MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE
SUMMARY
BACHELOR OF SCIENCE THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY
ISOLATION AND SELECTION THE
THERMOPHILIC KERATIN DEGRADATION
BACTERIA IN DONG THAP PROVINCE
SUPERVISOR
STUDENT
Dr. BUI THI MINH DIEU
PHAM MINH TRIET
Student’s code: 3082639
Session: 34 (2008- 2013)
Can Tho, 05/2013
APPROVAL
SUPERVISOR
Dr. BUI THI MINH DIEU
STUDENT
PHAM MINH TRIET
Can Tho, May , 2013
PRESIDENT OF EXAMINATION COMMITTEE
Abstract
Using of microorganisms, especially bacteria to process
keratin-containing waste source has demonstrated their potential
and advantages in many areas of life: food processing for cattle
and poultry, bio-fertilizer production with lower cost and
environmental friendly ... The temperatures in the waste graves
can raise highly and inhibit almost activities of the useful
bacteria. Therefore, the thesis was carried out for isolation and
selection the thermophilic keratinolytic bacteria strains which
have
high
degradation
capability
of
keratin-containing
wastes. Total of 18 aerobic keratinolytic bacteria strains were
isolated of five soil and two wastewater samples from animal and
poultry processing plants. The result showed that all the isolated
strains were capable of growth and degradation keratin at 45°C;
in which ten strains can grow at 50°C; and two strains can
o
survive at 55 C. All isolated strains expressed protease activity
with the halo zone creating on the skim milk medium after 24
hours incubation. The halo diameter ranged from 4.7mm to
10.3mm. The results of feathers degradation study was recorded
that 18 strains were capable of reducing 14.59% to 37.76%
poultry feather; and 25.12% to 42.18 goat hair. In which, V1, V2
and V9 strains showed the ability of keratin degradation
effectively than the others. The results of sequencing analysis of
partial 16S rRNA gene showed that the gene sequences of V1
shared 99% similarity with Bacillus megaterium species and V2
shared 99% similarity with Bacillus sp. P014.
Key words: Bacillus, Bacillus megaterium, keratinase, keratin
degradation, thermophilic bacteria
i
CONTENTS
ABSTRACT………………………………………………...........i
CONTENTS..………………………..……………...……....…...ii
1. INTRODUCTION ................................................................. 1
2. MATERIALS AND METHODS ............................................ 3
2.1. Materials ............................................................................ 3
2.2. Methods ............................................................................. 3
2.2.1. Collection and treatment samples method ................... 3
2.2.2. Method of isolation ..................................................... 3
2.2.3. Survey morphology and mobility of bacteria ............... 4
2.2.4. Gram staining ............................................................. 4
2.2.5. Study the biochemical properties ................................ 5
2.2.6. Count for bacteria cell density..................................... 5
2.2.7. Study protease activity of bacteria strains .................... 5
2.2.8. Study the poultry feather degradation of bacteria strains
............................................................................................ 6
2.2.9. Study the goat feather degradation of bacteria strains .. 6
2.2.10. Identification of the thermophilic bacteria with high
capable of keratin degradation by molecular technique ............... 7
3. RESULTS AND DISCUSSION ............................................ 8
3.1. Isolation and thermotolerance condition study .................. 8
3.2. Morphology of isolated bacteria strains............................. 8
3.3. Study of biochemical properties ...................................... 10
3.4. Study protease activity of bacteria strains ......................100
3.5. Study the poultry feather degradation of bacteria strains . 12
3.6. Study the goat feather degradation of bacteria strains ...... 14
ii
3.7. Identification of high keratin degradation thermophilic
bacteria strains ......................................................................... 15
4. CONCLUSIONS AND SUGGESTIONS ............................170
4.1. Conclusions ...................................................................190
4.2. Suggestions ...................................................................191
REFERENCES .......................................................................212
iii
1. INTRODUCTION
Today, the environmental problem is set many big challenges
for human. Using of microorganisms to treat environmental
pollution were showed their potential and benefits. In which
keratin degradation bacteria group are interested in many
scientists. Keratin is virtual resistant to the degradation of general
proteases as trypsin, pepsin…due to their stable structure. This
protein is major component of poultry feather and cattle feather
(up to 90%) (Gousterova et al., 2005.)
