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CHAPTER THREE:

ENRICHMENT, ISOLATION AND

IDENTIFICATION OF FEATHER

DEGRADING BACTERIA

Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria

School of Sciences, SVKM’S NMIMS (Deemed-to-be) University 49

3.1 INTRODUCTION:

3.1.1 Soil Microbiology (Pelczar et al., 1993; Bhatt and Kausadikar, 2010)

Soil is the outer material of the earth’s surface. It provides nutrition and

mechanical support to plants, thus supporting vegetation. Microbiologically, soil is one of

the most dynamic sites of biological interactions in the nature. It is the region where most

of the physical, biological and biochemical reactions related to decomposition take place.

Undoubtedly, soil is a universe of microorganisms. Living organisms of the soil

can be classified as: - Soil micro-flora which includes Bacteria, Fungi, actinomycetes,

algae and viruses, and Soil fauna which includes earthworms, protozoa, nematodes,

moles, ants, etc. Of all the different micro-flora present in the soil, bacteria are the most

numerous (80% of the total microbial population), followed by actinomycetes (13%),

fungi molds (3%) and others (algae and viruses 0.2-0.8%). Although occupying less than

1% of the total soil content, every microorganism is involved and responsible for

bringing about a specific change or transformation in the soil, thus giving a particular soil

its characteristic nature. Soil organisms convert complex organic nutrients into simpler

inorganic forms which can be easily utilized by plants for growth. Also, substances like

Indole Acetic Acid (IAA), gibberellins, antibiotics etc. which support plant growth are

produced by soil microorganisms.

Soil microorganisms which include fungi, actinomycetes, bacteria, protozoa etc.

play a vital role in the decomposition of organic matter, thereby bringing about release of

plant nutrients in soil. Soil microorganisms bring about oxidative decomposition of

organic matter into simpler, easily available materials that can be taken up by plants,

while the residue would eventually be transformed into humus. This process results in

increased fertility of the soil. Thus, soil microbes play an important role in maintaining

the fertility of the soil.

3.1.2 Soil organic matter:

. Organic matter present in the soil, which comes from debris of plants and

animals, could be cellulose, lignins and proteins (in cell wall of plants), glycogen (animal

tissues), proteins and fats (plants, animals). The soil of a poultry farm would have

physicochemical properties as compared to other soils. This is because, in a poultry farm,

the soil would contain several proteins of animal origin (Alabadan BA et al., 2009).



Keratin is one such protein which would be abundantly present in the poultry farm. The

origin of keratin in the poultry farm soil would be chicken feathers and other animal

wastes. Chicken feathers are a rich source of Beta-keratin i.e. they contain 90% keratin.

Keratin is structurally rigid protein, and is highly stabilized by hydrogen bonding,

disulfide linkages and hydrophobic interactions, thus making the feathers recalcitrant and

difficult to degrade (Martinez- Herandez and Velasco-Santos, 2012). Inspite of their

resistance, feathers do not tend to accumulate which confirms the existence of keratin-

degrading microorganisms (Onifade et al., 1998). Microorganisms present in the soil

would carry out decomposition of the keratin-containing wastes like other proteinacious

organic matter into simpler amino acids by virtue of extracellular enzymes: keratinases

(Onifade et al., 1998. Brandelli, 2008). Soil being a source of a variety of micro-flora,

keratin-utilizing bacteria that can degrade feather should be present indigenously. Also,

the abundant presence of keratin containing material in the poultry farm soil, leads to the

development of an ability to degrade keratin in microorganisms. This makes soil samples

from poultry farm an ideal source to obtain and isolate feather degrading bacteria.

3.1.3 Enrichment and isolation of bacteria from soil (Rao, 2009):

Collection of soil samples should be done carefully. Care should be taken not to

contaminate the soil samples with other soil or by exposing them too long to the

atmosphere. However, no absolute sterility in the process of sampling is required, since

the numbers of microorganisms in the soil are very large in comparison with any possible

contamination from a brief exposure. The samples are placed in sterile sampling bottles

or bags and brought to the laboratory as quickly as possible.

In order to isolate desired type of bacteria from a soil sample, the first step would

be to enrich those bacteria. Enrichment of soil samples is therefore the first and the most

crucial step to obtain the desired organisms. For enriching a selected group of bacteria

from soil sample, an enrichment medium should be defined accordingly. Various media

and different methods have to be used for the study of the different groups. In some

cases, special enrichment culture media favoring the development of particular organisms

have to be devised, so that the growth of the desired organisms will take place in

preference to that of all the other organisms. We thus often create artificial conditions

which are distinctly different from those of the soil. Also, to analyze the total micro-flora



of the soil, a general purpose medium can be used to study the bacteria that are originally

present in the soil.

In case of isolating feather-degrading bacteria from the soil, the enrichment

medium containing keratin as a sole source of carbon besides other elements, nitrogen

source and buffering agents is employed; thus limiting the enrichment of only those

bacteria that can utilize keratin and eliminating others. At least three successive

enrichments of this type would favor the enrichment of feather degrading bacteria. While

the enrichment takes place, a track of the enriched micro-flora can be maintained by

performing a viability check at regular intervals using a general purpose medium. While

doing this, potential feather degrading bacteria, i.e. those colonies which appear

consistently can be tracked. These colonies can then be selected for screening.

Screening of feather degrading bacteria can be performed using selective agar-

based medium that contains feathers as a sole source of carbon. Screening can be carried

out using a medium same as that for enrichment, except for the addition of agar. Thus,

the medium would selectively allow the growth of a feather degrading organism. These

bacteria while growing on such a selective medium would utilize the feathers and

produce a zone of clearance showing their feather-degrading ability and enabling their

selection. Selected colonies should be repeatedly tested for their feather degrading

potential by sub culturing on the selective media and only those colonies which are

consistently positive should be selected for identification.

3.1.4 Identification (Mackie & McCartney, 1996):

Identification of the selected isolates should be carried out stepwise, beginning

from studying the colony characteristics, microscopic characteristics and biochemical

characteristics which would involve performing several biochemical tests. A wide range

of techniques, based upon the specific characteristics of known bacteria are employed to

arrive at the identity of the specimen bacteria isolate. These tests are decided on the basis

of microscopic and cultural observations and as prescribed by the Bergey’s Manual. The

following characteristics aid in microbial identification:

a) Staining characteristics: Differential staining techniques enable the classification of

the specimen isolate into either Gram positive group or Gram negative group. Other

staining techniques such as Schaeffer and Fulton’s method determine the sporulation



character; Maneval’s method identifies the capsule formation, etc. Thus staining

techniques help in observing the bacterial structure, identifying special structures

such as spore, capsule formation, flagella, lipid granules, etc.

b) Media requirements: Most bacteria can grow in artificial laboratory media that

contain general sources of carbon, nitrogen, minerals, water and energy. Some

bacteria would require the presence of certain vitamins, amino acids, specific

carbon/nitrogen source or other special ingredients. There are few fastidious

pathogens which do not grow on artificial laboratory media. The presence or absence

of growth and the type of growth observed aid in identification.

c) Oxygen requirement: This criterion enables differentiation of bacteria into four

categories- aerobic, anaerobic, facultatively aerobic and micro-aerophilic.

d) Breakdown of a particular substrate: Every bacterium will utilize a specific profile

of substrates (carbon /protein/amino acid). This characteristic assists in identification

e) Enzymes: Tests which detect enzyme production also aid in the process of bacterial

identification.

Considering the above characteristics of bacteria, an array of biochemical tests are

performed which enable detection of certain characters and rule out certain possibilities.

Biochemical tests for identification can be categorized into following categories:

1. Tests to distinguish between aerobic and anaerobic breakdown of

carbohydrates.

2. Tests to show degradation of a range of carbohydrates and related compounds.

3. Tests for specific breakdown products

4. Tests to show ability to utilize particular substrate

5. Tests for metabolism of proteins and amino acids

6. Tests for detecting enzyme production

7. Combined tests-For example, Triple Sugar Iron (TSI) Agar test

Identification by 16S rRNA sequencing: Traditional methods for identification

have certain drawbacks when the specimen isolate has unique characteristics which do

not fit into patterns which have been used as a characteristic of a known species. This is

when molecular techniques for identification come into picture. In the past decade or so,

molecular techniques have proven beneficial in overcoming some limitations of



traditional phenotypic procedures for the detection and characterization of bacterial

phenotypes.

The rRNA gene is the most conserved (least variable) DNA in cells. Thus,

portions of rDNA sequence from distantly related organisms may be remarkably similar.

Sequences from distantly related organisms can be aligned to measure the differences.

Thus, the comparison of 16s rDNA sequence can show evolutionary relatedness among

microorganisms. The 16S rDNA has hypervariable regions, where sequences have

diverged with evolution. These hypervariable regions are often flanked by conserved

regions. Primers are designed to bind to these conserved regions, and amplify the variable

regions. The DNA sequence of the 16S rRNA gene has been sequenced for numerous

species, and are available on the internet through the NCBI (www.ncbi.nih.gov).

Comparison of the test sequence can be made with this database of sequences and a

distance based phylogenetic tree can be constructed to determine the isolate’s identity in

terms of % homology (Rodicio M and Mendonsa M, 2004).

The current chapter being the foundation of the project work deals with screening

soil samples from poultry farms for obtaining feather degrading isolates, followed by

their identification. The chapter gives a detailed account of the following objectives:

i. Collection of soil samples from different poultry farms

ii. Primary, secondary and tertiary enrichments of each soil sample, accompanied with

regular viability check

iii. Screening of enriched isolates to determine the feather degrading potential

iv. Identification of the selected isolates on the basis of cultural, morphological and

biochemical characterization

v. Identification by 16SrRNA sequencing



3.2 MATERIALS AND METHODS:

3.2.1 ENRICHMENT

3.2.1.1 Collection of Soil Samples:

Six soil samples were collected in sterile bags from different poultry farms of

Nasik, Nallasopara and Pune. The collection site had the presence of large amount of

feather waste due to the presence of poultry birds. The day temperature was

approximately 29-33oC, and night temperature was about 20-23

oC. Samples were

collected in sterile plastic bags, using sterile spatula and brought to the laboratory and

processed on the next day.

