Reporting Heavy Metal Resistance Bacterial Strains from Industrially Polluted Sites of Northern India Using Fatty Acid Methyl Ester (FAME) Analysis and Plasma-Atomic Emission Spectroscopy (ICP-AES) Manoj Kumar*, Navreet Kaur, Kamini Gautam, Ravi Kant Pathak, and Yogender Pal Khasa1 and Lovi Raj Gupta School of Biosciences; Lovely Professional University; Phagwara; Punjab, India * Dr. Manoj Kumar (corresponding author), Department of Biotechnology, Lovely Professional University, Phagwara, Punjab. email:
[email protected] /
[email protected]. Phone No.+919855030374. Mailing address :- Dr. Manoj Kumar (14302),Block No: - 28, Room No:- 202, Cabin No:02,Lovely Professional University, Jalandhar-Delhi G.T. Road (NH-1),Phagwara, Punjab (INDIA) -144402 Navreet Kaur, Department of Biotechnology, Lovely Professional University, Phagwara, Punjab. email:
[email protected] Kamini Gautam, Department of Biotechnology, Lovely Professional University, Phagwara, Punjab email:
[email protected]/
[email protected] .Phone no. +918968782993 Ravi kant Pathak, Department of Biotechnology, Lovely Professional University, Phagwara, Punjab. 1
email:
[email protected]. Phone No.+919501424486 Prof. (Dr. Lovi Raj Gupta), Department of Biotechnology, Lovely Professional University, Phagwara, Punjab. e.mail:
[email protected] PhoneNo. +911824506222
1
Dr. Yoginder Pal Khasa, Assistant Professor, Department of Microbiology, University
of Delhi (South Campus), New Delhi. email:
[email protected]
2
Abstract Microcosm experiments were performed using native sludge, sewage dumps and effluents from different contaminated sites of industry. Using Plasma-Atomic Emission Spectroscopy (ICP-AES) and serial inhibitory test, the microbial composition and activity were characterized in soil contaminated with arsenic (ranges from 1.15-4.62 mg/kg), nickel (ranges from 2.96-38.3mg/kg), cadmium (ranges from 0-18.72 mg/kg) and chromium (ranges from 7.715-33.28 mg/kg) which were sampled as “Heavy metal resistant bacterias” mentioned as HMR1, HMR2, HMR3 and HMR4 specifically. The growth inhibitory assay results for each of bacterial isolates from the tested heavy metals were performed. The maximum tolerance for HMR1 against arsenic was 500 µg/ml, HMR2 against cadmium was 700 µg/ml,
HMR3 against chromium was 250
µg/ml, of HMR4 against nickel was 175 µg/ml. Pure culture was raised from different samples (HMR1 to HMR4) and subjected for Fatty Acid Methyl Esters analysis to confirm them as Bacillus, Stenotrophomonas.
Key-words: Heavy metal resistant (HMR) bacteria, Maximum tolerance, Fatty Acid Methyl Esters (FAME), Plasma-Atomic Emission Spectroscopy (ICP-AES).
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Introduction: Conventional processes used for removal of heavy metals from industrial effluents include techniques like chemical precipitation, filtration, electrochemical techniques etc. Due to higher cost, all these processes stimulate the use of alternative biotechnological techniques which are affordable and less aggressive to the environment. In recent past extensive studies on environmental concern were reported wherein distribution of heavy metals and their toxicity to human physiology was noticed globally (Bailey et al., 1999, Kobya et al., 2005). Certain metals were found lethal to humans even at nano level amount which hamper the entire cell transport phenomenon by interfering with the cell membrane. Researchers reported the list of metals as cadmium, chromium, arsenic and mercury. (Bailey et al., 1999; Manahan, S.E 2004). Heavy metals may also stimulate the formation of free radicals and reactive oxygen species, which may lead cells into oxidative stress (Dietz et al., 1999). They are nonbiodegradable and tend to accumulate in the tissues of living organisms, a process called bio concentration (Bailey et al., 1999; Baird and Cann, 2005; Kobya et al., 2005). Heavy metal accumulation in soils is also of great concern in agricultural production due to the adverse effects on food quality (safety and marketability), crop growth (due to phytotoxicity) and environmental health (C. Augusto Costa, F. Pereira Duta.,2001). Heavy metal bioaccumulation in the food chain can especially be highly dangerous to human health. Heavy metals intake by human populations through the food chain has been reported in many countries with this problem receiving increasing attention from the public as well as governmental agencies, particularly in developing countries (C. Augusto Costa, F. Pereira Duta., 2001). Industrialization in northern India is accelerating 4
the deposition of heavy metals in soil and water irrespective of given regulatory parameters. This highlights the need of minimizing the effects of heavy metals through the use of suitable technology based methods. Now-a-days, microbial bioremediation of organic/inorganic pollutants is a promising method of environmental cleanup. This study highlights the level of soil damage through the evidences of certain heavy metals to be eradicated by specific growth of bacterial population.
