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Bioaccumulation of heavy metals by bacterial isolates

5.1 Introduction

5.1.1 Heavy metal pollution

Heavy metal pollution is a most important apprehension, as it leads to toxicity, risk

to human survival and disrupts ecological balance (WHO, 2007). The occurrence of toxic

heavy metals in the environment is geogenic or anthropogenic. The release of untreated

industrial wastewater into the environment has become a foremost concern in the

developing countries and is viewed as one of the most important environmental issues.

With rapid development of various industries such as mining and smelting of metals,

surface finishing industry, energy and fuel production, fertilizer and pesticide industry,

metallurgy, iron and steel, electroplating, electrolysis, electro-osmosis, leather industry,

photography, electric appliance manufacturing, metal surface treating, aerospace and

atomic energy installation, knowingly or unknowingly discharged the metal contaminated

pollutans to the surroundings causing serious threat to human beings and other life forms

(Volesky, 1990) and hence potent environmental stressors.

The metals are grouped into three kinds, including toxic metals (such as Hg, Cr,

Pb, Zn, Cu, Ni, Cd, As, Co, Sn, etc.), precious metals (such as Pd, Pt, Ag, Au, Ru etc.) and

radionuclides (such as U, Th, Ra, Am, etc.) (Wang and Chen, 2006). Toxic heavy metals

entering into the ecosystem leads to geo-accumulation, bio-accumulation and bio-

magnification processes which have intense ecological and public health implications

(Jarup, 2003). The heavy metal emission into the environment occurs via an array of

processes and pathways, to the air (during combustion, extraction and processing), to

surface waters (via runoff and releases from storage and transport) and to the soil (hence

into ground waters and crops) (Morgan and Stuum, 1991 and Ilyin and Travnikov, 2003).

Heavy metals are toxic to all groups of organisms from microbial to plant and animal. Due

to their smaller size and direct exposure to the environment, microorganisms are more

affected by toxic heavy metals (Patel et al., 2007 and Giller et al., 1999). Elevated levels

of heavy metals can result in decreased microbial community size and structure (Konopka

et al., 1999); decreased organic matter mineralization (Chander and Brookes, 1991) and

leaf litter decomposition (Strojan, 1978).

Chapter 5Chapter 5Chapter 5Chapter 5

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5.1.2 Treatment strategies

In mitigating heavy metal polluted environments several treatment strategies and

methods are available such as chemical precipitation, filtration, ion exchange,

electrochemical treatment, membrane technologies, adsorption on activated carbon,

evaporation, phytoremediation etc. and these processes are neither effective nor

economical when the heavy metal ion concentration is very low (Wilde and Beneman,

1993). These methods have disadvantages like partial metal removal, high reagent and

energy requirements and production of toxic sludge (Ahalya et al., 2003). Sheng et al.

(2006) reported that in bioremediation by phytoremediation, the synthetic chelators used

to amplify the phytoavailability of metals are highly expensive. Compared with the above

methods the removal of metals by indigenous microorganisms have advantages such as

low cost and without secondary pollution (Jun-xia et al., 2007), and biosorption is an

ideal candidate for the treatment of high volume and low concentration complex

wastewaters (Wang and Chen, 2006).

Among the different techniques employed for metal removal from multi-elemental

system, biosorption has been found to be highly selective depending on the typical binding

capacity of the biosorbents (Knauer et al., 1997). A number of bacteria and fungi that can

accumulate an abundant range of metal species have been described (Fourest et al., 1994

and Volesky and Holan, 1995). It also plays a benevolent role in reclamation of heavy

metal contaminated soil and wastewater. Microorganisms uptake metals either actively

(bioaccumulation) and/or passively (biosorption) (Hussein et al., 2003). Magyarosy et al.

(2002) reported that the high surface to volume ratio of microorganisms and their ability to

detoxify metals make them potential alternatives for remediation. The most important

benefit of microbial biosorption over conventional treatment methods are low cost,

minimization of chemical and biological sludge, high efficiency, regeneration of

biosorbent and possibility of metal recovery (Kratchovil and Volesky, 1998).

5.1.3 Bioaccumulation of heavy metals using bacterial isolates

Among the various methods, bioaccumulation and biosorption are potential for the

removal of metals (Volesky and Holan, 1995). The active mode of metal accumulation by

living cells is usually referred as bioaccumulation which is dependent on intrinsic

biochemical and structural properties, physiological and genetic adaptation, environmental

modification of metal specification, availability and toxicity (Blackwell et al., 1995).


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Bioaccumulation is defined as the uptake of toxicant by living cells and transport into the

cell (Malik, 2004) and is a growth dependent process mediated only by living biomass

(Karna et al., 1996). The process of biosorption is possible by both living and dead

biomasses (Brady et al., 1994). The ability of certain species of bacteria in accumulating

heavy metals was investigated thoroughly (Malekzadeh et al., 2002 and Chowdhury et al.,

2008). Hence the natural organisms, either indigenous or extraneous can be used for

bioremediation of heavy metals (Prescottet et al., 2002).

5.2 Review of literature

Even though certain heavy metals are required for various biochemical activities of

living organisms, their high concentration impose severe toxicity to living beings. The

microorganisms living in the heavy metal polluted environment are more resistant to

metals than the microbe from non-contaminated area (Frostegard et al., 1996; Kelly et al.,

1999; Macnaughton et al., 1999; Pennanen et al.,1996; Witter et al. 2000 and Bahig et al.,

2008). Das et al. (2006) reported that the microorganisms living in the marine extreme

environmental conditions have potential tolerance to heavy metals. Many researchers

reported the tolerance and environmental pollution of heavy metals (Hutchinson and

Symington, 1997 and Hernandez et al., 1998). The long term exposure of heavy metal

cause a selection pressure on bacterial population which enhances their tolerance level to

lead (Pb) (Lo et al., 1999 and Kasthuri and Chandran, 2009 ) and chromium (Cr) (De leo

and Ehrlich, 1994; McLean and Beveridge, 2001).

As heavy metal pollution is increasing day by day, the responses of microbial

communities are of serious concern (Rasmussen and Sorensen, 1998 and Nies, 1999).

Several researchers all over the world have noticed heavy metal resistance among many

bacterial species (Schmidt et al., 1991 and Liesegang et al., 1993). Goris et al. (2001)

isolated thirty one heavy metal resistant bacteria from industrial biotopes with

considerable variations in activity. Pseudomonas putida was able to remove 80% Cd+2,

Pb+2 , Zn+2, and Cu+2 from aqueous solution (Pardo, et al., 2003). Microbial populations in

metal polluted environments adapt to different concentrations of heavy metals and become

metal resistant (Prasenjit and Sumathi, 2005). Jankowska et al. (2006) isolated metal

resistant bacteria from coastal waters of Sopot beach, Gdańsk Bay (Poland). Congeevaram

et al. (2007) studied heavy metal resistant fungi and bacteria from the soil samples

adjoining electroplating industry and the bioaccumulations of Cr (VI) and Ni(II) by these


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isolates. The presence of heavy metal resistant bacterial strains in and around industrial

area in Kazanlı, Mersin, Turkey was investigated thoroughly (Sevgi et al., 2010). They

documented a total of 272 Pseudomonas sp. and 161 Bacillus sp. strains from soils.

Resistance to Cr was found in 73.9% of the bacterial strains, whereas 26% of the isolates

exhibited resistance to Ni, 18.4% to Zn, 11.5% to Cd, 9.2% to Co, and 7.3% to Cu.

Bacterial resistance to heavy metals might be chromosomal or plasmid mediated (Silver,

1996 and Gupta et al., 1999). Zolgharnein et al. (2003) reported that the occurrence of

plasmids in heavy metal resistant bacteria was more than that in common bacteria.

