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.