Page 1
Chapter VI: Microbial Remediation on Waste Dumps of
Magnesite and Bauxite Mines
6.1. Introduction
The treatment of mine waste is one of the most important issues created by
mining companies around the world (Garcia, et al., 2001). These mine wastes
containing metals are a significant toxic factor to biota in the environment
i.e. microbes and ecosystem processes (Giller, et al., 2009). There are several
remediation approaches which have been developed as excavation, land fill, thermal
treatment, electro reclamation and soil capping but all these are more expensive and
environmentally destructive (Ritcey, 1989). Bioremediation technology provides
an alternative to conventional methods for remediating the metal-polluted soils
(Khan, 2009). The microbiological processes are significant in determining metal
mobility and have actual potential application in bioremediation of metal pollution
(Gadd, 2004). According to Ge, et al., (2009) several approaches have been followed
for the reclamation of metal contaminated soil by bioremediation/biosorption process.
Biosorption technique appears to be suitable as secondary or polishing applications
for metal removal from metal polluted environment, which would be competitive with
ion-exchange resin, based on final cost-beneficial analysis and the greatest use for
biosorption may be in modular system for small companies.
Worldwide several researchers have been using microbes (bacteria, fungi etc.)
as an ideal agent for bioremediation process, due to their small size, their ubiquity,
their ability to grow under controlled condition and their resilience to a wide range of
environmental situations (Urrutia, 1997). The Acidothiobacillus sp were found most
abundantly in acid and metal containing environment. The most well-known species
is Thiobacillus ferrooxidans, for treating heavy metal contaminated tailing and soils,
which are industrially, exploited in bio-leaching of metal sulfide and uraninite ores
(Straube, et al., 2003). P. aeruginosa and P. putidaare previously reported as effective
Page 2
bacterial species for the reclamation of oil/metal contaminated soils by producing
surfactants and tolerant to certain heavy metals (Wong, et al., 1993; Mathiyazhagan
and Natarajan, 2011b). Garcia, et al., (2001), had used Sulfate Reducing Bacteria
(SRB) for bioremediation of mine effluents. Among the microbes, fungi also play an
important role in the biosorption of metals. The biomass of fungi (both live and dead
form) has been used as suitable biosorbent for metal biosorption (Sayed and
Morsey, 2004). The biomass from Rhizopus arrhizus has been extensively used for
the sorption of salts and complexes of different metals such as iron, nickel,
copper etc., present individually or in multi-component systems and polluted soils
(Aksu,et al., 1999; Yesim, et al., 2000; Subudhi and Kar, 2008). Harma, et al., (2009)
had used rumen fluid microorganisms for the bioremediation of sulphate rich mine
effluents.
The metal uptake by microorganisms is a rapid and reversible process and is
not mediated by metabolic processes. Hence, there is no difference in the metal
uptakes by death biomass and the living microorganisms. Several microbial
biomasses are proven to be good biological sorbents for heavy metals. A number of
mine tailing reclamation studies have emphasized a strong association between the
establishment of a stable plant community, abundance and composition of
microorganisms (Monica, et al., 2008). Some reports have also shown that indigenous
microbes and plant-microbe symbionts can tolerate high heavy metal concentrations
in different ways and might play a significant role in the restoration of contaminated
soils (Carrasco, et al., 2005; Ge, et al., 2009). Therefore, it is necessary to study the
occurrence of indigenous microorganisms in heavy metal polluted sites. It may
provide new insight into bacterial diversity under unfavourable conditions, new
isolates and probably new genetic information on heavy metal resistance, which could
be exploited in revegetation in future (Fabienne, et al., 2003). There are many
researchers who have been measuring the soil microbial activities as indicators of
heavy metals containing soil environment. The aim and objective of the present study
Page 3
was to evaluate the metal tolerant and bioremediation potential of the dominant
bacteria and fungus isolated from waste dump of magnesite and bauxite mines.
6.2. Material and methods
6.2.1. Metal adaptation and Minimum Inhibitory Concentration (MIC) of
dominant bacteria
Thiobacillus ferrooxidans and Pseudomonas aeruginosa were the dominant
bacteria which were isolated from bauxite and magnesite soils. These cultures were
adapted to metal tolerant test with some metals as per the modified method of
Alvarez, et al., (1999). 1ml culture of T. ferrooxidans (from 9K medium) was
inoculated on semisolid agar plates (4.5 - 5.5) and one ml of P. aeruginosa (from
nutrient broth) was inoculated on nutrient agar plate (6.8), it was supplemented
individually with 5 different metals (Cd, Mn, Cu, Cr and Hg) in the concentration
range of 20 to 100 g/ml respectively. The inoculated plates were incubated at
35oC ± 1
oC for 3 to 7 days. After the incubation, colonies from metal containing
plates were counted. MIC of heavy metals for each bacteria (T. ferrooxidans and
P. aeruginosa) was determined by a modified method of Luli, et al., (1983) and
Calomiris, et al., (1984) by spreading 250 l of each culture on plates containing
various concentrations (25 to 200 g/ml) of several heavy metals (Cd, Mn, Hg, Cu
and Cr). The plates were incubated at 35°C for 48 hours.
