4. OPTIMIZATION OF PROCESS
PARAMETERS & SELECTION
OF POTENTIAL BIOCONTROL
AGENTS
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4.1. INTRODUCTION
Enzymes are widely distributed in the environment in many biological systems. These
systems may be microbial (viruses, bacteria, fungal, protozoans, yeasts and algae),
animal or plant cells. They play an indispensable role in growth, maintenance and
propagation of living systems (Kulkarni & Deshpande 2007a).
Microbial cells form a major ‘workhouses’ for production of enzymes. In the past few
decades, it has become a common practice to use micro-organisms for isolating
enzymes. They are ideal candidates for isolating enzymes because of the variety of
advantages they possess. Some of them include growing on cheap media, thereby
reducing the cost of production, fast growth rates, which leads into lesser time in
isolation and lastly, economic processes of recovery in case of extracellular enzymes
(Kulkarni & Deshpande 2007b).
Chitinases (EC 3.2.1.14) are glycosyl hydrolases, which catalyse the degradation of
chitin to give monomers of GlcNAc. These enzymes are present in a wide range of
organisms such as bacteria, fungi, insects, plants, and animals (Patil et al. 2000).
Chitinases are principally gaining interest because of extensive areas of applications.
These include many industrial and agricultural applications (Zikakis 1989; Shaikh &
Deshpande 1993; Gooday 1995).
In order to attain the maximum amount of the enzyme, a detailed investigation was
required so as to establish the most suitable fermentation medium for individual
process. The impact of process parameters also play vital role in the production of
enzymes, but certain basic requirements must be met by production medium. The
basic requirements of all the micro-organisms include water, sources of energy,
carbon, nitrogen, trace elements, possibly vitamins and oxygen for aerobic organisms
(P. Stanbury et al. 1995).
The medium must be optimized such that it satisfies the elemental requirements for
cell biomass and metabolite production. An essential requirement for cell
maintenance and biosynthesis is sufficient energy supply (P. Stanbury et al. 1995).
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Carbon forms a fundamental medium component for any fermentation process as an
energy source. It influences the rate of biomass production as well as production of
primary or secondary metabolites. Another medium constituent which influences
enzyme production is nitrogen, which is supplied in the organic or inorganic form.
Some microbes can utilize inorganic nitrogen, which is supplied as ammonia gas,
ammonium salts or nitrates (Hutner 1972). Organic nitrogen is supplied as amino
acid, protein or urea.
Another essential requirement for all micro-organisms include certain mineral
elements for growth and metabolism (Hughes & Poole 1989). In many media,
elements such as magnesium, phosphorous, potassium, sulphur, calcium and chlorine
form essential components of the production media. Other minerals like cobalt, iron,
copper, manganese, molybdenum and zinc are also essential in micro concentrations,
but are usually present as impurities in other major ingredients (P. Stanbury et al.
1995).
Buffers are also added to the production media. The control of pH is essentially
important if optimal productivity is required. Often the media are buffered around pH
7 by incorporation of calcium carbonate. Many media have phosphates as their main
constituents, which play a major role in controlling the pH (P. Stanbury et al. 1995).
Along with carbon and nitrogen sources in the media, the addition of additives like
precursors, inhibitors and inducers favour the regulation of production of the desired
product rather than the supporting the growth of micro-organisms. Addition of
precursors like chemicals to the fermentation media result in incorporation of
precursors into the desired product. When inhibitors are incorporated into production
medium, it results in production of more specific product or accumulation of the
intermediate metabolic product which gets normally metabolized. Most industrially
important enzymes are inducible in nature. These enzymes are synthesized only in
response to the presence of an inducer in the environment (P. Stanbury et al. 1995).
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Along with medium optimization, one must also opt for optimization of process
parameters (pH and temperature) such that it meets the requirement of recovering the
maximum amount of the desired product. This is because efficient growth of biomass
may not necessarily relate to the optimal production of the desired product. Hence,
different combinations of process parameters need to be investigated in order to
produce biomass, which results in formation of maximum desired product (P.
Stanbury et al. 1995).
Following media optimization, in the view of maintaining sustainable agriculture,
there is a need to continuously screen for antagonists that are able to survive in the
soil. The development of locally isolated antagonistic organisms will ensure the
success of biocontrol agent, which will be commercially viable.
One of the most commonly used approaches in order to identify the potential
antagonistic strains is to screen the potential biocontrol agents against
phytopathogenic fungi in a simplified laboratory conditions. This approach makes the
examination more accessible in understanding the ability of the isolate to act as a
biocontrol agent. This is because although the isolation of antagonistic strains is easy
as they are ubiquitous, but the selection of effective strain is difficult since only a few
of them will be disease suppressors and potential biocontrol agent.
In vitro screening methods used to test the efficacy of the isolates include –
Dual culture plate technique
Antibiosis test for production of volatile compounds
Microscopic studies
Dual culture technique gives an overall ability of the isolates to inhibit the
phytopathogen through the production of lytic enzyme such as chitinase, production
of inhibitory compounds; while antibiosis test is used to determine the ability of the
isolates to produce volatile and non-volatile metabolites to inhibit phytopathogenic
fungi.
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Following isolation and identification of chitinase producers, the present chapter dealt
with the study of optimization of cultural conditions for maximum production of the
chitinases by the selected isolates. This was investigated by the classical method of
changing one independent variable while fixing all the others at a certain level. This
was followed by a screening of isolated strains for their ability to act as biocontrol
agents against phytopathogenic fungi – Rhizoctonia solani and Fusarium oxysporum.
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4.2. MATERIALS AND METHODS
4.2.1. Enzyme activity determination
Chitinase enzyme activity was determined using modified Schale’s procedure (Imoto
& Yagishita 1971). This procedure is widely used to determine the formation of end
product as the result of enzymatic degradation of chitin or modified chitin. This
method is based on the principle of ferricyanide which in the presence of reducing
sugars loses its color. The increased concentration of reducing sugars resulted in a
decrease in the colour of the assay.
The increased amount of reducing sugar yield in loss of colour intensities of the
Schale’s reagent that was measured using Perkin Elmer UV-Vis Spectrophotometer
Lambda 25 model set at wavelength 420 nm.
A standard curve was used to extrapolate the enzyme unit activity using N-Acetyl-D-
Glucosamine (GlcNAc) as reference compound. The enzyme activity was defined as
below:
Reagents used:
20 mM acetate buffer, pH 4.6
Schale’s Reagent
Compositions:
20 mM acetate buffer, pH 4.6
Solution A (Acetic acid): 1.5 ml of glacial acetic acid was made up to 100 ml using
D/W.
Solution B (Sodium acetate solution): 2.72 g of sodium acetate trihydrate was
dissolved in 100 ml D/W.
One unit of enzyme activity was expressed as the
amount of enzyme required to liberate one
microgram of GlcNAc per minute under assay
conditions
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For 100 ml of acetate buffer at pH 4.6, 51 ml of Solution A was mixed with 49 ml of
Solution B.
Schale’s reagent
Schale’s reagent was prepared by addition of 0.5 g of potassium ferricyanide to 1 litre
of 0.5 M sodium carbonate solution.
0.5 M Sodium carbonate (Molecular weight = 105.99)
52.99 g sodium carbonate dissolved in 1000 ml D/W.
Method:
All the reaction assays were done using 20 mM acetate buffer at pH 4.6. Cell-free
supernatants were used as crude enzyme source. Enzyme activity readings were
commenced without enzyme addition, to obtain blank values. Negative control tube
contained all components except substrate. The enzyme activity was assessed using
colloidal chitin as a substrate.
The test reaction mixture consisted of 1 ml of the crude enzyme solution added to 1 %
of substrate solution in acetate buffer. This mixture was incubated at 50 oC for 30
mins. After centrifugation at 10,000 rpm for 10 mins, the supernatant was transferred
to a fresh tube. The amount of reducing sugar in the supernatant was determined by
addition of 2 ml of Schale’s reagent. The mixture was heated in boiling water bath for
15 mins. The absorbance was read at 420 nm on a Perkin Elmer UV-Vis
Spectrophotometer Lambda 25 model and the amount of reducing sugars was
quantified using standard curve.
4.2.2. Determination of protein content
The protein content of the cell-free supernatant obtained by centrifugation of the
medium (MSM+C.C) used for cultivation of the isolates was estimated using the
Lowry’s method for quantification of proteins (Lowry et al. 1951). These proteins
were used to correlate the activity of the enzyme vis-à-vis the culture of the test
organisms. The Folin-Lowry method involves formation of a complex of the protein
with Cu2+
in alkaline solution.
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Bovine Serum Albumin (BSA) was used as a standard. The absorbance of BSA
solution was measured at 650 nm on Perkin Elmer UV-Vis Spectrophotometer
Lambda 25 model. The data obtained was used to generate a standard curve for BSA
and the linear equation of the curve was used to determine the concentrations of
protein content in unknown samples.
Reagents used:
Solution A = 2 % Na2CO3 in 0.1 N NaOH
Solution B = 1 ml of 1 % CuSO4 solution + 1 ml of 2 % Sodium potassium tartarate.
Solution C = 50 ml of Solution A + 1 ml of Solution B. This solution was prepared
freshly.
Folin- Ciocalteau phenol reagent
Compositions:
2 % Na2CO3 in 0.1 M NaOH
2 g of Na2CO3 was dissolved in 100 ml of 0.1 N NaOH which was prepared by
dissolving 0.4 g of NaOH in 100 ml D/W.
1 % CuSO4 solution
1 g of CuSO4 was dissolved in 100 ml D/W.
