List of abbreviations - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/28572/13/13... · 2018. 7. 2. · Atomic Absorption Spectrometer GBC 932 plus, Australia Double Beam UV

List of abbreviations

AAS Atomic Absorption Spectrophotometer

A Ampicillin

Am Amoxycillin

Cr Chromium

Cd Cadmium

Cu Copper

CFU Colony forming units

C Chloramphenicol

Co-tri Co-trimoxazole

DCM Dichloromethane

Do Doxycycline

DNA Deoxyribose nucleic acid

DNP Dinitrophenol

DMSO Dimethylsulphoxide

GC Gas Chromatography

G Gentamycin

g Gram

h Hour

Hg Mercury

K Kanamycin

Kg Kilogram

Km Kilometer

l Liter

M Molar

MIC Minimum Inhibitory Concentration

min Minutes

MS Mass Spectrometery

M Methicillin

mg Milligram

ml milliliter

Ni Nickel

NADP Nicotinamide adenine dinucleotide phosphate

NADH Nicotinamide adenine dinucleotide hydrogen

T Tetracycline

S Sptreptomycin

Zn Zinc

μ Micro

° Degree celsius

± plus-minus

μg Microgram

μl Microliter

μM Micromolar

% Percent

Materials used

1. Equipments/Instruments Manufacturer

Microscope CH-BI45-2, OLYMPUS, Japan

Spectrophotometer Spectronic 20D+ Thermo Spectronic, India

Atomic Absorption Spectrometer GBC 932 plus, Australia

Double Beam UV – VIS Spectrophotometer Electronic Corporation, India

Centrifuge C – 24 REMI, India

ANAMED Electronic Balance ANAMED Instruments, India

Cell Sonicator 250D, Branson Ultrasonics, USA

pH meter LI 120, ELICO, India

2. Chemicals Source

Acetic acid (glacial) Qualigens, India

Agar powder Hi Media, India

Agarose Hi Media, India

Ampicillin Ranbaxy, India

Biotin Hi Media, India

Cadmium chloride Hi Media, India

Chromic chloride Qualigens, India

Citric acid monohydrate Hi Media, India

Cupric chloride Hi Media, India

EDTA Hi Media, India

Glucose Hi Media, India

Histidine hydrochloride Hi Media, India

Hydrochloric acid Qualigens, India

Kovac’s reagent Hi Media, India

Magnesium sulphate Qualigens, India

Mercuric chloride Qualigens, India

Nickel chloride Hi Media, India

Nitric acid Qualigens, India

Perchloric acid Qualigens, India

Potassium chromate Qualigens, India

Di-potassium phosphate Hi Media, India

Mono-potassium phosphate Hi Media, India

Sodium ammonium phosphate E-Merck, Germany

Sodium chloride Qualigens, India

Sodium dodecyl sulphate Qualigens, India

Sodium hydroxide Qualigens, India

Tris HCl SRL, India

Zinc sulphate Qualigens, India

2, 4-Dinitrophenol Hi Media, India

Diphenylcarbazide Hi Media, India

Stannous chloride Hi Media, India

Arochlor 1254 Sigma, India

3. Solvents Source

Acetone SRL, India

Acetonitrile SRL, India

Chloroform SRL, India

Dichloromethane SRL, India

Dimethylsulphoxide SRL, India

n-Hexane SRL, India

Methanol SRL, India

Ethanol E. Merck, Germany

4. Reagents Composition

Crystal violet

Solution A

Crystal violet 2.0 g

Ethyl alcohol (95%) 20 ml

Solution B

Ammonium oxalate 0.8 g

Distilled water 80 ml

Safranin

Safranin O

(2.5% in 95% ethanol) 10 ml


Gram’s Iodine

Iodine 1.0 g

Potassium iodide 2.0 g


Barrit’s reagent

Solution A

α-naphthol 5.0

Absolute alcohol 95 ml

Solution B

Potassium hydroxide 40.0 g

Creatine 0.3 g


Hydrogen peroxide reagent

Hydrogen peroxide 3%

Diphenylamine indicator

Diphenylamine 0.25 g

Concentrated H2SO4 50 ml


Ferrous ammonium sulfate (0.4N)

Ferrous ammonium sulfate 156.8 g



Kovac’s reagent

p-Dimethylaminobenzaldehyde 5.0 g

Amyl alcohol 75 ml

Concentrated HCl 25 ml

Oxidase reagent

Tetramethyl-p-phenylenediamine

dihidrochloride 0.25 g


Methyl red solution

Methyl red 0.1 g

Ethyl alcohol (95%) 300 ml


Nitrate test solutions

Solution A

Sulphanilic acid 8.0 g

Acetic acid (5N) 1000 ml

Solution B

α-naphthylamine 5.0 g

Acetic acid (5N) 1000 ml

Sulfuric acid reagent

Ag2SO4 5.5 g

H2SO4 1000 g

Ferrous ammonium sulfate (0.1M)

Ferrous ammonium sulfate 39.2 g



Potassium dichromate solution (0.0167M)

