Construction of metagenomic clone library for microbial diversity assessment in an electronic waste polluted site and biosorption of heavy metals by isolated organisms

Thesis Submitted to Bharathidasan University for the Award of the Degree of Doctor of Philosophy in Environmental Biotechnology

By

Ramasamy RAJESHKUMAR, M.Sc., M.Phil., PGDBI

Under the Supervision of Dr. K. THAMARAISELVI Assistant Professor

Department of Environmental Biotechnology Bharathidasan University Tiruchirappalli - 620 024 Tamilnadu, India

December 2011 Department of Environmental Biotechnology School of Environmental Sciences BHARATHIDASAN Tiruchirappalli – 620 024. UNIVERSITY Tamilnadu, India.

Phone : +91-431-2407088 Fax : +91-431-2407045 Dr. K. Thamaraiselvi Email : [email protected] Assistant Professor [email protected]

CERTIFICATE

I hereby certify that the thesis entitled “Construction of metagenomic clone library for microbial diversity assessment in an electronic waste polluted site and biosorption of heavy metals by isolated organisms” submitted to Bharathidasan University, Tiruchirappalli, Tamilnadu, India, for the award of Doctor of Philosophy in Environmental Biotechnology is an authentic record of original work carried out by Mr. R. Rajesh kumar, MSc., MPhil., PGDBI., under my supervision at Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India.

I further certify that no part of the thesis has been submitted elsewhere for the award of any other degree, diploma or associate-ship of any other University.

K. Thamaraiselvi

Declaration

I do hereby declare that the research work on “Construction of metagenomic clone library for microbial diversity assessment in an electronic waste polluted site and biosorption of heavy metals by isolated organisms” has been originally carried out by me in the Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India and part of the work (Metagenomics) carried out in the Environmental Genomics Divison, National Environmental Engineering Research Institute (NEERI - CSIR), Nagpur, Maharashtra, India. I further declare that this work has not been submitted elsewhere for any other degree or other similar titles. To the best of my knowledge, this is the first metagenomic study for the assessment of microbial diversity in an e-waste recycling facility polluted surface soil. And this is the first study on employing of metal tolerant bacteria isolated from an e-waste recycling facility polluted surface soil for the removal of heavy metals.

R. Rajeshkumar Tiruchirappalli

Acknowledgement

I avail this opportunity to express my heartfelt thanks to helping hands behind this work. It is still great at this juncture to recall all the faces and spirits in the form of faculties, friends, near and dear ones. I would consider this work nothing more than incomplete without attending to the task of acknowledging the overwhelming help I received during this endeavor.

I express my thanks to research supervisor Dr. K. Thamaraiselvi, Assistant Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India

At the very outset, I feel inadequacy of words to express my profound indebtedness and deep sense of gratitude to my mentor, School chair and Head of the Department Dr. M. Krishnan, Professor, Department of Environmental Biotechnology, Bharathidasan University without whose appreciation, valuable advice and constant encouragement, this task would not have been accomplished. His interaction with students, freeness, helping nature, freedom to work and communication style really appreciable. He took care of my personnel problems also and helped me whenever I was in trouble.

Special thanks to Dr. Hemant J. Purohit, Scientist G and Head, Environmental Genomics Divison, NEERI(CSIR), Nagpur, Maharashtra, India for considering me as a visiting researcher to carry out metagenomics study under his valuable guidance, kind cooperation and providing all the facilities to work in his laboratory. I extend my thanks to Dr. Atya Kapley, Scientist, for her valuable guidance and critical suggestion during the study.

I acknowledge with sincere thanks to doctoral committee member Dr. Vasanthi Nachiappan, Associate Professor and Head, Department of Biochemistry, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for her constant help, guidance, co-operation throughout this Ph.D program.

I am overwhelmed with sincere feelings of indebtedness to Dr. M. Ravichandran, Professor and Head, Department of Environmental Management, Bharathidasan University, Thiruchirappalli, Tamilnadu, India for his help and support throughout this Ph.D program.

My thanks to Dr. R. Babu Rajendiran, Associate Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for encouragement during the period of investigation. I am very much inspired by his hardworking nature, affection towards the department and students.

I owe this opportunity to thank Dr. S. Achiraman, Assistant Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for his help, advice and constructive criticism.

I express my profound sense of gratitude to Dr. M. Vasanthy and Dr. T. Sivasudha, Assistant Professors, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, Tamilnadu, India for their fruitful guidelines and advice during this Ph.D program.

I feel grateful to University Grants Commission, Ministry of Human Resource Development, Government of India, New Delhi for awarding me Rajiv Gandhi National Research Fellowship and providing me financial assistance.

My untold sense of gratitude and profound indebtedness to my beloved parents Mr. P. Ramasamy and Mrs. Jelina Ramasamy without their blessings I would not have grown to this level, who are always with me and have taken a lot of pain to bring me up to this level.

I would like to express my sincere love and thanks to my younger sister Dr. R. Priyadarshini, Physician Assistant, Meenakshi Mission Hospital, Madurai, Tamilnadu, India for her continuous encouragement, helpful comments and suggestions in my life.

No words in this mortal world can suffice to express my feelings towards my uncles Er. K. Gandhi, BE, PGDCS, Pudhur M. Anbazhagan, Kavignar and Writer, and their family for their intangible encouragement, love and affection towards me. I am also thankful to Mr. Murugesan and Mr. K. Saravanan for their care, love and affection towards me. I am overwhelmed with lot of affection showered on me by all my family members, relatives and neighbors.

I express my thanks and indebtedness to my beloved lab mates and emerging Environmental Biotechnologists Ms. Valsala, Ms. Poornima Priyadarshini, Mr. Prakash and Ms. Elavarasi for their help and encouragement during this investigation. I would like to thank my friends from other departments Mr. T. Manikandan, Mr. S. P. Manivannan, Ms. Kavitha, Ms. Sugunadevi, Mr. Ganesan, Mr. Karthikeyan and others for the happiest moments they provided to me and for their constant encouragement which helped to sustain my interest and thereby accomplishing this task.

I would like to express my thanks to Mr. R. Rajesh Pal, Ms. Leena Agarwal and other Researchers, Environmental Genomics Division, NEERI (CSIR), Nagpur, Maharashtra, India.

I also express my thanks to the non-teaching staff Mr. Tharasingh, Mr. Muralidharan and Mr. Tirupathi, Department of Environmental Biotechnology and Mr. Logesh, Finance Section, Bharathidasan University, Thiruchirappalli, Tamilnadu, India for their generous and timely help.

Rajeshkumar. R

Affectionately Dedicated to

My Beloved Parents and Research Community

CONTENTS

Chapter No. Title Page No.

I General Introduction 1

II Review of Literature 9

III Materials and Methods 60

IV Results and Discussion 77

V Summary and Conclusion 129

References i - xxxvi

Appendices

LIST OF ABBREVIATIONS

% Percentage °C celcius µl microlitre bp base pairs bp basepair CFU Colony Forming Unit g grams h hours

H2SO4 Sulphuric Acid L litres L-1 per litre LB Luria Bertani M molar mg milligram min minutes ml millilitre N normality ng nano gram nm nano metre OD optical density pH concentration of hydrogen ions ppm parts per million rpm rotation per minute sec seconds TFTC too few to count TNTC too numerous to count V volts μg microgram μM Micro molar

Chapter I

1. Introduction

For much of the last century, microbiologists have been aware that the identity of only a tiny fraction of the inhabitants of the microscopic landscape. While most people are very familiar with the diversity of life in the plant and animal kingdoms, few actually realize the vast amounts of variability present in the bacterial populations.

Microorganisms represent the richest repertoire of molecular and chemical diversity in nature as they underlie basic ecosystem processes. The current inventory of the world’s biodiversity is very incomplete and that of viruses, microorganisms and invertebrates is especially deficient. Scientists have identified about 1.7 million living species on our planet. Studies indicate that the 5,000 identified species of prokaryotes represent only

1 to 10% of all bacterial species, therefore we have only a small idea of our true microbial diversity (Stanley, 2002).

Microorganisms constitute a huge and almost unexplained reservoir of resources likely to provide innovative applications useful to man. Microorganisms have been evolving for nearly 4 billion years and are capable of exploiting a vast range of energy sources and thriving in almost every habitat. For 2 billion years microbes were the only form of life on Earth. During this long history, all of the basic biochemistries of life evolved and all life forms have developed from these microbial ancestors. It is estimated that 50% of the living protoplasm on this planet is microbes. They underlie basic ecosystem processes such as the biogeochemical cycles and food chains, as well as maintain vital and often elegant relationships between themselves and higher organisms. Without microorganisms, all life on Earth would cease. Man has long

| 1 Introduction

exploited this metabolic wealth to produce food and to develop health applications.

They are used for food production and preservation, management of pests and pathogens, bioleaching of metals, increasing soil fertility, generating biofuels, monitoring pollutants, ridding coal mines from methane, cleaning up of oil spills, waste water treatment, assaying of chemicals and serving as tools for medical research.

Microorganisms are the major sources of antimicrobial agents and produce a wide range of other important medicinal compounds including enzymes, enzyme inhibitors, antihelminthics, antitumor agents, insecticides, vitamins, immune suppressants and immune modulators. These agents have all been discovered during the past 50 years and represent only a small portion of what is likely present in nature. Individual organisms that collectively make up the biota have specific properties that make them of direct value in satisfying the consumption or production needs of society (and hence the demand for particular species).

The biological diversity of the Indian subcontinent is one of the richest in the world owing to its vast geographic area, varied topography and climate and the juxtaposition of several biogeographical regions. Because of its richness in overall species diversity, India is recognized as one of the 12 mega diversity regions of the world. Nearly 72% of India’s biowealth is constituted by fungi (~18%), insects (~40%) and angiosperms (~13%). Thus, India’s contribution to the global diversity is around

8%. The most important mega-diversity centers are Western ghats, North-eastern hill regions, Bastar regions inhabited by tribals andaman Nicobar islands, mangrove forests of Sunderban area, silent valley of Kerala, playas of Rajasthan, Chilka lake of Orissa,

Sonar Lake of Maharashtra and the Himalayan region. Various types of diverse microenvironments and unique ecosystems such as boiling waters, deep sea vents, salt pans, acid mine drainage, cold environments are present in India that are home to

| 2 Introduction

diverse populations of microorganisms. It is interesting to note that extremely acidic soils (pH ~2.8) of Kerala are home to cyanobacteria. As many as 42 species were recorded in acidic soils of which 19 were recorded for the first time in Kerala. Hotspots are recognized on the basis of the presence of greatest number of endemic species.

Therefore, at the global level hotspots are the areas of high conservation priority because if unique species are lost they can never be replaced. The two major hotspots in the present scenario of India’s biodiversity are the Western ghats and the North-eastern region. The Western ghats are known to be tectonically active and an uplifted region. It has been reported that approximately 17% of a set of 2500 species are likely to be microbial in this region. The high biodiversity of this region therefore may be due to large nutrients the volcanism brought in, the relatively higher thermal gradients along this belt and widely varying elevations. The addition of new genera from diverse conditions especially from Indian North Western Himalayas is expected to add several new industrially important strains, which have better antibacterial potential with limited scope for the production of bioactive metabolites.

The diversity of microorganisms in an environment plays a key role in biogeochemical processes by contributing to plant nutrition and soil health (Nannipieri et al., 2003; Wardle et al., 2004; Arias et al., 2005), even in agricultural and extreme environments (Wall and Virginia, 1999; Mader et al., 2002) and the composition and/or activity of these microorganisms can indicate how well an ecosystem is functioning.

Soil microorganisms represent a considerable fraction of the living biomass on Earth

(Whitman et al., 1998), with surface soils containing 103 to 104 kg of microbial biomass per hectare (Brady and Weil, 2002). Despite this abundance and the importance of soil microorganisms for key ecosystem functions (Kent et al., 2002; Wardle et al., 2004;

Leininger at al., 2006), the diversity and structure of soil microbial communities remain

| 3 Introduction

poorly studied. With the advent of molecular techniques, we can now begin to survey the full extent of microbial diversity, including the vast majority of microorganisms which cannot be identified using traditional taxonomic approaches (Pace, 1997). Of the microbial groups that are abundant in soil, the bacteria have been the most extensively studied. With an estimated 103 to 107 bacterial “species” per individual soil sample

(Curtis et al., 2002; Torsvik et al., 2002; Gans et al., 2005; Tringe et al., 2005), they are often considered to be the most diverse group of soil microorganisms (Buckley and

Schmidt, 2002). For this reason, accessing and preserving the diversity of soil microorganisms is crucial, which contains a large pool of unknown genes that encode novel enzymes and proteins. Direct culture or molecular methods can be used to assess soil microbial diversity. Nevertheless, traditional microbiological approaches present severe limitations, as only small fraction of the soil bacteria is cultivable using standard methods (Torsvik et al., 1998). Therefore, in the last two decades, several molecular approaches have been proposed (Kirk et al., 2004; Kowalchuk et al., 2004; Bloem et al., 2006; van Elsas et al., 2007; Sorensen et al., 2009) and recently, the exploration of entire genomes present in a soil sample, metagenomics, has provided a new approach for detailed assessment (Handelsman, 2004; Daniel, 2005; Lorenz and Eck, 2005;

Schloss and Handelman, 2005; Langer et al., 2006, Mocali and Benedetti, 2010). Many researchers argue that it is now possible to explore the ‘‘black box’’ of soil microbial communities (Ritz et al., 1994; Tiedje et al., 1999).

Though the negative effects of bacteria such as disease are well known, their often subtle functions explain why their biodiversity positively affects humans. The most important ecological function of bacteria is bioremediation, a process by which contaminated regions are restored by means of bacterial biogeochemical processes. It is an economical, versatile, environment friendly and efficient treatment strategy and a

| 4 Introduction

rapidly developing field of environmental restoration. Bioremediation utilizes the microbial ability to degrade and/or detoxify chemical substances such as petroleum products, aliphatic and aromatic hydrocarbons, industrial solvents, pesticides and their metabolites and metals. The presence of a large number of diverse bacterial species in nature expands the variety of chemical pollutants that can be degraded as well as the extent to which pollutant sites can be decontaminated. The use of microorganisms for degradation of pollutants is now being increasingly applied as the technology of choice for cleans up or restoration of polluted sites as it can be self sustaining and inexpensive.

There is a general interest in studying the diversity of indigenous microorganisms capable of degrading different pollutants because of their various effects on the environment. Efforts have been made to characterize bacterial communities and their responses to pollutants, to isolate potential degraders and to identify the genes involved in particular degradation processes (Greene et al., 2000; Wantanabe et al., 2002). It has been established that contaminated environments harbor a wide range of unidentified pollutant degrading microorganisms that have crucial role in their bioremediation

(Margesin et al., 2003) that can be assessed only by the culture independent techniques.

The pollution of the environment with toxic heavy metals is spreading throughout the world along with industrial progress. Copper, chromium, cadmium and nickel are known to be the most commonly heavy metals used and the more widespread contaminants of the environment (Patterson, 1997; Aksu, 1998; Doenmez and Aksu,

1999). Traces of these heavy metals are necessary as co-factors of enzymatic reactions, but high levels of them may cause extreme toxicity to living organisms due to inhibition of metabolic reactions. The microorganisms respond to these heavy metals by several processes; including transport across the cell membrane, biosorption to the cell walls and entrapment in extracellular capsules, precipitation, complexation and

| 5 Introduction

oxidation-reduction reactions (Rai et al., 1981; Macaskie and Dean, 1989; Huang et al.,

1990; Brady and Duncan, 1994; Brady et al., 1994; Veglio et al., 1997). The bioremediation of heavy metals using microorganisms has received a great deal of attention in recent years, not only as a scientific novelty but also for its potential application in industry. Metal accumulative bioprocess generally falls into one of two categories, bisorptive (passive) uptake by nonliving, nongrowing biomass or biomass products and bioaccumulation by living cells (Clausen, 2000). Microbial survival in polluted soils depends on intrinsic biochemical and structural properties, physiological and/or genetic adaptation including morphological changes of cells, as well as environmental modifications of metal speciation (Wuertz and Mergeay, 1997). For example, high levels of heavy metals can affect the qualitative as well as quantitative composition of microbial communities. Several studies have found that metals influence microorganisms by harmfully affecting their growth, morphology and biochemical activities, resulting in decreased biomass and diversity (Baath, 1989;

Reber; 1992; Malik and Ahmed, 2002). Bahig et al., (2008) reported that the previous studies have shown that long term and short term stresses such as high temperature, extremes of pH or chemical pollution often result in altered metabolism, species diversity and plasmid incidence of soil bacteria populations. Bacteria are among the most abundant organism that occur every where on earth. Heavy metals are increasingly found in microbial habitats due to several natural and anthropogenic processes; therefore, microbes have evolved mechanisms to tolerate the presence of heavy metals by either efflux, complexation, or reduction of metal ions or to use them as terminal electron acceptors in anaerobic respiration (Gadd, 1990). Most mechanism studied till date involve the efflux of metal ions outside the cell and genes for tolerance mechanisms have been found on both chromosomes and plasmids. Bacteria that are

| 6 Introduction

resistant to and grow on metals play an important role in the biogeochemical cycling of those metal ions (Gadd, 1990).

Activities related to electronic waste (e-waste) is one of the emerging problems of the 21st century (Schmidt, 2002). E-waste refers to end of life electronic products such as computers, televisions and mobile phones made of plastics, metals, other trace elements (TEs), etc. About 50–80% of e-waste from the industrialized countries are exported to recycling centers in developing countries such as China, India, Pakistan,

Vietnam and the Philippines because of the lower wages for labor and less functional/lenient environmental regulations in these countries (UNEP, 2005).

Recently, e-waste trade has been increasing in African countries too (Schmidt, 2006).

The recycling and disposal of electronic waste (e-waste) in developing countries is causing an increasing concern due to its effects on the environment and associated human health risks.

To understand the contamination status, Ha et al., (2009) measured trace elements (TEs) in soil, air dust and human hair collected from e-waste recycling sites (a recycling facility and backyard recycling units) and the reference sites in Bangalore and

Chennai in India. Concentrations of Cu, Zn, Ag, Cd, In, Sn, Sb, Hg, Pb and Bi were higher in soil from e-waste recycling sites compared to reference sites. For Cu, Sb, Hg and Pb in some soils from e-waste sites, the levels exceeded screening values proposed by US Environmental Protection Agency (EPA). Concentrations of Cr, Mn, Co, Cu, In,

Sn, Sb, Tl, Pb and Bi in air from the e-waste recycling facility were relatively higher than the levels in Chennai city. High levels of Cu, Mo, Ag, Cd, In, Sb, Tl and Pb were observed in hair of male workers from e-waste recycling sites. Ha et al. (2009) results suggest that e-waste recycling and its disposal may lead to the environmental and

| 7 Introduction

human contamination by some TEs. This is the first study on TE contamination at e- waste recycling sites in Bangalore, India.

Fig 1. Route of e-waste transport and importing sites (Sources: Basel Action Network, Silicon Valley Toxics Coalition, Toxics Link India, SCOPE (Pakistan), Greenpeace China, 2002.)

In this study, e-waste recycling facility polluted surface soil was used for

Culture-independent approach to assess the microbial groups and for Culture-dependent approach to evaluate the sorption capacity of the isolated metal tolerant bacteria in a laboratory simulated environment.

| 8 Chapter II

2. Review of literature

2.1 Culture-independent approaches for microbial diversity assessment

Though cultivation of microorganisms is important and researchers have dedicated themselves to this task for several decades; most microorganisms have only been detected with culture-independent techniques. There are now many major groups of Bacteria and Archaea known only from sequences directly retrieved from environmental samples and, until these organisms can be cultivated, the only means of understanding their role in the environment is through culture-independent characterization linked to determination of in situ metabolic activity (Gray and Head,

2001). Although most microorganisms are uncultivable, unfortunately, growth conditions of most of them are not yet known. This is not only due to methodological limitations, but also due to a lack of taxonomic knowledge. It is difficult to study the diversity of a group of microorganisms when it is not understood how to categorize or identify the species present (Kirk et al., 2004). The recognition of the limitation to isolate a major part of the microbiota resulted in the development of a novel research area called ‘Molecular Microbial Ecology’ (Akkermans et al., 1996). The difference between this recent and classical microbial ecology is that microbial ecosystems are now being studied as a whole using culture-independent approaches (Zoetendal et al.,

2004a). The field of molecular microbial ecology is defined as the application of molecular technology, typically based on comparative nucleic acid sequence analysis, to identify specific microorganisms in a particular environment, to assign functional roles to these microorganisms and to assess their significance or contribution to environmental processes independent from cultivation (Zoetendal et al., 2004b).

Microbial diversity describes complexity and variability at different levels of biological

| 9 Review of Literature

organisation (Torsvik and Ovreas, 2002). It encompasses genetic variability within taxa

(species) and the number (richness) and relative abundance (evenness) of taxa and functional groups (guilds) in communities. Molecular techniques provide an excellent method for the rapid and quantitative monitoring of microbes in their communities and allow the investigation of spatial and temporal community changes within microbial ecosystems (Sekiguchi et al., 2001; O’Flaherty et al., 2006).

One of the most basic culture-independent microbiological techniques is microscopy, pioneered by Antonie van Leeuwenhoek (1632-1723). Although the first use was limited by microscope magnification, nowadays microorganisms can be morphologically studied with a whole range of microscopes (light and fluorescence microscopes, scanning and transmission electron microscopes (SEM and TEM), atomic force microscopes (AFM) and confocal laser scanning microscopes (CLSM). However, limited morphological variation renders the identification and differentiation of an estimated 1-10 million different species in the world merely based on structural features impossible (Pace, 1996).

With the help of specialized and powerful molecular techniques, specific microbial population dynamics and activities can be monitored. Microbial ecologists now have at their disposal a raft of complementary culture-independent methods that can be used to investigate microbial function, even in situ (Gray and Head, 2001).

Cultivation might be neither necessary nor the most effective approach for understanding of the roles of the incredibly vast number of microorganisms in the natural environment where comprehensive genomic information can be obtained and it could be possible to rely upon cultivation-independent methods to investigate physiology and activity directly (Tyson and Banfield, 2005). It has been shown that

| 10 Review of Literature

ecological interactions are essential element to be considered in studying microbial physiology and that functional genomics approaches can be used to complement classical microbiological methods for this purpose (Johnson et al., 2006). Although quantification is still difficult, several ambitious attempts to achieve this are conducted.

Significant biases are potentially introduced at various stages of molecular ecological approaches that make quantification difficult. Already during nucleic acid extraction, differences in lyses efficiency for different microbial populations can skew their representation in the nucleic acid pool (Vaughan et al., 2000). The degree of cell lysis therefore should be determined independently, which can be done by microscopic enumeration of cells in an environmental sample before and after lysis treatment

(Theron and Cloete, 2000). Typically, a direct cell lysis approach to DNA/RNA extraction and purification may be dissected into the following conceptual steps: washing the material to remove soluble components that may impair manipulation of subsequently isolated nucleic acids; disruption of cells in the material to release DNA or RNA; separation of the DNA or RNA from solids; and isolation and purification of the released nucleic acids for downstream molecular procedures (Moré et al., 1994;

Hirsch et al., 2010). Generally molecular approaches in assessing bacterial diversity are based on the analysis of total DNA isolated from environmental samples

(Metagenomics DNA). Analysis of PCR- amplified 16S rDNA from Metagenomics

DNA using universal bacterial primers cloned into a vector, followed by Restriction

Fragment Length Polymorphism (RFLP) and sequencing, is a basis for phylogenetic studies of bacteria communities (Guazzaroni et al., 2010; Dokic et al., 2010).

| 11 Review of Literature

Fig 2. General steps involved in culture-independent study (Rajendhran and Gunasekaran, 2008)

Metagenomic analyses can provide extensive information on the structure, composition and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification and if necessary, cloning methods for the intended downstream analyses.

The most important technical considerations in a metagenomic study include obtaining

| 12 Review of Literature

a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host (Kakirde et al., 2010).

Most culture-independent approaches for detection, identification and quantification make use of ribosomal RNA (rRNA) and the corresponding gene as a phylogenetic marker. Ribosomal RNA is present in all organisms and exhibits sufficient sequence variation to distinguish between different species, while providing regions of conservation at different levels of taxonomic resolution. Molecular tools based on 16S rRNA gene identification have revolutionised microbial ecology (Lane et al., 1985; Gray and Herwig, 1996; Dabert et al., 2002; Gill et al., 2006; Rajendhran and Gunasekaran, 2008). A limitation of the rRNA-based techniques, however, is the development of primers and probes, since specific probes are designed based on the comparison between sequences present in the database (Dabert et al., 2002). Another limitation of the use of the 16S rRNA gene is that it only gives an indication about the presence and abundance of microorganisms (http://rrndb.cme.msu.edu (Klappenbach et al., 2001)), but not about their activity. Simultaneous monitoring of process performance and variations in microbial community structure can solve this limitation.

It has been observed that the amount of rRNA per cell is roughly proportional to the metabolic activity of the cell, although this is not universal for all microorganisms and can vary with growth phase, since some microbial groups have a persistent high level of ribosomes, even in starving cells (Flärdh et al., 1992; Wagner, 1994; Amann et al.,

1995; Farrelly et al., 1995; Vaughan et al., 2000). Studies have applied a metagenomic approach to a number of different environments, such as soils (Rondon et al., 2000;

Voget et al., 2003; Tringe et al., 2005), the complex microbiome of the rumen

(Brulc et al., 2009), planktonic marine microbial assemblages (Beja et al., 2000;

| 13 Review of Literature

Breitbart et al., 2002), deep sea microbiota (Sogin et al., 2006), an acid mine site

(Tyson et al., 2004), arctic sediments (Jeon et al., 2009) and the Sargasso Sea (Venter et al., 2004).

2.1.1 PCR-based approaches

Polymerase Chain Reaction (PCR) is the basic technique in most molecular laboratories. With the advent of PCR (Saiki et al., 1988), a method became available to study genetic information in an efficient way. With specific primers, perfect amplification of virtually all nucleotide sequences is possible. Functional genes from different microorganisms can be amplified, but for molecular identification of microbes the 16S rRNA gene is mostly used. At a length of approximately 1500 nucleotides,

16S rRNA genes contain sufficient information for reliable phylogenetic analysis

(Amann et al., 1995). An advantage of the 16S rRNA sequences is the existence of a large database with currently about 500,000 (> 300 bp) small subunit rRNA sequences

(www.arb-silva.de), which can be used for the design of primers (Ludwig et al., 2004).

Since the 16S rRNA gene has conserved regions, with the same sequence in many microorganisms and variable regions, which sequences can be species specific, amplification and hence, detection at different levels of taxonomic specificity can be achieved. The use of conserved 16S rRNA sequences also allows detection of previously unknown and uncultured microorganisms. However, only limited conclusions regarding the biogeography and ecophysiology of uncultured bacteria can be drawn from the mere presence or absence of their 16S rRNA sequences in an ecosystem (Jaspers and Overmann, 2004). Moreover, it is known that PCR can cause biases, for example by preferential amplification of certain sequences (Head et al.,

1998). During exponential amplification of the mixture of DNA templates, ratio discrepancies between the amplified 16S rRNA gene fragments and the original

| 14 Review of Literature

mixture, caused by differences in Taq polymerase activity with varying G+C content may occur. While primers with 70% similarity to target sequences from pure cultures are sufficient for successful annealing and amplification, poor complementarity of

‘universal’ primers, especially at the 3’ end, will result in under representation of these sequences in the final PCR product (Baker et al., 2003; Polz and Cavanaugh 1998;

Stern and Holland 1993). Furthermore, chimeric sequences and other PCR-generated artefacts may arise during amplification, especially from complex microbial communities with several similar phylotypes (Hugenholtz and Huber 2003; Osborne et al., 2005). A Ligase Chain Reaction (LCR) can have greater specificity than PCR, since two oligonucleotides are used for each DNA strand and are ligated together to form a single primer (Barany, 1991a,b). LCR uses both a DNA ligase enzyme and a DNA polymerase to perform the reaction.

Real-time or quantitative PCR is based on the continuous monitoring of changes of fluorescence in the PCR tube during PCR and, in contrast to the conventional end- point detection PCR, quantification occurs during the exponential phase of amplification (Malinen et al., 2003). Thus, the bias often observed in the PCR template- to-product ratios can be largely avoided (Suzuki and Giovannoni, 1996). Real-time

PCR was developed in 1992 and made use of ethidium bromide and a fiber optic cable connected to a spectrofluorometer (Higuchi et al., 1992). Nowadays a broad spectrum of detection chemistries and machines with advanced software are available (Valasek and Repa, 2005). When compared to dot-blot hybridization, real-time PCR has superior sensitivity, more convenient and also less expensive for the quantification of selected bacterial populations (Malinen et al., 2003). Quantification of rRNA that is isolated directly from the ribosomes may be used to reveal the metabolically most active members of a bacterial community.

| 15 Review of Literature

Other quantitative PCR approaches are competitive (RT-)PCR and Most

Probable Number (MPN) PCR. With competitive PCR a specific standard of known concentration is added at different concentrations to the target followed by PCR amplification. The difference in size between the target and the standard allows discrimination and subsequent quantification on an agarose gel. The principle of

MPN-PCR is similar to MPN cultivation for the quantification of microorganisms.

Target DNA is diluted until extinction and used as template for PCR using species or group specific primers. The products from the MPN PCR are also analysed by agarose gel electrophoresis, which is one of the weak points of these quantitative PCR approaches, since agarose gels are not highly discriminative resulting in a low resolution. Truly quantitative information using molecular methods can only be obtained if cell lyses and extraction efficiency, as well as biases in the PCR step are under strict experimental control (Petersen and Dahllöf, 2005). Also the earlier mentioned disadvantages of PCR end-point detection play a role in competitive (RT-)

PCR and MPN PCR quantitative approaches.

2.1.2 Denaturing/ Temperature gradient gel electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE), or the nowadays less used

Temperature Gradient Gel Electrophoresis (TGGE), is based on the melting behavior of double stranded DNA fragments. The melting behavior, mostly described as the melting temperature (i.e. the temperature, where a double-stranded nucleic acid fragment dissociates), is dependent on the nucleotide composition of a fragment. Even in the case of a single-nucleotide substitution, fragments will potentially melt at a different temperature. By adding a GC-rich tail (GC-clamp) to one of the primers for the amplification, a fragment is produced that will partially melt, when it is electrophoresed in a denaturing gradient poly acryl amide gel. The GC-clamp will

| 16 Review of Literature

remain double stranded and the resulting fork-like structure causes the fragment essentially to stop migrating. This process will occur at a different position in the gel, when one or more base pair(s) are substituted or deleted. Therefore, mutations can be detected. Normally urea and formamide gradient is applied in DGGE and a temperature gradient in TGGE. A certain concentration of urea/formamide combined with e.g. 60ºC has the same effect as a much higher temperature without the presence of urea/ formamide. Muyzer et al., (1993) first applied DGGE to analyse complex bacterial communities. DGGE fingerprinting is an excellent and effective method to follow changes of microbial communities in time and space. In addition, DGGE is well suited for monitoring of complex communities, focusing on phylotypes for which the occurrence and/or the relative frequency are affected by any environmental change

(Akkermans et al., 2000; Fromin et al., 2002). By inter-sample comparison, dominant shifts in population dynamics can be studied in more detail. The intensity of an individual band is a semi-quantitative measure for the relative abundance of this sequence in the community (Vaughan et al., 2000). It has been reported that DGGE or

TGGE are sensitive enough to detect organisms that constitute upto 1% of the total microbial community (Muyzer et al., 1993; Zoetendal et al., 1998). This means that only the most dominant bacteria will be represented in the profiles when domain- specific primers are used, while with group-specific primers also minor microbial groups can be followed by DGGE analysis. For example, Heuer et al., (1997) generated

DGGE profiles with primers specific for Actinomycetes and Boon et al., (2002) used multiple group-specific primer pairs for the generation of a set of DGGE fingerprints characterising microbiota in different wastewater treatment plants. With improvements of statistical software, similarity indices can be calculated and cluster analysis of community profiles can be performed. Gafan et al., (2005), for example, analysed

DGGE profiles in three different ways: bacterial diversity by using the Shannon-

| 17 Review of Literature

Wiener index; hierarchical cluster analysis, expressed as a dendrogram; and individual

DGGE bands and their intensities were compared using logistic regression analysis.

Fromin et al., (2002) provided an excellent overview of currently used statistical analysis methods for DGGE data. In addition, Internal Standards in Molecular Analysis of Diversity (ISMAD) might be especially useful in comparative ecological and eco-toxicological DGGE experiments where differences between treatments from the same original community are studied and where changes due to treatment effects are sought (Petersen and Dahllöf, 2005). The use of ISMAD during PCR-DGGE makes it possible to analyse whether differences in bacterial abundance and diversity are due to differences in the original samples, or due to biases from experimental variability between samples introduced during the DNA extraction, PCR and DGGE (Petersen and

Dahllöf, 2005). Recently, a Denaturing High-Performance Liquid Chromatograph

(DHPLC) method was used and compared with DGGE for the analysis of natural bacterial communities and DHPLC analysis produced profiles of the community that described the composition and abundance of bacterial species (Barlaan et al., 2005).

The major advantage of DHPLC compared with gel-based approaches includes the use of automated instrumentation and convenience of analysis, especially the fraction collection of peaks for DNA isolation and sequencing. Sequencing of DGGE bands directly or via a cloning step, is important to identify the microorganisms behind the banding profiles.

2.1.3 Single strand conformation polymorphism based approaches

Another technique for the detection of differences in DNA sequences using separation by electrophoresis is Single Strand Conformation Polymorphism (SSCP).

Comparable to D(T)GGE, this technique was first developed in clinical research for the detection of known or novel polymorphisms of point mutations in DNA

| 18 Review of Literature

(Orita et al., 1989). Single-stranded DNA is separated in a polyacrylamide gel based on differences in mobility caused by their secondary structure. Even when DNA fragments are of equal size and no denaturant is present, folding and hence mobility will be dependent on the DNA sequences (Lee et al., 1996). By the use of a variable part of the

16S rRNA gene (for example the V3 region) from an environmental sample, each

SSCP peak corresponds to a distinct microbial sequence, indicating the presence of a microbial strain or species retrieved from the sample (Leclerc et al., 2001; Lee et al.,

1996). Lee et al., (1996) reported that SSCP can be applied to separate 16S rRNA gene fragments which are amplified from complex bacterial assemblages and that it was sensitive enough to detect a bacterial population that accounted for less than 1.5% of a bacterial community. In general, SSCP has the same limitations as DGGE. Some single-stranded DNA fragments can form more than one stable conformation and therefore one sequence may be represented by multiple bands (Tiedje et al., 1999).

However, SSCP does not require a GC-clamp or the construction of gradient gels and is therefore potentially more simple and straightforward than DGGE (Schwieger and

Tebbe, 1998). Hori et al., (2006) compared DGGE and SSCP based on the V3-V4

16S rRNA gene region and concluded that SSCP was superior compared to DGGE in detecting the bacterial dynamics of a methanogenic bioreactor. If one PCR primer is labelled with a fluorescent dye the detection can be automated and heteroduplex formation can be avoided. Furthermore, probes can be easily used for further detection and identification of certain bands. Denaturing gradient electrophoresis and SSCP fingerprinting patterns do not reflect only a tiny fraction of the real diversity, but correspond to a representation of the whole microbial community, when not only the number of visible bands or peaks are considered, but when the whole picture, including background is analysed (Loisel et al., 2006).

| 19 Review of Literature

2.1.4 Restriction analysis based approaches

A large number of molecular techniques make use of specific nucleic acid- modifying enzymes, initially purified and characterised from microorganisms. The thermo-stable Taq DNA polymerase, used for PCR, is a good example, but also restriction enzymes are widely used. Restriction enzymes recognize specific DNA sequences and cut in a reproducible way. The combination of PCR and restriction can, for example, be used for enhanced amplification of minor DNA templates (Green and

Minz, 2005). Unwanted or dominant DNA templates can be amplified in a first round of PCR, the produced double stranded products cut by restriction enzymes, resulting in the digested template no longer being available for PCR amplification (Green and

Minz, 2005).

In Amplified Ribosomal DNA Restriction Analysis (ARDRA), the ribosomal

RNA gene is amplified by PCR and digested into specific fragments by restriction enzymes (usually with 4-bp recognition sites). After the incubation with restriction enzymes, fragments are separated by high resolution gel electrophoresis, resulting in specific patterns from different sequences. ARDRA can be used for rapid comparison of rRNA genes (Laguerre et al., 1994; Moyer et al., 1994). The typical analysis of restriction digests for isolates or clones is performed on agarose gels, while for community analysis the potentially large number of fragments can be resolved by using polyacrylamide gels to produce a community-specific pattern (Martinez-Murcia et al.,

1995), but new high resolution matrices are nowadays available as well.

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is another derived fingerprinting technique and makes use of restriction enzymes as well, but only terminal restriction fragments (T-RF) are detected and used for qualitative and

| 20 Review of Literature

quantitative analysis (Liu et al., 1997). T-RFLP employs PCR, in which one of the two primers is fluorescently labelled at the 5’-end, for the amplification of a specific region of the 16S rRNA gene or for the amplification of functional genes. After amplification,

PCR products are cleaved with a site-specific restriction endonuclease to obtain genetic fingerprints of microbial communities or a specific product from a single micro- organism and the T-RF’s are precisely measured by using an automated DNA sequencer. Size markers bearing a different fluorophore can be included in every sample and complex communities can result in 60-80 unique T-RF (Marsh, 1999). The areas under the peaks of the obtained electropherograms indicate the relative proportions of the fragments. Several online tools have been developed for the performance of in silico hydrolysis of 16S rRNA gene sequences and recently an ARB- implemented tool was developed to predict the terminal restriction fragments of aligned small-subunit rRNA gene or functional gene sequences (Abdo et al., 2006; Ricke et al.,

2005). T-RFLP is a powerful tool for assessing the diversity of complex microbial communities and for rapid comparison of the community structure and diversity of different ecosystems. Like with DGGE, however, data obtained by T-RFLP should be cautiously interpreted, since microbial populations that are not numerically dominant are not represented, because the template DNA’s from these populations represent a small fraction of the total community DNA and consequently the species diversity of the microbial community is underestimated (Liu et al., 1997). Furthermore, determination of similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme may lead to erroneous conclusions. Hence, it is better to use multiple restriction enzymes individually, generating multiple data sets.

Osborne et al., (2006) introduced the variable threshold method for analysis of such multiple datasets, allowing more confident conclusions about the similarities of complex microbial communities.

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2.1.5 Cloning and sequencing approaches

The cloning and sequencing of 16S rRNA genes amplified directly from the environments through culture-independent approaches demonstrated that microbial diversity is far more extensive than we ever imagined from culture-based studies

(Handelsman, 2004). For proper phylogenetic characterization of microorganisms, the sequence of the marker gene used should be determined. Comparative sequencing of the 16S rRNA gene has become by far the most commonly used measure of environmental diversity (Vaughan et al., 2000). Ideally the whole genome should be sequenced and actually this is done more often nowadays. Even the metagenome of a whole community can be subjected to cloning and sequencing (Green Tringe et al.,

2005; Tyson et al., 2004; Venter et al., 2004). However, sequencing of whole community genomes is not practical (and economical), because most communities comprise atleast hundreds to thousands of species (Torsvik et al., 2002).

Pyrosequencing (Ronaghi et al., 1996) has been used to estimate the total diversity in soils and resulted in the detection of more than 50,000 OTU's (Roesch et al., 2007).

Ribosomal RNA sequences can be obtained by PCR amplification either from the encoding genes or directly from rRNA. In the case of direct amplification from rRNA, the sensitive reverse transcription-polymerase chain reaction (RT-PCR) has to be employed (Vaughan et al., 2000). Cloning consists in this case of ligation of the amplified rRNA (gene) fragments into a plasmid, usually with antibiotic resistance genes and the transformation of competent E. coli host cells with the resulting vector.

The 16S rRNA sequences that are naturally amplified in the obtained transformants can be screened, e.g. with (a combination of) ARDRA and DGGE, before isolation of the plasmids. The 16S rRNA inserts of these plasmids can be sequenced, for example with the Sanger dideoxy method (Sanger et al., 1977). Sequence comparisons of nucleic acids isolated from complex microbial ecosystems can be used to provide molecular

| 22 Review of Literature

characterization, while at the same time providing a classification system, which predicts natural evolutionary relationships (Pace, 1997; Woese et al., 1990). Several programmes for determining the most similar sequence are available at various internet sites (for example Genbank: http://www.ncbi.nlm.nih.gov/BLAST/ (McGinnis and

Madden 2004); or the RDP database: http://rdp.cme.msu.edu/ (Maidak et al., 2001;

Cole et al., 2005)) and databases containing sequences that are aligned according to their secondary structure paradigm are available (e.g. ARB (Ludwig et al., 2004)). By the use of these programmes phylogenetic relationships of microorganisms can be investigated. Furthermore, Kemp and Aller (2004a) suggested exploration of clone libraries in progressive stages by calculating phylotype richness estimators, before characterising a new subset of clones, until these estimators stabilise. They also developed a simple web based tool for these calculations (http://www.aslo.org/ lomethods/ free/ 2004/ 0114a.html) (Kemp and Aller, 2004b). However, the diversity and prevalence of individual variants within environmental bacterial populations has not been extensively explored and so questions regarding the ecological importance of genotypic variation remain unanswered (Thompson et al., 2005). Cloning is also not without bias, but sequence analysis of 16S rRNA genes has become a standard procedure in the identification of isolates and it is now impossible to adequately describe microbial communities without 16S rRNA sequence data (Leser et al., 2002;

Zoetendal et al., 2004a).

2.1.6 Fluorescence in situ hybridisation based approaches

Phylogenetically based oligonucleotide probes can be used directly for quantification via Fluorescence In Situ Hybridisation (FISH) and Dot-blot hybridization. FISH is a quantitative method on a cell by cell basis and was first developed in the late 1980s with radioactive labelled oligonucleotides, but soon

| 23 Review of Literature

fluorescently labelled oligonucleotide probes were demonstrated to allow detection of individual cells in situ much better (Giovannoni et al., 1988; De Long et al., 1989). This made whole-cell hybridisation with rRNA-targeted probes a suitable tool for determinative, phylogenetic and environmental studies in microbiology (Amann et al.,

1990). Nowadays it is even possible to detect up to seven microbial groups simultaneously with so called Rainbow-FISH (Sunamura and Maruyama, 2006).