2 % Sodium potassium tartarate
2 g of Sodium potassium tartarate was dissolved in 100 ml of D/W.
Folin-- Ciocalteau phenol reagent
This reagent was diluted with an equal volume of D/W just before use.
Method:
4 ml of Reagent C was added to all the tubes (standards/blank/unknown). The
solutions were mixed and incubated at room temperature for 15 mins. This was
followed by the addition of 0.5 ml of diluted Folin-Ciocalteau phenol reagent (1:1) to
all the tubes, followed by incubation at room temperature for 30 mins. The
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absorbance was read at 650 nm. The concentration of protein was quantified from the
standard curve.
4.2.3. Inoculum preparation for optimization of cultural condition experiments
The isolates were streaked onto NA plates to obtain 18 hours old culture of each of
the isolates under investigation. Saline suspensions of each isolates were prepared that
served as pre-cultures for inoculation in production media. 2 ml of the precultures
having 0.1 Optical Density (OD) at 600 nm was inoculated in the media which was
used for the subsequent experiments. These suspensions of the isolates were kept
constant and were used for inoculation throughout all experiments for optimization of
cultural conditions.
4.2.4. Time dependent chitinase production by selected isolates
The effect of time course on chitinase production was investigated for each selected
isolate. Pre-cultures of each of the isolate was inoculated in Minimal Salts Media
supplemented with 1 % Colloidal Chitin (MSM+C.C) and incubated at room
temperature on shaker condition (200 rpm). The enzyme activity was monitored after
every 24 hours, in terms of production of GlcNAc which was indicative of
degradation of colloidal chitin. This was done by removing 2 ml sample of culture
medium followed by centrifugation at 10,000 rpm for 20 mins at 4 oC. The resultant
supernatants obtained were used for subsequent enzyme assay and protein content
determination.
Media used:
Minimal Salts Media + 1 % Colloidal Chitin (MSM+C.C)
Table 4.1: Composition of MSM+C.C
Composition Quantity (gms/litre)
Potassium dihydrogen phosphate 7
Di potassium hydrogen phosphate 3
Magnesium sulphate 5
Ammonium sulphate 0.2
Colloidal Chitin 1
Ferrous sulphate 0.001
Zinc sulphate 0.001
Final pH (at 25 oC) 7.0 ± 0.2
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4.2.5. Production of chitinase in different media
Bacterial cells often change patterns of enzymes, in order to adapt themselves in a
specific environment. It is imperative to know the nature of the enzyme when one
aims to obtain the maximum amount of the enzyme from the isolate. Hence, this study
was carried out in an order to investigate the nature of chitinase enzyme i.e., whether
chitinase is an adaptive enzyme or constitutive enzyme.
Media used:
Minimal Salts Media (MSM)
Minimal Salts Media supplemented with 1 % Colloidal Chitin (MSM+C.C)
Nutrient Broth (NB)
Nutrient Broth supplemented with 1 % Colloidal Chitin (NB+C.C)
Compositions:
Minimal Salts Media (MSM)
Table 4.2: Composition of Minimal Salts Media (MSM)
Composition Quantity (gms/litre)
Potassium dihydrogen phosphate 7
Di potassium hydrogen phosphate 3
Magnesium sulphate 5
Ammonium sulphate 0.02
Ferrous sulphate 0.001
Zinc sulphate 0.001
D/w 1000ml
Final pH (at 25 oC) 7.0 + 0.2
Minimal salts media supplemented with 1 % Colloidal Chitin (MSM+C.C)
Refer to Table 4.1
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Nutrient Broth (NB)
Refer to Table 3.17
Nutrient Broth supplemented with 1 % Colloidal Chitin (NB+C.C)
1 g of Colloidal Chitin added to 100 ml of NB.
Method:
Each of the selected isolates were cultivated in four different types of media -
Minimal Salts Media (MSM), Minimal Salts Media supplemented with 1 % Colloidal
Chitin (MSM+C.C), Nutrient Broth (NB) and Nutrient Broth supplemented with 1 %
Colloidal Chitin (NB+C.C). 100 ml of the media were inoculated with 10 ml of
precultures of each of the isolates. The inoculated sample media was incubated at
room temperature on shaker condition (200 rpm) for five days. Post incubation period,
the media was subjected to centrifugation at 10,000 rpm for 20 mins at 4 oC and the
supernatant obtained from each of the isolate was used for performing enzyme assay.
4.2.6. Optimization of culture conditions for maximum chitinase production
Process parameters and media optimization is imperative in order to ensure optimum
growth of the organism and production of enzyme. The optimization experiments
were carried out using 25 ml MSM under shaker conditions. The factors were studied
in a sequential manner. One factor was optimized at a time. The optimal level of the
factor was incorporated into the next consequent step (Narayana & Muvva
Vijayalakshmi 2009; Paul et al. 2012) .
4.2.6.1. Investigation of effect of additional carbon source:
The effect of various additional carbon sources on production of chitinase was
investigated for each selected isolate. Glucose, Sucrose, Mannitol, GlcNAc were
different carbon sources that were supplemented at the concentration of 1 % (v/v) in
addition to the colloidal chitin in MSM. The media was inoculated with the pre-
cultures of each of the isolates. The inoculated production media (MSM) was
incubated at room temperature on shaker condition (200 rpm) for five days. After five
days of incubation, the media was subjected to centrifugation at 10,000 rpm for 20
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mins at 4 oC. The resultant supernatant from each isolate was used for subsequent
enzyme assay as mentioned earlier in Section 4.2.1.
Preparation of Carbon Sources-
10 % stock solution of the sugars - Glucose, Sucrose, Mannitol and N-acetyl-D-
Glucosamine (GlcNAc) was prepared by adding 2 g of sugar to 20 ml D/W. Each
sugar was subjected to sterilization by autoclaving at 10 psi for 10 minutes. Following
sterilization, each sugar was aseptically added to the production media such that the
final concentration of the sugar in the media was 1 %.
4.2.6.2. Influence of additional nitrogen source on chitinase production:
Different nitrogen sources, namely, two organic – peptone and tryptone; two
inorganic – ammonium chloride and ammonium sulphate were investigated for their
influence on chitinase production by each isolate. The additional nitrogen sources
were added at the concentration of 0.5 % (w/v) in the production medium along with
colloidal chitin as a sole source of carbon. The pre-culture inoculated production
media was incubated at room temperature on shaker condition (200 rpm) for five
days. Post incubation, the sample was subjected to centrifugation at 10,000 rpm for 20
mins at 4 oC. The cell-free supernatant from each isolate was used for enzyme assay
quantification (Section 4.2.1).
4.2.6.3. Optimization of MgSO4 concentration in the production media:
Trace elements play an important role in production of enzymes. Hence MgSO4
concentration was optimized in order to obtain maximum chitinase production from
each isolate. MgSO4 was added at the final concentrations of 0.04 %, 0.05 %, 0.06 %
and 0.07 % (w/v) to the production medium. The inoculated production media was
incubated at room temperature on shaker condition (200 rpm) for five days. Post
incubation, the sample was subjected to centrifugation at 10,000 rpm for 20 mins at 4
oC. The resultant supernatant from each isolate was further used for performing
enzyme assay as described in Section 4.2.1.
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4.2.6.4. Optimization of initial pH of the production media:
The initial pH optimization for each of the isolate was investigated by varying the pH
of the medium (pH 5-9) with the help of 1M NaOH or 1M HCl. The production media
was inoculated with precultures of selected isolates and incubated at room
temperature on shaker condition (200 rpm) for five days. Post incubation period, the
cell-free supernatant from each isolate was used for subsequent enzyme assay
described earlier (Section 4.2.1).
4.2.6.5. Optimization of incubation temperature:
The influence of temperature on chitinase production was assessed for each isolate by
varying the incubation temperature (25-45 oC). Each isolate was cultivated in
production media and the incubated at different temperatures on an incubator-shaker
(200 rpm) for five days. After which the sample was subjected to centrifugation at
10,000 rpm for 20 mins at 4 oC to obtain cell-free supernatant which was further used
for enzyme assay as described earlier (Section 4.2.1).
4.2.6.6. Optimization of substrate concentration:
To optimize the substrate (colloidal chitin) concentration of the production medium,
varying concentrations of colloidal chitin (0.10-2 % w/v) were supplemented to
production media. Incubation was carried out at an optimized temperature and pH on
shaker condition (200 rpm) for five days. The cell-free supernatant obtained after
incubation period from each isolate was used for enzyme assay (Section 4.2.1).
4.2.7. Statistical analysis
All the optimization studies were conducted in triplicate and the data was analysed for
significance of difference using single factor analysis of variance (ANOVA). This
was followed by a comparison of means by using Tukey’s Range test. The data is
graphically presented as the mean ± S.D. of triplicates. Analysis was performed using
GraphPad Prism Software version 5.0. P values < 0.05 were considered significant.
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4.2.8. Selection of potential bio-control agents
4.2.8.1. Fungal phytopathogens:
Two fungal phytopathogens were used in this study: Rhizoctonia solani and Fusarium
oxysporum. They were procured from Microbial Type Culture Collection (MTCC)
and Gene Bank, Institute of Microbiology (IMTECH), Chandigarh, India.
Fusarium oxysporum was procured in lyophilized form, whereas Rhizoctonia solani
was procured on Potato Dextrose agar slant. The lyophilized culture was revived on
solid Potato Dextrose agar and liquid broth medium and subsequently cultured on
Potato Dextrose agar slant.
Both the fungal cultures were maintained on Potato Dextrose medium (Hi Media,
India) at 4 oC, and sub-cultured every 30 days as recommended by MTCC
Chandigarh.
