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Chapter Title Bioremediation of Heavy Metals by Metagenomic Approaches

Copyright Year 2022
Copyright Holder The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.
Author Family Name Khan
Particle
Given Name Dibyendu
Suffix
Division UGC-Center for Advanced Study, Department of Botany
Organization The University of Burdwan
Address Bardhaman, West Bengal, India
Author Family Name Kabiraj
Particle
Given Name Ashutosh
Suffix
Division UGC-Center for Advanced Study, Department of Botany
Organization The University of Burdwan
Address Bardhaman, West Bengal, India
Author Family Name Roy
Particle
Given Name Rajendra Kr
Suffix
Division UGC-Center for Advanced Study, Department of Botany
Organization The University of Burdwan
Address Bardhaman, West Bengal, India
Author Family Name Let
Particle
Given Name Moitri
Suffix
Division UGC-Center for Advanced Study, Department of Botany
Organization The University of Burdwan
Address Bardhaman, West Bengal, India
Author Family Name Majhi
Particle
Given Name Krishendu
Suffix
 Division UGC-Center for Advanced Study, Department of Botany
Organization The University of Burdwan
Address Bardhaman, West Bengal, India
Corresponding Author Family Name Bandopadhyay
Particle
Given Name Rajib
Suffix
Division UGC-Center for Advanced Study, Department of Botany
Organization The University of Burdwan
Address Bardhaman, West Bengal, India
Abstract Deposition of heavy metals and other contaminating materials in the
environment is a ceaseless and inescapable process. Bioaccumulation of
heavy metals is extremely harmful to all domains of life. While dealing with
heavy metal pollutions, microbes, especially Proteobacteria and
Actinobacteria, acclimatize and adapt themselves to contaminated sites
through different metabolic activities. Microbial bioremediation through
biosorption, bioaccumulation, solubilization, immobilization,
transformation, etc. is an innovative, sustainable, cost-effective, and
efficient approach to reducing heavy metal toxicity. Since traditional
culturable approaches have limitations, metagenomic approaches help
uncover the microbes, associated genes, and their functions in heavy
metal bioremediation. Rapid improvements in molecular techniques, such
as high-throughput DNA sequencing, amplification, cloning, microarrays,
and other “-omic” tools, have shown promise in revealing the metabolic
diversity and nutrient versatility of microbes living in contaminated
environments. Study site selection, sample collection and nucleic acid
extraction, genome enrichment, and metagenomic library construction are
the key steps in metagenomic research. Starting with a complete
understanding of metagenomic screening, several function-based
techniques including phenotype-based screening, substrate-induced gene
expression (SIGEX), and metabolite-regulated expression (METREX) are
used to discover new classes of genes. Bioinformatic tools like MEGAN,
CAMERA, MG-RAST, and IMG/M play a variety of roles in the field of
metagenomic bioremediation data analysis. Although our findings are
moving forward in the right direction, many activities still lack adequate
screening methods and have poor expression systems for metagenomes. In
spite of these limitations, we have conducted our metagenomic research to
evaluate microbial community composition in polluted areas to select
effective microbes that can help in heavy metal remediation and to
determine how to apply them to enhance the nature-friendly elimination
processes.
Keywords Bioremediation - Sustainable - Heavy metals - Metagenomics -
(separated by ‘-’) Bioinformatics - Microbial community - MEGAN
Bioremediation of Heavy Metals by
Metagenomic Approaches 17 1

Dibyendu Khan, Ashutosh Kabiraj, Rajendra Kr Roy, Moitri Let, 3

Krishendu Majhi, and Rajib Bandopadhyay 4

Abstract 5

Deposition of heavy metals and other contaminating materials in the environment 6

is a ceaseless and inescapable process. Bioaccumulation of heavy metals is 7
extremely harmful to all domains of life. While dealing with heavy metal 8 AU2
pollutions, microbes, especially Proteobacteria and Actinobacteria, acclimatize 9
and adapt themselves to contaminated sites through different metabolic activities. 10
Microbial bioremediation through biosorption, bioaccumulation, solubilization, 11
immobilization, transformation, etc. is an innovative, sustainable, cost-effective, 12
and efficient approach to reducing heavy metal toxicity. Since traditional 13
culturable approaches have limitations, metagenomic approaches help uncover 14
the microbes, associated genes, and their functions in heavy metal bioremedia- 15
tion. Rapid improvements in molecular techniques, such as high-throughput 16
DNA sequencing, amplification, cloning, microarrays, and other “-omic” tools, 17
have shown promise in revealing the metabolic diversity and nutrient versatility 18
of microbes living in contaminated environments. Study site selection, sample 19
collection and nucleic acid extraction, genome enrichment, and metagenomic 20
library construction are the key steps in metagenomic research. Starting with a 21
complete understanding of metagenomic screening, several function-based 22
techniques including phenotype-based screening, substrate-induced gene expres- 23
sion (SIGEX), and metabolite-regulated expression (METREX) are used to 24
discover new classes of genes. Bioinformatic tools like MEGAN, CAMERA, 25
MG-RAST, and IMG/M play a variety of roles in the field of metagenomic 26
bioremediation data analysis. Although our findings are moving forward in the 27
right direction, many activities still lack adequate screening methods and have 28 AU3

D. Khan · A. Kabiraj · R. K. Roy · M. Let · K. Majhi · R. Bandopadhyay (*)

UGC-Center for Advanced Study, Department of Botany, The University of Burdwan, Bardhaman, AU1
West Bengal, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte

Ltd. 2022
V. Kumar, I. S. Thakur (eds.), Omics Insights in Environmental Bioremediation,
https://doi.org/10.1007/978-981-19-4320-1_17
 D. Khan et al.