Variables that influence the sensitivity and reproducibility of the in situ hybridisation technique include: the effects of cell fixation on target RNA preservation and accessibility to probes; the type and quality of probes; the efficiency of probe-target hybrid formation; the stability of hybrids formed in situ during post hybridisation treatments; the method of detection of hybrids; and the background noise masking the hybridisation signal (Pernthaler and Amann, 2004). Optimal fixation should result ingood probe penetration, retention of the maximal level of target RNA and maintenance of cell integrity and morphologic detail (Motor and Göbel, 2000). The accessibility of rRNA has been systematically investigated (Fuchs et al., 1998 and

2001; Behrens et al., 2003) and recently it has been shown that a rational probe design

0 strategy, involving ∆G overall, hybridisation kinetics and fluorophore quenching, resulted in no truly inaccessible target regions in the 16S rRNA of E. coli (Yilmaz et al., 2006). Cells of different species have different ribosome contents ranging roughly between 103 and 105 ribosomes per cell and even for one strain, cellular rRNA contents can vary significantly (at least over one order of magnitude), since they are directly correlated with the growth rate (Amann et al., 1995; DeLong et al., 1989).

However, the relative rRNA abundance should represent a reasonable measurement of the relative physiological activity of the respective population, since it is the product of the number of detected cells and the average rRNA content (Amann et al., 1995; de Vries et al., 2004; Wagner, 1994). FISH is a good technique for identification of

| 24 Review of Literature

specific microorganisms in complex communities and one of the best methods suited for quantification of these microorganisms in situ, while maintaining structural information. Probes for FISH have to be chosen wisely and in a nested approach to ensure the correct enumeration and identification (Schmid et al., 2005). Recently, a method for simultaneous FISH of mRNA and rRNA in environmental bacteria was published, which facilitates the simultaneous identification, activity detection and assessment of biogeochemical impact of individual organisms in situ (Pernthaler and

Amann, 2004). Such developments are possible by increasing the signal intensity and

Zwirglmaier published a comprehensive review on this topic, focussing mainly on two widely recognised varieties, tyramide signal amplification and multiply labelled polynucleotide probes (Zwirglmaier, 2005). In principle, five objectives of microbial ecological studies can be addressed with FISH: bypass cultivation problems; obtain information on community structure by using varying sets of probes; accurately enumerate target populations; identify sub-populations in natural systems and locate their niche; and determine the in situ cellular rRNA content (as a metabolic fitness measurement) (Vaughan et al., 2000). Without automation, however, FISH is very laborious and only a few probes can be used per analysis. Flow Cytometry (FCM)- based approaches could overcome this limitation and several studies have demonstrated the use of FCM in association with FISH (Davey and Kell 1996; Porter et al.,1996;

Wallner et al., 1995). As an extension to FCM, Fluorescent Activated Cell Sorting

(FACS) can be used to separate different populations from a mixed community, allowing for the physical enrichment or isolation even of yet uncultured organisms that can be used for subsequent molecular genetic studies and cultivation.

| 25 Review of Literature

2.1.7 Dot-blot hybridisation based approaches

A disadvantage of PCR based approaches is that they do not always provide unbiased quantitative data because of amplification biases. Quantification of certain

16S rRNA sequence type relative to the total 16S rRNA content of a given sample can be obtained by dot-blot hybridization of a directly isolated nucleic acid mixture with universal and specific oligonucleotide probes (Amann et al., 1995; Raskin et al., 1994).

For this purpose, total RNA is isolated from a sample, immobilised on a membrane and hybridised with labelled oligonucleotide probes. The relative abundance can be calculated as a ratio of the amount of specific probe bound to a given sample to the amount of hybridised universal probe (Amann et al., 1995). When radioactively labelled oligonucleotide probes were used in a dot-blot assay, rRNA sequences with a relatively low abundance between 0.1 and 1% could be quantified (Amann et al., 1995).

Although quantification is very accurate, these data of relative rRNA abundance cannot be directly translated into cell numbers, since cells of different species have different ribosome contents ranging roughly between 103 and 105 ribosomes per cell (Amann et al., 1995). However, because PCR or other amplification procedures are not involved, the quantification is very accurate and many oligonucleotide probes have been developed, validated and successfully used in the past 15 years. Recently, many of those probes have been collected in an interactive web-based database, ProbeBase

(http://www.microbial-ecology.net/probebase/) (Loy et al., 2003). Essentially all types of samples can be used for quantitative dot-blot hybridisation, which makes it the method of choice in those systems which are difficult for FISH (e.g. patchy environments) (Amann and Ludwig, 2000). Furthermore, von Wintzingerode et al.,

(1999) prepared specific PCR-made probes from a selection of bacterial clones to screen their bacterial clone library via dot-blot hybridisation.

| 26 Review of Literature

2.1.8 Phylogenetic microarrays

DNA microarrays (also called DNA chips, gene chips or biochips) typically consist of thousands of immobilised DNA fragments (PCR product, oligonucleotides or other DNA fragments) present on a surface, such as coated glass slide or membrane

(Ye et al., 2001). It can be considered as a reverse traditional dot-blot, since the identity of the spotted probes is known and the sample is labelled. Labelled sample nucleic acids will mark the exact positions on the array where hybridisation occurred. The microarray experiment output consists of a list of hybridisation events, indicating the presence or the relative abundance of specific DNA or RNA sequences present in the sample. Microarrays are already widely used for the detection of transcriptional profiles

(expression arrays) or the similarities and differences of genetic contents among different microorganisms and they can be used to subtype (fingerprint strains relative to the reference strain) bacterial isolates and for the identification of new diagnostic genetic markers (Call et al., 2003). Also mutation detection and the search of polymorphisms are done with microarrays, but the detection and identification of high numbers of different microbes, especially from complex microbial communities in environmental samples with microarrays is still very challenging. An annotated selection of World Wide Web sites relevant to environmental microbiological microarrays was recently published (Wackett 2006) as well as a number of excellent reviews (Letowski et al., 2003; Blaškovič and Barák 2005; Loy and Bodrossy 2006;

Wagner et al., 2007). The rRNA is still the most widely used marker gene, but a number of higher resolution and/or non-universal (i.e. focused on a narrower group of microbes) genes are also being used as markers (Bodrossy and Sessitsch, 2004;

Bodrossy et al., 2006). By using consensus primers to amplify and label targets, it is possible to differentiate a large number of organisms, provided that sufficient polymorphism exists within the amplified region (Nicolaisen et al., 2005).

| 27 Review of Literature

Use of microarrays for determinative studies provides several advantages over conventional hybridisation formats. Thousands of different oligonucleotides can be immobilised on a single array, allowing the simultaneous detection of a great variety of different microorganisms in a single sample. There is a low sample requirement, automation and high-throughput analysis with microarrays can be realised easily.

Microarrays, used in combination with high spatial resolution in situ measurements of concentrations of inorganic and organic ions and molecules, pH and redox potential, could allow linkage of gene expression or protein production to specific organisms and processes (Tyson and Banfield, 2005). Furthermore, DNA microarrays are promising for the quantification of microbial genes and therefore highly suited for molecular ecology studies (Cho and Tiedje 2002; DeSantis et al., 2005; Palmer et al., 2007;

Rajilic-Stojanovic, 2007). Direct detection and identification of rRNA from environmental samples, without PCR amplification is possible (Small et al., 2001;

Koizumi et al., 2002; Denef et al., 2003; El Fantroussi et al., 2003; Peplies et al., 2004;

Kelly et al., 2005). Two enzymatic signal amplification systems that have been employed are enzyme-linked fluorescence (ELF-97; Molecular Probes) and tyramide signal amplification (TSA) (Call et al., 2003). The sensitivity of microbial diagnostic microarrays is usually defined as the lowest relative abundance of the target group detectable within the analysed community and is in the range of 1-5% (Bodrossy and

Sessitsch, 2004). Currently the method of choice is the synergistic combination of microarray and real-time PCR, in which the screening is performed by microarrays and the precise quantification and high-throughput screening of selected target sequences is achieved by real-time PCR (Klein, 2002).

Although DNA microarrays have many advantages and are very promising, many optimisations have to be done. Adapting microarray hybridisation for use in environmental studies faces, besides quantification, several challenges associated with

| 28 Review of Literature

specificity and sensitivity (Zhou and Thompson 2002; Cook and Sayler, 2003).

Discrimination between perfect and mismatch binding between the probes on the array and the genetic content of the targets is needed (Liu et al., 2001; Urakawa et al., 2002;

Urakawa et al., 2003). In some studies oligonucleotide probes were designed to have almost identical melting temperatures, but there are indications that present models are only capable of predicting Tm values of free probes and not those of surface-bound probes (Bodrossy et al., 2003; Franke-Whittle et al., 2005). Thermal dissociation curves for each probe-target duplex and application of multiple probes for specific targets on the DNA microarray help to avoid false positive detection (El Fantroussi et al., 2003;

Liu et al., 2001; Urakawa et al., 2002; Urakawa et al., 2003). Understanding the principles governing nucleic acid hybridisations of short probe-target duplexes is necessary and fundamental for the application of microarray technology to routine environmental microbiology because it would facilitate the design of proper probes, minimize the changes of non-specific hybridisation and improve the ability for confident interpretation of microarray data (Urakawa et al., 2002). Urakawa et al.,

(2002) found for example that besides the formamide concentration in the hybridisation buffer (stringency of the hybridisation buffer), the position and type of mismatch are important for discrimination between perfect match and mismatch hybridisation

(Urakawa et al., 2002). He et al., (2005) investigated the effects of probe-target identity, continuous stretch (length of probe part without any mismatch), mismatch position and free energy on the design of 50-mer and 70-mer probes and then experimentally compared the designed probes to establish probe design criteria.

They found that a combination of similarity (85%), stretch (15 bases) and free energy (-30 kcal/mol) was able to exclude all non-specific probes for 50-mer probes and a combination of similarity (85%), stretch (20 bases) and free energy

(-40 kcal/mol) for 70-mer probes (He et al., 2005). The criteria for 50-mer oligonucleotides were recently reevaluated and a combination of 90% similarity,

| 29 Review of Literature

20 bases stretch and a free energy of – 35 kcal/mol were found to be predictive of probe specificity (Liebich et al., 2006). With this necessary background information and availability of three dimensional structures for large amount of probe binding and direct dissociation monitoring (e.g. polyacrylamide gel patches (Yershov et al., 1996; Guschin et al., 1997) or porous metal oxide (Beuningen et al., 2001)) and high-density microarrays (e.g. Affymetrix GeneChips (Brodie et al., 2006; Brodie et al., 2007;

DeSantis et al., 2005; DeSantis et al., 2007; Wilson et al., 2002a; Wilson et al., 2002b)), the routine use of this new technology is within reach, however, data handling and interpretation of data are still challenging. Also bead-based methods for multiplexed identification and quantification with a flow cytometer have great potential (Spiro et al.,

2000, Wireman et al., 2006). Completely integrated miniature systems, so called

‘laboratories-on-a-chip’, will further improve microbial monitoring by achieving detection and identification within minutes at the single-cell level (Liu and Zhu, 2005).

This will deepen the understanding of microbial community structure and diversity and correlate these to the spatial distribution of microorganisms.

2.2 Culture-dependent approaches for heavy metal removal The use of microbial communities to assess the impact caused by anthropogenic stress in natural habitats is increasing; however, there is considerable debate as to which approach is the most useful (Chapman, 1999). Culture-independent methods have received particular attention because it is commonly known that only a small proportion of the bacteria present in any given environment will form colonies on general laboratory media (Hugenholtz, 2002), but on the other hand, the traditional microbiological methods directly provide live bacteria and not merely a “molecular strain”.

| 30 Review of Literature

Culture-dependent approaches are used for the retrieval and detailed investigation of microorganisms present in ecosystems. Ideally also the activity and function of microorganisms can be investigated by cultivation and/or by in situ measurements. Physiological and biochemical properties can be used for the identification and characterization of microorganisms. Detailed physiological studies, which are often important in ecological research, almost always require cultivation of relevant microorganisms (Briones and Raskin, 2003). Cultivation techniques have seen amazing improvements in the last decennia, allowing the cultivation of numerous, previously uncultured, microorganisms. It is also possible to propagate defined co-cultures of syntrophic microorganisms and although it is complicated to study the metabolism of these microorganisms separately, these techniques have facilitated the generation of knowledge about complicated microbial assemblages (Boone and Bryant,

1980). The number of microbial isolates has increased considerably, in particular because of improved knowledge of microbial metabolism as well as characteristics of ecological niches at which specific populations are naturally occurring. This enabled researchers to design better isolation strategies. Also, information about the genetic potential of uncultivated organisms derived from genomic or metagenomic sequences can be used to predict metabolic interdependencies and nutritional requirements (Tyson and Banfield, 2005). Another important driving force in the development and successful application of novel cultivation approaches is the growing interest of industry in exploiting the metabolic capacity of yet unknown microorganisms and their enzymes (bioprospecting (Keller and Zengler, 2004)). For example, various processes for high cell density cultivation have been developed, which made the optimisation of formation of bioproducts possible (Park, 2004). Conventional isolation and cultivation methods are very sensitive techniques to detect microorganisms that easily grow in pure culture and that can be cultivated on selective media. Theoretically each viable cell can be multiplied and may be detected as a colony on a selective agar plate and by

| 31 Review of Literature

combining different cultivation techniques, it is possible to detect and identify very low numbers of microorganisms (Akkermans et al., 1994). The fraction of cells capable of multiplication (cultivability) on solid media can be quantified as colony forming units

(CFU) (Bruns et al., 2003). Samples of naturally occurring microbial communities are used as inoculum for laboratory prepared growth media that are designed to select a small subset of the initial community and therefore an enrichment of certain microorganisms is achieved. Since many microorganisms prefer liquid media, most probable number (MPN) dilution series are often used for the quantification and enrichment of target populations. The highest dilutions of MPN series with detectable growth provide enrichments or even pure cultures of abundant, but fastidious bacteria which are missed in conventional enrichment trials, since, dominant organisms are frequently overgrown by accompanying species which are less abundant, but grow faster under laboratory conditions. However, a principal limitation of the MPN method is that only a small number of parallel dilution series can be processed for each sample, which limits the number of potential isolates and causes a large statistical uncertainty.

To overcome these limitations, a novel approach for high throughput cultivation was developed. With a MicroDrop microdispenser system droplets of 150-200 pl are created at the nozzle of a glass micropipette by means of a computer driven piezo transducer and are dispensed automatically at predetermined positions with the aid of a XYZ positioning system, so that the inoculation of 96 samples takes less than a minute.

Bruns et al., (2003) compared the MicroDrop technique with the MPN technique and found that the MicroDrop technique tends to yield cultivation efficiencies which are a factor of 2.7 (r2 = 0.570, level of significance P < 0.001) lower. This may be due to an under estimation of cultivable bacteria by the MicroDrop technique and/or from an overestimation by the MPN method, caused by high surface tension and shear forces or clumps of microorganisms respectively. However, the MicroDrop technique makes high throughput cultivation possible, since many cultures can be started in parallel. In

| 32 Review of Literature

another study microbial cells were encapsulated in gel microdroplets for massive parallel microbial cultivation under low nutrient flux conditions, followed by flow cytometry to detect microdroplets containing microcolonies (Zengler et al., 2002). Gel microdroplets separate microorganisms from each other while still allowing the free flow of metabolites and signalling molecules between different microcolonies.

Therefore, this method might be also applicable for the analysis of interactions between different microorganisms under in situ conditions (Zengler et al., 2002).

Straightforward techniques like the micromanipulator or a Fluorescence Activated Cell

Sorter (FACS) can also be used for physical separation of microorganisms from mixed cultures (Fröhlich and König 1999; Park et al., 2005a).

Nevertheless, ideal growth conditions and growth requirements are not very well known for most microorganisms. The validity of measurements conducted on microbial communities removed from their original field setting is uncertain; because it is not sure that conditions imposed on the native microorganisms (post-sampling and incubation) have not quantitatively or qualitatively altered these populations and their physiological reactions (Madsen, 2005). Therefore, cultivation will always be selective for certain microorganisms and will be normally not comparable to the natural ecosystem from which the sample is derived (e.g. high concentrations of nutrients).

Only 27 out of 53 bacterial phyla contain previously cultivated microorganisms, with many phyla represented by only few isolates and some phyla containing only one described species (Keller and Zengler, 2004; Rappé and Giovannoni, 2003). Current inventories list about 8200 validly classified species of prokaryotes. The inability to isolate and cultivate many types of microbes has long been limited the range of microorganisms that are available for further analysis. Although the vast majority of microbes resist cultivation by traditional methods, it has been proposed that many more strains could be isolated using novel imaginative approaches. However, it is well

| 33 Review of Literature

known that many microorganisms do not grow under conditions routinely used in the laboratory and growth conditions of most microorganisms are actually not known.

Besides unknown growth requirements, stress caused by the cultivation procedures or obligatory interactions of microorganisms with each other may be additional reasons that hamper cultivation. Still, the ability to study individual strains from different environments under laboratory conditions is often essential to obtain insight in their metabolic function. If methods are found both to reveal the form of microbial interdependencies and to simulate them in the laboratory, cultivation of targeted strains can be achieved (Tyson and Banfield, 2005). The combination of methods for the direct description of microbial communities with traditional methods for enrichment and isolation of important strains is and will stay powerful in future research.

2.2.1 Heavy metal pollution and bacterial biosorption Heavy metal pollution is one of the most important environmental problems today. Various industries produce and discharge wastes containing different heavy metals into the environment, such as mining and smelting of metalliferous, surface

finishing industry, energy and fuel production, fertilizer and pesticide industry and application, metallurgy, iron and steel, electroplating, electrolysis, electro-osmosis, leather working, photography, electric appliance manufacturing, metal surface treating, aerospace and atomic energy installation etc. Thus, metal as a kind of resource is becoming shortage and also brings about serious environmental pollution, threatening human health and ecosystem. Three kinds of heavy metals are of concern, 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).

| 34 Review of Literature

Heavy metal pollution occurs directly by effluent outfalls from industries, refineries and waste treatment plants and indirectly by the contaminants that enter the water supply from soils/ground water systems and from the atmosphere via rain water

(Vijayaraghavan and Yun, 2008). Modern industry is, to a large degree, responsible for contamination of the environment. Lakes, rivers and oceans are being overwhelmed with many toxic contaminants. Among toxic substances reaching hazardous levels are heavy metals (Vieira and Volesky, 2000). Heavy metals are the group of contaminants of concern, which comes under the inorganic division. Some strong toxic metal ions such as Hg are very toxic even in lower concentration of 0.001- 0.1 mg L-1. Metals are extensively used in several industries, including mining, metallurgical, electronic, electroplating and metal finishing. The presence of metal ions in final industrial effluents is extremely undesirable, as they are toxic to both lower and higher organisms. Under certain environmental conditions, metals may accumulate to toxic levels and cause ecological damage (Jefferies and Firestone, 1984). Of the important metals, Mercury, lead, cadmium, Arsenic and Chromium (VI) are regarded as toxic; whereas, others, such as copper, nickel, cobalt and zinc are not as toxic, but their extensive usage and increasing levels in the environment are of serious concerns

(Brown and Absanullah, 1971; Moore, 1990; Volesky, 1990). There are many sources of water pollution, but two main general categories exist: direct and indirect contaminant sources. Direct sources include effluent outfalls from industries, refineries and waste treatment plants; whereas, indirect sources include contaminants that enter the water supply from soils/ground water systems and from the atmosphere via rain water. Comprising over 70% of the Earth's surface, water is undeniably the most valuable natural resource existing on our planet. Without this invaluable compound, the life on the Earth would be non-existent. Although this fact is widely recognized, pollution of water resources is a common occurrence. In particular, potable water has

| 35 Review of Literature

become greatly affected and in many instances has lost its original purpose

(Vijayaraghavan and Yun, 2008).

Methods for removing metal ions from aqueous solution mainly consist of physical, chemical and biological technologies. Conventional methods for removing metal ions from aqueous solution have been suggested, such as chemical precipitation,

filtration, ion exchange, electrochemical treatment, membrane technologies, adsorption on activated carbon, evaporation etc. However, chemical precipitation and electrochemical treatment are ineffective, especially when metal ion concentration in aqueous solution is among 1 to 100 mg L− 1 and also produce large quantity of sludge required to treat with great difficulty. Ion exchange, membrane technologies and activated carbon adsorption process are extremely expensive when treating large amount of water and wastewater containing heavy metal in low concentration, they cannot be used at large scale (Atkinson et al., 1998; Crini, 2006). Therefore, the search for efficient, eco-friendly and cost effective remedies for waste-water treatment has been initiated. In recent years, applying biotechnology in controlling and removing metal pollution has been paid much attention and gradually becomes hot topic in the

field of metal pollution control because of its potential application. Research attention has been focused on biological methods for the treatment of effluents, some of which are in the process of commercialization (Prasad and Freitas, 2003). There are three principle advantages of biological technologies for the removal of pollutants; first, biological processes can be carried out in situ at the contaminated site; Second, bioprocess technologies are usually environmentally benign (no secondary pollution) and third, they are cost effective. Of the different biological methods, bioaccumulation and biosorption have been demonstrated to possess good potential to replace conventional methods for the removal of heavy metals (Volesky and Holan, 1995;

Malik, 2004). Some confusion has prevailed in the literature regarding the use of the

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terms “bioaccumulation” and “biosorption” based on the state of the biomass. Herein, therefore, bioaccumulation is defined as the phenomenon of living cells; whereas, biosorption mechanisms are based on the use of dead biomass. To be precise, bioaccumulation can be defined as the uptake of toxicants by living cells. The toxicant can transport into the cell, accumulate intracellularly, across the cell membrane and through the cell metabolic cycle (Malik, 2004). Conversely, biosorption can be defined as the passive uptake of toxicants by dead/inactive biological materials or by materials derived from biological sources. Biosorption is due to a number of metabolism- independent processes that essentially take place in the cell wall, where the mechanisms responsible for the pollutant uptake will differ according to the biomass type.

Biosorption possesses certain inherent advantages over bioaccumulation processes, which are listed in (Table 1.) In general, the use of living organisms may not be an option for the continuous treatment of highly toxic organic/inorganic contaminants. Once the toxicant concentration becomes too high or the process operated f or a long time, the amount of toxicant accumulated will reach saturation

(Eccles, 1995). Beyond this point, an organism's metabolism may be interrupted, resulting in death of the organism. This scenario can be avoided in the case of dead biomass, which is flexible to environmental conditions and toxicant concentrations.

Thus, owing to its favorable characteristics, biosorption has, not surprisingly, received much attention in recent years.

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Table 1. Comparison of the features of biosorption and bioaccumulation

Features Biosorption Bioaccumulation

Cost Usually low. Most biosorbents used Usually high. The process involves living were industrial, agricultural and other cells and; hence, cell maintenance is cost type of waste biomass. Cost involves prone. mainly transportation and other simple processing charges. pH The solution pH strongly influences the In addition to uptake, the living cells uptake capacity of biomass. However, themselves are strongly affected under the process can be operated under a extreme pH conditions. wide range of pH conditions

Temperature Since the biomass is inactive, Temperature severely affects the process temperature does not influence the process. In fact, several investigators reported uptake enhancement with temperature rise.

Maintenance/ Easy to store and use External metabolic energy is needed for storage maintenance of the culture

Selectivity Poor. However, selectivity can be Better than biosorption improved by modification/processing of biomass

Versatility Reasonably good. The binding sites can Not very flexible. Prone to be affected by accommodate a variety of ions. high metal/salt conditions.

Degree of Very high. Some biomasses are reported Because living cells are sensitive to high uptake to accommodate an amount of toxicant toxicant concentration, uptake is usually nearly as high as their dry weight. low

Rate of uptake Usually rapid. Most biosorption Usually slower than biosorption. Since mechanisms are rapid. intracellular accumulation is time consuming.

Toxicant High under favorable conditions. Depends on the toxicity of the pollutant affinity

Regeneration High possibility of biosorbent Since most toxicants are intracellularly and reuse regeneration, with possible reuse over a accumulated, number of cycles. the chances are very limited

Toxicant With proper selection of elutant, Even if possible, the biomass cannot be recovery toxicant recovery is possible. In many utilized for next cycle. instances, acidic or alkaline solutions proved an efficient medium to recover toxicants.

Biosorbents for the removal of heavy metals mainly come under the following categories: bacteria, fungi, algae, industrial wastes, agricultural wastes and other

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polysaccharide materials. In general, all types of biomaterials have shown good biosorption capacities towards all types of metal ions. Potent metal biosorbents under the class of bacteria include genre of Bacillus (Nakajima and Tsuruta, 2004; Tunali et al., 2006), Pseudomonas (Chang et al., 1997; Uslu and Tanyol, 2006) and Streptomyces

(Mameri et al., 1999; Selatnia et al., 2004a)) etc. Important fungal biosorbents include

Aspergillus (Kapoor and Viraraghavan, 1997; Jianlong et al., 2001; Binupriya et al.,

2006), Rhizopus (Bai and Abraham, 2002; Park et al., 2005b) and Penicillium (Niu et al., 1993; Tan and Cheng, 2003), etc. Since these microorganisms are used widely in different food /pharmaceutical industries, they are generated as waste, which can be attained free or at low cost from these industries.

Heavy metals are highly persistent in the environment and are known to alter soil ecosystem diversity, structure and function (Sandaa et al., 2006; Ashraf and Ali,

2007). While the acute effect of heavy metals on the microbial community appears to lead to a subsequent shift in the community toward a more metal tolerant or metal resistant population (Ranjard et al., 2000; Sandaa et al., 2001), in chronically contaminated sites natural selection should have resulted in a pre dominantly metal tolerant community (Kandeler et al., 2000; Becker et al., 2006). Many studies have focused on the effects of heavy metals on bacterial community structure (Ranjard et al.,

2006; Ogilvie and Grant 2008; Khan et al., 2010; Pechrada et al., 2010) and relatively many bacteria have already been isolated from different heavy metal contaminated environments and their metabolic pathways for pollutant detoxification have been studied in detail. These studies included the mercury-reducing bacteria Bacillus megaterium MB1 (Huang et al., 1999); the cadmium-accumulating bacteria

Rhodospirillum rubrum (Smiejan et al., 2003); strains resistant to multiple heavy metals

(Schmidt and Schlegel 1994; Taghavi et al., 1994; Mergeay et al., 2003); bacteria

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specific to HgCl2 contaminated soils, such as Duganella violaceinigra, Lysobacter koreensis and Bacillus panaciterrae (Mera and Iwasaki, 2007); cadmium resistant bacteria, such as Alcaligenes xylosoxidans, Comamonas testosteroni, Klebsiella planticola, Pseudomonas fluorescens and Serratia liquefaciens (Chovanová et al.,

2004); Ochrobactrum sp. (Pandey et al.,2010); a Ralstonia pickettii strain, highly resistant to cadmium and a Sphingomonas sp. strain, highly resistant to zinc (Xie et al.,

2010); Aeromonas spp. and Pseudomonas spp., both highly resistant to copper (Matyar et al., 2010); and Geobacter daltonii sp. nov., an Fe(III) and uranium (VI) reducing bacterium (Prakash et al., 2010).

The most well characterized operons conveying resistance against heavy metals in Gram-negative bacteria are the czc operon from Cupriavidus metallidurans CH34

(Mergeay et al., 2003) and the ncc system from Achromobacter xylosoxidans 31A

(Schmidt and Schlegel 1994). In Gram-positive bacteria, the cad operon from Bacillus and Staphylococcus members has been well studied (Silver and Phung 1996). In both

Gram-positive and Gram-negative bacteria the ars operons from Escherichia coli

(Mobley et al., 1983; Saltikov and Olson, 2002) and Staphylococcus strains (Ji and

Silver 1992; Rosenstein et al., 1992) and the mer systems from Escherichia coli

(Nascimento and Chartone-Souza, 2003) and Bacillus populations (Bogdanova et al.,

1998) have been well characterized. In addition, the cyanobacterial smt locus from

Synechococcus PCC7942 also contains a well characterized heavy metal resistance system (Erbe et al., 1995).

2.2.2 History of bacterial biosorption

Early 1980 witnessed the capability of some microorganisms to accumulate metallic elements. Numerous research reports have been published from toxicological

| 40 Review of Literature

points of view, but these were concerned with the accumulation due to the active

metabolism of living cells, the effects of metal on the metabolic activities of the

microbial cell and the consequences of accumulation on the food chain (Volesky,

1987). However, further research has revealed that inactive/dead microbial biomass can

passively bind metal ions via various physicochemical mechanisms. With this new

finding, research on biosorption became active, with numerous biosorbents of different

origins being proposed for the removal of metals. Researchers have understood and

explained that biosorption depends not only on the type or chemical composition of the

biomass, but also on the external physicochemical factors and solution chemistry.

Many investigators have been able to explain the mechanisms responsible for

biosorption, which may be one or combination of ion exchange, complexation,

coordination, adsorption, electrostatic interaction, chelation and microprecipitation

(Vegliò and Beolchini, 1997; Volesky and Schiewer, 1999).

Table 2. Important results from the literature on metal biosorption by various bacterial species

Operating conditions Uptake Metal Organism Reference Temp (mg/g) pH (⁰C) Aluminum Chryseomonas luteola 5 NA 55.2 (L) Ozdemir and Baysal, 2004 Aeromonas caviae 2.5 20 284.4 (L) Loukidou et al., 2004 a, b Bacillus coagulans 2.5 28±3 39.9 (E) Srinath et al., 2002 Bacillus licheniformis 2.5 50 69.4 (L) Zhou et al., 2007 Bacillus megaterium 2.5 28±3 30.7 (E) Srinath et al., 2002

Chromium Bacillus thuringiensis 2 25 83.3 (L) Sahin and Öztürk, 2005 Chryseomonas luteola 4 NA 3.0 (L) Ozdemir and Baysal, 2004 Pseudomonas sp. 4 NA 95.0(L) Ziagova et al., 2007 Staphylococcus xylosus 1 NA 143.0 (L) Ziagova et al., 2007 Zooglea ramigera 2 25 27.5 (L) Saḡ and Kutsal, 1989 Bacillus sp (ATS-1) 5 25 16.3 (L) Tunali et al., 2006 Bacillus subtilis IAM 1026 5 25 20.8 (L) Nakajima et al., 2001 Copper Enterobacter sp. J1 5 25 32.5 (L) Lu et al., 2006 Micrococcus luteus IAM 1056 5 25 33.5 (L) Nakajima et al., 2001

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Pseudomonas aeruginosa PU21 5 NA 23.1 (L) Chang et al., 1997 Pseudomonas cepacia 7 30 65.3 (L) Savvaidis et al., 2003 Pseudomonas putida 6 NA 6.6 (L) Pardo et al., 2003 Pseudomonas putida 5.5 30 96.9 (L) Uslu and Tanyol, 2006 Pseudomonas putida CZ1 4.5 30 15.8 (L) Chen et al., 2005

Copper Pseudomonas stutzeri IAM 12097 5 25 22.9 (L) Nakajima et al., 2001 Sphaerotilus natans 6 NA 60 (E) Beolchini et al., 2006 Sphaerotilus natans b 5.5 30 5.4 (E) Beolchini et al., 2006 Streptomyces coelicolor 5 25 66.7 (L) Öztürk et al., 2004 Thiobacillus ferroxidans a 6 37 198.5 (L) Ruiz-Manriquez et al., 1997 Thiobacillus ferroxidans a 5 40 39.84(L) Liu et al., 2004 Aeromonas caviae 7 20 153.3 (L) Loukidou et al., 2004 a, b Bacillus circulans 7 30 26.5 (E) Yilmaz and Ensari, 2005 Enterobacter sp. J1 6 25 46.2 (L) Lu et al., 2006 Psedomonas aeruginosa PU21 6 NA 42.2(L) Chang et al., 1997

Cadmium Pseudomonas putida 6 NA 8.0(L) Pardo et al., 2003 Pseudomonas sp. 7 NA 278.0(L) Ziagova et al., 2007 Staphylococcus xylosus 6 NA 250.0(L) Ziagova et al., 2007 Streptomyces pimprina a 5 NA 30.4 (L) Puranik et al., 1995 Streptomyces rimosus a 8 NA 64.9 (L) Selatnia et al., 2004a

Iron Streptomyces rimosus a NA NA 122.0(L) Selatnia et al., 2004c Bacillus sp. (ATS-1) 3 25 92.3 (E) Tunali et al., 2006 Corynebacterium glutamicum 5 20±2 567.7 (E) Choi and Yun, 2004 Enterobacter sp. J1 5 25 50.9 (L) Lu et al., 2006 Pseudomonas aeruginosa PU21 5 NA 79.5 (L) Chang et al., 1997

Lead Pseudomonas aeruginosa PU21 b 5 50 0.7 (L) Lin and Lai, 2006 Pseudomonas putida 5.5 25 270.4 (L) Uslu and Tanyol, 2006 Pseudomonas putida 6.5 NA 56.2 (L) Pardo et al., 2003 Stretomyces rimosus a NA NA 135.0 (L) Selatnia et al., 2004b Streptoverticillium cinnamoneum a 4 28±3 57.7 (E) Puranik and Paknikar, 1997

Mercury Bacillus sp. 6 25 7.9 (L) Green-Ruiz, 2006 Bacillus thuringiensis 6 35 45.9 (L) Öztürk, 2007 Nickel Streptomyces rimosus a 6.5 NA 32.6 (L) Selatnia et al., 2004b Desulfovibrio desulfuricans 2 30 128.2 (L) de Vargas et al., 2004

Palladium Desulfovibrio fructosivorans 2 30 119.8 (l) de Vargas et al., 2004 Desulfovibrio vulgaris 2 30 106.3 (L) de Vargas et al., 2004 Desulfovibrio desulfuricans 2 30 62.5 (L) de Vargas et al., 2004

Platinum Desulfovibrio fructosivorans 2 30 32.3 (L) de Vargas et al., 2004 Desulfovibrio vulgaris 2 30 40.1 (L) de Vargas et al., 2004

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Arthrobacter nicotianae IAM 12342 3.5 30 75.9 (E) Nakajima and Tsuruta, 2004 Bacillus licheniformis IAM 111054 3.5 30 66.1 (E) Nakajima and Tsuruta, 2004 Bacillus megaterium IAM 1166 3.5 30 74.0 (E) Nakajima and Tsuruta, 2004 Bacillus subtilis IAM 1026 3.5 30 71.9 (E) Nakajima and Tsuruta, 2004 Thorium Corynebacterium glutamicum IAM 12345 3.5 30 36.2 (E) Nakajima and Tsuruta, 2004 Micrococcus luteus IAM 1056 3.5 30 77.0 (E) Nakajima and Tsuruta, 2004 Nocardia erythropolis IAM 1399 3.5 30 73.8 (E) Nakajima and Tsuruta, 2004 Zoogloea ramigera IAM 12136 3.5 30 67.8 (E) Nakajima and Tsuruta, 2004 Arthrobacter nicotianae IAM 12342 3.5 30 68.8 (E) Nakajima and Tsuruta, 2004 Bacillus lichenifromis IAM 111054 3.5 30 45.9 (E) Nakajima and Tsuruta, 2004 Bacillus megaterium IAM 1166 3.5 30 37.8 (E) Nakajima and Tsuruta, 2004 Bacillus subtilis IAM 1026 3.5 30 52.4 (E) Nakajima and Tsuruta, 2004

Uranium Corynebacterium equi IAM 1038 3.5 30 21.4 (E) Nakajima and Tsuruta, 2004 Corynebacterium glutamicum IAM 12345 3.5 30 5.9 (E) Nakajima and Tsuruta, 2004 Micrococcus luteus IAM 1056 3.5 30 38.8 (E) Nakajima and Tsuruta, 2004 Nocardia erythropolis IAM 1399 3.5 30 51.2 (E) Nakajima and Tsuruta, 2004 Zoogloea ramigera IAM 12136 3.5 30 49.7 (E) Nakajima and Tsuruta, 2004 Aphanothece halophytica 6.5 30 133.0 (L) Incharoensakdi and Kitjaharn, 2002 Pseudomonas putida 7 NA 6.9 (L) Pardo et al., 2003 Pseudomonas putida CZ1 5 30 17.7 (L) Chen et al., 2005 Streptomyces rimosus 7.5 20 30.0 (L) Mameri et al., 1999 Zinc Streptomyces rimosus a 7.5 20 80.0 (L) Mameri et al., 1999 Streptoverticillium cinnamoneum a 7.5 28±3 21.3 (E) Puranik and Paknikar, 1997 Thiobacillus ferroxidans a 6 25 82.6 (L) Celaya et al., 2000 Thiobacillus ferroxidans a 6 40 172.4 (L) Liu et al., 2004

Arsenic Bacillus antharcis 7 37 250 (L) Shakoori et al., 2010

Table 2 summarizes some of the important results of metal biosorption using

bacterial biomasses. A direct comparison of experimental data is not possible, due to

different systematic experimental conditions employed (pH, pH control, temperature

equilibrium time and biomass dosage). However, Table 2 provides basic information to

evaluate the possibility of using bacterial biomass for the uptake of metal ions. Also, it

should be noted that Table 2 is only comprised of biosorption studies that employed

either inactive or dead bacterial biomasses. Some variability in the results has been

observed when the same bacterium was employed for the same metal, but under

| 43 Review of Literature

different instances. A part from the different experimental conditions, this is due to the fact that the biomass was pretreated or immobilized to improve the biosorbent characteristics, as highlighted in Table 2. Also for most metal ions, weak acidic pH resulted in maximum biosorption. This is because of the involvement of carboxyl group and other acidic functional groups, which are responsible for binding metal cations through various mechanisms.

In addition, the formation of metal hydroxide and other metal-ligand complexes significantly reduce the amount of metal ions sorbed at high pH. However, the mechanisms for the biosorption have not always been confirmed or discussed in most studies; therefore, generalizations are not possible in these cases. The extent of biosorption not only depends on the type of metal ions, but also on the bacterial genus, due to variations in the cellular constituents. Very short contact times were generally sufficient to attain metal-bacterial biomass steady state. This is because biomass was either used in the form of fine powder or wet cells; where mass transfer resistances are usually negligible. The rapid kinetics observed with bacterial biomasses represents an advantageous aspect for the design of waste water treatment systems.

2.2.3 Bacterial structure and mechanism of bacterial biosorption

Bacterial structure a. Shape and size

Bacteria have simple morphology Bacteria are a major group of unicellular living organisms belonging to the prokaryotes, which are ubiquitous in soil and water and as symbionts of other organisms. Bacteria can be found in a wide variety of shapes, which include cocci (such as Streptococcus), rods (such as Bacillus), spiral (such as

Rhodospirillum) and filamentous (such as Sphaerotilus). Bacteria vary in size as much

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as in shape. For many prokaryotes, the cells remain together in groups or clusters after division (pairs, chains, tetrads, clusters, etc.). Cocci or rods may occur in long chains.

The gram-negative organism, Escherichia coli often as typical size of bacteria cell, is about 1.1 to 1.5 μm wide by 2.0 to 6.0 μm long. The smallest bacteria are about 0.3 μm and a few bacteria become fairly large, e.g. some spirochetes occasionally reach

500 μm in length and cyanobacterium Oscillatoria is about 7 μm in diameter. Cell size is an important characteristic for an organism. Small size of bacteria is very important because size affects a number of cell biological properties and ensures rapid metabolic processes.

b. Cell structure

A “typical” bacterial cell (e.g., E. coli), contains cell wall, cell membrane, cytoplasmic matrix consisting of several constituents, which are not membrane- enclosed: inclusion bodies, ribosomes and the nucleoid with its genetic material. Some bacteria have special structure, such as flagella, S-layer.

c. Cell wall

Main function of cell wall include: (1) The cell wall gives cell shape and protect it from osmotic lysis; (2) The wall can protects cell from toxic substances (3) The cell wall offers the site of action for several antibiotics. (4) The cell wall is necessary for normal cell division. The major classes of chemical constituents in the walls and envelopes of Gram-positive and Gram-negative bacteria are summarized by Salton and

Kwan shown in Table 3 (http://gsbs.utmb.edu/microbook/ch002.htm).

By Gram staining technique, the Gram-positive bacteria stained purple, whereas

Gram-negative bacteria were colored pink or red. The surface of Gram-negative cells is

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much more complex chemically and structurally than that of Gram-positive cells.

Because of the thicker peptidoglycan layer, the walls of Gram-positive cells are stronger than those of Gram-negative bacteria.

Table 3: The major classes of chemical constituents in the walls and envelopes of Gram-positive and Gram-negative bacteria. Source: Salton and Kwan 1996, http://gsbs.utmb.edu/microbook/ch002.htm

Gram- positive cell walls Examples Peptidoglycan All species Polysaccharides Streptococcus group A, B, C substances Teichoic acids Ribitol S. aureus B. subtilis

Lactobacillus sp. Glycerol B. licheniformis M. lysodeikticus Teichuronic acids (aminogalacluronic or aminoman nuronic acid polymers) Peptidoglycolipids (muramylpeptide– Corynebacterium sp. polysaccharide –mycolates Mycobacterium sp. Nocardia spp Glycolipids (“Waxes”) (polysaccharide –mycolates) Gram-negative envelopes Examples LPS (Lipoteichoic acids) All species Lipoprotein E. coli and many enteric bacteria Pseudomonas aeruginosa Porins (major outer membrane proteins) E. coli, Salmonella typhimurium Phospholipids and proteins All species Peptidoglycan Almost all species

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Cellular wall shape and strength is primarily due to peptidoglycan, which is a rigid, porous and amorphous material, the core of which is very similar in all bacteria.

Unique features of almost all prokaryotic cells are cell wall peptidoglycan and the specific enzymes involved in its biosynthesis. The amount and exact composition of peptidoglycan only found in cell walls vary among the major bacterial groups.

Peptidoglycan is a linear polymer of alternating units of two sugar derivatives,

N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (Figure 3).

Peptidoglycan also contain several different amino acids, three of which D-glutamic acid, D-alanine and meso- diaminopimelic acid are not found in proteins.

N-acetylglucosamine is also the main constituent of chitin. However, the three- dimensional structure differs from the crystalline structure of the chitin. A peptide chain of four or five alternating D-and L-amino acids is connected to the carboxyl group of N-acetylmuramic acid. The disaccharide-peptide units are joined by direct peptide bonds or by short peptides. The carboxyl group of the terminal D-alanine is often connected directly to the amino group of diaminopimelic acid. A common feature of bacterial cell walls is cross-bridging between the peptide chains. There are several types of peptidoglycan, depending on the nature and the localization of the peptide bridge. In a Gram-positive cell, the cross-bridging between adjacent peptides may be close to 100%, such as Staphylococcus aureus. By contrast, the frequency of cross- bridging in E. coli (a Gram-negative organism) may be as low as 30%. The peptidoglycan layer of a Gram-negative cell is generally a single monolayer, composed of phospholipids, lipopolysaccharides, enzymes and other proteins, including lipo proteins. Fig. 1 showed the peptidoglycan cross-links in a Gram-negative and a Gram- positive cell. Most Gram-negative cell wall peptidoglycans lack the peptide interbridge.