6.2.2. Metal tolerability test of fungi
The isolated dominant test fungi namely Rhizopus arrhizus, Mucor sp,
Trichoderma sp (both mine soil) and Scedosporium apiospermum (in magnesite soil)
were taken for this experiment. These cultures were adapted for metal tolerant
test to determine their metal tolerant efficiency as per the modified method of
Zafar, et al., (2007). The SDA medium was prepared with different concentrations
(20, 40, 60, 80 and 100 g/ml) of heavy metals (such as Cd, Cr, Hg, Mn, and Cu) and
the test fungi were inoculated on plates and control plates (without metals) were also
maintained. The inoculated plates were incubated at 27 ± 1oC for 3-5 days.
Page 4
6.2.3. Determination of Minimum Inhibitory Concentration (MIC) of fungi
The MIC of metals on test fungi was determined by performing the modified
method of Zafar, et al., (2007). The Sabouraud Dextrose Agar (SDA) medium was
prepared and amended with various amounts of heavy metals to achieve the desired
concentration (2 to 10 mg/ml). Each heavy metal containing plate was subdivided
into four equal sectors and inoculums of test fungi (Rhizopus arrhizus, Mucor sp,
Trichoderma sp and Scedosporium apiospermum) was spotted in triplicate on metal
and control plates. The plates were incubated at 27 1oC for 3 to 5 days to observe
the growth of fungi on the spotted area (Zafar, et al., 2007).
6.2.4. Bioremediation /Metal sorption by bacteria
The selected bacteria (T. ferrooxidans and P. aeruginosa) was adopted to
analyse their metal sorption or removal capabilities on waste soil of bauxite
and magnesite mines as per the modified method of Atac Uzel and Guven
Ozdemir, (2009). 100 ml of nutrient broth (pH 5 & 6.8) was taken in 250 ml
Erlenmeyer conical flasks and 5 grams of microbes free mine soil was added. About
5ml of young cultures of T. ferrooxidans and P. aeruginosa were inoculated at
exponential growth phase on both soils. The control medium (nutrient broth with soil
and without the culture) was also maintained (pH 6.3). The test and control medium
were continuously stirred and incubated on a rotary incubator (Everflow) on 160 rpm
at 34 ± 1oC for 8 days (Plates 23a and 23b). During the sorption process, pH was
observed for every 24 hours interval (24 - 192 hours).
After the incubation, the soil was sterilised and filtered by using filter papers
and filtered soils were dried and digested with acids (HCL and HNO3 in the ratio of
3:1) and the metals were analyzed in the treated and control soil by using Intensive
Coupled Plasma- Optical Emission Spectrophotometer (ICP-OES, Perkin Elmer,
USA).
Page 5
6.2.5. Fungal spore inoculums preparation
Based on the results of metal tolerability, R. arrhizus was selected for bio
remediation process. For large inoculums preparation, R. arrhizus was incubated three
times on SDA slants at 30°C for a week (Mulligan, et al., 1999). About seven days
old conidia were harvested from SDA surface using sterile distilled water for few
times under a sterile condition. Approximately 3.5×105
(spores/ml) of spore
suspension was prepared (Wan-Xia Ren, et al., 2008) for the bioremediation process.
6.2.6. Bioremediation on waste dumps (soil) of mines using fungi
The microbial bioremediation (Akhtar and Mohan, 1995; Mulligan, et al., 1999)
process was carried out, using 250ml autoclaved (at 121°C for 20 min and 15lbs)
conical flasks with 5% (w/v) of microbes free magnesite and bauxite mine soil in 100
ml of sucrose medium (pH 5.8) respectively. About 3 ml of R. arrhizus spore
suspension (approximately 3.5×105
spores/ml) was added aseptically to these
conical flasks and the experimental control was sucrose medium without soil
(Wan-Xia Ren, et al., 2008). All the flasks were incubated in a rotary shaking
incubator at 120 rpm/min and 30°C for 15 days (Plates 23a and 23b). At regular time
(2 days once) intervals over 15 days, samples from each conical flask were analysed
for pH variations using glass electrode.