Media used:
Potato Dextrose Broth
Potato Dextrose Agar (PDA)
Compositions:
Potato Dextrose Broth
Table 4.3: Composition of Potato Dextrose Broth
Composition (24 gms/1000 ml) Quantity (gms/liter)
Potatoes, infusion form 200
Dextrose 20
Distilled water 1000 ml
Final pH (at 25 oC) 5.1 ± 0.2
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Potato Dextrose Agar (PDA)
Table 4.4: Composition of Potato Dextrose Agar
Composition (39 gms/1000 ml) Quantity (gms/liter)
Potatoes, infusion form 200
Dextrose 20
Agar 15
Distilled water 1000 ml
Final pH (at 25 oC) 5.6 ± 0.2
4.2.9. In vitro dual culture technique
In vitro dual culture assay was performed to assess the potential of the selected
isolates to act as a biocontrol agent by virtue of its lytic action on the chitin
component of the cell walls of two fungal phytopathogens: Rhizoctonia solani and
Fusarium oxysporum.
This test was performed as described by Huang & Hoes (1976) with minor
modifications.
Media used:
Nutrient Agar
Luria Bertani Agar.
Compositions:
Nutrient Agar
Refer to Table 3.1
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Luria Bertani Agar
Table 4.5: Composition of Luria Bertani Agar
Composition (40 gms/1000 ml) Quantity (gms/liter)
Casein enzymic hydrolysate 10.0
Yeast extract 5.0
Sodium chloride 10.0
Agar 15.0
Final pH (at 25 oC) 7.5 ± 0.2
Method:
Each selected isolates were streaked onto sterile Nutrient agar plates and incubated
overnight at 37 oC. Saline suspensions of each of the isolate were prepared from 18
hour old culture as per the 0.5 McFarland turbidity standards to obtain approximate
cell density of 107-8
cells/ml. Each of the isolate was inoculated at the center of the
plates. Six mm of actively growing phytopathogen was aseptically placed on a sterile
LB agar plate in such a manner that they would lie opposite to each other at the
corners of the plate. The plates were incubated at room temperature for the period of
five days to allow sufficient growth of the colonies. The radial growth of fungus
towards the chitinolytic isolate was examined daily for the period of five days for both
test and control plates, and percentage inhibition was calculated. For the control plate,
phytopathogenic fungi were inoculated on the fresh LB agar plate without the selected
isolates. The experiment was conducted in triplicates.
KEY: C= radial growth of the fungi in absence of bacteria (control); T= radial
growth of the fungi in presence of bacteria (test).
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4.2.10. Microscopic studies
Microscopic studies were taken up so as to assess the damage caused by the
chitinolytic isolate to the fungal mycelia in the dual culture technique.
Stain used:
Lactophenol cotton blue (Hi Media, India)
Table 4.6: Composition of lactophenol cotton blue
* indicates toxic/corrosive material
Method:
The morphological changes in the fungal mycelia were observed using light
microscopy according to the procedure described by Saleem & Kandasamy (2002).
The fungal phytopathogen, inhibited by the growth of the chitinolytic isolate was
prepared for the microscopic examination. The mycelia present around the zone of
inhibition from the test plate and the mycelia from the control plate were taken onto
separate slides. They were stained using lactophenol cotton blue and observed under
the high power lens having a magnification of 400X.
4.2.11. Antagonism through production of volatile compounds
The antagonistic isolates were checked qualitatively for the ability to produce volatile
compounds. This investigation served to determine whether the antagonists isolates
were able to produce volatile compounds, since they are implicated to be involved in
antagonistic activity against phytopathogenic fungi along with lytic enzyme like
chitinase (Benítez et al. 2004; Ganesan & Sekar 2010). This was studied by
performing the protocol as described by Lahlali and Hijri (2010).
Composition Quantity (gms/20ml)
*Phenol crystals 20
Cotton Blue 0.05
*Lactic Acid 20 ml
Glycerol 20 ml
Distilled water 20 ml
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Media used:
Potato Dextrose Agar (PDA)
Composition of Potato Dextrose Agar
Refer to Table 4.4
Method:
Sterile PDA plates were inoculated in the center with actively growing 6 mm disc of
fungal phytopathogen and saline suspension (107-8
cells/ml) of overnight grown
culture of bacterial isolates (antagonist) separately. The lid of each of the plate was
removed and the base of the plates containing fungal phytopathogen and a bacterial
isolate were placed on top of the other plate facing each other. The two plate bases
were then sealed with a double layer of parafilm. All plates were incubated at room
temperature for the period of five days and observed for inhibition of the
phytopathogenic fungus by antagonist bacterial culture. Controls were prepared using
the same experimental setup, except that loopful of sterile D/W was streaked instead
of the antagonist (bacterial) culture.
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4.3. RESULTS AND DISCUSSION
Enzyme activity determination:
A standard curve was prepared to quantify the chitinase activity by extrapolation of
reaction data gathered. The experiment was carried out in triplicates and the readings
demonstrated good reproducibility of the N-Acetyl-D-Glucosamine in the
concentration range of 0-500 µg/ml.
The data obtained demonstrated a graph with regression of 0.9975 as observed in the
Figure 4.1. The equation obtained was used to determine the unit/ml activity of
chitinase enzyme.
The modified Schale’s procedure is reported to be widely used to determine the
formation of reducing product, which in the present case was N-Acetyl-D-
Glucosamine, formed during the enzymatic degradation of chitin or modified chitins
(Jarle Horn & Eijsink 2004). The assay resulted in the loss of the colour intensity as
the presence of reducing sugars increased in the test solution.
Figure 4.1: Standard graph for Schale’s procedure using GlcNAc as standard.
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Determination of Protein content:
A standard curve was plotted to quantify the protein content by extrapolation of the
data. The experiment was performed in triplicates and the data obtained demonstrated
good reproducibility of bovine serum albumin (BSA) between the ranges of 0 – 250
µg/ml.
The data obtained demonstrated a graph with regression of 0.9946 as observed in the
Figure 4.2. The equation obtained was used to determine the mg/ml protein in the
sample.
Figure 4.2: Standard graph for Folin-Lowry procedure using BSA as standard.
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Time dependent chitinase production by the selected isolates:
The profiles of enzyme production of each of the chitinase producing isolates were
assessed for the period of ten days. Each selected chitinolytic isolates were cultivated
in MSM+C.C medium and incubated under shaker conditions. The enzyme activity
was monitored in terms of production of N-Acetyl–D-Glucosamine (GlcNAc) which
was indicative of degradation of colloidal chitin in the medium. This was followed by
quantification of protein content.
The effect of time on chitinase production is represented in the Figures 4.3 and 4.4.
Previous reports reflect that the incubation time in order to achieve the maximum
enzyme level is governed by the characteristic of the culture. It is based on growth
rate and enzyme production by the culture (Sharmistha et al. 2012). The monitoring of
growth rate for each culture, however, was difficult because of the presence of
colloidal chitin in the medium which co-sedimented after centrifugation and resulted
in the inference with the actual biomass determination. Hence, extracellular protein
content was monitored which was used as an indirect measure of growth for each
culture.
The result obtained showed that the effect of incubation time influences enzyme
production, wherein, maximum enzyme production was observed on the fifth day of
incubation for all the selected isolates. The study demonstrated that each isolate
steadily produced chitinase which reached maximum level on the fifth day of
incubation after which it started decreasing. The level of chitinase steadily increased
in the exponential phase and was detected in the stationary phase for each isolate. A
sharp decline in the activity was observed in the late stationary phase of the cultures.
The plausible explanation for this phenomenon may be the depletion of nutrients in
the medium. The reduction in chitinase activity might also be caused by protein
degradation or inactivation by unclear mechanisms. The degradation of the product
may also be the likely reason for the decrease in chitinase activity.
As observed from the level of protein content, it was evident that the growth of
microbial culture was slow at the beginning which exponentially increased after 96 h
(i.e. 4 days) post incubation. This phenomenon also resulted in the exponential rise in
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chitinase activity which was detected in the maximum amount in the late log phase of
the culture, sharply declining at the late stationary phase. Thus chitinase production
correlated to the growth of the microbial culture in the medium. These findings were
in accordance with previous report by Priya et al. (2011) which exhibited the rise in
chitinase production by Streptomyces hygroscopicus culture from exponential growth
phase to stationary growth phase.
The investigation by Mane & Deshmukh (2009) reported detectable levels of
chitinase by M. brevicatiana at 4th
day post incubation. Similarly Wiwat et al. (1999)
have reported maximum production of chitinase by Bacillus circulans no. 4.1 at 4th
day. Highest chitinase production after 5 days of incubation has also been reported in
Streptomyces spp. NK1057 (Nawani & Kapadnis 2004) and Beauveria bassiana
(Suresh & Chandrasekaran 1998).
Nagpure & Gupta (2013) detected chitinase production in the culture broth of
Streptomyces violaceusniger after 2 days of incubation, which progressively increased
till the 4th
day after which it decreased. Wang & Hwang (2001) also reported
maximum chitinase production by B. cereus, B. alvei and B. sphaericus at 2 days of
incubation. A report by Shanmugaiah et al. (2008) demonstrated maximum chitinase
production by B. laterosporus MML2270 on the 4th
day post incubation.
The maximum chitinase production was observed on fifth day post incubation for all
the selected isolates under investigation, thus the incubation period of five days was
chosen to test the effect of different parameters on the production of chitinase. After
the required period of incubation, the cell free supernatant was taken and assessed for
enzyme activity. The findings from time course investigation, aided to study the
enzyme production profile of each selected isolate which assisted further in
optimization studies.