This cross-linking results in an enormous peptidoglycan sac which is actually a dense, interconnected network. These sacs are elastic and porous, molecules can penetrate them (Prescott et al., 2002).

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Fig 3. Peptidoglycan subunit composition. The peptidoglycan subunit of Escherichia coli, most other Gram-negative bacteria and many Gram-positive bacteria, NAG is N-acetylglucosamine; NAM is N -acetylmuramic acid (NAG with lactic acid attached by an ether linkage). The tetrapeptide side chain is composed of alternating D - and L-amino acids since meso-diaminopimelic acid is connected through its L-carbon. NAM and the tetrapeptide chain attached to it are shown in different shades of color for clarity. Source: Prescott et al., 2002

The Gram-positive cell wall consists of a single 20 to 80 nm thick homogeneous peptidoglycan or murein layer lying outside the plasma membrane. It also contains large amounts of teichoic acids, polymers of glycerol or ribitol joined by phosphate groups (Figure 4). Peptidoglycan of a Gram-positive cell wall accounts for 40 to 90% of the cell wall materials, containing a peptide interbridge. This peptidoglycan core is usually between 20 and 40 layers thick and adjacent glycan chains are cross linked

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through the amino acid stems forming a highly resilient, three-dimensional macromolecule that surrounds the cells. Amino acids such as D-alanine or sugars like glucose are attached to the glycerol and ribitol groups. The teichoic acids are connected to either the peptidoglycan itself by a covalent bond with the six hydroxyl of

N-acetylmuramic acid or to plasma membrane lipids (called lipoteichoic acids)

(Prescott et al., 2002). Lipoteichoic acids, only present in Gram-positive organisms are synthesized at the membrane surface and may extend through the peptidoglycan layer to the outer surface, are polymers of amphiphitic glycophosphates with the lipophilic glyco-lipid and anchored in the cytoplasmic membrane. They are antigenic, cytotoxic and adhesins (e.g., Streptococcus pyrogenes).

Teichoic acids appear to extend to the surface of the peptidoglycan and, because they are negatively charged, they are helpful to give the Gram-positive cell wall negative charge. The teichuronic acids are free of phosphate and made up of hexuronic acid linear chains. The proportion of teichoic acids and teichuronic acids depends on the cultural conditions, especially on the phosphate supply. The functions of these molecules are still unclear, but they may be important in maintaining the structure of the cell wall.

(a)

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(b)

Fig 4 a.b. Composition of the cell surfaces of Gram-positive and Gram-negative bacteria. Not all structures shown are found in all organisms. For example, M protein is only used to describe a structure in some of the streptococci. Also, not all organisms have flagella. (Source: Moat et al., 2002).

Teichoic acids are not present in Gram-negative bacteria. It is proved that the teichoic acids and teichuronic acids participate in metal tripping. Both the phosphoryl groups of the secondary polymers and the carboxyl groups of the peptide chains provide negatively charged sites in the Gram-positive cell wall (Moat et al., 2002;

Prescott et al., 2002; Remacle, 1990; Urrutia, 1997). Cell wall teichoic acids are found only in certain Gram-positive bacteria (such as Bacillus spp.) and their structures are illustrated in Figure 5. Teichoic acids are polyol phosphate polymers, with either ribitol or glycerol linked by phosphodiester bonds. Substituent groups on the polyol chains include D-alanine (ester linked), N-acetylglucosamine, N-acetylgalactosamine and glucose. They are strongly antigenic. These highly negatively charged polymers of the bacterial cell wall can serve as a cation-sequestering mechanism.

The cell wall of gram-negative is much more complex than the Gram-positive bacteria, about 30 to 80 nm thick and multilayered in structure. It has a 2 to 7 nm peptidoglycan layer surrounded by a 7 to 8 nm thick outer membrane. The

| 50 Review of Literature

peptidoglycan is sandwiched between the plasma membrane and the outer membrane, which is composed of phospholipids, lipopolysaccharides, enzymes and other proteins, including lipoproteins.

Fig 5. Structures of cell wall teichoic acids. Teichoic acid is a polymer of chemically modified ribitol (A) or glycerol phosphate (B). The nature of the modification (e.g. sugars, amino acids ) can de fine the serotype of the bacteria. Teichoic acid may be covalently attached to the peptidoglycan. Lipoteichoic acid is anchored in the cytoplasm membrane by a covalently attached fatty acid. Source: http://micro.digitalproteus.com/morphology3.php.

The thin peptidoglycan layer next to the plasma membrane may constitute not more than 5 to 10% of the cell wall weight. In E. coli it is about 2 nm thick and contains only one or two layers or sheets of peptidoglycan. Only one type of the peptide bridge occurs between the glycan chains. The space between the outer membrane and the inner membrane is referred to as the periplasmic space, which is the translucent region where various enzymes and proteins located. The peptidoglycan is covalently bound to the outer membrane by lipoproteins. The outer membrane is composed of lipopolysaccharide (LPSs), phospholipids and proteins.

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The Gram- negative bacteria have various types of complex macromolecular lipopolysaccharide (LPS). LPSs are probably the most unusual constituents of the outer membrane. LPSs structure was illustrated in Figures 6 and 7. LPSs contain both lipids and carbohydrates and consist of three parts: (1) lipid A, (2) the core polysaccharide and (3) the O side chain. The structure of lipid A required for insertion in the outer leaflet of the outer membrane bilayer; a covalently attached core composed of 2-keto-

3deoxyoctonic acid (KDO), heptose, ethanolamine, N-acetylglucosamine, glucose and galactose; and polysaccharide chains linked to the core. The polysaccharide chains constitute the O-antigens of the Gram-negative bacteria and the individual monosaccharide constituents confer serologic specificity on these components. LPS and phospholipids help confer asymmetry to the outer membrane of the Gram-negative bacteria, with the hydrophilic polysaccharide chains outermost. Each LPS is held in the outer membrane by relatively weak cohesive forces (ionic and hydrophobic interactions) and can be dissociated from the cell surface with surface-active agents

(http://gsbs.utmb.edu/micro-book/ch0 02.htm). The net negative charge of LPSs attributes to the negative surface charge of Gram-negative bacteria. The phosphate groups within LPSs and phospholipids have been proved to be the primary sites for metal interaction. However, only one of the carboxyl groups in LPSs is free to interact with metals (Moat et al., 2002; Prescott et al., 2002; Remacle, 1990; Urrutia, 1997).

Sherbert (1978) showed that the anionic functional groups present in the peptidoglycan, teichoic acids and teichuronic acids of Gram-positive bacteria and the peptidoglycan, phospholipids and lipopolysaccharides of Gram-negative bacteria were the components primarily responsible for the anionic character and metal-binding capability of the cell wall. Extracellular polysaccharides are also capable of binding metals (McLean et al.,

1992). However, their availability depends on the bacterial species and growth conditions and they can easily be removed by simple mechanical disruption or chemical washing (Yee and Fein, 2001).

| 52 Review of Literature

Fig 6. Lipopo lysaccharide Structure. (A) The lipopolysaccharide from Salmonella. This slightly simplified diagram illustrates one form of the LPS. Abbreviations: Abe, abequose; Gal, galactose; Glc, glucose; GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3-deoxyoctonate; Man, mannose; NAG, N-acetylglucosamine; P, phosphate; Rha, L -rhamnose. Lipid A is buried in the outer membrane. (B) Molecular model of an Escherichia coli lipopolysaccharide. The lipid A and core polysaccharide are straight; the O side chain is bent at an angle in this model. Source: Prescott et al., 2002

Fig 7. The three major, covalently linked regions that form the typical LPS. Source: Salton and Kwan 1996, http://gsbs.utmb.edu/microbook/ch002.htm

| 53 Review of Literature

d. Capsule and loose lime

Some bacterial cells can produce capsules or slime layer above the bacterial cell wall. They are highly hydrated (N 95% water) and loosely arranged polymers of carbohydrates and proteins. Capsules are composed of polysaccharides (high molecular-weight polymers of carbohydrates) and a few consist of proteins or polymers of amino acids called polypeptides (often formed from the D- rather than the L-isomer of an amino acid). The capsule of Streptococcus pneumoniae type III is composed of glucose and glucuronic acid in alternating β-1, 3- and β-1, 4- linkages (Moat et al.,

2002); consist of neutral polysaccharide, charged polysaccharide or charged polypeptide. Thus, capsule arrangement is considered to be important to metal binding

(Madigan et al., 2000; Moat et al., 2002; Urrutia, 1997).

Fig 8. Structure of glucose and glucuronic acid in alternating β-1, 3- and β-1, 4- linkages

Many prokaryotes contain a cell surface layer composted of a two-dimensional array of proteins, or glycoproteins, called S-layers or paracrystalline surface layer.

S-layers have a crystalline appearance in p1, p2, p4, p6 symmetry, such as hexagonal

(p6) and tetragonal (p4), depending on the number and structure of proteins or glycoproteins subunits of which they are composted. Non-covalent interactions, such as hydrogen bonding, electrostatic attraction and salt-bridging, are involved in the attachment between neighbouring subunits and the underlying wall. Commonly, divalent metal cations contribute to the correct assembly of the structure. Metals can

| 54 Review of Literature

also be bound after assembly. S-layers are associated with LPSs of Gram-negative or peptidoglycan of a Gram-positive cell (Madigan et al., 2000; Urrutia, 1997).

2.2.4 Mechanism of bacterial biosorption

The bacterial cell wall is the first component that comes into contact with metal ions, where the solutes can be deposited on the surface or within the cell wall structure

(Beveridge and Murray, 1976; Doyle et al., 1980). Since the mode of solute uptake by dead/inactive cells is extracellular, the chemical functional groups of the cell wall play vital roles in biosorption. Due to the nature of the cellular components, several functional groups are present on the bacterial cell wall, including carboxyl, phosphonate, amine and hydroxyl groups (Doyle et al., 1980; Van der Wal et al., 1997).

As they are negatively charged and abundantly available, carboxyl groups actively participate in the binding of metal cations. Several dye molecules, which exist as dye cations in solutions, are also attracted towards carboxyl and other negatively charged groups. Golab and Breitenbach (1995) indicated that the carboxyl groups of the cell wall peptidoglycan of Streptomyces pilosus were responsible for the binding with copper. Also, amine groups are very effective at removing metal ions, as it not only chelates cationic metal ions, but also adsorbs anionic metal species via electrostatic interaction or hydrogen bonding. Kang et al., (2007) observed that amine groups protonated at pH 3 and attracted negatively charged chromate ions via electrostatic interaction. In general, increasing the pH increases the overall negative charge on the surface of cells until all the relevant functional groups are deprotonated, which favors the electrochemical attraction and adsorption of cations. Anions would be expected to interact more strongly with cells with increasing concentration of positive charges, due to the protonation of functional groups at lower pH values.

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The solution chemistry affects not only the bacterial surface chemistry, but the metal speciation as well. Metal ions in solution undergo hydrolysis as the pH increases.

The extent of which differs at different pH values and with each metal, but the usual sequence of hydrolysis is the formation of hydroxylated monomeric species, followed by the formation of polymeric species and then the formation of crystalline oxide precipitates after aging (Baes and Mesmer, 1976). For example, in the case of nickel solution, López et al., (2000) indicated that within the pH range from 1 to 7, nickel

2+ 2+ 4+ existed in solution as Ni ions (90%); whereas at pH 9, Ni (68%), Ni4OH4 (10%) and Ni (OH)+ (8.6%) co-existed. The different chemical species of a metal occurring with pH changes will have variable charges and adsorb ability at solid–liquid interfaces. In many instances, biosorption experiments conducted at high alkaline pH values have been reported to complicate evaluation of the biosorbent potential as a result of metal precipitation (Selatnia et al., 2004b; Iqbal and Saeed, 2007).

It has generally been recognized that bacterial cell structure and composition offer much advantage in the interaction of heavy metals due to the presence of a variety of chemical moiety on the cell wall, plasma membrane, outer membrane, as well as exopolymers. The presence of many functional groups acts as binding site and heterogeneous nucleation site for the binding and formation of heavy metal crystals

(Veglio and Beolchini, 1997). The groups include amongst ethers, hydroxyl, carbonyl, carboxyl, sulfhydryl, thioether, sulfonate, amine, imine, amide, imidazole, phosphonate and phosphodiester groups. Furthermore, some morphological and physiological changes in bacteria have been observed when exposed to metals. The production of exopolymers or biopolymers, which promotes flocculation, is sometimes related to the cell’s defence mechanisms as it immobilizes toxic heavy metal ions thus inhibiting them from entering the cell (Schiewer and Volesky, 2000).

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2.2.5 Application of biosorption

A large amount of researches on metal biosorption have been published to elucidate the principles of this effective metal concentration phenomenon during the past 30 years. Biosorption is regarded as a potential cost-effective biotechnology for the treatment of high volume low-concentration complex wastewaters containing heavy metal(s) (Wang and Chen, 2006). Some efficient natural biosorbents have been identified that require little modification in their preparation. There have been few investigations on determining the compatibility of the biosorbent for real industrial effluents. However, several attempts to scaleup the biosorption process or to commercialize the process based on experiences from conventional sorption operations have not been successful so far. The biosorption has not been applied yet, while it seems that biosorption could hardly have any competition in many types of large-scale environmental metal removal applications (Volesky and Naja, 2005)

(http://biosorption.mcgill.ca/publication/BVibs05.pdf).

Some commercial biosorbents were reported, in the early 1980s, the first patents appeared, claiming the use of specific microbial biomass types as biosorbents for wastewater treatment (Tsezos, 2001). In the early 1990s, other biomaterials were developed and commercialized, including AlgaSORB™ (C. vulgaris), AMT-

BIOCLAIM™ (Bacillus biomass) (MRA), Bio-fix, etc., prepared by immobilization technology (Garnham, 1997; Veglio and Beolchini, 1997; Volesky, 1990b). The immobilization of the microbial biomass seems indispensable for biosorption application and also can make use of traditional chemical engineering reactor configurations, such as upflow or downflow packed bed reactors, fluidized bed reactors.

In the early 1990s, some enterprises in North America were mentioned in developing the biosorption system. Advance Mineral Technologies Inc. in Golden, Colorado,

| 57 Review of Literature

developed a broad range metal removal biosorbent based on Bacillus sp, but it stopped in late 1988 (Volesky, 1990b). AMT-BIOCLAIM™ (Visa Tech Ltd.) comprises of

Bacillus subtilis. It was treated with strong caustic solution, washed with water and immobilized as porous balls onto polyethyleneimine and glutaraldehyde, which can efficiently remove metal ions (Brierley, 1990; Garnham, 1997; Veglio and Beolchini,

1997; Vijayaraghavan and Yun, 2008). Brierley (1990) introduced the production and application of this kind of Bacillus-based biosorbent. AMT-BIOCLAIM™ based on

Bacillus biomass can accumulate 2.90 mmol Pb g−1, 2.39 mmol Cu g−1, 2.09 mmol

Zn g–1 , 1.90 mmol Cd g−1 or 0.8 mmol Ag g–1 metal cations with high efficiency of more than 99% from dilute solutions (Kuyucak, 1990). It is non-selective and metal(s) can be stripped using H2SO4, NaOH or complexing agents and the granules can be regenerated for repeated use (Gupta et al., 2000). AMT-BIOCLAIMTM is able to accumulate gold, cadmium and zinc from cyanide solutions and is therefore suitable for metal-finishing operations (Atkinson et al., 1998).

The biosorbent BIO-FIX is made up of a variety of biomasses, including

Sphagnum peat moss, algae, yeast, bacteria and/or aquatic flora immobilized in high density polysulfone. This biosorbent is selective for toxic heavy metals over that of alkaline earth metals (Vijayaraghavan and Yun, 2008). U. S. Bureau of Mines (Golden,

Colorado) produced the granular Bio-fix, which has been tested extensively for the treatment of acid mine waste (Garnham, 1997). The results showed the Zn binding to the biosorbent BIO-FIX is about 4-fold higher than the ion exchange resins. The metal affi nity followed: Al3+ > Cd2+ > Zn 2+ > Mn2+ and a much lower affinity for Mg2+ and

2+ Ca . Metal(s) can be eluted using HCl or HNO3 and the biosorbent can be used for more than 120 extraction– elution cycles (Gupta et al., 2000). The type of these systems employed is dependent on the amount of flow to be processed, its composition, its

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continuity and the regeneration conditions. From the process of application point of view, the design and operation of the biosorption are similar to the established technologies for ion exchange resin or activated carbon adsorption (Volesky, 1990b). In these systems, pre-treatment of liquor may be required in some cases, depending on the suspended-solids removal prior to biosorption (Volesky, 1990b). All the commercial biosorption enterprises, including both Bio-recovery Systems and B.V. Sorbet, offer small “ canisters” as flow-through fixed -bed systems, as well as large-scale fluidized- bed, pulsed-bed systems, multi-element large-scale treatment schemes capable of handling flows in excess of 100 m3d−1 (Volesky, 1990b). Kuyucak (1990) investigated the treatment of wastewater in the flow rate ranging from 3.8 to 30 L/min using 79 kg of MRA, the result showed that the fluidized-bed contactors would offer optimum removal process using large amount of MRA. The performance of the several biosorbents were summarized by Volesky (1990b), the major features are as follows: high versatility for wide-range of operational conditions, metal selectivity and not influenced by alkaline earth and common light metals, independent of concentration

(for ≤ 10 ppm or ≥ 100 ppm), high tolerance to organics and convenient and effective regeneration (Volesky, 1990b).

| 59 Chapter III

Materials and Methods

3.1. Materials

3.1.1 Chemicals and glassware

The metal salts, analytical grade reagents and chemicals were procured from

RANKEM, New Delhi, India. The molecular and microbiology chemicals were purchased from Himedia, Mumbai, India. Hydrochloric acid (HCl) and sodium hydroxide (NaOH ) were obtained from RANKEM. All glassware used in this study was obtained from Borosil, India.

3.1.2 Sterilization

All the media, buffers, microfuge tubes, tips and reagents etc., used in this study were sterilized at (15 lbs/inch2) for 20 min unless otherwise specified.

3.1.3 Samples

Soil sample was collected from an e-waste recycling facility polluted surface soil from Peenya Industrial Estate, Bangalore, Karnataka, India (Fig. 9) and stored at

-20°C. Soil was dried to remove the moisture content. Approximately 10 g of soil was mixed with 50 ml of sterile distilled water. The electrode was immersed into the soil suspension and the pH was measured.

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(a)

(b)

Fig. 9. Location of soil sampling (a) GIS map of soil sampling site at e-waste recycling facility (b) Google map of e-waste recycling facility

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3.2 Methods

I. Construction of metagenomic clone library (Culture-independent approach)

3.2.1 Soil microbe DNA Isolation

The DNA of soil microbe was isolated by using two different kits, the

FastDNA® SPIN Kit (MP Biomedicals, USA) and ZR Soil Microbe DNA MiniPrep™

(Zymo Research Corporation, USA) by following the manufacturer’s instructions. Soil sample was taken in six tubes in which S1, S2, S3 for MP BIO kit and S4, S5 and S6 for Zymo kit. The isolated DNA in each microfuge tube was quantified by using

Fluorospectrometer (NanoDrop 3300, Thermo, USA).

3.2.2 PCR amplification

The S2 and S5 soil DNA was diluted at 1: 20 concentration. Diluted soil DNA was amplified in the following conditions. Aliquots (5 μl) of the DNA, isolated from the soil, were used for subsequent PCR amplification of bacterial 16S rRNA gene fragments. 16S rRNA gene was amplified using universal eubacterial primers, the forward primer 5’-AGAGTTTGATCMTGGCTCAG-3’ and reverse primer

5’-TACGGYTACCTTGTTACGACTT-3’ (Genei, Bengaluru, India) as reported earlier

(Narde et al., 2004). The reaction mixture comprised of 2 μl of primers, 2.5 μl of each dNTPs, 6 μl of MgCl2, 5 μl of buffer and 0.5 μl of Amplitaq DNA polymerase (Perkin

Elmer, Turku, Finland) in a total volume of 50μl. PCR tubes were incubated in a thermal cycler (Gene AmpPCR System 9700, Applied Biosystems, USA). PCR reactions were performed under the following conditions: Initial denaturation at 95⁰C for1 min, followed by 35 cycles at 94⁰C for 1 min, annealing at 50⁰C, 55⁰C and 58⁰C for 1 min, 72⁰C for 1 min and a final extension at 72⁰C for 10 min. The Soil DNA S2 and S5 were amplified in triplicates. The presence and yield of PCR product was determined on 1% agarose gel electrophoresis at 200 V for 30 min.

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3.2.3 Gel elution of the amplified product

Amplified 16S rRNA products were extracted from the gel using QIA gel extraction kit (Qiagen, Hilden, Germany) by following the manufacturer’s instructions.

Eluted product was run on 1% agarose gel electrophoresis at 200 V for 30 min.

3.2.4 Cloning of bacterial 16S rRNA gene

The purified PCR product was cloned using QIAGEN PCR cloning kit, according to manufacturer protocol. The pDrive Cloning Vector is supplied in a linear form, ready-to-use for direct ligation of PCR products. This vector allows ampicillin and kanamycin selection, as well as blue/white colony screening. The vector contains several unique restriction endonuclease recognition sites around the cloning site, allowing easy restriction analysis of recombinant plasmids. The vector also contains a

T7 and SP6 promoter on either side of the cloning site, allowing in vitro transcription of cloned PCR products as well as sequence analysis using standard sequencing primers.

In addition, the pDrive Cloning Vector has a phage f1 origin to allow preparation of single-stranded DNA. A map of the pDrive Cloning Vector and the sequence of the region surrounding the cloning site are provided in Fig. 10.

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Fig. 10. Map of pDrive cloning vector

Ligation was performed in the following conditions as given in Table 4 and the mixture was incubated for 30 mins at 4-16⁰C.

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Table 4. Composition of ligation mixture

Component Volume/reaction pDrive Cloning Vector (50 ng/µl) 1 µl PCR product 1–4 µl MilliQ water variable Ligation Master Mix, 2x 5 µl Total Volume 10 µl

Transformation was done using QIAGEN PCR cloning kit Transformation protocol. This protocol is for use with QIAGEN EZ Competent Cells. Competent cells are extremely sensitive to temperature and mechanical stress. Hence the thawed cells kept on ice. Cells were mixed by gentle flicking. SOC medium (Super Optimal broth with Catabolite repression (SOC) is SOB with added glucose) was thawn and warm to room temperature. Stored at –20°C after use. Fresh LB agar plates were prepared containing Kanamycin (30 µg/ml LB agar) as a selection marker. Included IPTG

(Isopropyl β-D-1-thiogalactopyranoside) (50 µM) and X-gal (also abbreviated BCIG for 5-bromo-4-chloro-indolyl-β-D-galactopyranoside) (80 µg/ml) for blue/white screening of recombinant colonies and incubated overnight at 37⁰C. The single white colonies were picked and inoculated into test tubes containing LB broth 5 ml and

Kanamycin (30 µg/ml) and incubated overnight at 37⁰C.

3.2.5 Plasmid DNA isolation

The plasmids of the clones were isolated by using the QIAGEN Plasmid

Purification kit by following the manufactures protocol. The microfuge tubes containing isolated plasmids were dissolved in 30µl MilliQ water and used for digestion.

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3.2.6 Plasmid DNA digestion

The isolated plasmid was digested in the following conditions as given in Table 5.

Table 5. Composition of plasmid digestion mixture

Component Volume/reaction Plasmid 10 µl MilliQ water 16 µl Eco R1 Buffer 3 µl Eco R1 1µl Total volume 30 µl

The microfuge tubes containing the above mixtures were incubated at 37⁰C for

1.30 min. The digested product was run on 1% agarose gel electrophoresis at 200 V for

30 min.

3.2.7 DNA Sequencing and data analysis

The microfuge tube containing digested plasmid were than sequenced in ABI

3730 XL – 96 Capillary sequencer facility, Scigenom, Cochin, Kerala, South India.

Partial 16S rRNA gene sequences were initially analyzed using the BLASTn search facility and chimera were checked using CHECK_CHIMERA program of

(www.ncbi.nlm.nih.gov/blast/blast.cgi) RDP II analysis software (www.ce.msu.edu/

RDP/html/analyses.html) (Cole et al., 2005) and by a new chimera detect ion program

Bellerophon available at http://foo.maths.uq.edu.au/huber/bellerophon.pl (Huber et al.,

2004). Phylogenetic tree was constructed using software MEGA version 5.0. The sequences were submitted to the NCBI (National Centre for Biotechnology and

Information) and GenBank for obtaining accession numbers.

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II. Biosorption studies (Culture-dependent approach)

3.2.8 Metal adsorbates used

a. Aluminium potassium sulphate

b. Lead nitrate

c. Cadmium chloride

d. Copper sulphate

3.2.9 Preparation of metal adsorbates

Different metal concentrations were prepared by dissolving Aluminium potassium sulphate, Lead nitrate, Cadmium chloride and Copper sulphate in double distilled water to get metal concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg L-1 for aluminum, cadmium, copper and lead. A stock solution of 1000 mg L-1 was prepared; all other concentrations are obtained from it. Prior to experiment all the glasswares were treated with 0.1 M HCl before and after the biosorption experiments to avoid binding of metals to it.

3.2.10 Analytical estimation of metal Adsorbates

1. Estimation of Copper ( Bi-Cyclo Heaxazone Oxalyl Di Hydrazone Method)

To a series of Standard Measuring Flasks (SMF), 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ml of working copper standard was taken. As a blank 1.25 ml of copper reagent and 0.25 ml of ammonia were used. The SMF final volume was made up to 25 ml with distilled water. The SMF were incubated for 15 min at room temperature. The absorbance was measured at 600 nm. A Standard graph was plotted against the concentration of standard copper and absorbance. From the standard graph, the concentration of the sample can be calculated.

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2. Estimation of Cadmium (Pyronin G Method)

To a series of SMF different aliquots of standards were taken in the concentration range of 10– 100 mg/ml. To this 2.5 ml of citrate buffer (pH 4), 2.5 ml of potassium iodide, 2.5 ml of pyronine G and 1 ml of gelatin was added to the solution and mixed thoroughly and allowed to stand for 20 min at room temperature. To 1 ml of the sample, the above reagents were added. The absorbance was read at 470 nm in a spectrophotometer (Shimadzu, Pharmaspec, Japan). A standard graph was plotted against the concentration of cadmium and absorbance. From the standard graph, the concentration of the sample can be calculated.

3. Estimation of Aluminum (Erichrome Cyanine R Method)

To a series of SMF, 100 – 1000 µl of working aluminum standard solution was added and the final volume to 1000 µl was made using double distilled water. To the blank 1000 µl of double distilled water added. To each standard 0.1 ml of 0. 02 N

H2SO4 was added and mixed. 0.1 ml of Ascorbic acid solution was added and mixed well. Buffer solution 1 ml and 0.5 ml of working dye reagent was added. After mixing well all the SMFs were kept for 10 min at room temperature. Absorbance was measured at 535 nm. A Standard graph was plotted against the concentration of standard Aluminum and absorbance. From the standard graph, the concentration of the sample can be calculated.

4. Estimation of Lead (PAR Indicator Method)

To a series of SMF 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 ml working lead standard solution was taken. Distilled water was used as a blank. Ammonia buffer

1.25 ml was added to each SMF, followed by 1.25 ml of PAR indicator was added. The final volume was made up to 25 ml with distilled water. The SMF were kept for 10 min

| 68 Materials and Methods

at room temperature. Absorbance was measured at 520 nm. A Standard graph was plotted against the concentration of standard lead and absorbance. From the standard graph, the concentration of the sample can be calculated.

3.2.11 Enumeration and screening of metal tolerant bacteria

Metal tolerant bacterial (MTB) strains (biosorbents) were isolated from the soil samples using bacterial medium. Approximately 10 g of soil sample was serially diluted with peptone water and 100 μl of suspension from 10-4 dilutions from the broth was spreaded on to the nutrient agar plates (standard spread plate technique) amended with different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg L-1) of aluminium, cadmium, lead and copper to isolate MTB strains. The plates were incubated at room temperature (30–35⁰C) for 2-3 days. After incubation, the numbers of colonies were counted. Heavy metal resistant bacteria were screened by selecting the metal tolerant identical colonies from metal amended nutrient agar plate. The isolated colonies were grown in LB broth and these colonies were further characterized and employed for heavy metal tolerance studies. The pure cultures were isolated from the plate by inoculating the individual colonies into the sterile Luria Bertani Agar plates.

The plates were incubated at 37⁰C for 24 h.

3.2.12 Identification of the isolated MTB strains

Morphological, physiological and biochemical characteristics of the isolated

MTB strains were performed by following tests according to Bergey’s manual of systematic bacteriology.

(a) Gram Staining

The isolated MTB strains were smeared on glass slides, heat fixed and air dried.

The slides were flooded with Crystal violet staining solution for 1 min and washed

| 69 Materials and Methods

gently in tap water under direct flow. The slides were flooded with Gram’s iodine solution for 1 min and washed with distilled for 20 sec. The smears were immersed in decolourising solution (95 % ethanol) for 20 sec with gentle agitation and washed. The smears were immersed with Saffranin for 20 sec and washed under indirect stream of water until no colour appeared in the wash water. The smears blotted dry with absorbent paper and examined under microscope.

(c) Indole production test

Sterilized tryptone medium was prepared and inoculated with the isolated bacterial strain. The tubes were incubated along with a control tube at 37⁰C for 24 h.

After incubation, 2 to 3 drops of Kovac’s reagent were added. The formation of cherry red ring layer was observed.

(c) Methyl red test (MR test)

The isolated strains were inoculated in MR broth. The tubes were incubated at

37⁰C for 48 h. After incubation, 5 to 6 drops of methyl red solution was added and observed for colour change.

(d) Voges-proskauer test (VP test)

The isolated strains were inoculated in VP broth. The tubes were incubated at

37⁰C for 48 h. After 48 h, 12 drops of Barrit’s reagent A and 2 of 3 drops Barrit’s reagent B were added. The tubes shaken gently and the reaction were allowed to complete for 15 – 30 min and colour development was observed.

(e) Citrate test

The isolated strains were inoculated in Simmon citrate agar slants. The slants were incubated for 48 h. The slant cultures were observed for growth and colouration of the medium.

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(f) Triple Sugar Iron test (TSI test)

The isolated strains were inoculated in TSI agar slants. The slant cultures were incubated for 18 to 24 h at 37⁰C. The slants were observed for sugar fermentation, gas production and hydrogen sulphide production.

(g) Motility test

Sterilized plates of SIM medium were prepared. A loopful of isolated strains was transferred to the plates by single line streaking across the centre of the plates. The plates were observed for diffused growth or turbidity.

(h) Urease test

The isolated strains were inoculated into the urea agar slants. The slants were incubated for 24 to 48 h at 37⁰C. The slants were observed for colour changes.

(i) Catalase test

The isolated strains were inoculated in the medium and incubated at 37⁰C for

24 h. After incubation, the culture was placed on a non-metallic instrument and suspended in a 3% hydrogen peroxide solution. The formation of bubbles was examined.

(j) Oxidase test

The isolated strains were inoculated into the medium and incubated for 24 to

48 h at 37⁰C. Oxidase was reaction was carried out by touching and spreading a well isolated colony on oxidase disc. The reaction was observed within 5 to 60 sec at

25-30⁰C for development of colour.

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(k) ONPG Test

ONPG (Ortho-nitrophenyl-β-D-galactopyranoside) disc was placed into sterile test tube. 0.1 ml of 0.85% sodium chloride solution was added. The colonies isolated were picked with a sterile loop and emulsified in the tubes containing the disc and physiological saline. The tubes were incubated at 35⁰C for 6-12 h and observed for colour changes.

(l) Nitrate test

Nitrate disc was put aseptically in 5 ml peptone water. The isolated strain was inoculated into the medium and incubated ay 35⁰C for 18-24 h. Few drops of reagent were added and observed for colour change.

(m) Gelatin hydrolysis test

The isolated strains were spot inoculated on the agar and incubated at 37⁰C for

24-48 h. After incubation, the plates were flooded with the acidic mercuric chloride and allowed to stand for 5- 10 min. The solutions from the petridish were decanted and the results were observed.

(n) Starch hydrolysis test

Starch agar was heated till the contents melted and autoclaved. The medium was poured into sterilized plates. A loopful of isolated strain was taken and a single line streaking was performed across the centre of the petriplates and incubated at 37⁰C for

24 h. After incubation, the plates were flooded with iodine solution and observed for appearance of clear zone.

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3.2.13 Molecular identification of MTB by 16S rRNA

The ready to amplify DNA was isolated from the MTB by using the ZR

Fungal/Bacterial DNA MiniPrep™ kit (Zymo Research Corporation, USA) by following the manufacturer’s instructions. Isolated MTB DNA was run on 0.8%

Agarose Gel Electrophoresis to check the presence of DNA. MTB 16S rRNA genes were amplified by using the Universal primers (8 F’ 5’-AGAGTTTGATCCTGG

CTCAG-3’; 1492 R’ 5’-TACGGCTACCTTGTTGTTACGACTT-3’). PCR was performed using the following reaction mixtures: 1µl of DNA, 17µl of 1x Prime Taq™

DNA polymerase (Genet Bio, Republic of Korea), 1µl of each primer (Genei,

Bangalore, India), to give a final volume of 20 µl. Thermal cycling was carried out under the following conditions: Denaturation at 94⁰C for 3 min, Annealing at 92⁰C for

1 min, 52⁰C for 1 min, 72⁰C for 1.30 min followed by 30 cycles of 72⁰C for 5 min. The amplified MTB DNA was run on 1% Agarose Gel electrophoresis. The product was eluted and purified using HiYieldTM Gel/PCR Large DNA Extraction kit (Real Biotech

Corporation, Taiwan) by following the instructions manual. The purified MTB 16S rRNA was sequenced at Ocimum Biosolutions, Hyderabad, India. The MTB 16S rRNA gene sequences were initially analyzed using the BLASTn search facility. Phylogenetic tree was constructed using the software MEGA verison 5.0. The sequences were submitted to the NCBI (National Centre for Biotechnology and Information) and

GenBank for obtaining accession numbers.

3.2.14 Biosorbent quantification

The MTB biosorbents was quantified by withdrawing 2.5 ml of broth culture from the bacterial medium and the absorbance was measured using spectrophotometer at 595 nm. Uninoculated growth medium was used as blank (Prasenjit and Sumathi,

2005).

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3.2.15 Optimization of pH for heavy metal removal

The MTB biosorbents was inoculated into a series of 250 ml conical flasks containing heavy metal (100 mg L-1) at different pH (2, 4, 6, 8 and 10) and kept in orbital shaker (120 rpm) for 24 h incubation. After 24 h incubation, the biosorbent were separated by centrifugation at 3,000 rpm for 15 min and the heavy metal concentration was determined spectrophotometrically. The initial and the final concentration of heavy metal used in batch mode studies were calculated by estimating the concentration of heavy metal spectrophotometrically. From the difference in concentration the removal efficiencies of the biosorbent was calculated. Based upon the heavy metal removal and biosorbent data, the optimum pH was determined. To avoid precipitation of the heavy metal ions at high pH, all experiments were carried out only upto pH 10. The use of buffers was avoided to eliminate unknown effects of their components in the presence of metallic ions (Taniguchi et al., 2000). The pH was adjusted with 1 N NaOH and 1 N

HCl as required.

3.2.16 Optimization of temperature for heavy metal removal

The MTB biosorbents was inoculated into a series of 250 ml conical flasks containing different heavy metals (100 mg L-1). The flasks were incubated at different temperatures (25, 30, 35, 40 and 45⁰C) and kept in orbital shaker (120 rpm) for 24 h incubation. After 24 h incubation, the biosorbent were separated by centrifugation at

3,000 rpm for 15 min and the heavy metal concentration and bacterial biosorbent were determined by spectrophotometrically. From the heavy metal removal and biosorbent data, the optimum temperature was determined.

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3.2.17 Measurement of the kinetics of broth cellular growth and heavy metal

removal

The MTB bisorbent was inoculated into a 250 ml conical flask containing heavy metal (100 mg L-1). The flask was kept in orbital shaker (120 rpm) at optimum pH and temperature for 24 h. During the incubation period, heavy metal concentration and biosorbent were monitored for every two hours interval (2, 4, 6, 8, 10, 12, 14, 16, 18,

20, 22 and 24h) until heavy metal removal attains a saturation level.

3.2.18 Heavy metal tolerance assay

Bacterial isolates were added into a 250 ml flask containing nutrient broth amended with heavy metal (ranging from 100 to 1000 mg L−1). The biosorbents were shaken in a rotary shaker (120 rpm) in a temperature controlled water bath at pH 7 and

35ºC for 24 h. After 24 h incubation, the bisorbent was measured. The extent of tolerance was compared and the “normalized” bisorbent was calculated, i.e., biomass at each heavy metal concentration per biosorbent using a control. Bacterial biosorbent were quantified by spectrophotometer at 595 nm.

All the experiments were carried out in triplicates.

3.2.19 Sorption isotherms

The biosorption process involves a solid phase (sorbent, e.g. bacteria) and a liquid phase (solvent, normally water) containing a dissolved species to be sorbed

(sorbate, e.g. metal ions). Owing to the higher affinity of the sorbent for the sorbate species, the latter is attracted to the solid and bound by a number of different mechanisms. This process continues until equilibrium is established between dissolved and solid-bound sorbate.

| 75 Materials and Methods

Huang et al., (2001) proposed a formula to evaluate the metal removal capacity of the biosorbent. In their study they used “n” as removal rate (%). Tests were conducted with initial heavy metal level of 100 mg L-1.

n = [(Ci - Ce) / Ci] X 100 %, where ‘Ci’ is initial heavy metal level in solution (mg L-1), ‘Ce’ heavy metal level remaining in solutions after removal (mg L-1).

3.2.20 Maintenance of microorganisms

Bacterial cultures were maintained on LB agar slants and refrigerated in at 4⁰C for routine use. For long term storage, cells were suspended in sterile water and stored at 4⁰C.

| 76 Chapter IV

Results and Discussion

4.1 Soil analysis

The pH of the surface soil collected from e-waste recycling facility polluted soil was 6.67.

4.2 Construction of metagenomic clone library

4.2.1 Soil microbe DNA Isolation

The microbial DNA from surface soil of the e-waste recycling facility polluted was isolated for profiling metagenomic DNA by using two different kits. The maximum quantity of DNA was isolated from the ZR Soil Microbe DNA MiniPrep™

(Zymo Research Corporation, USA) than the FastDNA® SPIN Kit for Soil (MP

Biomedicals, USA). The higher yield of DNA was obtained from S5 and S2 of 79.5 ng and 20.6 ng respectively (Table 6) and run in 0.8% agarose gel electrophoresis (Fig 11).

The soil DNA isolated by ZR Soil Microbe DNA Miniprep kit shown higher yield of

79.5ng.

Table 5. Profile of metagenomic DNA yield from e-waste recycling facility polluted surface soil isolated by using kits

Name of the kit Sample no. DNA yield (ng) MP Bio kit S1 12.4 12.7 S2 21.3 20.6 S3 20.1 19.9 Zymo kit S4 9.5 7.2 S5 79.4 79.5 S6 30.6 10.5

| 77 Results and Discussion

Fig 11. Profile of metagenomic DNA isolated from soil sample. M- Marker; S2- DNA isolated by using MP Bio kit; S5- DNA isolated by using Zymo Kit

4.2.2 PCR amplification and cloning of soil microbe 16S rRNA

The agarose gel images revealed the amplified product was about ~1,500 bp in size (Fig 12). 16S rRNA amplified products were extracted from the gel. Eluted product was run on 1% agarose gel electrophoresis at 200 V for 30 min (Fig 13). The eluted products were also ~1500 bp in size. The eluted PCR products ES2 and ES5 were pooled and used for transformation.

| 78 Results and Discussion

Fig 12. Profile of PCR amplified 16S rRNA fragments of metagenomic DNA isolated from soil sample. M- Marker; S2- amplified DNA isolated by using MP Bio kit; S5- amplified DNA isolated by using Zymo Kit

Fig 13. Profile of eluted 16S rRNA fragments of PCR amplified product M- Marker; ES2- Eluted PCR amplified DNA isolated by using MP Bio kit; ES5- Eluted PCR amplified DNA isolated by using Zymo Kit

The eluted and pooled product was ligated with pDrive cloning vector and the transformants were grown on LB plates containing Kanamycin (30 µg/ml), IPTG

| 79 Results and Discussion

(50 µM) and X-gal (80 µg/ml). After overnight incubation at 37⁰C the plate was containing blue/white recombinant clones (Fig 14).

Fig 14. Plate of transformants (Blue/ white recombinant clones)

4.2.3 Plasmid DNA isolation and digestion

Plasmid DNA was extracted for 59 white colonies and one blue colony as control. The plasmid was digested to check the inserts. The plasmid was digested and run on 1% agarose gel electrophoresis at 200 V for 30 min. The agarose gel images revealed the presence of the inserts and the vector (Fig 15).

| 80 Results and Discussion

Fig 15. Profile of recombinant clones after plasmid digestion. Clones from HKT_RR1 to HKT_RR59; M- Marker

Fig 15 (Contd..) Profile of recombinant clones after plasmid digestion. Clones from HKT_RR1 to HKT_RR59; M- Marker

| 81 Results and Discussion

Fig 15 (Contd..) Profile of recombinant clones after plasmid digestion. Clones from HKT_RR1 to HKT_RR59; M- Marker; B- Blue colony as control

Fig 15 (Contd..) Profile of recombinant clones after plasmid digestion. Clones from HKT_RR1 to HKT_RR59 ; M- Marker

| 82 Results and Discussion

Fig 15 (Contd..) Profile of recombinant clones after plasmid digestion. Clones from HKT_RR1 to HKT_RR59; M- Marker

4.2.4 Computational analysis of the metagenomic clone library

Cloning revealed that almost 50% of the sequences obtained in the constructed libraries in this study were not related to the known bacteria. Since the percent similarity with the reported closest database matches are less than 97%, these may be categorized among the new bacterial species. The 49 clones were obtained after sequencing and phylogeny tree (Fig 16) was constructed using the open source software. Sequence information was deposited in NCBI database under the accession numbers, from JN030399 to JN030447 (Table 7) and the closest NCBI match their homology was listed in Table 8. A total of 10 major genara were observed from e-waste recycling facility polluted surface soil sample 16S rRNA gene libraries, based on their similarity among themselves. They were Alpha proteobacteria, Beta proteobacteria,

Gamma proteobacteria, Delta proteobacteria, Acidobacteriales, Actinobacteria,

Bacteroidetes, Planctomyceteria, Firmicutes and Fibrobacteres.

| 83 Results and Discussion

Table 7. E-waste recycling facility polluted surface soil metagenomic 16S rRNA genes deposited in NCBI database under the accession numbers JN030399 - JN030447. Cited as Rajeshkumar,R., Pal,R.R. and Purohit,H.J. Submitted on 26- MAY-2011.