Several industries discharge enormous amounts of chromium and lead into the

environment. Unlike many other pollutants, heavy metals are difficult to be removed from

the environment (Ren et al., 2009). Hence bioaccumulation/ biosorption was an effective

tool to remove the metals from the contaminated site. Microbs uptake metals either actively

(bioaccumulation) and/or passively (biosorption) (Anders et al., 1992; Hussein et al., 2001

and Hussein et al., 2003). Several authors reported the bioaccumulation/biosorption

property of microorganisms against the heavy metals in laboratory condition, particularly

against chromium (Cr VI) and lead (Pb II). Alam and Ahmad (2011) studied the chromium

removal by bacteria through biosorption and bioaccumulation from tannery effluents

contaminated soil. The studied isolates are Stenotrophomonas maltophilia,

Exiguobacterium sp., Pantoea sp., and Aeromonas sp. and the results were substantiated

by transmission electron microscopy which confirmed the accumulation of chromium in

cytoplasm. The biosorption and bioaccumulation of Cr and Pb were elucidated by many

researchers worldwide (Gadd and White, 1993; Sharma and Forster, 1993 and Fude et al.,

1994). The lead removing potential of Brevibacterium sp. from aqueous solutions are

reported earlier (Andreoni et al., 1997). Uslu et al. (2003) reported that the culture was

able to remove 54.9% and 30.5% total Pb (II) ions at 50 and 75 mg/L-1initial metal ion

concentrations. Rhizopus arrhizus M1 during lead biosorption indicated that the cell

surface functional groups of the fungus might act as ligands for metal sequestration

resulting in removal of the metals from the aqueous culture media (Pal et al., 2010).

Srinath et al. (2002) reported the bioaccumulation capacity of Bacillus circulans and

Bacillus megaterium by about 34.5 and 32.0 mg Cr/g dry weight, respectively and brought

the residual concentration of Cr(VI) to the permissible limit in 24 hrs when the initial

concentration was 50 mg Cr (VI)/l. Above 80% of Cr (VI) reduction achieved in

bioreactor with an initial chromium concentration of 50 mg/L at hydraulic retention time


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(HRT) of 8 hours (Rama Krishna and Philip, 2005). The bioaccumulation of heavy metals

by Gram negative bacteria are well established (Tohamy et al., 2006 and Noghabi et al.,

2007). El-Hendawy et al. (2009) studied the bioaccumulation of heavy metals by Vibrio

alginolyticus isolated from the wastes of Iron and Steel Factory, Helwan, Egypt. They

observed that the percentage reduction by V. alginolyticus was 20% for Cd, 31% for Cu+2,

40% for Pb+2 and 45% for Zn+2. A total of 90% chromium VI removal was obtained by

Micrococcus sp. at pH 7.0 (Shankar et al., 2007). Similrly S4 strain was able reduce

64.4% Cr (VI) at 160mg/L-1 within 72 hours (Farag and Zaki, 2010). It has been reported

that bioaccumulation for lead by B. licheniformis ranged from 0 to 1.1 mol metal/g

biomass (Issazadeh, 2011). Bioremediation of 74.5% Cr (VI) at 48 hours by B. cereus was

noted by Tripathi (2011).

5.3 Objectives of the study

1. To isolate and evaluate the metal resistance of bacterial strains from the sediment

samples of Vattakayal backwaters.

2. To evaluate the antibiotic sensitivity of the bacterial isolates.

3. To identify and characterise the metal resistant bacterial strains.

4. To study the bioaccumulation potentials of Bacillus cereus and Bacillus

lichiniformis against chromium and lead.

5.4 Materials and Methods

5.4.1 Sample collection

Surface sediment samples from Vattakayal backwaters (Station 6) were collected

using Peterson grab and transported on ice to the laboratory and processed within 5-7

hours of sample collection.

5.4.2 Isolation of heavy metal tolerant bacterial strains from the sediments

Isolation and enumeration of bacteria were carried out by standard serial dilution

plate technique. 10g of sediment was transferred to 90 ml normal saline and agitated

vigorously. Different aqueous dilutions, 10-1 to 10-7 of the suspensions were prepared.

Serially diluted samples were sown in Nutrient Agar (Hi-media India) ( beef extract 3gL-1,

Peptone 5gL-1, NaCl 5gL-1, Agar 20 gL-1), supplemented with 50µg/ml of heavy metals

individually and incubated at 37°C for 24- 48 hours (Ansari and Malik, 2007 and Narita et


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al., 2003). After incubation, morphologically distinct colonies were picked and purified.

Pure cultures of bacterial colonies were prepared and persevered at 4oC as slant cultures

for further analysis.

5.4.3 Preparation of metal stock solution

The stock solutions of chromium [Cr(VI)], copper [Cu(II)], Zinc [Zn(II)], cadmium

[(Cd(II)] and lead [Pb(II)] were prepared in deionized water and sterilized by filter

membrane (0.22 µm) and stored at 4°C. The salts used were potassium dichromate

(K2Cr2O7), copper (II) sulfate pentahydrate (CuSO45H2O), zinc sulphate hexahydrate

(ZnSO46H2O), Cadmium nitrate tetrahydrate (Cd(NO3)24H2O) and lead nitrate (PbNO3)2.

All working concentrations were obtained by diluting the stock solution with deionized

and steriled (Bahig et al., 2008) water and the pH were adjusted to the desired values

using 1M HCl and 1M NaOH solutions.

5.4.4 Determination of Minimal Inhibitory Concentration (MIC)

Resistance of the bacterial isolates to varying concentrations of heavy metals such

as lead, zinc, chromium, and cadmium were determined by agar dilution method (Luli et

al. 1983). Fresh overnight cultures of the isolates grown in peptone water were aseptically

inoculated into nutrient agar plates, which were supplemented with increasing

concentration of the aforesaid metals individually (5µg/ml to 500µg/ml). The plates were

incubated at room temperature and observed for bacterial growth. The lowest

concentration of heavy metals at which no growth occurred when compared with the

control plates was considered as the Minimal Inhibitory Concentration (MIC). All metal

salts were added to the medium after autoclaving and cooling to 45-50oC, from filter

sterilized stock solutions.

5.4.5 Sensitivity to antibiotics

Antibiotic sensitivity of heavy metal resistant isolates was determined by disc

diffusion method (Bauer et al., 1966). Antibiotic impregnated discs (6 mm diameter,

HiMedia) were placed on Muller - Hinton agar plates swabbed evenly with individual

isolates and incubated at 37oC for 24 hrs. The antibiotic diffuses out of the disk to form the

gradient; the test organism starts to divide, grow and progresses towards a critical mass of

cells. The so-called inhibition zone edge is formed at the critical time where a particular

concentration of the antibiotic is just able to inhibit the organism before it reaches an

overwhelming cell mass or critical mass (Acar and Goldstein, 1996). The diameter of the


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inhibition zones around the discs was measured in mm. Antibiotic resistance was

determined by comparing the diameter of inhibition zone around each antibiotic disk with

zone size interpretive chart supplied by Hi-media laboratories, Bombay. The

concentrations of the antibiotic present in the disc were ampicillin (10 µg), tetracycline (25

µg), chloramphenicol (25 µg), penicillin (1 µg), Tetracyline (25 µg), streptomycin (10µg)

and Sulphatriad (300 µg) respectively (Raja et al., 2006).