Material and Methods: Sampling and Isolation of bacterial strains Sludge and Grit samples were collected from sewage treatment plant. Effluent soil from automobile industry, battery industry & agricultural soil from various regions of northern India were also collected.
Fraction Distributions of Metals in Polluted Representative Samples: The qualitative and quantitative analyses were carried out keeping all the physicochemical properties in the account. pH of the samples were found to be variable depending upon the concentration levels. Moisture content had been calculated using the following formula: Wet weight of sample (g) – Dry weight of sample (g) % Moisture content = ----------------------------------------------------------------------------x 100 Dry weight of sample (g)
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The screening of metals and their concentration was determined by Inductively Coupled Plasma-Atomic
Emission
Spectroscopy
(ICP-AES)
with
Thermo
electron
spectrophotometer using the interference-free but most sensitive wavelengths. 1 gram of each air dried sample was weighed accurately in round bottomed flasks and 2 ml of 3:1 mixture of nitric acid and perchloric acid was added, followed by heating and refluxing at 95C until yellow and white fumes have been observed. The suspension was left at room temperature to cool, and then another 8 ml of 3:1 mixture was added and refluxed. The digestion process was continued until a clear solution was observed; thereafter entire process was rested with volatilization until residue in the flasks became clear and white. Whattman filter paper no. 1 was used to remove the residual particulates and then final volume was adjusted to 50 ml by adding distilled water. Stock solutions of As, Cd, Cr and Ni salts having concentration 10mg/ml were prepared in distilled water. All the solutions were sterilized by membrane filters of pore size 0.22 µm except for Cd which was prepared in autoclaved distilled water and stored at 4C until use.
Growth of isolates on different concentrations of heavy metals Each isolate growing in log phase on heavy metal supplemented nutrient agar media was inoculated into the test tubes containing 10 ml of nutrient broth with increasing concentrations of respective heavy metals, starting from 100 μg/ml. After 48 hours, the growth was observed for each isolates which were incubated at 37C. The growth was quantified spectrophotometrically by measuring the optical density (OD) at 600 nm.
Biochemical characterization of bacterial isolates: 6
FAME analysis: The FAME analysis was done by using Sherlock Microbial Identification System (MIS), developed and marketed by MIDI, Inc.,Newark, DE, USA which was used for identification of microorganisms isolated in pure culture on artificial media -Trypticase soy broth agar (TSBA). Sherlock uses a sample preparation procedure and gas chromatography (GC) to yield qualitatively and quantitatively reproducible fatty acid composition profiles. Fatty acids extracted from unknown microorganisms are automatically quantified and identified by the Sherlock software to determine the fatty acid composition. The fatty acid profile is then compared to a “library” (of profiles of reference strains) stored in the computer, to determine the identity of the unknown.