S.No Seq ID Acc No S.No Seq ID Acc No 1 HKT_RR1 JN030399 25 HKT_RR30 JN030423 2 HKT_RR2 JN030400 26 HKT_RR31 JN030424 3 HKT_RR3 JN030401 27 HKT_RR33 JN030425 4 HKT_RR4 JN030402 28 HKT_RR34 JN030426 5 HKT_RR5 JN030403 29 HKT_RR35 JN030427 6 HKT_RR6 JN030404 30 HKT_RR36 JN030428 7 HKT_RR8 JN030405 31 HKT_RR37 JN030429 8 HKT_RR9 JN030406 32 HKT_RR38 JN030430 9 HKT_RR10 JN030407 33 HKT_RR39 JN030431 10 HKT_RR11 JN030408 34 HKT_RR40 JN030432 11 HKT_RR12 JN030409 35 HKT_RR41 JN030433 12 HKT_RR13 JN030410 36 HKT_RR42 JN030434 13 HKT_RR14 JN030411 37 HKT_RR44 JN030435 14 HKT_RR15 JN030412 38 HKT_RR45 JN030436 15 HKT_RR16 JN030413 39 HKT_RR46 JN030437 16 HKT_RR17 JN030414 40 HKT_RR47 JN030438 17 HKT_RR19 JN030415 41 HKT_RR48 JN030439 18 HKT_RR23 JN030416 42 HKT_RR49 JN030440 19 HKT_RR24 JN030417 43 HKT_RR50 JN030441 20 HKT_RR25 JN030418 44 HKT_RR51 JN030442 21 HKT_RR26 JN030419 45 HKT_RR52 JN030443 22 HKT_RR27 JN030420 46 HKT_RR53 JN030444 23 HKT_RR28 JN030421 47 HKT_RR54 JN030445 24 HKT_RR29 JN030422 48 HKT_RR55 JN030446 49 HKT_RR58 JN030447

| 84 Results and Discussion

Archae Haloferax D11107 Aquifacae Aquifex aeolicus 15282445 28 Epsilonproteobacteria Campylobacter coli strain 299802547 44 999 HKT RR27 1000 972 51 1000 48 1000 838 >HKT RR30 1000 HKT RR53 HKT RR31 1000 29 685HKT RR37 1000 HKT RR45 HKT RR1 950 Acidobacteria Holophaga foetida strain TMBS4 310975027 HKT RR47 431 Gammaproteobacteria Vibrio AJ421444 628 891 GammaProteobacteria Pseudomonas AB037545 1000 34 920 Pseudomonas monteilii strain CIP 104883 NR 024910.1 835 1000HKT RR15 776 702HKT RR4 349HKT RR41 472HKT RR2 486 HKT RR52 840 HKT RR25 1000 1000 HKT RR50 Betaproteobacteria Burkholderia AB021369 543 Betaproteobacteria Alcaligenes faecalis AJ242986 1000 1000 HKT RR16 521 Betaproteobacteria Achromobacter AJ278451 450 998Betaproteobacteria Bordetella AJ249861 563 715 Betaproteo Bordetella hinzii LMG 13501 NR 027537.1 HKT RR23 1000 Beta Herbaspirillum seropedicae strain Z6 NR 029329.1 HKT RR9 HKT RR12 875 983 HKT RR6 854 HKT RR17 845 998 HKT RR19 49 845 HKT RR11 HKT RR54 580 DeltaProteobacteria Desulfovibrio sp. 110592250 903 DeltaProteobacteria Desulfuromonas alkaliphilus strain 343198800 40 679 HKT RR14 839 1000 Acetobacteria Stella vacuolata strain DSM 5901 NR 025583.1 600 565 HKT RR36 999 1000 33 895 alphaProteobacteria Rhizobium X87273 HKT RR24 1000 HKT RR26 Frmicute clostridium Thermacetogenium 926 Firmicute Clostridium thermobutyrecum 343206257 604 Firmicute Bacillus pumilus 343198673 1000 HKT RR3 1000 Thermoactinomyces vulgaris strain KCTC 9076 NR 041761.1 676 HKT RR5 1000 Iamia majanohamensis strain NBRC 102561 NR 041634.1 930 35 1000 845 Actinobacteria Streptomyces alboniger 343198668 558 42 845 55 845 39 952 HKT RR13 HKT RR8 988 HKT RR10 996 Chloroflexi Sphaerobacter thermophilus DSM 20745 strain DSM 20745 NR 042118.1 Cyanobacteria Prochlorococcus marinus 265678459 707 HKT RR38 610 HKT RR46 0.1

Fig 16. Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene clones from the e-waste recycling facility polluted surface soil

| 85 Results and Discussion

Clone Closest NCBI match (accsession number)/% homology Bacteroidetes (10 clones) HKT_RR27 Uncult. Bacteroidetes bacterium clone UMAB-cl-135 (FR749760.1) 94% HKT_RR29 Uncult. Bacteroidetes bacterium clone QZ-J10 (JF776919.1) 93% HKT_RR30 Uncult. Terrimonas sp. clone 7 (FJ713028.1) 90% HKT_RR31 Uncult. Bacteroidetes bacterium clone g49 (EU979058.1) 98% HKT_RR37 Uncult.Bacteroidetes bacterium clone g49 (EU979058.1) 98% HKT_RR44 Uncult. Bacteroidetes bacterium clone AS64 (EU283382.1) 92% HKT_RR45 Uncult. Bacteroidetes bacterium clone AKYH767 (AY921784.1) 95% HKT_RR48 Flavisolibacter sp. HY-50R 9 (HM130561.1) 89% HKT_RR51 Uncult. Bacteroidetes bacterium clone UMAB-cl-3 (FN811187.1) 91% HKT_RR53 Uncult. Bacteroidetes bacterium clone 2y-19 (FJ444670.1) 93% Acidobacteriales (9 clones) HKT_RR1 Uncult. Acidobacteriaceae bacterium clone T501B6 (HM438136.1) 99% HKT_RR6 Uncult. Acidobacteria bacterium clone NLS3.17 (HQ397104.1) 93% HKT_RR9 Uncult. Acidobacteria bacterium (FR749746.1) 89% HKT_RR10 Uncult. Chloroflexi bacterium clone AMHA11 (AM935396.1) 89% HKT_RR12 Uncult. Acidobacteria bacterium clone 34 (FJ713032.1) 94% HKT_RR17 Uncult. Acidobacterium sp. clone sw-xj73 (GQ302570.1) 95% HKT_RR19 Uncult. Acidobacteria bacterium clone UMAB-cl-123 (FR749748.1) 98% HKT_RR49 Uncult. Acidobacteria bacterium clone lhad18 (DQ648917.1) 96% HKT_RR58 Uncult. Acidobacteria bacterium clone UMAB-cl-123 (FR749748.1) 94%

| 86 Results and Discussion

Gamma proteobacteria (8 clones) HKT_RR2 Pseudomonas putida strain CDd-9 16S (GU248219.1) 99% HKT_RR4 Pseudomonas putida strain BM2 (DQ989291.1) 94% HKT_RR15 Pseudomonas putida (AM411058.1) 96% HKT_RR25 Stenotrophomonas maltophilia strain PSM-2 (FJ906801.1) 94% HKT_RR34 Pseudomonas pseudoalcaligenes strain A2 (GU447236.1) 94% HKT_RR41 Pseudomonas putida strain 75(GU828030.1) 97% HKT_RR50 Lysobacter sp. QT22 (GU385868.1) 90% HKT_RR52 Pseudomonas sp. IM4 (FJ211165.1) 98 % Actinobacteriae (7 clones) HKT_RR5 Uncult. actinobacterium clone B04-10E (FJ542993.1) 91% HKT_RR8 Uncult. Actinobacteria bacterium clone AKYH911 (AY922171.1) 89% HKT_RR13 Uncult. actinobacterium clone w1-42 (JF706672.1) HKT_RR35 Nocardioides oleivorans (AB365060.1) HKT_RR39 Uncult. Rubrobacteridae bacterium clone AMIG12 (AM935479.1) HKT_RR42 Uncult. actinobacterium clone UMAB-cl-58 (FN811242.1) HKT_RR55 Uncult. Solirubrobacter sp. clone w1-13 (JF706667.1) Alpha proteobacteria (6 clones) HKT_RR14 Uncult. Rhodospirillaceae clone AMEC5 (AM935304.1) HKT_RR24 Uncult. Sphingomonas sp. clone G2-18 (F703340.1) HKT_RR26 Novosphingobium sp. IAFILS9 (EU430056.1) HKT_RR33 Rhodoplanes sp. 303 (EU604755.1) HKT_RR36 Uncult. Rhizobiales bacterium clone A03-11G (FJ542846.1) HKT_RR40 Roseomonas sp. NML97-0121 (AF533359.1) Firmicutes (3 clones) HKT_RR38 Uncult. Firmicutes bacterium clone sw-xj33 (GQ302549.1) HKT_RR46 Uncult. Carnobacterium sp. clone Hg5-30 9EU344940.1) HKT_RR3 Thermoactinomyces vulgaris KCTC 9076 9AF138739.1) 97%

| 87 Results and Discussion

Beta proteobacteria (2 clones) HKT_RR16 Alcaligenaceae bacterium BZ45 (GQ246952.1) HKT_RR23 Uncult. proteobacterium clone R7C71 (DQ450172.1) Delta proteobacteria (2 clones) HKT_RR47 Uncult. Bdellovibrio sp. clone GR2-101 (DQ847445.1) HKT_RR11 Uncult. Proteobacterium clone F07_MO03 (EF220858.1) Planctomyceteria (1 clone) HKT_RR28 Uncult. Planctomycete clone EB1038 (AY395357.1) Fibrobacteres (1 clone) HKT_RR54 Uncult. Fibrobacteres bacterium clone GASP-MB2W2_C12 (EF665397.1)

Table 8. Phylogenetic assignment of gene library of amplified bacterial 16S rRNA genes from the e-waste recycling facility polluted surface soil

Fig 17. Distribution of different microbial community structure in the sequences of 16S rRNA gene clones obtained from the e-waste recycling facility polluted surface soil

| 88 Results and Discussion

Microbial diversity in an e-waste recycling facility polluted surface soil,

Bengaluru has been studied using culture independent methods. Soil is a very complex habitat dominated by the soil solid phase. In contrast to water systems, soil is relatively recalcitrant to mixing (Daniel, 2004). Our understanding of the links between microbial diversity and soil functions is poor because we cannot measure easily the microbial diversity, even if we can detect unculturable microorganisms by molecular techniques.

Comparative phylogenetic analysis revealed forty nine clones belonging to the phyla

Bacteriodetes, Acidobacteriales, Firmicutes, alpha, beta, gamma and delta

Proteobacteria, Plactomyceteria and fibrobacteres were identified. Most of the

16S rRNA clones represented unidentified unculturable bacteria. Cloning revealed that almost 50% of the sequences obtained in the constructed libraries in this study were not related to the known bacteria. Since the percent similarity with the reported closest database matches are less than 97%, these may be categorized among the new bacterial species. The 49 clones were obtained after sequencing and phylogeny tree (Fig 16) was constructed using the open source software. Sequence information was deposited in

NCBI database under the accession numbers, from JN030399 to JN030447 (Table 7) and the closest NCBI match their homology was listed in Table 8. A total of 10 major genera were observed from e-waste recycling facility polluted surface soil sample 16S rRNA gene libraries, based on their similarity among themselves. They were Alpha proteobacteria, Beta proteobacteria, Gamma proteobacteria and Delta proteobacteria,

Acidobacteriales, Actinobacteria, Bacteroidetes, Planetomyceteria, Firmicutes and

Fibrobacteres.

Bacteroidetes

Bacteroidetes represents 21% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. The Fibrobacteres, Chlorobi and

| 89 Results and Discussion

Bacteroidetes are presently recognized as three of the main divisions (or phyla) within

Bacteria. Species belonging to these groups exhibit enormous phenotypic and metabolic diversity. The bacteria belonging to Bacteroidetes division, previously known as the Cytophaga- Flavobacteria- Bacteroides (CFB) group, are widely distributed in different habitats ranging from Antarctic ice to fresh and salt water lakes, to terrestrial soil and hydrothermal Obsidian pool (Bacterial (Prokaryotic) Phylogeny

Webpage, 2007). CFB such as Bacteroidetes degrade polycyclic aromatic hydrocarbons and refractory biomacromolecules and Paludibacter consumes N-acetyl glucosamine.

Acidobacteriales

Acidobacteriales represents 12% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. Acidobacteria are a distinct group of bacteria that have recently be identified as a new group of bacteria that have been given their own phylum name (Acidobacteria) based on 16S rRNA based molecular surveys. Acidobacteria occur in diverse habitats around the world. Their abundant presence in soil habitats (up to 50%) suggest they play an important ecological role. This group of microbes is difficult to culture. It is believed that these microbes may be able to play a role in bioremediation

(http://www.denniskunkel.com/DK/Bacteria/28164A.html). The acidobacteria have been detected in nearly all soil samples analysed. This bacterial group contains at least six phylogenetic sub-groups (Dunbar et al., 1999). Although acidobacteria are widespread and abundant in soils, little is known about these microbes. Currently, a few strains of acidobacteria have been cultivated under laboratory conditions (Sait et al.,

2002; Joseph et al., 2003), providing new insights into the metabolic capabilities of this diverse group of microorganisms.

| 90 Results and Discussion

Gamma proteobacteria

Gamma proteobacteria represents 12% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. The Gamma proteobacteria is a large, diverse group that includes some of the most important microbial organisms

(e.g. Escherichia, Enterobacter, Francisella, Pasteurella). By and large, all organisms in this phylum are unicellular and most are rods. Usually they occur in environments where the conditions of anoxia and light both occur. This can be seen in particular clear lakes with anoxic bottom layers. In such conditions, anaerobic photosynthetic bacteria can be very abundant. The other great group tends to be heterotrophic and aerobic or facultatively anaerobic. The methane oxidizers (e.g. Methylococcus) feed on methane and other simple carbon compounds that do not have carbon-carbon bonds. Such organisms occur in highly reduced environments on the ocean floor and as symbionts with mytelid clams and pogonophorans which live in association with geothermal vents. Pseudomonads are motile rods, a combination of characters that is very common in the Proteobacteria and caused many unrelated taxa to be grouped together in the former artificial classification system. Molecular methods have demonstrated that the taxa of a group now called Pseudomonadales do cluster in the Gamma proteobacteria.

One of the most common species is Pseudomonas aeruginosa, normally a free-living organism. It is important in many environmental applications e.g, Bioremediation.

Actinobacteria

Actinobacteria represents 14% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. The phylum Actinobacteria is made up of gram-positive organisms with a high mole % G+C composition (> 55% G+C).

This group is comprised of 39 families and 130 genera making it one of the largest phyla within Bacteria. They encompass a wide range of morphology from

| 91 Results and Discussion

coccoid (e.g. Micrococcus) or rod-coccoid (e.g. Arthrobacter), fragmenting hyphal forms (e.g. Nocardia) to those with permanent and highly differentiated branched mycelium (e.g. Streptomyces). Many of the Actinobacteria are spore forming which range from motile zoospores to specialized propagules. They are also physiologically very diverse as evidenced by their production of numerous extracellular enzymes and by the thousands of metabolic products (including antibiotics) they synthesize and excrete. Actinobacteria are the major antibiotic producers in the pharmaceutical industry. Actinobacterial species are widely distributed in both terrestrial and aquatic ecosystems and they play an important role in decomposition and recycling of biomaterials. However, recent analyses of genomic sequences have led to identification of many conserved indels and unique proteins that are shared by either all

Actinobacteria, or different subgroups of them and provide important means for elucidating their taxonomy, phylogeny and unique biochemical and physiological characteristics (Bacterial (Prokaryotic) Phylogeny Webpage, 2007). Members of

Actinobacteria are high G + C contents Grampositive microorganisms and tend to be abundant in soil microbial communities. These bacteria are well represented in pure cultures and metabolically diverse. The coryneform bacteria and the filamentous actinomycetes are the Actinobacteria most commonly recovered in soil isolate collections. It is interesting to note that the Actinobacteria are recovered less frequently in clone libraries collected from soils than in soil isolate collections. This observation may be due to the over-representation of these organisms in culture collections or their under-representation in clone libraries owing to the difficulty in extracting nucleic acid from these resilient Grampositive cells (Janssen et al., 2002).

Proteobacteria

The Proteobacteria are a metabolically diverse group of microorganisms sub-divided into five groups, four of which, alpha, beta, gamma and delta

| 92 Results and Discussion

proteobacteria, are commonly detected in soils (Madigan and Martinko, 2005). The alpha proteobacteria appear to be one of the most abundant microbial groups in many soils, as assessed by both molecular and cultivation-dependent methods. This diverse microbial group contains many nitrogen-fixing bacteria and certain methylotrophic organisms. Members of beta and gamma proteobacteria, though generally not as abundant as the alpha proteobacteria, are also commonly detected in the soils. Microbes known to mediate nitrification are found among the beta proteobacteria, whereas organisms such as the fluorescent pseudomonads, which are well known for their ability to metabolise a diverse array of carbon compounds, are in the gamma proteobacteria. The gamma proteobacteria mainly consists of sulphate- and iron- reducing bacteria. These organisms are commonly found in the soils, although, because of their intolerance for atmospheric oxygen concentrations, they are rarely represented in isolate collections grown under aerobic conditions (Gupta, 2000).

Alpha proteobacteria

Alpha proteobacteria represents 12% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. Members of these genera are known for degradation of chlorophenol and dichlorophenoxy acetic acid, thiophene-

2-carboxylate metabolization of phenanthrene and sulphur containing. In the sample the populations were represented by Rhodospirillales and Sphingomonadales which degrades polycyclic aromatic hydrocarbons. The alpha proteobacteria comprise an important group which has contributed seminally too many aspects of the history of life. The origin of mitochondria via the endosymbitotic capture of an alpha proteobacteria is well established. There is also strong evidence indicating that the ancestral eukaryotic cell itself may have originated via a fusion, or long-term symbiotic association between one or more alpha proteobacteria and an archaebacteria.

| 93 Results and Discussion

The symbiosis between alpha proteobacteria (viz. Rhizobiaceae species) and plant root nodules plays a central role in the fixation of atmospheric nitrogen by plants. The alpha proteobacteria exhibit enormous diversity in their morphological and metabolic characteristics and they are presently recognized solely on the basis of their branching pattern in the 16S rRNA trees. This group is presently given the rank of a Class or subdivision within the Proteobacteria phylum. Besides their branching pattern in phylogenetic trees, no reliable phenotypic or molecular characteristic is known that is uniquely shared by these bacteria (Bacterial (Prokaryotic) Phylogeny Webpage, 2006).

Firmicutes

Firmicutes represents 6% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. Firmicutes are Gram-positive bacteria with a low mole % G+C content (less than 50%) and they constitute one of the main phyla within the Bacteria. This lineage is highly diverse in their morphology (rod, coccoid and spiral), physiology (anaerobic, aerobic), lifestyle (endospore-forming, nonspore-forming) and so on. However, these phenotypic characteristics tend to be less conserved above family level, thus they are not reliable indicators of phylogenetic relationships (Bacterial (Prokaryotic) Phylogeny Webpage, 2006). Firmicutes are involved in dechlorination and hydrocarbon degradation processes. The Firmicutes are low G + C Gram-positive bacteria which are well represented in pure cultures and are metabolically diverse. This group contains the endosporeforming bacteria, the lactic acid bacteria and Gram-positive cocci. The over-representation of this group can also be observed in culture collections (Ahmad et al., 2000).

Betaproteobacteria

Beta proteobacteria represents 4% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. The Beta proteobacteria are

| 94 Results and Discussion

chemoautotrophic bacteria is quite variable in form and grow on the energy supplied by small inorganic compounds, which they fix into the production of all necessary organic compounds. Thus, they convert species of inorganic elements from one form to another and come in three general forms as manifest by their metabolic requirements: nitrogen, sulfur and small organic molecules (e.g. methanol). Others require and oxidize complex organics. Alcaligenes, capable of biodegrading chlorpyrifos, 3,5,6-trichloro-2-pyridinol and production of polyhydroxyalkanoates. At least three groups include nitrogen fixers and some of the Burkerholderales can enter into nodule-forming symbioses with plants.

The Purple Sulfur Bacteria are pigmented with bacteriochlorophylls which allow them to capture light energy and use the hydrogens from hydrogen sulfide to fix carbon dioxide into food. Thus, the taxa in this phylum are very important in mediating the cycling of the elements nitrogen, sulfur and carbon through the ecosystem and biosphere (http://comenius.susqu.edu/bi/202/EUBACTERIA/PROTEOBACTERIAE/

BETAPROTEOBACTERIA/default.htm).

Delta proteobacteria

Delta proteobacteria represents 4% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. Delta proteobacteria are formed by two major and very different groups. One group tends to be unicellular and obligate anaerobes. Most of them use sulfate as the terminal electron acceptor and reduce it to sulfide. They occur in association with anoxic, sulfur-rich mud, geothermal springs and digestive tracts. Geobacter, first isolated from mud in the Potomac River, can pass off its electrons to metals, so NASA is testing it to see if it can be used to make a living battery that gets its energy through organic waste. The other major clade is made of obligate aerobes (there are no known facultatively anaerobic taxa in this phylum). The Bdellovibrios are predatory cells that feed on other bacteria. They have a

| 95 Results and Discussion

polar, sheathed flagellum, which allows them to swim at speeds up to 100 cell-lengths per second in their attack phase. After attaching to a bacterial cell, a bdellovibrio enters the intraperiplasmic space where they feed on the host and then reproduce

(http://comenius.susqu.edu/bi/202/EUBACTERIA/PROTEOBACTERIAE/DELTAPR

OTEOBACTERIA/default.htm).

Planctomycetes

Planctomycetes represents 2% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. Planctomycetes are an interesting group of bacteria. A group of bacteria that possess characteristics that are more commonly found in eukaryotic cells. They possess a form of intercellular compartments that appear to have specialized metabolic functions. One of these, called an anammoxosome (breaks down ammonia) that appears to have a similar function to the eukaryotic mitochondria. The DNA of a planctomycetes is contained within a membrane-bound nucleoid region-not quite a nucleus, but it definitely represents an internal compartment for the genetic material. Also, most of the planctomycetes lack peptidoglycans (a sugar-amino acid combination) in their cell walls. The presence of peptidoglycans is a defining characteristic of bacteria in general and is the target of many forms of antibiotics the fact that the planctomycetes are lacking this compound suggests that they are not a common form of bacteria. In addition, planctomycetes tend to (but not always) reproduce by budding instead of binary fission. The planctomyctes are found almost everywhere. They are found in aquatic and terrestrial environments, in caves and in fecal material and in both oxygen-rich and oxygen-poor environments.

Scientists do not yet know if the planctomyctes represent the link between the prokaryotes and eukaryotes, although most agree that the study of these organisms will definitely shed light on the processes that contributed to the evolution of the first

| 96 Results and Discussion

eukaryotic cells (http://ricochetscience.com/2011/06/11/bringing-plantomycetes.aspx).

Planctomycetes are aerobic organisms that grow best in dilute media. These organisms divide by budding and are one of the few bacterial groups that lack peptidoglycan in their cell walls. Though a number of strains are present in culture collections, few

Planctomycetes have been obtained from soil samples. It has been suggested that

Planctomycetes are both diverse and abundant members of soil microbial communities.

However, it seems that nothing is known about the role these organisms may be playing in soil systems (Griepenburg et al., 1999).

Fibrobacteres

Fibrobacteres represents 2% in the e-waste recycling facility polluted surface soil sample constructed metagenomic clone library. Fibrobacteres is a small bacterial phylum which includes many of the major rumen bacteria, allowing for the degradation of plant-based cellulose in ruminant animals. Members of this phylum were categorized in other phyla. The genus Fibrobacter (the only genus of Fibrobacteres) was divided from the genus Bacteroides in 1988 (http://flaksearch.com/s/Fibrobacteres).

Microbes are believed to be the common ancestors of all organisms. Microbes not only grow virtually everywhere but also are present in abundance. This is evident from major studies done exclusively on microbial diversity and its potential. Microbes are studied for their abilities such as novel antibiotic producers or clinically important drugs (Streit et al., 2004; Kalia et al., 2007b). Horizontal gene transfer (HGT) events, studied in detail exclusively in microbes involved in PHA production has revealed industrially important microbes that are involved in HGT such as B. cereus (Kalia et al., 2007a) and PHA are also exclusively studied in pathogenic strains of

Mycobacterium as novel drug targets (Purohit et al., 2007). Such landmark studies are

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indicative of large microbial potential and possibilities of exploring this untapped microbial wealth of India for many novel processes.

Microbial diversity is fundamental for the maintenance and conservation of global genetic resources. As extreme environments are explored, the richness of microbial diversity is increasingly evident. Measures were taken to estimate, record and conserve microbial diversity, not only to sustain human health but also to enrich the human condition globally through wise use and conservation of genetic resources of the microbial world. It has always been a very interesting and challenging area to explore.

Our perspective on microbial diversity has improved enormously over the past few decades. Estimating the diversity of life is a persistent challenge in biology (Schloss and Handelsman, 2005a). Advances in molecular techniques have given us a glimpse of the tremendous diversity present within the microbial world, but significant work remains to be done in order to understand the ecological and evolutionary dynamics that can account for the origin, maintenance and distribution of that diversity (Cohan and Perry, 2007).

Similar work by Chihching et al., in 2008 characterized 17 bacterial clones from agricultural field contaminated with heavy metals. This work is distinct on its own as e-waste polluted surface soil opens up a newer arena for metagenomic studies.

Pointelon et al., in 2009 assessed phylogenetic diversity of bacterial microflora in drinking water using serial analysis of ribosomal sequence tags. A considerable degree of diversity was observed in each sample, with an estimated richness ranging from 173 to 333 phylotypes. The community structure shows that there are differences in bacterial evenness between sampled sites. The taxonomic composition of the microbial communities was found to be dominated by members of the Proteobacteria

| 98 Results and Discussion

(57.2–77.4%), broadly distributed among the classes Alpha proteobacteria, Beta proteobacteria, Gamma proteobacteria and Delta proteobacteria. Additionally, a large proportion of sequences (6.3–36.5%) were found to be distantly related to database sequences of unknown phylogenetic affiliation.

Microbial characteristics in the anaerobic tank of a full-scale produced water treatment plant capable of anaerobic hydrocarbon removal were analyzed and compared to those in the influent produced water using cultivation-independent molecular methods. Clones related to methanogens including the methylotrophic

Methanomethylovorans thermophila and hydrogen- and the formate-utilizing

Methanolinea tarda were in abundance in both samples, but greater numbers of

M. tarda like clones were detected in the biofilm library. Both DGGE and cloning analysis results indicated that the archaea in the biofilm were derived from the influent produced water. Bacterial communities in the influent and biofilm samples were significantly different. Epsilon proteobacteria was the dominant bacterial group in the influent while Nitrospira and Delta proteobacteria were the predominant groups in the biofilm. Many clones related to syntrophic bacteria were found among the Delta proteobacteria (Liu et al, 2010). In a parallel study, a cultivation-independent approach based on polymerase chain reaction (PCR) amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons.

Pyrus communis L. cvs Blanquilla, Conference and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in

Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR denaturing gradient gel electrophoresis (DGGE) followed by cluster

| 99 Results and Discussion

analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars (Martínez-Alonso et al,

2010).

Culture-independent technologies can reveal more abundance than culture- dependent approaches in the terms of bacterial composition. However, there are several inherent biases regarding with PCR-based molecular methods, including primer preference; the efficiency of cell lyses; DNA extraction and purification (Snaidr et al,

1997). Therefore, isolation and characterization of bacteria from environmental samples is still essential. Previously, rep-PCR has been employed successfully to classify and differentiate among environmental strains (Sikora and Redzepovic, 2003;

Mohapatra and Mazumder, 2008).

Recently, Jin et al., in 2011 have analyzed bacterial community in bulking sludge using culture-dependent and culture-independent approaches. A total of

28 species were obtained from 63 isolates collected from six culture media. The most cultivable species belonged to Proteobacteria including Klebsiella sp., Pseudomonas sp., Aeromonas sp. and Acinetobacter sp. Further analysis of these strains by repetitive sequence based on polymerase chain reaction (rep-PCR) technology showed that rep-PCR yielded discriminatory banding patterns within the same genus using REP and

BOX primer sets. In this study, the bacterial community structure and composition in activated sludge obtained from aerobic zone of a wastewater treatment plant with A2/O process was investigated by cultivation techniques combined with 16S rRNA gene clone library in process of bulking. Phylogenetic analysis of the partial 16S rRNA gene sequences of 63 isolates showed that Proteobacteria were the dominant microorganisms among the collection, followed by Firmicutes, Bacteriodetes, Alpha proteobacteria and

| 100 Results and Discussion

Beta proteobacteria. However, 11 classes were found in 16S rRNA gene clone library.

While Beta proteobacteria were the most abundance phylogenetic group in these clones. Moreover, only Bacillus, Aeromonas and Pseudomonas species isolated by cultivation method were identified in the clone library. These results reinforced the conclusion that different approaches should be performed to improve the understanding of microbial diversity in bulking sludge (Rani et al., 2008). Firmicutes and

Proteobacteria are the predominant cultivable bacteria, a typical characteristic of the cultivation- based methods (Dunbar et al., 1999; Ellis et al., 2003; Jackson et al., 2005).

Zhang and Xu (2008) have described and compared the culture dependent and culture- independent methods in assessing bacterial diversity in soils and found that members of four major phylogenetic groups are ubiquitous to almost all soil types: class alpha proteobacteria and phyla Actinobacteria, Acidobacteria and Verrucomicrobia; phyla

Proteobacteria, Cytophagales, Actinobacteria and Firmicutes are well represented by cultivated organisms.

Ros et al., in 2008 utilized a DGGE based approach to evaluate microbial community activity, abundance and structure in a semiarid soil under cadmium pollution at laboratory level. They have demonstrated the occurrence of two

Cd concentrations on microbial community, entailing a reduction on biomass carbon and microbial activity as Cd pollution increased. Fungus diversity is more affected to

Cd pollution than several groups of bacteria. However, changes in fungus diversity are not necessarily related to changes in fungus abundance. A similar DGGE based study was done to assess bacterial diversity in heavy metal contaminated soil by Ellis et al., in

2003, However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2% and these values correlated negatively with metal concentrations. The culturable communities from each

| 101 Results and Discussion

sample were compared by 16S DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample.

These results reinforce the suggestion that culture independent need to be employed to uncover the total microbial diversity of these complex ecosystems and provides an opportunity for achieving efficient bioremediation.

4.3 Biosorption studies (Culture-dependent approach)

4.3.1 Heavy metals estimation (APHA, 1989)

The linear regression of the standard graph for the estimation of the heavy metals copper, lead, aluminium and cadmium was 0.992, 0.988, 0.991 and 0.992, respectively (Fig 18,19,20 and 21).

| 102 Results and Discussion

Fig 18. Calibration of Copper (Bi-Cyclo Heaxazone Oxalyl Di Hydrazone Method)

Fig 19. Calibration of Cadmium (Pyronin G Method)

Fig 20. Calibration of Aluminum (Erichrome Cyanine R Method)

| 103 Results and Discussion

Fig 21. Calibration of Lead (PAR Indicator Method)

4.4 Batch sorption studies on heavy metal removal

4.4.1 Enumeration and screening of MTB

The population of MTB was reduced due to the increasing concentration of metals (Tables 9, 10, 11 and12) that exerted more stress in the medium. Brown et al.,

(1994) concluded that the microbes continued to be metabolically active in the presence of higher concentration of heavy metals, but the number might be reduced. In general the maximum number of population was observed under control and as the concentration of heavy metals increased from 0 to 100 ppm, the population decreased sharply in all the isolates.

| 104 Results and Discussion

Table 9. Enumeration of copper resistant bacterial species at different concentrations

Concentration of No. of Colony forming copper (mg L-1) Colonies unit (CFU/ml) 0 TNTC - 4 10 TNTC 78X 10 4 20 TNTC 72 X 10 4 30 56 56 X 10 4 40 30 30 X 10 4 50 20 (TFTC) 20 X 10 4 60 15(TFTC) 15 X 10 4 70 12(TFTC) 12 X 10 4 80 9(TFTC) 9 X 10 4 90 7 (TFTC) 7X 10 4 100 3 (TFTC) 3X 10

Table 10. Enumeration of Cadmium resistant bacterial species at different concentrations

Concentration of No. of Colony forming cadmium (mg L-1) Colonies unit (CFU/ml) 0 TNTC - 10 TNTC - 20 TNTC - 30 TNTC - 4 40 28 28x10 4 50 20 20x10 4 60 14 14x10 4 70 9 9x10 4 80 8 8x10 4 90 5 5x10 4 100 3 3x10

| 105 Results and Discussion

Table 11. Enumeration of aluminium resistant bacterial species at different concentrations

Concentration of No. of Colony forming aluminium (mg L-1) Colonies unit (CFU/ml) 0 TNTC 4 100 20 (TFTC) 20X 10 4 200 15(TFTC) 15 X 10 4 300 12(TFTC) 12 X 10 4 400 7(TFTC) 7 X 10 500 - - 600 - - 700 - - 800 - - 900 - - 1000 - -

Table 12. Enumeration of lead resistant bacterial species at different concentrations

Concentration of No. of Colony forming lead (mg L-1) Colonies unit (CFU/ml) 0 TNTC - 10 TNTC - 20 TNTC - 30 TNTC - 40 TNTC - 4 50 32 32 X 10 4 60 29 29 X 10 4 70 16 (TFTC) 16X 10 4 80 11 (TFTC) 11 X 10 4 90 9 (TFTC) 9 X 10 4 100 4 (TFTC) 4 X 10

| 106 Results and Discussion

4.4.2 Identification of the isolated MTB strains

To determine the genus to which the strains belong, a series of biochemical tests were performed. Morphological, physiological, biochemical profile was subsequently examined according to Bergeys’ manual, the isolated potential organisms identified which is shown in Tables 13, 14, 15, 16. The MTB strains were identified as Bacillus sp (EWRR1), Bacillus sp (EWRR2), Paenibacillus sp (EWRR3), Pseudomonas sp

(EWRR4) for the heavy metals copper, cadmium, aluminium and lead respectively.

Table 13. Morphological, physiological and biochemical characteristics of the copper MTB bacterial species EWRR1

Morphological/Physiological/ Isolated Copper Biochemical characteristics MTB strain Gram’s staining + Cell shape Rod Indole production test - Methyl red test - Voges- Proskauer test - Citrate test - Triple sugar iron test K/A Gas production + Urease test + Motility test - Catalase test - Oxidase test + ONPG test - Nitrate test + Gelatin hydrolysis test + Starch hydrolysis test + Strain name Bacillus sp

| 107 Results and Discussion

Table 14. Morphological, physiological and biochemical characteristics of the isolated cadmium MTB bacterial species EWRR2

Morphological/Physiological/ Isolated Cadmium Biochemical characteristics MTB strain Gram’s staining + Cell shape Rod Indole production test - Methyl red test - Voges- Proskauer test + Citrate test + Triple sugar iron test K/A Gas production - Urease test - Motility test - Catalase test + Oxidase test - ONPG test - Nitrate test + Gelatin hydrolysis test + Starch hydrolysis test + Strain name Bacillus sp

Table 15. Morphological, physiological and biochemical characteristics of the isolated aluminium MTB bacterial species EWRR3

Morphological/Physiological/ Isolated Aluminium Biochemical characteristics MTB strain Gram’s staining + Cell shape Rod Indole production test - Methyl red test - Voges- Proskauer test + Citrate test - Triple sugar iron test K/A Gas production - Urease test + Motility test - Catalase test + Oxidase test - ONPG test -

| 108 Results and Discussion

Nitrate test + Gelatin hydrolysis test + Starch hydrolysis test - Strain name Paenibacillus sp

Table 16. Morphological, physiological and biochemical characteristics of the isolated lead MTB species EWRR4

Morphological/Physiological/ Isolated Lead MTB Biochemical characteristics strain Gram’s staining - Cell shape Rod Indole production test - Methyl red test - Voges- Proskauer test - Citrate test + Triple sugar iron test K/A Gas production - Urease test - Motility test - Catalase test + Oxidase test + ONPG test - Nitrate test + Gelatin hydrolysis test + Starch hydrolysis test - Strain name Pseudomonas sp

4.4.3 Identification of MTB strain by 16S rRNA The 16S rRNA of bacterial strains EW RR1, EW RR2, EW RR3 and EW RR4 were amplified with primers 1492R and 27F. The PCR amplified products were detected by 0.8% agarose gel electrophoresis with ultraviolet (UV). The length of object fragment is about ~1, 500 bp (Fig 22, 23, 24 and 25). Sequence analysis of the 16S rRNA gene has been considered a fast and accurate method to identify the phylogenic position of bacteria. Partial 16S rRNA of strains EWRR1, EWRR2, EWRR3 and EWRR4 were sequenced and used to construct phylogenetic development trees (Fig 26, 27, 28 and 29). Comparative analysis of the sequences with already

| 109 Results and Discussion

available database showed that the strains were closed to the members of genus and it was classified in the branch EWRR1- Bacillus anthracis, EWRR2- Bacillus cereus, EWRR3- Paenibacillus lactis and EWRR4- Pseudomonas aeruginosa.

Fig 22. Profile of PCR amplified 16S rRNA fragments of DNA isolated from Bacillus sp EWRR1. M- Marker

Fig 23. Profile of PCR amplified 16S rRNA fragments of DNA isolated from Bacillus sp EWRR2. M- Marker

| 110 Results and Discussion

Fig 24. Profile of PCR amplified 16S rRNA fragments of DNA isolated from Paeniacillus sp EWRR3. M- Marker

Fig 25. Profile of PCR amplified 16S rRNA fragments of DNA isolated from Pseudomonas sp EWRR4. M- Marker

| 111 Results and Discussion

Fig 26. Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene of MTB Bacillus sp EWRR1 isolated from the e-waste recycling facility polluted surface soil

| 112 Results and Discussion

Fig 27. Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene of MTB Bacillus sp EWRR2 isolated from the e-waste recycling facility polluted surface soil

| 113 Results and Discussion

Fig 28. Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene of MTB Paenibacillus sp EWRR3 isolated from the e-waste recycling facility polluted surface soil

| 114 Results and Discussion

Fig 29. Neighbor-Joining tree deduced from partial sequences of 16S rRNA gene of MTB Pseudomonas sp EWRR4 isolated from the e-waste recycling facility polluted surface soil

4.4.4 Optimization of pH for heavy metal removal by MTB strains

Hydrogen ion concentration in the adsorption is considered as one of the most important parameters that influence the adsorption behavior of metal ions in aqueous solutions (Goyal et al., 2003). It affects the solubility of the metal ions in the solution, replaces some of the positive ions found in the active sites and affects the degree of ionization of the adsorbate during the reaction (Nomanbhay and Palanisamy, 2004).

| 115 Results and Discussion

Tests were conducted with initial heavy metal levels of 100 mg L-1, at a temperature of

35⁰C and equilibration time of 24 h.

The effect of pH on the biosorption of heavy metal onto MTB strain was evaluated within the pH range of 2-10 (Fig 30, 31, 32 and 33). The copper MTB

Bacillus anthracis EWRR1, cadmium MTB Bacillus cereus EWRR2, aluminum MTB strain Paenibacillus lactis EWRR3 and lead MTB Pseudomonas aeroginosa EWRR4 reported a maximum removal of 86%, 82%, 84% and 92% respectively at pH 6 at a concentration of 100 mg L-1. The influence of pH on the percentage sorption of heavy metals by MTB strains was depicted in the Fig 30, 31, 32 and 33. This pH dependency of biosorption efficiency could be explained by the functional groups involved in metal uptake and metal chemistry. Above pH 5, the percent removal of all the metals increased rapidly by the isolated MTB strains. The low bioaccumulation capacity at pH values below six is attributed to the competition of hydrogen ion with metal ion on the sorption site. Thus, at lower pH, due to the protonation of binding site resulting from high concentration of proton, negative charge intensity on the site is reduced which results in the reduction or inhibition for the binding of metal ion. Most of the microbial surfaces are negatively charged due to the ionization of functional group, thereby contributing to metal binding. At low pH, some of the functional groups will be positive charged and may not interact with metal ions (Yan and Viraraghavan, 2003).

The increase in percent removal of metal with increase in pH from two to five is due to the strong relations of bioaccumulation to the number of surface negative charge, which depends on the dissociation of functional group (Yakup et al., 2004). As the pH is increased above the zeta potential of the biosorbent, there is a reduction in the electrostatic attraction between the heavy metals and the biosorbent surface, with a consequent decrease in percentage bioaccumulation. The rate of metal uptake and the

| 116 Results and Discussion

extent were enhanced as the pH increases up to certain pH range. At low pH negligible removal of metals ions noted may be due to the competition between hydrogen and metal ions. With further increase in pH, there is an increase in metal removal, which may be due to the ionization of functional groups and an increase in the negative charge density on the cell surface. At higher alkaline pH values (8 and above), a reduction in the solubility of metals contributes to lower uptake rates. At the pH of 6, similar metal removal was attained for copper by Pseudomonas putida (Pardo et al., 2003),

Sphaerotilus natans (Beolchini et al., 2006); Thiobacillus ferroxidans (Ruiz-Manriquez et al., 1997); Cadmium by Pseudomonas aeruginosa PU 21 (Chang et al., 1997);

Enterobacter sp J1 (Lu et al., 2006); Pseudomonas putida (Pardo et al., 2003);

Staphylococcus xylosus (Ziagova et al., 2007); Mercury by Bacillus sp (Green-Ruiz,

2006); Nickel by Bacillus thurigiensis (Öztürk, 2007); Arsenic by Bacillus anthracis

(Shakoori et al., 2010) and Zinc by Thiobacillus ferroxidans a (Celaya et al., 2000) and

Thiobacillus ferroxidans b (Liu et al., 2004). Heavy metal adsorption increased along with the increase of pH of the adsorbate solution which is shown in Fig 30, 31,

32 and 33.

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Fig 30. Cellular growth and copper removal by Bacillus anthracis EWRR1 in response to various pH

Fig 31. Cellular growth and cadmium removal by Bacillus cereus EWRR2 in response to various pH

| 118 Results and Discussion

Fig32. Cellular growth and aluminium removal by Paenibacillus lactis EWRR3 in response to various pH

Fig 33. Cellular growth and lead removal by Pseudomonas aeroginosa EWRR in response to various pH

| 119 Results and Discussion

4.4.5 Optimization of temperature for metal removal by MTB species

The range of optimal temperature values (30–35 C) were comparable to the range of room temperature that was used when isolating the microorganisms, suggesting that the selection of these isolates might have been influenced not only with the heavy metals but also with the temperature used in the isolation procedure. The temperature of the adsorption medium could be important for energy dependent mechanisms in metal removal by microorganisms. Temperature is known to affect the stability of the cell wall, its configuration and can also cause ionization of chemical moieties. These factors may simultaneously affect the binding sites on isolated fungal and bacterial species causing reduction in heavy metal removal. Energy-independent mechanisms are less likely to be affected by temperature since the processes responsible for removal are largely physiochemical in nature (Gulay et al., 2003).