5.4.6 Multiple Antibiotic Resistance (MAR) indexing of the isolates

The MAR index when applied to a single isolate is defined as a/b where ‘a’

represents the number of antibiotics to which the isolate was resistant and ‘b’ represents

the total number of antibiotics to which the isolate was exposed. Isolates with a MAR

index value higher than 0.2 is considered to have originated from high-risk source of

contamination such as human, commercial poultry farms, swine and dairy cattle where

antibiotics are often used.

5.5 Identification and characterization of heavy metal tolerant bacterial strains

5.5.1 Morphological

The morphological characteristics of the bacterial colonies including shape,

margin, elevation and pigment production were visually observed during isolation. Cell

shape of the bacterial strains were examined under microscope (1000x magnification) after

Gram staining. Motility of the isolates was tested by semisolid agar.

5.5.2 Biochemical characteristics

Bacterial isolates which are maintained as pure culture on Nutrient Agar were

characterized and identified by Biochemical tests of Oxidase, Catalase, Indole, Methyl

red, Voges Proakauer, Citrate, Urease, Nitrate reduction, D-Glucose gas production, D-

Glucose acid production, Lactose, Mannitol, Sucrose, Phenylalanine Deaminase, H2S from

LIA, Litmus milk reaction, Gelatine, Starch, Lipid etc. as per Bergey’s Manual of

Determinative Bacteriology: 9th edition (Holt et al., 1994) and 8th edition (Buchanan and

Gibbons, 1974).


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5.5.3 Molecular characterisation

5.5.3.1 Genomic DNA isolation from culture cells

Genomic DNA was isolated from the bacterial isolates. The pure bacterial colony

was inoculated in Luria Bertani broth. After incubation 1ml of the culture was transferred

to a sterile micro centrifuge tube and the cells were harvested by centrifugation (12000

rpm) for 10 minutes at room temperature. Supernatant was discarded and the pellet was

resuspended in 1 ml of 0.85% (w/v) NaCl solution and centrifuged as above. Discarded

the supernatant and added 600µl lysis buffer along with 7 µl of proteinase-K. Vortexed

the mixture and incubated at 65ºC for 1 hour. Added equal volume of chloroform: isoamyl

alcohol (24 : 1) with gentle mixing by inverting the tubes for 2-5 minutes. Centrifuged the

samples for 15 minutes (12000 rpm) at room temperature. The aqueous phase was

collected in another micro centrifuge tube without disturbing the interface and lower

phase. Repeated the steps of chloroform: isoamyl alcohol extraction. Again the aqueous

phase was collected and added 50µl volume of 3M Sodium Acetate (pH 5.2) followed by

equal quantities of ice cold isopropanol, so that the DNA gets precipitated and centrifuged

it again at room temperature for 5 minutes at 12000 rpm. The supernatant was discarded

and rinsed the pellet twice with 70% ethanol, followed by maintaining the tubes for 1hour

in vacuum desiccators. The desiccated DNA samples were completely resuspended in

50µl of DNA dissolving buffer (TE buffer) and stored at -20oC. The Ultraviolet (UV)

absorbance was checked at 260 and 280 nm for determination of DNA concentration and

purity. Concentration of DNA was estimated using the formula: Concentration of DNA

(mg/ml) = OD 260 ×50 × Dilution factor.

5.5.3.2 16S rDNA gene amplification

The bacterial 16S rDNA fragment was amplified from the extracted genomic DNA

by using 16S rDNA universal primer, 8F (5'-AGAGTTTGATCMTGG-3') and reverse

primer 1492r (5'-ACCTTGTTACGACTT-3'). PCR was performed in a final reaction

volume of 25 µl in 200 µl capacity thin wall PCR tube. PCR tubes containing the mixture

were tapped gently and spinned briefly at 10,000 rpm. The PCR tubes with all the

components were transferred to thermal cycler. The reaction mixture contain deionized

water - 16.5µl, Taq buffer without MgCl(10 X) - 2.5µl, MgCl22( 15 mM)- 1.0µl and

dNTPs mix (10 mM each) - 1.5µl. After the initial denaturation of 5 min at 95oC there

were 29 cycles consists of denaturation at 94oC for 30 seconds, annealing at 58oC for 30


213

seconds, extension at 72oC for 45 seconds and final extension at 72oC for 10 min. PCR

was carried out in ABI3730xl Genetic Analyzer (Applied Biosystems, USA) . The PCR

product were analysed by 1.5% (w/v) agarose gel electrophoresis in 1 X TBE (Tris-

Borate-EDTA; electrophoresis buffer) with Bromophenol blue loading dye. Viewed the

gels on UV transilluminator and photograph of the gel was taken. The PCR product was

sequenced by ABI3730xl Genetic Analyzer (Applied Biosystems, USA). Sequences were

matched with previously published bacterial 16S rDNA sequence in the NCBI database

using ADVACED BLAST (www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1997).

5.6 Phylogenetic identification

A phylogenetic tree was constructed using the neighbour-joining distance method

with the MEGA4 software (Tamura et al., 2007) and the reliability of the bootstrap

consensus inferred from 1000 replicates. Some reference sequence from the Gene Bank of

most closely related to that of Bacillus were used in generating phylogenetic tree (Yang et

al., 2009).

5.7 Bioaccumulation of two heavy metals (Cr and Pb) by bacterial

isolates

Determination of metal accumulation by bacterial isolates was done based on the

procedure of Hernandez et al. (1998) with some modifications. The bacterial strains

growing in high concentration of Cr and Pb were selected for Cr and Pb removal studies.

Bioaccumulation method at different pH (6, 7 and 8) and different temperature (25oC,

30oC and 35oC) with living bacterial cells was used for the removal of Cr and Pb. To study

heavy metal removal with live cells, nutrient broth amended with initial concentration of

chromium and lead (100µg/ml) were inoculated from overnight grown cultures of selected

bacterial isolates. The inoculated flasks were incubated at room temperature for 96 hrs in a

shaking condition (150rpm). Control flasks without bacterial biomass were running

simultaneously with the experiment flasks. An aliquot of 5ml sample was taken daily (24

hrs interval) from each flask. Samples were centrifuged to remove suspended biomass and

concentration of heavy metals was determined in the supernatant using Atomic Absorption

Spectrophotometer (Perkin Elmer-A Analyst 300, Atomic Absorption Spectrophotometer).

Bacterial growth in the experimental flask was monitored as a function of biomass by

measuring the absorbance at 595 nm against the blank. The average value was obtained

and put into the following formula to get the removal rate ( η) ( Xiao-xi, 2009).


214

( )100%

a b

aη

− = ×

Where a is the mass of metal in the solution without bacteria, and b is the mass of

metal in the solution with bacteria.

5.8 Results and Discussion

The present investigation was designed to determine the heavy metal tolerance of

the bacterial isolate from the heavy metal contaminated sediments of Vattakayal

backwaters near the industrial area of Chavara. It also aimed to determine the percentage

removal of heavy metal in the laboratory condition by selected bacterial strains. Therefore

in the present work heavy metal tolerant bacteria from the sediments were isolated,

identified and characterized. After isolation experiments were conducted to determine the

Minimum Inhibitory Concentration and the bioaccumulation property.

5.8.1 Isolation of heavy metal tolerant bacterial strains from the sediments

The bacterial strains were isolated from the heavy metal contaminated sediments

from station 6 (Plate II) where the wastewater from Kerala Minerals Metals Ltd.

(KMML) reaches the estuary. The microbes capable of tolerating individually 50µg/ml of

metals like chromium, copper, zinc, cadmium and lead were isolated. Totally 7 strains

(K1, K2, K3,K4, K5, K6 and K7) were isolated based on their difference in colony

morphology in the preliminary screening. Theses isolates were then subjected to further

determination of minimum inhibitory concentration.

