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BIOREMEDIATION OF CHROMIUM CONTAMINATED SOIL USING ORGANIC AMENDMENTS
PROJECT REFERENCE NO.: 39S_R_MTECH_003
COLLEGE : B.M.S. COLLEGE OF ENGINEERING, BENGALURU
BRANCH : DEPARTMENT OF CIVIL ENGINEERING
GUIDES : MRS. PRATHIMA.B
DR. SAVITHRI BHAT
STUDENTS : MS. KHUSHBU.K.BIRAWAT
KEYWORDS: Bioremediation; Chromium; Compost; Vermi-compost; Organic amendments.
INTRODUCTION: The population of world is growing at an alarming rate of 1.1% per year. This huge
population growth has given wind to the ever increasing fire of urbanization and
industrialization, with very less concern for the maintenance and safety of the environment we
live in. The air we breathe, the water we drink and the soil which we use for agriculture all are
being polluted and contaminated to a great extent, owing to the various anthropogenic
activities, most important of which is industrial activity. The conservation and protection of
all the attributes of the environment is a much needed necessity of today in order to live a
sustainable life on the planet. Compared to all the other attributes of the Earth such as air and
water, soil and prevention of its pollution has been neglected in the past. Soil represents a
significant aspect of the Earth. Soil is an interface between air, water and rock. 328.7 Million
hectares, is India’s total geographical area, out of which 146.8 Million hectares of soil is
degraded, i.e., approximately 44.66%, according to National Bureau of Soil Survey and Land
Use Planning, as assessed in 2004. If we see the amount of money India is losing per year
solely due to soil degradation then, 448.6 billion rupees is lost as a direct cost of land
degradation annually. The sources of soil pollution are both natural and anthropogenic, but the
latter posing greater threat to the environment. Soil pollution due to heavy metals has become
a global concern which needs to be dealt with utmost urgency.
One such heavy metal is chromium (Cr). Cr is an essential trace element and also
helpful in the metabolism of glucose tolerance factor. But it is also a heavy metal which has
both carcinogenic and mutagenic effects on humans if it enters and accumulates in living
organisms beyond a certain level. It has been quoted as a priority pollutant by the US EPA for
this reason. It is available in several oxidation states, however, the most stable ones are
trivalent chromium [Cr (III)] and hexavalent chromium [Cr (VI)]. Cr (III) and Cr (VI) have
unique properties. Cr (III) is less toxic compared to Cr (VI) as it is relatively insoluble in
water. Cr (III) presents lower mobility, and is mainly bound to organic matter in soil and
aquatic environments. Cr (VI) is found in the forms of chromate (CrO42-
), dichromate (CrO42-
),and CrO3 and is considered the most toxic forms of chromium, as it possess high oxidizing
potential, high solubility, and mobility across the membranes in living organisms and in the
environment. Chromium is used for chrome metal plating, as an additive for cooling towers,
as a metal-alloy in stainless steel and metal ceramics, as dye and pigment, wood preservative,
in tanning, etc. It is often found in soils around the industrial areas. Not only is that particular
land affected but by seepage into the lower layers of soil, the groundwater is also getting
poisoned.
Soil contamination and water pollution are the two globally recognized environmental
issues. Soil is found to be a chief sink for heavy metals. Bioavailable metals are mobile,
soluble and not easily sorbed and thus are taken up by the biological systems and hence
bioaccumulate in the food web. Bioaccumulation occurs due to inability of organisms to
easily metabolize and eliminate the chemical compounds. The heavy metal when comes in
contact with the soil is taken up by the plants and other smaller organisms. These plants are
then used as a source of food by the living beings in the higher trophic levels. This process of
transfer of heavy metal from lower trophic level to higher trophic level is known as
biomagnification. Heavy metals are elements that cannot be further degraded by organisms.
They are non-biodegradable, persistent inorganic chemical constituents. They generally
accumulate into the soil and the more soluble ones seep down into the water table.
The major concern lies in the biomagnification of chromium. Plants are under stress
due to chromium toxicity and these plants are food to various insects, animals, birds, and
humans too! Accumulation of chromium from one trophic level to another requires years but
repercussions irreversible.
The other problem which we are currently facing is that of solid waste management.