3.2.1.2 Preparation of Enrichment Media:

Enrichment of soil samples was carried out in a Minimal Salts Medium (MSM).

All chemicals, salts, media and media supplements were procured from Qualigens Fine

Chemicals, India and Himedia, India. For enrichment of the soil samples, 100 ml of

liquid Minimal Salts Medium (MSM) in 250ml flask was used. The composition of the

MSM is represented in table 3.1.

Table 3.1: Composition of Minimal Salts Medium (MSM) containing 1% Feathers

Components Quantity (Grams/liter)

NaCl 5

K2HPO4 1

KH2PO4 1

(NH4)2SO4 0.1

MgSO4 0.2

Feathers 10

pH 7.0 +0.2



Procurement and treatment of chicken feathers: Chicken feathers were

procured from a local poultry shop. They were first washed thoroughly under running tap

water to remove all the surface impurities, followed by thorough washing with distilled

water. After this, de-fatting was carried out using chloroform: methanol solution (1:1),

for 4 hours at room temperature with intermittent shaking (De Azeredo et al., 2006;

Mazzoto et al., 2009). The de-fatted feathers were then washed with distilled water and

dried and stored at 40oC for further use.

100 ml of Enrichment medium was prepared in 250 ml flask. The components

were weighed accordingly and dissolved in 100 ml of distilled water. Chicken feathers

were weighed and added, after which the media was sterilized by autoclaving at 15

psi/121oC for 20 minutes.

3.2.1.3 Initial Bacterial Count of the Soil Samples:

Before processing the soil samples for enrichment, an initial bacterial count of

these samples was determined by performing a viable count. The viable count was

performed by spread plate technique, using a general purpose medium i.e. Nutrient Agar

(NA, Himedia India).

For performing a viable count, 1 gram of soil sample was weighed and added

aseptically to 10 ml of sterile saline (in an assay tube). This suspension was vigorously

mixed by vortex in order to disperse the soil particles, after which it was allowed to stand

undisturbed for 20 minutes. This would allow all the soil particles to gradually settle

down while the microorganisms would remain suspended in the solution. After this, 1 ml

of the suspension was transferred to 9 ml of saline (in an assay tube) taken and proceeded

for serial dilution. Serial dilutions were carried out as presented in table 3.2. Viable count

was performed on NA plates wherein 0.1 ml of each of the dilutions was pipetted and

spread plated. The inoculated plates were incubated at room temperature and the counts

were taken after 24 and 48 hours.



Table 3.2: Serial dilutions of soil sample for viable count

Tube Number Volume of Stock Volume of saline (ml) Dilution Dilution Factor

1 1 gram soil 10 10-1

10

2 1 ml 9 10-2

102

3 1 ml 9 10-3

103

4 1 ml 9 10-4

104

5 1 ml 9 10-5

105

6 1 ml 9 10-6

106

7 1 ml 9 10-7

107

8 1 ml 9 10-8

108

9 1 ml 9 10-9

109

10 1 ml 9 10-10

1010

1 ml (Discard)

3.2.1.4 Enrichments

5 grams of each of this soil sample was weighed and inoculated into 100 ml of

Enrichment Media under aseptic conditions. The inoculated flasks were incubated at

room temperature under shaker conditions (150 rpm). The first enrichment was carried

out for approximately 25 days. For second enrichment, 1 ml of the enriched culture was

taken as inoculum and transferred aseptically into 100 ml fresh enrichment medium. The

second enrichment was carried out for 25 days. Similarly, third enrichment was carried



out using the enriched culture from the second enrichment as inoculum. The third

enrichment was also carried out for 25 days.

3.2.1.5 Viable count:

Viable counts were performed at regular intervals during the enrichments to

observe the outcome of enrichments in terms of the microbial count and the type of

bacteria which are getting enriched.

Viable count was carried out using the Spread Plate Technique. For performing

viable count, 1 ml of the enriched culture was aseptically taken from the enrichment flask

and serially diluted according to the above mentioned protocol. 0.1ml of each dilution

was spread plated on Nutrient Agar (NA, Himedia) plates. The plates were incubated at

room temperature for 24-48 hours. The final CFU (colony forming unit) value was

calculated as follows:

CFU/0.1ml = Number of colonies counted x dilution factor

CFU/1ml = Number of colonies x dilution factor x 10 (volume factor)

As a result, viable count by spread plate enabled to keep a track of the microbial

count during enrichment. The different types of colonies that were observed during viable

counts were noted in terms of their general characteristics such as- colony characteristics

and Gram nature, in order to maintain a track of the variety of microbes that are

flourishing during the enrichment process. The bacterial colonies that appeared

consistently during viable count were selected for screening.

3.2.2 ISOLATION:

3.2.2.1 Screening

The isolates which appeared consistently during viable counts were selected for

screening. Screening involved identifying microbial colonies which showed the ability of

feather degradation. Therefore, a selective medium allowing the growth of only keratin

degrading isolates was used. The selective medium used was Feather Agar Medium with

the following composition (Table 3.3):



Table 3.3: Composition of Feather Agar

Components Quantity (Grams/liter)

NaCl 5

K2HPO4 1

KH2PO4 1

(NH4)2SO4 0.1

MgSO4 0.2

Feathers 10

Agar 15

pH 7.0 +0.2

Feather Agar medium was prepared with the similar composition as that of

MSM. Additionally, it contained 1.5% agar and feathers were finely chopped, manually,

using scissors to enable their even dispersal.

The selected colonies obtained from enrichment were spot inoculated on feather

agar plates. The plates were incubated at room temperature for up to 5 days and were

observed for a zone of clearance around the spot inoculated culture. The zone of

clearance indicated the isolate’s feather degradation ability.

3.2.2.2 Selection of feather degrading isolates

On screening, isolates showing feather degrading ability were found. However, not all

isolates were proceeded for identification.

Factors considered for selection of isolates for identification:

Clearance zone observed during preliminary screening

Ability to retain the feather degrading potential on repeated sub-culturing

So, only those organisms that showed feather degrading potential consistently

were selected for identification.

3.2.2.3 CHARACTERIZATION OF GROWTH CONDITIONS:

The isolates selected were further subjected to characterize their growth

conditions. The parameters which were studied are as follows:

Study of optimum pH

Study of optimum temperature



Study of optimum salt concentration.

Study of comparative growth of the isolate in different media {Nutrient Broth

(NB), Soyabean Casein digest Broth (SCB) and Luria Bertani Broth (LB)}

Study of growth curve of each isolate under optimized growth conditions.

Preparation of inoculum:

Saline suspensions of each of the isolate were prepared as per the 0.5

McFarland turbidity standard to obtain approximate cell density of 107-8

cells/ml. 18

hour old culture was used to prepare the cell suspension. 10µL of this suspension was

used as inoculum. Negative control was included in each experimental batch. All the

flasks, tubes or plates were incubated at 37oC for 24 hours to 5 days, unless otherwise

mentioned.

3.2.2.3.1 Optimum pH determination:

The pH range was selected from 4.0 to 10.0 (4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and

10.0). The test was carried out using Nutrient Broth (N.B.). 10 ml Nutrient broth was

dispensed into assay tubes. The desired pH of N.B. was adjusted using 0.1N HCl and

0.1N NaOH. Test isolates were inoculated and incubated at 37oC. The tubes were

observed for the visual growth in terms of turbidity for 48 hours.

3.2.2.3.2 Optimum temperature:

The temperatures selected were as follows: 4oC, 25

oC, 37

oC, 40

oC and 50

oC.

10 ml Nutrient broth (adjusted to the optimum pH of the isolate) was dispensed into

assay tubes Test isolates were inoculated and incubated at respective temperatures.

The tubes were observed for the visual growth in terms of turbidity for 48 hours.

3.2.2.3.3 Study of growth at different NaCl concentrations:

To study the effect of different concentrations of NaCl, nutrient broth with

NaCl concentration ranging from 0.5%, 2%, 5%, 7%, 9%, 10% and 12% (w/v) was

prepared. Test organisms were inoculated and incubated at 37oC. The tubes were

examined for visual growth for 48 hours.

3. 2.2.3.4 Study of comparative growth of the isolate in different media:

The growth of the feather degrading isolates in different growth media i.e.

Nutrient Broth (NB, Himedia), SCB (Soyabean Casein digest Broth, Himedia) and



LB (Luria Bertani Broth, Himedia) was studied in terms of the amount of growth,

pellicle formation or even dispersal.

3.2.2.3.5 Study of generation time of each isolate under optimized growth

conditions (Pelczar et al., 1993):

1ml of 18-hour old cell suspension with an Optical Density (O.D) of 0.1 was

added to the broth aseptically. The contents of the flask were mixed thoroughly and

resulting absorbance was immediately read at 600nm (for 0 hour reading).The flask

was further incubated at 37oC on incubator cum shaker and absorbance was measured

at an interval of 30 minutes, for up to 8 hours. A graph of O.D. at 600nm vs. time was

plotted and the generation time was calculated for each selected keratin degrader.

3.2.3 IDENTIFICATION:

3.2.3.1 Identification by cultural and morphological characteristics: The selected

isolates were studied for their colony characteristics and microscopic

characteristics.

A) Cultural characteristics:

The cultural characteristics of the feather degrading bacteria were studied

on Nutrient Agar medium. This involved observation of the colony characteristics

with respect to size, shape, margin, elevation, color, texture, opacity and

consistency.