5.8.2 Heavy metal resistance patterns of bacterial isolates

In the present work a total of seven metal resistant bacterial strains were isolated

from the lake sediments. Most of the isolates showed resistance to one or more heavy

metals selected, however, the patterns of tolerance among the seven cultures varied (Table

5.1). Metal resistance studies of the bacterial isolates showed that about 57% of the

isolates showed comparatively high tolerance (≥300µg/ml) to lead, zinc and copper.

Resistance to cadmium and chromium was in between 95µg/ml - 300µg/ml and 100µg/ml

-250µg/ml respectively. Sampling environments that contain elevated concentrations of

heavy metals are a potential source of heavy metal tolerant bacteria. Many researchers

reported that indigenous organisms isolated from heavy metal contaminated sites had


215

tolerance to heavy metals toxicity (Kamala-Kannan et al., 2006; El-Hendawy et al., 2009

and Andreazza et al., 2011).

Among the heavy metals Zn, Cu and Pb were found to be less toxic, whereas Cd and

Cr were highly toxic to all strains in the order of resistance Pb>Cu>Zn>Cd>Cr indicating

the adaptation of the organism to a stressed environment. Similar observation has earlier

been reported (Raja et al., 2006). The metal resistance of Ralstonia in lake sediments and

industrial biotopes has previously been studied (Diels and Mergeay, 1990 and Yong et al.,

2008). Ralstonia sp. CH34, formerly Alcaligenes eutrophus (Brim et al., 1999) is one

among the best known example of heavy metal resistant bacterial strain. Duan and Min

(2004) isolated a strain DKC1 of Ralstonia eutropha from a long-term cadmium treated

soil sample and found that it had high resistance to cadmium, could grow well on solid

medium supplemented with 3 mM cadmium. Strain DKC1 showed high resistance to Cd

but lower resistance to other heavy metals. Sun et al. (2009) isolated copper tolerant

strains Sphingomonas sp. (JM14, YM22 and YM12) from copper contaminated soils.

According to Gupta et al. (1999) the bacterial resistance to heavy metals were

chromosomal or plasmid mediated. The plasmids are frequently present in heavy metal

resistant bacteria than in common bacteria (Zolgharnein et al., 2007). In the present study

it was found that the isolates K1 and K4 showed high tolerance to heavy metals

(Table.5.1) and hence they were selected for bioremediation studies in the laboratory

condition. The metals Cr and Pb are selected for bioremediation as they are present in

higher concentration in the wastewater coming from the factory as well as in the station 6

where the isolates were collected from the sediments.

Table 5.1 Heavy metal resistance patterns of bacterial strains isolated from Vattakayal Lake Sediments.

Isolates Heavy metal concentration (µg/ml)

Cr Cd Zn Cu Pb Mean K1 200 300 350 360 450 332 K2 150 120 450 300 275 259 K3 100 100 220 290 185 179 K4 250 240 110 380 375 271 K5 175 105 230 280 250 208 K6 100 140 350 300 300 238 K7 110 95 340 270 400 243

Mean 155 157.1 292.9 311.49 319.3


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5.8.3 Resistance to antibiotics

A total of seven bacterial strains were isolated from the sediment samples and

tested for their sensitivity to 6 common antibiotics (ampicillin, penicillin, sulphatriad,

chlorampenicol, streptomycin and tetracycline). 57.14% of the isolates were resistant to at

least one antibiotic tested. Resistance to ampicillin (57.14%) and penicillin (57.14%) was

noted most frequently. Resistance to sulphatriad (14.28%) and chlorampenicol (14.28%)

were relatively low (Table 5.2, Fig.5.1 & Plate XVI). A total of 3 resistant patterns were

observed among the isolates and the most frequent (50%) resistant pattern was AMP

(ampicillin- penicillin) (Table 5.3). Out of the 7 bacterial isolates many isolates showed

multiple metal and antibiotic resistances. Numerous isolates had acquired multiple drug

resistance (MDR). The present study identified the varying multiple antibiotic resistances

(MAR) index and resistance patterns of the isolates (Table 5.3).

The result of the present study, in general, agrees with those of other works in

which varying frequencies of antibiotic resistance were detected with bacteria isolated

from natural environments (Schmitt et al., 2006 and Patterson, et al., 2007). Diverse

antibiotic resistance patterns of bacterial isolates may be due to the disparity in the

selection pressure in the formation of resistant mutants. There has been substantial anxiety

about environment containing antibiotics due to the possibility of antibiotic-resistant

strains becoming dominant in the bacterial communities in such ecosystem (Wittwer et al.,

2005 and Heuer, and Smalla, 2007). Even though resistant organisms can be found

naturally in the environment, most resistance is linked with man made impacts of some

type, either agricultural or direct human impact.

Pseudomonas aeruginosa isolated from various soils in Spain were resistant to

many antibiotics and had higher levels of resistance than isolates from nearby surface

waters and these isolates were resistant to ampicillin, chloramphenicol, kanamycin,

nalidixic acid, tetracycline Marques et al.,1979). In the present study it was found that

the various bacterial isolates exhibited varying degrees of resistance to the different

antibiotics (Table 5.2).


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5.8.3.1 Association between metal tolerance and antibiotic resistance

Out of the 7 bacterial isolates many isolates showed multiple metal and antibiotic

resistance. The bacterial isolates K1 and K4 showed multiple antibiotic resistance against

3 antibiotics tested and they showed higher heavy metal resistance. The use of antibiotics

in medicine and agriculture clearly stimulates the proliferation of antibiotic resistance

(Neu, 1992). Heavy metals and other toxicants have also been suggested to play an

important role (Summers, 2002). Multiple genes encoding for metal and antibiotic

resistance are commonly found on the same plasmids and/or transposons, conferring

coresistance. In some cases, single enzymes function as efflux pumps for multiple metals

and antibiotics; this is defined as cross-resistance (Hayashi et al., 2000).

Table 5.2 Antibiotic sensitivity of bacterial strains isolated from Vattakayal lake

sediments.

Antibiotic disc Con. Isolates

(µg/disc) K1 K2 K3 K4 K5 K6 K7

Ampicillin 10 HR 20 (R) 36(S) 13 (R) 11(R) 21(I) 26(I)

Chloramphenicol 25 22(S) 17 (I) 29 (S) 11 (R) 14 (I) 16 (I) 20 S

Penicillin G 1 HR 19 (R) 30 (S) HR 11 (R) 22 (I) 26 (I)

Streptomycin 10 26(S) 26 (S) 22 (S) 17 (S) 19 (S) 22 (S) 21 (S)

Sulphatriad 300 HR 30 (S) 31 (S) 30 (S) 20 (S) 30 (S) 30 (S)

Tetracycline 25 21(S) 30 (S) HS 26 (S) 28 (S) 30 (S) 28 (S)

HR- Highly sensitive; I- Intermediate; S- sensitive; HS – Highly sensitive


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Plate XVI Antibiotic resistance pattern of the selected isolates

Table 5.3 MAR index and resistance pattern among the bacterial isolates from the

sediments of Vattakayal lake.

MAR Index Resistance Pattern No. of isolates showing the pattern

% of occurrence of pattern

0.5 AMPS3 1 25

0.5 AMCP 1 25

0.33 AMP 2 50 (AM- ampicillin, P- penicillin, S3 – sulphatriad, C - chlorampenicol,)


219

Fig. 5.1 Percentage of antibiotic resistance of bacterial isolates from Vattakayal lake

sediments.

0 10 20 30 40 50 60

AM

P

S3

C

S

T

% of resistance

An

tib

iotic

s

(AM- ampicillin, P- penicillin, S3 – sulphatriad, C - chlorampenicol, S - streptomycin and T - tetracycline).