Globally, approximately 1.3 billion tonnes of MSW is generated annually, and its predicted
that this will increase to a whooping 2.2 billion tonnes annually by the year 2025 . In India
alone, 68.8 million tonnes per year of waste is generated. The municipal waste generated in
India is of organic fraction and approximately comprises of 51%. If this fraction of waste is
converted to organic compost or vermicompost using appropriate methods and technology
with consideration for the environment, it can be used as an organic amendment for
bioremediation.
Thus, this approach of using compost to bioremediate heavy metal contaminated soil
can be cost-effective and an environmentally friendly way to treat heavy metal contaminated
soil. Using this approach we can possibly deal with two globally important issues - solid
waste management and heavy metal pollution of soil.
Figure 1: Concept in a nutshell: The organic fraction of municipal solid waste generated can
be converted to compost, and this compost can be in-turn used for bioremediation of
contaminated soil
• Municipal Solid Waste generation per day in India is 1,00,000 MT. Out of which 51% is organic fraction i.e, composta
ble.
Municipal solid waste generation
• Thus, 20,400 Kg of compost will be produced.
Compost production
• This compost can be used for cleaning up the contaminated soil.Bioremediatio
n
OBJECTIVE: The key thesis question is:
Can the addition of organic amendments such as compost and vermicompost reduce the
bioavailability of chromium present in the natural soil and thus bioremediate such soil?
To find the answer to the above question and consequently analyze the results obtained
through various experiments, following objectives were set:
1. Evaluate the presence of chromium in the soil obtained from one of the heavy metal
contaminated industrial areas in Bangalore.
2. Identify & isolate chromium resistant bacteria in the native soil by screening method.
3. Evaluate the efficiency of the chromium resistant microbial species in bio remediating
chromium contaminated soil.
4. Study various permutations and combinations of culture, compost and soil to check for
the best combination to bio remediate the contaminated soil, thus obtaining a tailored
mass of raw materials to treat chromium specifically.
METHODOLOGY: To achieve the above stated objectives, a set of experiments were conducted in
Biotechnology and Environmental Engineering laboratory of BMS College of Engineering.
Table 1: Name and application of the instruments used in laboratory
Sl. No. Name of the instrument Sl.
No.
Name of the instrument
1. Laminar Air Flow cabinet
(LAF)
9. Atomic absorption spectrophotometer
(AAS)- Chemito
2. BOD- incubator 10. Atomic absorption spectrophotometer-
Perkin Elmer
3. Shaking incubator 11. Hot air oven
4. Water bath 12. Rotary shaker
5. Autoclave 13. Electronic balance
6. Cooling centrifuge 14. pH meter
7. Refrigerator 15. Electrical conductivity ( EC) meter
8. Compound Microscope 16. Nephelometer
Figure 2: Materials and Methodology in a nutshell
Materials
& Methodology
Materials
Instruments & materials employed in Biotechnology
laboratory
Instruments & materials employed in Environmental
engineering laboratory
Methodology
Methodology of experiments conducted in
Biotechnology laboratory
Methodology of experiments conducted in
Environmental engineering laboratory
Experiment 2: Isolation of chromium
resistant bacteria from the soil samples
collected from industrial area
Experiment 1: Study conducted to assess
the effect of Bacillus subtilis on seed
germination
Experiment 3: Identification of the Cr
resistant bacteria using staining
techniques & biochemical tests
Experiment 4: DNA isolation from the
chromium resistant bacteria
Methodology of the sample
collection
Description of the study area
Preliminary analysis of samples
Study of effect of organic compost
on chromium contaminated soil
Study of effect of vermicompost on
chromium contaminated soil
Study of effect of chromium
resistant Bacillus subtilis on soil
Experiment 1: Study conducted to assess the effect of Bacillus subtilis on seed
germination
The chromium resistant Bacillus subtilis micro-organism was obtained from a PhD
student working on a similar project. The organism was used to check its capability to tolerate
chromium. An experiment was conducted to check the effect of chromium on seed
germination in presence of chromium resistant micro-organism.
Experiment 2: Isolation of chromium resistant bacteria from the soil samples collected
from an industrial area
Serial dilution, a pure culture technique was employed in order to obtain a pure culture
of micro-organisms that consists of only one type of organisms which are morphologically
and genetically similar to one another. This experiment was conducted in order to obtain a
pure culture that has grown from a single cell by serially diluting the soil sample and plating
the solutions thus obtained on a Potato Yeast Extract (PYE) Agar .
