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Imagine a situation: Year 2020. A primary school in
the industrialized city situated at the banks of river
meeting the sea. Dharitri - a young sweet school going
child, showing carcinogenic symptoms in her young age
itself, takes part in the Nature drawing competition
and paints soil with dark black, leaves of the trees
in brown and a river in dark blue! She is helpless because
she is unaware of the original colours of her environment
she lives in. Do we want this to happen? Nobody will
like to be a part of such a dreadful Nature!! Not to
wait for the year 2020, some industrial cities having
large number of alcohol distilleries releasing molasses
spent wash (MSW), or the paper and pulp mills, textiles
and dye-making industries or leather industries that
discharge highly coloured effluents are already experiencing
signs of this situation.
Industries are important for our economic growth, but
certainly not at the cost of the clean environment.
Take the stock of alcohol producing distilleries in
India alone. About 2.7 billion liters of alcohol is
produced by about 285 distilleries in India. MSW in
such effluents is nearly 15 times in volume of the total
alcohol production. This huge quantity, about 40 billion
liters of MSW effluent, if disposed untreated in water
courses can cause great stress on the aquatic life.
As such, alcohol serve as a basic chemical for a large
number of industries and therefore, the demand for alcohol
will see a great increase in future and so also the
distilleries producing alcohol. The known solutions
to clean the environment are extremely expensive. For
example, large volume of water is required to dilute
the concentration of these effluents before they are
released in the rivers. But where this clean water would
come from? Many of you know how scarce is this resource
in many developing countries.
Let us not paint only a dreadful picture. Scientists
all over the world are working to find a good solution
to this problem. Bioremediation technique - biological
control - is being tried to have a control over the
situation. This is found to be environmentally friendly.
The living, non-harmful, organisms are used in this
process. These organisms do the job of 'cleaning' the
environment by 'living' and 'growing' on the pollutants!
Several species of terrestrial fungi are being tried
as the source organisms. These remove about 70-80% of
the colour. These fungi produce extracellular enzymes
like manganese-dependent peroxidase (MNP), lignin peroxidase
(LIP) and laccase in low nitrogen medium and are helpful
in the decolourization. These enzymes lack substrate
specificity and are thus capable of degrading a wide
range of xenobiotics including industrial coloured wastewaters.
Scientists at the National Institute of Oceanography
(NIO), since the last few years, have been investigating
the possibility of using fungi from saline waters. Few
years earlier, they achieved decolourization of MSW
using free mycelia of Flavodon flavus, - a white-rot
fungus isolated from decomposing leaves of a seagrass
from a coral lagoon in India. This has provided an avenue
to substantially decolourize and detoxify the effluents
within single process. The story does not end here.
Continual research in this direction has given better
results achieving 100% decolourization too using another
fungi.
A team of scientists working on this problem
at NIO found that the use of immobilized fungus not
only helped in decolourization of MSW but also detoxified
the effluent to some extent. Flavodon flavus
immobilized in polyurethane foam cubes, removed up to
60% of the colour from MSW used at 10% concentration
within the first 5 days itself and showed a success
rate to about 73% if kept for subsequent days (Fig.
1).
.
It was interesting to understand why 10%
concentration of the MSW only was selected. Actually,
the test was initially carried out on different concentrations
of MSW and they found that the maximum decolourization
was achievable when MSW is used at 10% concentration
with live, immobilized fungus (Fig. 2).
While doing this experiment, they also
found that the same batch of immobilized fungus could
be used effectively up to three consecutive cycles of
decolourization of fresh MSW (Fig. 3) and if a second
round of new immobilized fungus is used over the treated
batch of MSW, then it further decolourizes to the extent
of 70-80%!
The scientists also found that the use of immobilized
white-rot fungus (F. flavus) helps even in removing
some of the toxic substances from the effluents. An
estuarine fish, Oreochromis mossambicus was exposed
to 10% MSW untreated and treated sample with immobilized
fungus. At the end of 4 days, based on serum sorbitol
dehydrogenase concentration in fish, they noticed that
the treatment of MSW has removed the damage-causing
factor to the extent of ca. 98%. This was further confirmed
by checking the damage to the hepatic cells of the fish.
85% of the cells in the fish exposed to untreated MSW
were found to be damaged as against only 9% in the treated
MSW indicating total removal of toxic factor after the
fungal treatment. One more parameter was used to confirm
the detoxification by checking the concentration of
polycyclic aromatic hydrocarbons (PAH). The PAH concentration
in the untreated MSW was 3.8 µg ml-1
and this was reduced by nearly 68% (1.2 g ml-1) after
treatment with the white-rot fungus.