6.2.7. Novel design for bioremediation
Obviously, the main objective of in vitro bioremediation process is to study
the remediation efficiency of our test microbes (bacteria/fungi) on specific pollution
either in solid or liquid form. The samples were sterilized before introducing for
remediation process to avoid the presence of non-targeted microbes on sample and to
get accurate results in remediation efficiency of test microbes. The sterilised
contaminated soil (5gm) was used in our newly designed plates under a sterile
condition by using P. aeruginosa and T. ferrooxidans bacteria (Plate 23c).
Page 6
The microbial pellets were obtained from overnight mass culture, by
centrifuging and collecting in sterile micro-centrifuge tube and diluted (depends upon
amount of pellet) with sterile distilled water (stock). From the stock, 0.5ml of
microbes and 2.5ml of nutrient broth (as nutrient for the microbes) was taken in a
sterile mini sprayer and the culture was sprayed over the soil containing plates evenly
and the plates were tightly closed. On the side wall of bottom of the plates, a scale
was drawn to check/monitor the sample value and at the lid of the plate, straw like
pipes (0.7 r-mm width and 5 cm in length) were molded to maintain the atmospheric
air equilibration. The bottom side of plate, one pipe was fitted with sterile cotton to
maintain aeration and another pipe was free i.e. without cotton (Plate 23c).
6.2.8. MATNAT (MAThiyazhagan NATarajan) Remediation Efficiency Index
(MREI)
This index is framed by us (Mathiyazhagan and Natarajan) (copyright reserved
© 2011, Govt. of India) to analyse and give a standard, accurate and quick
remediation efficiency value for the microbes by using the following formula.
MREI (mn)/o
pq
Where,
m = Amount of Pollutants before remediation
n = Amount of Pollutants after remediation
o = Amount of sample (Polluted soil/water) taken for remediation
p = Incubation time of the remediation process
q = Amount of Culture (or) Number of Plants (or) Amount of Biomass taken for
remediation process
6.2.9. Statistical analysis
The data of each treatment in this study was represented as mean average of
triplicates with standard deviation (X ± S.D.) which was calculated using SPSS
(Version 12.0) package (USA).
Page 7
6.3. Results and discussion
6.3.1. Bacterial analysis and Metal adaptation
The results of present investigation showed two dominant bacterial species
T. ferrooxidans (from bauxite soil) and P. aeruginosa (from magnesite soil) which
were isolated and characterized from waste dumps of mining industry.
T. ferrooxidans and P. aeruginosa were showed resistant to different heavy metals
(Cd, Mn, Cu, Cr and Hg) in the various concentrations (20 to 100 g/ml). The metals
tolerant potentiality were observed in the order of Cd > Mn > Cu > Cr > Hg, based on
the number of colonies observed in the plates. The number of colonies varied for each
metal at different concentrations; it decreased with increasing concentration of heavy
metals (Plate 20). These bacterial species are effectively used in bioleaching,
bioaccumulation of metal, ores beneficiation and desulfurisation of fossil fuel and
metal contaminated soils (Ehrlich, 1986).
The results highlighted that more number of well grown colonies of
P. aeruginosa (85 to 13 and 86 to 15 colonies) and T. ferrooxidans (86 to 31 and
75 to 26 colonies) were observed in various concentrations (20 to 100 g/ml) of
Cd and Mn (Figures 8a and b). The number of colonies obtained from Cu and Cr,
containing plates were in the range of 83 to 12 and 74 to 9 number for
P. aeruginosa and 69 to 20 and 81 to 11 colonies for T. ferrooxidans (Figures 8c and 8d).
Least number of colonies of P. aeruginosa (64 to 0) and T. ferrooxidans (72 to 0)
(Figure 8e) was obtained in Hg than T. ferrooxidans (Plate 20). This study was also
comparable to the work of Oliveira, et al., (2010) who reported the isolation of
mercury and chromate tolerant diazotrophic bacteria from the long term contaminated
soils and industrial effluents from arable field in the center of Portugal.
Page 8
Plate 21 Metal tolerant test for dominant bacteria
Legend: A-E: Colonies of T. ferrooxidans on Hg, Cr, Cu, Cd and Mn (100 g/ml concentration)
containing plates. F-J: Colonies of P. aeruginosa on Cd, Cr, Hg, Cu and Mn (100 g/ml
concentration) containing plates. Control: Sterile medium.
