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).
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
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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
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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.
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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):
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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.
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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.
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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
Distilled water 200 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
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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:
Composition Quantity (g/L)
Casein enzymic hydrolysate 20
Sodium chloride 5
Distilled water 1000 ml
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.
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Composition Quantity (g/L)
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
Distilled water 100 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
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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:
Composition Quantity (g/L)
Peptic digest of animal tissue 5
Meat extract 3
Sodium chloride 30
Potassium nitrate 1
Distilled water 1000 ml
Final pH 7.0+/- 0.2
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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:
Composition Quantity (g/L)
Peptic digest of animal tissue 1
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.
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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:
Composition Quantity (g/L)
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
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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:
Composition Quantity (g/L)
Peptic digest of animal tissue 10
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
Distilled water 1000 ml
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.
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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:
Composition Quantity (g/L)
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
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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
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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)
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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.
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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
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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
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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:
Components Volume (in µl)
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
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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:
Components Volume (in µl)
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
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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:
Components Volume (in µl)
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:
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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.
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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:
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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:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
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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:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
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3.3.1.2 Viability check during enrichments of Soil Sample 2:
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
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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: -
Table 3.25 represents the viable counts at different intervals and the types of bacteria that
appeared during 2nd
enrichment. An increase in viable count and a decrease in the types
of bacteria was observed (compared to 1st enrichment).
Table 3.25: Viable count during the second enrichment of Soil Sample 2:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
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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:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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3.3.1.3 Viability check during enrichments of Soil Sample 3:
Enrichment 1: Observations of regular viability check of the three successive
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.
Table 3.28: Viable count during the first Enrichment of Soil Sample 3
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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:
Table 3.29 represents the viable counts at different intervals and the types of bacteria that
appeared during 2nd
enrichment.
Table 3.29: Viable count during the second enrichment of Soil Sample 3:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
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Enrichment 3: -
Table 3.30 indicates the details of the third enrichment i.e. viable count observed at different intervals and the types of colonies
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:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Table 3.31 indicates the colony characteristics of the bacteria obtained from enrichment of soil sample 3.
Table 3.31: General characteristics of colony forming unit obtained from enrichment of soil sample 3:
Sr. No Size Shape Margin Elevation Color Texture Opacity Consistency Gram Nature Cell Morphology
1 Medium Irregular Irregular Convex Off-white Smooth Translucent Butyrous Gram positive Bacilli
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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3.3.1.4 Viability check during enrichments of Soil Sample 4:
Enrichment 1: Observations of regular viability check of the three successive
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.
Table 3.32: Viable count during the first Enrichment of Soil Sample 4
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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:
Table 3.33 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).
Table 3.33: Viable count during second enrichment of Soil Sample 4:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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Enrichment 3:-
Table 3.34 indicates the details of the third enrichment i.e. viable count observed at different intervals and the types of colonies
observed. By the end of the third enrichment, one type of colony was retained.
Table 3.34: Viable count of third Enrichment of Soil Sample 4:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Table 3.35 indicates the colony characteristics of the bacteria obtained from enrichment of soil sample 4.
Table 3.35: General characteristics of colony forming unit obtained from enrichment of soil sample 4:
Sr. No Size Shape Margin Elevation Color Texture Opacity Consistency Gram Nature Cell Morphology
1 Large Irregular Irregular Flat Off-white Rough Translucent Powdery Gram Positive Bacilli
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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3.3.1.5 Viability check during enrichments of Soil Sample 5:
Enrichment 1: Observations of regular viability check of the three successive
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.
Table 3.36: Viable count during the first Enrichment of Soil Sample 5:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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.
Table 3.37: Viable count during the second enrichment of Soil Sample 5:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
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Enrichment 3: -
Table 3.38 indicates the details of the third enrichment i.e. viable count observed at different intervals and the types of colonies
observed. Only one type of colony was retained at the end of the enrichment process.
Table 3.38: Viable count of third Enrichment of Soil Sample 5:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Table 3.39 indicates the colony characteristics of the bacteria obtained from enrichment of soil sample 5.