Soil samples were collected from an industrial area. Samples were collected in
polyethylene bags and were brought to laboratory. 10 grams of soil was kept in polythene
bags and stored in refrigerator for microbial analysis.
In order to isolate and enumerate bacteria, the collected samples were serially diluted
using sterile distilled water and were plated on Peptone Yeast Extract (PYE) agar
supplemented with hexavalent chromium (potassium dichromate). A control plate was used
for comparison, where hexavalent chromium was absent.
From the above experiment two morphologically different colonies were observed in the
plate containing 10-10
diluted soil sample solution. To obtain a pure culture, these two
microbial colonies were inoculated separately on PYE media containing 12.5 mg/l of
chromium solution and were incubated at 37 degree Celsius for 24 hours in BOD incubator.
Experiment 3: Identification of the Cr resistant bacteria using staining techniques &
biochemical tests
Staining techniques and biochemical tests were conducted to carry out preliminary
investigation of the isolated organisms- CVBT-01 and CVBT-02.
Simple and differential staining of bacteria was carried out. Following which Endospore
staining was done. The biochemical tests carried out were, Starch hydrolysis test, Gelatin
liquefaction and IMViC Test
Experiment 4: DNA isolation from the chromium resistant bacteria
The molecular characterization was done for both CVBT-01 and CVBT-02. For this
purpose bacterial genomic DNA isolation was carried out. Then for 3 µl of the extracted
DNA, PCR reaction was carried out using a primer designed for this experiment. The
resulting purified PCR product was subjected to 16s r RNA sequencing using an automated
DNA analyzer. The sequence generated was recorded and analysed.
Basic Local Alignment Search Tool (BLAST) tool from the NCBI database was used to
identify the nearest neighbour sequences. BLAST 2.3.1 tool was used to align the sequences
and based on the results the strains were identified.
Experiment 5: Study of effect of organic amendments on chromium contaminated soil
In order to reduce the bioavailability of chromium present in the soil and thus bioremediate it,
three cases were considered, which are:
1. Use of organic compost as organic amendment
2. Use of vermicompost as organic amendment
3. Use of chromium resistant Bacillus subtilis as a bioremediating micro-organism.
The effects of all three cases on bioremediating the contaminated soil and their efficiency
were analysed daily and observations were recorded.
10 pots of 8 inch diameter each were taken as reactors for this experiment. 250 grams of
the total material, which comprised of composite soil sample and organic compost was added
to each of the reactors. To ten reactors, organic amendment along with the composite soil
sample was added. The concentration of the soil and organic compost was varied from 0% to
90% in each set, where 0% means 250 grams of soil, which represented the control and 90%
means that the reactor has 25 grams of soil and 225 grams of organic amendment.
Plate 1: Bioremediation studies- Experimental set up- Front view
Plate 2: Soil and compost mixture in one the reactors
Methodology of bioremediation studies:
To assess the chromium concentration in all the samples, from day 1 to day-10 all the samples
were analyzed as follows
Figure 3: Methodology of sample analysis for bioremediation studies
RESULTS: Experiment 1: Study conducted to assess the effect of Bacillus subtilis on seed
germination
Shoot and root length measurements of green gram showed that the presence of chromium
affects its growth. Shoot and root were shorter in presence of chromium and longer in
presence of bacterial broth. The shoot length and root length for different combinations of
chromium and Bacillus subtilis is as shown in Fig 4 and Fig 5. From the graph it was
established that Bacillus subtilis helped in the bioremediation of chromium.
Sample was taken from each pot
, dried and ground.
10 grams of the sample was weighed and transferred to a conical flask
20 ml of DTPA extraction solution was added to each of the samples
The contents were mixed using a rotary shaker at 180 rpm for 2 hours
The samples were let to settle for five mintues.
The solution was passed through filter paper, thus separating water from soil particles.
This filtrate was used to analyse heavy metals using AAS.
The readings were given in ppm and they were recorded.
Shoot
length
in cm
Figure 4: Shoot length of green gram in each plate
Figure 5: Root length of green gram in each plate
Experiment 2: Isolation of chromium resistant bacteria from the soil samples collected
from industrial area
Two pure cultures of micro-organisms were obtained from the chromium contaminated soil as
shown in the figure below. They were designated as CVBT-01 and CVBT-02 respectively.