29 poor expression systems for metagenomes. In spite of these limitations, we have

30 conducted our metagenomic research to evaluate microbial community composi-
31 tion in polluted areas to select effective microbes that can help in heavy metal
32 remediation and to determine how to apply them to enhance the nature-friendly
33 elimination processes. AU4

34 Keywords
35 Bioremediation · Sustainable · Heavy metals · Metagenomics · Bioinformatics ·
36 Microbial community · MEGAN

37 17.1 Introduction

38 Heavy metal contamination throughout the world and its impact on living organisms
39 is not a new topic of discussion. Enormous numbers of studies have been conducted
40 in different continents in recent decades regarding “ambiguously” defined metals
41 and their perilous effects on different organisms either in low or high concentrations.
42 Loosely classifying these metals on the basis of their toxicity as either significantly
43 toxic (e.g., As, Cd, Pb, Hg, etc.) or certainly toxic (e.g., Zn, Cu, Ni, etc.), we observe
44 that the former (with no biological significance) would affect our health at lower
45 concentrations and the latter (sometimes acting as trace elements) at higher
46 concentrations (Su 2014). Land areas, water bodies, and the atmosphere are now AU5
47 highly under the risk of this pollution due to natural and anthropogenic activities.
48 Rapid industrialization and high chemical production were the highest in China in
49 2010, followed by the USA, Japan, and Germany, whereas more than 70 million
50 chemicals are produced throughout the world, which eventually get deposited either
51 in water bodies or soil (Wu et al. 2016). Biological organisms encounter these metals
52 directly or indirectly, thus producing reactive oxygen species (ROS). The attraction
53 of heavy metals toward thiol groups hampers proper protein function, which is one
54 of the causes of metabolic impairment.
55 Microbes, the silent leading scientists with the enormous potentiality to restrict
56 heavy metal pollution on the basis of their natural and/or adapted physical and
57 molecular attributes, are the pathfinders of this warfare through bioremediation, a
58 cheap and environment-friendly technology. Specific bacteria, fungi, and other
59 microbes may be able to transform (keeping in mind that heavy metals cannot be
60 eradicated or degraded totally from the environment) metals from one form to
61 another and alter their bioavailability. Enhancing the solubility of metals by
62 microbes results in metal leaching, whereas decreasing solubility reduces bioavail-
63 ability. So, both kinds of interactions show positive results with respect to different
64 biological organisms. Biosorption, a potent bioremediation technology adapted by
65 microorganisms, can be divided into metabolism-dependent (transport across the
66 membrane, precipitation, etc.) or metabolism-independent (ion exchange, complex-
67 ation, etc.) (Ojuederie and Babalola 2017). Extracellular polymeric substances
68 (EPSs), present outside the cells of prokaryotic and eukaryotic organisms, consist
17 Bioremediation of Heavy Metals by Metagenomic Approaches

of both micro and macromolecules (polysaccharides, proteins, phospholipids, 69

nucleic acids, etc.) and are responsible for biosorption of metallic compounds 70
using their negatively charged ions interacting electrostatically with positively 71
charged metal ions (Pal and Paul 2008; Kumar 2018). pH is an important parameter 72
for the biosorption process because altering the pH results in changing the solubility 73
of compounds (Ojuederie and Babalola 2017). 74
To date, we have been able to study very few microbes because of our lack of 75
knowledge about their environmental niche where they interact with different biotic 76
and abiotic factors. Simply put, they are unculturable, still playing pivotal roles in 77
the environment. To know about these microbes, recently, metagenomics with its 78
tremendous applicability has opened a window of hope for us. Metagenomics, 79
however, with some limitations, not only gives us insights into finding unculturable 80
microbes but also is a potent technology to decipher genes for proper functioning. 81
Function-, sequence-, and polymerase chain reaction (PCR)-based screenings are 82
well-known methods of metagenomics to study heavy metal bioremediation. Some 83
computational tools like MEGAN, MG-RAST, CAMERA, etc. may be mentioned 84
related to metagenomic analyses (Singh et al. 2009). 85
In this chapter, we discuss microbial interactions with heavy metals as well as 86
different bioremediation strategies, microbial community analysis of contaminated 87
areas through metagenomic library construction and different screening approaches, 88
and how different bioinformatic tools help analyze the metagenomic data. 89

17.2 Impacts of Heavy Metals on Living Systems 90

From prokaryotes to higher mammals, all are under the threat of heavy metal 91
toxicity. In human beings, various heavy metals are compartmentalized within 92
different body cells as well as cell organelles, leading to tissue damage, 93
malfunctioned protein production, and damage to organs like the kidneys, lungs, 94
liver, heart, etc. Long-time exposure accompanied by high accumulation of these 95
heavy metals in body cells cause neurotoxicity. The general enzymatic activities of 96
proteins are altered in the case of microbes. These may be involved in the reduction 97
of the diversity of microbes within a contaminated area. However, some microbes 98
are resistant to specific heavy metals, which will be discussed briefly. In the case of 99
plants, photosynthetic enzyme production, photosynthesis, and total metabolic 100
activities are drastically hampered. For crop plants, loss of crops leads to economical 101
imbalance. Heavy metals accumulate in different tissues of organisms. Meat we eat 102
like chicken, goat, etc. may also contain heavy metals within their tissues. On 103
consumption, these meats accumulate within our body and cause different diseases. 104 AU6
Heavy metals have negative impacts specifically on sex organ development in 105
different animals like birds, snakes, and mammals. Anemia and testicular hypoplasia 106
are common diseases among birds. Due to reduction of the production and functions 107
of ROS scavenging enzymes like superoxide dismutase, peroxidase, catalase, etc., 108
the growth, development, and reproduction of fishes are reduced. It is also related to 109
economic loss in society (Engwa et al. 2019). 110
 D. Khan et al.