B) Morphological characteristics:

This includes the determination of Gram’s character, cell morphology,

presence of endospore and its position in the cell (Endospore staining by

Schaeffer and Fulton’s method), cell arrangements and motility of the isolated

bacteria. Gram staining was performed using standard dyes and the motility was

observed by hanging drop method. Differential staining was performed to identify

special structures of the bacteria. These included endospore staining by Scaheffer

and Fulton’s method and capsule staining by Maneval’s method.

C) Test for oxygen requirement:

The isolates were streaked on anaerobic agar medium (Himedia). The

incubation was carried out inside an anaerobic jar where anaerobic conditions

were maintained by the presence of Anaero Gas Pack (Himedia). The plates were



observed for growth after one week. Growth in the form of colonies would

indicate the facultative anaerobic growth of the isolate.

3.2.3.2 Biochemical identification of the isolated bacteria

Bacteria differ widely in their ability to metabolize carbohydrates and related

compounds. Biochemical tests enable differentiation of bacteria on the basis of bacteria’s

ability to metabolize carbohydrates and related compounds. For the purpose of

identification, these differences can be demonstrated by four different tests which are

described below.

1. Tests to check the carbohydrate utilization- oxidatively and fermentatively.

2. Tests for specific breakdown products.

3. Tests for the ability of bacteria to utilize certain proteins and amino acids.

4. Tests to study the various enzymes produced by isolated bacteria.

5. Tests for metabolism of certain substrates

All the tests were performed as per standard techniques & media compositions

given in Mackie and McCartney, Practical medical microbiology by Collee et al., 1996.

The media used were autoclaved at 15 lb psi 121oC for 20 minutes, except

otherwise mentioned. The glass wares used for microbiological work were autoclaved

and dried in hot air oven at 160oC for 2 hours. All the media transfers and inoculation

were carried out under aseptic conditions.

Culture media: All the culture media (readymade dehydrated media) and reagents used

were procured from Himedia India, Mumbai. All media were prepared according to

manufacturer’s instructions. Weighed quantity of dehydrated medium was suspended in a

measured quantity of distilled water and mixed well. Medium was digested using

microwave to dissolve it completely and then autoclaved at 1210C under 15 psi for 20

minutes.

Inoculum preparation: Suspensions of isolates were made according to the 0.5 Mac

Farland’s turbidity standard from an 18-hours-old cultured plate. These suspensions of

isolates were used for inoculation throughout the experiments.

Incubation temperature and time: All the inoculated tubes, plates and slants were

incubated at 37°C for 24 – 48 hours unless and otherwise mentioned.



3.2.3.2.1 Carbohydrate metabolism: Oxidative/Fermentative (OF) test:

This method depends upon the use of a semi-solid medium containing

carbohydrates together with pH indicator placed in a tube. If sugar metabolism takes

place only at the surface where conditions are aerobic, acid production happens in that

region and it indicates that the attack on the sugar is oxidative. If acid is found throughout

the tube, including the lower layers where conditions are anaerobic, the breakdown is

considered to be fermentative.

Table 3.4: Oxidative fermentative medium (Hugh and Leifson medium)

Components Quantity (g/L)

Peptone 2

NaCl 2

K2HPO4 0.3

1% Bromothymol blue;

1% aqueous solution

3ml

Agar 3

Water 1000

pH 7.1

The pH adjusted to 7.1 before adding Bromothymol blue and medium is

autoclaved at 121°C for 15 min. The carbohydrate to be added was prepared as a 10%

stock solution and autoclaved separately at 10 psi for 10 minutes. The autoclaved sugar

solution was added to the sterile medium, under aseptic conditions to obtain a final

concentration of 1% sugar in the medium.

Method: Test organisms were stab inoculated in semi-solid medium with straight

nichrome wire loop, incubated at 37°C for 24 – 48 hours.

Interpretation:

1) Yellow coloration only at the surface of butt indicates oxidative utilization of

carbohydrates.

2) Yellow coloration throughout the butt indicates fermentative utilization of

carbohydrates.



3.2.3.2.2 Tests for specific breakdown products:

A) Methyl Red (MR) Test:

This test was employed to detect the production of sufficient acid during the

fermentation of glucose. The medium used is a buffered glucose broth, as represented in

table 3.5 A.

Table 3.5 A) Composition of MR – VP medium (Buffered Glucose Broth)

Composition Quantity (g/L)

Buffered peptone 7

Dipotassium phosphate 5

Dextrose 5

Distilled water 1000 ml

B) Methyl red indicator solution

Methyl Red 0.1

Ethanol 300 ml


Method: Glucose phosphate broth was inoculated with the test organisms and incubated

at 37oC for 24 hours. After incubation, about 5 drops of Methyl red indicator solution was

added to the broth culture, mixed well and was observed for the development of bright

red colour.

Interpretation: Bright red colour indicates positive MR test.

B) Vogues Prouskauer (VP)Test (test for acetoin production)

Many bacteria ferment carbohydrates with the production of acetyl methyl

Carbinol (CH3.CO.CHOH.CH3) or its reduction product 2, 3 butylene glycol (CH3.

CHOH.CHOH.CH3). It can be detected by chemical methods. This test is usually done in

conjunction with the methyl red test since the production of acetyl methyl Carbinol or

butylene glycol usually results in insufficient acid accumulation during fermentation to

give a methyl red positive reaction. An organism of the enterobacterial group is usually

either methyl red positive and Voges – Prouskauer negative or Voges – Prouskauer

positive and methyl red negative.

Method: As for the Methyl red test, Glucose phosphate broth were inoculated with the

test organisms and incubated at 37oC for 24 hours. To this broth culture 1 ml of Omera’s



reagent (40 % KOH and 3 ml of a 5 % solution of α – naphthol in absolute ethanol) was

added and incubated at 37 oC for 30 minutes and then observed for colouration.

Interpretation: A positive reaction is denoted by the development of an eosin pink color

after incubation.

3.2.3.2.3 Test for metabolism of certain protein and amino acids:

A) Indole test:

This test was employed to demonstrate the ability of certain bacteria to

decompose the amino acid, tryptophan to Indole, which accumulates in the medium. The

medium used is tryptone water, that contains tryptone. Indole, which is generated as an

end product of tryptone metabolism, is then tested for by addition of – dimethyl amino

benzyldehyde (Kovac’s reagent).

Table 3.6 A): Composition of Tryptone Water:


Casein enzymic hydrolysate 20

Sodium chloride 5


Final pH 7.5 +/- 0.2

B): Kovac’s reagent:

Amyl or isoamyl alcohol 150 ml

p – Dimethyl amino benzyldehyde 10 g

Concentrated hydrochloric acid 50 ml

Method: Sterile Tryptone water was seeded with test organism and incubated at 37oC for

24 hours. At the end of the incubation period 0.5 ml of Kovac’s reagent was added to the

culture and mixed well.

Interpretation: A positive reaction was indicated by formation of a reddish pink ring on

the surface of the inoculated media.



B) Gelatin liquefaction test

Some of the bacteria have ability to liquefy gelatin. Gelatin breakdown was

demonstrated by incorporating gelatin in buffered Nutrient agar, growing the culture on it

and flooding the medium with mercuric chloride solution that differentially precipitated

gelatin resulting in a zone of clearance around the spot inoculated culture. The medium

composition is as given below:

Table 3.7 A): Gelatin agar composition


Nutrient Agar 1000 ml

KH2PO4 0.5

K2HPO4 1.5

Gelatin 4

Glucose 0.05

B) Preparation of Mercuric chloride solution:

Mercuric chloride 15g

Hydrochloric acid 20 ml


Method: The plates were inoculated with test cultures and incubated at 37°C for 24

hours. They were then flooded with Mercuric chloride solution.

Interpretation: Clear zone around spot inoculated culture

3.2.3.2.4. Tests for studying the various enzymes produced by isolated bacteria

The isolate was checked for the production of enzymes like:

A) Oxidase

B) Catalase

C) Nitratase

D) Urease

E) Amylase



A) Oxidase

This test is based on oxidase enzyme production by certain bacteria in organism.

The enzyme is detected by a redox dye-tetra methyl p- phenylene Diamine hydrochloride.

The dye reduces to deep purple colour.

Method: Test organisms were inoculated on nutrient agar plates and incubated at 37°C

for 24 hours. A small filter paper strip soaked in a 1.0 – 1.5 % solution of tetra methyl p-

phenylene Diamine hydrochloride was laid in a petri plate. The colonies to be tested were

smeared on the paper by using flame sterilized glass slide or rod.

Interpretation: Positive reaction was indicated by deep purple hue, appearing with in 5-

10 seconds, a delayed positive reaction by coloration in 10-60 seconds and a negative

reaction by absence of coloration or by coloration later than 60 seconds.

B) Catalase

This test demonstrates the presence of Catalase, an enzyme that catalyzes the release of

oxygen from hydrogen peroxide.

Method: Test organisms were inoculated on Nutrient agar slant and incubated at 37° C

for 24 hours.10% Hydrogen peroxide was added drop wise to cultured slant with

substantial growth and checked for effervescence.

Interpretation: Effervescence within 30 seconds indicates a positive test.

C) Nitratase

This test was carried out to detect the presence of the enzyme Nitrate reductase (nitratase)

which causes the reduction of nitrates to nitrites. The media used for the test is nitrate

broth, the composition of which is given as follows (table 3.8):

Table 3.8: Composition of Nitrate Broth:


Peptic digest of animal tissue 5

Meat extract 3

Sodium chloride 30

Potassium nitrate 1


Final pH 7.0+/- 0.2



Test reagents and their composition:

1) Sulphanilic acid: 0.8 g of Sulphanilic acid dissolved in 100 ml of 5 mol of acetic acid.

