“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa” 1.1 Bioremediation: Bioremediation is a pollution control technology that uses biological systems to catalyze the degradation or transformation of various toxic chemicals to less harmful forms. The general approaches to bioremediation are to enhance natural biodegradation by native organisms (intrinsic bioremediation), to carry out environmental modification by applying nutrients or aeration (biostimulation) or through addition of microorganisms (bioaugmentation). Unlike conventional technologies, bioremediation can be carried out on-site. Bioremediation is limited in the number of toxic materials it can handle (Hart, 1996), but where applicable, it is cost- effective(Atlas&Unterman,1997). Biodegradation, mineralization, bioremediation, biodeterioration, biotransformation, bioaccumulation and biosorption are some terms with minor differences but often overlappingly used. Biodegradation is the general term used for all biologically mediated breaks down of chemical compounds and complete biodegradation leads to mineralization. Biotransformation is a step in the biochemical pathway that leads to the conversion of a molecule (precursor) into a product. A series of such steps are required for a biochemical pathway. In Dept of Biotechnology, BITM, Bellary. Page 1
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
1.1 Bioremediation:
Bioremediation is a pollution control technology that uses biological systems to catalyze
the degradation or transformation of various toxic chemicals to less harmful forms. The general
approaches to bioremediation are to enhance natural biodegradation by native organisms
(intrinsic bioremediation), to carry out environmental modification by applying nutrients or
aeration (biostimulation) or through addition of microorganisms (bioaugmentation). Unlike
conventional technologies, bioremediation can be carried out on-site. Bioremediation is limited
in the number of toxic materials it can handle (Hart, 1996), but where applicable, it is cost-
potato dextrose broth of LR grade were obtained from Hi-media Laboratory.
Acetate buffer, phosphate buffer and Tris HCL were prepared.
3.2 Collection of Microorganisms
For the study of various parameters such as optimum temperature, pH, incubation time
and degradation capability, we have collected the pure cultures of Pseudomonas aeruginosa and
aspergillus niger from Department of Microbiology, Vijayanagara Institute of Medical Sciences
(VIMS) Bellary.
Pseudomonas aeruginosa
FIG 1: PURE CULTURE OF P.Aeruginosa
Dept of Biotechnology, BITM, Bellary. Page 19
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
Aspergilllus niger
FIG 2: PURE CULTURE OF A.Niger
3.2 Methods:
Preparation of media:
100ml of Nutrient broth and nutrient agar was prepared by dissolving 1.3gms of nutrient
broth and 2.8gm of nutrient agar which contains; peptic digest of animal tissue(5 gm/lt),sodium
chloride(5 gm/lt),beef extract(1.5 gm/lt), yeast extract(1.5 gm/lt) and 15 gm/lt of agar was added
while preparing the nutrient agar in 100ml of double distilled water by maintaining pH 7.4±0.2
under aseptic conditions. A loop full of pure culture (p.aeruginosa)was inoculated in the prepared
media to obtain the sub culture under sterile conditions and was kept for incubation at 37c for 24
hours. Whereas for sub culturing of a.niger 2.4gm of potato dextrose broth and 4.1gms of potato
dextrose agar; which contains potatoes infusion(200 gm/lt), dextrose(20 gm/lt) and 15gm/lt of
agar was added while preparing potato dextrose agar dissolved in 100 ml of double distilled
water by maintaining pH of 5.1±0.2. For maintainence of pure cultures, sub culturing was done
every week.
Dept of Biotechnology, BITM, Bellary. Page 20
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
Preparation of congo red solution:
0.5% of congo red was prepared by adding 0.05 gms of congo red in 10 ml of distilled water.
FIG 3: CONGO RED DYE AND SOLUTION
3.2.1 pH optimization:
For the optimization of pH, buffers of different pH were prepared ranging from 4-9.