111 17.2.1 The General Mechanism of Heavy Metal Toxicity

112 Reactive oxygen species (ROS) production is one of the important consequences of
113 heavy metal toxicity, which, on entering the nucleus, not only interacts with DNA
114 and damages it but also interacts with the lipid bilayer of the cell and leads to lipid
115 peroxidation. Among the huge numbers of heavy metals, here, we discuss the
116 general impact of cadmium, arsenic, lead, mercury, and copper (a trace element).
117 Interestingly, some metals mimic other metals or compounds, for example, arsenate
118 mimics phosphate and impairs oxidative phosphorylation. Nonessential metallic
119 ions like Cd2+ interact with nonmetallic biomolecules and form complexes
120 (Fig. 17.1). This “misbonding” has a negative impact within cells.
121 Iron and copper are important metals, which have tremendous functions within
122 cells; however, excessive concentrations may produce ROS in the form of hydroxyl
123 ions (OH•), which react with biomolecules, such as proteins, lipids, and DNA. It may
124 be responsible for DNA methylation, histone structure alteration, and changes in the
125 expressions of cell cycle-regulating proteins like p53 and p21, leading to tumor
126 formation (Engwa et al. 2019).

Fig. 17.1 Heavy metal toxicity within a cell: (a) arsenic (As), cadmium (Cd), mercury (Hg), and
lead (Pb) enter through specific channels within the cell. Lead interacts with ALAD and induces the
production of the ΔALAD enzyme. By displacing Zn from the enzyme, ROS production is induced.
Cd and Hg interact with SH/SeH-containing molecules, which, in turn, induce GSH derivative
(GSHd) or ROS production. GSHd damages the mitochondria. Arsenic induces reactive nitrogen
species (RNS) production, and RNS causes DNA damage or lipid peroxidation. Cd and Hg may
damage the liver proteins of mammals. ROS may cause lipid peroxidation or DNA damage. (b) Pb
and Cd mimic Zn and Cu, respectively, and remove functional heavy metals
17 Bioremediation of Heavy Metals by Metagenomic Approaches

17.2.2 The Global Scenario of Heavy Metal Contamination 127

It is quite difficult to find out which biosphere is not contaminated by heavy metals. 128
For example, the polar regions are to some extent devoid of anthropogenic activities; 129
however, the Antarctic and Arctic regions of the world are contaminated with a 130
variety of heavy metals. Air-borne heavy metal particles (emitted through industrial 131
fossil fuel burning, forest fires, etc.), suspended in the atmosphere, come down 132
through rain, leading to the contamination of polar earth crusts. Oceans are one of 133
the contaminated zones of heavy metals, where coral reefs are also reported to have 134
high amounts of heavy metal deposition. As usual, urban areas are 10 times more 135
contaminated than are rural ones. For example, the average concentrations of arsenic 136
and copper in the rural and urban areas of the world are 3.2 and 20 ngm 3 and 7.9 137
and 155 ngm 3, respectively. Anthropogenic activities contaminate both the atmo- 138
sphere and aquatic ecosystems. Three decades ago, studies revealed that a total of 139
19 and 42 and 35 and 112 thousand tons of arsenic and copper were deposited in the 140
atmosphere and aquatic ecosystems per annum, respectively (Nriagu 1990). 141
Recently, Hoang et al. (2021) have extensively studied the impact and contamination 142
of heavy metals in different rivers of the world. They argued that different metals like 143
cadmium, arsenic, lead, copper, etc., which are enormously deposited within rivers, 144
result in chronic toxicity in aquatic plants and animals. Due to increased industriali- 145
zation, lead poisoning is a major problem in first-world countries and in the marine 146
environments of North America and Western Europe. Arctic regions are under heavy 147
metal stress due to Eurasian industrialization. Many Asian countries like India, 148
Taiwan, Bangladesh, Pakistan, etc. are highly under the risk of arsenic poisoning. 149
Heavy metal-contaminated foods are also one of the greatest concerns for 150
us. Sometimes, affected foods are also transported to different regions of the 151
world, which also causes health risks to different organisms (Hembrom et al. 152
2020). After studying the present scenario, we may conclude that there is not a 153
single biosphere where clutches of heavy metal contaminations are not present. 154

17.3 Reciprocal Interaction Between Microbes and Metals 155

Environmentally ubiquitous microorganisms are leading components that can inter- 156
act with heavy metals (Rahman and Singh 2020). Excessive concentrations of heavy 157
metals are toxic to microbial cells and could adversely affect the cellular activities of 158
microbes. In microbial cells, heavy metals mimic the essential elements of various 159
enzymes hampering metabolic processes, producing reactive oxygen species (ROS), 160
and, ultimately, altering RNA, DNA, and protein structure and function 161
(Prabhakaran et al. 2016). To overcome such toxic effects of heavy metals, 162
microorganisms have been acquired using various strategies like bioadsoption, 163
bioaccumulation, oxidation–reduction, biomineralization, precipitation, and 164
bioleaching. 165
In the biosorption process, heavy metals are strongly attached to the outer 166
structure of the bacterial cell surface with the aid of various functional groups like 167
 D. Khan et al.