Page 9
Nu
mb
er o
f co
lon
ies
(±3
) N
um
ber
of
colo
nie
s (±
3)
Figure 8 Metal tolerance potentiality of T. ferrooxidans and P. aeruginosa on
several metals
(a) CdCl2
T. ferrooxidans P. aeruginosa
100
80
60
40
20
0
20 40 60 80 100
Concentration of CdCl2 (g/ml)
(b) MnCl2
T. ferrooxidans P. aeruginosa
80
60
40
20
0
20 40 60 80 100
Concentration of MnCl2 (g/ml)
Page 10
Nu
mb
er
of
colo
nie
s (±
3)
Nu
mb
er
of
colo
nie
s (±
3)
Nu
mb
er
of
colo
nie
s (±
3)
(c) CuSo4
T. ferrooxidans P. aeruginosa
80
60
40
20
0
20 40 60 80 100
Concentration of CuSo4 (g/ml )
(d) K2Cr2O7
T. ferrooxidans P. aeruginosa
100
80
60
40
20
0
20 40 60 80 100
Concentration of K2Cr2O7 (g/ml )
(e) HgCl2
T. ferrooxidans P. aeruginosa
80
60
40
20
0
20 40 60 80 100
Concentration of HgCl2 (g/ml )
Page 11
M
M
Nu
mb
er
of
colo
nie
s (±
5)
Nu
mb
er
of
colo
nie
s (±
5)
6.3.2. MIC determination
The results of MIC test, showed that the bacterial populations decreased with
increasing concentration of various heavy metals (Cd, Mn, Cu, Cr and Hg), indicating
their sensitivity to higher level of heavy metals (Figures 9a and b). T. ferrooxidans
were resistant to various heavy metals (Cd, Mn, Cu, Cr and Hg) at various
concentrations (from 25 to 200 g/ml).
The MIC limit of P. aeruginosa in Cd and Mn was 150 g/ml and for
Cu, Cr and Hg it was 100 g/ml. Similar types of findings were reported earlier by
Dhakephalkar and Chopade, (1994) investigated on the high level of multiple metal
resistant bacteria isolated from the heavy metal contaminated enviro nment.
Figure 9a MIC of T. ferrooxidans on several metals
120
100
80
60
40
20
0
Cd
n
Cu
Cr
Hg
25 50 100 150 200
Concentration of metals (µg/ml)
Figure 9b MIC of P. aeruginosa on several metals
100
80
60
40
20
0
Cd
n
Cu
Cr
Hg
25 50 100 150 200
Concentration of metals (µg/ml)
Page 12
6.3.3. Metal tolerant and MIC of test fungi
The results of metal tolerant potential of test fungi (Rhizopus arrhizus,
Mucor sp, Trichoderma sp and Scedosporium apiospermum) showed the various
levels of resistant capabilities on several heavy metals (Cd, Cr, Hg, Mn and Cu)
(Table 11). Among the isolated fungi, R. arrhizus, expressed effective resistance (upto
80 µg/ml) potential to heavy metals followed by Mucor sp and Trichoderma sp for all
metals except Hg (Plate 22). S. apiospermum did not show any metal resistant
activity. Similar type of results were reported by Zafar, et al., (2007), with some soil
fungi (Aspergillus sp, Alternaria sp, Geotrichum sp, Fusarium sp, Penicillium sp,
Trichoderma sp, Rhizopus sp, Monilia sp and Mycelia sterilia group) which are able
to grow in the presence of some heavy metals (50 mg/ml).
The results of MIC values of test fungi are presented in Figure 10. The MIC
values suggest that the resistance level against individual metal was dependent on the
isolates. The two isolates (Mucor sp and Trichoderma sp) showed relatively low
tolerance to all metals than R. arrhizus (tolerate upto 10 mg/ml for Mn and 6 to 9 for
remaining metals except Hg). But S. apiospermum have lacked of metal resistance
potentiality. Effects of toxic metals on fungal growth have shown the intra and
interspecific variability and dependence on type of metal contaminants and speciation
(Plaza, et al., 1998). The tolerance among the isolated fungi were observed in
order of Mn > Cu > Cd > Cr > Hg. The occurrence of different kinds of fungi
(Aspergillus, Rhizopus, Penicillium, Fusarium, Chaetomium, Geomyces and
Paecilomyces species) in heavy metals polluted soil (Cu, Cd, Pb, As and Zn) has
also been reported by other workers from several parts of the world (Babich and
Page 13
Stotzky, 1985; Gadd, 1993). The variation in the metal tolerance might be due to the
presence of one or more types of tolerance strategies or resistance mechanisms
exhibited by different fungi (Gadd, 1993). One of the most important fungal metal
tolerant strategies is morphological strategy; it can be altered by toxic metals and
changes in mycelia density (Fomina, et al., 2005). For example Daedalea quercina
and Paxillus involutus exhibited increase hyphal branching, changes in mycelial
morphology, development of loops and connective filaments in Stereum hirsutum and
S. commune in response to cadmium polluted soil (Gabriel, et a., 1996). Another
important strategy of fungi are aggregated mycelia in metal contaminated soil, could
produce high local concentration of extracellular products such as complexing agents
(organic acids, siderophores and polyphenolic compounds), metal precipitating
agents (oxalates) and polysaccharides with pigments with metal binding abilities
(Baldrian, 2010).
