 belong to the genus Streptomyces.
Sequence similarity between isolate 196 with
Streptomyces albolongus was 100%, isolate 197
with Streptomyces griseorubens was 100%,
isolate 244 with Streptomyces parvulus was
100% and isolate 136 with Streptomyces
parvulus was 100%. Yassein et al., [39]
performed taxonomic study of the selected
cellulase producing Streptomyces isolate C188 by
16S rRNA gene analysis. The results of 16S
rRNA genes showed high similarity (98 to 100%)
with Streptomyces. Ninawe et al., [40] analyzed
16S rRNA gene sequences of three xylanase
producing strains and found that the strains
belong to the genus Strepomyces. Similar work
has been reported which includes the use of
16SrRNA gene sequence for taxonomic
characterization of actinomycete isolates and
pH
Figure 10 (a): Bell shaped curve of
activity at different pH shown by isolate
196
Temperature
Figure 10 (b): Bell shaped curve of
activity at different temperature shown
by isolate
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found that the strains belong to the genus
Streptomyces [1, 21-23, 33].
Based on the results of primary screening,
isolates 196 (for cellulase) and 197 (for xylanase)
were selected for quantitative analyses using
fermentation parameters of pH and temperature
so as to identify the conditions at which enzymes
show highest levels of activity. In case of isolate
196, maximum cellulase activity was 2.86
IU/ml/min at pH 6.8 and 2.92IU/ml/min activity
at 300C. Isolate 197 showed maximum xylanase
activity, 3.86IU/ml/min at pH 7.2 and
3.72IU/ml/min activity at 280C.
Golinska and Dahm, [41] estimated cellulase
activity in different Streptomyces sp. at different
pH and temperature and found enzyme activity
in the range of 8-13IU/ml using sugarcane
bagasse as substrate. Rawashdeh et al., [42]
estimated maximum xylanase activity of
5.12IU/ml/min at pH 6.5 and 5.52IU/ml/min at
temperature 600C using rice straw as substrate.
Statistical study of fermentation conditions
including temperature and pH was done using
Post-Hoc test (Turkey HSD) and ANOVA (one
way) test signified a notable pH and temperature
effect on enzyme activity of isolates 196 and 197.
Researchers used the same approach for
statistical analysis of fermentation conditions
and observed similar results [22, 43-45].
CONCLUSION
Actinomycete isolates from diverse habitats were
studied for their potential of waste degradation
during primary screening. It was observed that
isolates 196, 197, 136 and 244 are high
producers of cellulase, xylanase, chitinase and
phosphatase respectively and were found
efficient in degradation of sugarcane bagasse
and corn stover; rice straw and wheat bran;
crustacean shells; fruit pulp.Secondary
screening was performed for 196 (cellulase
producer) and 197 (xylanase producers) and with
the help of statistical analyses it was concluded
that there is a notable effect of pH and
temperature on enzyme activity. On increasing
pH and temperature, enzyme activity increases
initially, then attains a maximum level and
finally decrease gradually hence producing a bell
shaped curve. As a conclusion of our study,
actinomycete isolates were found efficient in
degrading various agro- industrial, fishery and
household wastes. Future studies involve
lyophilization of the bacterial cultures, using
their liquid formulations for degradation of
wastes kept either in compost bags under lab
conditions or alternatively degradation of wastes
directly in the field.
ACKNOWLEDGEMENT
The authors acknowledge University of Delhi for
granting the financial assistance for research
work under the Innovation Project scheme 2015-
2016. Acharya Narendra Dev College (ANDC),
University of Delhi is gratefully acknowledged
for providing infrastructural facilities.
CONFLICT OF INTEREST
The authors declare no conflict of interest
REFERENCES
1. Ventura M, Canchaya C, Tauch A, Chandra
G, Fitzgerald GF, Chater KF, Sinderen DV.
Genomics of Actinobacteria: Tracing the
Evolutionary History of an Ancient Phylum.
Microbiol Mol Biol Rev 2007; 71: 495-548.
2. Janaki T. Enzymes from actinomycetes-
Review. Int J Chemtech Res 2017; 10(2):
176-182.
3. Das P, Solanki R, Khanna M.
Characterization of Extracellular Enzymes
from Soil Actinomycetes: A Molecular
Approach. Int J Biotech Trends Technol
2015; 5(1): 36-46.
4. Anbu P, Gopinath SCB, Chaulagain BP,
Lakshmipriya T. Microbial Enzymes and
Their Applications in Industries and
Medicine. Biomed Res Int 2017; 1-3.
5. Sharma M. Actinomycetes: Source,
Identification, and Their Applications. Int
J Curr Microbiol App Sci 2014; 3(2): 801-
832.
6. Hassard F, Biddle J, Harnett R, Stephenson
T. Microbial extracellular enzyme activity
affects performance in a full-scale modified
activated sludge process. Sci Total Environ
2018; 625: 1527-1534.