168 hydroxyl, carboxyl, amino, ester, sulfhydryl, carbonyl, phosphate, etc. (Ali Redha
169 2020). These functional groups interact with heavy metals by covalent bonding,
170 electrostatic attraction, van der Waals interaction, ion exchange, complexation, and
171 precipitation, for example, carboxyl, phosphate, and sulfate functional groups pres-
172 ent in the polysaccharides and proteins of Oceanobacillus profundus KBZ 3-2 could
173 help in the ion exchange and complexation process of Pb (II) and Zn (II) ions
174 (Mwandira et al. 2020). Whereas in bioaccumulation, they are accumulated after
175 entering through various exporters (like ABC transporter, P-type ATPase, cation-
176 diffusion facilitator, etc.), ion channels, ion pumps, and endocytosis.
177 Microorganisms could also produce siderophores and metallothioneins, which help
178 facilitate the intracellular bioaccumulation of heavy metals (Sharma and Shukla
179 2021).
180 Microorganisms also catalyze the oxidation–reduction reactions of heavy metals,
181 where electrons are transferred from one elemental state to another (Rahman and
182 Singh 2020). Various heavy metals such as arsenic (As), chromium (Cr), etc. can be
183 effectively removed from the environment through the bacterial oxidation–reduction
184 process. In the case of enzymatic arsenate [As(V)] reduction, arsenate reductase
185 helps convert it to arsenite [As(III)], whereas in chromate [Cr(VI)] reduction, various
186 reducing enzymes including NAD(P)H-dependent chromate reductase,
187 oxidoreductases, nitroreductase, flavin mononucleotide (FMN) reductase, and lipoyl
188 dehydrogenase are involved (Thatoi et al. 2014).
189 However, in biomineralization, microorganisms naturally mineralize various
190 minerals like silicates, phosphates, sulfates, carbonate, and oxides via different
191 mechanisms. For instance, microbially induced calcium carbonate precipitation
192 (MICP) is a type of biomineralization process in which microorganisms induce
193 calcium carbonate precipitation through different mechanisms; among them, urea
194 hydrolysis is predominant. Various ureolytic bacteria could produce urease
195 enzymes, which help precipitate metal ions through urea hydrolysis and produce
196 carbonate and ammonia. For example, Sporosarcina pasteurii, a urease enzyme-
197 producing strain, was used to precipitate lead (Pb), cadmium (Cd), and zinc
198 (Zn) (Jalilvand et al. 2020). In the case of bioleaching, microorganisms can solubi-
199 lize metal sulfide into sulfate. In this regard, acidophilic bacteria, mostly
200 Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, are generally
201 involved in the oxidation of iron (Fe2+ to Fe3+) and sulfur (S0 to SO42 ) compounds,
202 which ultimately changes the pH and facilitates the bioleaching of heavy metals.

203 17.4 Bioremediation: A Future Outlook Toward Mankind

204 Environmental pollution is an increasing concern of mankind. Nonbiodegradable AU7

205 pollutants such as heavy metals, plastics, industrial dyes, pesticides, etc. are detri-
206 mental to human health on entering the food chain. Instead of using various physical
207 and chemical conventional techniques, bioremediation is a simple, nonconventional,
208 renewable, cost-effective, and eco-friendly approach to detoxify heavy metals from
209 contaminated sites (Kumar et al. 2018; Rastogi and Kumar 2020). Bioremediation is
17 Bioremediation of Heavy Metals by Metagenomic Approaches

not only engaged in environmental cleanup but also involved in wastewater treat- 210
ment, crop productivity, and production of some value-added products. Similarly, 211
Pseudomonas sp. WS-D/183 can be effectively used for textile wastewater treatment 212
as well as for heavy metal removal and plant growth promotion (Hussain et al. 2020). 213
Several recycled microalgal species are capable of pesticide degradation and pro- 214
duce some value-added products like biochar, biodiesel, etc. (Nie et al. 2020). These 215
culturable microbes have been efficiently studied for bioremediation purposes, but 216
the study on unculturable microbes (their growth, metabolism, and function) 217
provides new insights into the future bioremediation strategy. In this regard, 218
“omics” technologies, especially metagenomics and proteomics of the microbial 219
community, could be effectively used for the eradication of hazardous pollutants 220
from the environment. 221

17.5 Microbial Metagenomics: A Multi-potential Concept 222

Toward Heavy Metal Bioremediation 223

It is extremely useful to have prior information of the chemical, physical, and 224
ecological parameters of selected habitats to carry out a metagenomic analysis. 225
The extraction of nucleic acid from the sample site is the first step in any 226
metagenomic study. When a whole community is selected for metagenomic analy- 227
sis, its species composition must be considered to ensure affordable and high coding 228
gene densities. Therefore, not eukaryotic but only prokaryotic microbial 229
communities gain attention in most metagenomic studies. 230

17.5.1 Unraveling the Structure and Functional Potential 231

of Microbial Communities 232

Only a small amount of information regarding the indigenous microbial diversity in 233
contaminated sites has been obtained using culture-dependent approaches. We were 234
unable to properly investigate the microbial diversities, metabolic capacities, and 235
plasticity in heavy metal-contaminated locations using traditional culture-based 236
methodologies. To overcome these limitations, a new methodology called 237
“metagenomics” has evolved to investigate the genetic resources of both culturable 238
and nonculturable microbes from any environment. Metagenomics and high- 239
throughput sequencing methods have revealed the abundance, adaptation, ecology, 240
and evolution of microbes surviving in contaminated environments. 241

17.5.2 Dominant Microbial Community in the Metagenome 242

Heavy metal-contaminated areas are enriched with some special microbial phyla, 243
and microbial composition varies between contaminated sites. A study by Prakash 244
et al. (2021) reported that contaminated soil was dominated by bacteria (77.50%), 245
 D. Khan et al.

t:1 Table 17.1 List of major microbial phyla in contaminated areas AU8

Percentage
of presence
in
t:2 Dominant phylum community Metagenomic projects Source References
Gammaproteobacteria 22.88 Metagenomic analysis of soil Soil Czarny
t:4 Alphaproteobacteria 22.83 bacterial community and level et al.
t:3 of genes responsible for (2017)
t:5 Clostridia 11.11
biodegradation of aromatic
t:6 Bacilli 8.40
hydrocarbons
t:7 Betaproteobacteria 7.55
t:8 Planctomycetia 5.21
Proteobacteria 38.56 Metagenomic analysis of the Soil Feng et al.
t:9
t:10 Acidobacteria 18.13 microbial community and (2018)
t:11 Gemmatimonadetes 9.26 function involved in
cd-contaminated soil
t:12 Thaumarchaeota 5.45
Proteobacteria 48.63 Analysis of microbial Soil Hemmat-
t:14 Actinobacteria 17.28 communities in heavy metal- Jou et al.
t:13 contaminated soils using the (2018)
t:15 Acidobacteria 14.44
metagenomic approach
t:16 Gemmatimonadetes 8.92
t:17 Bacteroidetes 4.87
t:18 Gammaproteobacteria 12
t:19 Alphaproteobacteria 3