2) α – naphthylamine: 0.5 g of α – naphthylamine dissolved in 100 ml of 5 mol of acetic

acid.

Method: Nitrate broth was inoculated with the test organisms and incubated at 37oC for

24-48 hours. 2-3 drops of Sulphanilic acid and α- naphthylamine was added to nitrate

broth and colour change was observed.

Interpretation: Development of the red colour indicates the presence of nitrite and thus

the ability of an organism to reduce nitrate to nitrite was confirmed.

D) Urease

Bacteria, particularly those growing naturally in an environment with exposure to urine,

may decompose urea by means of the enzyme urease:

NH2.CO.NH2 + H2O 2NH3 +CO2

The presence or absence of this enzyme was detected by growing the organism in

the presence of urea. The production of alkali due to urea breakdown was detected by

means of pH indicator added to the medium. The medium used was Christensen’s Urea

medium that contains Phenol red as an indicator dye. The composition is given as follows

(table 3.9).

Table 3.9: Composition of Urea Agar Base:



Dextrose 1

Sodium Chloride 5

Potassium phosphate 2

Phenol Red 0.012

Agar 15

Distilled water 950

pH 6.8 +/- 0.2

After sterilization and cooling of Urea Agar Base approximately to 50oC, 50 ml of

sterile 40 % Urea was added, mixed well and dispensed aseptically in 5 ml aliquots in

sterilized tubes for the preparation of slants.



Method: The entire slope surface of urea agar slant was heavily inoculated with test

organism and incubated at 37°C for about 24 – 48 hours.

Interpretation: Urease positive reaction is indicated by the change in colour of the

media to purple pink.

E) Amylase

Starch agar was used to detect amylase production. Amylase enzyme acts on

starch and converts it into simple sugars. This could be detected by iodine solution, which

forms a dark blue colored complex with starch resulting in a zone of clearance at the site

of starch breakdown i.e. around the spot inoculated culture. The composition of starch

agar is as follows.

Nutrient agar 90 ml + 10 ml 10% starch solution (sterilized separately).

Method: Plates were inoculated with test organisms and incubated at 37° C for 48 hours.

After incubation, the plate was flooded with iodine solution.

Interpretation: Presence of amylolytic activity was indicated by clearance around the

colonies against the blue background.

3.2.3.2.5. Tests for metabolism of certain substrates:

A) Citrate utilization test:

This test was performed to detect the ability of an isolated test organism to utilize

Citrate as a sole carbon and energy source for growth and an ammonium salt as the sole

source of nitrogen.

Table 3.10: Composition of Simmons Citrate Agar:


Magnesium sulphate 0.2

Ammonium dihydrogen phosphate 1

Dipotassium phosphate 1

Sodium citrate 2

Sodium chloride 5

Bromothymol blue 0.08

Agar 15

pH 6.8 +/- 0.2



Method: Simmon’s citrate agar slope was streaked with the test organism and incubated

at 37°C for 24 hours.

Interpretation:

Positive = Change in colour from green to blue and the streak of growth.

Negative = Original green colour and no growth.

B) Triple Sugar Iron (TSI) Test:

This medium was used as a multi-test medium, which detects fermentation

reaction, gas production and ability of an organism to produce H2S.

Table 3.11: Composition of Triple Sugar Iron Agar:



Casein enzymatic digest 10

Beef extract 3

Yeast extract 3

Lactose 10

Sucrose 10

Dextrose 10

Sodium chloride 5

Ferrous sulphate 0.2

Sodium thiosulphate 0.3

Phenol red 0.024

Agar 12


pH 6.8 +/- 0.2

Method: A heavy inoculum was streaked over the surface of the slope and stabbed in the

butt. Medium was incubated at 37oC for 48 hours.

Interpretation: The results were interpreted as the fermentation reactions on the slope

and in the butt by change in colour to yellow due to acid production. The cracking of the

medium and cavities formed indicated gas production. The blackening of the medium

indicated H2S production.



3.2.3.3 Identification by 16SrRNA sequencing:

16SrRNA sequencing was carried out to confirm the results of

biochemical identification as well as for further identification up to the species

level. 16SrRNA sequencing would involve genomic DNA (gDNA) extraction,

followed by amplification of the 16SrRNA gene by Polymerase Chain Reaction

(PCR).

3.2.3.3.1 Genomic DNA extraction:

Reagents required and their composition:

1. Luria Bertani (LB) Broth (100ml):

Table 3.12: Composition of Luria Bertani (LB) Broth:


Casein enzymic hydrolysate 10

Yeast extract 5

Sodium Chloride 10

Final pH (at 25oC) 7.5 + 0.2

2.5 grams LB Broth powder was mixed in 100 ml distilled water. It was

sterilized by autoclaving at 15 psi pressure (121oC) for 15 minutes and

dispensed as desired.

2. 100 mM Tris.Cl buffer (pH 8.0, 1000ml):

12.1 g Tris base was dissolved in 800 ml H2O

Adjusted to pH 8.0 with concentrated HCl

Volume was made up to 1000 ml

It was sterilized by autoclaving at 15 psi (121oC) for 15 minutes and stored at

4oC.

3. Tris- EDTA (TE) buffer (pH 8.0):

10 mM Tris.Cl pH 8.0

1 mM EDTA, pH 8.0

In 800 ml distilled water, 1.21 grams of Tris base and 0.292 grams of EDTA

was dissolved. The pH was adjusted to 8.0 with concentrated HCl, and the



volume was made up to 1000ml using distilled water. It was sterilized by

autoclaving at 15 psi (121oC) for 15 minutes and stored at 4

oC.

4. 10% SDS (1ml):

0.1 gram SDS was dissolved in 1 ml distilled water and mixed to dissolve. It

was stored at room temperature.

5. 5 M NaCl (100ml):

29.22 g NaCl was dissolved in 80 ml distilled water, volume was made up to

100 ml. It was stored at room temperature.

6. CTAB- NaCl solution:

2% (w/v) CTAB

100mM Tris-Cl, pH 8.0

1.4M NaCl

In 100 ml 100mM tris-Cl buffer (pH 8.0), 2 grams of CTAB powder was

suspended. To this, 8.186 grams of NaCl was added. The solution was heated

to facilitate dissolving. It was prepared freshly before the experiment.

7. Chloroform/Isoamyl alcohol (24:1):

24 parts chloroform was mixed with 1 part isoamyl alcohol, stored at 4oC.

8. Phenol/Chlororfrom/Isoamyl alcohol (25:24:1):

25 parts phenol, equilibrated in 50 mM Tris-Cl (pH 8.0), 24 parts chlororform

and 1 part isoamyl alcohol were mixed and stored at 4oC.

9. 70% ethanol (100 ml):

70 ml ethanol was taken in a measuring cylinder and distilled water was added

to make up the volume to 100 ml.

Method:

Genomic DNA extraction was carried out using CTAB (cetyl trimethyl

ammonium bromide)/NaCl extraction method, according to the protocol by

Wilson K, 1995, elucidated in “Short Protocols in Molecular Biology, 3rd

edition,

1985. It is as depicted below:

1. Test isolate was inoculated in 10 ml sterile Luria Bertani (LB) broth, in an

assay tube, and grown up to saturation at the appropriate temperature under

shaker conditions



2. Bacterial culture was transferred into sterile centrifuge tube and centrifuged at

10,000 rpm, for 15 minutes. The supernatant was discarded.

3. The cell pellet was re-suspended in 600 µL of TE buffer.

4. To that, 60 µL of 10% SDS and 6 µL of Proteinase K were added. This was

incubated at 37oC for 1 hour.

5. After incubation, 200 µL of 5M NaCl was added and mixed thoroughly. 600

µL of CTAB/NaCl solution was added and the mixture was incubated for 10

minutes at 65oC.

6. To this mixture, equal volume of chloroform/isoamyl alcohol (24:1) was

added and centrifuged at 10,000 rpm for 10 minutes. The supernatant was

transferred to a fresh microfuge tube.

7. Equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added,

centrifuged at 10,000 rpm for 10 minutes. The supernatant was transferred to a

fresh tube.

8. To the supernatant, 0.6 volume of isopropanol was added and mixed gently, to

allow the DNA to precipitate. Precipitate was obtained by centrifugation at

10,000 rpm for 5 minutes.

9. The DNA precipitate was washed with 70% ethanol. After washing, the

ethanol was decanted and the DNA precipitate was dried at 37oC in an

incubator. The precipitate was re-suspended in Tris-EDTA buffer.

3.2.3.3.2 Qualitative Analysis of the gDNA (Daniel Voytas, 1995 “Short

Protocols in Molecular Biology”):

The extracted bacterial gDNA was subjected to Agarose Gel

Electrophoresis, for validation and for determination of the size of gDNA.

Reagents required:

1. Tris Borate EDTA (TBE) buffer

Composition of 5X TBE electrophoresis buffer

0.445 M Tris borate

0.01 M EDTA

pH 8.2 - 8.4 (at 25°C)



In 800ml distilled water, 54 grams of Tris base and 27.5 grams of Boric acid were

weighed and dissolved. To this 20 ml of 0.5 M EDTA (0.0146 grams EDTA in

100ml distilled water), was added. Mixing was carried out till all ingredients were

dissolved completely. The pH was adjusted to 8.3 using concentrated HCl. The

volume was adjusted to 1000 ml using distilled water.

2. 1% Agarose

0.4 grams agarose powder was weighed and taken in a conical flask of 100 ml

capacity. It was dissolved in 40 ml of TBE buffer and heated till completely

dissolved. The gel was allowed to cool till approximately 50 -55°C, and then

4µL of Gelstar dye solution was added to it, and mixed well.