K2Cr2O7 4.913 g

HgSO4 33.3 g

H2SO4 167 ml


Ferroin indicator

1,10-phenanthroline monohydrate 1.485 g

FeSO4 0.695 g


Iodine solution (0.025N)


Iodine 0.32 g


Sodium thiosulfate solution (0.025N)

Na2S2O3. 7H2O 6.205 g

6N NaOH 1.5 ml


Starch solution

Starch powder 1 g


Alkaline iodide-azide solution

a) Alkaline iodide solution

NaOH 5 g



b) Azide solution

Sodium azide 1 g


Mix both a & b

Manganous sulfate solution

MnSO4 5 g


3. Solutions and buffers

Glucose solution

Glucose 40.0 g


Histidine/biotin solution

D-Biotin 30.0 mg

L-Histidine HCl 24.0 mg


Magnesium sulphate solution (pH 8.5)

Magnesium sulphate 2.4 g


Normal saline solution

Sodium chloride 0.85 g


Lysing solution (pH 12.6)

SDS 3%

Tris HCl 50 mM


E buffer (pH 7.9)

Tris acetate 40 mM

Sodium EDTA 2 mM


TAE buffer (10X pH 8.0)

Tris HCl 48.4 g

Acetic acid (glacial) 11.4 ml

EDTA (0.5M) 20 ml


TE buffer (pH 8.0)

Tris HCl 10 mM

EDTA 1 mM


Tracking dye

Glycerol 50%

Bromophenol blue 0.05%

EDTA 40 mM

5. Media and Broths Composition

Medium for Ames test

50X VB salt 20.0 g

40% glucose 50 ml

Histidine HCl (2g/400ml) 10ml

0.5mM biotin 6 ml

Ampicillin solution

(8mg/ml 0.02M NaOH) 3.15 ml

Tetracycline solution

(8mg/ml 0.02N HCl) 0.25 ml

Agar 15.0 g

Distilled water 910.0 ml

Minimal glucose agar plates

50X VB salt 20 ml

40% glucose 50 ml

Agar 15.0 g


S9 mix (AROCLOR 1254 induced)

Rat liver S9 2.0 ml

MgCl2-KCl salt 1.0 ml

1M glucose-6-phosphate 0.25 ml

0.1M NADP 2.0 ml

0.2 M phosphate buffer

(pH 7.4) 25.0 ml

Distilled water 19.75 ml

MR-VP broth (pH 6.9)

Peptone 7.0 g

Dextrose 5.0 g

Potassium phosphate 5.0 g


Nitrate broth (7.2)

Peptone 7.5 g

Beef extract 3.0 g

Potassium nitrate 5.0 g


Nutrient agar (pH 7.4)

Peptone 5.0 g

Beef extract 1.5 g

Yeast extract 1.5 g


Agar 15.0 g


Nutrient broth (pH 7.4)

Peptone 5.0 g

Beef extract 1.5 g

Yeast extract 1.5 g



Carbohydrate fermentation broth base (pH 6.8)

Ammonium dihydrogen

Phosphate 1.0

Potassium chloride 0.2

Magnesium sulphate 0.2

Phenol red (5%) 0.7 ml

Sugar 1%


Simmon citrate agar (pH6.8)

Ammonium hydrogen

Phosphate 1.0 g

Dipotassium phosphate 1.0 g


Sodium citrate 2.0 g


Bromothymol blue 0.08 g

Agar 15.0 g


Starch agar (pH 6.9)

Peptone 5.0 g

Beef extract 3.0 g

Starch 2.0 g

Agar 15.0 g


Triple sugar iron agar (pH 7.4)

Peptone 10.0 g

Tryptone 10.0 g

Yeast extract 3.0 g

Beef extract 3.0 g

Lactose 10.0 g

Saccharose 10.0 g

Agar 15.0 g

Top agar


Agar 6.0 g

Histidine/biotin solution

(0.5mM) 10 ml


Vogel bonner medium E (50X)