Buffers of pH 4 and 5 were prepared using acetate buffer and ph 6, 7 and 8 were prepared by
using phosphate buffer and tris HCl buffer was used to prepare pH 9 buffer. Subcultures of
Pseudomonas aeruginosa and aspergillus niger were prepared. 6 test tubes were taken for each
organism for pH ranging from 4-9 1ml of buffer and 1ml of 0.5% congo red were added to all the
test tubes. To measure the OD, blank solutions for pH ranging from 4-9 were prepared i.e., for pH
Dept of Biotechnology, BITM, Bellary. Page 21
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
4 the blank was prepared by using 1ml of pH 4 buffer along with 1ml of 0.5% congo red dye,
likewise the blanks were prepared for the rest of the pH from 4-9.
3.2.2 Optimization of temperature:
For the optimization of temperature, take three test tubes for each organism and add 1ml
of buffer of Ph 4 and 1ml 0f 0.5 % congo red for all the test tubes. Then add 5ml of subcultured
organism to all test tubes i.e., P.Aureginosa with 3 test tubes and Aspergillus niger with other 3
test tubes. Incubate the test tubes for 24hrs at three different temperatures, first test tube at 37ºc,
second at 45ºc and third at 55ºc. After incubation all the test tubes will be centrifuged at
10,000rpm for 3 minutes and OD is measured at 497nm by preparing blank(1ml of Ph 4 buffer
and 1ml of dye).
3.2.3 Effect of incubation:
The specific time period for degradation of dye with the organisms was done by the
following procedure; test tubes with 1ml of pH 4 buffer, 1ml of 0.5% congo red and 5ml of
cultured organism were incubated at different time periods i.e., 1st test tube for 24 hours, 2nd test
tube at 48 hours , 3rd for 72 hours and 4th for 96 hours and were analysed for the degradation. The
24 hours incubated test tube was centrifuged and the OD was noted at 497nm. This process was
repeated for 48 hours, 72 hours and 96 hours test tubes and the OD was noted.
3.2.4 Study of media optimization:
Four different media were prepared i.e., one is free with beef extract, next media free
with peptone, other is free with sodium chloride and the last media is free with yeast extract.
Adjust the pH of all the media and sterilize. Four test tubes for each organism were taken and
Dept of Biotechnology, BITM, Bellary. Page 22
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
5ml of the different media was added to four different test tubes of both the organisms and
inoculated with the organisms for all the test tubes. These test tubes were incubated for optimum
temperature pH and incubation time and the OD was measured at 497nm to find the maximum
degradation.
3.2.5 Effect of UV exposure:
Nutrient agar and potato dextrose agar for P.aeruginosa and A.niger were prepared and
sterilized. The petriplates were prepared and allowed for solidification under aseptic conditions.
Then 200µl of the culture was poured to the petriplates by pour plate technique and incubated for
24 hours then exposed the petriplates under UV light for 10 min. Comparison of the maximum
degradation capacity of the UV unexposed organism along with the UV exposed organism was
done by the following procedure.
Six test tubes for each organism were taken and then the respective broth media of 5ml
was added to all the test tubes. Then three test tubes were inoculated with the UV unexposed
organism and the other three with the UV exposed organism and incubated for 24 hours (same
process with other organism).The OD was measured at 497nm and analyzed for the maximum
degradation capacity of the UV unexposed organism and UV exposed organism.
Dept of Biotechnology, BITM, Bellary. Page 23
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
4.1 Results:
The media and chemicals were prepared under the strict laboratory procedures and sterile
conditions as per chapter 3 and annexure. These were used for optimizing the parameters. The
media and chemicals prepared under such conditions gave 100% results without any
contamination.
4.1.1 Optimization of pH:
Buffers of different pH ranging from 4-9 and sub cultures of P.aureginosa and A.niger were
prepared. Six test tubes for each organism were taken. Add 1ml of the buffer, 1ml of 0.5%
congored and 5ml of sub cultured organisms for all the test tubes. All these test tubes were
incubated for 24 hours and OD was noted at 497nm to find the maximum degradation at a
particular pH. From the readings obtained by measuring the OD at 497nm, maximum
degradation for the organisms was found at pH 4 which was depicted from graph shown above.