Bioaccumulation of metal ions MTB species appears to be temperature dependent.

Maximum removal of all four metals was observed at 35⁰ for the isolated MTB strains

Bacillus anthracis EWRR1, Bacillus cereus EWRR2, Paenibacillus lactis EWRR3 and

Pseudomonas aeruginosa EWRR4. The temperature of the adsorption medium could be important for energy dependent mechanisms in metal biosorption. Energy- independent mechanisms are less likely to be affected by temperature, since the processes responsible for biosorption seems to be largely physicochemical (electrostatic forces) in nature (Scott and Palmer, 1988). A lot of experimental studies had been done on effects of temperature on heavy metal removal by microbes and the researches were reported on effects of different microbial species and conditions (Bengtsson et al.,

1995; Wu et al., 1999), temperature affects biosorption of heavy metals and bacterial acitivity as well. To study the effect of temperature on heavy metal removal, we conducted tests with different equilibration temperatures. Initial heavy metal levels are

100 mg L-1 for all four heavy metals. Experiments were carried out at pH 6.0 for 24 h

| 120 Results and Discussion

and results were shown as Fig 34, 35, 36, 37. As indicated by Fig, when the equilibration temperature ascended from 25°C to 35°C, bacterial biomass and removal of all four heavy metals also increased with temperature and afterwards declined when temperature became even higher. Therefore, the best temperature for maximum removal of all four metals namely copper by Bacillus anthracis EWRR1, cadmium by

Bacillus cereus EWRR2, aluminium by Paenibacillus lactis EWRR3, lead by

Pseudomonas aeruginosa EWRR4, at the tempearture 35°C and the metal removal is

86%, 87%,71% and 89% .

Fig 34. Cellular growth and copper removal by Bacillus anthracis EWRR1 in response to various temperatures

| 121 Results and Discussion

Fig 35. Cellular growth and cadmium removal by Bacillus cereus EWRR2 in response to various temperatures

Fig 36. Cellular growth and aluminium removal by Paenibacillus lactis EWRR3 in response to various temperatures

| 122 Results and Discussion

Fig 37. Cellular growth and lead removal by Pseudomonas aeruginosa EWRR4 in response to various temperatures

4.4.6 Kinetics of cadmium removal and cellular growth of the MTB species

Experiments were conducted to investigate heavy metal removal courses.

Results were shown as Fig 38, 39, 40 and 41. It was found from the Fig that within 24 h all four MTB isolates Bacillus anthracis EWRR1 shown 88% removal of copper,

Bacillus cereus EWRR2 shown 91% removal of cadmium, Paenibacillus lactis

EWRR3 shown 89% of aluminium, Pseudomonas aeruginosa EWRR4 shown 90% lead. After 12 h, bacterial biomass and removal of all four heavy metals also increased very slightly with time and removal equilibrium. The time-course data for heavy metal removal and cellular growth were observed for each isolate under optimal pH and temperature conditions. When these isolates are applied in removing heavy metal from industrial wastewater, information regarding the effect of growth phase will be important in designing solid (sludge) retention time (SRT) for continuous flow completely stirred (CFCS) bioreactor, which is a general reactor type for wastewater

| 123 Results and Discussion

treatment plants. In the MTB isolates, specific metal bioaccumulation (accumulative biosorption (removal) of each heavy metal per accumulative biomass) increased when cells were in stationary phases. Therefore, expanded SRTs (stationary phase) may be recommended using the MTB isolates in removing heavy metals from industrial wastewater however, a non-expanded SRT has to be designed for CFCS bioreactor so that a mid-log phase of cellular growth could be kept in the treatment system. The growth rate during the lag phase was very low because the isolated MTB isolates was adapting with the environment. After this stage, the isolates grew in logarithmic form using the nutrients. Similar studies have been reported by Enterobacter cloacae (Koji et al., 1992) and Bacillus circulans (Srinath et al., 2002).

Fig 38. Kinetics of cellular growth and copper removal by Bacillus anthracis EWRR1

| 124 Results and Discussion

Fig 39. Kinetics of cellular growth and cadmium removal by Bacillus cereus EWRR2

Fig 40. Kinetics of cellular growth and aluminium removal by Paenibacillus lactis EWRR3

| 125 Results and Discussion

Fig 41. Kinetics of cellular growth and lead removal by Pseudomonas aeruginosa EWRR4

4.4.7 Influence of initial metal concentration

The initial concentration of the cadmium in the solution remarkably influenced the equilibrium uptake. It was noticed that initial concentration increased the sorption of heavy metal as is generally expected due to equilibrium process (Fig 42). This increase in uptake capacity of the biosorbents with the increase in initial metal concentration is due to higher availability of metal ions for the sorption. Moreover, higher initial concentration provides increased driving force to overcome all mass transfer resistance of metal ions between the aqueous and solid phase resulting in higher probability of collision between metal ions and sorbents. This also results in higher metal uptake (Tewari et al., 2005).

| 126 Results and Discussion

Tolerance implies a large change in sensitivity between sets of organisms to a particular toxicant. Tolerance can be adaptive, constitutive, or induced. Adaptive tolerance is where the organism colonizing a contaminated site is less insensitive than the same species colonizing uncontaminated sites and where this change in sensitivity is caused by the selection of genes that confer enhanced insensitivity. Induced tolerance, for which there is less evidence, is where particular enzymes that cause decreased sensitivity are induced on exposure to metal ions (Meharg, 2003).

All the cultures exhibited growth even at higher levels of metal ions and the biomass production decreased with increase in the metal concentration. A significant reduction of mean growth was achieved in all the MTB isolates with the increasing concentration of heavy metals in the medium. Konopka et al., (1999) confirmed that the microbial biomass generation was decreased as the concentration of heavy metal increased. This is in agreement with the findings of Hussein et al., (2004) who reported that the total amount of biomass production decreased while increasing the concentration of heavy metals. Bridge et al., (1999) also confirmed that the microorganisms release a diverse range of specific and nonspecific metal binding compounds in response to high levels of toxic metals which can ameliorate the effect of toxic metals and mediate the uptake process.

| 127 Results and Discussion

Fig 42. Normalized biomass measure at 24h incubation time in response to varying initial concentrations of Cu, Cd, Al, Pb ions. Concentration range: 100-1000 mg L-1, temperature: 35°C

| 128 Chapter V

5. Summary and Conclusion

Biodiversity has been always a very attractive and challenging area to explore.

Modern molecular techniques, which enable us to assess microbial diversity without culturing them. In the Culture-independent approach - the soil DNA was isolated from polluted surface soil of e-waste recycling facility, Bangalore, Karnataka, India by using the MP Bio soil DNA isolation kit and Zymo Soil DNA isolation kit. The quantified

DNA was 20.6 ng by the kit MP Bio and 79.5 ng by the Zymo Research kit. The quantified DNA was used for the amplification by 50μl PCR mixture. The PCR product was pooled and used for cloning into the pDrive cloning vector which has ampicillin and kanamycin selection. The PCR product was then ligate in to the cloning vector.

Transformation done with QIAGEN EZCompetent Cells and plating was done on the plates containing LB agar (150 ml), Kan (150 µl), IPTG (250 µl), X-gal (500 µl).

About 60 colonies were picked up randomly and inoculated in LB tubes containing kanamycin. The plasmid was isolated and digested using EcoR1 restriction enzyme.

The stabs were prepared by using LB and kanamycin and the clones were stabbed into it sequenced. From the 60 clones only 49 clones were successfully sequenced. 16S rRNA gene sequences were initially analyzed using the BLASTn search facility

(www.ncbi.nlm.nih.gov/blast/blast.cgi). Chimera were checked using

CHECK_CHIMERA RDP II analysis software (www. ce. msu. edu/ RDP/ html/ analyses. html) and by new chimera detection program Bellerophon available at http://foo-maths.uq.edu.au/huber/bellerophon.pl. The sequences were submitted to the

NCBI and GenBank for obtaining accession numbers. The accession numbers from

JN030399 to JN030447 were assigned to the submitted 16S rRNA sequences.

| 129 Summary and Conclusion

The Phylogenetic tree based on 16S rRNA gene placed 49 clones. The major groups in microbial diversity in the 49 clones were Alpha proteobacteria, Beta proteobacteria, Gamma proteobacteria, Delta proteobacteria, Acidobacteriales,

Actinobacteria, Bacteroidetes, Planetomyceteria, Firmicutes and Fibrobacteres. Based on the fact that we have been targeting known eco-physiological niches, where bioremediation is a major issue, we could isolate a large number of bacterial members known for such metabolic activities including many members from uncultured groups.

In the Culture-dependent approach – the metal tolerant bacteria (MTB) was isolated from polluted surface soil of e-waste recycling facility, Bangalore, Karnataka,

India by serial dilution and plating on LB medium containing copper, cadmium, aluminium and lead in the concentration of 10ppm to 100ppm. The isolated MTB were initially identified based on gram staining and biochemical tests.Based on 16S rRNA analysis the MTB isolates were identified as Bacillus anthracis (EWRR1), Bacillus cereus (EWRR2), Paenibacillus lactis (EWRR3) and Pseudomonas aeuroginosa

(EWRR4). The partial 16S rRNA sequences of these four isolates were submitted to

NCBI and accession numbers were obtained as for Bacillus anthracis EWRR1 -

JN102340, Bacillus cereus EWRR2 - JN102338, Paenibacillus lactis EWRR3 -

JN102337 and Pseudomonas aeuroginosa EWRR4 - JN102339.

The batch sorption studies on heavy metal removal by the MTB revealed that the copper MTB Bacillus anthracis EWRR1, cadmium MTB Bacillus cereus EWRR2, aluminum MTB strain Paenibacillus lactis EWRR3 and lead MTB Pseudomonas aeroginosa EWRR4, were found a maximum removal of 84%, 82%, 92% and 86%, respectively in the optimised pH 6 at 35⁰C after 24 h incubation. In the optimized temperature 35°C the metal removal was 86%, 87%,71% and 89% respectively and

| 130 Summary and Conclusion

further the kinetics studies also revealed that the isolated MTB strains could remove

88% copper, 91% cadmium, 89% aluminium and 90% lead respectively after 24 h at optimized pH at 6 and temperature at 35°C. The effect pH, biomass dosage, incubation time and initial metal ion concentration during the biosorption process was carried out.

The observed effect(s) of pH on bioaccumulation was attributable mainly to organism specific physiology, as indicated by the observed positive correlation between biomass and heavy metal removal. Furthermore, the kinetic and tolerance experiments provided information for SRT design and the lethal tolerance limits, which are important in designing Chiral Selective Cation-exchange Membrane (CSCM) bioreactors for removing heavy metals of high concentrations. According to the kinetic data, a non- expanded SRT was recommended for designing Continuous Flow Completely Stirred

(CFCS) bioreactor so that a mid-log phase of cellular growth could be kept in the treatment system. The tolerance data with the extremely high range of heavy metal concentrations revealed the MTB resistant isolates can tolerate meatl toxicity up to

1000 mg L−1. The study demonstrated that the newly isolated MTB strains have potential application for the removal of copper, cadmium, aluminium and lead from industrial wastewaters. Further desorption studies can be carried out as the final approach for the management of heavy metal laden biomass as an environmental friendly method of disposal.

| 131 References

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| xxxvi APPENDIX A

Acidic mercuric chloride solution Mercuric chloride 15 g Concentrated Hydrochloric acid 20 ml Distilled water 100 ml

Aluminium stock Aluminium sulphate 0.8791gms Distilled water 100 ml

Ammonia Buffer solution Ammonia chloride 3.38gms Liquor ammonia 24.6ml

Ascorbic acid Ascorbic acid 0.01gm Distilled water 10ml

Bradford’s reagent Coomassie Blue G250 0.025 g Ethanol (95%) 12.5 ml Ortho Phosphoric acid (85%) 25 ml

Buffer reagent Sodium acetate 2.72gm 1N acetic acid 0.8ml Distilled water 20ml

Cadmium stock solution (100ppm) Cadmium chloride 0.0163 mg Distilled water 100 ml

Citrate buffer (pH 4) Ammonium citrate 58.8 g Citric acid 38.4 g Distilled water 500 ml

Copper solution Bicyclohexanone oxalyldihydrazone 0.025g Methylated ethanol 2.5ml Hotwater 2.5ml

Copper stock (100mg/L) Copper sulpahte 3.929mg Distilled water 100ml

Erichrome cyanine solution Erichrome cyanine 0.01ml Distilled water 10ml

Gelatin (1%) Gelatin 1 ml Distilled water 99 ml

Gelatin agar Gelatin 30 g Casein enzymatic hydrolysate 10 g Sodium chloride 10 g Agar 15 g Distilled water 1000 ml

Iodine solution Iodine 1 g Potassium iodide 2 g Distilled water 300 ml

Lead stock Lead nitrate 0.01599gm Distilled watert 10ml

Luria Bertani Broth/ Agar Casein enzymatic hydrolysate 10g Yeast extract 5 g Sodium chloride 10 g Distilled water 1000 ml

MR-VP broth Peptone 7 g Dextrose/ glucose 5 g Dipotassium hydrogen phosphate 5 g Distilled water 1000 ml

Nutrient Broth/ Agar Peptone 10g Beef extract 10g Sodium chloride 5g Distilled water 1000ml

PAR indicator (4-(2-pyridylazo) resorisol 0.03gms Distilled water 50ml

Pyronin G (0.024%) Pyronin G 0.024 g Distilled water 100 ml

SIM Medium (pH 7.3 ± 0.2) Peptic digest of animal tissue 30 g Beef extract 3 g Peptonized iron 0.20 g Sodium thiosulphate 0.025 g Agar 3 g Distilled water 1000 ml

Simmon Citrate Agar (pH 6.8 ± 0.2) Magnesium sulphate 0.20 g Ammonium dihydrogen phosphate 1 g Dipotassium phosphate 1 g Sodium citrate 2 g Sodium chloride 5 g Agar 15 g Distilled water 1000 ml

Sodium chloride solution (0.85%) Sodium chloride 0.85 g Distilled water 100 ml

Sulphuric acid (0.02 N) Sulphuric acid 5.8μl Distilled water 10 ml

Triple Sugar Iron Agar (pH 7.4 ± 0.2) Peptic digest of animal tissue 10 g Casein enzymatic hydrolysate 10 g Yeast extract 3 g Beef extract 3 g Lactose 10 g Sucrose 10 g Dextrose 1 g Sodium chloride 5 g Ferrous sulphate 0.20 g Sodium thiosulphate 0.30 g

Phenol red 0.024 g Agar 12 g Distilled water 1000 ml

Tryptone medium Tryptone 10 g Sodium chloride 5 g Distilled water 1000 ml

Urea agar (pH 6.8 ± 0.2) Peptone digest of animal tissue 1 g Dextrose 1 g Sodium chloride 5 g Disodium phosphate 1.20 g Monopotassium phosphate 0.80 g Phenol red 0.012 g Agar 15 g Distilled water 1000 ml APPENDIX - B

Uncultured bacterium clone HKT_RR1 16S ribosomal RNA gene, partial sequence GenBank: JN030399.1 FASTA Graphics PopSet

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LOCUS JN030399 1523 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR1 16S ribosomal RNA gene, partial sequence. ACCESSION JN030399 VERSION JN030399.1 GI:342328438 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1523) AUTHORS Rajeshkumar, R., Pal,R.R. and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1523) AUTHORS Rajeshkumar, R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1523 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR1" /environmental_sample /country="India: Bengaluru" rRNA <1..>1523 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gaatcaacgc tggcggcgtg cctaacacat gcaagtcgaa 61 cgcgaaaggg gcttcggccc tgagtagagt ggcgaacggg tgagaaacac gtgggtgatc 121 tacctccgag tgggggataa cgttccgaaa ggagcgctaa taccgcatga cgtcctgggt 181 ttgaatacct ggaaaccaaa gtcggggacc gcaaggcctg acgcttggag aggagcccgc 241 gcctgattag ctagttggtg gggtaatggc ccaccaaggc gacgatcagt agccggcctg 301 agagggcgga cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 361 tggggaattg atcgcaatgg gcgcaagcct gacgacgcaa cgccgcgtgg aggatgaagg 421 tcttcggatt gtaaactcct gttgaccggg aagaatggac cccgagctaa tactttgggg 481 tattgacggt accggttgag gaagccacgg ctaactctgt gccagcagcc gcggtaatac 541 agaggtggca agcgttgttc ggaattactg agcgtaaagg gccgcgtakg cggtcgcyta 601 aagtcggacg tgaaatcccc aagcttaact tgggaactgc gtccgatact gggtgacttg 661 agttcgggag aggaatgtgg aatttccagg tgtagcggtg aaatgcgtag atatctggag 721 gaacaccggt ggcgaaggcg gcattctgga ccgaaactga cgctgaggcg cgaaagccag 781 gggagcaaac gggattagat accccggtag tcctggccct aaacgatgag tgcttggtgt 841 ggcgggtatc gatccctacc gtgccgaagc taacgcatta agcactccgc ctggggagta 901 cggtcgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 961 ggttcaattc gacgcaacgc gaagaacctt acctgggctc gaaatgcaga cgacatccgg 1021 cgaaagtcgg ctcccgcaag ggcgtctgta taggtgctgc atggctgtcg tcagctcgtg 1081 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc tcgtcctctg ttgccatcag 1141 gttaagctgg gcactctgag gagactgccg gtgataaacc ggaggaaggt ggggatgacg 1201 tcaagtcagc atggccttta tgtccagggc cacacacgtg ctacaatggc ggatacaaag 1261 cgtcgcaatc tcgcaagagt gagctaatcg cataaagtcc gtctcagttc ggattgtagg 1321 ctgcaactcg cctgcatgaa gttggaatcg ctagtaatcg cggatcagca tgccgcggtg 1381 aatacgttcc cgggccttgt acacaccgcc cgtcacatca cgaaagctgg ctgtactaga 1441 agtagccccg ccaacccgca agggaggcag gttcctaagg tatggtcagt gattggggtg 1501 aagtcgtaac aaggtaaccg taa // Uncultured Pseudomonas sp. clone HKT_RR2 16S ribosomal RNA gene, partial sequence GenBank: JN030400.1 FASTA Graphics PopSet

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LOCUS JN030400 1426 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Pseudomonas sp. clone HKT_RR2 16S ribosomal RNA gene, partial sequence. ACCESSION JN030400 VERSION JN030400.1 GI:342328439 KEYWORDS ENV. SOURCE uncultured Pseudomonas sp. ORGANISM uncultured Pseudomonas sp. Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; environmental samples. REFERENCE 1 (bases 1 to 1426) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1426) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1426 /organism="uncultured Pseudomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:114707" /clone="HKT_RR2" /environmental_sample /country="India: Bengaluru" rRNA <1..>1426 /product="16S ribosomal RNA" ORIGIN 1 cttgattcag cggcggacgg gtgagtaatg cctaggaatc tgcctggtag tgggggacaa 61 cgtttcgaaa ggaacgctaa taccgcatac gtcctacggg agaaagcagg ggaccttcgg 121 gccttgcgct atcagatgag cctaggtcgg attagctagt tggtggggta atggctcacc 181 aaggcgacga tccgtaactg gtctgagagg atgatcagtc acactggaac tgagacmcgg 241 gtycmgaytc ctwmsggagg cagcaktggg gaatattgga caatgggcga aagcctgatc 301 cagccatgcc gcgtgtgtga agaagktctt cggattgtaa rgcacttwaa gttgggagga 361 rrggcagtaa gctaatacct tgctkktttg acgttacsra cagaataagc accggctaac 421 tctktgccag cagccgcggt aatacagagg gtgcaagcgt taatcggaat tactgggcgt 481 aaagcgcgcg tasgtggttt gttaaagttg gatgtgaaag ccccgggctc aacttgggaa 541 ctgcatccaa aactggcaag ctagagtacg gtagagggtg gtggaatttc ctgtgtagcg 601 gtgaaaatgc gtagatatag gaaaggaaca ccagtggccg aaggcgacca ccctgkactg 661 awwctgacac tgaggtgcra aagcgtggrg agcaaacagg attagataac ccctgktagt 721 ccmmgccgta aacgatgtym actwrccgtt ggaatccttg agattttagt ggcgcagcta 781 acgcattaag ttgaccgcct ggggagtacg gccgcaaggt taaaactcaa atgaattgac 841 gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 901 caggccttga catgcagaga actttccaga gatggattgg tgccttcggg aactctgaca 961 caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgtaacg 1021 agcgcaaccc ttgtccttag ttaccagcac gttatggtgg gcactctaag gagactgccg 1081 gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc atggccctta cggcctgggc 1141 tacacacgtg ctacaatggt cggtacagag ggttgccaag ccgcgaggtg gagctaatct 1201 cacaaaaccg atcgtagtcc ggatcgcagt ctgcaactcg actgcgtgaa gtcggaatcg 1261 ctagtaatcg cgaatcagaa tgtcgcggtg aatacgttcc cgggccttgt acacaccgcc 1321 cgtcacacca tgggagtggg ttgcaccaga agtagctagt ctaaccttcg ggaggacggt 1381 taccacggtg tgattcatga ctggggtgaa gtcgtaacaa ggtaaa // Uncultured Thermoactinomyces sp. clone HKT_RR3 16S ribosomal RNA gene, partial sequence GenBank: JN030401.1 FASTA Graphics PopSet

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LOCUS JN030401 1516 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Thermoactinomyces sp. clone HKT_RR3 16S ribosomal RNA gene, partial sequence. ACCESSION JN030401 VERSION JN030401.1 GI:342328440 KEYWORDS ENV. SOURCE uncultured Thermoactinomyces sp. ORGANISM uncultured Thermoactinomyces sp. Bacteria; Firmicutes; Bacillales; Thermoactinomycetaceae; Thermoactinomyces; environmental samples. REFERENCE 1 (bases 1 to 1516) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1516) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1516 /organism="uncultured Thermoactinomyces sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:478984" /clone="HKT_RR3" /environmental_sample /country="India: Bengaluru" rRNA <1..>1516 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tggcggcgtg cctaatacat gcaagtcgag 61 cgggagaatc atccttcggg atgaggatcc agcggcgaac gggtgagtaa cacgtgggca 121 acctgcccgc aagaccggga taactccggg aaaccggggc taataccgga tagtcctttt 181 ccccgcatgg ggaaaagggg aaaggcggct tcggctgtca cttgcggatg ggcccgcggc 241 gcattagctg gttggtgagg taacggctca ccaaggcgac gatgcgtagc cgacctgaga 301 gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 361 ggaattttct gcaatgggcg aaagcctgac ggagcaacgc cgcgtgagtg aggacggcct 421 tcgggttgta aaactctgtt cttgaggaag aattccttcc aggcgaacag cctggaaggt 481 tgacggtact caaggagaaa gccccggcta actacgtgcc ggcagccgcg gtaatacgta 541 gggggcgagc gttatccgga attattgggc gtaaagcgcg cgcagsgcgg ctgattaagt 601 caggtgtgaa aggctgcggc tcaaccgcag agcggcacct gaaactggtc agcttgagtg 661 caggagaggg gagcggaatt cccgggtgta scggtggaat gcgtagagat cgggaggaac 721 accagtggcg aaggcggctc tctggcctgt aactgacgct gaggcgcgaa agcgtgggga 781 gcaaacagga ttagataccc tggtagtcac gctgtaaacg atgagtgcta ggtgttgggg 841 ggctacgccc ctcagtgccg aaggtaaccc attaagcact ccgcctgggg agtacggccg 901 caaggctgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 961 attcgaagca acgcgaagaa ccttaccagg gcttgacatc ccgctgaccc ctccagagat 1021 ggaggtttcc ttcgggacag cggtaacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1081 gagatgttgg gttcagtccc gcaacgaacg caacccttgt cgttagttgc cagcatttcg 1141 gatgggcact ctaacgagac agccggtgaa agccggagga aggtggggat gacgtcaaat 1201 catcatgccc cttatgtcct gggctacaca cgtgctacaa tggctggtac aaagggcagc 1261 gaacccgcga gggggagcca atcccaaaaa gccagtctca gttcggatcg caggctgcaa 1321 ctcgcctgcg tgaagccgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1381 ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgcaacac ccgaagtcgg 1441 tgaggcaacc ttttaggagc cagccgccga aggtggggca gatgattggg gtgaagtcgt 1501 aacaaggtaa ccgtaa // Uncultured Pseudomonas sp. clone HKT_RR4 16S ribosomal RNA gene, partial sequence GenBank: JN030402.1 FASTA Graphics PopSet

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LOCUS JN030402 1507 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Pseudomonas sp. clone HKT_RR4 16S ribosomal RNA gene, partial sequence. ACCESSION JN030402 VERSION JN030402.1 GI:342328441 KEYWORDS ENV. SOURCE uncultured Pseudomonas sp. ORGANISM uncultured Pseudomonas sp. Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; environmental samples. REFERENCE 1 (bases 1 to 1507) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1507) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1507 /organism="uncultured Pseudomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:114707" /clone="HKT_RR4" /environmental_sample /country="India: Bengaluru" rRNA <1..>1507 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag 61 cggatgacgg gagcttgctc cttgattcag cggcggacgg gtgagtaatg cctaggaatc 121 tgcctggtag tgggggacaa cgtttcgaaa ggaacgctaa taccgcatac gtcctacggg 181 agaaagcagg ggaccttcgg gccttgcgct atcagatgag cctaggtcgg attagctagt 241 tggtggggta atggctcacc aaggcgacaa tccgtaactg gtctgagagg atgatcagtc 301 acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg aatattggac 361 aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa 421 gcactttaag ttggggggaa gggcagtaag ctaatacctt gctgttttga cgttaccgac 481 agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg tgcaagcgtt 541 aatcggaatt actgggcgta aagcgcggcg tagkkgkttg ttaagttgga tgtggaaagc 601 cccgggctca acctgggaac tgcatccaaa actggcaagc taggagtacg gtagagggtg 661 gtggaatttc ctgtgtagcg gtgaaaatgc gtaratatag raagggaaca ccagtggcga 721 aggcgaccac ctggactgat actgacactg aggtgcgaaa gcgtggggag caaacaggat 781 tagataccct ggtagtccac gccgtaaacg atgtcaacta gccgttggaa tccttgagat 841 tttagtggcg cagctaacgc attaagttga ccgcctgggg agtacggccg caaggttaaa 901 actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 961 acgcgaagaa ccttaccagg ccttgacatg cagagaactt tccagagatg gattggtgcc 1021 ttcgggaact ctgacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 1081 gttaagtccc gtaacgagcg caacccttgt ccttagttac cagcacgtta tggtgggcac 1141 tctaaggaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcatcatgg 1201 cccttacggc ctgggctaca cacgtgctac aatggtcggt acagagggtt gccaagccgc 1261 gaggtggagc taatctcaca aaaccgatcg tagtccggat cgcagtctgc aactcgactg 1321 cgtgaagtcg gaatcgctag taatcgcgaa tcagaatgtc gcggtgaata cgttcccggg 1381 ccttgtacac accgcccgtc acaccatggg ggtgggttgc accagaagta gctagtctaa 1441 ccttcgggag gacggttacc acggtgtgat tcatgactgg ggtgaagtcg taacaaggta 1501 accgtaa // Uncultured bacterium clone HKT_RR5 16S ribosomal RNA gene, partial sequence GenBank: JN030403.1 FASTA Graphics PopSet

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LOCUS JN030403 1496 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR5 16S ribosomal RNA gene, partial sequence. ACCESSION JN030403 VERSION JN030403.1 GI:342328442 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1496) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1496) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1496 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR5" /environmental_sample /country="India: Bengaluru" rRNA <1..>1496 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca ggacgaacgc tggcggcgtg cctaatgcat gcaagtcgag 61 cgacgaatag gtgcgggtaa ccaatcctat tacggagcgg cgaacgggtg agtaacacgt 121 gagcaacctg ccccgcagac cgggatagcc cagggaaact tggattaata ccggatattc 181 tcttagaacc gcatggcttc ctagaggaaa ggcttctgcg ctacgggatg ggctcacggc 241 ctatcagcta gttggtgggg taacggccta ccaaggcgtc gacgggtagc tggtctgaga 301 ggacgatcag ccacactggg actgagacac ggcccagact cctacgggag gcagcagcag 361 ggaattttgc gcaatgggcg aaagcctgac gcagcaacgc cgcgtggggg atgaaggcct 421 tcgggttgta aaccccttta gggagggacg aaattgacgg tacccccaga ataagccccg 481 gccaactacg tgccagcagc cgcggtgata cgtagggggc gagcgttgtc cggaatcatt 541 gggcgtaaag agctcgtagg cggcttagta agtcggtcgt gaaagcccga ggctcaactt 601 cgggactgcg gtcgatacta ctatagctag agtttggtag aggagaacgg aattcccggt 661 gtagcggtgg aatgcgcaga tatcgggagg aacacctgta gcgaaagcgg ttctctgggc 721 caatactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 781 ccacgctgta aacgttgggc actagtgtgg cgacccgacc taacggttgc ccgtgccgaa 841 gctaacgcat taagtgcccc gcctggggag tacggccgca aggctaaaaa ctcaaaggaa 901 ttgacgggtc ccccgcacaa gcggcggagc atgcggctta attcgatgcg acccgaaaga 961 accttacctg ggtttgacat tatgggaaaa gccgtagaga tacggtgtcc gaaagggccc 1021 ataacaggtg gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1081 aacgagcgca acccttgtcc tatgttacca gcgagttacg tcggggactc ataggagact 1141 gccggggaca actcggagga aggtggggat gacgtcaagt cctcatgccc cttatgccca 1201 gggctgcacg catgctacaa tggtcggtac aacgggctgc gatcccgcga gggtgagcga 1261 atccctaaaa gccggcctca gttcagattg gagtctgcaa ctcgactcca tgaagtcgga 1321 gtcgctagta atcgcggatc agcaaagccg cggtgaatgc gttctcgggg attgtacaca 1381 ccgcccgtca agtcacgaaa gctggcaaca cccgaagtca gtggcccaac catttggagg 1441 gagctgccga aggtggggtt ggtaattggg gctaagtcgt aacaaggtag ccgtaa // Uncultured bacterium clone HKT_RR6 16S ribosomal RNA gene, partial sequence GenBank: JN030404.1 FASTA Graphics PopSet

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LOCUS JN030404 1501 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR6 16S ribosomal RNA gene, partial sequence. ACCESSION JN030404 VERSION JN030404.1 GI:342328443 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1501) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1501) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1501 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR6" /environmental_sample /country="India: Bengaluru" rRNA <1..>1501 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gaatcaacgc tggcggcgtg cctcagacat gcaagtcgaa 61 cgattaagac tcccttcggg gagtgcataa agtggcgcac gggtgagtaa cacgtaagta 121 atctaccttc gagtggggaa taacatcggg aaaccgatgc taataccgca taacgcagcg 181 gcatcgcaag atgacagttg ttaaaggagc aatccgcttg aagaggagct tgcggcagat 241 tagcttgttg gtaaggtaac ggcttaccaa ggctacgatc tgtatccggc ctgagagggc 301 ggtcggacac actgacactg aataacgggt cagactccta cgggaggcag cagtcgggaa 361 ttttgggcaa tgggcgaaag cctgacccag caacgccgcg tgagggatga agtatttcgg 421 tatgtaaacc tcgaaagaat aggaagaata aatgacggta ctatttataa gctccggcta 481 actacgtgcc agcagccgcg gtaatacgta gggagcaagc gttgttcgga tttactgggc 541 gtaaaagggc gcgtaggcgg catattatag tcmrstgtga aatytccgag cttaactcgg 601 aamtgtcagc tggatactga tgtgctwtga gtgcagaagg ggcaatctgg aatttcttgg 661 tgtagcgggt gaaatggcgt agatatcaag aggaactacc tgaggtgaag acrggttgct 721 gggytgacta ctgacgctga ggcgcgaaag ccaggggagc aaacgggatt agataccccg 781 gtagtcctgg ccytaaacga tgaatacttg gtgtctggag tttcaaatac tccgggtgcy 841 gtcgctaacg ttttaagtta ttccgcctgg ggagtacgca cgcaagtgtg aaactcaaag 901 gaattgacgg ggacccgcac aagcgktgga gcatgtggtt tawtcgacgc aacgcgaaga 961 accttaccta rgctagaatg tgarggaatt ctgggtaatg ccagaagtcm gggaaacygr 1021 acccaaaaca aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgy wgggttwagt 1081 yccgcaacga gcscaaccct tatmmacagt tgccatcatt aagttgggaa mtctgttgag 1141 actgcsgttg ataaaacgga ggaaggtggg gatgatagtc arstcatsmt rgcctttaks 1201 ctayasgkct wcacaygtgc tacaatggat ggtacaaaac gtcgcaatcc cgcgaggggg 1261 agctaatcgc gaaaaccatc ctcagttcgg attgaagtct gcaactcgac ttcatgaagt 1321 tggaatcgct agtaatcgca aatcagcatg ttgcggtgaa tacgttcccg ggtcttgtac 1381 acaccgcccg tcacatcacg aaagtaggtt gtactagaag tagctgggcc aactcgcaag 1441 agaggcaggt taccacggta tgatttatga ttggggtgaa gtcgtaacaa ggtagccgta 1501 a // Uncultured bacterium clone HKT_RR8 16S ribosomal RNA gene, partial sequence GenBank: JN030405.1 FASTA Graphics PopSet

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LOCUS JN030405 1473 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR8 16S ribosomal RNA gene, partial sequence. ACCESSION JN030405 VERSION JN030405.1 GI:342328444 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1473) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1473) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1473 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR8" /environmental_sample /country="India: Bengaluru" rRNA <1..>1473 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctcg ggacgaacgc tagcggcgtg cctaaggtat gcaagtcgag 61 cgagaccttc gggtctagcg gcgaacggtc gagtaacacg tagacaacct gcctccgagt 121 gggggataac agcgggaaac tgctgctaat accgcatgtg gtggcctctg gcatcagagg 181 ttgactaaag gcgcaagtcg ctgggagatg ggtctgcggc ctatcaggta gttggtgggg 241 taacggccta ccaagccgac gacgggtagc tggtctgaga ggatgatcag ccggacgggg 301 actgagacac ggccccgact cctccgggag gcagcaatga gggatattgc acaatgggcg 361 aaagcctgat gcagcgacgc cgcgtgggtg aggaagttct tcggaatgta aagccctttt 421 gcaggggacg attcaagacg gtaccctgcg aataagcccc ggctaactac gtgccagcag 481 ccgcggtaat acgtaggggg caagcgttgt ccggatttac tgggcggtaa agaggcggcg 541 tagrcggaac gktaagtctg gcttgaaagc cccsggctca accggggaga gtggctggaa 601 actggcgktc ttgaggggga tagaggagag tggaattcct ggtgtagcgg tgaaatgcgt 661 agatatcagg aggaacaccc gtggcgaagg cggctctctg ggtccgcctg acgctgaggc 721 gcgaaagcgt ggggagcgaa cgggattaga taccccggta gtccacgccg taaacgatgg 781 tcactaggtg tgtgcggtat cgaccccgca cgtgccgcag ccaacgcaat aagtgacccg 841 cctggggagt acggtcgcaa ggttgaaact caaaggaatt gacgggggcc cgcacaagcg 901 gtggagcatg tggattaata cgtcactaac cgtagaacct tacccagact tgacatccca 961 ggaaccccgg tgaaagctgg gggtgctcgc aagagagcct ggagacaggt gttgcatggc 1021 tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aaccctcacc 1081 gtatgttgcc agcgtaaagt cgggcactct tacggaactg cccgggtaac cgggaggaag 1141 gtggggacga cgtcaagtca gcatggccct tacgtctggg gcttcacaca tgctacaatg 1201 gccggtacag agggttccaa aaccgcgagg tggtggcaat cccaaaaagc cggtctcagt 1261 tcggatcgca ggctgcaact cgcctgcgtg aagtcggaat cgctagtaac cgccggtcag 1321 ctaaacggcg gtgaatatgt tcccgggcct tgtacacacc gcccgtcacg tcacctgagt 1381 ctgttgcacc cgaagccgct ggcctaaccc gcaagggagg gaggcgtcga aggtgtggcc 1441 ggtaaggggg acgaagtcgt aacaaggtaa ccg //

Uncultured bacterium clone HKT_RR9 16S ribosomal RNA gene, partial sequence GenBank: JN030406.1 FASTA Graphics PopSet

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LOCUS JN030406 1494 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR9 16S ribosomal RNA gene, partial sequence. ACCESSION JN030406 VERSION JN030406.1 GI:342328445 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1494) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1494) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1494 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR9" /environmental_sample /country="India: Bengaluru" rRNA <1..>1494 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gaatcaacgc tggcggcgtg cctcagacat gcaagtcgaa 61 cgattaaact ttctttcggg aaagatataa agtggcgcac gggtgagtaa cacgtaggta 121 atctaccttt gagtggggaa taactcgggg aaactcgagc taataccgca taatgcagcg 181 gcaccagatg gtgacagttg ttaaagcgag caatcgtgct taaagaggag cctgcggcag 241 attaggtagt tggtagggta atggcttacc aagcctgcga tctgtaaccg acctgagagg 301 gtggtcggtc acactgacac tgaataacgg gtcagactcc tacgggaggc agcagtcggg 361 aattttgggc aatgggcgaa agcctgaccc agcaacgccg cgtgaaggat gaagtctttc 421 gggatgtaaa cttcgtaaga ataggaagaa taaatgacgg tactatttgt aaggtccggc 481 taactacgtg ccagcagccg cggtaatacg tagggaccaa gcgttgttcg gatttactgg 541 gcgtaaaggg cgcgtaggcg gcagatctag tcagctgtga aatctcaggg cttaaccctg 601 aacggtcagc tgatactgtt ttgctagagt gcagaggggg caatcggaat tcttggtgta 661 gcggtgaaat gcgtagatat caagaggaac acctgacgcg aagcgggttg ctgagctgac 721 actgacgctg agcgcgaaag ctagggtagc aaacgggatt agataccccg gtagtcctag 781 ccctaaacga tgaatacttg gtgtctggag ttttttattc tcccgggtgc cgtcgctaac 841 gtttttagta wtccgcctgg ggagtacgca cgcaagtgtg aaactcaaag gaattgacgg 901 gggacccgca caagcggtgg agcatgtggt ttaattcgac gcaacgcgaa gaaccttacc 961 tggactagaa tgtgagggaa ggttacttaa tcgtaaccgt ccgggcaacc ggacccgaaa 1021 caaggtgctg catggctgtc gtcagctcgt gtcgtgaggt gttgggttaa gtcccgcaac 1081 gagcgcaacc cctattaaca gttgccatcg ggtaaagccg ggaactctgt taagactgct 1141 gttgataaaa cggaggaagg tggggatgat gtcaagtcat catggccttt atgttcaggg 1201 ctacacacgt gctacaatgg caggtacaaa gcgctgcaaa ctcgtaagag ggagccaatc 1261 gcaaaaagcc tgtctcagtt cggattgaag tctgcaactc gacttcatga agctggaatc 1321 gctagtaatc gcagatcaga acgctgcggt gaatacgttc ccgggtcttg tacacaccgc 1381 ccgtcacatc acgaaagtgg attgtactag aagtagcagg gctaaccctt cgggggggca 1441 tgttaccacg gtatgattca tgattggggt gaagtcgtaa caaggtaacc gtaa // Uncultured bacterium clone HKT_RR10 16S ribosomal RNA gene, partial sequence GenBank: JN030407.1 FASTA Graphics PopSet

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LOCUS JN030407 903 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR10 16S ribosomal RNA gene, partial sequence. ACCESSION JN030407 VERSION JN030407.1 GI:342328446 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 903) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 903) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..903 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR10" /environmental_sample /country="India: Bengaluru" rRNA <1..>903 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcgtggctca ggatcaacgc tggcggcgtg cctaatgcat gcaagtcgag 61 cggggatcac ttcggtgatc ttacgcggcg gacgggtgcg taacacgtgg gcaacctgcc 121 ccgaggtggg ggatagccgt gggaaaccgc gggtaattcc gcatacgctc actggtcggc 181 aggacgagtg aggaaagggt cttcggactc gcctggggag gggcctgcgg ccgattagct 241 agttgggggg gtaacggcct cccaaggcga cgattggtag ctggtctgag aggacggcca 301 gccacacggg gactgagaca cggccccgac tcctacggga ggcagcagca aggaatcttc 361 cgcaatgggc gacagcctga cggagcaacg ccgcgtgcgg gatgacgccc ttcggggtgt 421 aaaccgcttt tcttccggaa gaacgtggcg caagccacct gacggtacgg gaggaagaag 481 gaccggctaa ctacgtgcca gcagccgcgg taatacgtag ggtccgagcg ttgtccggag 541 ttactgggcg taaagcgcgc gcaggcggcg gtgtcagcat ggcgtgaaag ccccaggctc 601 aacctgggag ggtcgtcgtg gactgcaccg cttgagggcg gtaggggctg gtggaatgcc 661 tggtgtagtg gtgaaatgcg tagagatcag cggaacaccc gtggcgaaag cggccagctg 721 ggccgtccct gacgctgagc gcgaagcgtg ggagcgaacg ggatagatac ccggtagtcc 781 acgcagttaa ccgatgccga ctaagcgtgg gggagtgacc ccctccgtgc cggaacccac 841 gcgggaagtc taccgccttg agagttacgc ccctaggcta tactccaaga attgactggg 901 gac // Uncultured bacterium clone HKT_RR11 16S ribosomal RNA gene, partial sequence GenBank: JN030408.1 FASTA Graphics PopSet