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Plate 22 Metal tolerant test for selected fungi
Legend: 1: Rhizopus arrhizus; 2: Mucor sp; 3: Trichoderma sp; 4: Scedosporium apiospermum
(numbers mentioned on the plate) and plate depicts the results of fungus on
lowest (20 µg/ml: eg. Cu 1) and highest (80 µg/ml: eg. Cu 2) concentration of metals.
Control: Sterile medium
Page 15
Met
al
con
cen
trati
on
(in
mg
/ml)
Figure 10 Minimum inhibitory concentration (MIC) of heavy metals against
test fungi
12
10
Rhizopus sp
8
Mucor sp 6
Scedosporium 4
apiospermum
2 Trichoderma sp
0
Mn Cu Cr Cd Hg Heavy metals
Page 16
Table 11 Heavy metal tolerant potentiality of isolated test fungi
Name of test
fungus
Concentration of metals (g/ml)
Cd
Cr
Hg
Mn
Cu
20 40 60 80 100
20 40 60 80 100
20 40 60 80 100
20 40 60 80 100
20 40 60 80 100
R. arrhizus
Mucor sp
S. apiospermum
Trichoderma sp
+++ +++ + + +/-
+++ ++ + + -
- - - - -
++ + + + -
++ ++ + - -
++ + - - -
- - - - -
++ + - - -
++ + - - -
+ + - - -
- - - - -
- - - - -
+++ +++ ++ + -
+++ ++ ++ + -
++ ++ - - -
+++ ++ ++ + -
+++ ++ + + -
++ + - - -
- - - - -
+ + - - -
Legend :
+++ : Effective growth
++ : Good growth
+ : Normal growth
+/- : Trace growth
- : No growth
105
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6.3.4. Microbial bioremediation/metal sorption on magnesite and bauxite mine
soils
The microorganisms (bacteria, fungi, algae and actinomycetes) are highly
effective in sequestering heavy metals remediation (Wong and So, 1993) and these
have been used to remove metals from polluted industrial sites and domestic effluents
on a large scale. The biosorption potentiality of present investigation showed that the
test bacteria possessed effective metal sorption efficiency within a short time of
treatment process (Plates 23a and 23b). The results of bioremediation process by
T. ferrooxidans and P. aeruginosa, on mine waste soil were found to be considerable
amount of portions of the metals which were removed from the mine soil
horizons and their residual concentrations, with the exception of Fe, Cu, Mg and
Hg (Table 12). The highest rates of heavy metals were reduced by P. aeruginosa
(Cd 384 & 370, Ca 49 & 427, Zn 95.24 & 82, Cr 36.99 & 27.99, Mn 26 & 27 and
Pb 19 & 16 mg/kg from bauxite and magnesite mine soil) followed by T. ferrooxidans
(Cd 294 & 280, Zn 102 & 82, Cr 38.84 & 17.99, Mn 31& 39 and Pb 26 & 24 mg/kg
on bauxite and magnesite mine soil).
The metal sorption variations among these two bacteria as well as fungi
are depicted in Figures 11 and 12. It was well recognized by some researchers
(Ritcey, 1989) who have reported that the microorganisms have a high affinity for
metals and can accumulate both heavy and toxic metals by a variety of mechanisms
(Silver, 1991; Simmons, et al., 1995). Wong, et al., (1993) isolated Pseudomonas
putida from electroplating effluent, showing that it accumulated Cu, upto 6.5% of its
dry weight, from a Cu containing solution. Murugesan and Vasanthy, (2003) reported
that the Pseudomonas species have high metal sorption efficiency on Cr metal
containing environment. Other investigators have demonstrated the capabilities of
several bacteria in removal of Cd, Pb and other toxic metals from polluted soil
Page 18
(Mullen, et al., 1989; Lovely, et al., 1991; Phillips,et al., 1995). The rate of
biodegradation of such waste contaminants are more dependent on biological (number
of bacteria, conditions of culture, etc.), physicochemical factors (insufficient oxygen,
nutrient availability, water availability, etc.) and their relatedness to the pollutants
(volatility, polarity, etc.) and bioavailability (Antizar-Ladislao, et al., 2006).