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Isolate 1- Bacillus cereus
0 1 2 3 4 5 6 7 8 9 100
5
10
15
20
0.00
0.02
0.04
0.06
0.08
14.21 U/ML
Time (days)
En
zym
e a
cti
vit
y U
/ML
Pro
tein
co
nte
nt m
g/m
l
Isolate 2- Cellulosimicrobium cellulans
0 1 2 3 4 5 6 7 8 9 100
5
10
15
0.00
0.02
0.04
0.06
0.0813.07 U/ML
Time (days)
En
zym
e a
cti
vit
y U
/ML
Pro
tein
co
nte
nt m
g/m
l
A
B
Figure 4.3: Time course study for maximum chitinase production.
Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans (B). The
values are represented as Mean ± S.D. Each of the experiment was performed in
triplicates.
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Isolate 3 - Bacillus cereus strain NOC2011
0 1 2 3 4 5 6 7 8 9 100
2
4
6
8
10
0.00
0.02
0.04
0.068.88 U/ML
Time (days)
En
zym
e a
cti
vit
y U
/ML
Pro
tein
co
nte
nt m
g/m
l
Isolate 4- Bacillus licheniformis
0 1 2 3 4 5 6 7 8 9 100
5
10
15
20
0.00
0.01
0.02
0.03
0.04
0.05
15.04
U/ML
Time (days)
En
zym
e a
cti
vit
y U
/ML
Pro
tein
co
nte
nt m
g/m
l
A
B
Figure 4.4: Time course study for maximum chitinase production.
Isolate 3: Bacillus cereus strain NOC2011 (A); Isolate 4: Bacillus licheniformis (B).
The values are represented as Mean ± S.D. Each of the experiment was performed in
triplicates.
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Production of chitinase in different media:
In order to investigate the nature of chitinase enzyme, the isolates were cultivated in
different media and the production of chitinase was examined. The results of this
investigation have been presented in Figure 4.4 and have been depicted in the Table
4.7.
Previous works on regulation of chitinase in different organisms have shown that
chitinase enzymes can be induced, suggesting the inducive nature of chitinase
(Monreal & Reese 1969; Bassler et al. 1991; Robbins et al. 1992; Melentiev et al.
2014). Similar observations were recorded in the present investigation; all the isolates
under investigation produced maximum chitinase when cultivated in the medium
containing colloidal chitin, implying that colloidal chitin acted as an inducer which
increased the production of chitinase enzyme.
Low levels of chitinase were also detected in the MSM devoid of colloidal chitin by
the isolates 1, 2 and 3. This phenomenon suggested that chitinase was produced
constitutively in low levels by these isolates. The plausible explanation of this
occurrence remains to be established, it appears probably due to the stress conditions
inducing the enzyme production or the conditions of carbon starvation and poor
growth of the culture activating the production of hydrolytic enzyme. Similar
observation has been reported by Tweddell et al. (1994) who investigated the
production of chitinases and β-1,3-glucanases by Stachybotrys elegans under various
culture conditions. This phenomenon was also observed by Gupta et al. (1995) who
investigated the chitinase production by S. viridificans, they reported minimum levels
of constitutive production of chitinase with both simple and complex carbon substrate.
Sharmistha et al. (2012) stated the nature of chitinases as both constitutive and
adaptive enzyme, producing chitinase in the absence or presence of substrates. They
also reported, however, the addition of chitin in the media to significantly increase
enzyme production. Several isolates of the Aeromonas species have been shown to
produce constitutive as well as inducible chitinase (Singh & Sanyal 1992).
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Techkarnjanaruk et al. (1997) reported the induction of the chitinase gene promoter
by starvation conditions in the marine bacterium Pseudoaltermonas spp. strain S9.
Chitinase synthesis induction in Trichoderma harzianum has also been reported by
Ulhoa & Peberdy (1991) in a chitin containing medium, suggesting the inducible
nature of the enzyme. The absence of chitinase activity by all the isolates cultivated
in the nutrient broth medium indicated towards the catabolite repression of the
enzyme by the nutrient composition of the medium.
The information derived from this investigation highlighted the nature of chitinase
enzyme produced by each selected isolate which can be further employed while
formulating an optimum medium for maximum enzyme production on a large scale
basis.
Table 4.7: Chitinase production (U/ML) of selected isolates in different media.
MSM MSM + C.C NB NB + C.C
Isolate 1
B. cereus 8.033 ± 0.15 14.91 ± 0.67 0 ± 0.00 15.76 ± 0.46
Isolate 2
C. cellulans 9.43 ± 1.11 15.66 ± 0.05 0.00 ± 0.00 11.93 ± 0.47
Isolate 3
B. cereus strain
NOC2011
11.66 ± 0.32 15.55 ± 0.25 0.00 ± 0.00 15.36 ± 0.12
Isolate 4
B. licheniformis 0.00 ± 0.00 14.38 ± 0.22 0.00 ± 0.00 14.27 ± 0.26
* The values are expressed as Mean ±S.D.
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Isolate 1- Bacillus cereus
MSM
MSM
+C.C N
B
NB+C
C
0
5
10
15
20
a aE
nzym
e a
cti
vit
y U
/ML
Isolate 2- Cellulosimicrobium cellulans
MSM
MSM
+C.C N
B
NB+C
.C
0
5
10
15
20
En
zym
e a
cti
vit
y U
/ML
Isolate 3 - Bacillus cereus strain NOC2011
MSM
MSM
+C.C N
B
NB+C
.C
0
5
10
15
20
a a
En
zym
e a
cti
vit
y U
/ML
Isolate 4- Bacillus licheniformis
MSM
MSM
+C.C N
B
NB+C
.C
0
5
10
15
20
a aE
nzym
e a
cti
vit
y U
/ML
A B
C D
Figure 4.5: The study of chitinase production in different media.
Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans (B); Isolate 3:
Bacillus cereus strain NOC2011 (C); Isolate 4: Bacillus licheniformis (D). The values
are represented as Mean ± S.D. Each of the experiment was performed in triplicates.
Columns marked with same letters denote the means are not significantly different
from each other.
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Optimization of process parameters for maximum chitinase production:
Medium optimization is imperative in order to ensure optimum growth of the
organism so as to achieve maximum production of enzyme. Media optimization
ensures the optimal supply of nutrients to the organisms in order to meet its nutritional
requirements. An ideal production medium must sufficiently supply energy for cell
growth and biosynthesis (P. Stanbury et al. 1995). Earlier reports suggest that carbon
and nitrogen source amendments differentially influence bacterial growth, which in
turn affects production of enzymes and secondary metabolites and subsequently
biocontrol activity (Slininger & Shea-Wilbur 1995; Meidute et al. 2008).
The optimization of media and process parameters for maximum chitinase production
from each isolate was investigated by adopting component replacing for medium
constituents and one factor at a time approach for process parameters. There are
several statistical and non-statistical methods for optimization of media amongst
which Plackett-Burman and Response Surface Methodology (RSM) are most widely
used. They offer the advantage of reducing time and expense, however, the use of
Plackett-Burman design is either decided by literature survey or by random selection.
Before statistical optimization of medium for the production of desired product from a
new source of bacterium, it is essential to screen the possible medium constituents.
One factor at a time approach generates information on medium components in order
to form the desired product from the organism under study and can also identify new
components affecting its production (Singh 2010).
The optimization of the cultural conditions for maximum chitinase production was
carried out using Minimal Salts Media (MSM) and gradually incorporated with the
ingredients that were investigated. The optimized parameter was incorporated into
subsequent experiments.
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Investigation of effect of additional carbon source:
The production of chitinase was investigated for all the selected isolates under the
influence of different additional carbon sources and colloidal chitin alone. Glucose,
sucrose, mannitol, and GlcNAc (1 % w/v) were supplemented along with colloidal
chitin and chitinase production was investigated. A control flask was maintained,
which contained medium supplemented with colloidal chitin as a sole source of
carbon.
Amongst all the carbon sources investigated, it was observed that addition of simple
sugars like glucose, mannitol, GlcNAc and sucrose in the medium suppressed
chitinase activity as relatively little or no chitinase activity was observed from all the
selected isolates. Thus, it was confirmed that chitinase is an inducible enzyme and the
suppressed chitinase activity is due to the availability of easily metabolized sugars in
the production medium which may have resulted in catabolite repression or carbon
competition. Maximum chitinase production was observed for all the isolates when
the medium was supplemented with colloidal chitin alone. Similar observation has
been reported for Paenibacillus spp. D1 (Singh 2010), M. timonae (Faramarzi et al.
2009), Aeromonas spp. (Ahmadi et al. 2008) and Serratia marcescens (Sharmistha et
al. 2012). High levels of chitinase production in M. verrucaria were observed with
chitin supplementation and no detectable activity was observed when the medium was
supplemented with lactose, maltose, sucrose, chitosan, starch and cellulose (Vyas &
Deshpande 1989). Recently, Melentiev et al. (2014) reported repression of chitinase
production by Bacillus spp. IB-OR-17 in the presence of 1 % glucose as carbon
source.
A previous investigation of St Leger et al. (1986) reported repression of chitinase
production under the influence of GlcNAc. Another report by Tweddell et al. (1994)
also reported no chitinase production when glucose, sucrose or GlcNAc was used as
carbon sources. Induction of chitinase by colloidal chitin was reported previously by
Dhar & Kaur (2010). Colloidal chitin contains minute amounts of GlcNAc that helps
to induce chitinase production initially, but it has been reported that high
concentration of GlcNAc in medium causes catabolite repression (Campos et al.