Importantly, a substantial number of reports suggest that metal contamination in natural

environments could have an important role in the maintenance and proliferation of

antibiotic resistance (Alonso et al., 2001). This is of particular concern considering that

anthropogenic levels of heavy metals are currently several orders of magnitude greater

than levels of antibiotics (Stepanauskas et al., 2005). Berg et al. (2005) found that soil

microbes isolated from a copper-amended field were more resistant to copper and

antibiotics than strains isolated from control plots 21 months after copper amendment.

Additionally, copper resistant strains were significantly more resistant to ampicillin and

sulfonamide than copper sensitive isolates, which strengthened the argument that the traits

are co-selected. Elevated frequencies of microbial resistance to various antibiotics have

been observed in metal contaminated freshwater streams (McArthur and Tuckfield, 2000)

and metal contaminated ash settling basins (Stepanauskas et al., 2005).

Nakahara et al. (1977) have suggested that the combined expressions of antibiotic

resistance and metal tolerance may not be a fortuitous phenomenon but rather is caused by

selection resulting from metals present in an environment. Jon et al. (1984) reported that

there was a positive association of tolerance to Cu, Pb, and Zn with multiple antibiotic

resistances of bacterial isolates from drinking water sources, which is further supported by

the results of the present work.


220

5.8.4 Identification and characterization of heavy metal tolerant bacterial

strains

5.8.4.1 Morphological and Biochemical characteristics

The morphological and biochemical tests were carried out for the identification of

seven heavy metal tolerant bacterial isolates. These tests were performed following the

Bergey’s Manual of Determinative Bacteriology: 9th edition (Holt, et al., 1994) and 8th

edition (Buchanan and Gibbons, 1974. Based on the analysis the isolates were identified as

Bacillus cereus, Enterobacter aerogens, Pseudomonas putida, Bacillus licheniformis,

Klebsiella pneumonia , Alcaligens faecalis, and Citrobacter kosari (Table 5.4).

5.8.4.2 Molecular characterization of heavy metal tolerant bacterial strains

The Genomic DNA was isolated from the pure culture pellets of isolates K1 and

K4 and 16S rDNA fragment was amplified by PCR from the above isolated genomic DNA

using16S rDNA universal primers. The PCR amplicon was purified by column

purification in order to remove contaminants (Figs. 5.2a & b). The sequencing of purified

gene segment was done using ABI3730xl Genetic Analyzer (Applied Biosystems, USA)

(Figs. 5.3 a & b). The comparative analysis of the sequences of isolates with already

available database using BLAST (Basic Local Alignment Search Tool) showed that the

strains were close to the members of genus Bacillus. The highest sequence similarity of

the sediment bacteria are as follows: K1, Bacillus cereus (showed 99% similarity with

Bacillus cereus Accession No: 346665.1) and isolate K4 (96% similarity with Bacillus

licheniformis strain CICC 10180, accession No: AY859478.1).The 16S rDNA sequences

were submitted in the National Center for Biotechnology Information (NCBI) data bank

under the accession numbers JQ863364 (Bacillus cereus) and JQ863365 (Bacillus

licheniformis )


221

Table 5.4 Morphological and biochemical characteristics of metal tolerant bacterial isolates from the sediments of Vattakayal backwaters.

Organisms Bacillus cereus (K1) Enterobacter aerogens (K2)

Pseudomonas Putida (K3)

Bacillus licheniformis(K4)

Klebsiella pneumonia(K5)

Alcaligens faecalis (K6)

Citrobacter kosari (K7)

Morphology Abundant,opaque,white,waxy growth

Abundant, thick, glistening growth

Thin viscous growth

Irregular with lobate margine White transluscent

Thin viscous growth

Thin abundant growth

Gram staining +R -B -B +R -B -B -B Motility at 37 o C + + + + - - + Oxidase - - + - - + - Catalase + + + + + + + Indole - - - - - - + Methyl red - - - - - - + Voges Proakauer +/- + - + - - - Citrate - + + + + + + Urease - - - - + - + Nitrate reduction + + + + + - - D-Glucose gas production

A + - A + - +

D-Glucose acid production

- + - + + - +

Lactose - + - + + - + Mannitol - + - A + - + Sucrose A + - + + - + Phenylalanine Deaminase

+ - - - - - -

H2S from LIA - - - - - - - Litmus milk reaction

Peptonization Peptonization Peptonization Alkaline

Gelatine + - - + - - - Starch + - - + - - - Lipid +/- - + + - - -

Bioaccum

ulation of heavy metals using bacterial isolates

221


222

Gel Image

Lane 1: 1 Kb •

Lane 2: 16S rDNA amplicon

Fig. 5.2a Gel image for K1 Fig. 5.2b Gel image for K1 Fig. 5.3a Consensus Sequence Data Culture K1: (877bp) TGCAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGGCCTGGGGAGTACGGGCCGCAAGGCTGAAACTCAAAGGAATTTGACGGGGG Fig. 5.3b Consensus Sequence Data Culture K4: (458 bp) TGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGGCTTTTACCTACCACTTACAAATGGACCCCCGGCGCATTACCTATTTGGGGAGGTAACGGCTCACCAAGGCAACAATGCTTACCCAACCTGAAAGGTGGATCGGCCACCCTGGAACTGAAACACGGCCCAAACTCCTACGGAAGGCACCATTAGGAAATCTTCCGCATGGAACAAAAGTCTGACGAACCACCCCCCCGGGATTGATGAAGGTTTTCGAATCGAAAAACTCTGTTGTTAGGAAAAAACAATTACCGTTCAATTAGGGGGGTACCTTGACGGTACTTAACCAAAAAGCAACGG


223

5. 9 Phylogeny

Phylogenetic tree was constructed by using MEGA4 software and the evolutionary

history was inferred by neighbour-joining method (NJ) (Fig. 5.4) by using published

sequence from Gene Bank. The phylogenetic relationship was illustrated based on the

alignments of 16S rDNA from 8 different Bacillus species namely, B. cereus, B.

thuringiensis, B. subtilis, B. anthracis, B. licheniformis, B. olivae, B. aerius, and B.

sonorensis and the strains K1 and K4. The bootstrap consensus was inferred from 1000

replicates. It was found that the strain K1 classified in the branch of Bacillus cereus and

K4 in the branch of Bacillus licheniformis where the bootstrap value is 100.

Fig. 5.4 Phylogenetic tree

5.10 Bioaccumulation

5.10.1 Bioaccumulation of chromium (VI) by Bacillus cereus JQ863364 and

Bacillus licheniformis JQ863365

In the present investigation Bacillus cereus JQ863364 and Bacillus licheniformis

JQ863365 were used for studying the potential of chromium (VI) removal property with

the initial concentration 100mgL-1. The experiment was conducted at pH 6, 7 and 8 and at

the temperature of 25oC, 30oC and 35oC. Biosorbent free solutions were used as control.

All experiments were conducted in triplicates and the mean values for the percentage of

metal removal, optical density for bacterial biomass corresponding to each time interval

were represented in the Tables 5.5, 5.6 and 5.7 and Figs. 5.5, 5.6 and 5.7 respectively.