Citric acid monohydrate 100.0 g

Potassium phosphate

(anhydrous) 500.0 g

Sodium ammonium

Phosphate 175.0 g


Phenol (saturated) SRL, India

GGAGGCGCTGTCGGATATTGGGCGTAAGCGCGCGCAGGCGGCCTCTTAA

GTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGCCATTGGAAACTG

GGAGGCTTGAGTATAGGAGAGAAGAGTGGAATTCCACGTGTAGCGGTGA

AATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGC

CTATAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGA

TACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTT

TCCGCCCTTCAGTGCTGAAGCTAACGCATTAAGCACTCCGCCTGGGGAGT

ACGGTCGCAAGGCTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGC

GGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAACT

CTTGACATCCCCCTGACCGGTACAGAGATGTACCTTCCCCTTCGGGGGCA

GGGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG

GGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCATTTG

GTTGGGCACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGG

ATGACGTCAAATCATCATGCCCCTTATGAGTTGGGCTACACACGTGCTAC

AATGGACGGTACAAAGGGCAGCGAAGCCGCGAGGTGGAGCCAATCCCA

GAAAGCCGTTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGT

CGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCG

GGTCTTGTACAC

a) 16s rDNA sequence of Exiguobacterium sp. ZM-2

GGGAGTAGGATATGGCGTAAGCGCGCGTAGTGGTTCAGCAAGTTGGATG

TGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTACTGAGCTA

GAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTA

GATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACT

GACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG

TAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCT

TAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGC

AAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGC

ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATG

CTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAG

GTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC

CGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCAC

TCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCA

AGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGG

TACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGAT

CGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCT

AGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTAC

ACACCG

b) 16s rDNA sequence of Stenotrophomonas maltophilia ZA-6

GGATGACGTATCGGATATTGGGCGTAAGCGCGCGTAGGCGGTTTCTTAAG

TCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGG

GAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAA

ATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTC

TGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGAT

ACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTT

CCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTA

CGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCG

GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCT

TGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCCTTCGGGGGACAA

AGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGG

TTAAGTCCCGCAACGAGCGCAACCCTTAAGCTTAGTTGCCATCATTAAGT

TGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT

GACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAA

TGGACAATACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAA

AGTTGTTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGG

AATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGT

CTTGTACA

c) 16s rDNA sequence of Staphylococcus gallinarum W-61

TTTTGGACGTATCGGATCTGGGCGTAAGCGCACGCAGGCGGTCTGTCAAG

TCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGC

AGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAA

TGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACA

AAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATA

CCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTG

AGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTAC

GGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGG

TGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTT

GACATCCAGAGAACTTACCAGAGATGCTTTGGTGCCTTCGGGAACTCTGA

GACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTA

AGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCG

GGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATG

ACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAAT

GGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAA

GTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGA

ATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCC

TTGTACACACCG

d) 16s rDNA sequence of Pantoea sp. KS-2

AAATTGGGAGTAGGATCTGGGCGAAGCCACGCGGCGGTTGGATAAGTTA

ATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTAAAACTGTCCAGC

TAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG

TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGA

CTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT

GGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGCTGTGTCCTTGAGAC

GTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCC

GCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGA

GCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACA

TGTCTGGAATCCTGTAGAGATACGGGAGTGCCTTCGGGAATCAGAACAC

AGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT

CCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCAGCACGTAATGGTGGG

AACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGAC

GTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGG

CGCGTACAGAGGGCTGCAAGCTAGCGATAGTGAGCGAATCCCAAAAAGC

GCGTCGTAGTCCGGATTGGAGTCTGCACTCGACTCCATGAAGTCGGAATC

GCTAGTAATCGCAAATCAGAATGTTGCGGTGAATACGTTCCCGGGCCTTG

TACACA

e) 16s rDNA sequence of Aeromonas sp. KS-14

Genotoxic and Mutagenic Potential of Agricultural Soil Irrigatedwith Tannery Effluents at Jajmau (Kanpur), India

Mohammad Zubair Alam Æ Shamim Ahmad ÆAbdul Malik

Received: 18 September 2008 / Accepted: 1 January 2009 / Published online: 20 January 2009

� Springer Science+Business Media, LLC 2009

Abstract It is a common practice in India to irrigate

agricultural fields with wastewater originating from indus-

tries and domestic sources. At Jajmau (Kanpur), India,

tannery effluent is used for irrigation purposes. This practice

has been polluting the soil directly and groundwater and

food crops indirectly. This study is aimed at evaluating the

mutagenic impact of soil irrigated with tannery effluent. Soil

extracts were prepared using four organic solvents

(dichloromethane, methanol, acetonitrile, and acetone) and

tested with Ames Salmonella/microsome test and DNA

repair-defective E. coli k-12 mutants. Gas Chromatography-

mass spectrometric analysis of soil samples revealed the

presence of a large number of organic compounds including

bis(2-ethylhexyl)phthalate, benzene, 1,3-hexadien-5-yne,

2,4-bis(1,1-dimethyl)phenol, Docosane, 10-methylnonade-

cane, and many higher alkanes. The soil extracts exhibited

significant mutagenicity with Ames tester strains. TA98 was

found to be the most sensitive strains to all the soil extracts,

producing maximum response in terms of mutagenic index

of 14.2 (–S9) and 13.6 (?S9) in the presence of dichloro-

methane extract. Dichloromethane-extracted soil exhibited

a maximum mutagenic potential of 17.3 (–S9) and 20.0

(?S9) revertants/mg soil equivalent in TA100. Methanol,

acetonitrile, and acetone extracts were also found to be

mutagenic. A significant decline in the survival of DNA

repair-defective E. coli K-12 mutants was observed com-

pared to their isogenic wild-type counterparts when treated

with different soil extracts. PolA mutant was found to be the

most sensitive strain toward all four soil extracts.