2005). The data obtained from this investigation suggested that colloidal chitin was
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indispensable and served as an inducer for chitinase production. In order to
investigate and identify the regulatory elements of chitinolytic enzymes further
studies are required.
Investigation of additional nitrogen source:
The influence of different nitrogen sources on chitinase production was investigated
for all the chitinolytic isolates, since nitrogen regulation is crucial in industrial
microbiology as it affects the synthesis of enzymes.
The results of the study indicated that peptone was the best nitrogen source for highest
chitinase production by all the selected isolates (Figure 4.6). The study reflected that
in comparison to inorganic sources such as ammonium chloride and ammonium
sulphate, the organic nitrogen sources – peptone and tryptone served as superior
supplements for chitinase production. The increase in the chitinase activity due to
peptone is likely because crude organic peptone contains nitrogenous compounds,
growth factors and oligomers of GlcNAc in minute amounts which may have a
stimulatory effect on cell growth (Nawani & Kapadnis 2005). It is assumed that the
nitrogenous compounds and growth factors support cell growth, increasing the initial
cell growth leading to excretion and accumulation of chitinase in the medium. The
enzyme productions of the selected isolates have been summarized in the Table 4.8.
In confirmation to present finding, similar results have been previously reported. The
increase in chitinase activity under the influence of peptone has been reported in
Bacillus licheniformis strain (Mohd Akhir et al. 2009), Streptomyces spp. Da11 (Lee
et al. 2008) and Pantoea dispersa (Gohel, Chaudhary, et al. 2006). Earlier other
organic nitrogen source like yeast extract has been reported to enhance chitinase
production in Beauveria bassiana (Suresh & Chandrasekaran 1998), Serratia
marcescens (Monreal & Reese 1969), Alcaligens xylosoxydans (Vaidya et al. 2003)
and Trichoderma harzianum (Nampoothiri et al. 2004); urea has been reported to
increase chitinase production in Myrothecium verrucaria (Vyas & Deshpande 1989).
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In contrast to present findings, a report by Chaiharn et al. (2013) demonstrated that
ammonium sulphate was most favourable nitrogen source for chitinase production.
Enhanced chitinase production due to the addition of inorganic source such as
ammonium sulphate and sodium nitrate has been reported for Aspergillus spp. SI-13
(Rattanakit et al. 2002) and Stachybotrys elegans (Tweddell et al. 1994).
Table 4.8: Enzyme production (U/ML) by selected isolates under the influence of
different nitrogen sources
Ammonium
sulphate
Ammonium
chloride
Peptone Tryptone
Isolate 1
B. cereus 11.2 ± 0.87 12.66 ± 0.95 17.08 ± 0.10 14.58 ± 0.47
Isolate 2
C. cellulans 12.62 ± 0.67 10.32 ± 1.10 16.54 ± 0.51 14.45 ± 0.40
Isolate 3
B. cereus strain
NOC2011
12.33 ± 0.30 8.59 ± 0.47 14.19 ± 0.16 11.69 ± 0.26
Isolate 4
B. licheniformis 13.5 ± 0.35 7.30 ± 1.11 15.97 ± 0.46 13.93 ± 0.51
* The values are expressed as Mean ± S.D
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Isolate 1- Bacillus cereus
Am
moniu
m s
ulphat
e
Am
moniu
m c
hloride
Pep
tone
Trypto
ne
0
5
10
15
20
aa
En
zym
e a
cti
vit
y U
/ML
Isolate 2- Cellulosimicrobium cellulans
Am
moniu
m s
ulphat
e
Am
moniu
m c
hloride
Pep
tone
Trypto
ne
0
5
10
15
20
a
a
En
zym
e a
cti
vit
y U
/ML
A B
Am
moniu
m s
ulphat
e
Am
moniu
m c
hloride
Pepto
ne
Trypto
ne
0
5
10
15
20
aa
Isolate 3 - Bacillus cereus strain NOC2011
En
zym
e a
cti
vit
y U
/ML
Isolate 4- Bacillus licheniformis
Am
moniu
m s
ulphat
e
Am
moniu
m c
hlorid
e
Pepto
ne
Trypto
ne
0
5
10
15
20
a a
En
zym
e a
cti
vit
y U
/ML
C D
Figure 4.6: The effect of different nitrogen sources on chitinase production.
Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans (B); Isolate 3:
Bacillus cereus strain NOC2011 (C); Isolate 4: Bacillus licheniformis (D). The values
are represented as Mean ± S.D. Each of the experiment was performed in triplicates.
Columns marked with same letters denote the means are not significantly different
from each other.
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Optimization of MgSO4 concentration:
Trace elements play a crucial role in the production of enzymes, divalent cations like
Mg act as a co-factor for the production of several enzymes; they also play an
important role in the cell mass build-up, enzyme activity and stability. MgSO4
concentrations in the production medium were optimized for all the isolates by
cultivating each isolate in presence of different concentrations of MgSO4
concentrations i.e., 0.04 %, 0.05 %, and 0.06 % (w/v).
Chitinase activity of selected chitinolytic isolates at different MgSO4 concentrations
have been summarized in the Table 4.9 and depicted in Figures 4.7 & 4.8. The study
demonstrated the effect of different MgSO4 concentration on chitinase production.
Each isolate exhibited a different preference of MgSO4 concentration which favoured
maximum chitinase production.
Isolates 1 (Bacillus cereus) and 4 (Bacillus licheniformis) produced maximum
chitinase in the range of 0.05-0.06 % MgSO4 whereas 0.06 % MgSO4 concentration
favoured maximum chitinase production for Isolate 3 (Bacillus cereus strain
NOC2011). Isolate 2 (Cellulosimicrobium cellulans) produced maximum chitinase at
0.05 % of MgSO4.
Scientific data regarding the effect of MgSO4 concentrations on chitinase activity is
limited; however, some studies report the effect of MgSO4 on chitinase production.
Previous reports have stated the positive effect of MgSO4 on chitinase production.
The observations recorded in the present investigation were in agreement with
previous work by Tasharrofi et al. (2011) which stated that MgSO4 concentration can
assist in increase in chitinase production. Nawani & Kapadnis (2004) reported trace
elements as an important factor that affected chitinase production in strains of
Streptomyces spp. such as NK1057, NK528 and NK951. Lee et al. (2008) and Gohel,
Singh, et al. (2006) also reported the positive effect of MgSO4 on chitinase production
by Streptomyces and P. dispersa respectively.
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Table 4.9: The influence of different MgSO4 concentrations on enzyme
production (U/ML) by the isolates
MgSO4 Concentrations
0.04% 0.05% 0.06%
Isolate 1
B. cereus 13.68 ± 0.73 17.02 ± 0.01 16.53 ± 0.47
Isolate 2
C. cellulans 12.65 ± 1.29 16.20 ± 0.43 13.57 ± 0.55
Isolate 3
B. cereus strain
NOC2011
11.78 ± 0.43 13.52 ± 0.71 16.96 ± 0.054
Isolate 4
B. licheniformis 11.36 ± 0.60 15.97 ± 0.46 16.3 ± 0.96
*The values are expressed as Mean ± S.D.
Isolate 1- Bacillus cereus
0.04
0.05
0.06
0
5
10
15
20a a
MgSO4 Concentrations (%)
En
zym
e a
cti
vit
y U
/ML
Isolate 2- Cellulosimicrobium cellulans
0.04
0.05
0.06
0
5
10
15
20
a a
MgSO4 Concentrations (%)
En
zym
e a
cti
vit
y U
/ML
A B
Figure 4.7: The effect MgSO4 concentrations on chitinase production.
Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans (B). The
values are represented as Mean ± S.D. Each of the experiment was performed in
triplicates. Columns marked with same letters denote the means are not significantly
different from each other.
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Isolate 3 - Bacillus cereus strain NOC2011
0.04
0.05
0.06
0
5
10
15
20
MgSO4 Concentrations (%)
En
zym
e a
cti
vit
y U
/ML
Isolate 4- Bacillus licheniformis
0.04
0.05
0.06
0
5
10
15
20
aa
MgSO4 Concentrations (%)
En
zym
e a
cti
vit
y U
/ML
A B
Figure 4.8: The effect MgSO4 concentrations on chitinase production.
Isolate 3: Bacillus cereus strain NOC2011 (A); Isolate 4: Bacillus licheniformis (B).
The values are represented as Mean ± S.D. Each of the experiment was performed in
triplicates. Columns marked with same letters denote the means are not significantly
different from each other.
Optimization of initial pH of the production media:
Micro-organisms are sensitive to the concentration of H+ ions present in the medium.
The initial pH of the production media has an impact over the availability of
metabolic ions. Hence, the initial pH of the production media was optimized by
cultivating each selected isolates in the medium with different pH ranging from 5 to 9.
The results obtained on the effect of pH on chitinase production of the selected
chitinolytic isolates have been summarized in the Table 4.10 and illustrated in Figure
4.9. The investigation revealed that each selected isolate preferred different pH for
maximum chitinase synthesis.
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Isolates 1 and 3 which were identified as B. cereus and B. cereus strain NOC2011
favoured maximum chitinase production between a pH range of 6-7; isolate 4 which
was identified as B. licheniformis preferred the pH range of 7-8 whereas isolate 2, C.
cellulans demonstrated pH 7 as optimum pH for chitinase production. Previous report
state that a majority of bacteria produce maximum chitinase at neutral or slightly
acidic pH (Mathivanan et al. 1998). This statement was supported by the observations
recorded in the current study wherein Isolates 1 and 3 preferred slightly acidic pH for
chitinase production. These results were in agreement with previous report by Patil et
al. (2000) which state that chitinase production from B. circulans WL-12 and Bacillus
strain MH-1 was optimum at acidic conditions. Rattanakit et al. (2002) also reported
pH 5 or 6 for maximum chitinase productivity by Aspergillus spp. S1-13. It is also
known from previous reports which states that the enzyme binds very specifically to
colloidal chitin at low pH values, resulting in high levels of enzyme activity (Aziz et
al. 2012).