Identification of the metal resistant isolates by 16S rDNA sequencing Genomic DNA was isolated and purified from the biochemically and morphologically identified bacterial isolates by phenol chloroform extraction method ( Chomczynski, P. & Sacchi, N., 1987). PCR of genomic DNA from bacterial isolates was performed using the universal bacterial 16S rDNA primers, [ F
(5’-AGAGTTTGATCCTGGCTCAG-3’) and R
(5’ACGGCTACCTTGTTACGACTT-3’)]. The total reaction mixture volume was 25-μl which contains 1.5μl of DNA template (50ng/μl), primers at a concentration of 3.46 pmol/μl and 3.62 pmol/μl respectively, dNTPs at a concentration of 0.8 mM, as well as 0.5 μl of Taq polymerase (500 units/μl) and 1X buffer with MgCl2. After the initial denaturation for 5 min at 95⁰C, there was 29 cycles consisting of denaturation at 94⁰C for 1min, annealing at 55⁰C for 1 min, elongation at 72⁰C for 2 min has been performed 7
along with final extension at 72⁰C for 10 min. PCR was carried out in a 48 well microtitre plate in the make of Biometra, T 3000 thermocycler. PCR products were analyzed by 1% (w/v) agarose gel electrophoresis in 1.5X TBE buffer with ethidium bromide (0.5 μg/ml).
Sequence analysis: Sequencing of 16S rDNA amplicon was performed using the Applied Biosystems 3130xl Genetic Analyser. The sequenced products were submitted to NCBI.
Results Physicochemical Characterization: Different pH for different samples were shown in table 1.The majority of samples was found to be alkaline in nature. The maximum alkalinity was observed in the contaminated soil collected from the waste disposal region of a battery industry (pH-8.5) followed by the grit from the waste water treatment plant (pH-8.0).
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Sample
Sample
pH
1.
Sludge from STP, Bhatian
7.0
2.
Effluent soil of automobile industry, Jalandhar
7.5
3.
Effluent soil of battery industry, Jalandhar
8.5
4.
Agricultural soil, Phagwara
6.5
5.
Grit from STP, Bhatian
8.0
No.
Table 1: showing pH of the samples Moisture content The moisture content for obtained samples used is presented in table 2. Agricultural soil as well as the sludge from the sewage treatment plant (STP) showed the maximum moisture level (25%) followed by the grit (20%) which was also collected from sewage treatment plant. While the minimum moisture content was observed in the effluent soil collected from the waste disposal region of a battery industry (10%).
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Sample no.
Sample
Moisture content
1.
Sludge from STP, Bhatian
25%
2.
Effluent soil of automobile industry,
15%
Jalandhar 3.
Effluent soil of battery industry, Jalandhar
10%
4.
Agricultural soil, Phagwara
25%
5.
Grit from STP, Bhatian
20%
Table 2: showing moisture content (%) for collected samples from different sites Heavy metal content The results of the elemental analysis obtained for the samples by using ICP-AES were shown in figure 1 as bar graph representation indicating different heavy metal contents in different samples. The standard value of amount of these heavy metals set by WHO, 2005 was 0.007 mg/Kg for these industrial sites.
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Figure 1: showing amount of heavy metals plotted against various industrial regions Isolation and Purification of Bacterial Strains from Contaminated Samples: Morphologically distinct colonies were observed after 24hrs of culturing. Four heavy metal resistant bacterial isolates were raised against arsenic, cadmium, chromium and nickel. The selected isolates were designated as HMR1 to HMR4. HMR1 was resistant against As, HMR2 was resistant against Cd, HMR3 was resistant against Cr and HMR4 was resistant against Ni. Growth Inhibitory concentration was determined and had been represented in the Table 3.
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Particular heavy metal
Maximum tolerance limit to
against which the strain is
respective heavy metal
resistant
(µg/ml)
HMR1
Arsenic
500±0.5
HMR2
Cadmium
700±0.5
HMR3
Chromium
250±0.3
HMR4
Nickel
175±0.5
Strain
Table 3: showing tolerance of bacterial isolates (Strains) against conferred heavy metals on semi-solid media as well as the studies in liquid media.
