2011 2nd International Conference on Environmental Science and Technology
IPCBEE vol.6 (2011) © (2011) IACSIT Press, Singapore
BIOREMEDIATION OF SOIL BY REMOVING HEAVY METALS USING
Saccharomyces cerevisiae
Dilna Damodaran
Gummadi Suresh
Research student
Department of Chemical Engineering
National Institute of Technology
Surathkal, Karnataka, India.
[email protected]
Post Graduate Student
Department of Chemical Engineering
National Institute of Technology
Surathkal, Karnataka, India
[email protected]
Raj Mohan B
Assistant Professor
Department of Chemical Engineering
National Institute of Technology
Surathkal, Karnataka, India
[email protected]
Abstract—-Heavy-metal pollution represents an important
environmental problem due to the toxic effects of metals, and
their invasion in to the food chain leads to serious ecological
and health problems. Metal remediation through common
physico-chemical techniques is expensive and unsuitable in
treating large contaminated area effectively. Bioremediation
offers a promising means to reclaim such contaminated soil in
an economical and eco friendly way. Bioremediation employs
microorganisms capable of degrading toxic contaminants or
have the ability to accumulate it in their cells. This
concentrated end product can afterwards be directed for a
controlled way for recovery of metals. In this study
Saccharomyces cerevisiae was used for the removal of heavy
metals like Lead and Cadmium from contaminated soil .The
tolerance of Saccharomyces cerevisiae against the metals was
found to be upto 250 ppm and for Pb2+, 500 ppm for Cd2+. The
parameters affecting the biosorption of heavy metals; such as
time, metal concentration and biomass concentrations have
been investigated. The results revealed that biosorption of
about 67-82% of Pd2+ and 73-79 % of Cd2+ was attained
within 30 days. The time taken for maximum sorption of Pb2+
and Cd2+ was 30 days for soil containing 100 and 300 ppm of
Pb2+and Cd2+ respectively. Biosorption rate are higher when
the cells are in stationary phase. The biosorption and the
growth of the microorganism in aerated soil were found to be
more comparing to non-aerated soil.
Keywords-Bioremediation, biosorption,
Saccharomyces cerevisiae, soil pollution.
heavy
metals,
I. INTRODUCTION
The quality of environmental components of major cities
in India has been rated in form of Comprehensive
Environmental Pollution Index (CEPI) by the Central
Pollution Control Board (CPCB), which has drawn the
attention of the research communities in devising techniques
to down the levels of pollution in the environment. The
CEPI for land (Soil and Ground water) of Mangalore had
scored 54 out of which heavy metal content belonging to
Group -C had contributed 20 units. And any score of range
50 - 60 shows severe level of pollution. Heavy metals like
Cu, Pb, Fe, Zn, Cd, Mn, Ni, Cr and Co are major among
them. The industrial complexes of Mangalore are situated
much closer to the urban areas and areas with rich flora and
fauna. Among the environmental components, soil being the
least preferred, it has been contaminated in the areas of
industrial complexes and many other urban areas. The
concept of soil cleaning has little importance where as the
consequences of the contaminated soils over environment
measures large, viz. deforestation, poor vegetation, soil
erosion, groundwater contamination, loss of habitation etc.
Thus there emerges a major threat to the life forms in the
biodiversity of Mangalore. Hence it is necessary to analyze,
control and remediate / recover this environmental
component (soil) for the betterment of the people living in
this fast growing city. In the industrial complexes of
Mangalore there are nearly 1039 industries ranging from
small scale to large scale industries, out of which 156 of
metal processing industries are present. Thus, these
industries have the potential to contaminate the soil
component that would have lead to high score CEPI for soil
in and around Mangalore city. According to [1] heavy
metals are normally regarded as metals with an atomic
number 22 to 92 in all groups from period 3 to 7 in the
periodic table. Some of the metals such as Cu, Zn, Cd, Pb,
Fe, Cr, Co, Ni, Mn, Mo, V, Se are essential in trace
quantities for the general well being of living organism but
an excess of these can be lethal. According to recent report
of Comprehensive Environmental Response, Compensation,
and Liability Act (CERCLA) section 104 (i), as amended by
the Superfund Amendments and Reauthorization Act
(SARA), on top 20 priority chemicals, Pb gets 3rd and Cd
gets 7th place. Heavy metals at higher concentrations are
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toxic in nature to higher life forms because of their
recalcitrant nature which can lead to biomagnifications.
