OPEN JOURNAL OF WATER POLLUTION AND TREATMENT
ISSN(Print): 2374-6343 ISSN(Online): 2374-6351
DOI: 10.15764/WPT.2014.02010
Volume 1, Number 2, September 2014
OPEN JOURNAL OF WATER POLLUTION AND TREATMENT
Bioremoval Of Cadmium Using
Pseudomonas fluorescens
M. Sankarammal1 , A. J. Thatheyus2 *, D. Ramya2
1
PG Department of Immunology and Microbiology, The American College, Madurai 625 002, India.
PG and Research Department of Zoology, The American College, Madurai 625 002, India.
*Corresponding author: jthatheyus@yahoo.co.in
2
Abstract:
Electroplating industries discharge heavy metals such as cadmium, chromium, copper, nickel,
lead and zinc in their effluents. Cadmium in high concentration in the effluent cause direct
hazards to human and animals. Pseudomonas fluorescens isolated from soil samples collected
from contaminated sites was inoculated in 250, 500, 750 and 1000 ppm concentrations of
cadmium for a period of eight days. Atomic absorption spectrophotometric (AAS) analysis was
carried out for the samples at an interval of two days to determine the amount of cadmium
removed. Maximum cadmium removal was found at 1000 ppm concentration. Experiments
were also designed to study the effect of dead cells and sugars on the biosorption of cadmium
ions. Among glucose, sucrose, lactose, fructose and dextrose supplemented, sucrose exhibited
the highest biomass. The results of this study indicated the cadmium removal capacity of P.
fluorescens and hence it can be exploited in the bioremediation of cadmium.
Keywords:
Cadmium; Pseudomonas fluorescens; Biosorption; Dead Cells and Sugars
1. INTRODUCTION
Electroplating industry is one of the industries where a variety of heavy metals including lead, cadmium,
copper, chromium, zinc and mercury are being used. The industry uses nitric acid, sulphuric acid chromic
acid and hydrochloric acids in stripping and cleaning processes, Chromic acid is used in cleaning as a
result of which free-acids are released into effluents [1]. Effluents discharged from the electroplating
industries are the cause of serious ground water and soil contamination in vicinity area which pose a
significant threat to human health and ecology [2]. The harmful effects of cadmium include a number
of acute and chronic disorders, such as itai-itai disease, renal damage, emphysema, hypertension, and
testicular atrophy [3]. Cadmium is one of the most toxic metals affecting the environment. Mining and
metallurgy of cadmium, cadmium electroplating, battery and accumulator manufacturing, pigments and
ceramic industrial waste water discharge undesired amounts of cadmium ions. The main techniques which
have been utilized for treatment of cadmium bearing waste streams include precipitation, evaporation,
adsorption, ion exchange, membrane processing and solvent extraction. These methods have been found
to be limited, since they often involve high capital and operational costs and may also be associated with
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Bioremoval Of Cadmium Using Pseudomonas fluorescens
the generation of secondary wastes which pose treatment problems [4].
Microbial and other biomass types have been shown to possess good biosorption potential and they
have been proposed as the basis for treatment for metal-bearing industrial wastewaters [5, 6]. Compared to
techniques such as precipitation and ion exchange, biosorption as a polishing process has the advantages
of low cost, good efficiency and it does not produce sludge of high metal content. Furthermore, the
potential exists for metal recovery from loaded biosorbents through elution or incineration treatment [7].
Hence in the present work an attempt has been made to study the biosorption of cadmium by the natural
isolate, Pseudomonas fluorescens. Experiments were also designed to study the effect of dead cells and
sugars on the biosorption of cadmium.
2. MATERIALS AND METHODS
The soil samples were collected from an electroplating industry near Jaihindpuram at Madurai. Samples
were collected in sterile containers. The bacterial strains isolated from such soil samples by serial dilution
method were maintained in agar slants. Among them, one strain was selected and tentatively identified
according to morphological and biochemical criteria such as the Gram straining, Indole, Methyl Red,
Voges Proskauer, Citrate utilization and Catalase tests as Pseudomonas fluorescens.
The tolerance of P. fluorescens for cadmium was determined by its inoculation onto the nutrient agar
medium containing wide range of cadmium concentrations (50, 100, 500, 1000, 2000, 3000 and 4000
ppm). The plates were incubated at 37o C and observed for growth after 24 hours. Based on the growth,
250, 500, 750 and 1000 ppm concentrations of cadmium were selected for further experiments.
From the overnight culture maintained in nutrient broth the organism was inoculated (0.1 ml) into 100
ml minimal broth containing the selected concentrations of cadmium (250, 500, 750 and 1000 ppm) in
250 ml Erlenmeyer flasks. The flasks were incubated at room temperature on a shaker for intermittent
mixing and the samples were then subjected to the estimation of residual cadmium concentration in
Atomic Absorption spectrophotometer (AAS) after every two days up to eight days. 2 ml of the sample
from the culture flask was taken and with the colorimeter optical density readings were taken at 600 nm.