The temperature and pH of the soil was essential parameters/index for
bioremediation, because they were affecting the microbial growth, metal utilisation
and activity on soil of mining industries (Ehrlich, 1986). In the present study during
the metal sorption process showed that the pH was reduced/increased from 8.32 to
8.10 and 5.22 to 6.75 by P. aeruginosa and T. ferrooxidans in magnesite and bauxite
mine treatments. The temperature applied in this study was equal to the fi eld profile
(in the range of 25oC to 35
oC). The metal sorption efficiency of bacteria was
effectively occurred at 38 ± 2oC and in acidic environment the activity was enhanced
by the water solutions of soluble organic compounds. The metal sorption by this
bacterial biomass mainly depends on the cell, especially through cell surface and the
spatial structure of the cell wall. Peptidoglycan, teichoic acids and lipoteichoic acids
are all important chemical components of bacterial surface structures. Various
polysaccharides, (cellulose, chitin, alginate, glycan, etc.) on the fungi cell walls, have
been proved to play a vital role in metal binding. Various proteins are also reported to
involve in metal binding for certain kinds of biomasses. Some functional groups have
been found to bind metal ions, especially carboxyl group (Wang and Chen, 2009).
This results was agrees with report of Muller, et al., (2001), who stated that the acidic
environment and average temperature effectively enhanced the metal sorption.
Page 19
Plate 23a Microbial bioremediation on bauxite soil
Legend: A: T. ferrooxidans; B: P. aeruginosa; C: R. arrhizus and D: Control soil
Plate 23b Microbial bioremediation on magnesite soil
Legend: A: Control; B: P. aeruginosa; C: T. ferrooxidans and D: R. arrhizus treated soil
Page 20
6.3.5. Novel approach for bioremediation
The novel bioremediation method showed highly effective results rather than
the conventional method (Table 13). The T. ferrooxidans and P. aeruginosa
effectively reduced the metals such as Cd (481.8 & 417.4), Cr (84.3 & 31.27),
Mn (595.7 & 146.8), Pb (55.2 & 54.6) and Zn (118.8 & 162.2 mg/kg) in bauxite and
magnesite waste soils. It was higher when compared with the results of traditional
methods (Cd 294 & 370; Cr 38.84 & 27.99; Mn 31 & 27; Pb 26 & 16 and
Zn 102 & 82 mg/kg). In traditional method (in a conical flask) the bacteria cannot
survive for a long period, due to lack of fast multiplication process, more gas
production (metabolic process) and absence of frequent aeration (Plate 23c). Further,
there is a chance for elements present in soil to react with the liquid (medium) with
the help of bacterial secondary metabolites and it may create unfavourable
environment for bacteria. The remediation process may stop in a particular time, but
these complications are rectified using the present novel method, by providing
continuous clean aeration and removal of gases produced by the bacteria during the
bioremediation process in the vessel. In addition, maintained the moisture content of
the soil; which are significantly supported or enhanced the bioremediation rate on
contaminated soil. This statement was agreed by Rhykerd, et al., (1999)
concomitantly. The overall results indicate that this novel bioremediation
(vessel/design) process is very useful for the in vitro bioremediation process of
polluted soils.
Page 21
Plate 23c Newly designed petriplate method for bioremediation process
Legend: Culture inoculated newly designed plate on sterile laminar air flow chamber
Legend: Incubation of inoculated newly designed plate on open environment with control
110
Page 22
Table 12 Metal analyzation in pre and post treated (T. ferrooxidans, P. aeruginosa and R. arrhizus) bauxite and magnesite soils
S.No Metals
Pre treatment
(mg/kg)
Post treatment (mg/kg)
Bauxite Magnesite
Bauxite Magnesite T. ferrooxidans P. aeruginosa R. arrhizus T. ferrooxidans P. aeruginosa R. arrhizus
1 pH 5.22 8.32 6.75 6.24 5.50 8.15 8.10 8.12
2 Cd 1060 2070 766 (27.7) 676 (36.2) 562.7 (46.9) 1790 (13.52) 1700 (17.8) 1470 (28.9)
3 Cu 105.9 65.96 105.9 (0.0) 106.02 (0.0) 131.1 (0.0) 65.96 (0.0) 77.96 (0.0) 97.95 (0.0)
4 Fe 2222 2222 2222 (0.0) 2222 (0.0) 2222 (0.0) 2222 (0.0) 2222 (0.0) 2221 (0.0)
5 Ca 85.95 4907 56.95 (33.7) 36.95 (57.0) 67.75 (21.1) 4670 (4.82) 4480 (8.70) 3530 (28.0)
6 Mg 2610 5330 2620 (0.0) 2610.80 (0.0) 2624 (0.0) 5380 (0.0) 5380 (0.0) 5360 (0.0)
7 Zn 827.5 1141 725.5 (12.3) 732.23 (11.5) 780.1 (5.7) 1059 (7.18) 1059 (7.18) 1073 (5.95)
8 Cr 553.7 69.96 514.86 (7.0) 516.71 (6.68) 541.6 (2.1) 51.97 (25.7) 41.97 (40.0) 65.96 (5.71)
9 Mn 6674 3173 6643 (0.46) 6648 (0.38) 6286.7 (5.8) 3134 (1.2) 3146 (0.85) 2905 (8.46)
10 Pb 742.6 443 716.6 (3.50) 723.6 (2.55) 725.7 (2.27) 419 (5.41) 427 (3.61) 432.6 (2.34)
11 Hg 15 NDA 15 (0.0) 15 (0.0) 15 (0.0) NDA NDA NDA
Legend: NDA: Not Detectable Amount. The values are the mean values of triplicates. The value mentioned in the parenthesis are denotes percentage (%) of metal
absorption.