246 followed by unknown organisms (12.50%) and archaea, viruses, and Eukarya
247 (<10%). Proteobacteria (Gammaproteobacteria, Alphaproteobacteria,
248 Betaproteobacteria, and Deltaproteobacteria) are the keystone group of bacteria,
249 whereas most abundant genera—Nocardioides and Pseudomonas—are dominated.
250 They have great morphological and metabolic diversities that play a pivotal role in
251 heavy metal bioremediation (Xavier et al. 2019). Proteobacteria, Acidobacteria, and
252 Actinobacteria are the major groups of terrestrial bacteria (Hur and Park 2019). From
253 different metagenomic studies (Table 17.1), we found that contaminated sites are
254 dominated by the same bacterial groups, which postulate that these groups of
255 bacteria are able to handle extreme environments. Alphaproteobacteria and
256 Deltaproteobacteria are able to bioremediate chromium compounds,
257 Betaproteobacteria can degrade aromatic, aliphatic, and hydrocarbon compounds,
258 and Gammaproteobacteria play a role in oxidation of sulfide compounds. Firmicutes
259 under environmental stress like hypersalinity and low oxygen level can eradicate
260 pollutants. Bacteriodetes can efficiently degrade various organic pollutants under
261 low oxygen concentration. A previous report mentioned that Planctomycetes are
262 able to remove dissolved organic matter, whereas Gemmatimonadetes can accumu-
263 late phosphate and remove phosphate from wastewater. Alphaproteobacteria,
264 Gammaproteobacteria, and Firmicutes possess specific functional genes for sulfate
265 reduction that might help in the conversion of metals into inert metal sulfides.
266 Members of Sphingomonadaceae can remediate heavy metals from sewage. Fungal
267 species reported from contaminated sites have the potential to enzymatically convert
17 Bioremediation of Heavy Metals by Metagenomic Approaches

toxic compounds, hazardous substances, and metal ions into nontoxic simpler forms. 268
The cell walls of fungi and bacteria interact electrostatically, resulting in biosorption 269
of cadmium, copper, zinc, uranium, and cobalt (Liaquat et al. 2021). Heavy metals 270
such as lead and copper have been found to be tolerated by marine fungi, and 271
filamentous fungi have the capacity to remediate Cd, Cu, and Ni. Enzymes like 272
catalase and peroxidase produced by bacteria and fungi have heavy metal tolerance 273
capacity, whereas phytochelatin synthase (PCS) catalyzes the formation of 274
phytochelatin from glutathione, protecting cells from nonessential heavy metal 275
toxicity. Algal biomass is also used to remove heavy metals through biosorption, 276
an ecologically safe and cheaper process. An earlier report suggested that the 277
Phaeophyceae group of algae contains alginate, which acts as a good candidate for 278
biosorption of heavy metal ions (Shine et al. 2015). 279

17.5.3 The Importance of Metagenomic Library Construction 280

Metagenomic DNA is directly isolated from the polluted environment using culture- 281
independent methods. Various methods have now been discovered to isolate DNA 282
from environmental samples. To represent all microbial genomes, entire 283
metagenomic DNA must be extracted and purified followed by construction of a 284
metagenomic library. Isolation of metagenomic DNA from environmental sources 285
has been accomplished using two methods: cell recovery and direct lysis. Both 286
physical and chemical methods, involved in the extraction process, have indepen- 287
dent advantages regarding metagenome extraction. In physical methods, more 288
microbial diversities are recovered, whereas in chemical methods, high-molecular- 289
weight DNA is obtained. The choice of cloning vectors is dependent on the size of 290
interested genes. Plasmid cloning vectors need small DNA fragments, whereas 291
larger inserts require cosmids (25–35 kb), fosmids (25–40 kb), or bacterial artificial 292
chromosomes (BACs) (100–200 kb). It is preferable to use clones with high- 293
molecular-weight DNA inserts for the evaluation of genes and metabolic pathways, 294
as this increases the likelihood of obtaining positive hits during library screening. 295
More clones are required for optimal coverage of the metagenome when the cloned 296
inserts are smaller. 297

17.5.4 Screening of Detoxifying Genes from the Polluted 298

Environment 299

17.5.4.1 Function-Based Screening 300

This method is highly useful for discovering new metabolic activities, new classes of 301
genes, functional gene products, and enzymes relevant to bioremediation (Fig. 17.2). 302
Prior sequence information or sequence similarity is not required for the function- 303
based approach. One of the major disadvantages of the function-based approach is 304
that it requires production of functional gene products by the bacterial host (Daniel 305
2005). To investigate these rich genetic resources, a range of function-based 306
 D. Khan et al.

Fig. 17.2 A workflow of different metagenomic techniques

307 metagenomic screening approaches have been devised, culminating in the discovery
308 of a huge number of new enzymes with a distinct metabolic activity.

309 17.5.4.1.1 Phenotype-Based Screening

310 Because intact cells and their native cellular milieu are employed, phenotypic
311 screening is usually more biologically realistic and less artificial. In this method, a
312 metagenomic DNA library was created using metagenomes obtained directly from
313 contaminated locations. The primary hits found in phenotypic screening could be
314 used to target a variety of proteins (receptors, enzymes, transcription factors, and so
315 on) as well as signaling cascades. Several detoxifying or biodegradative enzymes
316 such as naphthalene dioxygenase, nitrilase, styrene monooxygenase, and extradiol
317 dioxygenase (EDO) have been identified from DNA libraries through a function-
318 based approach (Pushpanathan et al. 2014).