Exposure to these metals at lower concentrations results
various disorders.
II.
MATERIALS AND METHODS
There are many methods available for bioremediation
soil like excavation, In-situ-vitrification, soil dressing,
washing and bioremediation. Bioremediation methods have
drawn the attention of the researchers as chemical
detoxification methods failed to handle the issue of soil
remediation economically. Specific microbial species are
used to assimilate, precipitate, oxidize or reduce heavy
metals to reduce their toxicity [6 and 13]. A wide variety of
fungi, algae and bacteria are used as biosorbents for heavy
metal remediation [3and15] and some of them cause metal
transition and reduce toxicity [5, 7,15 and16].
Many microorganisms have proved to be efficient in
removing toxic heavy metals [4, 7 and 15] Among the
microorganism being used in bioremediation process of
metal contaminated environment Saccharomyces cerevisiae
was known for its versatility and efficiency in removing the
metals from both soil and liquid medium [11and 19] and
hence, Saccharomyces cerevisiae has been subjected for
biosorption of few heavy metals in the present study.
Saccharomyces cerevisiae also show similarities to humans
in the molecular level is a preferred modal organism for
toxicological studies as its complete genome is sequence and
apparent conservation of molecular mechanism between
yeast and human cells. The live cells of Saccharomyces
cerevisiae have proved to be efficient in bioaccumulation of
heavy metals [9, 13 and 15].
A. Soil sampling and culture of microorganisms:
Soil samples were collected from potential sites of metal
contamination near areas of Kudremukh Iron Ore Company
Ltd (KIOCL), Gurupur River, lay bye areas of NH-17 and
Bikampady Industrial estate areas. The soil sample from
college premises is taken as the blank and it is subjected to
the analysis of its characteristics with emphasis on metal
contamination [1]. Soil samples were sieved to attain ≤ 2
mm grain size and then stored in clean sterile plastic packets
and stored at 280C. Physical characters like bulk density,
specific gravity and water holding capacity (WHC) were
determined and chemical characters of soil like pH,
electrical conductivity and alkalinity were determined based
on standard protocols for soil analysis. Saccharomyces
cerevisiae (ATCC-9763) used in the study was obtained
from
National
Chemical
Laboratory,
Pune,
India.S.cerevisiae is grown on glucose as sole carbon and
energy source in the minimal medium containing the
nutrients at concentrations as defined in the medium. For
metal tolerance experiments cultures were grown at 30oC at
pH 5.6 in an agar medium comprised of (in gL-1): yeast
extract, 3.0; Peptone, 5.0; nutrient agar, 28.0; dextrose, 20.0
(YPAD) medium. The constituents of YPAD medium were
obtained from literature [4, 8, 12 and 21]. Effect of pH,
glucose concentration and aeration on the growth of
S.cerevisiae in the soil troughs and its role in biosorption of
heavy metal is studied. Soil pH is maintained in the range 5
to 7.5 by spraying 2 N acids and lime solutions and biomass
of the organism is determined at 600 nm by
spectrophotometer. Effect of aeration on growth rate of
S.cerevisiae is studied by determining the biomass from soil
samples from non aerated and aerated soil troughs both
continuous and discontinuous (every 2 hours).To determine
the optimum glucose concentration for the growth of
S.cerevisiae, growth rate studies are performed at different
concentrations of glucose (0 to 5%). The stock solutions
containing the 1000 mg/ml concentration of Pb+2 and Cd+2
were prepared by dissolving the Lead Nitrate and Cadmium
Sulphate in the distilled water.
B. Metal tolerance test
YPAD medium was prepared and amended with various
amounts of heavy metals viz., 3CdSO4.8H2O and Pb (NO3)2
to achieve the desired concentration of 5, 10, 25,
50,100,150,200 and 250 ppm for Pb2+ and 30, 75, 150, 225,
300,350 and 400 ppm for Cd2+ .pH was maintained at 5.6 by
adding 3 molar solution of NaOH. Inoculums of
S.cerevisiae were spread in the metal and control plates
without metal. The plates were incubated at 29 ± 1oC for 2-5
days to observe the growth of
S.cerevisiae . The
S.cerevisiae colonies were counted using a colony counter.