Thousands of chemicals are released and find their way

into the environment, i.e., air, land, groundwater, and

surface water, by industrial activity, agricultural practices,

domestic activity, etc. Soil contaminants are widespread in

industrialized countries, causing direct pollution of the soil

and indirect pollution of the groundwater and food.

In most towns in India, the treated industrial and

domestic wastewater is being used for the irrigation of

agricultural land. The agronomic and economic benefits of

wastewater irrigation are obvious; however, pollutants

could be introduced and accumulated in the soil environ-

ment after long-term application, which contributes

significantly to the contamination of the soil in wastewater

receiving areas (Bansal 1998; Aleem et al. 2003). Among

the toxic compounds, particular attention should be paid to

soil mutagens and carcinogens due to their potentially

detrimental effects on animal populations and human health

(Monarca et al. 2002). Genotoxic compounds in soil may

have an effect on human health through inhalation of dust,

ingestion of plants that absorbed the compounds from the

soil, and leaching of the compounds from soil to ground-

water and surface water used for drinking (Watanabe and

Hirayama 2001).

The physical and chemical nature of soil is very complex

and standard chemical and pedological analyses are limited

in their ability to characterize the chemical composition of

genotoxicants in soil. On the other hand, genotoxicological

and mutagenicity bioassays provide a means of assessing

the genotoxicity of complex mixtures without the need for

M. Z. Alam (&) � A. MalikDepartment of Agricultural Microbiology,

Faculty of Agricultural Sciences,

Aligarh Muslim University, Aligarh 202 002, India

e-mail: [email protected]

S. Ahmad

Microbiology Division, Institute of Ophthalmology,

Faculty of Medicine, J.N. Medical College,

Aligarh Muslim University, Aligarh 202 002, India

123

Arch Environ Contam Toxicol (2009) 57:463–476

DOI 10.1007/s00244-009-9284-0

Author's personal copy

Mutagenicity and genotoxicity of tannery effluents used for irrigation atKanpur, India

Mohammad Zubair Alam a,n, Shamim Ahmad b, Abdul Malik a, Masood Ahmad c

a Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, Indiab Microbiology Division, Institute of Ophthalmology, Faculty of Medicine, JN Medical College, Aligarh Muslim University, Aligarh 202002, Indiac Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India

a r t i c l e i n f o

Article history:

Received 29 September 2009

Received in revised form

11 May 2010

Accepted 11 July 2010Available online 3 August 2010

Keywords:

Mutagenicity

Genotoxicity

Tannery effluent

Ames Salmonella test

XAD

GC–MS

Dichloromethane

Solvent

a b s t r a c t

The tannery effluents at Kanpur (India) have been in use for irrigation since last many years, polluting

soil directly while ground water and food crops indirectly. Gas chromatography–mass spectrometric

analysis of the test samples revealed the presence of organic compounds including diisooctyl phthalate,

phenyl N-methylcarbamate, dibutyl phthalate, bis 2-methoxyethyl phthalate, and higher alkanes.

Tannery effluent extracts were prepared using XAD-4/8 resins, dichloromethane, chloroform, and

hexane and tested with Ames Salmonella test and DNA repair-defective Escherichia coli K-12 mutants. In

the presence of XAD-concentrated tannery effluent, TA98 found to be the most sensitive strain in terms

of mutagenic index followed by TA97a whereas in terms of mutagenic potential TA102 was most

responsive. The extracts were also found genotoxic as determined in terms of survival of E. coli K-12

mutants, suggesting the presence of DNA damaging compounds in the tannery effluents. In the light of

results, precautious use of tannery effluents for irrigation is suggested.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

The use of industrial or municipal wastewater in agriculture isa common practice in many parts of the world (Sharma et al.,2007). The major objectives of wastewater irrigation are that itprovides a reliable source of water supply to farmers and has thebeneficial aspects of adding valuable plant nutrients and organicmatter to the soil (Liu et al., 2005b; Horswell et al., 2003).Untreated or partially treated wastewater can introduce a hugeamount of inorganic and organic contaminates into agriculturallands (Wang and Tao, 1998). Hence, continual use of wastewaterover extended periods can exert adverse impacts on quality of soiland plants grown on it (Madyiwa et al., 2002; Sinha et al., 2006).Therefore, indiscriminate use of untreated wastewater can beconsidered as one of the significant sources of environmentalpollution that may affect the human health via crops and soil(Wang and Tao, 1998; Butt et al., 2005). However, with carefulplanning and management, the positive aspects of wastewaterirrigation can be achieved (WHO, 2006).

The Indian leather industry being a major contributor to thenational economy is unfortunately also one of the major polluters.The leather processing units in India are more than 1900 out of

which 75% are in the small scale sector. The inherent nature of thetanning process is such, that large quantities of water areconsumed (Khwaja et al., 2001). Around 30 litres of liquid effluentis produced per kilogram of leather processed. Thus, a substantialamount of effluent is discharged from tanneries, which affects theaquatic life and makes the water hazardous for human consump-tion. The composition of organic pollutants in tannery wastewateris complex. Proteins, mainly collagen and their hydrolysisproducts – amino acids derived from the skin – are predominant,while others such as fats are in low concentrations. The mostimportant organics used in tanning of skin are tannins bothnatural and synthetic, fatty aldehydes and quinones. Tanneriesalso use compounds like aliphatic amines, non-ionic surfactants,oils, and pigments. Most of these pollutants are in a soluble form,but a lot of them exist in suspension and only a few are colloids(Ates et al., 1997; Cassano et al., 2001; Di Iaconi et al., 2002).