319 17.5.4.1.2 Agar Plate Screening

320 This functional approach is useful to identify novel enzymes (hydrolytic and
321 nonhydrolytic) that function under diverse environmental conditions. This method-
322 ology has led to the discovery of a number of new hydrolytic enzymes, including
323 lipases, esterases, cellulases, proteases, laccases, glycosylases, nitriles, and halides.
324 This method is frequently used to look for genes that cause resistance to harmful
325 elements such as antibiotics, high salt concentrations, high pH, and heavy metals.
326 Enzymes like β-glycosidases, dioxygenases, dichlorophenol hydroxylases,
327 polyhydroxyalkanoate synthases, etc. are identified through this method (Ngara
328 and Zhang 2018).

329 17.5.4.1.3 Fluorescence-Activated Cell Sorting (FACS)-Based Screening

330 This technique is useful for library screening based on the size, shape, and fluores-
331 cence properties. FACS is easily related to a number of different high-throughput
17 Bioremediation of Heavy Metals by Metagenomic Approaches

screening approaches, such as droplet sorting and reporter-based screening, because 332
of its excellent sorting properties. Reporter gene expression is a fundamental step in 333
reporter-based screening where a metagenomic library is transformed into host cells 334
harboring reporter genes. Substrate-induced gene expression (SIGEX) is a FACS- 335
based intracellular screening method, which sorts green fluorescent protein (GFP)- 336
expressing cells and responds to the presence of aromatic and hydrocarbon 337
degrading genes. It is possible to identify phenol degradative operons and genes 338
involved in benzoate and catechol pathways by this approach. SIGEX has 339
drawbacks, for example, it is sensitive to the structure and orientation of genes 340
associated with desirable attributes. During the screening technique, catabolic genes 341
that are constitutively expressed are missed (Wakamatsu et al. 2018). Another 342
modified reporter-based screening system is called product-induced gene expression 343
(PIGEX), in which a transcriptional activator is highly sensitive to the product of the 344
desired enzyme. Metabolite-regulated expression profiling (METREX) is also a 345
FACS-based intracellular screening strategy that improves sensitivity while screen- 346
ing poorly expressed gene products (Williamson et al. 2005). This system is used to 347
screen biodegradative genes as well as quorum-sensing signal molecules using 348
biosensors from the microbial community. 349

17.5.4.1.4 Microfluidics-Based Screening 350

This is a high-throughput screening technology, coupled with FACS, which gives an 351
advantage over other methods due to its appropriateness for cell-based assays, 352
minimal analytical costs, and ease of handling liquids in picoliter amounts. A single 353
droplet functions as a reaction chamber where cells, enzymes, substrates, and 354
products are confined (Ngara and Zhang 2018). The detection method, which is 355
usually confined to a fluorescent signal, is the key limitation of this technology. 356
In addition to these screening methods, microtiter plate screening, genetic 357
enzyme screening systems (GESSs), microfluidic gel microdroplets (GMDs), and 358
microfluidic absorbance-activated droplet sorters (AADSs) (Ngara and Zhang 2018) 359
are also available for screening microbial communities from the metagenomic 360
library. 361

17.5.4.2 Sequence-Based Screening 362

This screening-based approach is not only restricted to discovering new classes of 363
genes but also to exploring microbial ecology, transposon in bacteria, and phyloge- 364
netic profiles. A previous knowledge of nucleotide information from databases is 365
required for the screening of detoxifying genes. Massive amounts of sequence data 366
are collected and combined into larger fragments until a sufficient level of coverage 367
is achieved. Degenerate primers or high-density arrays can be used to improve the 368
detection frequency of a variety of target genes. Deep sequencing with great 369
coverage provides for high resolution, allowing even rare single-nucleotide 370
polymorphisms (SNPs) to be detected (Pushpanathan et al. 2014). Some of the 371
sequence-based methods include primer-based screening through polymerase 372
chain reaction (PCR), microarray profiling, probe hybridization, and high- 373
throughput sequencing technologies. 374
 D. Khan et al.

375 17.5.4.2.1 Screening by PCR

376 This method of approach is useful in microbial community analysis as well as in
377 biotechnological fields for characterization of enzymes, antibiotics, or resistant
378 genes. For the identification of a desired gene fragment, conserved nucleotide
379 sequences are taken into consideration. Using this screening approach, we can
380 identify members of a particular environment, catabolic genes, and their phyloge-
381 netic relationships. The advantage of using this procedure is that it can bypass the
382 processes of cloning, transformation, and clone maintenance. For proper screening,
383 there are some crucial steps including primer design, annealing temperature, ampli-
384 fication cycles, template concentration, and the DNA polymerase used (Sze and
385 Schloss 2019). Full-length environmental genes from various metagenomic samples
386 have been successfully amplified and expressed using primer sequences based on
387 specific enzyme-encoding sequences. By analyzing transcripts and the quantity of
388 ecologically relevant core genes in environmental samples, quantitative PCR
389 methods can be used to investigate the specific metabolic activity. Although a
390 major drawback of PCR-based strategies is unequal amplification of mixed template
391 DNA, it sometimes prevents accurate microbial diversity estimation in environmen-
392 tal samples (Kalle et al. 2014).

393 17.5.4.2.2 Hybridization-Based Screening

394 In this approach, target-specific probes are also used to screen metagenomic
395 libraries. PCR amplification and further sequencing help recover full-length targeted
396 genes. The amplification of conserved sequences from target genes using
397 biotinylated primers is one of the hybridization techniques that use magnetic bead
398 separation and subtractive hybridization. The probe binds to full-length gene
399 sequences, which are subsequently isolated from the rest of the metagenome using
400 a magnetic field. This screening approach has been used to identify 16S rRNA genes
401 and highly conserved domains like polyketide synthases, gluconic acid reductases,
402 and nitrile hydratases (Daniel 2005).