The dismission of poultry feather and cattle feather directly to
the environment not only polluted the environment but also
wasted the protein sources. Therefore, there are many researches
to process this waste source. However, almost of physical
methods and chemical methods were consumed big energy and
environmental unfriendly; and also reduces the nutritional value
of the product due to loss of several amino acids (Wang and
Parsons, 1997). Use of the thermophilic keratinolytic bacteria
strains is the new way to handle the problem above. This novel
method of treatment is potential and wide application. It helps to
enrich the nutritional value of feather products and environment
friendly (Lee et al., 1991).
The temperatures in the waste graves can rise highly and
inhibit almost activities of the useful bacteria. For that reason, the
thesis "Isolation and selection thermophilic keratin degradation
bacteria in Dong Thap province" was conducted to select the
thermophilic keratinolytic bacteria strains which have high
degradation capability of keratin-containing wastes.
1
Objectives
Isolation and selection of thermophilic bacteria strains with
high keratin degradation capability.
2
2. MATERIALS AND METHODS
2.1. Materials
Five soil and two wastewater samples were collected from
Cao Lanh district, Lai Vung district and Cao Lanh city, Dong
Thap province. These samples were stored at Molecular
Biotechnology laboratory, Can Tho University.
Medium: skim milk medium (peptone 5g/l, yeast extract 3g/l,
non-fat milk 100ml/l, agar 12g/l) (Riffel và Brandelli, 2006),
medium with poultry feather addition (NaCl 0.5g/l, KH2PO4
0.4g/l, K2HPO4 0.3g/l, NH4Cl 0.5g/l, Agar 20g/l, poultry feather
powder 10g/l) (Daniel et al., 2008), Luria Bertani medium (Bacto
tryptone 10g/l, Bacto yeast extract 5g/l, Nacl 10g/l).
Chemicals and equipments in Molecular Biotechnology
laboratory.
2.2. Methods
2.2.1. Collection and treatment samples method
- Soil samples: took soil 20cm deep from the surface at
poultry or cattle farm and slaughter places.
- Water samples: collected directly at wastewater sources.
Samples were stored at laboratory until the experiments are
carried out.
2.2.2. Method of isolation
Add 1ml (water sample) or 1g (soil sample) of sample to
erlen 250ml containing 100ml medium (NaCl 0.5g/l, KH2PO4
0.4g/l, K2HPO4 0.3g/l, NH4Cl 0.5g/l, poultry feather powder
3
10g/l) sterilized at 121ºC in 10 minutes. Growing bacteria at
37°C, 120rpm for 24h to 48h.
Dilute from 10-6 to 10-8 times and streak on skim milk
medium. Incubate at 45ºC for 24h. After that select the colonies
with variant morphologies and halo zone creating to grow on
medium with feather powder addition for 24h at 45ºC until they
get pure strains.
Study the thermotolerance of bacteria strains by growing
them at 50ºC, 55ºC, 60ºC… Record their development in 24h to
48h.
2.2.3. Survey morphology and mobility of bacteria
Prepare bacteria samples as:
Drop 5 l of sterilized distilled water on lame take a
little of colony and stretch out regularly on water drop put
lamen on the lame and observe their morphology and mobility
under x400 microscope.
2.2.4. Gram staining
Drop 20 l sterilized distilled water on lame take a little
of colony and stretch out regularly on water drop place the
lame on fire (not over heat) put one or two drops of Crystal
Violet for two minutes wash with sterilized distilled water,
dried slightly slower Instil one or two drops of iodine solution
for a minute Wash with sterile distilled water, dried slightly
slower Instil one or two drops Fusin for a minute Wash
with sterilized distilled water. Observe sample in optical
microscope at magnification x1000 and record by Gram bacteria.
Bacteria is Gram-positive if sample has blue purple color of
4
Crystal violet, Gram-negative if sample is red color of Fusin (Cao
Ngoc Diep and Nguyen Huu Hiep, 2008).
2.2.5. Study the biochemical properties
Biochemical properties was performed for identification
the bacteria.
Study catalase activity: Add one to three drops of H2O2 3%
to Petri dish containing bacteria. Record the results.