Pollutants can affect organisms at various levels of biologicalorganization, from molecular to community levels (Theodoraskiset al., 2000). The composite effects of mixtures cannot be readilyassessed by way of analytic methods. Rather, toxicity is oftenevaluated by means of tests like bacterial genotoxicity tests,which do not require a priori knowledge of toxicant identity and/or physicochemical properties. Several studies have been carriedout on industrial and domestic wastewater and have been foundgenotoxic and mutagenic in various short-term test systems(Houk, 1992). There are many assays for detecting mutagenicity

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/ecoenv

Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2010.07.009

n Corresponding author. Fax: +91 571 2703516.

E-mail address: [email protected] (M. Zubair Alam).

Ecotoxicology and Environmental Safety 73 (2010) 1620–1628

Environ Monit Assess (2011) 178:281–291DOI 10.1007/s10661-010-1689-8

Prevalence of heavy metal resistance in bacteria isolatedfrom tannery effluents and affected soil

Mohammad Zubair Alam · Shamim Ahmad ·Abdul Malik

Received: 5 January 2010 / Accepted: 23 August 2010 / Published online: 8 September 2010© Springer Science+Business Media B.V. 2010

Abstract In the present study, a total of 198bacteria were isolated, 88 from the tanneryeffluents and 110 from agricultural soil irrigatedwith the tannery effluents. Tannery effluents andsoils were analyzed for metal concentrations byatomic absorption spectrophotometer. The tan-nery effluents and soil samples were found to becontaminated with chromium, nickel, zinc, cop-per, and cadmium. All isolates were tested fortheir resistance against Cr6+, Cr3+, Ni2+, Zn2+,Cu2+, Cd2+, and Hg2+. From the total of 198isolates, maximum bacterial isolates were foundto be resistant to Cr6+ 178 (89.9%) followed byCr3+ 146 (73.7%), Cd2+ 86 (43.4%), Zn2+ 83(41.9%), Ni2+ 61 (30.8%), and Cu2+ 51 (25.6%).However, most of the isolates were sensitive toHg2+. Among the isolates from tannery effluents,97.8% were resistant to Cr6+ and 64.8% were re-sistant to Cr3+. Most of the soil isolates were resis-tant against Cr6+ (83.6%) and Cr3+ (81.8%). Allisolates were categorized into Gram-positive and

M. Z. Alam (B) · A. MalikDepartment of Agricultural Microbiology,Faculty of Agricultural Sciences,Aligarh Muslim University, Aligarh 202 002 Indiae-mail: [email protected]

S. AhmadMicrobiology Division, Institute of Ophthalmology,Faculty of Medicine, J.N. Medical College,Aligarh Muslim University, Aligarh 202 002 India

Gram-negative bacteria. In a total of 114 Gram-positive isolates, 91.2% were resistant to Cr6+ fol-lowed by 73.7% to Cr3+, 42.1% to Zn2+, 40.4% toCd2+, and 32.5% to Ni2+. Among Gram-negativeisolates, 88.1% were found showing resistance toCr6+, 75.0% to Cr3+, and 47.6% were resistantto Cd2+. Majority of these metal-resistant isolateswere surprisingly found sensitive to the ten com-monly used antibiotics. Out of 198 isolates, 114were found sensitive to all antibiotics whereasonly two isolates were resistant to maximum eightantibiotics at a time. Forty-one and 40 isolateswhich constitute 20.7% and 20.2% were resistantto methicilin and amoxicillin, respectively.

Keywords Metal resistance · Tannery effluents ·Heavy metals · Antibiotics · Prevalence

Introduction

Heavy metals are ubiquitous and persist as en-vironmental pollutants that are introduced intothe environment through anthropogenic activities,such as mining and smelting, as well as throughirrigation and other sources of industrial waste.The use of industrial or municipal wastewater inagriculture is a common practice of irrigation inmany parts of the world (Sharma et al. 2007). Themajor objectives of wastewater irrigation are thatit provides a reliable source of water supply to

416 Journal of Basic Microbiology 2008, 48, 416–420

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Short Communication

Chromate resistance, transport and bioreduction by Exiguobacterium sp. ZM-2 isolated from agricultural soil irrigated with tannery effluent