Other studies report nearly neutral pH as optimum pH for chitinase produced by
certain Bacillus strains and Pseudomonas aeruginosa K-187 (Wang & Chang 1997;
Chang et al. 2003; Yuli et al. 2004; Ghorbel-Bellaaj et al. 2011).
Although chitinase production was observed at a slightly acidic pH, in case of Isolate
4 (B. licheniformis), it favoured maximum chitinase production between pH 7-8. The
results of the present investigation were in accordance with the report, which states
pH 7 and 8 to be optimum for chitinase production by Bacillus subtilis (Karunya et al.
2011). Frändberg & Schnürer (1994) have reported pH 8 as optimum pH for
maximum chitinase production from B. pabuli K1. Another investigation by
Shanmugaiah et al. (2008) reports optimum pH for chitinase production by B.
laterosporus to be 8. Alkaline conditions favouring maximum chitinase production
has been recently reported from A. hydrophilia strain (Saima & Roohi 2013). Previous
reports also suggested that Micrococcus spp. AG84 (Annamalai et al. 2010), A.
xylosoxydans (Vaidya et al. 2001), Serratia marcescens XJ-01 (Xia et al. 2011) and
Aeromonas spp. JK1 (Ahmadi et al. 2008) are proficient of maximum chitinase
synthesis at alkaline condition.
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Isolate 2, identified as C. cellulans produced maximum chitinase at pH 7. This result
coincides with previous report by Fleuri et al. (2009) which stated that the pH of the
medium oscillated between 6.6 and 7.3 during chitinase production by C. cellulans
191.
Slight acidic pH has been reported to be favourable for chitinase production by
Streptomyces strain and T. harzianum (Ulhoa & Peberdy 1993; Jagadeeswari &
Selvam 2012) whereas neutral pH has favoured chitinase synthesis in S. marcescens
and S. thermoviolaceus (Tsujibo et al. 1993; Sharmistha et al. 2012)
The study revealed that the pH of the medium strongly affects the growth and activity
of microorganisms. It was also apparent that pH maintenance plays a crucial role in
conserving the enzymatic activity in the medium.
Table 4.10: Enzyme production (U/ML) by chitinolytic isolates under the
influence of various pH
pH
5 6 7 8 9
Isolate 1
B. cereus
10.90
±
0.85
15.72
±
0.67
16.14
±
0.13
14.52
±
1.08
8.98
±
0.04
Isolate 2
C. cellulans
8.59
±
0.38
14.18
±
0.72
14.88
±
0.79
12.69
±
0.94
10.46
±
0.41
Isolate 3
B. cereus strain
NOC2011
13.57
±
0.52
15.06
±
0.78
16.14
±
0.13
13.85
±
0.91
12.65
±
0.55
Isolate 4
B. licheniformis
0.00
±
0.00
10.74
±
0.42
12.21
±
0.70
12.86
±
0.92
5.80
±
0.71
* The values are represented as Mean ± S.D.
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Isolate 1- Bacillus cereus
pH 5
pH 6
pH 7
pH 8
pH 9
0
5
10
15
20
aa
En
zym
e a
cti
vit
y U
/ML
Isolate 2- Cellulosimicrobium cellulans
pH 5
pH 6
pH 7
pH 8
pH 9
0
5
10
15
20
aa
En
zym
e a
cti
vit
y U
/ML
Isolate 3 - Bacillus cereus strain NOC2011
pH 5
pH 6
pH 7
pH 8
pH 9
0
5
10
15
20
ab
a,c a,c
b
En
zym
e a
cti
vit
y U
/ML
Isolate 4- Bacillus licheniformis
pH 5
pH 6
pH 7
pH 8
pH 9
0
5
10
15
aa,b
b
En
zym
e a
cti
vit
y U
/ML
A B
C D
Figure 4.9: The effect of various pH on chitinase production
Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans (B); Isolate 3:
Bacillus cereus strain NOC2011 (C); Isolate 4: Bacillus licheniformis (D). The values
are represented as Mean ± S.D. Each of the experiment was performed in triplicates.
Columns marked with same letters denote the means are not significantly different
from each other.
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Optimization of incubation temperature:
The influence of temperature on chitinase production was investigated by cultivating
isolates under study at different temperatures such as 25 oC, 30
oC, 35
oC, 40
oC and
45 oC in order to obtain maximum chitinase. The results of the effect of temperature
on chitinase production by selected isolates have been presented in Table 4.11 and
depicted in Figure 4.10.
This examination demonstrated the mesophilic preference of each isolate for
production of chitinase. The incubation temperature of 35 oC was found to be
beneficial for Isolates 1 (B. cereus) and 3 (B. cereus strain NOC2011) whereas the
range of 30-40 oC was optimum for production of chitinase by Isolate 2 (C. cellulans)
and Isolate 4 (B. licheniformis) favoured maximum chitinase productions in the range
30-35 oC. Increase in temperature resulted in no chitinase production. It was assumed
that impact of temperature on chitinase production is related to the growth of the
organisms. Temperature is also responsible for influencing protein denaturation, cell
growth and enzyme inhibition, thus playing significant role in biological processes
(Sharmistha et al. 2012).
The optimum temperature for chitinase production by B. cereus and B. cereus strain
NOC2011 was 35 oC whereas B. licheniformis demonstrated maximum chitinase
synthesis in the range 30-35 oC. These observations were in complete agreement with
previous report by Gomaa (2012) which state 30 oC as optimum temperature for
chitinase production by B. licheniformis and B. thuringiensis. Another study by
Shanmugaiah et al. (2008) also state 35 oC as the optimum temperature for B.
laterosporus to produce maximum chitinase. Das et al. (2012) also reported 35 oC as
the optimum temperature for chitinase production by B. amyloliquefaciens SM3
strain. Wang et al. (2006) reported 37 oC as optimum for chitinase synthesis by B.
subtilis W-118.
Chitinase synthesis significantly increased in the temperature range of 30-40 oC by C.
cellulans (isolate 2). Presently there are no scientific reports available which
investigate the influence of temperature on chitinase production by C. cellulans.
However, Fleuri et al. (2009) reportedly studied the production of extracellular
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chitinase from Cellulosimicrobium cellulans strain 191 which gave the maximum
yield of chitinase after 72h of cultivation at 25 oC. Saima & Roohi (2013) have stated
37 oC as optimum temperature for maximum chitinase production by A. hydrophila
HS4 and A. punctata HS6. Other studies report maximum chitinase synthesis at 35 oC
by Streptomyces spp. ANU6277 (Narayana & Muvva Vijayalakshmi 2009),
Streptomyces spp. strain S242 (Saadoun et al. 2009) and T. harzianum (Sudhakar &
P.Nagarajan 2011) respectively. The optimum growth temperature for chitinase
production was reported to be at 25-30 oC by M. timonae (Faramarzi et al. 2009).
It was observed that chitinase production from all the selected isolates was quite
stable between the temperature ranges of 30-40 oC. This makes the isolates, especially
suitable for field applications as it will ensure its stability on the culture and chitinase
production under different field conditions.
Table 4.11: The effect of different temperatures on chitinase production (U/ML)
by selected isolates
Temperature (oC)
25 30 35 40 45
Isolate 1
B. cereus
11.44 ± 0.38 14.13 ± 1.24 16.26 ± 0.68 12.97 ± 0.50 0.00 ± 0.00
Isolate 2
C. cellulans
12.77 ± 0.32 14.79 ± 0.93 15.63 ± 0.50 15.06 ± 0.56 0.00 ± 0.00
Isolate 3
B. cereus strain
NOC2011
14.27 ± 0.47 15.19 ± 0.64 16.68 ± 0.15 15.58 ± 0.18 0.00 ± 0.00
Isolate 4
B. licheniformis
13.27 ± 0.31 14.76 ± 0.22 15.24 ± 0.49 12.37 ± 0.50 0.00 ± 0.00
* The values are expressed as Mean ± S.D.
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Isolate 1- Bacillus cereus
Co
25Co
30Co
35Co
40Co
45
0
5
10
15
20
a
a,bb
En
zym
e a
cti
vit
y U
/ML
Isolate 2- Cellulosimicrobium cellulans
Co
25Co
30Co
35Co
40Co
45
0
5
10
15
20
a a a
En
zym
e a
cti
vit
y U
/ML
Isolate 3 - Bacillus cereus strain NOC2011
Co
25Co
30Co
35Co
40Co
45
0
5
10
15
20
aa a
Temperature
En
zym
e a
cti
vit
y U
/ML
Isolate 4- Bacillus licheniformis
Co
25Co
30Co
35Co
40Co
45
0
5
10
15
20
aa
b bE
nzy
me a
cti
vit
y U
/ML
A B
C D
Figure 4.10: The effect of different temperatures on chitinase production
Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans (B); Isolate 3:
Bacillus cereus strain NOC2011 (C); Isolate 4: Bacillus licheniformis (D). The values
are represented as Mean ± S.D. Each of the experiment was performed in triplicates.
Columns marked with same letters denote the means are not significantly different
from each other.
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Optimization of substrate concentration:
The substrate (colloidal chitin) concentration of the production medium was
optimized for each isolate by supplementing 0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 % (w/v)
of colloidal chitin in the production medium.