The scientists also claim that the F. Flavus
not only decolourize the MSW but also effectively act
on other synthetic dyes and bleach plant effluent of
pulp and paper mills. Being economical, easier to handle
and its advantage of reusability this seem to be the
best method as of today - opine the scientists.
Above experiments decolourized the effluents to the
extent of 50-80% while 100% is the target! Knowing the
importance of lignin degrading enzymes, the scientists
further tried to find out the effect of a major lignin-degrading
enzyme - Laccase - from an isolate (numbered as NIOCC#2a)
of an unknown basidiomycetous fungus collected from
decaying wood pieces in the mangrove swamps of Chorao
Islands at Goa, India. To start with, the effect of
various salinities on the growth of fungus and production
of lignin-degrading enzymes viz, LIP, MNP and laccase
were determined to have the best product. The salinity
in the mangrove environment fluctuates between 5 to
35 parts per thousand (ppt). Therefore this range was
considered to be ideal for the test. They found maximum
fungal biomass at 34ppt salinity, whereas, best laccase
production at 25ppt salinity (Fig. 4) . The MNP and
LIP production in this fungus was, however, negligible.
Laccase is an extracellular enzyme. If it is required
for decolourization in large quantity, its enhanced
production is essential. The relevant experiments revealed
enhanced production of laccase in the presence of coloured
effluents and other synthetic dyes collected from distilleries,
paper mills and textile dyeing industries. This was
also tested with other known inducers. Among the various
inducers, Copper sulphate and Guaiacol induced the maximum
laccase production individually as well as in combination
in 21 days. Among the dyes, brilliant green and among
the effluents, textile effluent B induced good laccase
activity within 18 days. In general, it was noticed
that the dyes that acted as inducers of laccase production
in the culture medium were in turn decolourized by the
enzyme so produced!
The scientists further tried with two forms of cultures
of the fungus for the decolourization - fungus free-culture
supernatant and exopolymeric substances (EPS) produced
by the fungus. And interestingly, they noticed almost
total decolourization of some of the dyes and effluents
within 24 hours of incubation with the EPS produced
by the fungus.
TABLE 3. decolourization of dyes and effluents
using fungus free- culture supernatant and EPS produced
by the fungus
|
% decolourization by the culture
supernatant |
% decolourization by the EPS of
the fungus |
| Dye |
Hours
|
Hours
|
|
6
|
12
|
12
|
24
|
| Trypan Blue (0.04%) |
22
|
25
|
20
|
79
|
| Aniline Blue (0.04%) |
55
|
40
|
46
|
75
|
| Methylene Blue (0.02%) |
3
|
5
|
4
|
6
|
| RBBR (0.04%) |
67
|
46
|
19
|
100
|
| Crystal Violet (0.02%) |
44
|
54
|
45
|
80
|
| Brilliant Green (0.02%) |
72
|
79
|
2
|
90
|
| Poly-R 478 (0.02%) |
21
|
43
|
33
|
90
|
| Congo Red (0.02%) |
54
|
47
|
18
|
29
|
| RO 176 (0.015%) |
ND
|
ND
|
35
|
100
|
| Effluents |
|
|
|
|
| Textile effluent A (10 %) |
9
|
11
|
11
|
100
|
| Textile effluent B (10 %) |
14
|
22
|
35
|
100
|
| Molasses spent wash (10 %) |
34
|
33
|
12
|
100
|
| Black liquor (10 %) |
71
|
59
|
41
|
100
|
| 500 µl of culture supernatant
having 18 U ml -1 laccase activity was
incubated with 500 µl of dye solution at pH
6.0 and 60°C. The absorbance was measured at
appropriate wavelengths to calculate the % of decolourization
after 6 and 12 hours. 10 mg of freeze-dried EPS
of the fungus was incubated with dye solutions prepared
in phosphate buffer pH 6.0 at 60°C. decolourization
was measured at 6 and 12 h at the absorbance maxima
specific to the dye and effluents. The % decolourization
was calculated based on the initial readings. All
the values are mean of 2 replicates. |
The efforts to identify better fungi and further processes
for bioremediation that not only maximize the decolourization
process in terms of percentage and shortest time but
also remove the toxicity to the highest level will go
on. Putting the research results in practice, we all
will live healthily in the Nature that is gifted to
us and refrain from the situation described in the early
part of this story.
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