Mohammad Zubair Alam and Abdul Malik

Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India

Bacterial strain Exiguobacterium sp. ZM-2 isolated from agricultural soil irrigated with tannery

effluents, was examined for its resistance to hexavalent chromium. Exiguobacterium sp. ZM-2

could resist 12.37 mM of potassium chromate. The isolate was also found resistant to other

heavy metal ions. Exiguobacterium sp. ZM-2 was able to reduce 500 µM hexavalent chromium

completely within 56 h under in vitro conditions. Chromate reduction was severely affected in

presence of metabolic inhibitors, sodium cyanide and sodium azide. No chromate reduction

was observed in presence of 1 mM sodium cyanide while only 17% of 250 µM chromate was

reduced when medium contained 1 mM sodium azide. A 10 mM sodium sulphate inhibited

hexavalent chromium reduction up to 35%. On the other hand, use of 1 mM 2, 4-dinitro-

phenol, an uncoupling agent, stimulated the chromate reduction, indicating that the

respiratory-chain-linked electron transport to Cr (VI) was limited by the rate of dissipation of

the proton motive force. Cell free extract of Exiguobacterium sp. ZM-2 readily reduce Cr (VI) to Cr

(III). The kinetics of chromate reductase fit well in the linearized Lineweaver-Burk plot and

showed a Km of 106.1 µM Cr (VI) and Vmax of 1.24 µmol/min per mg of protein.

Keywords: Exiguobacterium / Chromate reduction / Metabolic inhibitor / Cell free extract

Received: February 04, 2008; accepted: March 14, 2008

DOI 10.1002/jobm.200800046

Introduction*

One of the most common polluting metals is chro-

mium, arising from discharged effluents from leather

tanning, chromium plating, wood preservation and

alloy preparation. Nearly 80% of the tanneries in India

are engaged in the chrome tanning process. Most of

them discharge untreated wastewater into the envi-

ronment [1]. Chromium exists in a wide range of oxida-

tion states from –2 to +6. Trivalent and hexavalent

forms are the dominant oxidation states of chromium

that exist in the environment. The toxicity of chro-

mium is dependent on its oxidation state. Trivalent

chromium is most stable, less toxic and is an essential

Correspondence: Mohammad Zubair Alam, Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim Univer-sity, Aligarh-202 002, India E-mail: [email protected] Phone: +91 571 2703516 Fax: +91 571 2703516

micronutrient for many higher organisms while chro-

mium (VI) is highly toxic, readily crossing the mem-

branes of eukaryotic and prokaryotic cells and causing

oxidative cellular damage. In the presence of organic

matter, Cr (VI) is reduced to Cr (III), but higher concen-

tration of Cr (VI) may overcome the reducing capability

of the environmental conditions and thus it persists [2].

Moreover under particular environmental conditions, a

part of chromium (III) can be transformed into Cr (VI)

[3]. The presence of Cr (VI) in the environment plays a

selective pressure on microflora. Reduction of Cr (VI) to

Cr (III) is therefore a potentially useful process for

remediation of Cr (VI) contaminated environments.

Bioreduction of Cr (VI) can occur directly as a result of

microbial metabolism (enzymatic) or indirectly, medi-

ated by a bacterial metabolite such as H2S [4].

Many bacteria including Pseudomonas spp. [5, 6], E. coli

[7], Brucella [8] and Bacillus [9, 10] can reduce Cr (VI) to

less toxic Cr (III), which readily precipitates as Cr (OH)3.

Mohammad Zubair Alam1

Shamim Ahmad2

1Department of Agricultural

Microbiology, Faculty of Agricultural

Sciences, Aligarh Muslim University,

Aligarh, India2Microbiology Division, Institute of

Ophthalmology, Faculty of Medicine,

J.N. Medical College, Aligarh Muslim

University, Aligarh, India

Research Article

Chromium Removal through Biosorption andBioaccumulation by Bacteria from TanneryEffluents Contaminated Soil

Four bacterial isolates (two resistant and two sensitive to chromium) were isolated

from soil contaminated with tannery effluents at Jajmau (Kanpur), India, and were

identified by 16S rDNA gene sequencing as Stenotrophomonas maltophilia, Exiguobacterium

sp., Pantoea sp., and Aeromonas sp. Biosorption of chromium by dried and living

biomasses was determined in the resistant and sensitive isolates. The effect of pH,

initial metal concentration, and contact time on biosorption was studied. At pH 2.5

the living biomass of chromium resistant isolate Exiguobacterium sp. ZM-2 biosorbed

maximum amount of Cr6þ (29.8mg/g) whereas the dried biomass of this isolate

biosorbed 20.1mg/g at an initial concentration of 100mg/L. In case of chromate

sensitive isolates, much difference was not observed in biosorption capacities between

their dried and living biomasses. The maximum biosorption of Cr3þ was observed

at pH 4.5. However, biosorption was identical in resistant and sensitive isolates. The

data on chromium biosorptionwere analyzed using Langmuir and Freundlich isotherm

model. The biosorption data of Cr6þ and Cr3þ from aqueous solution were better fitted

in Langmuir isotherm model compared to Freundlich isotherm model. Metal recovery

through desorption was observed better with dried biomasses compared to the living

biomasses for both types of chromium ions. Bioaccumulation of chromate was found

higher in chromate resistant isolates compared to the chromate sensitive isolates.