The tolerance limit was studied by comparing the growth
patterns of S.cerevisiae in the samples amended with
various metals concentrations with that of the control plate.
In liquid medium growth was determined using a
spectrophotometer at 590 nm.
C. Biosorption Experiments:
To study the biosorption rate the cultures were grown at
30oC at pH 5.5 in a liquid minimal medium comprised of (in
gL-1): KH2PO4, 2.72; K2HPO4, 5.22; (NH4)2SO4, 2.0;
MgSO4 7H2O, 0.5; FeSO4·7H2O, 0.0022; ZnSO4·7H2O,
0.004, MnSO4 4H2O, 0.004; CuSO4·5H2O, 0.004; d-glucose,
20.0; and yeast extract, 1.0. The constituents of basal
medium were based on the study by [8 and 12]. For soil
experiments cultures were grown at 30oC at pH 5.5 in a
liquid medium comprised of (in gL-1): yeast extract, 3.0;
Peptone, 5.0; dextrose, 20.0. Lateritic soil (blank) of 2 mm
size was taken [3, 19 and 25] and artificially contaminated
with solutions of metals salts Pb(NO3)2 to achieve 5, 10, 25,
50 and 100 ppm in the soil of 2 kg in each sample placed in
the troughs. The soil was well mixed with the solution to
achieve the expected uniform concentration of metal ions in
the sample. The samples were dried in atmospheric
condition for 48 hrs to remove the solvent (water) so that the
metal salt remains and the contaminated soil at the
predetermined concentration. A 24 hrs old culture of
S.cerevisiae in yeast peptone dextrose medium (YPD) was
taken and inoculated in the soil samples of various metal
concentrations. The inoculation was done by spraying 30 ml
of the pre cultured liquid media in the soil samples [2].
Bioaccumulation was determined as the difference between
the initial and the final concentration (after every 24 hrs of
incubation with the cells). Two types of controls were made;
one without cells, plus the quantity of the corresponding
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heavy metal concentration, and another one, without cells
and the corresponding heavy metal concentration. The
objective of the latter is to maintain moisture a layer of
sponge is provided and water is sprayed manually using a
sprayer at regular intervals. An out let is provided at the
bottom of troughs for leachate collection. 5 g of soil sample
is removed using a sterile spatula at regular intervals and
residual heavy metal content in soil is analyzed using
different parameters influencing the growth on soil and
biosorption of heavy metals like effect of glucose and
aeration of soil.
D. Metal analysis
The soil samples taken at different intervals of time were
subjected for metal analysis in order to calculate the
efficiency of bioaccumulation of heavy metal by
S.cerevisiae [14]. The microorganism is separated from the
soil media by gradient centrifugation. The metal
concentration in both microorganism and soil (residual metal
concentration) was determined using atomic absorption
spectrophotometer (Atomic Absorption Spectroscopy GBC
905 model) [1, 6, 15 and17].
III.
RESULTS AND DISCUSSION:
The physical and chemical characterization of the soil
samples from sample sites were analyzed using the standard
analytical methods and their significance in bioaccumulation
is analyzed. Metals are more soluble in acidic pH, hence
toxicity problems are moresever in acidic soil than alkaline
soil [19 and 10]. The concentration of heavy metals in the
samples are given in Table 1.1.
A. Metal tolerance tes
Interaction between S.cerevisiae and heavy metal were
experimented as it has higher level of resistance and
detoxification to heavy metals. The S.cerevisiae. showed
maximum growth till 50 ppm of Pb2+and scanty growth till
250 ppm, where as in case of Cd2+ contaminated medium
maximum growth till 250 ppm and scanty growth till 500
ppm .It was reported that, S cerevisiae shows high levels of
tolerence to various metals and metalloids and hence
associated in accumulating these metals to higher capacities
compared other microorganisms [2]. The high incidence of
heavy metal resistance detected in this work indicates the
potential of S. cerevisiae as bioremediation agents. Thus
S.cerevisiae has been chosen to biosorb heavy metals from
contaminated soils in this study
TABLE 1.1 CONCENTRATIONS OF HEAVY METALS OF DIFFERENT SOIL
SAMPLES.