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LOCUS JN030408 650 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR11 16S ribosomal RNA gene, partial sequence. ACCESSION JN030408 VERSION JN030408.1 GI:342328447 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 650) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 650) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..650 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR11" /environmental_sample /country="India: Bengaluru" rRNA <1..>650 /product="16S ribosomal RNA" ORIGIN 1 gagtttgatc atggctcaga acgaacgctg gcggcgtgcc taacacatgc aagtcgcacg 61 agaaagtcct tcggggcgag tagagtggcg cacgggtgag taacacatgg gaacattcct 121 ataggtgggg gataaccacg ggaaactgtg gctaataccg catggtctcg agagagtaaa 181 ggtggcttaa aacccattat caaggggctt catagcaggc ctcttgggta gtgggtatag 241 ctatcgccaa tagattggcc catgtcccat tagcttgttg gcggggtaac ggcccaccaa 301 ggcgacgatg ggtagctggt ctgagaggat gatcagccac actggaactg agacacggtc 361 cagactccta ctggaggcag cagtggggaa tattgcacaa tgggggaaac cctgatgcag 421 caacgccgcg tgagtgaaga aggctcttgg gtcgtaaagc tctttcggct ggaaagaagg 481 ttggcaaggt aaacaatctt gtcagctgac ggtaccagaa gaagaaggac ctgctaactg 541 tgtgccagca gcagcggtaa tacgtggggt ccgagcgttg ttcggaatta ctgggcgtaa 601 agcgcacata ggcgggtgag attgtcgatt gtgaaatccc tgggctaaac // Uncultured bacterium clone HKT_RR12 16S ribosomal RNA gene, partial sequence GenBank: JN030409.1 FASTA Graphics PopSet

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LOCUS JN030409 1488 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR12 16S ribosomal RNA gene, partial sequence. ACCESSION JN030409 VERSION JN030409.1 GI:342328448 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1488) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1488) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1488 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR12" /environmental_sample /country="India: Bengaluru" rRNA <1..>1488 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gaatcaacgc tggcggcgtg cctcagacat gcaagtcgaa 61 cgattaagac ttccttcggg aagtgtataa agtggcgcac gggtgagtaa cacgtaagta 121 atctaccctc gagtggggaa taacatcggg aaaccgatgc taataccgca taacgcagcg 181 gcaccgcaag gtgacagttg ttaaaggagt aattcgcttg aggaggagct tgcggcagat 241 tagctagttg gtaaggtaac ggcttaccaa ggctacgatc tgtatccggt ctaagaggac 301 ggtcggacac actgacactg aataacgggt cagactccta cgggaggcag cagtcgggaa 361 ttttgggcaa tgggcgaaag cctgacccag caacgccgcg tgaaggatga agtatttcgg 421 tatgcagact tcgaaagaat gggaagaata aatgacggta ccatttataa gctccggcta 481 actacgtgcc agcagccgcg gtaatacgta gggagcaagc gttgttcgga tttactgggc 541 gtaaagggcg cgtaggcggc gcggtaagac acttgtgaaa tctctgagct taactcagaa 601 cggccaagtg atactgcagt gctagagtgc agaaaggggc aatcggaatt cttggtgtag 661 cggtgaaatg cgtagatatc aagaggaaca cctgagtgaa gacgggttgc tgggctgaca 721 ctgacgctga ggcgcgaaag ccaggggagc aacgggatta gataccccgg tagtcctggc 781 cctaaacgat gaatacttgg tgtctggagt ttcaagactc caggtgccgt cgctaacgtt 841 ttaagtattc cgctggggag tacgctcgca agagtgaaac ctcaaaggaa ttgaccggaa 901 cccgcacagc ggtggagcat ggtggtttaa ttycgacgca macgcgaaga ayctacctgg 961 actarawkgt gaggggatgt cgggtaatgc cggcagtccg ggaaaccgga cccaaaacaa 1021 ggtgctgcat ggctgtcgtc agctggtgtc gtgagatgtt gggttaagtc ccgcaacgag 1081 cgcaaccctt atcaacagtt gccatcatta agttgggaac tctgttgaga ctgccgttga 1141 taaaacggag gaaggtgggg atgatgtcaa gtcatcatgg cctttatgtt cagggctaca 1201 cacgtgctac aatggatggt gcaaaacgtc gcaatcccgc gagggggagc taatcgcgaa 1261 aaccatcctc agttcggatt gaagtctgca actcgacttc atgaagttgg aatcgctagt 1321 aatcgcaaat cagcatgttg cggtgaatac gttcccgggt cttgtacaca ccgcccgtca 1381 catcacgaaa gtaggttgta ctagaagtag ctgggccaac tcgcaagagg ggtaggttac 1441 cacggtatga tttatgattg gggtgaagtc gtaacaaggt aaccgtaa // Uncultured bacterium clone HKT_RR13 16S ribosomal RNA gene, partial sequence GenBank: JN030410.1 FASTA Graphics PopSet

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LOCUS JN030410 1517 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR13 16S ribosomal RNA gene, partial sequence. ACCESSION JN030410 VERSION JN030410.1 GI:342328449 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1517) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1517) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1517 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR13" /environmental_sample /country="India: Bengaluru" rRNA <1..>1517 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca ggacgaacgc tggcggcgtg cttaacacat gcaagtggag 61 cgacgaacca gggcttgccc tggggcaaag ccgcgaacgg gtgagtaaca cgtgggtaac 121 ctgcctcgat gatcgggaca acccggggaa acccgggcta ataccgaatg tgccccgtcc 181 acatcagtgg acggtgtaaa ggaagcttcg gcctccgctt cgagatgggc ccgcggccca 241 ttagcttgtt ggtggggtaa aggcctacca aggctccgat gggtagctgg tctgagagga 301 cgatcagcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 361 atcttgcgca atgcgcgaaa gcgtgacgca gcaacgccgc gtgggggaag acggccttag 421 ggttgtaaac ccctttcaag agggacgaag gtcggcgcgt caatagcggg tccgactgac 481 ggtaacctcc acaagaagcc ccggctaact acgtgccagc agccgcggta atacgtaagg 541 ggcaagcgtt gtccggaaat tattgggcgt aaagaggcgt gtargcggtc cggtaagtca 601 gctgtgaaar tcaarggctc aacccttgaa agccggttga tactgtcggg ctagagtccg 661 gaagaggcga gtggaattcc ccggtgtagc ggtgaaatgc gcagatatcg ggaggaacac 721 caatggcgaa ggcagctcgc tgggacggta ctgacgctga gacgcgaaag cgtggggagc 781 aaacaggatt agataccctg gtagtacacg ccgtaaacga tgggcactag gtgttggggg 841 tgtcgactcc cccggcgccg tagctaacgc attaagtgcc ccgcctgggg agtacggccg 901 caaggctaaa actcaaagga attgacgggg gcccgcacaa gcagcggagc atgtggttta 961 attcgacgca acgcgaagaa ccttacctgg gcttgacatg ttcgtgacag gtgtggaaac 1021 acaccctccc ttcggggcac gatcacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg 1081 agatgttggg ttaagtcccg caacgagcgc agcccccgtc gcatgttgcc agcatttagt 1141 tggggactca tgcgagactg ccggtgacaa accggaggaa ggtggggatg acgtcaagtc 1201 atcatgcccc ttatgtccag ggctacacac gtgctacatt ggcgcataca aagggctgcg 1261 ataccgcgag gtggagcgaa tcccaaaaag tgcgtctcgg ttcggattgg aggctgaaac 1321 ccgcctccat gaaggtggag ttgctagtaa tcccggatca gcaacgccgg ggtgaatacg 1381 ttcccgggcc ttgtacacac cgcccgtcac accacgaaag cgggcaacac ccgaagccgg 1441 tggcctaacc cttacgggag ggagccgtcg aaggtggggc tcgtgattgg ggtgaagtcg 1501 taacaaggta accgtaa // Uncultured bacterium clone HKT_RR14 16S ribosomal RNA gene, partial sequence GenBank: JN030411.1 FASTA Graphics PopSet

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LOCUS JN030411 1468 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR14 16S ribosomal RNA gene, partial sequence. ACCESSION JN030411 VERSION JN030411.1 GI:342328450 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1468) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1468) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1468 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR14" /environmental_sample /country="India: Bengaluru" rRNA <1..>1468 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gaacgaacgc tggcggcatg cctaacacat gcaagtcgaa 61 cgcaccttcg ggtgagtggc ggacgggtga gtaacgcgtg ggaacctgca cttcagtggg 121 ggataacgac gggaaactgt cgctaatacc gcgtacgccc tacgggggaa agatttatcg 181 ctgaaggatg ggcccgcgtc ggattagcta gttggtgagg taatggctca ccaaggcgac 241 gatccgtagc tggtctaaga ggatgatcag ccacactggg actgagacac ggcccagact 301 cctacgggag gcagcagtgg ggaatattgg acaatggggg aaaccctgat ccagcaatgc 361 cgcgtgagtg atgaaggcct tagggttgta aagctctttc ggtggtgacg atgatgacgg 421 taaccacaga agaagccccg gctaacttcg tgccagcagc cgcggtaaga cgaagggggc 481 tagcgttgtt cggaattact gggcgttaaa gcgcacaggt aggcggcctt tcaagttccr 541 ratgtgaarg cccckggctw aactcgggaa ctgcattkrw tactgttggg ctagagrmck 601 ggagagkata gcggaatttc ccagtgtaga rgtgaaattc gtagaatatk ggrargaaca 661 ccagtggcga argcggctat cttggaccgg tactgacgct gargttgcga aagcgtgggg 721 agcaaacagg attwgatacc cttggtagtm cacgccgtaa actatgagtg ctagacgtcg 781 ggaagcttgc tttttcsgtg tcgcagctaa cgcaattaag cactccgcct ggggagtacg 841 gccgcaaggt taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 901 tttaattcga tgcaacgcga agaaccttac cagcccttga caatactagg tttcgactcc 961 gagagattgg agtttttcag ttcrgctggg mctartacag gtgctgcatg sctgwcktca 1021 gctcgtgtys tgagatgttg ggttaagtcc cgcaacsagc gcaaccctmm tcttcagttg 1081 ccawcaggtt atgctgggca myctgaagaw actgccggtk acaagccgga ggaagktggg 1141 gatgacktca agtcctcatr gcmcttacgg gctggtkcta cacacgtgct acaatggcgg 1201 tgacagtggg atgcgatggc gcaagccgga gcgaatctcc aaaagccgtc tcagttcgga 1261 ttgcactctg caactcgggt gcatgaagtc ggaatcgcta gtaatcgtgg atcagcatgc 1321 cacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagttggttt 1381 taccttaaga cggtgcgcta accgcaagga ggcagccggc cacggtaagg tcagcgactg 1441 gggtgaagtc gtaacaaggt aaccgtaa // Uncultured Pseudomonas sp. clone HKT_RR15 16S ribosomal RNA gene, partial sequence GenBank: JN030412.1 FASTA Graphics PopSet

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LOCUS JN030412 1508 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Pseudomonas sp. clone HKT_RR15 16S ribosomal RNA gene, partial sequence. ACCESSION JN030412 VERSION JN030412.1 GI:342328451 KEYWORDS ENV. SOURCE uncultured Pseudomonas sp. ORGANISM uncultured Pseudomonas sp. Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; environmental samples. REFERENCE 1 (bases 1 to 1508) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1508) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1508 /organism="uncultured Pseudomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:114707" /clone="HKT_RR15" /environmental_sample /country="India: Bengaluru" rRNA <1..>1508 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag 61 cggatgacgg gagcttgctc cttgattcag cggcggacgg gtgagtaatg cctaggaatc 121 tgcctggtag tgggggacaa cgtttcgaaa ggaacgctaa taccgcatac gtcctacggg 181 agaaagcagg ggaccttcgg gccttgcgct atcagatgag cctaggtcag attagctagt 241 tggtggggta atggctcacc aaggcgacga tccgtaactg gtctgagagg atgatcagtc 301 acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg aatattggac 361 aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa 421 gcactttaag ttgggaggaa gggcagtaag ctaatacctt gctgttttga cgttaccgac 481 agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg tgcaagcgtt 541 aatcggaatt actgggcgta aagcgcgcgt aggtggtttg ttaagttgga tgtgaaaagc 601 cccgggctca acctgggaac tgcatccaaa actggcaagc tagagtacgg tagagggtgg 661 tggaattttc ctgtgtagcg gtgaaatgcg tagatatagg aagggaacac cagtggcgaa 721 ggcgaccacc tggactgata ctgacactga ggtgcgaaag cgtggggagc aaacaggatt 781 aagatacyct ggtagtccac gcygtaaacg atgtcaacta gccgttggaa tcctttgaga 841 ttttagtggc gcagctaacg cattaagttg accgcctggg gagtacggcc gcaaggttaa 901 aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 961 aacgcgaaga accttaccag gccttgacat gcagagaact ttccagagat ggattggtgc 1021 cttcgggaac tctgacacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1081 ggttaagtcc cgtaacgagc gcaacccttg tccttagtta ccagcacgtt atggtgggca 1141 ctctaaggag actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcatcatg 1201 gcccttacgg cttgggctac acacgtgcta caatggtcgg tacagagggt tgccaagccg 1261 cgaggtggag ctaatctcac aaaaccgatc gtagtccgga tcgcagtctg caactcgact 1321 gcgtgaagtc ggaatcgcta gtaatcgcga atcagaatgt cgcggtgaat acgttcccgg 1381 gccttgtaca caccgcccgt cacaccatgg gagtgggttg caccagaagt agctagtcta 1441 accttcggga ggacggttac cacggtgtga ttcatgactg gggtgaagtc gtaacaaggt 1501 aaccgtaa // Uncultured bacterium clone HKT_RR16 16S ribosomal RNA gene, partial sequence GenBank: JN030413.1 FASTA Graphics PopSet

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LOCUS JN030413 1499 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR16 16S ribosomal RNA gene, partial sequence. ACCESSION JN030413 VERSION JN030413.1 GI:342328452 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1499) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1499) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1499 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR16" /environmental_sample /country="India: Bengaluru" rRNA <1..>1499 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tagcgggatg ctttacacat gcaagtcgaa 61 cggcagcgcg ggctttcggg ccaggcggcg agtggcgaac gggtgagtaa tgtatcggaa 121 cgtgcccagt agcgggggat aactacgcga aagcgtagct aataccgcat acgccctacg 181 ggggaaaggg ggggatcgca agacctctca ctattggagc ggccgatatc ggattagcta 241 gttggtgagg tataggctca ccaaggcgac gatccgtagc tggtttgaga ggacgaccag 301 ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaattttgg 361 acaatggggg caaccctgat ccagccatcc cgcgtgtgcg atgaaggcct tcgggttgta 421 aagcactttt ggcagggaag aaaaggcgcc ggctaatacc tggcgccgct gacggtacct 481 gcagaataag caccggctaa ctacgtgcca gcagccgcgg taatacgtag ggtgcaagcg 541 ttaatcggaa ttactgggcg taaagcgtgc gcaggcggtt cggaaagaaa gatgtgaaat 601 cccagggctt aaccttggaa ctgcattttt aactgccggg ctagagtatg tcagaggggg 661 tagaattcca cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg atggcgaacg 721 cagccccctg ggataatact gacgctcatg cacgaaagcg tggggagcaa acaggattag 781 ataccctgtt agtccacgcc ctaacgattg tcaactagct tttggggcct tcaggcctta 841 gtagcgcagc taacgcgtga agttgaccgc cttggggagt acggtcgcaa gattwaaact 901 ymaargawtt gacggggacc cgcacaagcg gtggatgatg tkgattwatt cgatgcaacg 961 cgaaaaacct tacctaccct tgacatgtct ggaatgccga agagatttgg cagtgctcgc 1021 aagagaaccg gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcatg agatgttggg 1081 ttaagtcccg caacgagcgc aacccttgtc attagttgct acgaaagggc actctaatga 1141 gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttatg 1201 ggtagggctt cacacgtcat acaatggtcg ggacagaggg tcgccaaccc gcaaggggga 1261 gccaatccca gaaacccgat cgtagcccgg attggagtct gcaactcgac tccatgaagt 1321 cggaatcgct agtaatcgcg gatcagaatg tcgcggtgaa tacgttcccg ggtcttgtac 1381 acaccgcccg tcacaccatg ggagtgggtt ttaccagaag tagttagcct aaccgcaagg 1441 agggcgatta ccacggtagg attcatgact ggggtgaagt cgtaacaagg tagccgtaa // Uncultured bacterium clone HKT_RR17 16S ribosomal RNA gene, partial sequence GenBank: JN030414.1 FASTA Graphics PopSet

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LOCUS JN030414 1489 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR17 16S ribosomal RNA gene, partial sequence. ACCESSION JN030414 VERSION JN030414.1 GI:342328453 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1489) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1489) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1489 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR17" /environmental_sample /country="India: Bengaluru" rRNA <1..>1489 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gaatcaacgc tggcggcgtg cctcagacat gcaagtcgaa 61 cgattaaact tcccttcggg gaagatataa agtggcgcac gggtgagtaa cacgtaagta 121 atctaccccc gagtggggaa taacgtccgg aaacggacgc taataccgca taatgcagcg 181 gcaccgcaag gtgacagttg ttaaagattt atcgcttgag gaggagcttg cggcagatta 241 gctagttggt aaggtaatgg ctyaycrags ctaacratct gtaaccsgty wmagaggacs 301 gtcggtcaca mtgacactga wtaacsggtc aractcctay sggaggcagc aktcgggaat 361 tttgggcmat gggcgaaagc ytgaycyagc awcgccgckt gaaggatgaa gtatttcggt 421 atgtaaactt cgaaagawtg ggaagaatta atgacggtac catttataar gtccggctaa 481 ctacgtgcca kcagccgcgg taawacgtak ggaccaagcg ttgttcggat twctgggcgt 541 aaarggcggc gtakgcggca tgacaagtca cttkttgaaa wctctgggct taaacccaga 601 acggccaagt gatactgtcg cgctagagtg cggaaggggc aatcggattc ctcggtgtag 661 cggtgaaatg cgtagatatc gagaggaaca ctgaggtgaa gacgggttgc tgggccgaca 721 ctgacgctga ggcgcgaaag ccaggggagc aaacgggatt agataccccg gtagtcctgg 781 ccctaaacga tgaatacttg gtgtctggag ttattagtgc tccgggtgcc gtcgctaacg 841 tttttagtat tccgcctggg gagtacgctc gcaagagtga aactcaaagg aattgacggg 901 gacccgcaca agcggtggag catgtggttt aattcgacgc aacgcgaaga accttaccta 961 ggctagaatg cgagggaagg aagggtaata ccgaccgtcc gggaaaccgg acccaaaaca 1021 aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1081 gcgcaacccc tattgatagt tgccatcatt aagttgggaa ctctatcaag actgctgttg 1141 ataaaacgga ggaaggtggg gatgatgtca agtcatcatg gcctttatgc ttagggctac 1201 acacgtgcta caatggccgg tacaaaacgt cgcgatcccg taagggggag ctaatcgcaa 1261 aaaccggtct cagttcggat tggagtctgc aactcgactc catgaagttg gaatcgctag 1321 taatcgcgaa tcagaacgtc gcggtgaata cgttcccggg tcttgtacac accgcccgtc 1381 acatcacgaa agtaggttgt actagaagta gctgggccaa cccgtaaggg aggcaggtta 1441 ccacggtatg atttatgatt ggggtgaagt cgtaacaagg taaccgtaa // Uncultured bacterium clone HKT_RR19 16S ribosomal RNA gene, partial sequence GenBank: JN030415.1 FASTA Graphics PopSet

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LOCUS JN030415 1491 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR19 16S ribosomal RNA gene, partial sequence. ACCESSION JN030415 VERSION JN030415.1 GI:342328454 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1491) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1491) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1491 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR19" /environmental_sample /country="India: Bengaluru" rRNA <1..>1491 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gaatcaacgc tggcggcgtg cctcagacat gcaagtcgaa 61 cgattaaagc tctcttcggg gagtgcatag agtggcgcac gggtgagtaa cacgtaagta 121 atctaccttc gagtggggaa taacgtcggg aaaccgacgc taataccgca taatgcagcg 181 gcatcgcaag atgacagttg ttaaaggagc aatccgcttg aagaggagct tgcggcagat 241 tagctagttg gtaaggtaat ggcttaccaa ggctacgatc tgtaaccggt cttggaggac 301 ggtcggtcac actgacactg aataacgggt cagactccta cgggaggcag cagtcgggaa 361 ttttgggcaa tgggcgaaag cctgacccag caacgccgcg tgaaggatga agtatctcgg 421 tatgtaaact tcgaaagaat gggaagaatc aatgacggta ccatttataa ggtccggcta 481 actacgtgcc agcagccgcg gtaatacgta gggaccaagc gttgttcgga tttactgggc 541 gtaaagggcg cgtaggcggc aattcaagtc agctgtgaaa tctctgggct taacccagaa 601 cggccagctg atamtgcttt gctagagtgc agaaggggca atysgaattc tcggkgtagy 661 ggtgaaatgc gtagatatcg agaggaacac ytgaggtgaa gacgggttgc tgggctgaca 721 ctgacgctga ggcgctgaaa gtcaggggag caaacgggat tagatacccc ggtagtcctg 781 gcccttaaac gatgaatact tggtgtctgg agttattagt gctccgggtg ccgtcgctaa 841 cgtttttagt attccgcctg gggagtacgc tcgcaagagt gaaactcaaa ggaattgacg 901 gggacccgca caagcggtgg agcatgtggt ttaattcgac gcaacgcgaa gaaccttacc 961 taggctagaa tgtgagggaa gaaagggtaa ttccaatcgt ccgggaaacc ggacccaaaa 1021 caaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1081 gagcgcaacc cctattgata gttgctaaca ttaagttgag aactctatca agactgctgt 1141 tgataaaacg gaggaaggtg gggatgatgt caagtcatca tggcctttat gcttagggct 1201 acacacgtgc tacaatggat ggtacaaaac gtcgcgatcc cgtaaggggg agctaatcgc 1261 aaaaaccatt ctcagttcgg attgaagtct gcaactcgac ttcatgaagt tggaatcgct 1321 agtaatcgcg gatcagaacg ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg 1381 tcacatcacg aaagtaggtt gtactagaag taggagggct aacccgcaag ggaggcatct 1441 taccacggta tgatttatga ttggggtgaa gtcgtaacaa ggtaaccgta a // Uncultured bacterium clone HKT_RR23 16S ribosomal RNA gene, partial sequence GenBank: JN030416.1 FASTA Graphics PopSet

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LOCUS JN030416 1503 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR23 16S ribosomal RNA gene, partial sequence. ACCESSION JN030416 VERSION JN030416.1 GI:342328455 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1503) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1503) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1503 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR23" /environmental_sample /country="India: Bengaluru" rRNA <1..>1503 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tggcggcatg ccttacacat gcaagtcgaa 61 cggcagcgcg ggggcaaccc tggcggcgag tggcgaacgg gtgagtaata tatcggaacg 121 tgccctagag tgggggataa cgtagcgaaa gttacgctaa taccgcatac gatctatgga 181 tgaaagtggg ggatcgcaag acctcatgct cctggagcgg ccgatatctg attagctagt 241 tggtgaggca aaggctcacc aaggcgacga tcagtagctg gtctgagagg acgaccagcc 301 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aattttggac 361 aatgggggca accctgatcc agcaatgccg cgtgagtgaa gaaggccttc gggttgtaaa 421 gctcttttgt caggaacgaa acggtgaagg ctaatatcct ttgctaatga cggtacctga 481 agaataagca ccggctaact acgtgccagc agccgcggta atacgtaggg tgcgagcgtt 541 aatcggaatt actgggcgta aagcatgcgc aggcggtttg gtaagacaga tgtgaaatcc 601 ccgggcttaa cctgggaatt gcatttgtga ctgcccggct agagtgtgtc agagggaggt 661 agaattccac gtgtagcagt gaaatgcgta gatatgtgga ggaataccga tggcgaaagc 721 agcctcctgg gataacactg acgctcatgc acgaaagcgt ggggagcaaa caggatttag 781 ataccctggt agtccacgcc ctaaaacgat gtctactagt tgtcgggtct taattgactt 841 ggtaacgcag ctaacgcgtg aagtagatcc gcctggggag tacggtcgca agatttaaaa 901 ctcaaaggaa ttgacgggga cccgcacaag cggtggatga tgtggattaa tttcgatgca 961 acgcgaaaaa ccttacctac ccttgacatg tctggaaccc tgctgaragg tgggggtgct 1021 cgaaagagaa ccagaacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1081 gggttaagtc ccgcaacgag cgcaaccctt gtcattagtt gctacgaaag ggcactctaa 1141 tgrractksc gkkkacaaac cggaggaagg tggggatgay rkcwagtcct catggycctt 1201 atggrtagrg cttacacasg tcatacaatg gtacatacag agggctgccg acccgcgagg 1261 gggagctaat cccagaaagt gtatcgtagt ccggattgta gtctgcaact cgactacatg 1321 aagttggaat cgctagtaat cgcggatcag catgtcgcgg tgaatacgtt cccgggtctt 1381 gtacacaccg cccgtcacac catgggagcg ggttttacca gaagtaggta gcttaaccgc 1441 aaggagggcg cttaccacgg taggattcgt gactggggtg aagtcgtaac aaggtaaccg 1501 taa // Uncultured Sphingomonas sp. clone HKT_RR24 16S ribosomal RNA gene, partial sequence GenBank: JN030417.1 FASTA Graphics PopSet

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LOCUS JN030417 1460 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Sphingomonas sp. clone HKT_RR24 16S ribosomal RNA gene, partial sequence. ACCESSION JN030417 VERSION JN030417.1 GI:342328456 KEYWORDS ENV. SOURCE uncultured Sphingomonas sp. ORGANISM uncultured Sphingomonas sp. Bacteria; Proteobacteria; Alphaproteobacteria; Sphingomonadales; Sphingomonadaceae; Sphingomonas; environmental samples. REFERENCE 1 (bases 1 to 1460) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1460) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1460 /organism="uncultured Sphingomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:158754" /clone="HKT_RR24" /environmental_sample /country="India: Bengaluru" rRNA <1..>1460 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gaacgaacgc tggcggcatg cctaacatat gcaagtcgaa 61 cgagaccttc gggtctagtg gcgcacgggt gcgtaacgcg tgggaatctg cccttgggtt 121 cgggataaca gttggaaacg actgctaata ccgaatgatg actccggtcc aaagatttat 181 cgcccaggga cgagcccgcg tcggattagc tagttggtga ggtaaaggct caccaaggcg 241 acgatccgta gctggtctga gaggatgatc agccacactg ggactgagac atggcccaga 301 ctcctacggg aggcagcagt ggggaatatt ggacaatggg cgaaagcctg atccrgmmak 361 gccgcgtgag tgatgaaggc cttaggkttg taaagctctt twacccggga tgatwatgac 421 agtaccgrga gaataagccc cggctaactc cgtaccagca gccgcgktaa tacggagggg 481 gctagcgttg wtcggaatta ctgggcgtaa agcggcgcgt aggcggcttt gcaagttaga 541 ggtgaargcc cggagcttaa ctcsggaact gcctttaaaa ctgcatcgct agaatcatgg 601 agaggttagt ggaaattccg agtgtaggag gtgaaattcg tagatattcg gaagaacacc 661 agtggcgaag tcgactaact gracatgcat tkracgctga rgtggcgaaa gcgtggggag 721 caaacargat tagatacyct kgataktcca cgycgtaaaa cratgatgac tagctgtcgg 781 ggctcatgga gtttcggtgg cgcagctaac gcgttaagtc atccgcctgg ggagtacggc 841 cgcaaggtta aaactcaaag aaattgacgg gggcctgcac aagcggtgga gcatgtggtt 901 taattcgaag caacgcgcag aaccttacca gcgtttgaca tgccaggacg gtttccagag 961 atggattcct tcccttacgg gacctggaca caggtgctgc atggctgtcg tcagctcgtg 1021 tcgtgagatg ttgggttaag tcccgcaacg ggcgcaaccc tcgtctttag ttgccaccat 1081 tcagttgggc actctaaaga aactgccggt gataagccgg aggaaggtgg ggatgacgtc 1141 aagtcctcat ggcccttacg cgctgggcta cacacgtgct acaatggcgg tgacagtggg 1201 cagcaaactc gcgagagtga gcaaatcccc aaaagccgtc tcagttcgga ttgttctctg 1261 caactcgagg gcatgaaggc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1321 acgttcccag gccttgtaca caccgcccgt cacaccatgg gagttggttt cacccgaagg 1381 cgctacgcta acccgtaagg gaggcaggcg accacggtgg gatcagcgac tggggtgaag 1441 tcgtaacaag gtaaccgtaa // Uncultured Stenotrophomonas sp. clone HKT_RR25 16S ribosomal RNA gene, partial sequence GenBank: JN030418.1 FASTA Graphics PopSet

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LOCUS JN030418 1522 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Stenotrophomonas sp. clone HKT_RR25 16S ribosomal RNA gene, partial sequence. ACCESSION JN030418 VERSION JN030418.1 GI:342328457 KEYWORDS ENV. SOURCE uncultured Stenotrophomonas sp. ORGANISM uncultured Stenotrophomonas sp. Bacteria; Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Stenotrophomonas; environmental samples. REFERENCE 1 (bases 1 to 1522) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1522) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1522 /organism="uncultured Stenotrophomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:165438" /clone="HKT_RR25" /environmental_sample /country="India: Bengaluru" rRNA <1..>1522 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gagtgaacgc tggcggtagg cctaacacat gcaagtcgaa 61 cggcagcaca gaggagcttg ctccttgggt ggcgagtggc ggacgggtga ggaatacatc 121 ggaatctact ctgtcgtggg ggataacgta gggaaactta cgctaatacc gcatacgacc 181 tacgggtgaa agcaggggat cttcggacct tgcgcgattg aatgagccga tgtcggatta 241 gctagttggc ggggtaaagg cccaccaagg cgacgatccg tagctggtct gagaggatga 301 tcagccacac tggaactgag acacggtcca gactcctacg ggaggcagca gtggggaata 361 ttggacaatg ggcgcaagcc tgatccagcc ataccgcgtg ggtgaagaag gccttcgggt 421 tgtaaagccc ttttgttggg aaagaaatcc atctggttaa tacccgggtg ggatgacggt 481 acccaaagga taagcaccgg ctaacttcgt gccagcagcc gcggtaatac gaagggtgca 541 agcgctactc ggaattactg ggcgtaaagc gtgcgtaggt ggtcgtttaa gtccgttgtg 601 aaagccctgg gctcaacctg ggaactgcag tggatactgg gcgactagaa tgtgggtaga 661 gggtagcgga attcctggtg tagcagtgaa atgcgtagag atcagaagga acattccatg 721 gcgaaggcag ctayctttgg accamcattt gacactgagg caygaaagcg tggggagcaa 781 acaggattag ataccctgkt agtccacgcc cttaaacgat gcgaactgga tgttgggtgc 841 aatttgggca cgcagtawyc aagctaacgc gttaaagttc gccgcytggg gaktacggtc 901 gcaagactkg aaaactcaaa ggaattgacs ggggcccgac wcaagcggtg gagtatgtgg 961 tttaattcga tgcaacgcga agaaccttac ctggccttga catgtcgaga actttccaga 1021 gatggattgg tgccttcggg aactcgaaca caggtgctgc atggctgtcg tcagctcgtg 1081 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgtccttag ttgccagcac 1141 gtaatggtgg gaactctaag gagaccgccg gtgacaaacc ggaggaaggt ggggatgacg 1201 tcaagtcatc atggccctta cggccagggc tacacacgta ctacaatggt agggacagag 1261 ggctgcaagc cggcgacggt gagccaatcc cagaaaccct atctcagtcc ggattggagt 1321 ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg cagatcagca ttgctgcggt 1381 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagttt gttgcaccag 1441 aagcaggtag cttaaccttc gggagggcgc ttgccacggt gtggccgatg actggggtga 1501 agtcgtaaca aggtaaccgt aa // Uncultured bacterium clone HKT_RR26 16S ribosomal RNA gene, partial sequence GenBank: JN030419.1 FASTA Graphics PopSet

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LOCUS JN030419 871 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR26 16S ribosomal RNA gene, partial sequence. ACCESSION JN030419 VERSION JN030419.1 GI:342328458 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 871) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 871) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..871 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR26" /environmental_sample /country="India: Bengaluru" rRNA <1..>871 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gaacgaacgc tggcggcatg cctaacacat gcaagtcgaa 61 cgagaccttc gggtctagtg gcgcacgggt gcgtaacgcg tgggaacctg cctttaggtt 121 cggaataact ccccgaaagg ggtgctaata ccggatgatg tcttcggacc aaagatttat 181 cgccttaaga tgggcccgcg taagattagg tagttggtgg ggtaaaggcc taccaagccg 241 acgatcttta gctggtctga gaggatgatc agccacactg ggactgagac acggcccaga 301 ctcctacggg aggcagcagt ggggaatatt ggacaatggg cgaaagcctg atccagcaat 361 gccgcgtgag cgatgaaggc cttagggttg taaagctctt ttaccaggga tgataatgac 421 agtacctgga gaataagctc cggctaactc cgtgccagca gccgcggtaa tacggaggga 481 gctagcgttg ttcggaatta ctgggcgtaa agcgcacgta ggcggcgact caagtcaggg 541 gtgaaagccc ggggctcaac cccggaactg cccttgaaac taggttgcta gaatcttgga 601 gaggtcagtg gaattccgag tgtagaggtg aaattcgtag atattcggaa gaacaccagt 661 ggcgaaagcg actgactgga caagtattga cgctgagtgc gaaagcgtgg ggagcaaaca 721 ggattagata ccctgatagt ccacgcgtaa acgatgataa ctagctgtcc gggcacttgg 781 tgcttgggtg gcgcagctaa cgcattaagt tatccgcctg gggagtaccg tcgcaagatt 841 aaactccaaa agaattgacg ggggcctgcc c // Uncultured bacterium clone HKT_RR27 16S ribosomal RNA gene, partial sequence GenBank: JN030420.1 FASTA Graphics PopSet

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LOCUS JN030420 1505 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR27 16S ribosomal RNA gene, partial sequence. ACCESSION JN030420 VERSION JN030420.1 GI:342328459 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1505) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1505) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1505 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR27" /environmental_sample /country="India: Bengaluru" rRNA <1..>1505 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagcggcagg cttaatacat gcaagtcgaa 61 cggtaacagg tctgtagcaa tacagatgct gacgagtggc agacgggtga ggaacacgta 121 cacaactttc ctttatctgg tgaatagccc cgagaaatcg ggattaatac accatagtat 181 catgaagggt catctctttg tgattaaagc tcaggcggat aaagatgggt gtgcgtctga 241 ttagctagtt ggttgaggta acggctcacc aaggcgacga tcagtracys stgtgtggag 301 agmacrrcac agtcacacgg scaykgagat acgrkcccga ytcctacggg argcagcagt 361 waggaatatt ggtcawtgga cgcmagtctg aaccagscat gccgcgtgca ggatgaaggc 421 cytctgggtc gtaaactgct tttatcaggg aagaaatcac twatttctat gagkgttgac 481 ggtacctgag gaataagcac yggctaactc cgtgccagca gccgcggtaa tacgrrgggt 541 gcaagcgtta tccggattta ctgggtttaa agggtgcgta gsmagscttt aagtcagtgg 601 tgaaaagctc cgagcttaac tcggaaactg ccaattgata ctattggtct tgaattcrgt 661 cgaggtttgg cggaatgtgt cstgtarccg gtgaaaatgc ttaratatga cacagaacaa 721 ccgatttgcg aaggcagcta gctagacgac attgacgctg aggcacgaaa gcgtggggat 781 caaacaggat tagaaaccct ggtagtccac gccctaaact atgattactc gatgttggcg 841 atacactgcc agcgtctaag cgaaagcatt aagtaatcca cctggggagt acgatcgcaa 901 gattgaaact caaaggaatt gacgggggtc cgcacaagcg gtggagcatg tggttcaatt 961 cgatgatacg cgaggaacct tacctgggct agaatgcaag ggggccgtgg gtgaaagctc 1021 actttccagc aatggaccgc ttgcaaggtg ctgcatggct gtcgtcagct cgtgccgtga 1081 ggtgttgggt taagtcccgc aacgagcgca acccccatct ttagttgcca gcgagtaatg 1141 tcggggactc tagagaaact gcctgcgtaa gcagtgagga aggaggggac gacgtcaagt 1201 catcacggcc tttatgtcca gggctacaca cgtgctacaa tggcgggtac aatgggttgc 1261 tacacggcga cgcgacgcta atcccaaaaa acccgtctca gttcggattg gagtctgcaa 1321 ctcgactcca tgaagctgga atcgctagta atcgcgcatc agcaacggcg cggtgaatac 1381 gttcccggac cttgtacaca ccgcccgtca agccatggaa gccgggtgta cctgaaggcg 1441 ataaccgcaa ggagtcgctc aaggtaaaat cggtaactgg ggctaagtcg taacaaggta 1501 accga //

Uncultured bacterium clone HKT_RR28 16S ribosomal RNA gene, partial sequence GenBank: JN030421.1 FASTA Graphics PopSet

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LOCUS JN030421 1469 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR28 16S ribosomal RNA gene, partial sequence. ACCESSION JN030421 VERSION JN030421.1 GI:342328460 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1469) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1469) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1469 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR28" /environmental_sample /country="India: Bengaluru" rRNA <1..>1469 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gcctgaacgc tggcggcgtg gctaagacat gcaagtcgca 61 cggacgtagc aatacgttag tggcgaacgg gtgagtaatg aatcgctaat gtgccctgga 121 ctttgggata gctacccgaa agggtaggta ataccagatg atatgcagct gtcgcatgac 181 agttgcctca aaggtccgcc ggtctgggag caggcgattt cgtatcagct agttggtgag 241 gtaacggctc accaaggcga tgacgcgtag ccggactgag aggttggccg gccacattgg 301 gactgagaca ctgcccagac tcctacgggg ggctgcagta acgaattttc cgcaatgggc 361 gaaagcctga cggagcgacg ccgcgtgtag gagaagccct tcggggtgta aactactgtc 421 aggggttaga aagttccgat caaccccaga ggaaggcacg gctaactctg tgccagcagc 481 cgcggtaaga cagaggtgcc aagcgttagg cggaatcact gggcttaaag cgtgtgtagg 541 cgggtcgtta agtaccttgt gaaatcccac ggcttaaccg tggaactgct tggtatactg 601 gcgatcttga gccacttagg ggttacygga acaaacggtg gagcggtgaa atgcgtagat 661 atcgtttgga acgccaatgg tgaaaacagg taactgggag tgcgctgacg ytgagacacg 721 aaagccaggg gagcgaacgg gattagatac cccggtagtc ctggcmgtaa acgatgtcga 781 ctagatcgtg gcagctctga cgttgtcacg gtcggaagca aargtgctaa gtcggaccgc 841 ctgggaagta cggtcgcaag gctaaaactc aaaggaattg acgggggctc acacaagcgg 901 tggagcatgt ggttcaattc gaagcaacgc gcagaacctt acctgggctt gacatgcttg 961 gattatctcc atgaaagtgg ggtaggccct tcggggtaca acaagtacag gtgctgcatg 1021 gctgtcgtca gctcgtgtcg tgagatgtcg ggttaagtcc cttaacgagc gaaaccttta 1081 cccttagtta ccagcgggtt atgccgggga ctctaagggg actgccggtg tcaaaccgga 1141 ggaaggtgga gatgacgtca agtcctcatg gcctttatgc ccagggccac acacgtgcta 1201 caatggggtg cacagagcga accgagagcg caagctggag gaaatcgcat aaatcacccc 1261 ccagttcaga tcggaggctg caactcgcct ccgtgaagtt ggaatcgcta gtaatcgcgg 1321 atcagctacg ccgcggtgaa tgtgttcctg agccttgtac acaccgcccg tcaagtcatg 1381 ggagccggaa atggccgaag tcgtctcacc actgagatgc ctacgccagg ttcggtgact 1441 gggactaagt cgtaacaagg taaccgtaa // Uncultured bacterium clone HKT_RR29 16S ribosomal RNA gene, partial sequence GenBank: JN030422.1 FASTA Graphics PopSet

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LOCUS JN030422 996 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR29 16S ribosomal RNA gene, partial sequence. ACCESSION JN030422 VERSION JN030422.1 GI:342328461 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 996) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 996) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..996 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR29" /environmental_sample /country="India: Bengaluru" rRNA <1..>996 /product="16S ribosomal RNA" ORIGIN 1 cagcagccgt cgtaattccg aggggtgcga ggcggttatc cggaaattac tggtttaaag 61 gggcgtaagg cggctttgtt aagtcaagag ggaaagtttg cggctcaacc gtaaaaattg 121 cttttgatac tgcaaagctg gaattaggat gaagttcagc ggaatgtggc agtagcggtg 181 aaatgcatag atattgccat agaacaccaa ttgcgaaggc agcttgctag acctggattg 241 acggtgaggc acgaaagcgt ggggagcgaa caggattaga taccctggta gtgcacgccc 301 taaacgatgc tcgctcgacg tatgatgcta gacattgtgc gtccaaggga aaccgttaag 361 tgagccacct ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggtccgc 421 acaagcggtg gagcatgtgg tttaattcga tgatacgcga ggaaccttac ctgggctaga 481 atgcgagtga cgtcctgtga aagcaggatt cccttcgggg cacaaagcaa ggtgctgcat 541 ggctgtcgtc agctcgtgcc gtgaggtgtt gggttaagtc ccgcaacgag cgcaacccct 601 gtccttagtt gccaacccat cgcaagatgg agggactcta aggagactgc cggcgtaagc 661 cgtgaggaag gtggggatga cgtcaagtca tcatggcctt tatgcccagg gcgacacacg 721 tgctacaatg gccggtacag agggttgcca agccgcaagg tggagccaat cccttaaagc 781 cggtctcagt tcggattgga gtctgaaacc cgactccatg aagttggaat cgctagtaat 841 cgcgcatcag ccatggcgcg gtgaatacgt tcccggacct tgtacacacc gcccgtcaag 901 ccatggaagc cgggggtacc tgaagacggt gactttactg ggagctgtct aaggtaaaac 961 tggtgactgg ggctaagtcg taacaagtaa ccgtaa // Uncultured bacterium clone HKT_RR30 16S ribosomal RNA gene, partial sequence GenBank: JN030423.1 FASTA Graphics PopSet

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LOCUS JN030423 850 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR30 16S ribosomal RNA gene, partial sequence. ACCESSION JN030423 VERSION JN030423.1 GI:342328462 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 850) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 850) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..850 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR30" /environmental_sample /country="India: Bengaluru" rRNA <1..>850 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagcggcagg cttaatacat gcaagtcgag 61 gggcagcgcg gtgtagcaat acactggcgg cgaccggcaa acgggtgcgg aacacgtaca 121 gaaccttcct tcgagcgggg aatagcccag agaaatttgg attaataccc catagtatat 181 tagagtggca tcactttgat attaaagatt tatcacttga agatggctgt gcggctgatt 241 aggtagttgg cggggtaacg gcccaccaag cctacgatca gtaactggtg tgagagcacg 301 accagtcaca cgggcactga gacacgggcc cgactcctac gggaggcagc agtaaggaat 361 attggtcaat ggacgaaagt ctgaaccagc catgccgcgt ggaggatgaa ggtcctctgg 421 attgtaaact tcttttatat gggacgaaaa agggcttttc taagtcaact gacggtacca 481 tatgaataag caccggctaa ctccgtgcca gcagccgcgg taatacggag ggtgcaagcg 541 ttatccggat tcactgggtt taaagggtgc gtaggtgggt tggtaagtca gtggtgaaat 601 ccccgagctt aacttgggaa ctgccattga tactatcagt cttgaatacc gtggatgtca 661 gcagattatg tcatgtagcg gtgaaatgct taaatatgac atagaacacc cattgcgaag 721 gcagctggct acacgaatat tgacactgag cacgaaagcg tggggatcaa acgggattaa 781 aataccctgg tagtccacgc cctaaactaa tggaatactc gactatacgc gaatacactg 841 tgtgttgtct // Uncultured Bacteroidetes bacterium clone HKT_RR31 16S ribosomal RNA gene, partial sequence GenBank: JN030424.1 FASTA Graphics PopSet