Transmission electron microscopy confirmed the accumulation of chromium in

cytoplasm in the resistant isolates.

Keywords: Bioaccumulation; Biosorption; Chromium; Tannery effluents; Transmission electronmicroscopy

Received: July 2, 2010; revised: September 27, 2010; accepted: October 5, 2010

DOI: 10.1002/clen.201000259

1 Introduction

The existence of heavy metals in the environment represents a

significant and long-term environmental hazard. They are non-bio-

degradable and tend to accumulate in living organisms, leading to

various diseases and disorders [1, 2]. Chromium is a heavy metal,

dissipates into the environment as a result of various industrial

activities such as leather tanning, dyes, paints and pigments man-

ufacturing, wood preservation, and electroplating industries [3, 4].

Chromium can exist in oxidation states ranging from –2 to þ6.However, Cr6þ and Cr3þ are the two stable oxidation states, which

have widely contrasting toxicity and transport characteristics. Cr6þ

is a known mutagen, teratogen, and carcinogen [5, 6]. In aqueous

environments Cr6þ exists as oxyanions which are soluble, highly

mobile, and can permeate through bacterial and eukaryotic cell

membranes. In contrast, Cr3þ is less toxic, less soluble, and less

mobile, mostly found as oxides, hydroxides or sulfates, generally

bound to organic matter in soils. However, long-term exposure to

Cr3þ is known to cause allergic skin reactions and cancer [7, 8].

The removal of heavy metals from aqueous solutions has received

considerable attention in recent years. However, the practical appli-

cation of physicochemical technology such as chemical precipi-

tation, membrane filtration, and ion exchange is sometimes

restricted due to technical or economical constraints. For example,

the ion exchange method is very effective but requires expensive

adsorbent materials [9]. In recent years, applying biotechnology in

controlling and removing metal pollution has been paid much

attention, and gradually becomes hot topic in metal pollution con-

trol practices because of its potential application. A practical and

dynamic approach is the use of biological agents, i.e., microorgan-

isms which include bacteria, fungi, yeast, and their products [10].

The ability of microbial biomass to remove metals can be described

as active (energy dependent) or passive (energy independent) pro-

cesses, commonly known as bioaccumulation and biosorption,

Correspondence: M. Zubair Alam, Department of AgriculturalMicrobiology, Faculty of Agricultural Sciences, Aligarh MuslimUniversity, Aligarh 202 002, India.E-mail: [email protected]

Abbreviation: DB, dried biomass; MIC, minimum inhibitoryconcentration; NA, nutrient agar.

226 Clean – Soil, Air, Water 2011, 39 (3), 226–237

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com

ORIGINAL ARTICLE

Toxic chromate reduction by resistant and sensitive bacteriaisolated from tannery effluent contaminated soil

Mohammad Zubair Alam & Shamim Ahmad

Received: 15 September 2010 /Accepted: 11 February 2011# Springer-Verlag and the University of Milan 2011

Abstract Bacterial strains ZA-6, W-61, KS-2 and KS-14were isolated from agricultural soil irrigated with tanneryeffluents and subsequently identified by 16S rDNAsequencing as Stenotrophomonas maltophilia, Staphylococcusgallinarum, Pantoea sp. and Aeromonas sp., respectively. Allisolates were examined for their resistance to hexavalentchromium and other heavy metal ions. The bacterialisolate S. maltophilia ZA-6 and S. gallinarum W-61 wereresistant to 16.5 and 12.4 mM of potassium chromate,respectively, whereas Pantoea sp. KS-2 and Aeromonassp. KS-14 were found to be sensitive to potassiumchromate. S. maltophilia ZA-6 and S. gallinarum W-61completely reduced 500 μM Cr6+ to Cr3+ within 56 h,while chromate-sensitive isolates Pantoea sp. KS-2 andAeromonas sp. KS-14 exhibited poor chromate-reducingactivity. Chromate reduction was severely affected in thepresence of the metabolic inhibitors sodium cyanide andsodium azide. Sodium cyanide completely inhibitedchromate reduction in each isolate, whereas 1 mM sodiumazide and 10 mM sodium sulfate affected the inhibition ofchromate reduction to varying degrees. The use of 1 mM2,4-dinitrophenol, an uncoupling agent, stimulated the

chromate reduction. The cell-free extract (CFE) of chromate-resistant isolates readily reduced Cr6+ to Cr3+, with that ofS. gallinarum W-61 showing a Km value of 121.7 μMchromate and a Vmax of 1.12 μmol/min per milligramprotein in the presence of NADH. The chromate-resistantisolates displayed lower Michealis–Menton constant (Km)values and higher maximum velocity (Vmax) than chromate-sensitive isolates. These results suggest that chromateresistance and reduction in these bacteria are related.

Keywords Chromate reduction . Hexavalent chromium .