Table 3.39: General characteristics of colony forming unit obtained from enrichment of soil sample 5:
Sr. No Size Shape Margin Elevation Color Texture Opacity Consistency Gram Nature Cell Morphology
1 Medium Irregular Irregular Convex White Smooth Opaque Mucoid Gram Positive Bacilli
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3.3.1.6 Viability check during enrichments of Soil Sample 6:
Enrichment 1: Observations of regular viability check of the three successive
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.
Table 3.40: Viable count during the first Enrichment of Soil Sample 6:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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.
Table 3.41: Viable count during the second enrichment of Soil Sample 6:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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Enrichment 3:-
Table 3.42 indicates the details of the third enrichment i.e. viable count observed at different intervals and the types of colonies
observed. Not much increase was observed in the viable count and one type of bacteria was retained.
Table 3.42: Viable count of third Enrichment of Soil Sample 6:
Time of Viable Count
(during Enrichment)
Average Viable Count
(Colony forming units/ml)
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
Table 3.43: General characteristics of colony forming unit obtained from enrichment of soil sample 6:
Sr. No Size Shape Margin Elevation Color Texture Opacity Consistency Gram Nature Cell Morphology
1 Small Round Entire Low-convex Yellow Smooth Translucent Butyrous Gram negative Coccobacilli
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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.
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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 + + +
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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.
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
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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
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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
utilization of sugar
Oxidative and
fermentative
utilization of sugar
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
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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.
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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
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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
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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.
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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
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16SrRNA sequence of isolate RM02:
CTAGCGGGCTGGCCTTAATAATGCCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTG
ATGTTAGCGGCGGACGGGTGTGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATA
ACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTAT
AAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGG
TGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGC
CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCT
TCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG
GATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCT
TGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA
CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTC
TTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGG
AACTTGAGTGCAGAAGAGGAGAGTGAATTCCACGTGTAGCGGTGAAATGCGTAGAG
ATGTGAGGACACCAGTGGCGAAGGCGACTCTCTGGTCTGTACTGACGCTGAGGCGCG
AAAGCGTGGGGAGCGACAGATTAGATACCCTGTAGTCACGCGTAACGATGATGCTAT
GTAGAGGGTTTCCGCCTTAATGCTGCAGCACGCATAGCACTCGGCCTGGGAGTACGT
TCGCAGACTGGAACTCAGATGACGGGTCCGCAACGGTGACATGGGATTATTCG(903bp
)
Figure 3.8: Phylogenetic tree of isolate RM02: The isolate shows maximum homology
with B. licheniformis
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16SrRNA sequence of isolate RM03:
TAAACGGGCTTCCCAATAAAGACAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTG
ATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGAT
AACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTA
TAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTG
GTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGG
CCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATC
TTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG
GATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCT
TGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA
CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTC
TTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAAACTGGG
GAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAG
AGATGTGGAGGGAACACCAGTGGCGAAGCGACTCTCTGGGTCTGTAACTGACGCTG
AGCGCGAAGCGTGGGGGGAGCGAACAGGATTAGATACCCTGTAGTCCACCGCCCGT
AAACGATGATTGCTTAGTGTAGAGGTTTCCGCCCATTAGTTGCTGCAGCAAACGCAA
TTAAGCACTCCGCCTGGGGGAGTACCGCTCGCAAGACTTGAACTCAAGGAATGACCG
GGGTCCA (910bps)
Figure 3.9: Phylogenetic tree of isolate RM03: The isolate shows maximum homology
with B. subtilis
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
School of Sciences, SVKM’S NMIMS (Deemed-to-be) University 108
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.
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
School of Sciences, SVKM’S NMIMS (Deemed-to-be) University 109
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
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
School of Sciences, SVKM’S NMIMS (Deemed-to-be) University 110
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).
Chapter 3: Enrichment, Isolation and Identification of Feather Degrading Bacteria
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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.