Study methyl red test: Add five drops of methyl red to 1ml
of bacteria suspension. Record the results.
Study amylase activity: Inoculate 1ml of bacteria solution
to 1ml of salt solution 0.1%. After that add 1ml of starch solution,
incubate at 30°C for 30 minutes. Record the results.
2.2.6. Count for bacteria cell density
Add 100 l of bacteria suspension to eppendorf containing
900 l sterilized distilled water to have diluted sample 10-1.
Dilute the sample to 10-10. Take 5 l of bacteria suspension and
drop on feather powder addition medium. Record the results after
24h to 48h at conditions of study.
2.2.7. Study protease activity of bacteria strains
Protease activity can determine by the ability of creating
halo zone on milk medium.
Drop 5 l of bacteria suspension on skim milk medium.
Record the results after 24h at different conditions of study. The
degrading diameter was calculated by:
Degrading diameter = Halo diameter – Diameter of
bacteria growing drop
5
2.2.8. Study the poultry feather degradation of bacteria
strains
Prepare a Erlenmeyer flask 250ml contain 100ml medium
(NaCl 0.5 g/l, KH2PO4 0.4 g/l, K2HPO4 0.3 g/l, NH4Cl 0.5 g/l and
1g of poultry feather powder) sterilize at 121°C for 10 minutes.
Inoculate 5ml of bacteria suspension to Erlenmeyer flask;
incubate at 120rpm at study temperature for one week. Filter
medium by filter paper and dry at 75°C until weight is constant.
Record the weight. The percentage of poultry feather degradation
A(%) are calculated by:
A (%) = (mBĐ - mC) x 100/ mBĐ
mBĐ: weight at beginning
mC: weight after experiment
* Study the degradation of whole chicken feather
Put whole chicken feather (same size and weight) to test
tube containing 20ml of (NaCl 0.5 g/l, KH2PO4 0.4 g/l, K2HPO4
0.3 g/l, NH4Cl 0.5 g/l), sterilized at 121°C for 10 minutes.
Inoculate 4ml of bacteria suspension and incubate at different
temperature of study, 120rpm for 10 days. Record the results.
2.2.9. Study the goat feather degradation of bacteria strains
Prepare an Erlenmeyer flask 250ml contain 100ml medium
(NaCl 0.5 g/l, KH2PO4 0.4 g/l, K2HPO4 0.3 g/l, NH4Cl 0.5 g/l and
1g of goat feather powder) sterilize at 121°C for 10 minutes.
Inoculate 5ml of bacteria suspension to Erlenmeyer flask;
incubate at 120rpm at study temperature for one week. Filter
medium by filter paper and dry at 75°C until weight is constant.
6
Record the weight. The percentage of goat feather degradation
A(%) are calculated as the form of experiment 2.2.8.
2.2.10. Identification of the thermophilic bacteria with high
capable of keratin degradation by molecular technique
The bacterial isolate was identified by sequence analysis of
partial 16S rRNA gene. A couple of primer including forward
primer
27F:
5’-AGAGTTTGATCCTGGCTC-3’and
reverse
primer 1492R: 5’-TACGGTTACCTTGTTACGACT-3’ were
employed to amplify the target gene through PCR technique. PCR
product was sequenced and analyzed at Molecular Biology
laboratory, Research and Development Biotechnology Institute,
Can Tho University.
7
3. RESULTS AND DISCUSSION
3.1. Isolation of thermolytic keratinolytic bacteria
Total 18 aerobic keratinolytic bacteria strains were isolated
from five soil and two wastewater samples using poultry feather
supplemented medium. The bacteria strains were named Vx with
x stand for ordinal number of the isolated bacteria strains.
The results of thermotolerance condition study showed that
all of 18 strains can grow and degrade keratin at 45°C (V1 to
V18); 10 strains can grow at 50°C (V1, V10, V11, V12, V13,
V14, V15, V16, V17, V18) and two strains can survive at 55°C
(V17, V18).
3.2. Morphology of isolated bacteria strains
Colony of 18 isolated strains has circle form, white color.