7. Joutey NT, Bahafid W, Sayel H, Ghachtouli
NE. Biodegradation: Involved
Microorganisms and Genetically
Engineered Microorganisms. Intech Open
Science, Open Minds 2013; p 11.
8. Karigar CS, Rao SS. Role of Microbial
Enzymes in the Bioremediation of
Pollutants: A Review. Enzyme Res 2011; 1-
11.
Kapur et al 248
J Pharm Chem Biol Sci, September - November 2018; 6(3):237-249
9. Awasthi MK, Wong JWC, Kumar S,
Awasthi SK, Wang Q, Wang M, Ren X,
Zhao J, Chen H, Zhang Z. Biodegradation of
food waste using microbial cultures
producing thermostable α-amylase and
cellulose under different pH and
temperature. Bioresour Technol 2018; 248:
160-170.
10. Karimi K, Taherzadeh J. A critical review
of analytical methods in pretreatment of
lignocelluloses: Composition, imaging, and
crystallinity. Bioresour Technol 2016; 200:
1008-1018.
11. Kumar P, Barrett DM, Delwiche MJ,
Stroeve P. Methods for pretreatment of
lignocellulosic biomass for efficient
hydrolysis and biofuel production. Ind Eng
Chem Res 2009; 48: 3713–3729.
12. Brodeur G, Yau E, Badal K, Collier J,
Ramachandran B, Ramakrishnan S.
Chemical and Physicochemical
pretreatment of lignocellulosic biomass: A
review. Enzyme Res 2011; 1-17.
13. Das P, Solanki R, Khanna M. Isolation and
Screening of Cellulolytic Actinomycetes
from Diverse Habitats. Int J Adv Biotechnol
Res 2014; 5: 438-451.
14. Shirling EB, Gottlieb D. Methods for
characterization of Streptomyces species.
Int J Syst Bacteriol 1996; 16(3): 313-340.
15. Ramakrishnan J, Narayanan M. Studies on
xylanase producing thermophilic
Streptomyces sp.from compost soil. Int
J Pharmtech Res 2013; 5: 1386-1392.
16. Hsu SC, Lockwood JL. Powdered chitin
agar as a selective medium for enumeration
of actinomycetes in water and soil. Appl
Microbiol 1975; 29: 422-426.
17. Priya CS, Jagannathan N, Kalaichelvan
PT. Production of chitinase by Streptomyces
hygroscopicus VMCH2 by optimisation of
cultural conditions. Int J Pharma Bio Sci
2011; 2(2): 210-219.
18. Sabarathnam B, Manilal A, Sujith S, Kiran
GS, Selvin J, Thomas A, Ravji R. Role of
sponge associated actinomycetes in the
marine phosphorous biogeochemical cycles.
Am Eurasian J Agric Environ Sci 2010;
8(3): 253-256.
19. Salcedo LDP, Prieto C, Franco M. Screening
phosphate solubilizing actinobacteria
isolated from the rhizosphere of wild plants
from the Eastern cordillera of the
Colombian Andes. Afr J Microbiol Res 2014,
8(8):734-742.
20. Khanna M, Solanki R. Streptomyces
antibioticalis: a novel species from sanitary
landfill soil. Indian J Microbiol 2012; 52:
605-611.
21. Patagundi BI, Shivasharan CT, Kaliwal
BB. Isolation and Characterization of
Cellulase producing bacteria from Soil. Int
J Curr Microbiol App Sci 2014; 3(5): 59-69.
22. Zhang D, Luo Y, Chu S, Zhi Y, Wang B,
Zhou P. Biological pretreatment of rice
straw with Streptomyces griseorubens JSD-
1 and its optimized production of cellulase
and xylanase for improved enzymatic
saccharification efficiency. Prep Biochem
Biotechnol 2015; 46(6): 575-85.
23. Kampfer P, Schafer J, Lodders N, Martin K.
Murinocardiopsis flavida gen. nov., sp. nov.,
an actinomycete isolated from indoor walls.
Int J Syst Evol Microbiol 2010; 60: 1729–
1734.
24. Wood TM, Bhat KM. Method for measuring
cellulase activities. M enzymol cellulo
hemicellulo, Aca P NY 1998; 87-112.
25. Mohanta YK. Isolation of cellulose
degrading actinomycetes and evaluation of
their cellulolytic potential. Bioeng Biosci
2014; 2(1): 1-5.
26. Emimol A, Ganga G, Parvathy R, Radhika
G, Nair GM. Screening of Microbes
producing extracellular hydrolytic enzyme
from corporation waste dumping site and
house hold waste for the enhancement of
bioremediation methods. IOSR J Pharm
Biol Sci 2012; 4: 54-60.