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LOCUS JN030424 1507 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Bacteroidetes bacterium clone HKT_RR31 16S ribosomal RNA gene, partial sequence. ACCESSION JN030424 VERSION JN030424.1 GI:342328463 KEYWORDS ENV. SOURCE uncultured Bacteroidetes bacterium ORGANISM uncultured Bacteroidetes bacterium Bacteria; Bacteroidetes; environmental samples. REFERENCE 1 (bases 1 to 1507) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1507) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1507 /organism="uncultured Bacteroidetes bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:152509" /clone="HKT_RR31" /environmental_sample /country="India: Bengaluru" rRNA <1..>1507 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagctggagg cttaatacat gcaagtcgag 61 cgggattggc ccagcaatgg gccatgagag cggcgcacgg gtgagtaacg cgtacacgac 121 ctacctctaa cagggggata gccctgggaa accgggatta ataccgcata ttatttggat 181 aaataaagga ttcaaatgaa agctgaggcg gttagagatg ggtgtgcgtc tgattagcta 241 gttggtaggg taacggccta ccaaggcgat gatcagtagg gggcgtgaga gcgtggtccc 301 ccacacgggt actgagacac ggacccgact cctacgggag gcagcagtaa ggaatattgg 361 acaatggccg caaggctgat ccagccatcc agcgtgcagg aagaaggccc tatgggttgt 421 aaactgcttt tgtcagggaa gaaacctctt catttatgag gagctgacgg tacctgaaga 481 ataagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc gagcgttatc 541 cggaattact gggtttaaag ggtgcgtagg cggcgcaagt aagtcaggag tgaaagtttg 601 cggctcaacc gtaaaattgc ttttgatact gctgtgctag aattatgatg atgtcagcgg 661 aatgtggcat gtagcggtga aatgcataga tatgccatag aacaccaatt gcgaaagcag 721 cttggctaga cctggattga cgctgaagca ccgaaaagcg ttgggtagcg aacaaggatt 781 arataccctg gtagtccacg ccctaaacga ttgctcactc gacgatatga tactarawaw 841 tgtgcgtcca ggggaamccg ttwagtgagc cacctggggg agtacgaccg caaggttgaa 901 actcaaagga attgacrggg gkycgcacaa tgcggtgtga gcwkktggtt taattcgatg 961 atacgcgagg aaccttacct gggctagaat gcgagtgacg gaccgtgaaa gcggtcttcc 1021 cttcggggca caaagcaagg tgctgcatgg ctgtcgtcag ctcgtgccgt gaggtgttgg 1081 gttaagtccc gcaacgagcg caacccctgt ccttagttgc caactctccc gtaagggaga 1141 agggactcta aggagactgc cggcgtaagc cgtgaggaag gtggggatga cgtcaagtca 1201 tcatggcctt tatgcccagg gcgacacacg tgctacaatg gccggtacag agggttgcca 1261 agccgcaagg tggagccaat cccttaaagc cggtctcagt tcggattgga gtctgaaacc 1321 cgactccatg aagttggaat cgctagtaat cgcgcatcag ccatggcgcg gtgaatacgt 1381 tcccggacct tgtacacacc gcccgtcaag ccatggaagc cgggggtacc tgaagacggt 1441 gactttactg ggagctgtct aaggtaaaac tggtgactgg ggctaagtcg taacaaggta 1501 accgtaa // Uncultured bacterium clone HKT_RR33 16S ribosomal RNA gene, partial sequence GenBank: JN030425.1 FASTA Graphics PopSet

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LOCUS JN030425 1456 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR33 16S ribosomal RNA gene, partial sequence. ACCESSION JN030425 VERSION JN030425.1 GI:342328464 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1456) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1456) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1456 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR33" /environmental_sample /country="India: Bengaluru" rRNA <1..>1456 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gagcgaacgc tggcggcagg cttaacacat gcaagtcgag 61 cgccccgcaa ggggagcggc agacgggtga gtaacacgtg ggaacgtgcc ttttggttcg 121 gaacaaccca gggaaacttg ggctaatacc ggataagccc taacggggaa agatttatcg 181 ccaaaagatc ggcccgcgtc tgattagcta gttggtgggg taatggccca ccaaggctac 241 gatcagtagc tggtctgaga ggatgatcag ccacattggg actgagacac ggcccaaact 301 cctacgggag gcagcagtgg ggaatattgg acaatggggg caaccctgat ccagccatgc 361 cgcgtgagtg atgaaggcct tagggttgta aagctctttt agcggggaag ataatgacgg 421 tacccgcaga aaaagccccg gctaacttcg tgccagcagc cgcggtaata cgaagggggc 481 tagcgttgct cggaatcact gggcgtaarg sgcacagtar gcrgcttctw arktcagggr 541 tgaaaagcyt ggagctcaam tycagaactg cctttgwtac tgaaggaggc tkragtccgg 601 gagaggtgag tggaactkcg agtktakagg tgaaattcgt agwtattcgc aagaacacca 661 gtggcgaarg cggctcactg kcccggtact gacgctgarg tgsgaaarcg tggggagcaa 721 acargattat kataycwytg ktagtccacg cygtaaacga tgaatgctag ccgttggcga 781 gcttgctcgt cagtggcgca gctaacgctt taagcattcc gcctggggag tacggtcgca 841 agattaaaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggttcaa 901 ttcgacgcaa cgcgaagaac tttaccagcc cttgacatgc cacgacggtt tccagagatg 961 gattcctccc cgcaaggggc gtggacacag gtgctgcatg gctgtcgtca gctcgtgtcg 1021 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg cccttagttg ccatcattca 1081 gttgggcact ctaaggggac tgccggtgat aagccgcgag gaaggtgggg atgacgtcaa 1141 gtcctcatgg cccttacggg ctgggctaca cacgtgctac aatggcggtg acagtgggat 1201 gcaatggagt aatcctgcgc aaatctcaaa aagccgtctc agttcggatt gtgctctgca 1261 actcgagcac atgaagttgg aatcgctagt aatcgcagat cagcacgctg cggtgaatac 1321 gttcccgggc cttgtacaca ccgcccgtca caccatggga gttggtttta cctgaagacg 1381 gtgcgctaac cgcaagggag cagccggcca cggtagggtc agcgactggg gtgaagtcgt 1441 aacaaggtaa ccgtaa // Uncultured bacterium clone HKT_RR34 16S ribosomal RNA gene, partial sequence GenBank: JN030426.1 FASTA Graphics PopSet

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LOCUS JN030426 1506 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR34 16S ribosomal RNA gene, partial sequence. ACCESSION JN030426 VERSION JN030426.1 GI:342328465 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1506) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1506) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1506 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR34" /environmental_sample /country="India: Bengaluru" rRNA <1..>1506 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag 61 cggatgaagg gagcttgctc cctgatttag cggcggacgg gtgagtaatg cctaggaatc 121 tgcctggtag tgggggataa cgttccgaaa ggaacgctaa taccgcatac gtcctacggg 181 ggaaagcagg ggaccttcgg gccttgcgct atcagatgag cctaggtcgg attagctagt 241 tggtgaggta atggctcacc aaggcgacga tccgtaactg gtctgagagg atgatcagtc 301 acactggaac tgggacacgg tccagactcc tacgggaggc agcagtgggg aatattggac 361 aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa 421 gcactttaag ttgggaggaa gggcgtttgg ctaatatcca agcgttttga cgttaccgac 481 agactaagca ccggctaact tcgtgccagc agccgcggta atacgaaggg tgcaagcgtt 541 aatcggaatt actgggcgta aagcgcgcgt aggtggttcg ttaagttgga tgtgaaagcc 601 ccgggctcaa cctgggaact gcatccaaaa ctggcgagct agagtacggt agagggtagt 661 ggaatttcct gtgtagcggt gaaatgcgta gatataggaa ggaacaccag tggcgaaagc 721 gactaccctg gactgatact gacactgaag tgcgaaagcg tgggggagca aacaggatta 781 gaataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttgggtt csttgagaac 841 ttagtggcgc asctaacgca ttaagttgac cgcctgggga gtacggccgc aaggttaaaa 901 ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 961 cgcgaagaac cttacctggc cttgacatgc tgagaacttt ccagagatgg attggtgcct 1021 tcgggaactc agacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1081 ttaagtcccg taacgagcgc aacccttgtc cttagttacc agcacgttat ggtgggcact 1141 ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatggc 1201 ccttacggcc agggctacac acgtgctaca atggtcggta caaagggttg ccaagccgcg 1261 aggtggagct aatcccataa aaccgatcgt agtccggatc gcagtctgca actcgactgc 1321 gtgaagtcgg aatcgctagt aatcgtgaat cagaatgtca cggtgaatac gttcccgggc 1381 cttgtacaca ccgcccgtca caccatggga gtgggttgct ccagaagtag ctagtctaac 1441 cttcgggggg acggttacca cggagtgatt catgactggg gtgaagtcgt aacaaggtag 1501 ccgtaa // Uncultured Nocardioidaceae bacterium clone HKT_RR35 16S ribosomal RNA gene, partial sequence GenBank: JN030427.1 FASTA Graphics PopSet

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LOCUS JN030427 1488 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Nocardioidaceae bacterium clone HKT_RR35 16S ribosomal RNA gene, partial sequence. ACCESSION JN030427 VERSION JN030427.1 GI:342328466 KEYWORDS ENV. SOURCE uncultured Nocardioidaceae bacterium ORGANISM uncultured Nocardioidaceae bacterium Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales; Propionibacterineae; Nocardioidaceae; environmental samples. REFERENCE 1 (bases 1 to 1488) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1488) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1488 /organism="uncultured Nocardioidaceae bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:253824" /clone="HKT_RR35" /environmental_sample /country="India: Bengaluru" rRNA <1..>1488 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggacgaacgc tggcggcgtg cttaacacat gcaagtcgag 61 cggaaaggcc acttcggtgg tactcgagcg gcgaacgggt gagtaacacg tgagtaatct 121 gcccctggct ttgggatagc caccggaaac ggtgattaat accggatatg accacttcac 181 gcatgtgatg gtggtggaaa gtttttcggc cagggatgtg ctcgcggcct atcagcttga 241 tggtgaggta atggctcacc atggcttcga cgggtagccg gcctgagagg gtgaccggtc 301 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattggac 361 aatgggcgga agcctgatcc agcaatgccg cgtgagggat gacggccttc gggttgtaaa 421 cctctttcgc ctgtgacgaa gcgcaagtga cggtaacagg taaagaagca ccggccaact 481 acgtgccagc agccgcggta atacgtaggg tgcgagcgtt gtccggaatt awtgggcgta 541 aagggcttcg taggcggttw gtygcgtcgg ragtgaaaac maggtgctta acagctgksy 601 tgctttcgat acgggcrrrc tagaaggtat tcagggraga acggaattyc tggtgtagcg 661 gtgaaatgcg cagattatca agraggaaca mcggtggcga argcrgttct ctgggaaatg 721 acytgacgct gaggagcgga aagtgtgggg agcgaacagg attagatacc ttggtagttc 781 acactgtaaa cgttgggcgc taggtgtggg gtccattcca cggattccgt ggtgcagcta 841 acgcataaag cgccccgcct ggggagtacg gccgcaaggc taaaactcaa aggaattgac 901 gggggcccgc acaagcggcg gagcatgcgg attaattcga tgcaacgcga agaaccttac 961 ctgggtttga catacaccct gccgctccag agatggggct tcttttgggg gtgtacaggt 1021 ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1081 aaccctcgtt ctatgttgcc agcacgtaat ggtggggact cataggagac tgccggggtc 1141 aactcggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgtcc agggcttcac 1201 gcatgctaca atggccggta caaagggctg cgatcccgta agggggagcg aatcccaaaa 1261 agccggtctc agttcggatt ggggtctgca actcgacccc atgaagtcgg agtcgctggt 1321 aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1381 acgtcacgaa agtcggcaac acccgaagcc ggtggcccaa cccttgtgga gggagccgtc 1441 gaaggtgggg ctggcgattg ggacgaagtc gtaacaaggt aaccgtaa // Uncultured bacterium clone HKT_RR36 16S ribosomal RNA gene, partial sequence GenBank: JN030428.1 FASTA Graphics PopSet

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LOCUS JN030428 1465 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR36 16S ribosomal RNA gene, partial sequence. ACCESSION JN030428 VERSION JN030428.1 GI:342328467 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1465) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1465) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1465 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR36" /environmental_sample /country="India: Bengaluru" rRNA <1..>1465 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gagcgaacgc tggcggcagg cctaacacat gcaagtcgag 61 cgccccgcaa ggggagcggc agacgggaga gtaacacgtg ggaacgtgcc cttcagttcg 121 gaacaaccaa gggaaacttt ggctaatacc gaatacgtcc gtaaggagaa agatttatcg 181 ctgaaggatc ggcccgcgtc tgattagcta gttggtgggg taatggctca ccaaggcgac 241 gatcagtagc tggtctgaga ggrtrrtsag cctcactggg aytgagacac ggcccagkct 301 mctacgggag gcagcagtgg ggaatattgg rcawtggrcg caagcctgat ccagccatgc 361 cgcgtgagtg atkaargcct tagggttgta aagctctttc gtcagggaag wtaatgacgg 421 tacctgaaga agaagycccg gctaacttcg tgccagcagc cgcgktaata cgragggggg 481 ctaagcgttg ctcsgaatca ctgggcgtwa agcgccacgt aaggcggctt tytaagtcag 541 gggkkraatc cttggagctc caactccaga actgcctttg atacttggag agcttgagtt 601 cggragaggt gagtggaact gcgagtggta gaggtgaaat tcgtagatat tcgcaagaac 661 accagtggcg aarggcggct cactggccct gatactgacg ctgaggtgcg aaagcgtggg 721 gagcaaacar gattagatac cctggtagtc cacgcygtaa acgatggatg ctagccgttg 781 gcgagctcgc tcgtcagtgg cgcagctaac gcattwaggc atcccgcctg gggagtacgg 841 tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 901 tcaattcgaa gcacacgcgc araaccttac caacccttga catgtccagt atgggyttca 961 gagatgarat ccttcagttc sgctggctgg aacacaggtg cygcatggcy kkcgtcasst 1021 ckkgtcgtga gatgttgggt taagtcccgc aacgagcgca accctcgccc ttagttgcca 1081 tcatttagtt gggcactcta gggggactgc cggtgataag ccgcgaggaa ggtggggatg 1141 acgtcaagtc ctcatggccc ttacgggttg ggctacacac gtgctacaat ggcggtgaca 1201 atgggatgca agggcgcgag cctacgcaaa tctcaaaaag ccgtctcagt tcggattggg 1261 gtctgcaact cgaccccatg aagtcggaat cgctagtaat cgtggatcag catgccacgg 1321 tgaatacgtt cccgggcctt gtacacaccg cccgtcatac catgggagtt ggctttacct 1381 gaaggcgttg cgctaacccg caagggaggc agacgaccac ggtagggtca gcgactgggg 1441 tgaagtcgta acaaggtaac cgtaa // Uncultured Bacteroidetes bacterium clone HKT_RR37 16S ribosomal RNA gene, partial sequence GenBank: JN030429.1 FASTA Graphics PopSet

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LOCUS JN030429 1495 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Bacteroidetes bacterium clone HKT_RR37 16S ribosomal RNA gene, partial sequence. ACCESSION JN030429 VERSION JN030429.1 GI:342328468 KEYWORDS ENV. SOURCE uncultured Bacteroidetes bacterium ORGANISM uncultured Bacteroidetes bacterium Bacteria; Bacteroidetes; environmental samples. REFERENCE 1 (bases 1 to 1495) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1495) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1495 /organism="uncultured Bacteroidetes bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:152509" /clone="HKT_RR37" /environmental_sample /country="India: Bengaluru" rRNA <1..>1495 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagctggagg cttaatacat gcaagtcgag 61 cgggatccgc ccttcggggc ggtgagagcg gcgcacgggt gagtaacgcg tacacgacct 121 acctctaaca gggggatagc cctgggaaac tgggattaat accgcatata atttagattt 181 taaaagttct aaatgaaagt tcaggcggtt agagatgggt gtgcgtctga ttagctagtt 241 ggtaaggtaa cggcttacca aggcgatgat cagcaggggg cgtgagagcg tggtccccca 301 cacgggtact gagacacgga cccgactcct acgggaggca gcagtaagga atattggaca 361 atggccgcaa ggctgatcca gccatccagc gtgcaggaag aaggccctat gggttgtaaa 421 ctgcttttgt cagggaagaa acctttggat ttactccgga gctgacggta cctgaagaat 481 aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcga gcgttatccg 541 gaattactgg gtttaaaggg tggcgtaggc ggctttgtaa gtcaggagtg aaagtttgcg 601 gctcaaccgt aaaattgctt ttgatactgc gaggctagaa ttaggatgas gtcagcggaa 661 tgtggcatgt agcggtgaaa tgcatagata tgccatagaa caccmatttg cgaasgcagc 721 tggctagacc tggattgacg ctgaggcacg aaagcgtggg ggagcgaaca ggattagata 781 ccctgktagt ccacgccyta aacgatgctc actcgacgta tgaccctaga rgttgtgckt 841 ccaagggaaa ccgttaagtg gakccacctg gggagtacga ccgcaaggtt gaamytcaaa 901 ggaattgacg ggggtccgca caagcggtgg agcatgtggt ttaattcgat gatacgcgag 961 gaaccttacc tgggctagaa tgcgagtgac gtcctgtgaa agcaggattc ccttcggggc 1021 acaaagcaag gtgctgcatg gctgtcgtca gctcgtgccg tgaggtgttg ggttaagtcc 1081 cgcaacgagc gcaacccctg tccttagttg ccatctcccc gtaaggggaa gggactctaa 1141 ggagactgcc ggcgcaagcc gtgaggaagg tggggatgac gtcaagtcat catggccttt 1201 atgccagggc gacacacgtg ctacaatggc cggtacagag ggttgccaag ccgcaaggtg 1261 gagccaatcc cttaaagccg gtctcagttc ggattggagt ctgaaacccg actccatgaa 1321 gttggaatcg ctagtaatcg cgcatcagcc atggcgcggt gaatacgttc ccggaccttg 1381 tacacaccgc ccgtcaagcc atggaagccg ggggtacctg aagacggtga ctttactggg 1441 agctgtctaa ggtaaaactg gtgactgggg ctaagtcgta acaaggtaac cgtaa // Uncultured soil bacterium clone HKT_RR38 16S ribosomal RNA gene, partial sequence GenBank: JN030430.1 FASTA Graphics PopSet

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LOCUS JN030430 1469 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured soil bacterium clone HKT_RR38 16S ribosomal RNA gene, partial sequence. ACCESSION JN030430 VERSION JN030430.1 GI:342328469 KEYWORDS ENV. SOURCE uncultured soil bacterium ORGANISM uncultured soil bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1469) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1469) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1469 /organism="uncultured soil bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:164851" /clone="HKT_RR38" /environmental_sample /country="India: Bengaluru" rRNA <1..>1469 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca ggatgaacgc tagcggtacg tctgacacat gcaagtcgaa 61 cggtaagcca ccttcgggtg gcctagagtg gcgaacggct gagtaacacg taggaaccta 121 cccctcagtt cgggacaacg ggacgaaagc cccgctaata ccggataata tcgaaagatt 181 gaaaggttta ctgctgaggg aggggcctgc gggctattag cttgatggtg gggtaacggc 241 tcaccatggc gatgatggct agctggtctg agrggatgat ywgccagact gggactgaga 301 ctgacggycc agactcctac gggaggcarc rgttcagaat cttgcacaat gsgcgaaagc 361 ctgatgcags gataccgcgt kggtgaagaa ggccctcggg ttcgtaarkc cctgtcasag 421 ggaaagataa tgacsgtacc ytctaargaa gccacggcta attacgtgcc agcagccgcg 481 gtaatacgta rkkggcgagg cgttatccgg aattactggg cgtaaagcgt gcgtaggcgg 541 tttagtgtgt gggatttgaa agcccggggc tcaactccgg atccggatcc caaactgcta 601 gactcgagag tatcagagga gagcggaatt cccggtgtag cggtaatatg cgtagatatc 661 gggaggaaca ccagtggcga asgcggctct ctggggtwtt ttytgacgct gaggcacgaa 721 agccagggga gcgamcggga ttagataccc cggtagtcct grccataaac gatgatcact 781 aggtgtagga ggcttcgacc ccttctgcsc cgccgctwac gcawtwagtt gatccgcctg 841 ggrartacgg ccgcaagktt aaaactcaaa kgaattgacr gkggcccgca caagcagtgg 901 asmwtgtggt ttaawtcgat gctacsygaa aaacyttacm agggtttgac wtccsggtgt 961 aagcccctag aawtaggggc ccccttcggr caccggtsac aggtggtgca wggttgtcgt 1021 casctcgtgc cgtgaggtgt tggattaagt tccgcaacga gcgcaaccct cgttgctagt 1081 tgccatcatt aagttgggca ctctagcgag actgccagcg tcaagctgga ggaaggtggg 1141 gatgacgtca aatcatcatg ccccttacac tctgggctac acacgtgcta caatggccgg 1201 tacaacgggt tgctacttcg cgagaagatg cgaatcccca aagccggtct cagttcggat 1261 tgcaggctgc aactcacctg catgaagctg gaattgctag taaccgtgga tcagcatgcc 1321 gcggtgaata cgtactcggg ccttgtacac accgcccgtc acaccacgaa agccggaaac 1381 gcccgaagtc cgtgagctaa cccgcaaggg aagcagcggc cgaaggtgga gtcggtgatt 1441 ggggtgaagt cgtaacaagg taaccgtaa // Uncultured bacterium clone HKT_RR39 16S ribosomal RNA gene, partial sequence GenBank: JN030431.1 FASTA Graphics PopSet

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LOCUS JN030431 1515 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR39 16S ribosomal RNA gene, partial sequence. ACCESSION JN030431 VERSION JN030431.1 GI:342328470 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1515) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1515) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1515 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR39" /environmental_sample /country="India: Bengaluru" rRNA <1..>1515 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca ggacgaacgc tggcggcgtg cttaacacat gcaagtggag 61 cgacgaacca gggcttgccc tggggcagag ccgcgaacgg gtgagtaaca cgtgggtaac 121 gtgccccaat gatcgggaca acccggggaa acccgggcta ataccgaatg tgctgtccca 181 acatcagttg ggattggaaa ggaagcttcg gcctccgcat tgggatcggc ccgcggccca 241 ttagcttgtt ggtgaggtaa cggctcacca aggctgcgat gggtagctgg tctgagagga 301 cgatcagcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 361 atcttgcgca atgcgcgaaa gcgtgacgca gcaacgcygc gtggkggrag aargccttmg 421 gkttgtaaam cccttycagg agggasgarr ycagkgcggt taatagccga yccgggtgac 481 ggtamctcca gaagaagccc ckgctaacta cgtgccagca gscgskgtaa tacgtagggg 541 gcaagcgttg tccggaatca ttgggcgtwa agcgygtgta kgcsgtccgg taagtcggct 601 gtgaarktcc agggctcaac cctgggatgc cggtcgatac tgccggamta gagttcggaa 661 gaggcgagtg gaattccccg gkgkagcggt gaaatgcgca grtatcggga ggaacaccaa 721 tggcgaargc agctcgctgg gacgttactg acgctgagac gcgaaagcgt ggggagcaaa 781 caggattaga taccctggta gtccacgccg taaacgatgg gcactaggtg tgggaggtgt 841 cgactcctcc cgtgccggag ctaacgcatt aagtgccccg cykggggagt acggccgcaa 901 ggctaaaact caaaggratt gacggggacy cgcacaagca gcggagcatg tggtttgaty 961 cgacgcaacc gcgaagaacc ytacctkggw ttgacattgt tcctgaccgc cctkgaaaca 1021 gggcttycct tcgggkcagg atcacaggtg gtgcatggct gtcgtcagct cgtgtcgtga 1081 gatgttgggt taagtcccgc aacgagcgca acccctgtcg catgttgcca gcatttagtt 1141 ggggactcat gcgagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca 1201 tcatgcccct tatgtccagg gctacacacg tgctacattg gccggtacag agggctgcga 1261 taccgcgagg tggagcgaat cccaaaaagc cggtctcggt tcggattgga ggctgaaact 1321 cgcctccatg aaggcggagt tgctagtaat cccggatcag caacgccggg gtgaatacgt 1381 tcccgggtct tgtacacacc gcccgtcaca ccacgaaagc gggcaacacc cgaagccggt 1441 gacccaaccc tctgggaggg agccgtcgaa ggtggggctc gtgattggtg tgaagtcgta 1501 acaaggtaac cgtaa // Uncultured bacterium clone HKT_RR40 16S ribosomal RNA gene, partial sequence GenBank: JN030432.1 FASTA Graphics PopSet

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LOCUS JN030432 1455 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR40 16S ribosomal RNA gene, partial sequence. ACCESSION JN030432 VERSION JN030432.1 GI:342328471 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1455) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1455) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1455 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR40" /environmental_sample /country="India: Bengaluru" rRNA <1..>1455 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gagcgaacgc tggcggcatg cttaacacat gcaagtcgca 61 cggtcggcgc aagtcggcag tggcggacgg gtgaggaacg cgtaggaacg tgtccagggg 121 tgggggataa cgctgggaaa ctggcgctaa taccgcatgt gagctgaggc tcaaagccga 181 gaggcgcctt tggagcggcc tgcgtccgat taggtagttg gtggggtaat ggcctaccaa 241 gcctgcgatc ggtagctggt ctgagaggac gaccagccac actgggactg agacacggcc 301 cagactccta cgggaggcag cagtggggaa tattggacaa tgggcgcaag cctgatccag 361 caatgccgcg tgggtgaaga aggtcttcgg attgtaaagc cctttcgaca gggacgatga 421 tgacggtacc tgtagaagaa gccccggcta acttcgtgcc agcagccgcg gtaatacgaa 481 gggggctagc gttgctcgga attactgggc gtaaagggcg cgtaggcggc gccacaagtc 541 aggcgtgaaa ttcctgggct taacctgggg gctgcgcttg agactgtggt gctagaggac 601 ggaagagggt cgtggaattc ccagtgtaga ggtgaaattc gtagatattg ggaagaacac 661 cggtggcgaa rgcggcgacy tggtccgtta ctgacgctga ggcgcgacag cgtggggagc 721 aaacaggatt agataccctg gtagtccacg cygtaaacga tgtgcgctgg atgttggggc 781 tcttaragcs tcagtgtcgt agccaacgcg gtaagcgcac cgcctgggga gtacggccgc 841 aaggttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 901 ttcgaagcaa cgcgcagaac cttaccagcc cttgacatgg tcacggccgg tccagagatg 961 ggctttcccc gcaaggggcg tggcgcacag gtgctgcatg gctgtcgtca gctcgtgtcg 1021 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg cctccagttg ccagcattga 1081 gttgggcact ctggaggaac tgccggtgac aagccggagg aaggtgggga tgacgtcaag 1141 tcctcatggc ccttatgggc tgggctacac acgtgctaca atggcggtga cagtgggaag 1201 ccaggcagtg atgtcgagcc gatcccaaaa agccgtctca gttcagattg cactctgcaa 1261 ctcgggtgca tgaaggcgga atcgctagta atcgcgcatc agcatggcgc ggtgaatacg 1321 ttcccgggcc ttgtacacac cgcccgtcac accatgggag ttggttctac cttaagtggc 1381 tgcgccaacc gcaaggaggc aggtcaccac ggtagggtca gcgactgggg tgaagtcgta 1441 acaaggtaac cgtaa // Uncultured Pseudomonas sp. clone HKT_RR41 16S ribosomal RNA gene, partial sequence GenBank: JN030433.1 FASTA Graphics PopSet

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LOCUS JN030433 1504 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Pseudomonas sp. clone HKT_RR41 16S ribosomal RNA gene, partial sequence. ACCESSION JN030433 VERSION JN030433.1 GI:342328472 KEYWORDS ENV. SOURCE uncultured Pseudomonas sp. ORGANISM uncultured Pseudomonas sp. Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; environmental samples. REFERENCE 1 (bases 1 to 1504) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1504) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1504 /organism="uncultured Pseudomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:114707" /clone="HKT_RR41" /environmental_sample /country="India: Bengaluru" rRNA <1..>1504 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag 61 cggatggcgg gagcttgctc cttgattcag cggcggacgg gtgagtaatg cctaggaatc 121 tgcctggtag tgggggacaa cgtttcgaaa ggaacgccaa taccgcatac gtcctacagg 181 agaaagcagg ggaccttcgg gccttgcgct atcagatggg cctaggtcgg attagctagt 241 tggtggggta atggctcacc aaggcgacga tccgtaactg gtctgagagg atgatcagtc 301 acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg aatattggac 361 aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa 421 gcactttaag ttgggaggaa gggcagtaag ctaatacctt gctgttttga cgttaccgac 481 agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg tgcaagcgtt 541 aatcggaatt actgggcgta aagcgcgcgt aggtggtttg ttaagttgga tgtgaaagcc 601 ccgggctcaa cttgggaact gcatccaaaa ctggcaagct agagtacggt agagggtggt 661 ggaatttyct gtgttagcgg tgaaatgcgt agatatagga aaggaacayc agtkgcgaag 721 gcgaccacyt gsactgataa ctgacactga gktgcgaaag cttggggagc aaacaggatt 781 agatacctgg tagtcacgct gtaaacgatg tcaaatagcc gttggaatcc ttgagatttt 841 agtggcgcag ctaacgcatt aagttgaccg cttggggagt acggccgcaa ggttaaaact 901 caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggttttaat tcgaagcaac 961 gcgaagaacc ttaccaggcc ttgacatgca gagaactttc cagagatgga ttggtgcctt 1021 cgggaactct gacacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1081 taagtcccgt aacgagcgca acccttgtcc ttagttatca gcacgttatg gtgggcactc 1141 taaggagact gccggtgaca aaccggagga aggtggggat gacgtcaagt catcatggcc 1201 cttacggcct gggctacaca cgtgctacaa tggtcggtac agagggttgc caagccgcga 1261 ggtggagcta atctcacaaa accgatcgta gtccggatcg cagtctgcaa ctcgactgcg 1321 tgaagtcgga atcgctagta atcgcgaatc agaatgtcgc ggtgaatacg ttcccgggcc 1381 ttgtacacac cgcccgtcac accatgggag tgggttgcac cagaagtagc tagtctaacc 1441 ttcgggagga cggttaccac ggtgtgattc atgactgggg tgaagtcgta acaaggtagc 1501 cgta // Uncultured bacterium clone UHKT_RR42 16S ribosomal RNA gene, partial sequence GenBank: JN030434.1 FASTA Graphics PopSet

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LOCUS JN030434 1515 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone UHKT_RR42 16S ribosomal RNA gene, partial sequence. ACCESSION JN030434 VERSION JN030434.1 GI:342328473 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1515) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1515) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1515 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="UHKT_RR42" /environmental_sample /country="India: Bengaluru" rRNA <1..>1515 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca ggacgaacgc tggcggcgcg cttaacacat gcaagtcgag 61 cgagaaccgg tccttcggga ccggggacag cggcgaacgg gtgagtaaca cgtgggtaat 121 ctgccctcga ttctgggata gcccggggaa acccggatta ataccggata gcctctcgag 181 ctttcgagct cgtgagaaaa ggtagcttcg gcctccgatc gaggatgagc ccgcggcgga 241 ttagcttgtt agtggggtaa tggcctacca aggcgacgat ccgtagctgg tctgagagga 301 cgatcagcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 361 atcttgccca atgggcgaaa gcctgaggca gcgacgccgc gtgggggaag aaggccttcg 421 ggttgtaaac ctctttcagc agggacgaag ctactcgggt gaatagccca gagggtgacg 481 gtacctgcag aagaagcccc ggctaactac gtgccagcag ccgcggtaat acgtaggggg 541 caagcgttgt ccggatttat tgggcgtaaa gagcgtgtag gcggccaggc aggtccgttg 601 tgaaaactag aggctcaacc tctagacgtc gatggaaacc gtctggctag agtccggaag 661 aggagagtgg aattcctggt gtagcsgtga aatgcgcaga tatcagraga acacccgtgg 721 ctaaagcggc tctctagtac ggtactgacg ctgagacgcg aaagcgtggg gagcgaacag 781 gattagatac cytggtagtc cacgccgtaa acgatgggtg ctaggtgtgg ggggggtgtc 841 gactccctcc gtgctgaagc taacgcatta agcaccccgc ctggggagta cggccgcaag 901 gctaaaactc aaaggaattg acgggggccc gcacaagcag cggagcatgt ggtttaattc 961 gacgcaacgc gaagaacctt accaaggctt gacatgcact ggaaagccct agaaataggg 1021 cctcccttcg gggccagtgc acaggtggtg catggctgtc gtcagctcgt gtcgtgagat 1081 gttgggttaa gtcccgcaac gagcgcaacc cctgtcctat gttgccagcg agttatgtcg 1141 gggactcata ggagactgcc ggtgacaaat cggaggaagg tggggatgac gtcaagtcat 1201 catgcccctt atgtcttggg ctacacacgt gctacattgg ccggtacaaa gggctgcgat 1261 gccgcgaggc ggagcgaatc ccaaaaagcc ggtcccggtt cggattggag gctgaaactc 1321 gcctccatga aggcggagtt gctagtaatc gcgaatcagc aacgtcgcgg tgaatacgtt 1381 cccgggcctt gtacacaccg cccgtcacac cacgaaagtc ggcaataccc gaagccggtg 1441 ggctaacccg caagggaggc agccgtcgaa ggtagggtcg atgattgggg tgaagtcgta 1501 acaaggtaac cgtaa // Uncultured Bacteroidetes bacterium clone HKT_RR44 16S ribosomal RNA gene, partial sequence GenBank: JN030435.1 FASTA Graphics PopSet

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LOCUS JN030435 1500 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Bacteroidetes bacterium clone HKT_RR44 16S ribosomal RNA gene, partial sequence. ACCESSION JN030435 VERSION JN030435.1 GI:342328474 KEYWORDS ENV. SOURCE uncultured Bacteroidetes bacterium ORGANISM uncultured Bacteroidetes bacterium Bacteria; Bacteroidetes; environmental samples. REFERENCE 1 (bases 1 to 1500) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1500) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1500 /organism="uncultured Bacteroidetes bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:152509" /clone="HKT_RR44" /environmental_sample /country="India: Bengaluru" rRNA <1..>1500 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagcggcagg cttaatacat gcaagtcgaa 61 cggtaacagg tccagcaatg gatgctgacg agtggcgcac gggtgcgtag cacgtatgca 121 acctaccttc aactggagaa tagcccgaag aaattcggat taatactcca tagttttatg 181 aaacggcatc gttttataaa taaagctcag gcggttgaag atgggcatgc gcgacattag 241 ctagttggtg aggtaacggc tcaccaaggc tacgatgtct aggggatctg agaggattaa 301 cccccacact ggtactgaga cacggaccag actcctacgg gaggcagcag taaggaatat 361 tggtcaatgg acgcgagtct gaaccagcca mtrctcgcgt gcmrgatgaa ggccctatsg 421 gwtgtaaact gckywtgtac gggaaarawm cyctgatygt gatcsgggct gatgktaccg 481 twagaataag gatcggctwa ctccstgcca gcagccgcgg tawtacggag gatycmagcg 541 ttattccsgg attcattggg tttaaagggt gcgtaggcgg gatagtaagt cagtggtgaa 601 gacctgcagc ttwactgcag aactgccatt gatactgcta acctagagta acagctgatt 661 gtkggssgaa tgkgttcgtg tagcggtgaa atgcttagat atgacacaga acacmcgatt 721 gcgaakgcag ctcacaaaac ctgtaactga cgctgasgca cgaaagcgtg gggatcgaac 781 aggattagat accctkgtag tccacgctgt aaacgatgat cactcgatgt tggcgatata 841 ccgtcagcgt ccaaggcgaa atgcgataag tgatccacct ggggagtacg atcgcaagat 901 tgaaactcaa aggaattgac gggggcccgc acaggcggar garcatgtgg tttaawtcga 961 tgatacrcga rgaaccttac mrgggcttga aagttagcga cggtccgtga aagcggactt 1021 cccttcgggg cgcgaaacta ggtgctgcat ggctgtcgtc agctcgtgcc gtgaggtgtt 1081 gggttaagtc ccgcaacgag cgcaacccct atcattagtt gccaacaggt tatgctgggg 1141 actctaatga aactgcccgc gcaagcggtg aggaaggtgg ggatgacgtc aagtcagcac 1201 ggcccttacg tcctgggcta cacacgtgct acaatggcaa atacaatggg ctgctacaca 1261 gtaatgtgat gcaaatctct aaagtttgtc tcagttcgga ttgaggtctg caactcgacc 1321 tcatgaagct ggattcgcta gtaatcggat atcagcaatg atccggtgaa tacgttcccg 1381 ggccttgtac acaccgcccg tcaaacaatg gaagttgggg gtacctgaag tcggtcaccg 1441 caaggagccg cctagggtaa aaccgataac tggtgttaag tcgtaacaag gtagccgtaa // Uncultured bacterium clone HKT_RR45 16S ribosomal RNA gene, partial sequence GenBank: JN030436.1 FASTA Graphics PopSet

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LOCUS JN030436 1492 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR45 16S ribosomal RNA gene, partial sequence. ACCESSION JN030436 VERSION JN030436.1 GI:342328475 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1492) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1492) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1492 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR45" /environmental_sample /country="India: Bengaluru" rRNA <1..>1492 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagctggagg cttaatacat gcaagtcgag 61 cgggatccgc ccttcggggc ggtgagagcg gcgcacgggt gagtaacgcg tacacgacct 121 acctctaaca gggggatagc cctgggaaac tgggattaat accgcatata atttagattt 181 taaaagttct aaatgaaagt tcaggcggtt agagatgggt gtgcgtctga ttagctagtt 241 ggtaaggtaa cggcttacca aggcgatgat cagcaggggg cgtgagagcg tggtccccca 301 cacgggtact gagacacgga cccgactcct acgggaggca gcagtaagga atattggaca 361 atggccgcaa ggctgatcca gccatccagc gtgcaggaag aaggccctat gggttgtaaa 421 ctgcttttgt cagggaagaa acctttggat ttactccgga gctgacggta cctgaagaat 481 aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcga gcgttatccg 541 gaattactgg gtttaaaggg tgcgtaggcg gctttgtaag tcagagtgaa agtttgcggc 601 tcaaccgtaa aattgctttt gatactgcga ggctagaatt aggatgasgt cagcggaatg 661 tggcatgtag cggtgaaatg catagatatg ccatagaaca ccmatttgcg aasgcagctg 721 gctagacctg gattgacgct gaggcacgaa agcgtggggg agcgaacagg attagatacc 781 ctgktagtcc acgccytaaa cgatgctcac tcgacgtatg accctagarg ttgtgcktcc 841 aagggaaacc gttaagtgag ccacctgggg agtacgaccg caaggttgaa actcaaagga 901 attgacgggg gtccgcacaa gcggtggagc atgtggttta attcgatgat acgcgaggaa 961 ccttacctgg gctagaatgc gagtgacgtc ctgtgaaagc aggattccct tcggggcaca 1021 aagcaaggtg ctgcatggct gtcgtcagct cgtgccgtga ggtgttgggt taagtcccgc 1081 aacgagcgca acccctgtcc ttagttgcca tctccccgta aggggaaggg actctaagga 1141 gactgccggc gcaagccgtg aggaaggtgg ggatgacgtc aagtcatcat ggcctttatg 1201 ccagggcgac acacgtgcta caatggccgg tacagagggt tgccaagccg caaggtggag 1261 ccaatccctt aaagccggtc tcagttcgga ttggagtctg aaacccgact ccatgaagtt 1321 ggaatcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg gaccttgtac 1381 acaccgcccg tcaagccatg gaagccgggg gtacctgaag acggtgactt tactgggagc 1441 tgtctaaggt aaaactggtg actggggcta agtcgtaaca aggtaaccgt aa // Uncultured bacterium clone HKT_RR46 16S ribosomal RNA gene, partial sequence GenBank: JN030437.1 FASTA Graphics PopSet

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LOCUS JN030437 1496 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR46 16S ribosomal RNA gene, partial sequence. ACCESSION JN030437 VERSION JN030437.1 GI:342328476 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1496) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1496) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1496 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR46" /environmental_sample /country="India: Bengaluru" rRNA <1..>1496 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggacgaacgc tggcggcgtg gattaggcat gcaagtcgag 61 cgacggacac ccttcggggt gttacggagc ggcgaacggg tgagtaacac gtgaataacc 121 tgcccatatg ttccggataa ttggccgaaa ggccttgtaa tacgggggag gatcggtctt 181 tggcatctta gaccgaggga agtcgcaaga cgcatatgga ggggttcgcg gattatcagg 241 tagttggtgg ggtaacggcc taccaagcct acgacgatta gctgagtmtg ctagaggawg 301 gtsmgccacw ttggaactga gacactgtyc mgamtccwwc gggargctgc aktcgagaat 361 yttggtcaat gcacgaaagt gtkaaccagc gacsycgcgt gaaggatgaa ggccytctgg 421 gtygtaaact tcttttktgr gagargaacm caatgacgkw wtctcasgaa taagcmmcgg 481 ctaactackk kccagccagy cgssgtaata tcgtaggtgg caagcgtttg tcsggattta 541 ctgggcgtaa agcgaacgca aggcggattt ttaagtagaa agtgaaargt tggagctcaa 601 ctctaacact gctccctatt actggaaatc tttgagtgcc grrraggaaa acggaatcaa 661 gttgtktagc ggtgaaatgc gttgatataa cttggaacac caatggcgaa agcagttttc 721 tggacggatw ctgacgctca tgttcgaagg ccaaggtagc maacaggatt agataccctg 781 gtagtcttgg cyytwaacga tgctcactag ktgttggggg gtaaccctcg gtgccgcagc 841 ctaacgccat taagtgagcc gcctggggaa ctacggcsgc aagkttgaaa ctcwaaggra 901 ttggcsgggg aaccgcacag gcgtgtggas yatatggctt aattcgatks macgcgaaga 961 accttaccag ggcttgacat gcacctgcaa gctgccgaaa agcagtcgct ttcgaaggtg 1021 gtgcacaggt ggtgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg 1081 caacgagcgc aaccctcatg ttttgttacc agcgagtaat gtcggggact cgaaacaaac 1141 tgcctctgta aagaggagga aggcggggat gacgtcaagt cagcatgccc cttacgccct 1201 gggctgcaca tatgctacaa tggccggcac aaagagttgc caactcgcga gagtgagcta 1261 atctcaaaaa accggtctca gttcggatcg aagtctgcaa ctcgacttcg tgaagtcgga 1321 atcgctagta accgccggtc agcaatacgg cggtgaatac gttcccggtc cttgcacaca 1381 ccgcccgtca cgccacgaaa gtctgtctca cccgaagtcg ctgcgctaac cgcaaggaag 1441 caggcgccga aggtggggcc gatgattggg acgaagtcgt aacaaggtaa ccgtaa // Uncultured bacterium clone HKT_RR47 16S ribosomal RNA gene, partial sequence GenBank: JN030438.1 FASTA Graphics PopSet