Therefore pH 4 was maintained for all the other parameters.
P.Aeruginosa
Dept of Biotechnology, BITM, Bellary. Page 24
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
FIG 4: PH OPTIMIZATION (pH 4 - pH 9)
Table 6 : PH Optimization for P.Aeruginosa
Sl no
Buffer
(ml)
Dye
( ml)
Subculture of P.aeruginosa
(ml)
Incubation
For 24 hrs
At 37oC
OD at
497nm
1 pH 4 1 1 5 0.06
2 pH 5 1 1 5 0.04
3 pH 6 1 1 5 0.02
4 pH 7 1 1 5 0.04
5 pH 8 1 1 5 0.01
6 pH 9 1 1 5 0.02
A.niger
Dept of Biotechnology, BITM, Bellary. Page 25
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
FIG 5: PH OPTIMIZATION (4 -9)
Table 7: PH Optimization for A.Niger
Sl no
Buffer
(ml)
Dye
( ml)
Subculture of A.niger
(ml)
Incubation
For 24 hrs
At 37oC
OD at
497nm
1 pH 4 1 1 5 0.11
2 pH 5 1 1 5 0.01
3 pH 6 1 1 5 0.01
4 pH 7 1 1 5 0.02
5 pH 8 1 1 5 0.01
6 pH 9 1 1 5 0.08
Dept of Biotechnology, BITM, Bellary. Page 26
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
4 5 6 7 8 90
0.02
0.04
0.06
0.08
0.1
0.12pH optimization
p.aeruginosaa.niger
pH
OD at497nm
4.1.2 Optimization of temperature:
Three test tubes for each organism were taken and 1ml of buffer of pH 4,1ml of 0.5% congored and 5ml of the subcultured organisms were added to all the test tubes. These test tubes were incubated for 24 hours at three different temperatures i.e., 370c, 450c and 550c and OD was noted at 497nm to analyse the maximum degradation at a particular temperature. From the readings obtained, maximum degradation was found at 450c for A.niger and 550c for P.aeruginosa which is shown in the graph. Therefore these temperatures were maintained for the respective organisms for the optimization of other parameters.
P.aeruginosa
Dept of Biotechnology, BITM, Bellary. Page 27
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
FIG 6: OPTIMIZATION OF TEMPERATURE
Table 8: Optimization of temperature for P.Aeruginosa
Sl no
Temperature
(0C)
OD at
497nm
1 37 1.39
2 45 1.44
3 55 1.45
4 65 1.35
A.niger
FIG 7: OPTIMIZATION OF TEMPERATURE
Dept of Biotechnology, BITM, Bellary. Page 28
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
Table 9: Optimization of temperature for A.Niger
Sl no
Temperature
(0C)
OD at
497nm
1 37 1.46
2 45 1.48
3 55 1.42
4 65 1.41
37 45 55 651.25
1.3
1.35
1.4
1.45
1.5 Temperature optimization
p.aeruginosaA.niger
Temperature
OD at 497nm
4.1.3 Effect of incubation time:
To know the specific time for the degradation of the dye, four test tubes were taken and 1ml of pH4 buffer, 1ml of 0.5% congored and 5ml of sub cultured organisms was added for all the test tubes. Incubate all these test tubes for different time periods i.e., at 24 hours,48 hours,72 hours and at 96 hours by maintaining specific temperatures for the particular organisms. From the readings and the graph, maximum degradation was found at 48 hours for A.niger and 96 hours for P.aeruginosa. Therefore these time periods were maintained to get the maximum degradation of the dye.
