International Journal of Research in Engineering, Science and Management 450

Volume-2, Issue-12, December-2019

www.ijresm.com | ISSN (Online): 2581-5792

Isolation of Metal Tolerant Microorganisms

from the Municipal Solid Waste of Kolhapur
Dige Komal Tanaji1, Mrunal Dattatray Ulape2
1
Student, Department of Biochemistry, Shivaji University, Kolhapur, India
2
Assistant Professor, Department of Biotechnology, Vivekanand College, Kolhapur, India

Abstract: Waste generation and its control have taken on 1. Introduction

important role in our environment. With the doubling of
population and changing lifestyle pattern of the inhabitants the A. Applications
quantity of municipal waste generating is increasing at alarming  The isolated species can be used to treat waste water.
rate. Most of this waste is subjected to dumping in specified  Waste can be used as a perfect advancement media for
disposal yard. The greatest challenge to the environmentalists is
the eco-friendly management of this waste and application of development of various microorganisms.
microorganisms in this context has got an age over other available  By fermenting waste various enzymes can be isolated.
technologies. Organic waste is consumed by the bacteria and is no  Bioremediation for chemical and petroleum industrial
longer present to produce orders, sludge, pollution or unsightly solid wastes.
mess.
When bacteria consume waste, they convert the waste into safe
2. Aim and objective of the work
by products and in due course of this conversion they actually
produce several metabolites to break down the complex waste into A. Aim
simple compounds. Soil microorganisms are increasingly
becoming an important molecule. Extent of microbial diversity in
“Isolation of metal tolerant microorganisms from the
nature is still largely unknown, thus there might be many more municipal solid waste of Kolhapur.”
useful products yet to be identified from soil microorganisms. In
B. Objective
soil 80- 90% of microorganisms remain undented whereas these
biological communities are known to play a dominant role in  To Collect the Municipal waste sample and isolate the
maintaining a sustainable biosphere. Today both academic and bacteria from this sample.
industrial interest in soil bacteria (due to their several advantages  To study the morphological characters, Gram nature,
over other microorganisms) is on the rise in search of deriving
and Motility of isolated bacteria.
these unique biologically active metabolites and novel
commercially important products from them. Bacteria are present  To study heavy metal tolerance of isolated bacteria.
in diverse ecological habitats.  To study the degradation of waste from polluted water
They are considered highly valuable as they are used in by organisms.
Fermentation processes much as brewing, baking cheese and
butter manufacturing, chemical manufacturing such as ethanol, 3. Review of literature
acetone, organic acid, enzymes, perfumes etc. Microbial mining
and they produce various antibiotics vaccines steroids as well as  Golamari Siva Reddy et.al/ J. pharm. sci & vol.9 (9), 2017,
other therapeutically useful compound useful components with 1490-1497. Isolation and characterisation of bacteria from
diverse biological activities. Hence there is an immense possibility compost for municipal solid waste from Gunter and
to screen effective bacterial strains from waste dump sites with Vijayawada. In this paper the bacterial strains from waste
valuable applications. To cope up with the demand for new
organisms with properties of production of unique enzymes or dump destinations with a definitive goal of waste corruption
molecules for industrial application and waste degradation there and finding new valuable bioactive mixes for modern
have been a constant effort in isolating novel bacteria from diverse application was studied.
environment. Accordingly, the present study was aimed to  Journal of microbiology and experimentation in this the
investigate bacterial strains from, waste dump site with the study of isolation and characterisation of bacteria isolated
ultimate objective of waste degradation and discovering novel
from municipal solid waste for production of industrial
bioactive compounds for industrial application. In this a heavy
metal tolerance of isolated bacteria is seen and also the effect of enzymes and waste degradation was carried out.
this isolated strain on the waste water was studied.  In research gate the study of isolated and characterization of
effective bacteria for solid waste degradation for organic
Keywords: Metal Tolerant Microorganisms, Municipal Solid manure was studied.
Waste  In international journal of science inventions today,
isolation of metal resistant bacteria from municipal solid
 International Journal of Research in Engineering, Science and Management 451
Volume-2, Issue-12, December-2019
www.ijresm.com | ISSN (Online): 2581-5792

waste dumpsite, Madurai was studied. technique. The cleaned Nutrient Agar (NA) medium (it contain
 In open access international journal of science and 0.5% peptone, 0.3% yeast separate, 0.5% NaCl, 0.25% glucose,
engineering a critical review of biotechnology for solid 1.5% agar, D/W and pH was adjusted to 7 at room temperature.)
waste management was studied. was sit up for bacterial development. For this a spread plate
method was used. On this with the help of sterile glass borer
4. Material prepare a wells and on each well mark with particular metal and
add 0.01ml of solution of respective metals. After that keep it
A. Study Area
them in deep freezer for diffusion of metal for 10 minutes. After
Soil test was gathered from a site of Kolhapur Green energy diffusion put this plate in incubator at 370 C temperature for 24
PVT.LTD (municipal solid wastes management project, Client- hours. The inhibitory zones around the well was observed on
Kolhapur municipal corporation.) in Kolhapur district of the plate for some metals.
Maharashtra in India. 2) Waste water degradation potential of selected bacterial
1) Collection of samples samples
An aggregate of ten waste examples was gathered from For this a 24 hours of old bacterial colony was used. In this
dumping site of Garbage in the Kolhapur Green energy PVT. method take 50ml of waste water sample in five conical flasks
LTD site. Test sample was gathered in sterile zip-lock plastic respectively. Take pH and O.D (optimum density) of the
bag kept in aseptic conditions at 40C and labelled with respect sample. Add the isolated bacterial colony in each Flask of the
to their source and area. The gathered examples were conveyed water sample. Put all this tube at the room temperature for 96
to the lab for selection of soil microscopic organisms. hours and record the O.D. at 530nm and pH after 24 hours, 72
hours and 96 hours.