The study reflected that each isolate preferred different colloidal chitin concentration
in the production medium for maximum chitinase synthesis (Table 4.12 & Figure
4.11). B. cereus (Isolate 1) produced maximum chitinase in the range of 1-2 % of
colloidal chitin. C. cellulans (Isolate 2) exhibited maximum chitinase at 1 % of
colloidal chitin. B. cereus strain NOC2011 (Isolate 3) required 0.5-1 % while B.
licheniformis (Isolate 4) preferred 1.5-2 % colloidal chitin respectively for maximum
chitinase production.
The results were in accordance with previous report by Gomaa (2012) which state that
1.5 % colloidal chitin increased chitinase production by B. licheniformis. Several
workers have reported different concentrations of colloidal chitin for maximum
chitinase production by different organisms. 1.5 % chitin amended medium increased
chitinase production by Streptomyces viridificans (Gupta et al. 1995). 0.3 % of
colloidal chitin is reported to increase chitinase production by B. subtilis (Karunya et
al. 2011). Streptomyces spp. S242 reportedly produced maximum chitinase at 1.6 %
of colloidal chitin supplemented in the medium whereas B. laterosporous MML2270
preferred 0.3 % colloidal chitin concentration for maximum chitinase synthesis
(Shanmugaiah et al. 2008; Saadoun et al. 2009).
The experiment revealed that concentration of colloidal chitin in the medium was an
important factor affecting the production of chitinase by the selected isolates. It
served to induce chitinase production by the selected isolates. The results of the
investigation demonstrated that chitinase production increased with increasing
concentrations of colloidal chitin in the medium, only to drop soon after optimum
concentration is reached. This observation was in complete agreement with literature
which state the fact that most of chitinolytic systems reported are inducible (Ulhoa &
Peberdy 1991).
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Table 4.12: The effect of substrate concentration on enzyme production (U/ML) of selected isolates
Colloidal chitin concentration (%)
0.10 0.25 0.50 0.75 1.0 1.50 2.0
Isolate 1
Bacillus cereus
4.93
±
0.15
7.73
±
0.38
10.28
±
.29
12.27
±
0.29
12.99
±
0.18
12.41
±
0.25
11.75
±
0.26
Isolate 2
Cellulosimicrobium cellulans
0.00
±
0.00
4.71
±
0.28
10.25
±
0.41
12.97
±
0.24
16.05
±
0.08
14.64
±
0.36
15.01
±
0.10
Isolate 3
Bacillus cereus strain NOC2011
0.00
±
0.00
0.00
±
0.00
15.27
±
0.67
16.18
±
0.53
13.88
±
0.65
10.73
±
0.47
11.22
±
0.66
Isolate 4
Bacillus licheniformis
0.00
±
0.00
0.00
±
0.00
11.35
±
0.51
12.01
±
0.01
12.86
±
0.35
14.29
±
0.25
13.92
±
0.19
* The values are expressed as Mean ± S.D.
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Isolate 1- Bacillus cereus
0.10
%
0.25
%
0.50
%
0.75
% 1%
1.50
% 2%
0
5
10
15a
a,ba,b
a,b
En
zym
e a
cti
vit
y U
/ML
Isolate 2- Cellulosimicrobium cellulans
0.10
%
0.25
%
0.50
%
0.75
% 1%
1.50
% 2%
0
5
10
15
20
a a
En
zym
e a
cti
vit
y U
/ML
Isolate 3 - Bacillus cereus strain NOC2011
0.10
%
0.25
%
0.50
%
0.75
% 1%
1.50
% 2%
0
5
10
15
20
aa
a
bb
En
zym
e a
cti
vit
y U
/ML
Isolate 4- Bacillus licheniformis
0.10
%
0.25
%
0.50
%
0.75
% 1%
1.50
% 2%
0
5
10
15
20
a a
bb
En
zym
e a
cti
vit
y U
/ML
A B
C D
Figure 4.11: The effect of different substrate concentrations on chitinase
production. Isolate 1: Bacillus cereus (A); Isolate 2: Cellulosimicrobium cellulans
(B); Isolate 3: Bacillus cereus strain NOC2011 (C); Isolate 4: Bacillus licheniformis
(D). The values are represented as Mean ± S.D. Each of the experiment was
performed in triplicates. Columns marked with same letters denote the means are not
significantly different from each other.
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With the rise in the population and limited natural resources, enzyme technology can
prove to be constructive to industries in order to overcome the problems. Large-scale
production of micro-organisms for their products will ensure supply of surplus
amounts of economical, profitable and commercial value added products. Hence, one
of the goals of the present study was to optimize cultural as well as process
parameters in order to obtain maximum amount of enzyme from the chitinolytic
isolates. The outcome of optimization of culture conditions for each selected
chitinolytic isolates has been summarized in the Table 4.13.
From the results obtained from the optimization of the cultural conditions in order to
obtain maximum chitinase from each selected isolate, it can be concluded that the
organisms isolated during the present study produced increased chitinase production
when cultured in appropriate culture media and conditions. The study suggested an
avenue for production of chitinase by selected isolates having application in
agricultural industry since the isolates were capable of producing enzymes at
temperatures prevalent in agricultural fields.
Further, a pilot scale study can be adopted in order to harness the potential of the
selected isolates to produce the maximum amounts of chitinase.
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Table 4.13: Summary of optimized process parameters for chitinase production of each isolates
ISOLATE CARBON
SOURCE
NITROGEN
SOURCE
OPTIMUM MgSO4
CONCENTRATION
OPTIMUM
pH
OPTIMUM
TEMPERATURE
OPTIMUM
SUBSTRATE
CONCENTRATION
ISOLATE 1 -
Bacillus cereus
Colloidal
chitin
Peptone 0.05-0.06 % 6-7 35 oC 1-2%
ISOLATE 2 -
Cellulosimicrobium
cellulans
Colloidal
chitin
Peptone 0.05 % 7 30- 40 oC 1 %
ISOLATE 3 -
Bacillus cereus
strain NOC2011
Colloidal
chitin
Peptone 0.06 % 6-7 35 oC 0.5- 1 %
ISOLATE 4 -
Bacillus
licheniformis
Colloidal
chitin
Peptone 0.05-0.06 % 7-8 30-35 oC 1.5- 2 %
*All the optimization experiments were conducted in triplicates and the data was analysed using single factor analysis of variance (ANOVA)
followed by Tukey’s Range test. The statistical analysis was carried out using GraphPad Prism 5 software version 5.0. P values < 0.05 were
considered significant.
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Selection of potential bio-control agents
Following optimization of cultural conditions, the chitinolytic isolates under
investigation were evaluated in order to select the potential biocontrol agents against
the fungal phytopathogens selected for the present study, i.e., Rhizoctonia solani and
Fusarium oxysporum. This experiment allowed selection of the organisms with
noticeable antifungal activity and also served the purpose to streamline the project.
The selection of potential biocontrol agents was done on the basis of the following
studies:
Dual culture plate technique
Microscopic studies
Antibiosis test for production of volatile compounds
In vitro Dual culture assay:
In vitro dual culture assay was performed to test the potential of the isolates to act as a
biocontrol agent by virtue of its lytic action on the chitin component of the cell walls
of two fungal phytopathogens: Rhizoctonia solani and Fusarium oxysporum. Dual
culture assay is a well-known and widely used assay for detection of antagonistic
bacteria towards pathogenic fungi. The method allowed determination of the isolates
to inhibit the growth of phytopathogenic fungi. The evaluation of in vitro antifungal
activity is a pre-requisite for in planta evaluation of antifungal activity (Susilowati et
al. 2011). This method has been successfully employed to select different biocontrol
agents (BCA’s) (Viterbo et al. 2002).
Amongst the 4 chitinolytic isolates tested for dual culture assay, two isolates
demonstrated appreciable biocontrol ability against both R. solani and F. oxysporum.
The confrontation between the phytopathogenic fungal cultures and the chitinolytic
isolates exhibited clear inhibition zones (Figures 4.12-4.19) and also displayed
different inhibition rates (Table 4.14). The highest inhibition rates were observed with
B. cereus (Isolate 1) and B. cereus strain NOC2011 (Isolate 3). These two promising
isolates from the current study were selected for further investigations.
The isolates which most effectively inhibited the growth of phytopathogenic fungal
cultures in the dual culture experiment resulted in a major zone of inhibition. It was
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observed that the selected isolates made no physical contact with the phytopathogenic
fungi suggesting that the selected isolates could be producing lytic enzyme like
chitinase and/or antifungal metabolites which resulted in the inhibition of the growth
of phytopathogenic fungi. The involvement of antifungal volatile metabolites was
investigated by performing additional experiment which is discussed later.
The absence of physical contact between isolates and the fungal cultures suggested
the plausible mode of mechanism of inhibition due to antibiosis i.e. inhibition of fungi
by virtue of production of lytic enzymes, antifungal antibiotics, metabolites etc. This
assay employed the use of LB agar as culture medium; being rich in nutrients ruled
out the possibility of competition as mode of action for these isolates. Chitinolysis is a
common trait in bacteria that exhibit antifungal activity and hence it was assumed that
production of this lytic enzyme resulted in the inhibition of the phytopathogenic
fungal cultures (De Boer et al. 2004; Hoster et al. 2005; Ajit et al. 2006).
In the present study, it was observed that the inhibition rate against R. solani was
higher than that recorded for F. oxysporum. This observation suggested that the
hyphal walls of R. solani were more susceptible to the chitinases as opposed to F.
oxysporum which seemed to be more resistant. Sivan & Chet (1989) have debated that
cell wall of Fusarium species contain more protein compared to the cell wall of other
fungi. The observations recorded in the present investigation seemed to confirm this
hypothesis.