Metal resistant . Cell-free extract . Metabolic inhibitors

Introduction

Industrialization is a hallmark of civilization. However, thefact remains that industrial emissions have been adverselyaffecting the environment, leading to the large-scaledestruction of agricultural land and water bodies world-wide and thereby becoming a matter of great concern(Poopal and Laxman 2009). When toxic substancesaccumulate in the environment and in food chains, theycan profoundly disrupt biological processes. Chromium(Cr) is an important metal that is employed ubiquitously inmany industrial processes, including chrome leather tanning,chrome plating, ceramics, dyes, paints and pigments manu-facturing, textile processing, metal finishing, wood processingand photographic sensitizer manufacturing (Thacker et al.2006; Cheung and Gu 2007; Desai et al. 2008). It can existin oxidation states ranging from −2 to +6. The mostpersistent forms of chromium in the environment is thesoluble and mobile—and most toxic—hexavalent species

M. Z. Alam (*)Department of Agricultural Microbiology,Faculty of Agricultural Sciences, Aligarh Muslim University,Aligarh 202 002 Uttar Pradesh, Indiae-mail: [email protected]

S. AhmadMicrobiology Division, Institute of Ophthalmology, Facultyof Medicine, J.N. Medical College, Aligarh Muslim University,Aligarh 202 002 Uttar Pradesh, India

Ann MicrobiolDOI 10.1007/s13213-011-0235-4

ORIGINAL ARTICLE

Multi-metal biosorption and bioaccumulationby Exiguobacterium sp. ZM-2

Mohammad Zubair Alam & Shamim Ahmad

Received: 6 June 2012 /Accepted: 6 November 2012# Springer-Verlag Berlin Heidelberg and the University of Milan 2012

Abstract The present work deals with the biosorption per-formance of dried and non-growing biomasses of Exiguo-bacterium sp. ZM-2, isolated from soil contaminated withtannery effluents, for the removal of Cd2+, Ni2+, Cu2+, andZn2+ from aqueous solution. The metal concentrationsstudied were 25 mg/l, 50 mg/l, 100 mg/l, 150 mg/l and200 mg/l. The effect of solution pH and contact time wasalso studied. The biosorption capacity was significantlyaltered by pH of the solution. The removal of metal ionswas conspicuously rapid; most of the total sorption oc-curred within 30 min. The sorption data have been ana-lyzed and fitted to the Langmuir and Freundlich isothermmodels. The highest Qmax value was found for the bio-sorption of Cd2+ at 43.5 mg/g in the presence of the non-growing biomass. Recovery of metals (Cd2+, Zn2+, Cu2+

and Ni2+) was found to be better when dried biomass wasused in comparison to non-growing biomass. Metal re-moval through bioaccumulation was determined by grow-ing the bacterial strain in nutrient broth amended withdifferent concentrations of metal ions. This multi-metalresistant isolate could be employed for the removal ofheavy metals from spent industrial effluents before dis-charging them into the environment.

Keywords Biosorption . Bioaccumulation . Metal .

Bioremediation . Adsorption isotherms

Introduction

Heavy metal pollution is one of the most important environ-mental problems today. Various industries, such as mining,metallurgy, electroplating, leather working, photography,electric appliance manufacturing, metal surface treatingetc., produce and discharge waste containing variousheavy metals into the environment. The metals are notbiodegradable and tend to accumulate in living organisms,leading to various diseases and disorders (Kobya et al.2005; Liao et al. 2008). Thus metals, a valuable resource,can also cause serious environmental pollution, threateningecosystem and human health through their extreme toxic-ity. Low concentrations of certain transition metals such ascobalt, copper, nickel and zinc are essential for manycellular processes of bacteria, since they provide vitalcofactors for metalloproteins and enzymes. (Nies 1999;Li et al. 2004) However, higher concentrations of thesemetals are often toxic (Nies 1999). On the other hand,cadmium is extremely toxic, and to date no biologicalfunction has been ascribed to it (Gadd 2004). The removalof heavy metal ions from industrial wastewater is a prob-lem of increasing concern that has been addressed primar-ily by chemical and physical treatments. However, theseprocedures have significant disadvantages, such as incom-plete metal removal, high reagent or energy requirements,or generation of toxic sludge or other waste products, andare generally very expensive (Volesky and Naja 2005).Therefore, there is a need for the development of lowcost and easily available materials able to adsorb toxicheavy metals from wastewater. Microorganisms are potentbioremediators, removing heavy metals via active or passiveuptake mechanisms.

Metal accumulative bioprocesses generally fall into oneof two categories, biosorptive uptake by non-living and/ornon-growing biomass and bioaccumulation by living cells.

M. Z. Alam (*)King Fahd Medical Research Center, King Abdulaziz University,P. O. Box 80216, Jeddah 21589, Saudi Arabiae-mail: [email protected]

S. AhmadMicrobiology Division, Institute of Ophthalmology,Faculty of Medicine, J.N. Medical College,Aligarh Muslim University,Aligarh 202 002, India

Ann MicrobiolDOI 10.1007/s13213-012-0571-z

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