It was performed from two to eight days of treatment.
The pH of the medium after treatment was determined using a pH meter and pH 7 was observed
throughout the treatment period. Pellet from the above step was collected and poured in a Petri dish.
Then the Petri dish containing the pellet was dried in a hot air oven at 80o C for three hours. The final
dried biomass was weighed and the dry biomass was determined. For obtaining dead cells, the bacterial
culture (24 hours) in nutrient broth was autoclaved at 121o C for thirty minutes and used for the study.
For testing the biosorption of dead cells, 100 ml of minimal broth containing 250, 500, 750 and 1000
ppm of cadmium in 250 ml Erlenmeyer flasks was prepared. To such flasks, dead cells were inoculated
individually and samples were taken after five minutes up to eighty minutes.
10 ml of the sample each from the 250, 500, 750 and 1000 ppm concentrations of cadmium was
centrifuged at 2500 rpm for fifteen minutes, after five minutes up to eighty minutes. The clear supernatant
was used for AAS analysis. The values obtained by AAS analysis represent the residual concentrations
of cadmium. The efficiency of the bacterium for the sorption of cadmium was tested by supplementing
different carbon sources like dextrose, fructose, glucose, lactose and sucrose at 10% concentration in
minimal broth containing 500 ppm concentration of cadmium and the inoculum (109 cells). The flasks
were incubated at 37o C on a shaker and the optical density was estimated after two days while biomass
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OPEN JOURNAL OF WATER POLLUTION AND TREATMENT
Table 1. Results of Microbiological and Biochemical Tests for the Isolated Bacterial Strain
Biochemical tests
Colony character
Colony size
Cell type
Gram reaction
Methyl Red
Voges Proskauer
Indole
Catalase
Citrate
Isolated bacteria
Round and cream color
Medium
Rod
+
+
*Note: + Positive; - Negative
was determined by performing centrifugation at 2500 rpm for fifteen minutes, followed by drying in a hot
air oven at 80o C for three hours.
Two way analysis of variance (ANOVA) was performed for the factors residual cadmium concentration,
percent removal of cadmium and biomass of P. fluorescens during cadmium treatment for the two
variables namely cadmium concentration and treatment period. It was also performed for the factors
residual cadmium concentration and percent removal of cadmium for dead cell preparations with two
variables namely treatment period and cadmium concentration, using Microsoft MS- Excel Package
(Table 4).
3. RESULTS AND DISCUSSION
The bacterial strain Pseudomonas fluorescens was tested for its cadmium tolerance with wide range of
cadmium concentrations (50, 100, 500, 1000, 2000, 3000 and 4000 ppm). The results indicated that after
twenty four hours of incubation, the strain grew well up to 1000 ppm concentration of cadmium. Based
on the tolerance level, P. fluorescens was subjected to 250, 500, 750, 1000 ppm cadmium for eight days.
The bacterial strain was isolated from electroplating industrial effluent and identified as P. fluorescens
on the basis of biochemical tests which are shown in Table 1. The organism was positive to Citrate
and Catalase and negative for Gram reaction, Indole, Methyl red and Voges Proskauer tests. Figure 1
illustrates the percent removal of cadmium after treatment with P. fluorescens. Among the cadmium
concentrations highest percent removal was for 1000 ppm concentration of cadmium throughout the
treatment period. The optical density values obtained during the treatment of P. fluorescens are shown
in Figure 2. Increase in the optical density values during the treatment period was observed. Highest
optical density was observed after six days for 1000 ppm of cadmium concentration. Figure 3 illustrates
the biomass of P. fluorescens during cadmium treatment. Highest biomass was obtained for all the
concentrations after eight days of treatment.
Figure 4 illustrates the percent removal of cadmium after treatment with the dead cells of P. fluorescens.
It indicates highest percent removal for 1000 ppm concentration of cadmium after eighty minutes.
Influence of sugars at 10% concentration on the biomass of P. fluorescens during cadmium treatment
is exhibited in Figure 5. It indicates that the biomass was the highest for sucrose followed by glucose,
lactose, fructose and dextrose. Figure 6 exhibits the optical density values obtained during treatment with
P. fluorescens after two days of cadmium treatment. Highest value was obtained for sucrose followed by
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Bioremoval Of Cadmium Using Pseudomonas fluorescens
Figure 1. Percent removal of cadmium after treatment with Pseudomonas fluorescens
Figure 2. Optical density values obtained during cadmium treatment with Pseudomonas fluorescens
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OPEN JOURNAL OF WATER POLLUTION AND TREATMENT
Figure 3. Biomass of Pseudomonas fluorescens during cadmium treatment
Figure 4. Percent removal of cadmium after treatment with the dead cells of Pseudomonas fluorescens
lactose, fructose, glucose and dextrose.
Two way analysis of variance for the percent removal of cadmium with the variables treatment period
and cadmium concentration for P. fluorescens is given in Table 2. The variations in the percent removal
of cadmium and biomass due to treatment period and cadmium concentration were statistically significant.