The observations made during the present study were in accordance with previous
report by Montealegre et al. (2003) which demonstrated antifungal ability of B.
subtilis and B. lentimorbus isolates against R. solani isolate.
Previously, in vitro studies have reported biological control ability of Bacillus spp.
against different phytopathogens. Korsten & Jager (1995) demonstrated inhibitory
activity of the strains of B. subtilis, B. cereus and B. licheniformis against C.
gloeosporioides, P. perseae, D. setariae, P. versicolor and F. solani when tested with
dual culture technique.
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Huang et al. (2005) have reported the chitinolytic ability of B. cereus 28-9 strain. This
strain demonstrated inhibitory activity against Botrytis elliptica in in vitro dual culture
assay. El-Tarabily et al. (2000) demonstrated the antifungal activity of chitinolytic S.
marcescens, S. viridodiasticus and M. carbonacea against S. minor in vitro. Basha &
Ulaganathan (2002) also reported inhibition of Curvularia lunata by Bacillus species
(strain BC121) in dual cultures. The inhibitory antifungal activity of B. subtilis strains
against R. necatrix and other soil-borne phytopathogenic fungi has also been reported
earlier (Cazorla et al. 2007).
The observation recorded in this experiment was further confirmed by assessing the
physical damage caused by the selected isolates by performing microscopic studies.
Table 4.14: Percent (%) inhibition of the phytopathogenic fungi – Rhizoctonia
solani and Fusarium oxysporum by each chitinolytic isolates
ISOLATE % INHIBITION
Rhizoctonia solani Fusarium oxysporum
ISOLATE 1 33.33 ± 5.6 32.89 ± 4.0
ISOLATE 2 23.87 ± 3.0 20.17 ± 2.0
ISOLATE 3 32.56 ± 3.4 41.27 ± 2.0
ISOLATE 4 25.31 ± 4.4 26.31 ± 2.6
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Figure 4.12: Antagonistic activity of the Bacillus cereus (Isolate 1) against
phytopathogenic fungi - Rhizoctonia solani where A= Control plate, growth of R.
solani in absence of the isolate; B = Test plate, growth of R. solani in presence of the
isolate.
Figure 4.13: Antagonistic activity of the Bacillus cereus (Isolate 1) against
phytopathogenic fungi – Fusarium oxysporum where A= Control plate, growth of
F. oxysporum in absence of the isolate; B = Test plate, growth of F. oxysporum in
presence of the isolate.
A B
A B
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Figure 4.14: Antagonistic activity of the Cellulosimicrobium cellulans (Isolate 2)
against phytopathogenic fungi - Rhizoctonia solani where A= Control plate,
growth of R. solani in absence of the isolate; B = Test plate, growth of R. solani in
presence of the isolate.
Figure 4.15: Antagonistic activity of the Cellulosimicrobium cellulans (Isolate 2)
against phytopathogenic fungi – Fusarium oxysporum where A= Control plate,
growth of F. oxysporum in absence of the isolate; B = Test plate, growth of F.
oxysporum in presence of the isolate.
B
B A
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Figure 4.16: Antagonistic activity of the Bacillus cereus strain NOC2011 (Isolate
3) against phytopathogenic fungi - Rhizoctonia solani where A= Control plate,
growth of R. solani in absence of the isolate; B = Test plate, growth of R. solani in
presence of the isolate.
Figure 4.17: Antagonistic activity of the Bacillus cereus strain NOC2011 (Isolate
3) against phytopathogenic fungi – Fusarium oxysporum where A= Control plate,
growth of F. oxysporum in absence of the isolate; B = Test plate, growth of F.
oxysporum in presence of the isolate.
A B
B
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Figure 4.18: Antagonistic activity of the Bacillus licheniformis (Isolate 4) against
phytopathogenic fungi - Rhizoctonia solani where A= Control plate, growth of R.
solani in absence of the isolate; B = Test plate, growth of R. solani in presence of the
isolate.
Figure 4.19: Antagonistic activity of the Bacillus licheniformis (Isolate 4) against
phytopathogenic fungi – Fusarium oxysporum where A= Control plate, growth of
F. oxysporum in absence of the isolate; B = Test plate, growth of F. oxysporum in
presence of the isolate.
B
A B
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Microscopic studies:
In order to evaluate the physical damage caused by the selected chitinolytic isolates
towards the fungal phytopathogens, the fungal mycelium from the test and control
plates of the dual culture assay were studied microscopically. The mycelium around
the zone of inhibition in the test plate served as test mycelium. Mycelium from the
control plates without the chitinolytic isolates influence were also taken and assessed.
The light microscopic examination revealed severe deformities in the mycelium in the
presence of selected chitinolytic isolates. The results of this investigation are
presented in the Figures 4.20 & 4.21 below. Both the isolates (B. cereus and B. cereus
strain NOC2011) were able to induce deformities in the mycelial and hyphal
structures of both the fungal phytopathogens-R. solani and F. oxysporum respectively.
In case of R. solani, mycelium swelling was observed. The thin vegetative hyphae of
R. solani observed in control plates, exhibited abnormalities such as swelling and
condensation of the hyphae in the test plate. The transverse septae in the hyphae of R.
solani completely disappeared in the case of mycelium observed from test plates. In
case of F. oxysporum, similar observations were recorded. The mycelium from the
test plates showed abnormalities like condensation, thickening of the walls and
vacuolisation of the hyphae.
The observations recorded in the present study clearly confirmed the mycolytic
activity of the selected isolates. The deformities of the fungal cell walls were
attributed to chitinase produced by the selected isolates. Previous report demonstrated
the degraded appearance of fungal hyphae after treatment with chitinolytic
Streptomyces strain (Quecine et al. 2008). The authors indicated the inhibitory role of
chitinase to plant pathogenic fungi. Another study by Saleem & Kandasamy (2002)
also suggested the ability of Bacillus species (strain BC121) to produce chitinase
which induced abnormal hyphal structures in Curvularia lunata.
The morphological abnormalities recorded in the present investigation were in
accordance with previous study, which demonstrated hyphal deformities of F.
oxysporum caused by the presence of B. subtilis (Chaurasia et al. 2005). Similar
results have also been reported by Getha & Vikineswary (2002) that relates to
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antibiotic substances which induced malfunctions such as stunting, distortion,
swelling, hyphal protuberances etc. Someya et al. (2000) reported abnormal forms of
R. solani mycelia in the presence of S. marcescens strain B2. Another investigation
also reported morphological changes in hyphal structures of R. solani in the presence
of Streptomyces spp. AM-S1 strain (Sowndhararajan & Kang 2012). A similar
observation has been reported for dissolution of fungal mycelium of A. niger by B.
subtilis AF1 strain (Podile & Prakash 1996).
Figure 4.20: Light microscopic (400X) observation of mycelium of R. solani
where A: R. solani mycelium from the control plate, B: R. solani mycelium from the
test plate around the inhibition zone.
Figure 4.21: Light microscopic (400X) observation of mycelium of F. oxysporum
where A: F. oxysporum mycelium from the control plate, B: F. oxysporum mycelium
from the test plate around the inhibition zone.
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Antagonism through production of volatile compounds
The test for antagonism through the production of volatile compounds was conducted
in order to evaluate the role of volatile compounds produced by the selected isolates
which may have resulted in inhibition of the growth of phytopathogenic fungi.
Volatile compounds are organic compounds that easily evaporate into gas due to their
high vapour pressure resulting from a low boiling point at ordinary room temperature
under normal atmospheric pressure (Lee et al. 2013).
Previous literature suggests the mechanisms by which antagonistic organisms act
includes direct parasitism, competition for nutrients or by production of volatile, non-
volatile compounds and lytic enzymes (Ganesan & Sekar 2010). Hence, volatile
compounds such as hydrocarbons, halogenated hydrocarbons, and nitrogen- and
sulphur-containing hydrocarbons were qualitatively evaluated for antagonistic
properties against R. solani and F. oxysporum.
This investigation revealed that the volatile compounds produced by the selected
isolates were incapable of inhibiting the growth of phytopathogenic fungi. This
observation suggested the predominant inhibitory role of lytic enzyme chitinase in
antagonism of test fungi, R. solani and F. oxysporum. Although the role of lytic
enzyme was evident, the involvement of non-volatile compounds in biocontrol against
the test fungi could not be ruled out in the present study. This was confirmed by
performing an additional investigation.
Chaurasia et al. (2005) reported deformities in six pathogenic fungi. This effect was
attributed to the production of diffusible and volatile antifungal compounds by B.
subtilis. Yuan et al. (2012) reported the ability of B. amyloliquefaciens to produce
volatile compounds that resulted in inhibition of growth and spore germination of F.
oxysporum f. sp. cubense.
The information gained from the present investigation enabled to screen potential
biocontrol agents against phytopathogenic fungi-R. solani and F. oxysporum. The
preliminary tests enabled to gain information on the possible mechanisms involved in
Chapter 4: Optimization of process parameters & selection of biocontrol agents
School of Science, SVKM’S NMIMS (Deemed-to-be) University Page 174
antagonism. The results indicated the role of lytic enzyme and other non-volatile
compounds in the inhibitory role against phytopathogens.
One of the key goals of this research included to evaluate the antifungal ability of the
chitinolytic isolates towards fungal phytopathogens selected for the study. This goal
was achieved by purifying chitinases from the selected isolates which enabled to
comprehend the characteristics of the chitinases from each isolate, followed by
employment of chitinases for antifungal studies. The purification and study of enzyme
characteristics is discussed in the following chapter.
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