6. Results and Discussion

 For the isolation of bacteria from municipal solid waste site
of Kolhapur serial dilution method is used. In this a nutrient
agar medium a well isolated colonies were seen. In this a
five specimen namely A, B, C, D and E is selected for the
further studies in this the morphological characters and gram
nature of the specimen was studied which is as follows:
A. Isolated Species

5. Methods
A. Isolation of bacteria from waste samples
For isolation of bacteria serial dilution method is used. In this
method take 10 test tubes mark it as 10-1, 10-2…,10-10 and add
10ml sterile distilled water in first tube and 9ml distilled water
in remaining tubes. After this 1gm of waste soil sample is
weighed and added in the first tube. Shake this tube for some
time. Allow the soil to settle and pickup 1ml of water sample
and transfer into 2nd tube. The all procedure was repeated up to
the last tube. After dilution take 0.1ml of dilution of each tube
spread on Nutrient agar plate (It contain 0.5% peptone, 0.3%
yeast separate, 0.5% NaCl, 0.25% glucose, 1.5% agar, D/W and B. Morphological characters, gram nature and motility of
pH was adjusted to 7 at room temperature.) and incubate at 370 selected species
C for 24 hours. After incubation well isolated colonies were 1) Species A
seen from these 5 well isolated colonies were selected and
further studied morphological characters, Gram nature and Size Shape Colour Elevation
motility. Further this selected colony was restriking on the 4mm Round Red Concave
Nutrient agar slant (stock) and preserve it in Freezer for further
Margin Opacity Surface Consistency
studies of this organisms.
Entire Opaque Rough Smooth
1) Metal tolerance assay
Investigation of MIC (Minimum Inhibitory concentration) of Gram Nature Motility
substantial metals viz, lend (Pb), zinc (Zn) and Mercury (Hg) Gram Negative Actively Motile Cells
was helped out for the bacterial strains through cup Assay
 International Journal of Research in Engineering, Science and Management 452
Volume-2, Issue-12, December-2019
www.ijresm.com | ISSN (Online): 2581-5792

2) Species B
5) Species E
Size Shape Colour Elevation
Size Shape Colour Elevation
3mm Irregular Creamy Pink Concave
3mm Round Orange Concave
Margin Opacity Surface Consistency
Margin Opacity Surface Consistency
Irregular Opaque Rough Smooth
Entire Opaque Smooth Smooth

Gram Nature Motility

Gram Nature Motility
Gram Negative Actively Motile Cells
Gram Negative Actively Motile Cells

3) Species C
C. For this three heavy metals such as lead (Pb), Zinc (Zn),
Size Shape Colour Elevation and mercury (Hg) was used. In this cup assay technique is used.
3mm Round Pink Concave The results obtain were as follows:
Margin Opacity Surface Consistency
Entire Opaque Smooth Sooth Specimen Metal Tolerance
Lead(Pb) Zinc(Zn) Mercury(Hg)
Gram Nature Motility A Positive Positive Negative
Gram Negative Actively Motile Cells B Positive Positive Negative
C Positive Negative Negative
D Positive Positive Negative
E Positive Positive Negative

4) Species D
D. Waste water degradation potential of selected bacterial
samples
Size Shape Colour Elevation
2mm Round Yellow Concave
For this 50ml of waste water sample is placed in conical
Margin Opacity Surface Consistency flask. Check the O.D (optimum density) and PH of the sample.
Entire Opaque Smooth Sooth The result obtained were as follows. In this it is prove that the
at the incubation period increases isolated species having the
Gram Nature Motility
Gram Negative Actively Motile Cells
ability for the degradation of waste water. The results obtain
were as follows:
 International Journal of Research in Engineering, Science and Management 453
Volume-2, Issue-12, December-2019
www.ijresm.com | ISSN (Online): 2581-5792
TIME SPECIMEN OPTIMUM DENSITY PH 7. Conclusion
After 24 Hours Control 0.03 7
A 0.02 7  Results of the studies shows that the waste contain more
B 0.01 7 number of bacterial species.
C 0.03 7
D 0.05 7
 Isolated species has capacity to tolerate some heavy metals
E 0.03 7 like Lead (Pb) & Zinc (Zn).
 Isolated species do not show metal tolerance towards
After 72 Hours Control 0.03 7 Mercury (Hg).
A 0.02 7
B 0.01 7  Isolated species has potential to degrade waste water.
C 0.02 7
D 0.02 7 References
E 0.01 7
[1] Golamari Siva Reddy e.t al, J. Pharm. Science and Research, 2017, 1490-
1495, Isolated and characterization of Bacteria from compost for
After 96 Hours Control 0.03 7 Municipal solid water from Guntur and Vijaywada.
A 0.01 7 [2] Saha A, Santra SC. Isolation and characterization of bacteria isolated from
B 0.01 7 municipal solid waste for production of industrial enzymes and waste
C 0.01 7 degradation. J Microbiol Exp. 2014;1(1):12-19.
D 0.02 7 [3] Deepak Dille, K. Archana, “Biotechnology for solid waste management-
E 0.01 7 A Critical Review,” Open Access International Journal of science and
Engineering, vol. 2, no. 9, pp. 53-57, 2017.
[4] D. M Chetam. (2017). Isolation and Characterization of Bacteria from
Solid Waste. Isolation and Characterization of Bacteria from Solid Waste,
vol. 4, 63-68.
[5] S. Anjanapriya and S. Lalitha, “Isolation of metal resistant bacteria from
municipal solid waste dumpsite, Madurai, IJSIT, vol. 56, no. 5, pp. 659-
672, September-October 2017.