Plate 3: Pure cultures of CVBT-01 and CVBT-02
Petri plate number
Root
length
cm
Petri plate number
Experiment 3: Identification of the Cr resistant bacteria using staining techniques &
biochemical tests
The table below shows the results for the strains CVBT-01 and CVBT-02. This indicates that
the strains belong to the Bacillus species.
Table 2: Results of the Staining and Biochemical tests
Name of the Experiment Result for CVBT-01 Result for CVBT-01
Gram staining Gram positive Gram positive
Simple staining Oval shaped, Cells arranged
in clustered chain
Oval shaped, Cells arranged
in chain
Starch hydrolysis Positive Positive
Indole Production Negative Negative
Methyl Red test Negative Negative
Voges Proskauer Test Positive Positive
Experiment 4: DNA isolation from the chromium resistant bacteria
The DNA was isolated and stored in TE buffer for PCR amplification to check the presence of
ycnD in CVBT-01. Upon running the gel, the result of the DNA isolated from the bacteria
was seen as shown in the figure.
Plate 4: DNA Isolation (0.8% agarose gel, slit casting tray) Lane4- CVBT-01 (3µl)
When the PCR was carried out, it further confirmed the presence of the ycnD gene, by the
appearance of bands during gel electrophoresis.
Plate 5: Result for CVBT-01 templated PCR (loaded on gel-3ul)
Plates 4 and 5 show that the isolated bacterias CVBT-01 and CVBT-02 contains
oxidoreductase which helps in chromium metabolism. Thus two novel strains were identified.
Experiment 5: Study of effect of organic amendments on chromium contaminated soil
The heavy metal chromium present in the reactor mixture was analyzed for ten days. The
chromium results obtained from the reactors having mixture of contaminated soil and organic
compost are shown in the table.
Table 3: Bioremediation studies using organic compost- Chromium analysis in reactors
Organic
compost
percentage
Control 10% 20% 30 % 40 % 50 % 60 % 70% 80 % 90
%
Day 1 90.24 73.7 69.74 60.5 47.74 45.32 13.92 9.68 7.32 6.08
Day 2 90.33 73.48 67.32 55.88 43.12 36.3 12.92 9.38 6.54 2.96
Day 4 90.27 71.5 65.78 53.9 37.4 28.82 10.14 8.22 4.88 2.58
Day 5 90.32 71.28 63.8 46.2 31.46 25.96 10.03 7.87 4.47 1.6
Day 6 90.46 69.3 61.16 44.22 28.6 25.08 9.26 7.17 4.03 1.59
Day 7 90.44 67.98 57.42 42.46 26.84 23.54 8.66 5.71 3.24 1.59
Day 8 90.31 65.8 50.6 41.14 23.32 21.78 8.34 5.36 3.03 1.49
Day 9 90.54 59.18 45.3 35.86 22.16 18.04 7.2 4.9 2.72 1.46
Day 10 90.26 57.86 42.6 34.36 21.4 15.18 6.42 4.86 2.23 1.42
Figure 6: Graphical representation of results obtained from bioremediation studies using
organic compost
From Figure 6 it is observed that as the percentage of compost increases chromium
concentration in the soil samples were decreasing. The minimum percentage reduction in
chromium was recorded as 21.5% at 10% compost combination and maximum reduction is
found to be 76% at 90% compost combination. Hence the percentage increase in compost
decreases the bioavailablilty of the chromium.
020406080
100
Cr.