The variations in the percent removal of cadmium by dead cells due to treatment period were statistically
not significant, while for cadmium concentration they were statistically significant.
Cadmium which is listed as Known to be Human Carcinogens in the Eleventh Report on Carcinogens
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Bioremoval Of Cadmium Using Pseudomonas fluorescens
Figure 5. Influence of sugars on the optical density values obtained at 500 ppm cadmium after two days with
Pseudomonas fluorescens
Figure 6. Influence of sugars on the biomass (g dry wt/ml) of Pseudomonas fluorescens at 500 ppm cadmium
treatment after two days
receives increasing attention as one of the most toxic heavy metals [8, 9]. Most physico - chemical methods
for cadmium removal appear to be expensive, inefficient and labor-intensive [9]. Bioremediation, which
involves the use of living microbes to remove heavy metals, has been considered to be a safe and economic
alternative to physico-chemical strategies due to their ability of self-replenishment, continuous metabolic
uptake of metals after physical adsorption, and the potential for optimization through development of
resistant species and cell surface modication [10, 11]. The employment of bacterial biomass for the metal
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OPEN JOURNAL OF WATER POLLUTION AND TREATMENT
Table 2. Two way analysis of variance for the various factors with the variables, treatment period and cadmium
concentration for Pseudomonas fluorescens
Factor
Source of variation
Percent removal
of cadmium with live cells
Treatment period
3 9.25
Cadmium concentration 3 0.072
df MS
Biomass
Percent removal of cadmium
with dead cells
Calculated F value Table F value Level of Significance
5.55
43
3.86
3.86
Significant
Significant
Treatment period
3 0.000056 71.65
Cadmium concentration 3 0.000024 30.027
3.86
3.86
Significant
Significant
Treatment period
3 0.0041
Cadmium concentration 3 0.082
3.26
3.49
Not Significant
Significant
2.026
40.47
removal of industrial effluents is a strategy suggested by many researchers dealing with metal-bacteria
interactions [12]. Polarizable groups present on bacterial surfaces are capable of interacting with and they
are responsible for reversible metal binding capacity. Such groups include phosphate, carboxyl, hydroxyl
and amino-groups [13].
Many researchers have reported the efficiency and mechanisms of bacteria to remove different metal
ions and many of their statements are comparable to the present study. Richard et al., [14] reported that
copper and lead bind to materials on their cell surface. Lead is precipitated in an insoluble form that is
localized to the cell membrane or cell surface [15]. This could be generally explained by the fact that the
negatively charged groups (carboxyl, hydroxyl and phosophryl) of the bacterial cell wall adsorb metal
cations through various mechanisms such as electrostatic interaction, Van der Waals forces, covalent
bonding or the combination of such processes [16].
In the present study, Pseudomonas fluorescens was able to tolerate the concentration upto 1000 ppm of
cadmium. The percent removal of cadmium by P. fluorescens was maximum at 1000 ppm and least at
250ppm of cadmium. This indicates that P. fluorescens was more efficient in the removal of cadmium
when the concentration of cadmium is high. The concentration of cadmium was high, P. putida was
tolerant [17]. The biomass of P. fluorescens during cadmium treatment increased on the subsequent days
and this confirms that P. fluorescens was capable of tolerating the cadmium concentrations up to 1000 ppm.
The capacity of living cells to remove metal ions from aqueous solutions is also significantly influenced
by environmental growth conditions, such as temperature, pH and biomass concentrations [18].
The highest optical density value for P. fluorescens was observed at 1000 ppm cadmium concentration
after six days of treatment. The percent removal of cadmium was the highest at 1000 ppm after eighty
minutes with dead cells of P. fluorescens. The highest biomass of P. fluorescens during cadmium treatment
was noticed in sucrose supplemented flask.
The key aspects in the remediation of metals are that metals are non-biodegradable. But they can be
transformed through sorption, methylation, complexation, and changes in valency state. These transformations affect the mobility and bioavailability of metals. At low concentrations, metals can serve
as important components in life processes, often serving important functions in enzyme productivity.
However, above certain threshold concentrations, metals can become toxic to many species [19]. Fortunately, microorganisms can affect the reactivity and mobility of metals. Thus microorganisms that
affect the reactivity and mobility of metals can be used to remove heavy metals and prevent further metal
contamination.
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Bioremoval Of Cadmium Using Pseudomonas fluorescens
4. CONCLUSION
Pseudomonas fluorescens isolated from cadmium contaminated soil was identified based on biochemical
tests. Maximum cadmium removal was observed at 1000 ppm cadmium concentration. Dead cells
exhibited 99% removal in five minutes. Supplementation of sugars enhanced the biomass of the bacterium
and maximum biomass was observed for sucrose followed by glucose, lactose, fructose and dextrose.
ACKNOWLEDGEMENTS
The authors thank the authorities of the American College for facilities and encouragement.
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