403 17.5.4.2.3 Microarray Profiling

404 Microarray technology has been successfully used to know the composition and
405 activity of the soil microbial community as well as to identify functional genes. Short
406 oligonucleotide probes are fixed as dots on the surface of a slide in microarrays. As a
407 result, each point in the array corresponds to the complementary sequence of a
408 specific gene from the genomes and/or transcripts of interest. Only those sequences
409 that effectively hybridize with correct complimentary probes are immobilized on the
410 slide after the metagenomic nucleic acids are extracted and tagged with fluorescent
411 dyes, followed by hybridization and washing procedures. However, gene detection
412 through the microarray approach shows a 100- to 10,000-fold lower sensitivity than
413 that through PCR (Daniel 2005). The “GeoChip” technology has been developed
414 based on the microarray approach to explore direct linkages between biogeochemi-
415 cal processes and functional activities of microbial communities (He et al. 2007).
416 Another microarray-based metagenome profiling has been discovered in which the
417 metagenomic DNA itself can be used to develop the microarray. This gateway does
17 Bioremediation of Heavy Metals by Metagenomic Approaches

not require any previous sequence knowledge about microbial communities and 418
opens a door to uncover novel detoxifying genes (Pushpanathan et al. 2014). 419

17.6 Bioinformatic Tools in Metagenomic Data Analysis 420

Several computational tools are now gaining popularity in multiple fields of science. 421
Bioinformatics is used in the field of metagenomic bioremediation to conduct a 422
variety of tasks, the most important of which is the processing of metagenomic data. 423
Multiple metagenomic studies are generating large amounts of sequence data, thus 424
putting pressure on bioinformatics to develop more reliable and accurate methods 425
(Table 17.2) for interpreting metagenomic sequence data. 426

17.7 Other Applications of Metagenomics 427

17.7.1 Fluorescent in Situ Hybridization (FISH) 428

Fluorescent in situ hybridization is a cyto-molecular technique that usually relies on 429

the principle of binding of a fluorescent probe to highly similar nucleic acid 430
sequences. Small probes are generally 18–30 nucleotides long, capable of easily 431
passing the biological membrane, and flourishing on binding to the sequence of 432
interest. This technique is extremely useful for sorting out a particular group of 433
microbes from environmental samples using FACS. Cells expressing the target gene 434
and the level of expression can also be quantified using FISH. Actively growing 435
microbial populations contain a higher number of ribosomes than do slower growing 436
ones and will have a higher quantity of rRNA. So, if the probe is targeting the rRNA 437
of a microbe, then the growth rate of the community can be quantified by correlating 438
the intensity of the fluorescent under a confocal laser scanning microscope. The 439
abundance of microbial communities and their capability to bioremediate in soil- 440
contaminated areas are reflected using FISH as a key approach (Pushpanathan et al. 441
2014). 442

17.7.2 Ribosomal Intergenic Spacer Analysis (RISA) 443

RISA is a PCR-based application in which the intergenic space sequence and length 444
between the 16S and 23S rDNA is amplified in order to identify the bacterial 445
community in aquatic environments, land systems, and the human gut. The length 446
in between the two rRNA genes is significantly variable among microbes and is thus 447
highly useful to generate phylogeny. It is widely used as an important part of 448
metagenomic tools as it has no need to culture bacteria but to generate a library, 449
which is further utilized to reveal unculturable microbes (Mahomed et al. 2021). 450
 D. Khan et al.

t:1 Table 17.2 List of computational tools for metagenomic data analysis
Computational
t:2 tools Characteristics References
MEGAN 1. Singh et al. (2009), Bharagava
Accepts a variety of data (taxonomic et al. (2019), Logares et al.
t:3 and functional) input types and may (2012), Lanzén et al. (2012)
export analysis (geographical and
statistical) results in a variety of text-
based and graphical formats
2.
Focuses primarily on taxonomic
profiles
SmashCommunity 1.
Suitable for data derived from sanger
and 454 sequencing
2.
Estimates metagenomes’
quantitative, phylogenetic, and
t:4 functional components
CAMERA 1.
Provides a powerful cross-analysis of
environmental samples
2.
A genome analysis tool that allows
users to query, analyze, annotate, and
compare data from metagenomes and
t:5 genomes
MG-RAST 1.
Offers quantitative insights into
microbial communities
2.
Accepts different phylogenetic and
metabolic data
3.
Performs protein prediction,
clustering, and similarity-based
t:6 annotation on nucleic acid sequences
IMG/M 1.
Able to handle larger metagenome
datasets
2.
Provides a platform for function-
based comparison including
abundant protein families, functional
families, or functional categories
t:7 across metagenomic samples
UniFrac Compares microbial community
t:8 diversity in a phylogenetic approach
FOAM 1.
Classifies gene functions relevant to
environmental microorganisms
(continued)
17 Bioremediation of Heavy Metals by Metagenomic Approaches

Table 17.2 (continued)

Computational
tools Characteristics References t:10
2.
Easy to organize results into different
functional categories t:9
JANE 1.
Allows you to quickly get a sense of
the genome’s structure and functions
2.
Expressed sequence tags (ESTs) and
variable length sequences can be
mapped and assembled quickly t:10
COGNIZER 1.
Provides multiple workflow options
2.
A stand-alone annotation platform
that allows users to functionally
annotate sequences from
metagenomic datasets t:11
RDP 1.
Used in the fields of microbial
ecology, environmental
microbiology, nucleic acid
chemistry, and phylogenetics
2.
Provides aligned and annotated
rRNA gene sequence data to the
research community as well as tools
to allow researchers to evaluate their
own rRNA gene sequences in the
RDP framework t:12
myPhyloDB Microbial community populations
may now be stored, processed,
analyzed, and distributed more easily t:13
WebMGA 1.
A quick and unique tool as well as a
lot of flexibility when it comes to
analyzing complex metagenomic
data
2.
ORF calling, sequence grouping, raw
read quality control, elimination of
sequencing artifacts and
contaminations, taxonomic analysis,
and functional annotation are only a
few of the tools included in
WebMGA t:14
WebCARMA 1.
Using short reads that encode for
known proteins, this characterizes the
(continued)
 D. Khan et al.