3. 6X loading buffer

Glycerol (10%)

Bromophenol blue (0.25%) in water

0.5% stock Bromophenol blue

Dissolve 50 mg of bromo phenol blue in 10 ml of distilled water.

344 µL of available glycerol (87%) + 500µL of 0.5% Bromophenol blue

solution were mixed in a microfuge. The total volume was made to 1 ml with

sterile distilled water, mix well.

Store at 4°C.

Method:

1. The Horizontal Electrophoresis assembly (Technosys) was set up.

2. 1% Agarose solution was prepared by adding 0.4 g Agarose powder to 40 ml of

TBE buffer and heated using microwave till it had completely dissolved.

3. The gel was cooled to approximately 50 -55°C, and then 4µL of Gelstar dye

solution (Lonza) was added to it, mixed well and poured carefully into the tray.

The comb was inserted.

4. The gel was allowed to solidify. After the gel solidified, the tray was placed into

the chamber, and working solution of TBE buffer was poured into it, such that it

covers the gel. The comb was carefully removed.

5. Next, the chamber electrodes were connected to the power pack.



6. For loading, firstly 10 µL of DNA samples were mixed well with 2 µL of 6X

loading buffer (i.e. 1:6 dilution of the loading buffer to obtain final

concentration of 1X buffer). Further 5µL of the DNA ladder (Lambda DNA/Eco

RI/Hind III Marker) was mixed with 1 µL of loading buffer. Then the total

volume was loaded into the respective wells with the help of a micropipette.

7. The power supply was switched on; constant voltage was set to 50V and

constant current to 30 mA. The run was monitored by observing the movement

of the tracking dye.

8. The gel was observed using UV Transilluminator and GELDOC. The

flouroscent bands represent the gDNA contained in the sample. The molecular

weights could be determined by comparison with the DNA ladder.

3.2.3.3.3 Amplification of the gDNA using by Polymerase Chain Reaction (PCR)

(Sambrook J et al., 1989):

Reagents:

1. Sterile distilled Water,

2. 10X Polymerase chain reaction buffer, (Applied biosystems)

3. 25 mM MgCl2, (Applied biosystems)

4. 10 m M dNTPs mix, (Fermentas Lifesciences)

5. Forward primer (8F), (Applied biosystems)

6. Reverse primer (1391R), (Applied biosystems)

7. Target DNA (g DNA template),

8. Taq Polymerase enzyme, (Applied biosystems).

9. Ice pack

10. Sterile gloves

11. Applied Biosystems Thermocycler (ABI2400).

Consumables: Sterile Microfuge tubes, PCR tubes, microtips, PCR tips, etc.

Method:

The procedure for Polymerase chain reaction was followed as mentioned in

Molecular cloning - A laboratory manual by J. Sambrook, P.Maniatis, E.F.

Fritsch; 2nd

edition. Amplification reactions were carried out using Applied



Biosystems Thermocycler (ABI2400). The amplification was catalyzed by the

enzyme Taq Polymerase (Applied Biosystems).

Preparation of gDNA template: The extracted gDNA was subjected to

amplification by Polymerase Chain Reaction (PCR). The gDNA was treated with

RNases for elimination of RNA contamination prior to amplification. Briefly, the

gDNA template was incubated with 2µl of RNases, at 37oC for 1 hour.

Primers: The primers used were Universal bacterial primers. The sequences of

the primers used were:

Forward primer: 8F: (5'-AGAGTTTGATCCTGGCTCAG-3').

Reverse primer: 1391R: (5'-GACGGGCGGTGTGTRCA-3').

For each isolate, three PCR reaction mixtures were set up as follows:

1. PCR Reaction Mixture 1: A regular reaction mixture was prepared as indicated

in table 3.13.

Table 3.13: Regular PCR reaction mixture:

Components Volume (in µL)

10X PCR Buffer 2.5

25 mM MgCl2 1.5

10 mM dNTP Mix 2.5

Taq Polymerase 0.25

Sterile Distilled Water 15.25

Forward Primer (8F) 1

Reverse Primer (1391R) 1

Template DNA 1

Total Volume 25



2. PCR Reaction Mixture 2: A reaction mixture containing Dimethyl Sulfoxide

(DMSO) as a reaction enhancer was prepared as follows (Table 3.14):

Table 3.14: PCR reaction mixture with DMSO:

Components Volume (in µl)

10X PCR buffer 2.5

25 mM MgCl2 1.5

10 mM dNTP 2.5

Taq polymerase 0.25

Sterile distilled water 14.75

Forward primer 1

Reverse primer 1

2% DMSO 0.5

Template DNA 1

Total Volume 25

3. PCR Reaction Mixture 3: A reaction mixture containing Betaine, as enhancer

was prepared as indicated in the below table 3.15:

Table 3.15: PCR reaction mixture with Betaine:


10X PCR buffer 2.5

25 mMMgCl2 1.5

10mM dNTP 2.5

Taq polymerase 0.25

Sterile distilled water 10.25

Forward primer 1

Reverse primer 1

1.5 M Betaine 5

Template DNA 1

Total Volume 25



In the present study, three types of reaction mixtures were prepared- a

regular PCR reaction mixture, a PCR reaction mixture with DMSO and a PCR

reaction mixture with Betaine. DMSO and Betaine are PCR reaction enhancers,

i.e. they enhance the reactions for DNA templates with high GC content or DNA

templates forming secondary structures. The above reaction mixtures were

prepared for a single reaction, having a total volume of 25 µl. For multiple

reactions, a common PCR mixture is prepared, according to the number of

reactions, and then split into equal parts for individual reactions, followed by

addition of the target template DNA and the required enhancer (either DMSO or

Betaine or none). The PCR product obtained from all three reaction mixtures was

then compared. The best suited reaction mixture was then employed for scale-up

of reaction to obtain amplicon volume of 100 µl.

In this study, the PCR reaction of gDNA of isolates RM01 and RM02 was

enhanced best by the addition of 2% DMSO i.e. the best PCR product was

obtained by using a PCR reaction mixture containing DMSO. So the reaction

mixture used for gDNA amplification for isolates RM01 and RM02 was as per

table 3.14. In order to scale up the reaction to 100 µL, the PCR reaction mixture

was set up as described in table 3.16.

Table 3.16: PCR reaction mixture with DMSO for isolate RM01 and RM02:


10X PCR buffer 10

25 mM MgCl2 6

10 mM dNTP 10

Taq polymerase 0.8

Sterile distilled water 59

Forward primer 3

Reverse primer 3

2% DMSO 2

Template DNA 4

Total Volume 97.8



For isolate RM03, neither 2% DMSO nor 1.5 M Betaine was required as

enhancer. So a regular PCR reaction mixture was scaled up as represented in table

3.17:

Table 3.17: Regular PCR reaction mixture for isolate RM03:


10X PCR buffer 10

25 mM MgCl2 6

10 mM dNTP 10

Taq polymerase 0.8

Sterile distilled water 61

Forward primer 3

Reverse primer 3

Template DNA 4

Total Volume 97.8

A negative control tube was also maintained along with the other PCR

tubes. This tube comprises of all the components of a PCR mix except the

template DNA. The negative control helps to keep a check on the purity of the

components of the PCR mix. A negative control thus confirms the absence of any

nonspecific DNA template strands, which could be a source of contamination in

the process, leading to amplification of the nonspecific contaminant DNA.

The reaction mixtures were set up and placed in the thermocycler, which

was programmed for the cycle parameters as follows:



Table 3.18: PCR cycle parameters:

Number of

cycles

Cycle

duration/temperature

What happens?

1 cycle 5 mins at 94oC Initial cell breakage and DNA

denaturation.

35 cycles 1 min at 94oC

45 seconds at 55oC

2 mins at 72oC

DNA denatures into single strands

Primers anneal to ssDNA

Primers are extended from 3’ end by

Taq.

1 cycle 10 mins at 72oC Final extension to make sure all products

are full length.

--- 4 oC Storage

The qualitative analysis of the PCR product was carried out by 1%

Agarose gel by electrophoresis, along with ECO RI/HINDIII DNA ladder. After

observing the PCR product, the PCR reaction was scaled up to obtain amplicon

volume of 100 µL. The PCR product was sent to GeneOmbio laboratory for

sequencing. The sequencing data obtained was processed for nucleotide BLAST.

It was submitted to NCBI- BLAST, online software used for aligning and

comparing sequences. This software would align the test sequence with the

sequences present in the GenBank. The most closely related sequences listed in

the BLAST report are selected, and processed for CLUSTAL for generating a

phylogenetic tree using CLUSTAL W2 software. The phylogenetic tree is

generated using the neighbor joining (NJ) algorithm.



3.3 RESULTS:

3.3.1 Results of Enrichment process of soil samples:

After collection of the soil samples from poultry farms, they were brought to the

laboratory and processed for enrichment. Initial count of these soil samples was

determined. The below table (Table 3.19) represents the initial viable count of all the soil

samples.

Table 3.19: Details of soil samples and the initial bacterial count from each soil

sample:

Sr.No Soil Sample Initial Count (cells/ml)

S1 Poultry farm, Nasik 106

S2 Poultry farm, Pune 105

S3 Poultry Farm 1, Nallasopara 105

S4 Poultry Farm 2, Nallasopara 105

S5 Local Poultry farm, Malad 105

S6 Local poultry farm, VilePalrle 105

After inoculating the soil sample into the enrichment flask, the culture was immediately

processed for day 0 (initial) viable count. After inoculation and initial viable count,

regular viability checks every 3-5 days were performed. These viable counts tracked the

increase/decrease in the bacterial counts and also the types of bacteria that were being

favoured by the enrichment treatment.