Dept of Biotechnology, BITM, Bellary. Page 29
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
24 hours 48 hours
72 hours 96 hours
FIG 8: EFFECT OF INCUBATION
Table 10: Effect of incubation time for P.Aeruginosa
Sl no Time OD at 497 nm
1 24 0.06
2 48 0.05
3 72 0.02
4 96 1.06
Dept of Biotechnology, BITM, Bellary. Page 30
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
Table 11: Effect of incubation time for A.Niger
Sl no Time OD at 497 nm
1 24 0.04
2 48 0.2
3 72 0.04
4 96 0.03
Graph:
24 48 72 960
0.2
0.4
0.6
0.8
1
1.2Incubation time
p.aeruginosa
a.niger
Time in hrs
OD at 497nm
4.1.4 Study of media optimization:
Four different media were prepared i.e., one is free with beef extract, next media free with peptone, other is free with sodium chloride and the last media is free with yeast extract. Adjust the pH of all the media and sterilize. Four test tubes for each organism were taken and 5ml of the different media was added to four different test tubes of both the organisms and inoculated with the organisms for all the test tubes. These test tubes were incubated for optimum temperature Ph and incubation time and the OD was measured at 497nm to find the maximum degradation. From
Dept of Biotechnology, BITM, Bellary. Page 31
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
the readings obtained and the graph plotted the maximum degradation was found in the sodium chloride free media for A.niger and peptone free media and yeast free media for P.aeruginosa.
P.Aeruginosa
Yeast NaCl Peptone Beef
FIG 9: OPTIMIZATION OF MEDIA
Table 12: Optimization of media for P.Aeruginosa
Media OD at 497nm
Beef free 0.15
Peptone free 1.6
NaCl free 0.30
Yeast free 1.54
A.Niger
Dept of Biotechnology, BITM, Bellary. Page 32
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
Yeast NaCl Beef Peptone
FIG 10: OPTIMIZATION OF MEDIA
Table 13: Optimization of media for A.Niger
Media OD at 497 nm
Beef free 0.03
Peptone free 0.04
NaCl free 0.15
Yeast free 0.06
beef free peptone free nacl free yeast free0
0.20.40.60.8
11.21.41.61.8
media optimization
p aeruginosaa.niger
MEDIA
OD at 497nm
Dept of Biotechnology, BITM, Bellary. Page 33
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
4.1.5 Effect of UV exposure:
Nutrient agar and potato dextrose agar for P.aeruginosa and A.niger were prepared and
sterilized. The petriplates were prepared and allowed for solidification under aseptic conditions.
Then 200µl of the culture was poured to the petriplates by pour plate technique and incubated for
24 hours then exposed the petriplates under UV light for 10 min. Comparison of the maximum
degradation capacity of the UV unexposed organism along with the UV exposed organism was
done by the following procedure.
Six test tubes for each organism were taken and then the respective broth media of 5ml
was added to all the test tubes. Then three test tubes were inoculated with the UV unexposed
organism and the other three with the UV exposed organism and incubated for 24 hours (same
process with other organism).The OD was measured at 497nm and analyzed for the maximum
degradation capacity of the UV unexposed organism and UV exposed organism. From the
readings obtained and the graph plotted maximum degradation was found in UV exposed
organism compared to UV unexposed organism for P.aeruginosa, and for A.niger maximum
degradation was found in UV unexposed organism compared to that of the UV exposed
organism.