111
Page 23
Soil samples
Cd
Cr
Mn
Pb
Zn
Magnesite
2070
69.96
3173
443
1141
Bauxite
1060
553.7
6674
742.6
827.5
Magnesite
1700 (17.87)
41.97 (40.0)
3146 (0.85)
427 (3.61)
1059 (7.18)
Bauxite
766 (27.73)
514.86 (7.01)
6643 (0.46)
716.6 (3.51)
725.5 (12.3)
Magnesite
1652.6 (20.1)
38.69 (44.69)
3026.2 (4.5)
388.4 (12.3)
974.8 (14.5)
Bauxite
548.2 (48.28)
469.4 (15.11)
6078.3 (8.9)
687.4 (7.4)
708.7 (14.3)
Table 13 Comparison of bioremediation efficiency of newly designed plate method with conventional method (using T. ferrooxidans on
bauxite soil and P. aeruginosa on magnesite soil)
Metal treatment/analysis
and methods
Name of metals
Metal content in pre treated soil
(mg/kg)
Traditional method
Novel (Plate) method
Legend: The values mentioned in the parenthesis are denotes percentage (%) of metal absorption.
112
Page 24
6.3.6. Bioremediation on magnesite and bauxite dump soil using fungi
The importance of metallic ions to fungal and yeast metabolism has been
known for a long time (Gadd, 1993). The presence of heavy metals affects the
metabolic activities of fungal species which created the interest in relating to study of
metal absorption behaviour in fungi. The results of this study led to a concept of using
fungi and yeasts for the removal of toxic metals (lead and cadmium) and recovery of
precious metals (gold and silver) from metal contaminated environment (Kapoor and
Viraraghavan, 1997). The metal-tolerant Rhizopus arrhizus was evaluated for their
biosorption potential for heavy metals present in the waste dump of magnesite and
bauxite mining industries (Plates 23a and 23b). Out of ten metals, seven metals were
reduced in the waste soil of magnesite and bauxite mines by R. arrhizus i.e. Cd
(600 & 497.3), Fe (1), Ca (1377 & 18.2), Zn (68 & 47.4), Cr (4 & 12.1),
Mn (268 & 387.3) and Pb (10.4 & 16.9 mg/kg) and the variations of heavy metal
sorption was observed in Ca, Mg and Cr (Table 12, Figures 11 and 12). Similarly, the
earlier findings reported that varying levels of metal biosorption through different
fungi (Rhizopus nigricans, Mucor sp, Penicillium sp, A. fumigatus, etc.) on
metal contaminated soils (Bai and Abraham, 2001; Rama Rao, et al., 2005;
Ahmad, et al., 2005). These metal sorption variation is due to the pH and
physicochemical nature of the soil and efficiency of the fungi on metal.