224

Bioaccumulation studies revealed that with increase in time, the biomass of the

selected bacterial isolates increased. Correspondingly, with increase in biomass, the heavy

metal bioaccumulation was also increased. The percentage removal of chromium (VI) by

Bacillus cereus at 25oC was minimum (3.1%) at pH 6 and maximum (13.5%) at pH 8

(Table 5.5). Table 5.6 represents the percentage removal of chromium (VI) by Bacillus

cereus at 30oC and pH 6, 7 and 8. The maximum chromium (VI) removal (57.33%) was

observed at pH 8 and minimum (48.93%) at pH 7. At pH 6 and 8 the chromium (VI)

removal was above 50% compared to pH 7. Table 5.7 represents the mean value of cell

growth and percentage removal of chromium (VI) by Bacillus cereus at pH 6, 7, and 8 and

temperature 35oC. The percentage removal of chromium (VI) was very low at 25oC and

35oC compared to 30oC (Table 5.6, Fig.5.6). The analysis of variance of bioaccumulation

of chromium between temperature and between time were significant (P<0.01). The

Duncan test (Table 5.8) showed significant variation in bioaccumulation except at 25oC

and 35oC.

The data on percentage of chromium removed by Bacillus licheniformis at pH 6, 7,

and 8 at temperatures 25oC, 30oC and 35oC are represented in Tables 5.9, 5.10, 5.11 and

Figs. 5.8 and 5.9, 5.10. At temperature 25oC the percentage of removal of chromium (VI)

was maximum for pH 8 (35.33%) and minimum at pH 6 (6.83%). Similarly at temperature

30oC the percentage removal of chromium (VI) was high at pH 8 (57.73%) and

temperature 30oC and at pH 6 and 7 it was below 50% (Table 5.10). At 35oC chromium

removal was maximum (27%) at pH 8. Analysis of variance between temperature, pH and

time showed that there was significant difference at 1% level. Duncan test (Table 5.12) for

temperature showed no significant difference between temperatures 25oC and 35oC.

Duncan test (Table 5.12 & 5.13) for temperature and pH, indicates notable variation

between temperature and between pH. From the analysis it can be concluded that at pH 8

and temperature 30oC the percentage removal of chromium was maximum(Table 5.10).

Chromium exists in several oxidative states but the most stable forms are

chromium (III) and chromium (VI) and they have dissimilar chemical characteristics and

biological effect (Cervantes et al., 2001). The hexavalent chromium (Cr VI) is generally

recognized as mutagen and carcinogen, mainly respiratory tract and pancreatic cancer

(Ganguli and Tripathi, 2002). It is evident from the above observation that the microbe

Bacillus cereus has the ability to accumulate maximum chromium (VI) at 30oC when

compared with results at 25oC and 35oC. It was also found that the percentage of removal


225

was increased with increase in time, as observed by Kaushik et at. (2008). The maximum

uptake of chromium (57.33%) was observed at pH 8 and it is in agreement with the

observation made by Kocberber and Donmez (2007). Similarly Yang et al. (2009)

reported that Intrasporangium sp. Q5-1 grew in pH 6 to 10 and the maximum Cr (VI)

reduction observed at pH 8 and the removal was 98 % .

The heavy metal tolerance pattern of bacterial strain isolated from the metal

contaminated sediments of Vattakayal backwaters (station 6) showed a maximum

tolerance of 200 µg/ml for chromium. The resistance of the present strains may be due to

the presence of high concentration of heavy metals in the collection site, frequent presence

of plasmids or due to mutation (Ohta et al., 1971 and McLean and Beveridge., 2001).

Earlier studies revealed that Bacillus species can grow in medium containing high

concentration of heavy metals and have high ability to remove heavy metals (Kim, 2005).

Sultan and Hasnain (2007) isolated several groups of chromium resistant strains from

tannery effluent. Many researchers reported the metal resistance and metal removal

capabilities of Bacillus sp. (Mythili and Karthikeyan, 2011; Ashok Kumar, 2010 and

Rajkumar et al., 2012). The walls of Gram positive bacteria are efficient metal chelators

and have thicker peptidoglycan layer and Bacillus cereus has similar cell wall properties.

In B. subtilis, the carboxylic group of glutamic acid of peptidoglycan was the major site of

metal deposition (Gadd, 1990).

In the environment chromium (III) and chromium (VI) are the most prevalent

species and the environmental impact of chromium is at higher level, and its

environmental behavior pattern lies in its oxidation states (Losi et al., 1994). The present

study was conducted for analyzing the heavy metal resistance pattern of B. licheniformis

and its possible strategies for the bioaccumulation at varying pH and temperatures at

regular time intervals. Kavitha et al. (2011) reported that B. licheniformis exhibits growth

and tolerance (225mg/L) in the presence of higher concentration of chromium (VI). B.

licheniformis isolated from the sediments of Vattakayal backwaters contaminated with

heavy metals also exhibited high tolerance to chromium (VI) (250µg/ml) compared with

the Minimum Inhibitory Concentration (150mg/L) (Srinath et al., 2002). Similar trend was

shown by chromium resistant bacteria isolated from tannery effluent 250 µg/mL of Cr

(Basu et al., 1997).

The microbes survived in polluted soil are prone to show higher tolerance to heavy

metals as compared to the strains from non-contaminated sites (Zouboulis et al., 2004).


226

The bacterial isolates with high chromium resistantce can be used for biosorption of

chromium VI contaminated environment (Polisak et al., 2009). At pH 8 and temperature

30oC the percentage removal of chromium by B. licheniformis isolated from the heavy

metal contaminated sediments of Vattakayal was 57.73%. The pH was a significant factor

to determine the adsorption of metal ion by bacterial strains. This result is in tune with the

adsorption of chromium by Staphylococcus sp. where the optimum pH was 8 (Mythili and

Karthikeyan, 2011). According to Udandi Boominadhan et al. (2009) the protease activity

of B. licheniformis was high at pH 8. B. licheniformis showed 31.31% chromium removal

within 24 hours and is comparable to Bacillus fumus RH109 (38.85%) (Rani et al., 2007).

The higher uptake of metals by B. licheniformis may be due to the presence of teichoic

and teichuronic acids, which are acting as important binding sites in B. licheniformis

(Gadd, 1990). The present study shows that B. licheniformis is an effective agent for the

removal of hexavalent chromium from aqueous solution and the results may be helpful for

the development and design of indigenous bioreactors for removal of chromium VI from

the industrial wastewater.


227

Table 5.5 Bioaccumulation of Cr (VI) by Bacillus cereus in response to various pH and

time intervals at temperature 25oC and concentration of chromium at 100 mgL-

1.

Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595 Removal % Cr mgL

-1 OD595 Removal % Cr mgL

-1 OD595 Removal %

0 100 0.019 0.0 100 0.02 0 100 0.023 0 24 98.7 0.95 1.3 98.8 1.063 1.17 93.4 1.103 6.57 48 97.8 1.124 2.23 96.6 1.193 3.43 90.2 1.437 9.77 72 97.3 1.131 2.7 95.2 1.26 4.83 87.4 1.49 12.6 96 96.9 1.142 3.1 94.8 1.287 5.2 86.5 1.467 13.5

Exp. Experimental

Table 5.6 Bioaccumulation of Cr (VI) by Bacillus cereus in response to various pHand

time intervals at temperature 30oC and concentration of chromium at 100 mgL-1.

Time

hrs.

pH 6 pH 7 pH 8


-1 OD595 Removal % Cr mgL-1 OD595 Removal %

0 100 0 0 100 0.001 0 100 0.037 0 24 69.8 0.83 30.2 72.3 0.85 27.67 80.8 0.907 19.23 48 61.5 1.051 38.47 62.6 1.06 37.37 65.1 1.373 34.93 72 51.6 1.09 48.37 56.5 1.097 42.47 50.8 1.54 49.17 96 43.4 1.12 56.63 51.1 1.177 48.93 42.7 1.617 57.33

Table 5.7 Bioaccumulation of Cr (VI) by Bacillus cereus in response to various pH and

time intervals at temperature 35oC and concentration of chromium at 100 mgL-

1.

Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595

Removal % Cr mgL

-1 OD595 Removal %

Cr mgL-1 OD595

Removal %

0 100 0.01 0 100 0.005 0 100 0 0 24 88.9 0.923 11.1 100 0.853 0 99.3 1.025 0.73 48 86.3 1.01 13.67 92.2 1.037 7.77 94.8 1.27 5.2 72 85.2 1.023 14.87 90.8 1.037 9.2 93 1.42 7 96 83 1.027 17.03 90.3 1.037 9.73 91.7 1.52 8.33


228

Fig. 5.5 Cr (VI) removal by B. cereus at Fig. 5.6 Cr (VI) removal by B. cereus at

various pH and at temperature 25oC . various pH and at temperature 30oC.

0.0

5.0

10.0

15.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs

Temp 25 oC pH 6 Temp 25 oC pH 7

Temp 25 oC pH 8

0.0

20.0

40.0

60.0

80.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 30 oC pH 8 Fig. 5.7 Cr (VI) removal by B. cereus in at various pH and at temperature 35oC . ANOVA

0.0

5.0

10.0

15.0

20.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 35 oC pH 8

Table 5.8 Duncan test of Cr (VI) removal by Bacillus cereus at various temperatures

Subset

Temperature 1 2

25 OC 4.427

30 OC 32.718

35 OC 6.976

Temperature p< 0.01 Significant

pH P> 0.05 Not significant

Time P< 0.01 Significant


229

Table 5.9 Bioaccumulation of Cr (VI) by Bacillus licheniformis in response to various pHand time intervals at temperature 25oC and concentration of chromium at 100 mgL-1.

Time

hrs.

pH 6 pH 7 pH 8



0 100 0.011 0 100 0.03 0 100 0.011 0 24 95.4 0.22 4.57 93.8 0.477 6.23 85.1 0.987 14.93 48 94.1 0.337 5.87 92.8 0.64 7.23 71.7 1.263 28.33 72 93.8 0.433 6.17 92.3 0.803 7.7 69.2 1.311 30.77 96 93.4 0.51 6.83 91.8 0.91 8.17 64.7 1.550 35.33

Table 5.10 Bioaccumulation of Cr (VI) by Bacillus licheniformis in response to various pH

and time intervals at temperature 30oC and concentration of chromium at 100 mgL-1.

Time

hrs.

pH 6 pH 7 pH 8



0 100 0.016 0 100 0.021 0 100 0.016 0 24 78.3 0.133 21.6 71.9 0.427 28.13 68.9 1.231 31.13 48 73.1 0.237 26.87 64.6 0.852 35.37 58.7 1.57 41.33 72 69.1 0.31 30.87 57.4 1.013 42.63 51.5 1.686 48.53 96 63.3 0.353 36.67 53.5 1.127 46.53 42.3 1.704 57.73

Table 5.11 Bioaccumulation of Cr (VI) by Bacillus licheniformis in response to various pH and


Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595

Removal % Cr mgL

-1 OD595 Removal %

Cr mgL-1 OD595

Removal %

0 100 0.011 0 100 0.013 0 100 0.013 0 24 96.5 0.043 3.5 96.4 0.107 3.63 93.5 0.31 6.5 48 94.1 0.06 5.93 91.6 0.15 8.37 84.2 0.437 15.83 72 92.8 0.067 7.23 86.1 0.167 13.87 76.2 0.583 23.77 96 92.7 0.063 7.27 82.7 0.173 17.3 73 0.63 27


230

Fig. 5.8 Cr (VI) removal by B. licheniformis Fig. 5.9 Cr (VI) removal by B. licheniformis at various pH and at temperature 25oC . at various pH and at temperature 30oC .

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 25 oC pH 8

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 30 oC pH 8

Fig. 5.10 Cr (VI) removal by B. licheniformis at various pH and at temperature 35oC . ANOVA

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 35 oC pH 8

Table 5.12 Duncan test of Cr (VI) removal by Bacillus licheniformis at various

temperatures

Subset

Temperature 1 2 25 OC 10.809 30 OC 29.827 35 OC 9.347

Table 5.13 Duncan test of Cr (VI) removal by Bacillus licheniformis at various pH

Subset

pH 1 2 3 6 10.891 7 15.011 8 24.08


pH P< 0.01 Significant



231

5.10.2 Bioaccumulation of lead (II) by Bacillus cereus JQ863364 and Bacillus

licheniformis JQ863365

Effect of temperature and pH on the removal of lead by Bacillus cereus are

presented in Table 5.14, 5.15, 5.16 and Figs. 5.11 and 5.12, 5.13. The percentage removal

of lead was maximum (88.6%) at pH 6 and temperature 25oC. At pH 7 and 8 also the

percentage of lead removal was found above 50%. At temperature 30oC and 35oC and pH

6 the maximum removal of lead was 68.27% and 76.83% respectively (Tables 5.15 and

5.16). ANOVA showed that the variation between temperature, pH and time intervals

were significant at 1% level. Duncan test for temperature and pH showed two subsets in

the study signifying notable variations. But no significant variation noticed between

temperature 25oC and 35oC and pH 7 and 8 (Tables 5.17 and 5.18). The study thus

revealed that the percentage of lead removal by Bacillus cereus was maximum (88.6 %) at

pH 6 and temperature 25oC (Table 5.14 and Fig. 5.11).

Bioaccumulation of lead by Bacillus licheniformis at pH 6,7, and 8 and

temperature 25oC, 30oC and 35oC in a medium amended with 100 mgL-1 lead at various

time intravels 0, 24, 48, 72 and 96 hours are presented in Tables 5.19, 5.20, 5.21 and Figs.

5.14, and 5.15, 5.16. At temperature 25oC the percentage of removal was maximum at pH

6 (71.17%) and minimum (27.13%) at pH 8 (Table 5.19). Table 5.20 represents the

percentage of lead removal at 30oC with a maximum of 65.53% at pH 7 and minimum of

35.87% at pH 8 for 96 hours. At temperature 35oC the concentration of lead reduced to

75.23% for 96 hours at pH 6 (Table 5.21). In all cases the percentage of reduction in the

concentration of lead in the experimental medium increases with the increase in time.

Barring the pH 8 at temperature 25oC and 30oC, B. licheniformis exhibited above 50%

removal of lead. The analysis of variance of bioaccumulation of lead between temperature,

pH and time intervals were found significant at 1% level (P<0.01). Duncan test also

showed significant variations (Table 5.22 & Table 5.23) but there was no significant

variation between pH 6 and 7. The result clearly indicated that in Bacillus licheniformis,

the percentage of accumulation was maximum at pH 6 and temperature 35oC.

The microorganisms have the ability to accumulate metallic elements and this has

been studied at first from toxicological point of view (Volesky, 1990a, b, c). In the present

study the Bacillus cereus isolated from the heavy metal contaminated sediments ( Station

6) of Vattakayal backwaters exhibited a high percentage of metal uptake, agreeing with


232

the report that the strains isolated from polluted soil showed the capacity of biosorption

(Ozdemir, et al., 2004 and YU Xia, et al.,2003). The metal binding property may be due

to the phosphoryl groups and the carboxyl group of peptide chains which provide

negatively charged sites in the Gram positive bacterial cell wall (Urrutia et al., 1997 and

Moat et al., 2002). Ray et al. (2005) reported that the removal of Pb (II) ions with growing

cells of Bacillus Cereus M116 was maximum (85%) when initial lead concentration was 50

mg/L at pH 6 and temperature 30oC. Similarly in the case of Bacillus circulans only 78 %

lead uptake from 500mg/L-1 in 72 hours and also it increases with increase in pH upto 6

(Khanafari, et al., 2008). Parungao (2007) reported that Stenotrophomonas maltophilia

was able to remove only 42.75 % of lead from the primary solution and immobilized cells

of Bcillus sp. adsorbed 84.27% lead from the industrial wastewater (Rani, 2010). It may be

noted that in the present study the metal accumulation by Bacillus cereus was found

higher when compared to the results of other similar studies. Hence, it is found to be a

potential agent for the removal of lead from the wastewater in the industrial area.