3.3.1.1 Viability check during enrichments of Soil Sample 1:

Enrichment 1:

Observations of regular viability check of the three successive enrichments, for Soil

sample 1 (S1), from poultry farm of Nasik have been represented below:



Table 3.20 indicates the viable counts obtained at the regular intervals (every three days),

and the different types of colonies observed at each viable count. An overall increase in

the viable count was observed from day 0 to day 24, while the types of bacteria gradually

decreased throughout the primary enrichment.

Table 3.20: Viable count during the first Enrichment of Soil Sample 1:

Time of Viable Count

(during Enrichment)

Average Viable Count

(Colony forming units/ml)

Different types of

colonies observed

Day 0 3.03 x 106 6

Day 3 2.45 x 107 5

Day 6 6.2 x 107 5

Day 9 1.44 x 107 5

Day 12 1.03 x 108 5

Day 15 1.40 x 108 5

Day 18 1.18 x 108 3

Day 21 2.03 x 108 3

Day 24 1.83 x 108 3

Enrichment 2: -

Table 3.21 represents the viable counts at different intervals and the types of bacteria that

appeared during 2nd

enrichment. While the total viable count increased, less types of

colonies were observed (compared to 1st enrichment), which indicates that the passage

from primary to secondary enrichment favored certain bacteria to flourish, while

inhibiting the others.

Table 3.21: Viable count during the second enrichment of Soil Sample 1:


(during Enrichment)



Types of colonies

observed

Day 0 4.01 x 107 3

Day 7 8.5 x 108 3

Day 14 7.9 x 108 2

Day 21 6.53 x 108 2

Day 28 6.86 x 108 2



Enrichment 3: -

Table 3.22 indicates the details of the third enrichment i.e. viable count observed at different intervals and the types of

colonies observed. However, only one type of colony was retained. Table 3.23 indicates the colony characteristics of the

bacteria during third enrichment.

Table 3.22: Viable count during the third Enrichment of Soil Sample 1:


(during Enrichment)



Types of colonies

observed

Day 0 5.21 x 107 2

Day 7 4.39 x 108 2

Day 14 5.63 x 108 2

Day 21 5.19 x 108 1

Table 3.23: General characteristics of colony forming units obtained from enrichment of soil sample 1:

Sr. No Size Shape Margin Elevation Color Texture Opacity Consistency Gram

Nature

Cell

Morphology

1. Large Irregular Flat Butyrous Off-white Smooth Translucent Translucent Gram

Positive

Bacilli




Enrichment 1: Observations of regular viability check of the three successive

enrichments, for Soil sample 2 (S2), from poultry farm of Pune have been represented

below:

Table 3.24 represents the viable count during the first enrichment of soil sample 2.

Table 3.24: Viable count during the first Enrichment of Soil Sample 2


(during Enrichment)



Different types of

colonies observed

Day 0 2.81 x 105 4

Day 3 5.98 x 105 4

Day 6 3.25 x 106 4

Day 9 6.68 x 106 4

Day 12 9.5 x 106 4

Day 15 4.69 x 107 3

Day 18 8.29 x 107 3

Day 21 5.76 x 108 3

Day 24 2.19 x 108 3

Enrichment 2: -


appeared during 2nd

enrichment. An increase in viable count and a decrease in the types

of bacteria was observed (compared to 1st enrichment).



(during Enrichment)



Types of colonies

observed

Day 0 5.24 x 107 3

Day 7 4.69 x 108 3

Day 14 8.94x 108 2

Day 21 8.01 x 108 2

Day 28 7.66 x 108 2



Enrichment 3: -

Table 3.26 indicates the details of the third enrichment i.e. viable count observed at different intervals and the types of colonies

observed. An increase in viable count was observed and only one type of bacteria was retained.

Table 3.26: Viable count during the third Enrichment of Soil Sample 2:


(during Enrichment)



Types of colonies

observed

Day 0 2.11 x 107 2

Day 7 2.35 x 108 1

Day 14 5.02 x 108 1

Day 21 4.6 x 108 1

Table 3.27 indicates the colony characteristics of the bacteria obtained from enrichment of soil sample 2.

Table 3.27: General characteristics of colony forming unit obtained from enrichment of soil sample 2:

Sr. No Size Shape Margin Elevation Color Texture Opacity Consistency Gram Nature Cell Morphology

1. Medium Circular Entire Convex White Smooth Opaque Butyrous Gram Positive Bacilli





enrichments, for Soil sample 3 (S3), from poultry farm 1 of Nallasopara (Mumbai) have

been represented below. Table 3.28 represents the viability details of the first enrichment

carried out for soil sample 3 i.e. viable count and the type of colonies observed at each

viable count.



(during Enrichment)



Different types of

colonies observed

Day 0 1.09 x 105 7

Day 3 4.08 x 105 5

Day 6 4.33 x 106 5

Day 9 9.70 x 106 4

Day 12 1.3x 107 4

Day 15 3.21 x 107 3

Day 18 5.21 x 107 3

Day 21 5.91 x 107 3

Day 24 4.62 x 107 3

Enrichment 2:


appeared during 2nd

enrichment.



(during Enrichment)



Types of colonies

observed

Day 0 3.07 x 106 3

Day 7 6.22 x 107 2

Day 14 7.09 x 107 2

Day 21 7.88 x 107 2

Day 28 6.71 x 107 2



Enrichment 3: -


observed. An increase in the viable count was observed, while only one type of colony was retained.

Table 3.30: Viable count of third Enrichment of Soil Sample 3:


(during Enrichment)



Types of colonies

observed

Day 0 1.04 x 107 2

Day 7 8.96 x 107 1

Day 14 4.01 x 108 1

Day 21 4.35 x 108 1




1 Medium Irregular Irregular Convex Off-white Smooth Translucent Butyrous Gram positive Bacilli





enrichments, for Soil sample 4 (S4), from poultry farm 2 of Nallasopara (Mumbai) have

been represented below. Table 3.32 represents the details of viability check, of the first

enrichment for soil sample 4.



(during Enrichment)



Different types of

colonies observed

Day 0 3.08 x 105 6

Day 3 6.81 x 106 6

Day 6 7.12 x 106 4

Day 9 2.08 x 107 4

Day 12 3.92 x 107 3

Day 15 4.78 x 107 3

Day 18 8.23 x 107 3

Day 21 6.65 x 107 3

Day 24 5.98 x 107 3

Enrichment 2:


appeared during 2nd

enrichment. A further increase in viable count and a further decrease

in the types of bacteria was observed (compared to 1st enrichment).

Table 3.33: Viable count during second enrichment of Soil Sample 4:


(during Enrichment)



Types of colonies

observed

Day 0 5.46 x 106 3

Day 7 4.58 x 107 2

Day 14 5.39 x 107 2

Day 21 4.19 x 107 2

Day 28 5.57 x 107 2



Enrichment 3:-


observed. By the end of the third enrichment, one type of colony was retained.



(during Enrichment)



Types of colonies

observed

Day 0 3.29 x 106 2

Day 7 6.18 x 107 1

Day 14 7.28 x 107 1

Day 21 7.13 x 107 1




1 Large Irregular Irregular Flat Off-white Rough Translucent Powdery Gram Positive Bacilli





enrichments, for Soil sample 5 (S5), from local poultry farm of Malad (Mumbai) have

been represented below. Table 3.36 represents the details of viability check of first

enrichment for soil sample 5. As it can be observed, the viable count gradually increased

from day 0, followed by saturation and then a marginal decrease towards the end of the

enrichment. There was also a decrease in the type of colonies.



(during Enrichment)



Different types of

colonies observed

Day 0 8.95 x 105 4

Day 3 7.96 x 106 4

Day 6 4.16 x 107 3

Day 9 5.68 x 107 3

Day 12 7.65 x 107 3

Day 15 8.66 x 107 3

Day 18 7.91 x 107 3

Day 21 7.01 x 107 3

Day 24 7.64 x 107 3

Enrichment 2:-

Table 3.37 represents the viable counts at different intervals and the types of bacteria

that appeared during 2nd

enrichment. A further increase in viable count was observed,

while the types of bacteria were retained.



(during Enrichment)



Types of colonies

observed

Day 0 4.6 x 106 2

Day 7 6.14 x 107 2

Day 14 6.08 x 107 2

Day 21 6.45 x 107 2



Enrichment 3: -


observed. Only one type of colony was retained at the end of the enrichment process.



(during Enrichment)



Types of colonies

observed

Day 0 5.85 x 106 2

Day 7 5.66 x 107 1

Day 14 7.93 x 107 1

Day 21 8.94 x 107 1




1 Medium Irregular Irregular Convex White Smooth Opaque Mucoid Gram Positive Bacilli





enrichments, for Soil sample 6 (S6), from local poultry farm of Vile Parle (Mumbai) have

been represented below. Table 3.40 represents the viability check of first enrichment

carried out for S6.



(during Enrichment)



Different types of

colonies observed

Day 0 6.18 x 105 4

Day 3 3.54 x 106 4

Day 6 1.93 x 107 3

Day 9 3.44 x 107 3

Day 12 6.48 x 107 3

Day 15 8.39 x 107 3

Day 18 8.01 x 107 3

Day 21 8.19 x 107 3

Day 24 7.96 x 107 3

Enrichment 2:-

Table 3.41 represents the viable counts at different intervals and the types of bacteria

that appeared during 2nd

enrichment. A further increase in viable count and a further

decrease in the types of bacteria was observed (compared to 1st enrichment), which

indicates that the passage from primary to secondary enrichment favored certain

bacteria to flourish, while inhibiting the others.



(during Enrichment)



Types of colonies

observed

Day 0 7.9 x 106 2

Day 7 9.61 x 107 2

Day 14 8.95 x 107 1

Day 21 8.46 x 107 1



Enrichment 3:-


observed. Not much increase was observed in the viable count and one type of bacteria was retained.