The high metal sorption efficiency was recorded in R. arrhizus on mine soil, in
relation to the cell wall nature of fungus. The cell wall of R. arrhizus contains higher
amount of chitin. The ability of chitin to form complex metal ions has been confirmed
by Dursun, et al., (2003). The assessing of metal binding sites in fungi is more
difficult. However, Tobin, et al., (1990) have reported two non equalent binding sites
in R. arrhizus during the Cu absorption process. Viable R. arrhizus could remove the
Cu with the maximum specific uptake capacity of 10.76 mg/g at 75 mg/l of initial
Cu concentration (Dursun, et al., 2003) and R. arrhizus were reported to absorb Pb
Page 25
g
Red
uce
d v
alu
e of
meta
ls
(mg
/kg
)
Cd
Cu
Fe
Ca
Mg
Zn
Cr
Mn
Pd
Hg
(Naja, et al., 2005), Cd, Ni, Cr (Bhattacharyya, et al., 2002). At the time of metal
sorption process, pH of the medium was measured on both mine effluents and the
results in magnesite varied from 8.32 to 8.12 and bauxite soils from 5.22 to 5.50
respectively. The results indicated that the effective metal sorption was occurred at
acid pH (5) than alkaline pH (8). It was interrelated with the statement of Okoronkwo,
et al., (2005) who stated that the abundance and activities of soil microflora are
assessed by the several physical parameters including pH. Other factors such as
contact time, biomass dosage and temperature are known to influence the biosorption
of metals which were reported by several investigators (Fourest, et al., 1994; Kapoor
and Viraraghavan, 1997; Dhami, et al., 1998; Zhou, 1999; Yan and Viraraghavan,
2000; Bai and Abraham, 2001). Another study has reported that the Mucor rouxii has
effectively absorbed Cd (6.94 mg/g biomass and 31 mg/g) and Cr (30 mg/g) (Yan and
Viraraghavan, 2000; Yesim, et al., 2000) from metal polluted soil. Sag, et al., (1999)
have reported that the metal absorption efficiency of R. arrhizus isolated from the
heavy metal contaminated soils.
Figure 11 Metal absorption variation among P. aeruginosa, T. ferrooxidans
and R. arrhizus on bauxite mine soil
500
400
300
T. ferrooxidans 200
P. aeru
inosa
100
R. arrhizus
0
-100
Heavy metals
Page 26
e
Red
uce
d v
alu
e of
met
als
(m
g/k
g)
Figure 12 Metal absorption variation in P. aeruginosa, T. ferrooxidans and
R. arrhizus on magnesite mine soil
1400
1200
1000
800
600
400
200
0
-200
Cd Cu
Fe Ca Mg Zn Cr Mn Pd
Heavy metals
T. f rrooxidans
P. aeruginosa
R. arrhizus
6.3.7. MATNAT index
The MATNAT Remediation efficiency index (MREI) values support these
two bacteria and single fungus which showed the effective remediation in both metal
polluted mine soils. These values show the range in-between 0.017 to 6.120 (good
metal sorption efficiency) of T. ferrooxidans, P. aeruginosa and R. arrhizus. The
maximum value was notified in Cd, Cr, Pb and Zn in both mine soil (Table 14),
followed by other metals.
Page 27
Table 14 MATNAT Remediation Efficiency Index of mines
Name of
the
sample
MREI value of R. arrhizus
Cd Cu Fe Ca Mg Zn Cr Mn Pb Hg
Bauxite 2.210 -0.111 0 0.080 -0.062 0.210 0.053 1.721 0.075 0
Magnesite 2.666 -0.142 0.004 6.120 -0.133 0.302 0.017 1.191 0.046 0
MREI value of T. ferrooxidans
Bauxite 2.45 0.0 0.0 0.241 -0.08 0.849 0.323 0.258 0.216 0.0
Magnesite 3.2 -9.999 0.0 0.408 -0.006 0.785 0.308 0.216 0.158 0.0
MREI value of P. aeruginosa
Bauxite 2.333 0.0 0.0 1.975 -0.416 0.683 0.149 0.325 0.220 0.0
Magnesite 3.083 -0.1 0.0 3.558 -0.416 0.683 0.233 0.225 0.133 0.0
Legend: The efficiency is considered from 0.010 onwards (based on the assessment of metal reduced value
in non treated soil, which was maintained at same incubation time and condition as like treatment)
Page 28
6.4. Conclusion
The results obtained from the study revealed that the above-said
bioremediation (in vitro) method can be very efficient for the treatment of soils
contaminated with toxic heavy metals. The dominant bacterial flora (T. ferrooxidans
and P. aeruginosa) and fungus (Rhizopus arrhizus, Mucor sp and Trichoderma sp)
isolated from the metal containing effluents of mining industries possess metal
tolerant potential for several heavy metals. These metal resistant potential are made
by the metal containing environment (waste dumps of mining industry) and they
possess effective bioremediation or metal sorption potentiality. Among these two
bacterial strains P. aeruginosa absorb heavy metals very effectively (in the order of
Cd, Zn, Cr and Ca) followed by T. ferrooxidans in a very short period, even growing
at pH 5 to 9.5 at temperature of 38oC. The novel bioremediation method has given
presumptive results than conventional method in a short period. Rhizopus arrhizus
reported high metal tolerance ability than Mucor sp and Trichoderma sp and had
effective metal sorption efficiency on magnesite and bauxite mine waste dumps . Due
to heavy metal tolerant and metal absorption potentiality of these bacteria and fungus
are more prominent choice for bioleaching and bioremediation processes in future.