Heavy metals are highly toxic to all biological systems from microbs to plants and

animals (Patel et al., 2007). They exerts a selective pressure on microbial population

leading to the emergence of resistant strains (McGrath et al., 2001). These microorganisms

can play a key role in the bioremediation of heavy metal contaminated soil and

wastewater. In the present study B. licheniformis accumulates a maximum of 75.23% lead

from 100mg/L-1 aqueous solution at pH 6 and temperature 35oC. Similar study had been

conducted by Issazadeh (2011) where there was 1.1 mol/g biomass for bioaccumulation of

lead by B. licheniformis. Gram-positive bacteria, such as B.licheniformis, B. subtilis,

Brevibacterium helovolum, and Rhodococcus elythropolis, exhibited a particularly high

capacity for accumulating samarium (Sm) and the accumulation increased with increase

in pH (Tsuruta, 2007). El-Hendawy, (2009) reported that the bioaccumulation of heavy

metals by Vibrio alginolyticus isolated from wastes of Iron and Steel factory could reduce

40% Pb2+. The high accumulation ability in B.licheniformis may be due to the presence of

teichoic and teichuronic acids which are the principal sites of metal binding in B.

licheniformis walls (Beveridg, 1982). From the above results it can be concluded that the

Gram positive bacteria B. licheniformis, is a potential agent for lead removal.


233

Table 5.14 Bioaccumulation of Pb(II) by Bacillus cereus in response to various pH and time intervals at temperature 25oC and concentration of chromium at 100 mgL-1

.

Time

hrs.

pH 6 pH 7 pH 8



0 100 0.033 0 100 0.023 0 100 0.023 0 24 76.1 1.087 23.9 52.1 0.867 47.93 62.9 0.82 37.13 48 19.5 1.8 80.5 60.1 1.633 39.93 44.8 1.473 55.17 72 14.2 1.99 85.83 62.1 1.907 37.77 43.9 1.71 56.1 96 11.4 2.13 88.6 44.9 1.953 55.13 40.4 1.747 59.63

Table 5.15 Bioaccumulation of Pb(II) by Bacillus cereus in response to various pH and

time intervals at temperature 30oC and concentration of chromium at 100 mgL-1

.

Time

hrs.

pH 6 pH 7 pH 8



0 100 0.04 0 100 0.021 0 100 0.017 0 24 91.9 1.193 7.93 91.4 1.197 8.63 81.6 1.227 18.43 48 62.5 1.707 37.63 77.2 1.443 22.83 66.9 1.49 33.13 72 35.6 1.743 64.43 63.8 1.477 36.2 55.2 1.507 44.83 96 31.7 1.78 68.27 55.5 1.493 44.53 50.1 1.533 49.87

Table 5.16 Bioaccumulation of Pb (II)by Bacillus cereus in response to various pH and time intervals at temperature 35oC and concentration of chromium at 100 mgL-

1.

Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595

Removal % Cr mgL

-1 OD595 Removal %

Cr mgL-1 OD595

Removal %

0 100 0.053 0 100 0.018 0 100 0.017 0 24 62.5 1.607 37.53 70.7 1.61 29.27 82.4 1.507 17.63 48 33.3 1.93 66.73 46.2 1.857 53.83 54.2 1.837 45.77 72 26.1 2.027 73.9 42. 1.963 57.23 48.1 1.927 51.87 96 23.2 2.093 76.83 40.6 2.033 59.37 44.8 1.937 55.17


234

Fig. 5.11 Pb (II) removal by B. cereus at Fig.5.12 Pb (II) removal by B. cereus at various pH and at temperature 25oC. various pH and at temperature 30oC

0.0

20.0

40.0

60.0

80.0

100.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 25 oC pH 8

Fig 5.13 Pb (II) removal by B. cereus at various pH and at temperature 30oC. ANOVA

Table 5.17 Duncan test of Pb removal by Bacillus cereus at various temperatures

Subset

Temperature 1 2

25 OC 44.509

30 OC 29.116

35 OC 41.676

Table 5.18 Duncan test of Pb removal by Bacillus cereus at various pH

Subset

pH 1 2

6 47.473

7 32.844

8 34.982





235

Table 5.19 Bioaccumulation of Pb(II) by Bacillus licheniformis in response to various pH and


Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595

Removal % Cr mgL

-1 OD595 Removal %

Cr mgL-1 OD595

Removal %

Exp. Exp. Exp.

0 100 0.027 0 100 0.02 0 100 0.055 0 24 71.7 1.147 28.33 81.2 0.943 18.77 98.9 0.937 1.13 48 50.8 1.723 49.23 60.9 1.31 39.07 95.4 1.223 4.57 72 36.7 1.78 63.33 47.8 1.453 52.23 84.4 1.273 15.57 96 28.8 1.803 71.17 44.9 1.547 55.13 72.9 1.297 27.13

Table 5.20 Bioaccumulation of Pb(II) by Bacillus licheniformis in response to various pH and


Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595

Removal % Cr mgL

-1 OD595

Removal % Cr mgL

-1 OD595

Removal %

Exp. Exp. Exp.

0 100 0.097 0 100 0.127 0 100 0.147 0 24 63.7 0.913 37.27 76.5 1.01 23.53 84.1 1.353 15.87 48 53.8 1.21 46.23 48.5 1.233 51.53 73.7 1.673 26.33 72 49.7 1.427 50.33 43.2 1.407 56.83 69.2 1.76 30.77 96 49.1 1.457 50.87 34.5 1.447 65.53 64.1 1.763 35.87

Table 5.21 Bioaccumulation of Pb (II) by Bacillus licheniformis in response to various pH and


Time

hrs.

pH 6 pH 7 pH 8

Cr mgL-1 OD595

Removal % Cr mgL

-1 OD595 Removal %

Cr mgL-1 OD595

Removal %

Exp. Exp. Exp.

0 100 0.027 0 100 0.04 0 100 0.026 0 24 75.1 1.323 24.87 71.5 1.033 28.47 65.3 1.317 34.73 48 51.1 1.71 48.93 46.2 1.363 53.83 47.6 1.75 52.37 72 34.1 1.927 65.93 42 1.413 57.97 43 2.033 57.03 96 24.8 1.973 75.23 41 1.47 58.97 41.5 2.073 58.4


236

Fig. 5.14 Pb (II) removal by B. licheniformis Fig.5.15 Pb (II) removal by B. licheniformis at various pH and at temperature 25oC . at various pH and at temperature 30oC .

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 25 oC pH 8

0.0

20.0

40.0

60.0

80.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 30 oC pH 8

Fig.5.16 Pb (II) removal by B. licheniformis at various pH and at temperature 35oC . ANOVA

0.0

20.0

40.0

60.0

80.0

0 24 48 72 96

% o

f R

em

ov

al

Time hrs


Temp 35 oC pH 8

Table 5.22 Duncan test of Pb removal by Bacillus licheniformis at various temperatures

Subset

Temperature 1 2 3

25 OC 32.664

30 OC 28.378

35 OC 41.116

Table 5.23 Duncan test of Pb removal by Bacillus licheniformis at various pH

Subset

pH 1 2

6 40.716

7 37.458

8 23.984





237

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