(during Enrichment)



Types of colonies

observed

Day 0 7.51x 106 2

Day 7 2.54 x 107 1

Day 14 2.67 x 107 1

Day 21 2.8 x 107 1

Table 3.43 indicates the colony characteristics of the bacteria obtained from enrichment of soil sample 6



1 Small Round Entire Low-convex Yellow Smooth Translucent Butyrous Gram negative Coccobacilli



Table 3.44 represents the colony characteristics of the colony forming units obtained after three consecutive enrichments of each of

the six soil samples:

Table 3.44: Colony characteristics of isolates after enrichment of all six soil samples:

Sr. No. Size Shape Margin Elevation Colour Texture Opacity Consistency Gram nature Motility

1 Large Irregular Irregular Flat Off-white Smooth Translucent Butyrous Gram

Positive

Motile

2 Small Round Entire Low-convex Yellow Smooth Translucent Butyrous Gram

negative

Non-motile

3 Medium Circular Entire Convex White Smooth Opaque Butyrous Gram

Positive

Motile

4 Medium Irregular Irregular Convex Off-white Smooth Translucent Butyrous Gram

positive

Motile

5 Large Irregular Irregular Flat Off-white Rough Translucent Powdery Gram

Positive

Motile

6 Medium Irregular Irregular Convex White Smooth Opaque Mucoid Gram

Positive

Non-

motile



3.3.2 RESULTS OF SCREENING AND ISOLATION:

Isolates showing the ability of feather degradation in the screening step were

preceded for identification. However, only those isolates that consistently showed feather

degrading activity even on repeated sub-culturing were considered to be "true"

keratinolytic strains. Isolates which were temporary feather degraders were ruled out in

this step. From the six isolates obtained after enrichment, Isolates 1, 3 & 4, were selected

on the basis of their feather degrading potential and their ability to retain this activity.

They were named as Isolate RM01, Isolate RM02 and Isolate RM03, respectively were

subjected to biochemical identification and confirmation by 16S rRNA sequencing.

Figure 3.1: Screening, Clearance zone by isolate on an agar based medium

containing finely chopped feathers. Clearance indicates feather- degradation.



3.3.3 RESULTS OF CHARACTERIZATION OF GROWTH CONDITIONS:

3.3.3.1 Determination of optimum pH for growth:

Table 3.45 represents the growth response observed at different pH levels and it was

observed that the isolates showed optimum growth at neutral pH i.e. between 7 and 8.

Table 3.45: Study of optimum pH of Feather degrading isolates:

pH Isolate 1

(RM01)

Isolate 3

(RM02)

Isolate 4

(RM03)

4.0 - - -

5.0 + + -

6.0 + + +

7.0 +++ +++ +++

8.0 ++ +++ +

9.0 + + +

10.0 + + +

3.3.3.2 Determination of optimum temperature for growth:

The growth response of the isolates at different temperatures is shown in Table 3.46.

Optimum growth was observed at moderate temperatures, i.e. 35- 40oC.

Table 3.46: Study of optimum temperature of Feather degrading isolates:

Temperature Isolate

RM01

Isolate

RM02

Isolate

RM03

4oC - - -

25oC + + +

30 o

C + +++ +

37oC +++ ++ +++

40oC ++ ++ +

50oC + + +



3.3.3.3 Determination of optimum NaCl concentration for growth:

Table 3.47 represents the growth of the isolates observed in the presence of different

NaCl levels. Optimum growth was observed at 0.5% NaCl concentration.

Table 3.47: Study of optimum NaCl concentration of Feather degrading isolates:

NaCl

concentration

Isolate

RM01

Isolate

RM02

Isolate

RM03

0.5% +++ +++ +++

2% ++ + +

5% + + -

7% + + -

9% + + -

10% + - -

12% - - -

3.3.3.4 Optimum medium for growth:

On studying the growth of the feather degrading isolates in different growth media i.e.

Nutrient Broth (NB), Soya Casein Broth (SCB) and Luria Bertani (LB), it was found that

SC broth was optimum for growth and for studying the growth curve, since it showed

even turbidity throughout the broth.



3.3.3.5 Study of Growth Curve:

Table 3.48 shows the optical density of the three isolates at different time intervals.

Table: 3.48: Growth Curve study: Absorbance of the culture broth at 600 nm:

OD at 600 nm Isolate RM01 Isolate RM02 Isolate RM03

0 minutes 0.01 0.0245 0.0124

30 minutes 0.146 0.0594 0.0249

60 minutes 0.173 0.0485 0.1027

90 minutes 0.187 0.0938 0.1374

120 minutes 0.176 0.0931 0.224

150 minutes 0.1851 0.1434 0.2531

180 minutes 0.2075 0.2278 0.2731

210 minutes 0.1966 0.4334 0.2666

240 minutes 0.2154 0.6356 0.3279

270 minutes 0.2458 0.8952 0.3419

300 minutes 0.3246 1.0442 0.4146

330 minutes 0.3994 1.0145 0.5689

360 minutes 0.6076 1.1652 0.7418

390 minutes 0.6738 1.1215 0.8447

420 minutes 0.8752 1.1371 1.0522

450 minutes 0.9769 1.2464 1.1784

480 minutes 1.20008 1.2007 1.1518

510 minutes 1.2438 1.0825 1.1398



Figures 3.2, 3.3 and 3.4 show the graphical representation of the growth curves for

isolates RM01, RM02 and RM03 respectively.

Figure 3.2: Growth curve of isolate RM01 Figure 3.3: Growth curve of isolate RM02

Figure 3.4: Growth Curve of Isolate RM03

Table 3.49 summarizes the results of the growth curve study with respect to

generation time (in minutes) of each isolate.

Table 3.49: Generation time in minutes of feather degrading isolates:

Isolate RM01 Isolate RM02 Isolate RM03

Generation

time

90 minutes 60 minutes 90 minutes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 200 400 600

Ab

sorb

ance

Time in minutes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 200 400 600

Ab

sorb

ance

Time in minutes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 200 400 600

Ab

sorb

ance

Time in minutes



3.3.4 RESULTS OF IDENTIFICATION:

3.3.4.1 Biochemical Identification of Isolates:

Identification of the isolated organisms was carried out on the basis of

microscopic, cultural, and biochemical characteristics as prescribed by the Bergey's

manual of Systematic Bacteriology (Volume 2, 1986). Table 3.50 indicates the general

characteristics of the isolates, while Table 3.51 represents the biochemical characteristics

of the isolates.

Table 3.50: General Characteristics of the Feather degrading Isolates:

Characteristics Isolate 1 Isolate 2 Isolate 3

Gram Nature Gram Positive Gram Positive Gram Positive

Microscopic

Morphology

Endospore forming

Bacilli

Endospore forming

Bacilli

Endospore forming

Bacilli

Sporulation Sub-terminal

Endospore

Central Endospore Sub-terminal

Endospore

Motility Motile Motile Motile

O2 Requirement Facultative

Anaerobic

Facultative

Anaerobic

Facultative

Anaerobic



Table 3.51: Biochemical Characteristics of the Feather degrading Isolates:

Isolates Isolate 1 Isolate 2 Isolate 3

Tests to distinguish between aerobic and anaerobic breakdown of carbohydrates

O/F test

(glucose)#

Oxidative and

fermentative

utilization of sugar

Oxidative and

fermentative


Oxidative and

fermentative


Tests to show degradation of range of carbohydrates and related compounds

Glucose Acid Production Acid Production Acid Production

Sucrose Acid Production Acid Production Acid Production

Lactose Acid Production No Acid/Gas

Production

No Acid/Gas

Production

Maltose Acid Production Acid Production No Acid/Gas

Production

Mannitol No Acid/Gas

Production

Acid Production Acid Production

Xylose No Acid/Gas

Production

No Acid/Gas

Production

No Acid/Gas

Production

Tests for Specific Breakdown Products

Methyl Red Positive Negative Positive

Vogues-

Proskauer

Negative Positive Negative

Tests to show Ability to utilize particular Substrate

Starch Positive Positive Positive

Citrate Negative Negative Negative

Tests for Metabolism of Proteins and Amino-acids

Indole

Production

Negative Negative Negative

Arginine

dihydrolyase

Positive Positive Negative

Gelatin

hydrolysis

Positive Positive Positive



Table 3.51: Biochemical Characteristics of the Isolates (continued)

Tests for Enzymes

Catalase Positive Positive Positive

Oxidase Negative Negative Negative

Urease Positive Positive Positive

Nitrate Reduction Positive Positive Positive

Combined Tests

Triple Sugar Iron

(TSI) reaction

K/A** K/A**

K/A**

Key:

# Test to check the ability to metabolize a carbohydrate under aerobic and

anaerobic conditions

** Reaction in a TSI medium K/A indicates alkaline slant and acid butt

Table 3.52 represents the results of the biochemical identification of the isolates. On the

basis of the microscopic, cultural, and biochemical characteristics and reference of the

Bergey’s Manual, the isolates were identified upto the genus level and they were all

found to belong to the Bacillus spp.

Table 3.52: Results of biochemical identification of Feather degrading isolates:

ISOLATES BIOCHEMICAL

IDENTFICATION

RM01 Bacillus spp.

RM02 Bacillus spp.

RM03 Bacillus spp.



3.3.4.2 Identification by 16S rRNA sequencing:

To confirm the results of biochemical identification and also to carry out further

identification, 16S rRNA sequencing needed to be performed.

Genomic DNA extraction:

The below image (figure 3.5) represents the qualitative assessment of the extracted

gDNA from the test isolates. As observed in the figure, gDNA was successfully extracted

using the CTAB-NaCl protocol.