27. Selvam K, Vishnupriya B, Yamuna M.
Isolation and description of keratinase
producing marine actinobacteria from
South Indian Coastal Region. Afr J
Biotechnol 2013; 12(1): 19-26.
28. Masih H, Singh S. Degradation of
Keratinous Waste Products by Keratinolytic
Bacteria Isolated from soil. Int J of Eng
Comp Sci 2014; 3: 7588-7595.
29. Alves PD, Siqueira FF, Facchin S,
Campolina C, Horta R, Victoria JMN,
Kalapothakis E. Survey of Microbial
Enzymes in Soil, Water, and Plant
Microenvironments. Open Microbiol J 2014;
8: 25-31.
30. Shah D, Soni A. Isolation and screening of
actinomycetes from mangrove soil for
Kapur et al 249
J Pharm Chem Biol Sci, September - November 2018; 6(3):237-249
enzyme production and antimicrobial
activity. Int J Res Sci Inno 2016; 3(3): 54-
59.
31. Shanthi V, Roymon MG. Isolation and
screening of alkaline thermostable xylanase
producing bacteria from soil in Bhilai Durg
region of Chhattisgarh, India. Int J Curr
Microbiol App Sci 2014; 3(8): 303-311.
32. Santhi R. Isolation of chitinase producing
Streptomyces albus FS12, production and
optimization of extracellular chitinase. Int J
Adv Res Biol Sci 2016; 3(4): 229-237.
33. Bhardwaj S, Bhattacharya S, Das A.
Phosphate solubilizing activity of a
mangrove isolate of Streptomyces badius
from Muthupettai mangrove, Tamil Nadu,
India. J Chem Bio Phy Sci 2012; 2(2): 868-
876.
34. Nayaka S, Vidyasagar GM. Occurrence and
extracellular enzyme potential of
Actinomycetes of a thermotolerant,
northern region of Karanataka, India. Int
Multidiscip Res J 2012; 2(12): 40-44.
35. Porsuk I, Ozakin S, Bali B, Ince-yilmaz EA.
Cellulase-free, thermoactive, and alkali
xylanase production by terrestrial
Streptomyces sp. CA24. Turkish J Biol
2013; 37: 370-375.
36. Deepthi MK, Sudhakar MS, Devamma MN.
Isolation and screening of Streptomyces sp.
from Coringa mangrove soils for enzyme
production and antimicrobial activity. Int J
Pharm Chem Biol Sci 2012; 2(1): 110-116.
37. Hamedani K, Nazanin Soudbakhsh M, Das
A, Prashanthi K, Bhattacharya S, Suryan S.
Enzymatic screening, antibacterial
potential and molecular characterization of
streptomycetes isolated from Wayanad
District in Kerala, India. Int J Pharm Biol
Sci 2012; 2(1): 201-210.
38. Kuddus SM, Ahmad RIZ. Isolation of novel
chitinolytic bacteria and production
optimization of extracellular chitinase. J
Gen Eng Biotech 2013; 11:39–46.
39. Yassien MAM, Jiman-Fatani AAM, Asfour
HZ. Production, purification and
characterization of cellulase from
Streptomyces sp. Afr J Microbiol Res 2014;
4: 348-354.
40. Ninawe S, Lal R, Kuhad RC. Isolation of
three xylanase producing strains of
actinomycetes and their identification using
molecular methods. Curr Microbiol 2006;
53: 178-182.
41. Golinska P, Dahm H. Enzymatic activity of
actinomycetes from the genus Streptomyces
isolated from the bulk soil and rhizosphere
of the Pinus sylvestris. Dendrobiology 2011;
65:37–46.
42. Rawashdeh R, Saadoun I, Mahasneh A.
Effect of cultural conditions on xylanase
production by Streptomyces sp. (strain Ib
24D) and its potential to utilize tomato
pomace. Afr J Biotechnol 2005; 4 (3): 251-
255.
43. Sivakumar T, Shankar T, Vijayabaskar P,
Ramasubramanian V. Statistical
optimization of keratinase production by
Bacillus cereus. Glob J Biotechnol Biochem
2011; 6(4): 197-202.
44. Sreenivasan N, Kumar P, Suneetha V. One
way analysis of variance of temperature for
efficient pharmaceutically exploited
microbes used in Bioremediation. Int J Drug
Dev Res 2015; 7(1): 194-200.
45. Immaneul G, Dhanusha R, Prema P,
Palavesam A. Effect of different growth
parameters on endoglucanase enzyme
activity by bacteria isolated from coir
retting effluents of estuarine environment.
Int J Environ Sci Technol 2006; 3(1): 25-34.
Cite this article as:
Monisha Khanna Kapur, Payal Das, Prateek Kumar, Munendra Kumar, Renu Solanki. Role of
Microbial Extracellular Enzymes in the Biodegradation of Wastes. J Pharm Chem Biol Sci 2018;
6(3):237-249