4. optimization of process parameters & selection of...

4. OPTIMIZATION OF PROCESS

PARAMETERS & SELECTION

OF POTENTIAL BIOCONTROL

AGENTS

Chapter 4: Optimization of process parameters & selection of biocontrol agents

School of Science, SVKM’S NMIMS (Deemed-to-be) University Page 119

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).



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).



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.



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.



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



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.



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



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



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



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


School of Science, SVKM’S NMIMS (Deemed-to-be) University 9

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.


School of Science, SVKM’S NMIMS (Deemed-to-be) University 0

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.



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




Table 4.4: Composition of Potato Dextrose Agar


Potatoes, infusion form 200

Dextrose 20

Agar 15


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



Luria Bertani Agar

Table 4.5: Composition of Luria Bertani Agar


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).



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




Media used:


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.



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.



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.



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



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.



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.



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.



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).



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.




MSM

MSM

+C.C N

B

NB+C

C

0

5

10

15

20

a aE

nzym

e a

cti

vit

y U

/ML


MSM

MSM

+C.C N

B

NB+C

.C

0

5

10

15

20

En

zym

e a

cti

vit

y U

/ML


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


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.



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.



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



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).



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




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


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


En

zym

e a

cti

vit

y U

/ML


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.





from each other.



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.



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.


0.04

0.05

0.06

0

5

10

15

20a a

MgSO4 Concentrations (%)

En

zym

e a

cti

vit

y U

/ML


0.04

0.05

0.06

0

5

10

15

20

a a


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.




0.04

0.05

0.06

0

5

10

15

20


En

zym

e a

cti

vit

y U

/ML


0.04

0.05

0.06

0

5

10

15

20

aa


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.



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.



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.




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


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


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


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





from each other.



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



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.




Co

25Co

30Co

35Co

40Co

45

0

5

10

15

20

a

a,bb

En

zym

e a

cti

vit

y U

/ML


Co

25Co

30Co

35Co

40Co

45

0

5

10

15

20

a a a

En

zym

e a

cti

vit

y U

/ML


Co

25Co

30Co

35Co

40Co

45

0

5

10

15

20

aa a

Temperature

En

zym

e a

cti

vit

y U

/ML


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





from each other.



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).



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.




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


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


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


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.



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.



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.



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



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.



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



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



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


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



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


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



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


B

A B



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



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.



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



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.

4. optimization of process parameters & selection of...

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