Heavy
Metals
Cadmi
um
Cobalt
Sample
1
Nil
Sample
2
Nil
Sample
3
Nil
Sample
4
Nil
Sample
5
Nil
11
Nil
46.2
80.3
126.5
Chrom
ium
Coppe
r
Iron
39.6
15.4
44
33
31.9
28.6
55
18.7
15.4
Nil
14187.8
5533
>20000
15598
6378.9
Manga
nese
Nickel
47.3
75.9
886.6
280.5
398.2
Nil
Nil
Nil
Nil
0.11
Lead
Zinc
23.1
259.6
Nil
92.4
14.3
180.4
25.3
136.4
12.1
88
The S.cerevisiae cells were observed to have tolerance
up to 250 ppm of Pb2+ contaminated YPD agar media. At
lower metal concentrations, these metals act as stimulants
and maximize the growth of the microorganism. A
maximum growth of the microorganism was observed
within 24 hrs by the colony counter and the lawn of the
growth is shown in the Fig. 1.2. For concentrations above 50
ppm of Pb2+contaminated YPD agar media, the strain found
to take about 4-6 days to attain a visible growth. These
results clearly show that above 50 ppm of Pb2+ is found to be
lethal to microorganisms and hence it requires longer growth
periods to achieve a lawn on YPD agar media. S.cerevisiae
multiplies faster at log phase and hence no of cell dyeing
due to Pb2+ will be out grown and a visible colony is
observed after 2nd day of inoculation. The strain,
S.cerevisiae showed better growth in YPD agar media
samples contaminated with Cd2+ up to 300 ppm even before
48hrs shown in Fig.1.3. Thus the strain S.cerevisiae
expressed better tolerance towards Cd2+ than Pb2+ in this
study and hence regarded as better tool to remediate Cd
2+
.This shows that S.cerevisiae have a better Cd 2+ tolerant
metabolic pathways and hence higher growth rate at higher
concentrations than Pb2+. Similar kinds of observations were
reported by [14] in their study on metal tolerance of
S.cerevisiae with heavy metals the scale of Cd 2+ & Pb2+
tolerance by S.cerevisiae .
B. Standardization of parameters for higher biomass
Studies on the effect of various parameters like aeration,
carbon source and soil pH that can influence the growth rate
of S.cerevisiae in metal contaminated soil were performed.
A better growth of S.cerevisiae is observed in soil samples,
which is continuously aerated at 6.5 LPM and soil pH is
maintained at 5.5 than other acidic and basic soil troughs. It
is mainly due to higher metal solubility. Results are shown
in Fig. 1.4. and 1.5. Similar observations were made by [13]
in their study on metal solubility, where maximum growth
rates were reported up to a pH 5.8. The study also reported
that S.cerevisiae at low concentrations of both Pb2+ and
Cd2+ metals had stimulatory effect on organism during the
metal tolerance test. Experiments conducted on the effect of
glucose as carbon source on S.cerevisiae showed a better
growth as the concentration of glucose increases. At 1.5 %
glucose solution, supplied as carbon source, a maximum
growth of the microorganismon both Pb2+ and Cd2+
V2-24
LEGEND
A – Air inlet
D – Leachate outlet
S
C – Compressor
N
V – Valve
D
R- Rota meter
A
S – Sponge
N - Nylon woven mat
R
V
C
Fig. 1.2 Systematic diagram of experimental setup
was observed as shown in the Fig. 1.6. Fig. 1.6 also clearly
indicates that an inhibitory effect on growth rate of the
organism was observed at higher glucose concentrations
above 1.5 %, this phenomenon is the Crab tree effect which
is a common inhibitory effect in the growth phase of
microorganisms with respect to substrate concentration.
C. Biosorption studies using S.cerevisiae
Biosorption studies carried out to study the effect of
heavy metal biosorption on growth rate with respect to
aeration and carbon source in metal contaminated soil.
S.cerevisiae is inoculated in to 2 sets of soil samples
containing different concentrations of Pb2+ (5, 10, 25, 50,
100 ppm) and Cd2+ (30, 75, 150, 225, 300 ppm) respectively.