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LOCUS JN030438 1350 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR47 16S ribosomal RNA gene, partial sequence. ACCESSION JN030438 VERSION JN030438.1 GI:342328477 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1350) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1350) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1350 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR47" /environmental_sample /country="India: Bengaluru" rRNA <1..>1350 /product="16S ribosomal RNA" ORIGIN 1 accgaaaggt cagctaatac cgcatgagac cacagttcct gcgggaacag aggttaaaga 61 tttatcgctg aatgatcagt ctgcgtaaga ttagctagtt ggtgaggtaa tggctcacca 121 aggcaacgat ctttaggtgg tctgagagga tgaccaccca cactggaact gagacacggt 181 ccagactcct acgggaggca gcagtwggra cgtcgatmty cgagcrcawt gggggaaayy 241 stsatgsasc kaskccgacc cgctgagtga traaggcctt cgggtcgtaa agctctgttg 301 aacsggaaga aaaaaatgac ggtaccgttt aagaaaggat cggctaactt cgtgccagca 361 gccgcgktaa atacgaggga tcctagcgtt gktcggaatc attgggcgta aagagtwwgt 421 aggcggccta gtaagtcagt tgcgaaagcc cygggctcaa cycgggaagt gcaattgata 481 ctgcttggct tgaatgtgga agagggcagt agaattccag gtgtagtggt gaaatacgta 541 gatatctgga ggaataccgg tggcgaagsm gsctgcctgk tcctacattg acgctgagat 601 acgaaagccg tggggagcaa acaggatwag ataccctggt wrwycacgcy gtaaacgatg 661 aaccamcttg ttgttgragr tattgayccs ttcagtgacs aarctaacgc gttaagtgtt 721 ccrcctgggg agtwcgrtct gcamrattaa aactcaaaga aattgacggg ggcccgcaca 781 agcggtggag catgtggttt aattcgatgc aacgcgaaaa accttacctg ggctcgaaat 841 gcagtggaag taagcagaga tgtttacgcc ttcgggccgc tatataggtg ctgcatggct 901 gccgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgcct 961 ttagttgcca gcatttagtt gggcactcta aagggactgc cggtgttaaa ccggaggaag 1021 gtggggatga cgtcaagtcc tcatggccct tatgtccagg gctacacacg tgctacaatg 1081 ggtggtacag agagctgcga actcgcaaga gggtgctaat ctcataaaac cattctaagt 1141 tcggattgag gtctgcaact cgacctcatg aaggtggaat cgctagtaat cgcggatcag 1201 catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgaaagtc 1261 ggctgtacca gaagtcgctg tgctaaccgt aaggaggcag gcgcccaagg tatggtcgat 1321 gattggggtg aagtcgtaac aaggtaaccg // Uncultured bacterium clone HKT_RR48 16S ribosomal RNA gene, partial sequence GenBank: JN030439.1 FASTA Graphics PopSet

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LOCUS JN030439 1505 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR48 16S ribosomal RNA gene, partial sequence. ACCESSION JN030439 VERSION JN030439.1 GI:342328478 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1505) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1505) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1505 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR48" /environmental_sample /country="India: Bengaluru" rRNA <1..>1505 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagcggcagg cttaatacat gcaagtcgag 61 gggcagcagg actgtagcaa tacagttgct ggcgaccggc aaacgggtgc ggaacacgta 121 cgcaaccctc ctttaagtga gggatagccc agggaaactt ggattaatac ctcgtaatat 181 ttgagactgg catcactttc taattatagc tccggcgctt aaagatgggc gtgcgactga 241 ttaggtagtt ggcggggtaa cggcccacca agcctgcgat cagtaactgg tgtgagagca 301 cgaccagtca cacgggcact gagacacggg cccgactcct acgggaggca gcagtaagga 361 atattggtca atggacgcaa gtctgaacca gccatgccgc gtggaggatg aaggtcctct 421 ggattgtaaa cttcttttat ctgggaagaa acccatattt tctagtgtgg ttgacggtac 481 cagatgaata agcaccggct aactccgtgc cagcagccgc ggtaatacgg agggtgcaag 541 cgttatccgg attcactggg tttaaagggt gcgtaggcgg gttggtaagt ccgtggtgaa 601 atctccgagc ttaacccgga aactgccgtg gatactatcm atcttgaaat atcgtggagg 661 taagcggaat aatgtcatgt agcggtgaag ttgcttaaga tatgacatag aaccaccaat 721 tgcgtaggca gcttactaca cgatcattga cgctgaggca ygaaagcgtg gggagcaaac 781 agggattaga taccctggtt agtccacgcc ctaaacgaat ggatactcga catcagcgat 841 acactgttgg tgtctgagcg agagcattaa gtatcccacc tgggaagtac gatcgcaaga 901 ttgaaactca aaggaattgg cgggggtccg cacaagcggt ggagcatgtg gtttaattcg 961 atgatacgcg aggaacctta cctgggctag aatgctggaa gaccgagggt gaaagctctc 1021 tttgtagcaa tacacttcca gtaaggtgct gcatggctgt cgtcagctcg tgtcgtgagg 1081 tgttgggtta agtcccgcaa cgagcgcaac ccccatcact agttgccatc aggtaatgct 1141 gggaactcta gtgaaactgc cgtcgcaaga cgcgaggaag gaggggatga tgtcaagtca 1201 tcatggcctt tatgcccagg gctacacacg tgctacaatg gggaggacaa agggctgcca 1261 cttagtgata aggagctaat cccaaaaacc tcttctcagt tcagattgga gtctgcaact 1321 cgactccatg aagctggaat cgctagtaat cgtatatcag caatgatacg gtgaatacgt 1381 tcccggacct tgcacacacc gcccgtcaag ccatggaagc cgggtgtacc taaagtcggt 1441 aaccgtaagg agccgcctag ggtaaaactg gtgactgggg ctaagtcgta acaaggtaac 1501 cgtaa // Uncultured bacterium clone HKT_RR49 16S ribosomal RNA gene, partial sequence GenBank: JN030440.1 FASTA Graphics PopSet

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LOCUS JN030440 759 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR49 16S ribosomal RNA gene, partial sequence. ACCESSION JN030440 VERSION JN030440.1 GI:342328479 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 759) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 759) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..759 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR49" /environmental_sample /country="India: Bengaluru" rRNA <1..>759 /product="16S ribosomal RNA" ORIGIN 1 gaggcgcgaa aggccagggg agcaaacggg ataggtaccc cgttagttct tgccctaaaa 61 cgatgaatac tggttttgga ggttcaagac tccggtgccg tcgttaacgt tttaagtatc 121 cgcctgggga gtacgctcgc aatgagtgaa actcaaagga attgacgggg acccgcacaa 181 gcggtggagc atgtggttta attcgacgca acgcgaaaga accttacctg gactagaatg 241 tgaggggatg tcgggtaatg ccggcagccc gggaaaccgg acccaaaaca aggtgctgca 301 tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 361 tatcaacagt tgccatcatt aagttgggaa ctctgttgag actgccgttg ataaaacgga 421 ggaaggtggg gatgatgtca agtcatcatg gcctttatgt tcagggctac acacgtgcta 481 caatggatgg tacaaaacgt cgcaatcccg cgagggggag ctaatcgcga aaaccatcct 541 cagttcggat tgaagtctgc aactcgactt catgaagttg gaatcgctag taatcgcaaa 601 tcagcatgtt gcggtgaata cgttcccggg tcttgtacac accgcccgtc acatcacgaa 661 agtaggttgt actagaagta gctgggccaa ctcgcaagag gggtaggtta ccacggtatg 721 atttatgatt ggggtgaagt cgtaacaagg taaccgtaa // Uncultured bacterium clone HKT_RR50 16S ribosomal RNA gene, partial sequence GenBank: JN030441.1 FASTA Graphics PopSet

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LOCUS JN030441 1519 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR50 16S ribosomal RNA gene, partial sequence. ACCESSION JN030441 VERSION JN030441.1 GI:342328480 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1519) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1519) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1519 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR50" /environmental_sample /country="India: Bengaluru" rRNA <1..>1519 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gagtgaacgc tggcggcagg cctaacacat gcaagtcgaa 61 cggcagcaca ggagagcttg ctctctgggt ggcgagtggc ggacgggtga ggaatgcgtc 121 ggaatctgcc tatttgtggg ggataacgta gggaaactta cgctaatacc gcatacgacc 181 tacgggtgaa agtgggggac cgcaaggcct cacgcagata gatgagccga cgccggatta 241 gctagttggc ggggtaaagg cccaccaagg cgacgatccg tagctggtct gagaggatga 301 tcagccacac tggaactgag acacggtcca gactcctacg ggaggcagca gtggggaata 361 ttggacaatg ggcgcaagcc cgatccagcc atgccgcgtg tgtgaagaag gccttcgggt 421 tgtaaagcac ttttgtccgg aaagaaaagc attcggctaa taaccgggtg ttatgacggt 481 accggaagaa taagcaccgg ctaacttcgt gccagcagcc gcggtaatac gaagggtgca 541 agcgttactc ggaattactg ggcgtaargc gtggcgtagg tkgtttgtta agtccgatgt 601 gaaagctcct gggctcaacc ttgggaatgg catttggata ctggcasgct agagkgcggt 661 agamggggtg tggaatttcc cggtgtagca gtgaaatgcg tagatatcgg gaggaacatc 721 tgtggcgaag gcgacaccct ggamcagcac tgacaytgag gcacgaaagc gtggggagca 781 aacaggatta gataccctgr tagtccacgc cctaaacgat gcggaactgg aatgttgggt 841 gcaacttggc actcagtatc gaagctaacg cgttaagttc gccgcctggg aagtacggtc 901 gcarractga aactcaaagg aattgacggg ggcccgcaca agcggtggag tatgtggttt 961 aattcgatgc aacgcgaaga accttacttg gccttgacat gcacggaact ttccagagat 1021 ggattggtgc cttcgggaac cgtgacacag gtgctgcatg gctgtcgtca gctcgtgtcg 1081 tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg tccttagttg ccagcacgta 1141 atggtgggaa ctctaaggag accgccggtg acaaaccgga ggaaggtggg gatgacgtca 1201 agtcatcatg gctcttacgg ccagggctac acacgtacta caatggtggg gacagagggc 1261 tgcgatgccg cgaggcggag ccaatcccag aaaccctatc tcagtccgga ttggagtctg 1321 caactcgact ccatgaagtc ggaatcgcta gtaatcgcag atcagcattg ctgcggtgaa 1381 tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtttgtt gcaccagaag 1441 caggtagctt aaccttcggg agggcgcttg ccacggtgtg gccgatgact ggggtgaagt 1501 cgtaacaagg taaccgtaa // Uncultured bacterium clone HKT_RR51 16S ribosomal RNA gene, partial sequence GenBank: JN030442.1 FASTA Graphics PopSet

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LOCUS JN030442 1518 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR51 16S ribosomal RNA gene, partial sequence. ACCESSION JN030442 VERSION JN030442.1 GI:342328481 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1518) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1518) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1518 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR51" /environmental_sample /country="India: Bengaluru" rRNA <1..>1518 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagcggcagg cttaatacat gcaagtcgtg 61 gggcagcatg gtggtagcaa taccactgat ggcgaccggc aaacgggtgc ggaacacgta 121 cgcaatctac ccagaactgg cgaatagccc tccgaaagga ggattaatac gccgtaacat 181 tataaagtgg catcacttaa taattaaagc tccggcggtt ttggatgagc gtgcgcccca 241 ttaggtagtt ggttgaggta atggctcacc aagcctgcga tgggtaactg gtgtgagagc 301 acgaccagtc acacgggcac tgagacacgg gcccgactcc tacgggaggc agcagtaagg 361 aatattggtc aatggacgca agtctgaacc agccatgccg cgtggaggat gaaggccctc 421 tgggttgtaa acttctttta tgggggacga aatcacttat tcttaagtgt ctgacggtac 481 cccaggaata agcaccgact aactccgtgc cagcagccgc ggtaatacgg agggtgcaag 541 cgttatccgg attcactggg tttaaagggt gcgtaggagg gtgagtaagt cagtggtgaa 601 atcttcgagc ttaactcgga aactgccgtt gatactactt gtcttgaata tcgtggaggt 661 gggcggaata tgtcatgtag cggttgaaat gcttagatac gacatagaaa caccaaattg 721 cgaaagcagc tcgctacacg aatatttgac ttctgaggca cgaaagcgtg gggatcaaac 781 aaggattaaa tacccttggt tagttcacgc cctaaactta tgggatatct cgaccatacg 841 cgattacatg gtgttgtgtc tgagcgaaag gcatttagta tcccaacctt gggaaggtac 901 gaccggcagg ttgaaacytc aatggawttg gcgggggtcc scmcaagcgg tgcgarcwtg 961 tggtttaatt cgatgatacg cgaggaacct tacctggggc tagaatgctg gtggaccgtg 1021 ggtgaaagct cactttgtag caatacaccg ccagtaaggt gctgcatggc tgtcgtcagc 1081 tcgtgccgtg aggtgttggg ttaagtcccg caacgagcgc aacccccatc actagttgcc 1141 atcaggtaac gctgggaact ctagtgaaac tgccgtcgta agacgcgagg aaggagggga 1201 tgatgtcgag tcatcatggc ctttatgccc agggctacac acgtgctaca atgggaggga 1261 caaagagctg ccacttagcg ataaggagcc aatctcaaaa accctctctc agttcagatc 1321 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aaccgtatat cagcaatgat 1381 acggtgaata cgttcccgga ccttgcacac accgcccgtc aagccatgga agccgggtgt 1441 acctaaagtc ggtaaccgag aggagccgcc tagggtaaaa ctggtaactg gggctaagtc 1501 gtaacaaggt agccgtaa // Uncultured Pseudomonas sp. clone HKT_RR52 16S ribosomal RNA gene, partial sequence GenBank: JN030443.1 FASTA Graphics PopSet

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LOCUS JN030443 1502 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Pseudomonas sp. clone HKT_RR52 16S ribosomal RNA gene, partial sequence. ACCESSION JN030443 VERSION JN030443.1 GI:342328482 KEYWORDS ENV. SOURCE uncultured Pseudomonas sp. ORGANISM uncultured Pseudomonas sp. Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; environmental samples. REFERENCE 1 (bases 1 to 1502) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1502) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1502 /organism="uncultured Pseudomonas sp." /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:114707" /clone="HKT_RR52" /environmental_sample /country="India: Bengaluru" rRNA <1..>1502 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag 61 cggatgacgg gagcttgctc cttgattcag cggcggacgg gtgagtaatg cctaggaatc 121 tgcctggtag tgggggacaa cgtttcgaaa ggaacgctaa taccgcatac gtcctacggg 181 agaaagcagg ggaccttcgg gccttgcgct atcagatgag cctaggtcgg attagctagt 241 tggtggggta atggctcacc aaggcgacga tccgtaactg gtctgagagg atgatcagtc 301 acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg aatattggac 361 aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa 421 gcactttaag ttgggaggaa gggcagtaag ctaatacctt gctgttttga cgttaccaac 481 agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg tgcaagcgtt 541 aatcggaatt actgggcgta aagcgcgcgt acgtggtttg ttaagttggt tgtgaaagcc 601 ccgggctcaa cttgggaact gcatccaaaa ctggcaagct agagtacagt agagggtggt 661 ggaatttcct gtgtagcggt ggaaatgcgt agatatagga argaacacca gtggcgaagg 721 cgacacttgg actgatactg acactgaggt gcgaaagcgt ggggagcaaa caggattaga 781 taccctggta gtcacgccgt aaacgatgtc aactagccgt tgaatccttt gagattttag 841 tggcgcagct aacgcattaa agttgaccgc ctggggagta cggccgcaag gttaaaactc 901 aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 961 gaagaacctt accaggcctt gacatgcaga gaactttcca gagatggatt ggtgccttcg 1021 ggaactctga cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 1081 agtcccgtaa cgagcgcaac ccttgtcctt agttaccagc acgttatggt gggcactcta 1141 aggagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca tcatggccct 1201 tacggcctgg gctacacacg tgctacaatg gtcggtacag agggttgcca agccgcgagg 1261 tggagctaat ctcacaaaac cgatcgtagt ccggatcgca gtctgcaact cgactgcgtg 1321 aagtcggaat cgctagtaat cgcgaatcag aatgtcgcgg tgaatacgtt cccgggcctt 1381 gtacacaccg cccgtcacac catgggagtg ggttgcacca gaagtagcta gtctaacctt 1441 cgggaggacg gttaccacgg tgtgattcat gactggggtg aagtcgaaca aggtaaccgt 1501 aa // Uncultured Bacteroidetes bacterium clone HKT_RR53 16S ribosomal RNA gene, partial sequence GenBank: JN030444.1 FASTA Graphics PopSet

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LOCUS JN030444 1517 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured Bacteroidetes bacterium clone HKT_RR53 16S ribosomal RNA gene, partial sequence. ACCESSION JN030444 VERSION JN030444.1 GI:342328483 KEYWORDS ENV. SOURCE uncultured Bacteroidetes bacterium ORGANISM uncultured Bacteroidetes bacterium Bacteria; Bacteroidetes; environmental samples. REFERENCE 1 (bases 1 to 1517) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1517) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1517 /organism="uncultured Bacteroidetes bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:152509" /clone="HKT_RR53" /environmental_sample /country="India: Bengaluru" rRNA <1..>1517 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcatggctca ggatgaacgc tagcggcagg cttaatacat gcaagtcgag 61 gggcagcagc tctgtagcaa tacagaggct ggcgaccggc aaacgggtgc ggaacacgta 121 cacaaccttc ctttgagcgg ggaatagccc ggggaaaccc ggattaatac cccatagtat 181 attgaaaagg catcttttta atattaaaga tttatcactt aaagatgggt gtgcggctga 241 ttaggtagtt ggcggggtaa cggcccacca agcctacgat cagtaactgg tgtgagagca 301 cgaccagtca cacgggcact gagacacggg cccgactcct acgggaggca gcagtaagga 361 atattggtca atggacgaaa gtctgaacca gccatgccgc gtggaggatg aaggtcctct 421 ggattgtaaa ctccttttat atgggacgaa aaaagggttt tctaactcgt ctgacggtac 481 catatgaata agcacyggyy atamtcckkg ccasgtcagc cggckgtata tacgrrrggt 541 gcaagcktta tccggattca ytgggtttaa wgggtgcgta kgwgggttgg taagtcartg 601 gtgaaaatyc ccgagcttaa cttgggaaac tgccattgat actatcagtc ttgaataccg 661 tggaggtcag cggaatwwgt catgtagcgg tgaawtgctt agatatgaac atagaacacc 721 aattgcgaag gcagctkgct acamgaatat tgacamtgag gcacgaaagc gtggggatca 781 aacaggatta raataccctg gtagtccacg ccytaaacta tggaatactc gacataacgc 841 gataacactg ttgtggtgtc tgagcgaars cattaagtat cccacctggg gaagtacgat 901 csgccaagat tgraactcaa aggaattggc gggggtcccg cammagcggt ggaarcatgg 961 tggkttaatt cgatgatacg cgaggaacct tacctgggct agaatgctgg gggaccgaga 1021 gtgaaagctc tctttgtagc aatacaccgc cagtaaggtg ctgcatggct gtcgtcagct 1081 cgtgccgtga ggtgttgggt taagtcccgc aacgagcgca acccccatca ctagttgcca 1141 tcaggtaacg ctgggaactc tagtgaaact gccgtcgtaa gacgtgagga aggaggggat 1201 gatgtcaaga catcatggcc tttatgccca gggctacaca cgtgctacaa tggggcgtac 1261 aaagggctgc aacacagcga tgtgaagcta atcccaaaaa acgcctctca gttcagattg 1321 gagtctgcaa ctcgactcca tgaagctgga atcgctagta atcgtatatc agcaatgata 1381 cggtgaatac gttcccggac cttgcacaca ccgcccgtca agccatggaa gctgggtgta 1441 cctaaagtcg gtaaccgtaa ggagccgcct agggtaaaac tagtgactgg ggctaagtcg 1501 taacaaggta accgtaa // Uncultured bacterium clone HKT_RR54 16S ribosomal RNA gene, partial sequence GenBank: JN030445.1 FASTA Graphics PopSet

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LOCUS JN030445 1035 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR54 16S ribosomal RNA gene, partial sequence. ACCESSION JN030445 VERSION JN030445.1 GI:342328484 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1035) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1035) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1035 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR54" /environmental_sample /country="India: Bengaluru" rRNA <1..>1035 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gaacgaacgc tgatggcgtg cctcatacat gcaagtcgag 61 cgggtagcaa taccagcggc gaacgggtga gtaacgcgta aacaatctgc ctcaaggtgg 121 gggataacta tcccaacgga tagctaatac cgcatagagc aacgggactg catggttttg 181 ttgttaaaga tttatcgcct tgagatgagt ttgcgtccca ttagctagtt ggcggggcaa 241 cggcccacca aggcgacgat gggtagccgg cctgagaggg tgtccggcca cattgggact 301 gagatacggc ccagactcct acgggaggca gcagtaggga attttgcgca atgggggaaa 361 ccctgacgca gcaacgccat gtgtgggatg aagcatttag gtgtgtaaac cactgtcggc 421 agggaataaa ggccctgaat agggatttga acgtacctgc agaggaagcc ccggcaaact 481 tcgtgccagc agccgcggta atacgagggg ggcaagtgtt gttcggaatc actgggcgta 541 aagggagcgt aggcgggagc ctaagttgga tgtttaagac cggggcccaa ccccgggagg 601 gcatccaaaa ctgggtttct tgaatgggac agacgtcgat ggaattggac gtgtagcggt 661 ggaatgcgta gatatctcca agaacaccga ttgcgaacgc agtcgactgg ggtccacatt 721 gacgctgagc tcgaaagcgt ggggagcaaa caggattaga taccctggtt agtccacgcc 781 ataaacgatg aatactagta ttgtgctatt caacggtgca gtgccgcagc taacgcgtta 841 agtaattccg cctggggagt acgcccgcta gggtgaaact caaaggattt gaacggggcc 901 cgcacagacg tggacatggt gtttaattcg acgtcaccgc aaaaccttac aaggcttgac 961 atggaatgac ctgctctgag aatccgctct cgccaagcac ctctccacag atgcgcagcc 1021 tgacctactc tcgtt // Uncultured bacterium clone HKT_RR55 16S ribosomal RNA gene, partial sequence GenBank: JN030446.1 FASTA Graphics PopSet

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LOCUS JN030446 801 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR55 16S ribosomal RNA gene, partial sequence. ACCESSION JN030446 VERSION JN030446.1 GI:342328485 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 801) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 801) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..801 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR55" /environmental_sample /country="India: Bengaluru" rRNA <1..>801 /product="16S ribosomal RNA" ORIGIN 1 caatcggcga aggcaagctg gctgggacga ttacgacgct gagacgcgaa agcgtgggga 61 gcaatcagga ttagataccc tggtagtcac gccgtaaacg atgggcacta gatgtggggg 121 gtgtcgactc cccccgtgtc gtagctaacg cgttaaagtg ccccgcctgg ggagtacggc 181 cgcaagggct aaaactcaaa ggaattgacg gggacccgca caagcagcgg agcatgtgga 241 ttaattcgac gcaacgcgaa gaaccttacc tgggcttgac atgctgctga cctccctgga 301 aacagggatt cccttcgggg cagcaataca ggtggtgcat ggctgtcgtc agctcgtgtc 361 gtgagatgtt gggttgagtc ccgcaacgag cgcaaccccc gtcctatgtt gccagcggat 421 aatgccgggg actcatggga tactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 481 aagtcatcat gccccttatg tccagggctt cacacgtgct acattggcgc atacagaggg 541 ctgcgatacc gcgaggtgga gcgaatccca aaaagtgcgt ctcggttcgg attggaggct 601 gaaactcgcc tccatgaagg cggagttgct agtaatcccg gatcagcaat gccggggtga 661 atacgttccc gggtcttgta cacaccgccc gtcacaccac gaaagcaagc aacacccgaa 721 gccggtgagc taaccctctg ggaggcagcc gtcgaaggtg gggctcgtga ttggggtgaa 781 gtcgtaacaa ggtaaccgta a // Uncultured bacterium clone HKT_RR58 16S ribosomal RNA gene, partial sequence GenBank: JN030447.1 FASTA Graphics PopSet

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LOCUS JN030447 1499 bp DNA linear ENV 22-SEP-2011 DEFINITION Uncultured bacterium clone HKT_RR58 16S ribosomal RNA gene, partial sequence. ACCESSION JN030447 VERSION JN030447.1 GI:342328486 KEYWORDS ENV. SOURCE uncultured bacterium ORGANISM uncultured bacterium Bacteria; environmental samples. REFERENCE 1 (bases 1 to 1499) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Partial 16S rDNA sequence of bacterial clones from an electronic waste dumping site at Bengaluru, India JOURNAL Unpublished REFERENCE 2 (bases 1 to 1499) AUTHORS Rajeshkumar,R., Pal,R.R., and Purohit,H.J. TITLE Direct Submission JOURNAL Submitted (26-MAY-2011) Environmental Genomic Division, National Environmental Engineering Research Institute (NEERI), Dept of Environmental Biotechnology, Bharathidasan University, Nehru Marg, Nagpur, Maharashtra 440020, India FEATURES Location/Qualifiers source 1..1499 /organism="uncultured bacterium" /mol_type="genomic DNA" /isolation_source="electronic waste dumping site" /db_xref="taxon:77133" /clone="HKT_RR58" /environmental_sample /country="India: Bengaluru" rRNA <1..>1499 /product="16S ribosomal RNA" ORIGIN 1 tagagtttga tcctggctca gaatcaacgc tggcggcgtg cctcagacat gcaagtcgaa 61 cgattaaagc tctcttcgga gagtgcatag agtggcgcac gggtgagtaa cacgtaagta 121 atctaccttc gagtggggaa taacgtcggg aaaccgacgc taataccgca taatgcagcg 181 gcatcgcaag atgacagttg ttaaaggagc aatccgcttg aagaggagct tgcggcagat 241 tagctagttg gtaaggtaat ggcttaccaa ggctacgatc tgtaaccggt cttagaggac 301 ggtcggtcac actgacactg aataacgggt cagactccta cgggaggcag cagtcgggaa 361 ttttgggcaa tgggcgaaag cctgacccaa caacgccgcg tgaaggatga agtatctcgg 421 tatgtaaact tcgaaagaat gggaagaatc aatgacggta ccatttataa ggtccggcta 481 actacgtgcc agcagccgcg gtaatacgta gggaccaagc gttgttcgga tttactgggc 541 gtaaagggcg sktargsggc aattcaagtc agctgtgaaa tytykgggyt waacccagaa 601 cggsctagct gatactgctt tgstrgakkg crgaaggggc aatcggaatt cttcggkgta 661 gcggtgaaat gcgtaggata tcggagagga acamcttgag ktggargacg ggttgctggg 721 ctgacactga cgctgaggcg cgaaaggcca ggggagcaaa cgggactaga taccccggta 781 gtcctggccc taaacgaatg aatacttggt gtcttggagt tattagtgct ccgggtgccg 841 tcgctaamsk ttttwagtat tccgcctggg gagtacgctc gcaagagtga aactcaaagg 901 aattgacggg gacccgcaca agcggtggag catgtggttt aattcgacgc aacgcgaaga 961 accttaccta ggctagaatg tgagggaaga aagggtaatt ccgatcgtcc gggaaaccgg 1021 acccaaaaca aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1081 cccgcaacga gcgcaacccc tattgatagt tgctaacatt aagttgagaa ctctatcaag 1141 actgctgttg ataaaacgga ggaaggtggg gatgatgtca agtcatcatg gcctttatgc 1201 ttagggctac acacgtgcta caatggatgg tacaaaacgt cgcgatcccg taagggggag 1261 ctaatcgcaa aaaccattct cagttcggat tgaagtctgc aactcgactt catgaagttg 1321 gaatcgctag taatcgcgga tcagaacgcc gcggtgaata cgttcccggg tcttgtacac 1381 accgcccgtc acatcacgaa agtaggttgt actagaagta ggagggctaa cccgcaggga 1441 aggcatctta ccacggtatg atttatgatt ggggtgaagt cgtaacaagg taaccgtaa //

APPENDIX- C

Bacillus anthracis strain EWRR1 16S ribosomal RNA gene, partial sequence GenBank: JN102337.1 FASTA Graphics

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LOCUS JN102337 761 bp DNA linear BCT 23-SEP-2011 DEFINITION Bacillus anthracis strain EWRR1 16S ribosomal RNA gene, partial sequence. ACCESSION JN102337 VERSION JN102337.1 GI:344953290 KEYWORDS . SOURCE Bacillus anthracis ORGANISM Bacillus anthracis Bacteria; Firmicutes; Bacillales; Bacillaceae; Bacillus; Bacillus cereus group. REFERENCE 1 (bases 1 to 761) AUTHORS Rajeshkumar,R. TITLE Heavy metal tolerant bacteria isolated from electronic waste contaminated site JOURNAL Unpublished REFERENCE 2 (bases 1 to 761) AUTHORS Rajeshkumar,R. TITLE Direct Submission JOURNAL Submitted (09-JUN-2011) Department of Environmental Biotechnology, Bharathidasan University, Palkalaiperur, Tiruchirappalli, Tamilnadu 620024, India FEATURES Location/Qualifiers source 1..761 /organism="Bacillus anthracis" /mol_type="genomic DNA" /strain="EWRR1" /isolation_source="electronic waste contaminated surface soil" /db_xref="taxon:1392" /country="India" /collected_by="Ramasamy Rajeshkumar" /identified_by="Ramasamy Rajeshkumar" rRNA <1..>761 /product="16S ribosomal RNA" ORIGIN 1 gaactgggcg gcgtgctaat acatgcaagt cgagcgaatg gattaagagc ttgctcttat 61 gaagttagcg gcggacgggt gagtaacacg tgggtaacct gcccataaga ctgggataac 121 tccgggaaac cggggctaat accggataac attttgaacc gcatggttcg aaattgaaag 181 gcggcttcgg ctgtcactta tggatggacc cgcgtcgcat tagctagttg gtgaggtaac 241 ggctcaccaa ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 301 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 361 tctgacggag caacgccgcg tgagtgatga aggctttcgg gtcgtaaaac tctgttgtta 421 gggaagaaca agtgctagtt gaataagctg gcaccttgac ggtacctaac cagaaagcca 481 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggaatta 541 ttgggcgtaa agcgcgcgca ggtggtttct taagtctgat gtgaaagccc acggctcaac 601 cgtggagggt cattggaaac tgggagactt gagtgcagaa gaggaaagtg gaattccatg 661 tgtagcggtg aaatgcttag agatatggag gaacaccagt ggcgaaggcg actttctggt 721 ctgtaactga cactgaggcg cgaaaagcgt gggagcaaaa c //

Bacillus cereus strain EWRR2 16S ribosomal RNA gene, partial sequence GenBank: JN102338.1 FASTA Graphics

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LOCUS JN102338 1019 bp DNA linear BCT 23-SEP-2011 DEFINITION Bacillus cereus strain EWRR2 16S ribosomal RNA gene, partial sequence. ACCESSION JN102338 VERSION JN102338.1 GI:344953310 KEYWORDS . SOURCE Bacillus cereus ORGANISM Bacillus cereus Bacteria; Firmicutes; Bacillales; Bacillaceae; Bacillus; Bacillus cereus group. REFERENCE 1 (bases 1 to 1019) AUTHORS Rajes hkumar,R. TITLE Heavy metal tolerant bacteria isolated from electronic waste contaminated site JOURNAL Unpublished REFERENCE 2 (bases 1 to 1019) AUTHORS Rajes hkumar,R. TITLE Direct Submission JOURNAL Submitted (09-JUN-2011) Department of Environmental Biotechnology, Bharathidasan University, Palkalaiperur, Tiruchirappalli, Tamilnadu 620024, India FEATURES Location/Qualifiers source 1..1019 /organism="Bacillus cereus" /mol_type="genomic DNA" /strain="EWRR2" /isolation_source="electronic waste contaminated soil" /db_xref="taxon:1396" /country="India" /collected_by="Ramasamy Rajeshkumar" /identified_by="Ramasamy Rajeshkumar" rRNA <1..>1019 /product="16S ribosomal RNA" ORIGIN 1 gcaacctggc ggcgtgccta atacatgcaa gtcgagcgaa tggattaaga gcttgctctt 61 atgaagttag cggcggacgg gtgagtaaca cgtgggtaac ctgcccataa gactgggata 121 actccgggaa accggggcta ataccggata acattttgaa ccgcatggtt cgaaattgaa 181 aggcggcttc ggctgtcact tatggatgga cccgcgtcgc attagctagt tggtgaggta 241 acggctcacc aaggcaacga tgcgtagccg acctgagagg gtgatcggcc acactgggac 301 tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa 361 agtctgacgg agcaacgccg cgtgagtgat gaaggctttc gggtcgtaaa actctgttgt 421 tagggaagaa caagtgctag ttgaataagc tggcaccttg acggtaccta accagaaagc 481 cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggaat 541 tattgggcgt aaagcgcgcg caggtggttt cttaagtctg atgtgaaagc ccacggctca 601 accgtggagg gtcattggaa actgggagac ttgagtgcag aagaggaaag tggaattcca 661 tgtgtagcgg tgaaatgcgt agagatatgg aggaacacca gtggcgaagg cgactttctg 721 gtctgtaact gacactgagg cgcgaaagcg tggggagcaa acaggattag ataccctggt 781 agtccacgcc gtaaacgatg agtgctaagt gttagagggt ttccgccctt tagtgctgaa 841 gttaacgcat taagcactcc gcctggggag tacggccgca aggctgaaac tcaaaggaat 901 tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc 961 cttaccagtc ttgacatcct ctgacaaccc tagagatagg gcttctcctt cgggaagca //

Paenibacillus lactis strain EWRR3 16S ribosomal RNA gene, partial sequence GenBank: JN102339.1 FASTA Graphics

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LOCUS JN102339 836 bp DNA linear BCT 23-SEP-2011 DEFINITION Paenibacillus lactis strain EWRR3 16S ribosomal RNA gene, partial sequence. ACCESSION JN102339 VERSION JN102339.1 GI:344953324 KEYWORDS . SOURCE Paenibacillus lactis ORGANISM Paenibacillus lactis Bacteria; Firmicutes; Bacillales; Paenibacillaceae; Paenibacillus. REFERENCE 1 (bases 1 to 836) AUTHORS Rajeshkumar,R. TITLE Heavy metal tolerant bacteria isolated from electronic waste contaminated site JOURNAL Unpublished REFERENCE 2 (bases 1 to 836) AUTHORS Rajeshkumar,R. TITLE Direct Submission JOURNAL Submitted (09-JUN-2011) Department of Environmental Biotechnology, Bharathidasan University, Palkalaiperur, Tiruchirappalli, Tamilnadu 620024, India FEATURES Location/Qualifiers source 1..836 /organism="Paenibacillus lactis" /mol_type="genomic DNA" /strain="EWRR3" /isolation_source="electronic waste contaminated soil" /db_xref="taxon:228574" /country="India" /collected_by="Ramasamy Rajeshkumar" /identified_by="Ramasamy Rajeshkumar" rRNA <1..>836 /product="16S ribosomal RNA" ORIGIN 1 acttgcgggg tgcctataat gcaagtcgag cggacttgag gaggagcttg cttcactgat 61 agttagcggc ggacgggtga gtaacacgta ggcaacctgc cctcaagact gggataacta 121 ccggaaacgg tagctaatac cggataatta aattcgctgc atggcggagt tatgaaaggc 181 ggagcaatct gtcacttgag gatgggcctg cggcgcatta gctagttggt gaggtaacgg 241 ctcaccaagg cgacgatgcg tagccgacct gagagggtga acggccacac tgggactgag 301 acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg ggcgaaagcc 361 tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgccagg 421 gaagaacgtc tcatagagta actgctatga gagtgacggt acctgagaag aaagccccgg 481 ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttgtcc ggaattattg 541 ggcgtaaagc gcgcgcaggc ggttctttaa gtctggtgtt taaacccgag gctcaacttc 601 gggacgcact ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 661 gcggtgaaat gcgtagatat gtggaggaac accagtggcg aaggcgactc tctgggctgt 721 aactgacgct gaggcgcgaa agcgtgggga gcgaacagga ttagataccc tggtagtccc 781 ccgccgtaac gatgaatgcc aggtgttagg ggtttccaca cccttggtcg ccgaat //

Pseudomonas aeruginosa strain EWRR4 16S ribosomal RNA gene, partial sequence GenBank: JN102340.1 FASTA Graphics

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LOCUS JN102340 952 bp DNA linear BCT 23-SEP-2011 DEFINITION Pseudomonas aeruginosa strain EWRR4 16S ribosomal RNA gene, partial sequence. ACCESSION JN102340 VERSION JN102340.1 GI:344953331 KEYWORDS . SOURCE Pseudomonas aeruginosa ORGANISM Pseudomonas aeruginosa Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas. REFERENCE 1 (bases 1 to 952) AUTHORS Rajeshkumar,R. TITLE Heavy metal tolerant bacteria isolated from electronic waste contaminated site JOURNAL Unpublished REFERENCE 2 (bases 1 to 952) AUTHORS Rajeshkumar,R. TITLE Direct Submission JOURNAL Submitted (09-JUN-2011) Department of Environmental Biotechnology, Bharathidasan University, Palkalaiperur, Tiruchirappalli, Tamilnadu 620024, India FEATURES Location/Qualifiers source 1..952 /organism="Pseudomonas aeruginosa" /mol_type="genomic DNA" /strain="EWRR4" /isolation_source="electronic waste contaminated soil" /db_xref="taxon:287" /country="India" /collected_by="Ramasamy Rajeshkumar" /identified_by="Ramasamy Rajeshkumar" rRNA <1..>952 /product="16S ribosomal RNA" ORIGIN 1 ctggggggca ggctaacaca tgcagtcgag cggatgaagg gagcttgctc ctggattcag 61 cggcggacgg gtgagtaatg cctaggaatc tgcctggtag tgggggataa cgtccggaaa 121 cgggcgctaa taccgcatac gtcctgaggg agaaagtggg ggatcttcgg acctcacgct 181 atcagatgag cctaggtcgg attagctagt tggtggggta aaggcctacc aaggcgacga 241 tccgtaactg gtctgagagg atgatcagtc acactggaac tgagacacgg tccagactcc 301 tacgggaggc agcagtgggg aatattggac aatgggcgaa agcctgatcc agccatgccg 361 cgtgtgtgaa gaaggtcttc ggattgtaaa gcactttaag ttgggaggaa gggcagtaag 421 ttaatacctt gctgttttga cgttaccaac agaataagca ccggctaact tcgtgccagc 481 agccgcggta atacgaaggg tgcaagcgtt aatcggaatt actgggcgta aagcgcgcgt 541 aggtggttca gcaagttgga tgtgaaatcc ccgggctcaa cctgggaact gcatccaaaa 601 ctactgagct agagtacggt agagggtggt ggaatttcct gtgtagcggt gaaatgcgta 661 gatataggaa ggaacaccag tggcgaaggc gaccacctgg actgatactg acactgaggt 721 gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatgt 781 cgactagccg ttgggatcct tgagatctta gtggcgcacc taacgcgata agtcgaccgc 841 ctggtgagta cggccgcagg gttaaaactc aaatgaattt gacagggggc ccgcacaagc 901 ggtggagcct gtggtttaat tcgaagcaac gcgaaagaat cataacctgt cc // IMMUNOLOGY, HEALTH, AND DISEASE 

Study on acquisition of bacterial antibiotic resistance determinants in poultry litter

 

..  .

ABSTRACT                           .  .  .  .      .  .. .  . .  .       .      .           . .. ...  .  .   .     . ..        .   .   .. . 

INTRODUCTION   ..          .            . .       . .        .         .. ..         .            .   . .  ..

           .   .           MATERIALS AND METHODS           Bacterial Identification   .         .     . Isolation and Detection of Plasmid DNA              .    .    ...            .   . ..                      .     .       .       Curing of Plasmid DNA .    .   Antimicrobial Susceptibility Testing .  Disk Diffusion Method.    .             ... ..  .    .    . .       .               Transfer of Drug Resistance                    .   .   ...   .             .           .           .  Determination of Minimum Inhibitory Concentra- Conjugation tion.  .    . . .           .       

RESULTS AND DISCUSSIONS Screening and Identification of Microorganisms in Poultry Litter

. .    .              .        . . .      .... .. ..  .   .      .     .  .     . . .  . Antimicrobial Susceptibility Testing

Disk Diffusion Method.              .  .    .                .  .  ..   ..       .  . .   .

    .

 

.         .         .    . 



   .

.    .    ..        .    .           

.       . .  . ..             .             .            .. .        .              .     ..   ..    . MIC. ..            . Isolation and Curing of Plasmid .   .       .  ..         .     .      

.

 

    

.     .     ..          .     .                 .

 

      

.     .     ..          .     .              

.              .  ....  .  . .                                          ..  . . Conjugation Analysis         .    Transformation  .                . .           .. .   . . .        .  .    



  .     

     ..     .          .     .     .            . .        .   ..     .              .        . .  .    . . .  . .   .           . .             ACKNOWLEDGMENTS  . .  .          .  .. REFERENCES  . .   ..   ...   ..   .   .               .      . ...       .        . ..             . .  ..    .  .                     .              .  .    .           . ..     .     .       . . .   .              .       .  .  .             .    

.        .    . . . .      .     . .    .             .                                . .  . .