Co
nc
in p
pm
Increasing percentage of organic amendment
Day 1
Day 2
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Table 4: Bioremediation studies using vermi compost- Chromium analysis in reactors
Comp
ost % Control 10 20 30 40 50 60 70 80 90
Cr concentration in mg/lt
Day 1 90.24 95.92 72.16 58.74 51.26 39.82 32.12 11.66 7.32 5.16
Day 2 90.33 84.92 63.36 51.04 46.64 38.94 31.46 11.12 6.74 5.02
Day 4 90.27 82.72 57.86 50.16 44.88 38.28 30.36 9.9 5.18 3.06
Day 5 90.32 79.2 55.66 48.62 44 34.98 22.22 7.58 3.34 2.93
Day 6 90.46 65.56 54.56 47.74 37.62 34.32 16.94 7.01 3.02 2.56
Day 7 90.44 65.56 50.82 45.32 33.88 26.4 16.66 6.8 2.74 1.72
Day 8 90.31 63.8 46.42 44.44 33.22 25.96 16.5 6.78 1.98 1.67
Day 9 90.54 60.06 40.04 38.06 23.32 22.44 15.4 5.66 1.94 0.97
Day
10 90.26 51.7 39.18 37.84 14.74 17.49 12.76 3.93 1.68 0.9
Figure 7: Graphical representation of results obtained from bioremediation studies using
vermi compost
From Figure 7 it is observed that as the percentage of vermicomposting increases chromium
concentration in the soil samples were decreasing as in case of organic compost. The
minimum percentage reduction in chromium was recorded as 46% at 10% vermicompost
combination and maximum reduction is found to be 82% at 90% compost combination.
Hence the percentage increase in vermicompost decreases the bioavailability of the
chromium.
The percentage reduction in chromium is more in soil with vermi compost application
compared to organic compost.
0102030405060708090
100
Cr.
Co
nc
in p
pm
Increasing quantity of vermicompost
Day 1
Day 2
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Table 5: Bioremediation studies using Bacillus subtilis- Determination of chromium in
reactors
Concentration of
nutrient broth
containing B.
subtilis
Nutrient
broth- 0
ml
(Control)
Nutrient
broth- 20
ml
Nutrient
broth- 40
ml
Nutrient
broth- 60
ml
Nutrient
broth- 80
ml
Nutrie
nt
broth-
100 ml
Day 1 90.24 92.18 90.34 91.2 90.82 91.44
Day 2 90.33 90.42 83.38 52.58 47.3 40.7
Day 3 90.27 84.64 81.4 47.96 42.9 36.74
Day 4 90.32 84.93 80.52 40.26 40.7 31.02
Day 5 90.46 84.64 74.36 39.83 36.74 24.86
Day 6 90.44 80.34 73.7 33.78 31.24 21.34
Day 7 90.31 79.32 60.72 32.17 26.4 20.02
Figure 8: Graphical representation of results obtained from bioremediation studies using
Bacillus subtilis
From figure 8 the percentage removal of chromium is increased with increase in concentration
of Bacillus subtilis. In control the chromium concentration in soil is found to be 90.24 –
90.46 ppm in soil . After addition of various concentration of Bacillus subtilis, the maximum
removal efficiency was recorded as 78% at nutrient broth of 100ml. The reduction in
chromium was also noticed with respect to time in days.
CONCLUSIONS: The soil samples collected from contaminated area showed presence of heavy metals viz.,
Chromium, Nickel and copper.
1. The minimum concentration of 0.08 ppm and maximum concentration of 197.88 ppm
were recorded in the collected soil samples.
2. Two novel micro-organisms were isolated which were chromium resistant and were
found to be Bacillus cereus
3. The isolated organisms were found to have oxidoreductase which helps in chromium
metabolism.
0102030405060708090
100
Cr
con
c in
pp
m
Nutrient broth- 0 ml (Control)
Nutrient broth- 20 ml
Nutrient broth- 40 ml
Nutrient broth- 60 ml
Nutrient broth- 80 ml
Nutrient broth- 100 ml
4. As the percentage of organic compost and vermi compost increases chromium
concentration in the soil samples were decreasing.
5. The minimum percentage reduction in chromium was recorded as 21.5% at 10%
compost combination and maximum reduction is found to be 76% at 90% compost
combination. Hence the percentage increase in compost decreases the bioavailablilty
of the chromium.
6. The minimum percentage reduction in chromium was recorded as 46% at 10%
vermicompost combination and maximum reduction is found to be 82% at 90%
compost combination.
7. Bacillus subtilis, showed the maximum removal efficiency of 78% at addition of 100
ml nutrient broth.
FUTURE WORK: The current study was carried out at lab scale. A step forward can be taken in the
future to study the bioremediation of the heavy metal contaminated soil at a site level. A site
can be chosen in an industrial area for remediation purpose and the organic amendments can
be applied to it at the field and the observations made can be recorded. This approach will
yield results that will be be more real time, as the effect of organic amendment of soil
structure and also its effect on ground water can be measured in long-term.
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