Experimental setups were aerated continuously and 1.5%
glucose solution is sprayed at regular intervals of every 2
hrs .Soil in troughs are maintained at pH 5.5. It is found that
about 67 - 82% of Pd2+ (82% was reached for 5 ppm,76%
for 10 ppm, 73% for 25 ppm and 67% for 50 ppm and 67%
for 100 ppm) and 73-79% of Cd2+ (79% for 30 ppm, 77%
for 75ppm, 50ppm, 72ppm for 225ppm and 73% for 300
ppm) was removed within 30 days. Similar results were also
observed by [7 and 18]. In his studies were 81% Pb2+, 84%
Zn2+ and 78% Cd2+ degradation was obtainedusing
Alcaligenes eutrophus Ch34. Chemical soil remediation
methods like soil washing using chelating agents removes
only 50.3% Cu2+, 38.4% of Cd2+ and 31% of Pb2+ as per
Zhan et al., [19]. From these biosorbtion studies it is evident
that biosorption and biomass are directly related to aeration
and carbon source supply as it favors budding of yeast cells
and we know biosorption rate is found to be higher in log
phase than in stationary phase [12 and 19] and live cell
absorb more than dead cells using live cells than dead cells
[14]. Figure 1.9 and 2.0 represents sorption percentage of
both Pb2+ and Cu2+ and its shows a drastic increase in the
rate of biosorption with respect to time and it is observed
that recovery of metal was not rapid in the initial stages and
it gradually increased with lapse of time. S.cerevisiae took
only 160 hrs and about 94-99%of Pb2+ and 88- 99 % of Cu2+
was biosorbed form the aqueous phase at similar conditions,
This shows it’s potential to be used in waste water treatment
similar results have been observed by [11] using
Saccaromyces sp.Spirullina biosorb about 50% of Cr2+ as
per [14] studies.
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Fig: 1. 2. S.cerviceae lawn on Fig: 1.3. S.cerviceae lawn on YPAD
YPAD media 50ppm of Pb
at media at 150 ppm of Cd
Fig 1.4 Effect of aeration on Biomass
Pb2+forconcentration upto 300 ppm and 250 ppm
respectively.
• The results also revealed that S.cerevisiae has good
potential of accumulating about 82 – 87 % of Pb2+
and 75 – 78 % of Cd2+ within 30 days from metal
contaminated soil.
Thus bioremediation of metal contaminated soils using
S.cerevisiae can be considered as the most cost effective
way to remediate soil provided better techniques aid to
isolate the organism from the soil.
Fig 1.5 Effect of Soil pH on growth of S.cerevisiae
REFERENCE
[1]
[2]
Fig 1.6
Effect of various Glucose concentrations on S.cerevisiae
[3]
[4]
[5]
Fig: 1.9 Bioaccumulation rate of Cd2+ by S. cerevisiae at constant
conditions.
[6]
[7]
[8]
Fig: 2.0. Bioaccumulation rate of Pb2+ by S. cerevisiae at constant
conditions
IV.
[9]
CONCLUSION:
[10]
The experiments conducted on the biosorption of heavy
metals from the synthetic soil contaminated with metal salts
of lead and cadmium by S.cerevisiae revealed that the
organism has high potential of removing heavy metals from
the soil through biosorption mechanism. Certain important
physical and chemical characteristics of the biosorption
process were analyzed and reported in this paper. The
following significant observations were made during the
study:
• The rate of bioaccumulation is observed increase
with increase in aeration and an optimum rate was
observed at 6.5 LPM.
• The availability of glucose as carbon source found
to facilitate the bioaccumulation process indirectly
by increasing the biomass. An optimum
concentration of 1.5 % glucose was found to support
the biosorption to a maximum level.
•
S.cerevisiae showed better tolerance with soil
than
samples
contaminated
with
Cd2+
[11]
[12]
[13]
[14]
[15]
[16]
V2-26
A. Sharm aand M. B. Rehman, “Laboratory scale approach,” Indian
Journal of Microbiology, Vol. 48,pp. 142–146,Aug 2009.
D.Smet, Diels, L. M. Hooyberghs, L. Kinnaer, L. Corbisier, P.and G.