FAME Analysis: Sample Profiling was the key information at fatty acid composition level. The results of FAME analysis are presented in the form of “Library Matches” which compares the fatty acid composition of the samples to the Sherlock Libraries by considering the values of Similarity index (SI). Similarity index is based on numerical value which expressed proximity to the fatty acid composition of an unknown strain compares with the mean fatty acids composition of the strains used to create the library enlisted as its match. Assumption of SI indicated that the fatty acid distributions for species of microorganisms which had normal Gaussian distribution (the classic “bell shaped curve”) and the mean of the population in the series of traits (e.g., fatty acid 12
percentages) characterized the group. Most of the population was fallen near the mean, but samples had differences in their compositions and thus may showed considerable variance from the mean. Parallel visualization of SI was done by looking at the Gaussian distribution of the population level (in this case, the fatty acid composition). In Figure 2, the perfect mean percentage for all fatty acids in a single species entry (no variance on any fatty acid) was indicated by the line at the centre. The SI for unknown strain was found on the line at 1.000. As the variance increases, the SI of the strain started to fall down from the line < 0.600. Samples with an SI of 0.500 or higher and with a separation of 0.100 between the first and second choice were considered good library comparisons. SI was indicated between 0.300 and 0.500 and got well separated from the second choice (>0.100 separation), it was a perfect match with the existing library.
S.I = 1.000
S.I = 0.600
Figure 2: showing similarity index as a function of population distribution 13
Therefore, FAME analysis confers the samples with Similarity Index (SI) of 0.500 or higher and with a separation of 0.100 between the first and second choice are considered accurate library comparisons. Name of the
Values of Similarity
Entry Name
bacteria
Index
HMR1
0.412
Bacillus cereus
HMR2
0.310
Bacillus cereus
HMR3
0.390
Stenotrophomonas maltophia
HMR4
0.543
Bacillus alcalophilus
Table 4: showing similarity index obtained by Fatty acid methyl esters analysis
Discussion Physico-chemical properties of the soil have a partial effect on the resistance of the microorganisms. The pH and moisture content of the collected samples were determined and found in the range of 6.5-8.5 and 10-20% respectively. Samples from the waste water treatment plant were found to be alkaline (pH-8.0). Effluent samples from the industries had neutral pH-7.0 while the agricultural soil was found to be slightly acidic in nature (pH-6.5). Neutral as well as slightly acidic pH values were observed for the contaminated soil samples. Several researchers have reported the soil texture and pH range for such contaminated regions stating the physic-chemical properties under the scan of microbial population (Abshkharon et al. 2009; Kermani et al. 2009) 14
A multi-element study was performed for metal-tolerant bacterial population and the associated soils collected from four contaminated areas in northern India (Punjab) using ICP-AES and ICP-MS. Similarly plants were found acclimatized and developed resistance against the multi-element (heavy metal tolerance) It was determined in the roots, stems and leaves of the three metallophytes, Blepharis aspera, Blepharis diversispina and Helichrysum candolleanum, previously known to accumulate Cu and Ni (Bonang B. M. Nkoane et al., 2007) The amount of cadmium in the samples ranges from 0-18.72 mg/kg of the sample. Absence of cadmium was noted in the effluent soil of a battery industry. Nickel was found to be minimum in the effluent sample (2.96 mg/kg). Although chromium is naturally found in soil but the levels detected in these studies are much higher than the safe limits. The amount of chromium found falls in the range 7.715-33.28 mg/kg of the sample. This study resulted in the isolation and purification of four bacterial isolates (HMR1 to HMR4) which have the high ability to resist and tolerate the respective heavy metals from the collected samples. These bacterial isolates were screened for their heavy metal resistance in both nutrient agar and nutrient broth medium containing different metal salt concentrations of each of As2+, Cd2+, Cr3+ and Ni2+ respectively. FAME analysis was found to very accurate and contemporary tool to detect the closely related bacterial species (Bacillus and Stenotrophomonas species)
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A new library was successfully created using Sherlock Library Generation Software (MIDI). In recent past FAME technology was recognized by several researchers (Peltroche-Llacsahuanga et. al., 2000) The present work suggests that the environmental impact of heavy metal toxicity should be rapidly handled by using the bioremediation processes in order to reduce the toxic levels of heavy metals. To stop pollution & to prevent metal toxicity, there is a clear need for a strategy which has the goal of elimination of priority pollutants at source. By the help of these As2+, Cd2+, Cr3+ and Ni2+ resistant bacterias, we can easily achieve our goal of squeezing out the hazardous heavy meals from the environment.
Acknowledgment This research was supported by the Lovely Professional University and The Department of Microbiology, South Campus, Delhi University, New Delhi, India. Authors are grateful to the Universities for the support.
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