Table 17.2 (continued)

Computational
t:16 tools Characteristics References
species diversity and genetic
t:15 potential of microbial samples
provide Used for accurate estimation of viral
t:16 diversity in metagenomic samples
Treephyler 1.
Used for fast taxonomic profiling of
metagenomes
2.
Applicable to both nucleotide and
t:17 protein input data
PhyloPhytias Allows the accurate classification of
most sequence fragments across all
t:18 considered taxonomic ranks
TACOA The taxonomic origin of genomic
segments as short as 800 bp can be
t:19 predicted with great accuracy
Phymm/ Phylogenetic classification of
PhymmBL metagenomic data with interpolated
t:20 Markov models
GAAS 1.
In both textual and graphical modes,
it delivers improved estimates of
community composition and average
genome length for metagenomes.
2.
Produces a more accurate
representation of community
t:21 composition for larger genomes
Meta-pipe Provides preprocessing, assembly,
taxonomic classification, and
functional analysis of marine
t:22 metagenomes
Xipe 1.
Normalizes sequencing efforts by
repeated random subsampling of the
datasets under scrutiny
2.
Pairwise tests between all feasible
pairs of metagenomes can be used to
t:23 compare numerous metagenomes
LefSe 1.
Provides a platform for metagenomic
biomarker discovery
2.
Determines features like organisms,
clades, operational taxonomic units
(OTUs), genes, or functions in an
t:24 effective manner
(continued)
17 Bioremediation of Heavy Metals by Metagenomic Approaches

Table 17.2 (continued)

Computational
tools Characteristics References t:26
STAMP 1.
A comparative metagenomic tool that
operates statistical analysis and
reporting
2.
Allows to conduct statistical studies
in a graphical environment t:25
AMPHORA Phylogenetic analysis of
metagenomic data t:26
ARB Metagenomic data analysis
(sequence alignment, primary and
secondary structure editing,
phylogenetic analysis) and
maintenance t:27
GOLD Genetic data management system that
stores and monitors huge
metagenomic data worldwide t:28
Megx.net Stores and analyzes marine
metagenomic data based on
oligonucleotide signatures t:29
RefSeq Sequences representing genomic
data, transcripts, and proteins from
more than 2400 organisms are
included in this nonreductive
collection t:30
SILVA Contains rRNA gene database from
bacteria, archaea, and eukaryotes and
provides high-throughput
classification of data t:31
SINA Alignment and taxonomical
classification of 16S rRNA gene
sequences from metagenomes t:32
XplorSeq Allows for the easy collection,
management, and phylogenetic
analysis of DNA sequences t:33
PhyloSift Phylogenetic analysis of protein
coding and RNA sequences in
metagenomic datasets t:34
VSEARCH A versatile tool for processing and
preparing metagenomic sequence
data by global sequence alignment of
the query against potential target
sequences t:35
METAREP Helps analyze and compare
annotated metagenomic datasets at
various functional and taxonomic
levels t:36
(continued)
 D. Khan et al.

Table 17.2 (continued)

Computational
t:38 tools Characteristics References
MetaBAT Able to analyze and reconstruct
synthetic and real metagenome
datasets with accuracy and
t:37 computational efficiency
Tax4Fun Based on 16S rRNA information,
determines the functional capabilities
t:38 of microbial communities
CREST Environmental sequence
classification tools (alignment-based)
for creating and using custom
t:39 taxonomies and reference datasets
LotuS Analyzes microbial 16S data and can
generate phylogenetic trees from
t:40 next-generation sequencing data

451 17.7.3 Amplified Ribosomal DNA Restriction Analysis (ARDRA)

452 Amplified ribosomal DNA restriction analysis is a PCR-based DNA fingerprinting

453 technique in which amplification of 16S rDNA-conserved regions is performed.
454 Both culturable and unculturable microbes are subjected to DNA isolation. All
455 samples are amplified by 16S rDNA-specific primers to be digested by restriction
456 endonucleases. Gel electrophoresis is performed to identify the microbial
457 populations having unique patterns. This technique is of interest nowadays as it
458 can reveal the microbial community of a habitat before and after heavy metal
459 contamination. ARDRA is a useful tool for microbial community typing in
460 copper-contaminated areas. Smit et al. (1997) successfully demonstrated the
461 eubacterial community shifting after copper contamination in the soil.

462 17.8 Conclusions

463 In microbial ecology, metagenomic and bioinformatic approaches have already been
464 utilized to analyze all the genetic resources present in heavy metal-contaminated
465 sites comprising a wide range of uncultivable bacteria. Functional analysis focuses
466 on screening libraries that allow identification of novel genes, whereas sequence-
467 based metagenomic analyses rely on comparisons with databases of known genomic
468 sequences. Despite significant breakthroughs in metagenomic approaches, there are
469 a number of constraints that limit microbial community analysis. The two major
470 restrictions are the inefficient expression of some metagenomic genes in the host
471 bacteria used for screening and the lack of effective screening methods for many
472 activities. On the one hand, we must take steps to limit anthropogenic activities,
473 whereas on the other, we must create high-throughput technologies and screening
474 methodologies to investigate the uncultured microbial community.
17 Bioremediation of Heavy Metals by Metagenomic Approaches

Acknowledgments All authors are thankful to UGC Centre for Advanced Study, Department of 475
Botany and DST-FIST, The University of Burdwan, Burdwan, for pursuing all the research 476
facilities. D.K. is thankful to “Swami Vivekananda Merit-Cum-Means Scholarship” (non-NET 477
fellowship). A.K. is thankful to DHESTBT (WB-DBT) (Memo No. 30 (Sanc.)-BT/ST/P/SandT/ 478
2G-48/2017). R.K.R., M.L., and K.M. are thankful to UGC-JRF for providing fellowship. 479

Funding No funding has been received for this study. 480

Compliance with Ethical Standards Conflict of Interest: The authors declare that they have no 481 AU9
conflict of interest. 482
Statement on the welfare of animals. No animal was used in this study. 483

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