Brox, “Bioremediation of heavy metal contaminated soils with a
biometal sludge reactor (BMSR). Hydrometallurgy,”Unpublished
D.Hooyberghs, L. Diels, M. Smet and L.Corbisier, “Heavy Metals
Bioremediation of Soil.Molecular Biotechnology,”Vol.12,pp.149 –
158,April 1992.
G. M. Gadd,J.C.Gadd,G.M.Herbert,R.A. Jones, C. W. Watson and
I.A Craik, “Microbial Control of Pollution,” Cambridge University
Press, Cambridge, United Kingdom,Vol.2,pp.59– 87,Aug 1992.
Hall , P. Swash and S. Kotilainen, “The importance of biological
oxidation of iron in the aerobic cells of the Wheal Jane pilot passive
treatment system,” Science Total Environment. Vol. 338,pp. 41–
48,June 2005.
I. Suciu, C. Cosma, D. Sorana, Bolboacă and L. Jäntschi, “Analysis
of Soil Heavy Metal Pollution and Pattern in Central Transylvania,”
International Journal of Molecular Sciences Vol.9,pp. 434-453.Dec
2008.
Johnson and K. Hallberg, “Acid mine drainage: remediation options:
a review,” Science Total Environment,Vol.338, pp. 3–14,June 2004.
[8] K. J. Blackwell and J. M. Tobin, “Cadmium accumulation and
its effects on intracellular ion poolsin a brewing strain of
Saccharomyces cerevisiae”Journal of Industrial Microbiology and
Biotechnology,Vol.23,pp. 204–208.March 1999.
Leusch, Z.R. Holan and B. Volesky, “Solution and particle leaf facts
on the biosorption of heavy metals by seawead biomass,”Journel of
Appliedbiochem-biotechnology,Vol .61,pp. 231- 249,Aug 1996.
M.Rahatgaonkar and N. R. Mahore,.A Selective Bioreduction of
Toxic heavy metals. Journal of experimental biotechnology,Vol 47,
pp.766-769. Jan 2008.
Parvathi, R. Nagendran and R. Nareshkumar, “ Lead biosorption onto
waste beer yeast byproduct,a means to decontaminate effluent
generated from battery manufacturing industry” Electronic Journal of
Biotechnology,Vol.10,pp.13-15.Jan 2007.
P. R. Norris and D.Kelly, “Accumulation of Cadmium and Cobalt by
Saccharomyces cerevisiae,” Journal of General Microbiology,Vol.
99,pp.317-324,Oct 1977.
Paula,B. Adams, D. Anita, G.F Selma, and Leite, “Factors involved
withCd
Absorbtion
by
a
wild
type
strain
of
Saccharomyceserevisiae,” Brazillian Journal of Microbiology,Vol
34,pp.55-60.Jan 2003.
R. A. Wuana, F. E. Okieimen and J. A. Imborvungu, “Removal of
heavy metals from a contaminated soil using organic chelating
acids,” Int. J. Environ. Sci. Tech.Vol.7 (3),pp. 485-496,Jan 2010.
S.Iram,I. Ahmad and S. Doris, “Analysis Of Mines And
Contaminated Agricultural soil samples for fungal diversity and
tolerance to heavy metals,” Pakistan journal of botany,Vol 41,
pp.885-895, Nov 2009.
V.Valentina and Umrania, “Bioremediation of toxic heavy metals
using
acido
thermophilic
autotrophies,”
Bioresource
technology,Vol.97,pp. 1237–1242.April 2006.
[17] Volesky,and Z. S. Holan, “Biosorption of heavy metals,” Journal of
Biotechnology,Vol .3, pp.11235–11250, Jan 1995.
[18] Whitehead,B. J. Cosby and H. Prior, “The Wheal Jane wetlands
model for bioremediation of acid mine drainage. Science Total
Environment,Vol. 338,pp.125-135. Aug 2005.
[19] Y. Zhan, C. Fan , Q. Meng,Z. Diao , L. Dong, X. Peng and Shuyan
Ma and Q. Zhou, “Biosorption of Pb2+ by Saccharomyces
Cerevisiae in static and dynamic adsorption tests bulletin”Journel of
environment contamination and toxicology,” Vol. 83, pp. 708712.June 2009.
V2-27