Colony diameter ranged from 0.5mm to 6.0mm. The results of
Gram staining at x1000 microscope showed that the cell of 14
bacteria strains were Gram negative (4 strains were Gram
positive: V1, V2, V5 and V9). Almost bacteria strains were rod
shaped (except strain V3 was sphere) (Table 8’).
Table 3’: Colony properties of bacteria strains
Colony properties
Name
Form
Elevation
Margin
Color
Diameter
(mm)
V1
Circular
Raised
Entire
Light pink
V2
Circular
Crateriform
Entire
Opaque white
8
1.5
6
V3
Circular
Raised
Entire
Opaque white
2
V4
Circular
Raised
Entire
Light yellow
1.5
V5
Circular
Raised
Entire
Light green
4
V6
Circular
Raised
Entire
Opaque white
0.8
V7
Circular
Crateriform
Entire
Opaque white
2.5
V8
Circular
Raised
Entire
Opaque white
3
V9
Circular
Crateriform
Waxy
Light yellow
5
V10
Circular
Crateriform
Waxy
Opaque white
1.2
V11
Circular
Raised
Entire
Opaque white
0.7
V12
Circular
Crateriform
Waxy
Opaque white
0.8
V13
Circular
Raised
Entire
Light yellow
1
V14
Circular
Raised
Entire
Opaque white
1.1
V15
Circular
Raised
Entire
Opaque white
0.9
V16
Circular
Raised
Entire
Opaque white
0.5
V17
Circular
Crateriform
Entire
Opaque white
3
V18
Circular
Raised
Entire
Light yellow
0.5
9
3.3. Study of biochemical properties
3.3.1. Catalase activity
The results of experiment showed that 14 strains had
catalase activity (bubble grew up, about 77.8%); four strains were
negative (including V1, V7, V10, V11).
3.3.2. Methyl red test
Four strains were considered as high acid creation as ++
value of three scale of value: 0, +, ++ (V3, V7, V13, V14) and 14
strains were low acid production as + value.
3.3.2. Amylase activity
Almost bacteria strains, except strain V16 showed amylase
activity with the ability to discolor blue color of mixed iodine and
starch.
3.4. Study protease activity of bacteria strains.
All isolated strains expressed protease activity with the
halo zone creating on the skim milk medium after 24h incubation.
Halo diameter ranged from 4.7mm to 10.3mm. In which, the
strains V1, V2, V9, V16 had halo circular diameter more than
9mm (difference was statistically significant at the 5% level
compared with other strains in each group of thermotolerant
study). Degrading halo diameters in this research were higher
than the research of Le Dinh Quyen and Tran Thi Lan Phuong
(2001) with 9.5mm degrading halo diameter creation by Bacillus
strain.
10
Halo diameter (mm)
12.0
10.3
10.2
10.0
7.3
8.0
6.3
6.0
5.0
5.3
v3
v4
6.0
4.7
4.0
2.0
0.0
v2
v5
v6
v7
v8
v9
Strain
Figure 7: Degrading halo diameter of bacteria strains on skim
milk medium after 24h incubation at 45°C
The result of Figure 7 showed that strains V2 and V9 could
create degrading halo diameter more than 9mm (difference was
statistically significant at the 5% level compared with the others).
The others also expressed protease activity. However, the result
Halo diameter (mm)
was not high.
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
9.3
8.3
9.3
8.7
7.7
7.7
6.0
4.7
v1
v10
v11
v12
v13
v14
v15
v16
Strain
Figure 8: Degrading halo diameter of bacteria strains on skim
milk medium after 24h incubation at 50°C
Figure 8 represented the result of degrading halo diameter
creation on skim milk medium after 24h incubation at 50°C. In
general, the result of this group was higher than the previous
11
group. However, the maximum halo diameter creation was lower
(9.3mm of V1 compared with 10.3mm of V2).
The results of study on degrading halo diameter creation of
bacteria strains at 55°C showed that this group could create
degrading diameter. However, the results were not important.
3.5. Study the poultry feather degradation of bacteria strains
After one week inoculation, the results demonstrated that
total 18 strains have keratin degradation capability by degradation
poultry feather powder (ranged from 14.59% to 37.76%).
Difference was statistically significant at 5% level compared with
the control treatment. The study group at 45°C gave better results.