Figure 3.5: Qualitative analysis of gDNA of feather degrading isolates:

Lane 1: gDNA of Isolate RM01,

Lane 2: gDNA of Isolate RM02

Lane 3: gDNA of Isolate RM03,

Lane 4: Negative control

1 2 3 4

gDNA

samples



Amplification of 16S rRNA gene: The below image (figure 3.6) represents the PCR

products i.e. the amplified 16S rRNA gene of the isolates. The molecular weights of the

PCR products were determined to be approximately 1.3 kbps.

Figure 3.6: PCR products:

Lane 1: PCR product of Isolate RM01

Lane 2 PCR product of Isolate RM02

Lane 3: PCR product of Isolate RM03

Lane 4: Marker

Lane 5: Negative control

1 2 3 4 5

PCR

product



Sequencing results:

The 16S rRNA sequences of the three isolates were analysed using ncbi BLAST tool to

obtain a list of closely related species from the GenBank database. The isolates which

showed maximum homology (the first 20 closest relatives) were selected from BLAST

report, and they were aligned with the test sequence using CLUSTAL W2. A distance

based phylogenetic tree was constructed by the Neighbour Joining (NJ) algorithm using

CLUSTAL W2.



16SrRNA sequence of isolate RM01:

GCGCCCGTCCTAATAATGCAGTCGAGCGGAAGATGGGAGCTTGCTCCCTGATGTTAG

CGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG

GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGG

TGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGT

AACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACT

GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCA

ATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGT

AAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGG

TACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT

GGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTC

TGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGA

GTGCAGAGAGGAGAGTGAATTTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGAG

GACACCAGTGCGAGGCGACTCTCTGTCTGTACTGACGCTGAGGCGCGAAAGCGTGGA

GCGACAGATAGATACCTGTAGTCACGCGTAACGATGAATGCTATGTAGAGGTCCGCC

TTTAATGCTGCAGCAACGCATTAGCACTCGCTTGGGGAGTACGTCCAGACTGACTCA

GGATGACGGGGCGCAACGTGGACTTGGTTATCGCAGCATCG (892bp)

Figure 3.7: Phylogenetic tree of isolate RM01: The isolate shows maximum homology

with B. sonorensis




CTAGCGGGCTGGCCTTAATAATGCCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTG

ATGTTAGCGGCGGACGGGTGTGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATA

ACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTAT

AAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGG

TGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGC

CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCT

TCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG

GATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCT

TGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA

CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTC

TTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGG

AACTTGAGTGCAGAAGAGGAGAGTGAATTCCACGTGTAGCGGTGAAATGCGTAGAG

ATGTGAGGACACCAGTGGCGAAGGCGACTCTCTGGTCTGTACTGACGCTGAGGCGCG

AAAGCGTGGGGAGCGACAGATTAGATACCCTGTAGTCACGCGTAACGATGATGCTAT

GTAGAGGGTTTCCGCCTTAATGCTGCAGCACGCATAGCACTCGGCCTGGGAGTACGT

TCGCAGACTGGAACTCAGATGACGGGTCCGCAACGGTGACATGGGATTATTCG(903bp

)


with B. licheniformis




TAAACGGGCTTCCCAATAAAGACAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTG

ATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGAT

AACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTA

TAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTG

GTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGG

CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATC

TTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG

GATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCT

TGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA

CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTC

TTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAAACTGGG

GAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAG

AGATGTGGAGGGAACACCAGTGGCGAAGCGACTCTCTGGGTCTGTAACTGACGCTG

AGCGCGAAGCGTGGGGGGAGCGAACAGGATTAGATACCCTGTAGTCCACCGCCCGT

AAACGATGATTGCTTAGTGTAGAGGTTTCCGCCCATTAGTTGCTGCAGCAAACGCAA

TTAAGCACTCCGCCTGGGGGAGTACCGCTCGCAAGACTTGAACTCAAGGAATGACCG

GGGTCCA (910bps)


with B. subtilis



Table 3.53 summarizes the results of biochemical identification and 16S rRNA

sequencing.

Table 3.53: Summary of results of identification of Feather degrading Isolates:

Isolates Biochemical

identification

Shows Homology

with:

Origin

RM01 Bacillus spp. Bacillus sonorensis Poultry farm soil,

Nallasopara

RM02 Bacillus spp. Bacillus

licheniformis

Poultry farm soil,

Nallasopara

RM03 Bacillus spp. Bacillus subtilis Poultry farm soil,

Nasik

Thus, on the basis of Biochemical Identification and 16SrRNA sequencing,

isolates RM01, RM02 and RM03 were identified as Bacillus sonorensis, Bacillus

licheniformis and Bacillus subtilis respectively.



3.4 DISCUSSION:

3.4.1 Enrichment of soil samples:

Soil having a variety of microorganisms, serves as an important source of

industrially applicable bacteria and fungi. Although feather degrading bacteria have been

isolated from a variety of ecosystems (Gessesse et al., 2003; Giongo et al., 2007; Ionata

et al., 2008; Pillai and Archana, 2008) poultry farm soil is one of the most obvious

sources for isolating such bacteria, due to the presence of abundant amount of keratin

containing material in that environment (Yamamura S. et al., 2002; Coello and Vidal,

2002; Thys RCS et al., 2004, Cai et al., 2008).

The enrichment process resulted in an overall increase in viable count, however,

the types of enriched bacteria gradually decreased at the end of three consecutive

enrichments. This indicated that the enrichment medium, that contained minimal salts

and feathers as a sole source of carbon and nitrogen, encouraged the bacteria of interest to

flourish, which would be feather degrading bacteria. Simultaneously, other bacteria

which appeared transiently, during viable counts, were ultimately inhibited. Thus, the

enrichment media and enrichment process resulted in selective proliferation of potential

feather degrading isolates. On processing six soil samples for three successive

enrichments, six isolates were enriched and they were proceeded for screening.

3.4.2 Screening:

Six potential feather degrading isolates that were obtained after enrichment were

screened using feather agar plates. Certain isolates were capable of feather degradation

on repeated sub-culturing, while others lost this property. Thus, out of the six potential

keratinolytic isolates obtained after enrichment, three isolates lost the feather degrading

property due to repeated subculturing. These may be transient keratin degraders, and they

were eliminated, due to their weak keratinolytic potential. Consequently, three isolates

were retained and they were selected for biochemical identification. The selected isolates

were consistent and capable of feather degrading ability on repeated sub-culturing.

The optimum growth conditions for the selected isolates were determined with

respect to pH, temperature and NaCl concentration. The isolated feather degrading

bacteria showed growth at a wide temperature range- 25 to 40oC, with optimum growth



between 30-37oC, thus they belong to “mesophilic group” (Pelczar et al., 1993).

Keratinolytic bacteria have been reported to show optimal growth at thermophilic

temperatures (Lin et al., 1999; Kim et al., 2001). The observations made in the current

study are in agreement with previous findings wherein keratinolytic bacteria belonging to

Gram negative family have optimum growth at mesophilic temperatures such as Vibrio

spp. Kr2 (Sangali and Brandelli, 2000) and Stenotrophomonas sp. D-1 (Yamamura et al.,

2002). A Gram positive B. megaterium has also been reported to have optimum growth

within the temperature range of 25-40oC (Park and Son, 2009). The isolates preferred a

moderate pH range i.e. 7-8, which is in agreement with previous data on most

keratinolytic bacilli (Wang and Shih, 1999, Park and Son, 2009). Optimum growth was

observed at 0.5% NaCl concentration. The selected isolates were studied for their

cultural, morphological and microscopic characteristics.

The isolates were Gram positive, endospore forming bacilli, which have neutral

growth requirements; however, they could withstand higher temperatures. Keratinolytic

bacteria have been reported to show growth at mesophilic and thermophilic temperatures.

For example, in the study carried out by Williams et al., 1990, the isolate Bacillus

licheniformis PWD-1, showed feather degrading activity at 50oC, thus withstanding high

temperature. Similarly, Kim et al., 2001 and Cortezi et al., 2008, have reported Bacillus

spp., that could withstand temperature as high as 50oC. Other studies have mentioned

keratinolytic bacteria having preference for mesophilic temperatures (El- Refai et al.,

2005, Ni et al., 2011).

Biochemical Identification:

By identification on the basis of morphological, cultural and microscopic

characteristics, the identity of the isolates was determined to be Bacillus spp. The current

findings are in agreement with previous reports about keratinolytic organisms since most

keratinolytic bacteria, which have been previously reported are Gram positive and belong

to the Bacillus spp. (Williams et al., 1990, Lin et al., 1992, Kim et al., 2001, Brandelli,

2008, Cai et al., 2008).



16SrRNA sequencing:

For further identification using 16SrRNA sequencing, gDNA extraction was

carried out, followed by its amplification by PCR. During standardization of PCR

reaction mixtures, DMSO and Betaine were examined as PCR enhancers, and the PCR

products obtained were compared. gDNA of isolates RM01and RM02 was amplified by

using DMSO as an enhancer in the PCR reaction mixture, while neither DMSO nor

Betaine were required to enhance the gDNA amplification for isolate RM03. While most

gDNA amplifications are carried out using a regular PCR mixture, certain isolates which

have GC rich DNA, would require enhancers like DMSO or Betaine (Jensen et al., 2010).

16S rRNA sequencing further revealed the identity of the isolates as B.

sonorensis, B. licheniformis and B. subtilis. Previous studies have mentioned about

keratinolytic bacteria belonging to the Bacillus spp. (Williams et al., 1990; Kim et al.,

2001; Cai et al., 2008). However, according to the background reports on keratinolytic

bacteria, B. sonorensis has not yet been reported as a feather degrading bacterium.

The isolates would then be taken further for quantifying their feather degrading

potential, followed by optimizing their media conditions to achieve maximum feather

degradation and enzyme production.

chapter three: enrichment, isolation and …shodhganga.inflibnet.ac.in/bitstream/10603/73110/10/09....

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