P.Aureginosa
UV Exposed Normal
FIG 11: COMPARING DEGRADATION CAPACITY OF NORMAL & UV EXPOSED
Table 14: Comparing degradation capacity of normal and UV exposed P.Aeruginosa
Sl no UV unexposed OD at 497 nm
UV exposed OD at 497 nm
Dept of Biotechnology, BITM, Bellary. Page 34
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
1 0.02 0.04
2 0.02 0.03
3 0.02 0.04
A.Niger
UV Exposed Normal
FIG 12: COMPARING DEGRADATION CAPACITY OF NORMAL & UV EXPOSED
Table 15: Comparing degradation capacity of normal and UV exposed A.Niger
Sl no UV unexposed OD at 497 nm
UV exposed OD at 497 nm
1 0.08 0.04
2 0.06 0.05
3 0.06 0.05
4.2 Discussion:
Earlier works has been done on the biodegradation of congo red, azo carmine, direct orange dyes by organisms like bacillus sp, pseudomonas sp, citrobacter sp candidazeylanoides, etc
Dept of Biotechnology, BITM, Bellary. Page 35
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
(1) Decolorization of direct orange 39(Orange TGLL) by an isolated bacterium Pseudomonas aeruginosa was done by Jyothi.P.Jadhav, Swapnil S. Phugare1, Rhishikesh S. Dhanve1
and Shekhar B. Jadhav
(2) Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium Pseudomona sp. SU-EBT Amar A. Telke Swati M. Joshi Sheetal U. Jadhav Dhawal P. Tamboli , Sanjay P. Govindwar
In the present study, we have taken two organisms namely P.aeruginosa and A.Niger for the degradation of the congo red dye which is generally used in the textile industry for coloring purpose.
We have taken few parameters like PH, temperature, incubation time, dye concentration, media optimization, effect of carbon and nitrogen sources, and effect of exposure to UV light for stabilizing the P.aeruginosa and A.Niger for effective degradation.
In our project, we first stabilized the PH by checking maximum degradation in range PH
4-9. After taking the OD it was concluded that PH 4 was optimum for both the organism.
Then in the temperature optimization, the maximum degradation was checked for different temperature like 370c, 450c, 550c. After the OD results, graphs were plotted and concluded that P.aeruginosa optimum temperature was450c and A.Niger was 550c.
For incubation time, the maximum degradation was checked by incubating for different time periods like 24hr, 48hr, 72hr, and 92hrs. After the OD results, graphs were plotted and concluded that P.aeruginosa incubation time was 96hrs and A.Niger was 48hrs.
Media optimization: The maximum dye degradation was checked by using the media lacking yeast or peptone or beef or NaCl subsequently. By the OD results, it was concluded that the nutrient media lacking peptone free was suited for P.aeruginosa and NaCl was suited for A.niger.
Further the work can be done on the following:
• Analysis of metabolites obtained after decolorization:After the degradation of the congo red dye by the micro organisms the
metabolites can be identified and studied.
Dept of Biotechnology, BITM, Bellary. Page 36
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
• Analysis of the degradation pathway:For the degradation of the congo red dye the pathways so followed by the
organisms can be found and studied to further follow the appropriate pathway to obtain maximum degradation.
• The enzyme responsible for the degradationAspergillus sp and pseudomonas sp have many enzymes which are responsible
for the degradation of the dye at different pH, these enzymes can be identified and purified for degradation of the dyes.
• Effect of mutation due to UV exposure.After the exposure to UV light, confirmation of mutation can be done and the
mutated gene can be analysed to further improve the degradation capacity.
The industrial pollution has grown as one of the most potent challenges for the
environment. The removal of the pollutants from the nature has become one of the prime areas
of research. The bio based removal of pollutants has gained niche because of permanent, short
period and safe bioconversions. Various microorganisms have been reported to have such
capability and the present study is on microbial degradation of congo red, a prominently used azo
dye in textile industry. Congo red at high concentration is toxic to microorganisms, plants and
animals including human beings. Naturally pseudomonas occurring are found to be well suited to
congo red degradation process in which temperature, can be monitored and controlled easily.
The congo red degradability of the isolates can be maintained and used at large scale effluent
treatment. . The pseudomonas sp. isolate was found to have a better degradation capability than
the aspergillus niger.
Dept of Biotechnology, BITM, Bellary. Page 37
“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”
Annexure :
Table 5: Preparation of buffers:
1. Acetate buffer:Solution A: 0.2 mol/lt of acetic acid(11.55ml/1000ml)Solution B: 0.2 mol/lt of sodium citrate(16.4gm/1000ml)