Strain V9 showed the best degradation result than other strains
(37.76%). These results were lower than the research of Nguyen
Huy Hoang et al. (2010) with degradation results reached from
52.40% to 98.45% after one week inoculation. Optimum the
condition as temperature and medium components can investigate
degrading rate. The results were showed in Figure 10, Figure 11,
Degrading percentage (%)
and Figure 12.
37.76
40.00
32.29
35.00
30.00
24.79
22.96
25.00
20.00
14.59
15.88
16.96
18.44
v5
v6
v7
15.00
10.00
5.00
2.52
0.00
C
v2
v3
v4
v8
v9
Strains
Figure 9: Degradation of poultry feather powder by bacteria
strains after one week inoculation at 45°C
C: control treatment
12
Total eight strains of this group could degrade poultry
feather powder. Difference was statistically significant at 5%
level compared with the control treatment. In which V2 and V9
strains expressed degradation capability more effectively than the
Degrading percentage (%)
others (32.29% of V2 and 37.76% of V9).
40.00
35.64
35.00
30.67
30.68
30.00
28.28
29.82
26.44
28.81
28.96
v15
v16
25.00
20.00
15.00
10.00
5.00
2.29
0.00
C
v1
v10
v11
v12
v13
v14
Strains
Figure 10:
Degradation of poultry feather powder by
bacteria strains after one week inoculation at 50°C
C: control treatment
Figure 11 represented the result of poultry feather powder
degradation at 50°C. All strains of this group degraded feather
powder more than 26% after one week inoculation. The strain V1
showed the best result (35.64%).
The result of degradation capability of bacteria strains at
55°C was statistically significant difference at the 5% level
compared with control treatment (27.75% of V17 and 26.75% of
V18 compared with 2.38% of control treatment). However, the
maximum degrading percentage was lower than two previous
groups.
13
* Study chicken shaft degradation
Total 18 isolated bacteria strains were showed the capable
of chicken feather degradation in time study, except the feather
shaft. The study group at 45°C gave better result than other group.
Strain V2 and V9 were broken down whole chicken feather after
10 days inoculation. Strain V13 and V1 degraded well with more
than 70% broken up of chicken feather. The others also degraded
feather. However, the rate was low.
3.6. Study degradation of goat hair powder by bacteria strains
The aim of this experiment was selection bacteria strain
with high keratin degradation capability. The results of each
group were similar to poultry feather degradation test. The best
result was recorded by strain V9 with 42.18% degrading feather
weight. Strain V2 showed good result with 40.85%. Remainder
strains showed the degradation capability ranged from 25% to
40.47%. Figure 14, Figure 15, Figure 16 represented the goat hair
Degrading percentage (%)
degradation capability.
45.00
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
42.18
40.85
36.95
34.50
29.91
31.15
30.32
v5
v6
v7
25.12
2.38
C
v2
v3
v4
v8
v9
Strains
Figure 12: Degradation of goat hair powder by bacteria
strains after one week inoculation at 45°C
C: control treatment
14
Figure 14 showed the result of goat hair powder
degradation of bacteria strains at 45°C. Almost strains could
degrade goat hair powder more than 30%. Strains V2 and V9
Degrading percentage (%)
gave effective results with 40.85% of V2 and 42.18% of V9.
45.00
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
40.48
31.65
31.07
30.65
34.29
37.32
33.53
28.19
2.18
C
v1
v10
v11
v12
v13
v14
v15
v16
Strains
Figure 13: Degradation of goat hair powder by bacteria
strains after one week inoculation at 50°C
C: control treatment
Total eight strains of this group showed goat hair powder
degradation capability with degrading percentage ranged from
28.19% to 40.48%. Strain V1 showed the best result with 40.48%
goat hair powder degradation.
3.7. Identification of the high thermophilic keratinolytic
bacteria strains
Based on the study of keratin degradation, tow strains V1
and V2 were selected to sequence 16S rRNA gene and BLAST on
NCBI website. The result showed that the gene sequences of
isolated V1 shared 99% similarity with Bacillus megaterium;
isolated V2 shared 99% similarity with Bacillus sp. P014.
The result of 16S rRNA gene sequencing of strain V1
15
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