Environmental Technology & Innovation 14 (2019) 100369

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Environmental Technology & Innovation

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

Bioremediation of heavy metals by microbial process

∗
Samakshi Verma, Arindam Kuila
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan 304022, India

highlights

• Bioremediation of heavy metals using microbes.

• Bioremediation using genetically engineered microbes.
• Biosorption and bioaugmentation of toxic heavy metals.

article info a b s t r a c t

Article history: Bioremediation is an inventive and optimistic technology which is applicable for the
Received 15 December 2018 retrieval and reduction of heavy metals in water and polluted lands. Microorganism
Received in revised form 3 April 2019 plays an essential part in bioremediation of heavy metals. By using genetic engineering,
Accepted 8 April 2019
genetically modified organisms can be generated which can likely reduce different types
Available online 12 April 2019
of polycyclic hydrocarbons (PAHs). Flavobacterium, Pseudomonas, Bacillus, Arthrobacter,
Keywords: Corynebacterium, Methosinus, Rhodococcus, Mycobacterium, Stereum hirsutum, Nocardia,
Bioremediation Methanogens, Aspergilus niger, Pleurotus ostreatus, Rhizopus arrhizus, Azotobacter, Alcali-
Heavy metals genes, Phormidium valderium, Ganoderma applantus are some microbial species that help
Microorganisms in bioremediation of heavy metals. This review not only discussed about the importance
Biosorption of microbes for bioremediation of heavy metals but also discussed about the challenges
Genetically engineered bacteria and limitations of native and engineered bacteria for bioremediation. Significance of
Polycyclic hydrocarbons
bioremediation with the help of genetically engineered bacteria is in light because of
its eco-friendly nature and minimum health hazards other than the physio-chemical
dependent strategies, which are less eco friendly and dangerous to life.
© 2019 Elsevier B.V. All rights reserved.

Contents

1. Introduction............................................................................................................................................................................................... 2
2. Role of biotechnology in bioremediation .............................................................................................................................................. 2
3. Mechanism of action of microbial remediation.................................................................................................................................... 4
4. Microbes dependent cleaning system.................................................................................................................................................... 4
4.1. Bioremediation by adsorption .................................................................................................................................................... 5
4.2. Bioremediation by physio-bio-chemical mechanism............................................................................................................... 5
4.3. Bioremediation by biosorption................................................................................................................................................... 5
4.4. Bioremediation by bioaugmentation ......................................................................................................................................... 6
5. Microbial bioremediation ........................................................................................................................................................................ 6
6. Genetic engineering of microbes involved for enhanced bioremediation......................................................................................... 7
7. Problems that are faced by genetically engineered microbes in bioremediation ............................................................................ 7
8. Conclusion and future aspects ................................................................................................................................................................ 8

∗ Corresponding author.
E-mail address: arindammcb@gmail.com (A. Kuila).

https://doi.org/10.1016/j.eti.2019.100369
2352-1864/© 2019 Elsevier B.V. All rights reserved.
2 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369

Acknowledgement .................................................................................................................................................................................... 8
References ................................................................................................................................................................................................. 8

1. Introduction

Today human needs and activities are increasing day by day because of the enhanced population all over the
world due to which our environment has been polluted with a huge amount of hazardous contaminants from various
sources (Raghunandan et al., 2014, 2018). There are some consequences of industrialization which are affecting human
health and they are also harmful for every global region i.e. reduction of natural resources, an expansion in carbon
emissions and pollution (Ahuti, 2015). Industrialization has some limiting effects like it affects economic and social
transformation of human societies and it also requires hi-tech renovations (Mgbemene et al., 2016). According to the
industrial revolution, large scale utilization of contaminants results in causing hazardous health problems. Industrial
and technological modifications not only bring their unacceptable partners with them but it also causes environmental
pollution and degradation. And because of these revolutions, there is accidental and intentional release of xenobiotic,
chemicals and toxic gases into the environment.
Environmental pollution is a persistent issue which will affect human health. Even though several strategies have been
utilized to record and tackle this issue but it remains a distressing problem. Environment and humans both are affected
by these hazards globally. A novel technology should be designed in order to protect environment and humans from
the disadvantageous reactions of environmental pollution, and bioremediation is one of those methods (Raghunandan
et al., 2014, 2018). Bioremediation, which has emerged as a comparatively economical process, can prove to be an
effective tool to counter the ill-effects of pollution and render the contaminated soil less polluted and free of toxic or
recalcitrant compounds (Singh and Gupta, 2016). Microbial population utilizes toxic heavy metals as a source of nutrition
in bioremediation. There are two sites at which bioremediation can be performed: on site contamination (in situ) or
on contamination which is brought away from its native place (ex situ). In situ bioremediation contains the therapy of
heavy metals on the site at which they are detected. Subsequently, there is an increased interest regarding microbial
bioremediation of contaminants amongst people as they aspire to remediate polluted environments by discovering
sustainable methods (Raghunandan et al., 2014, 2018; Kumar et al., 2016).
The vast amount of inorganic and organic chemicals that are dumped onto the earth’s surface, either deliberately
through industrial processes or accidentally through spillage results their accumulation beyond the permissible limits.
Magnification of such heavy metals or chemicals contaminated soil remnant with various organic modifications such as
biosolids, MSW and manure composts guides to enhance the physical properties and potency of the soils and also to
expand the bioavailability of nutrients for microbes (Jin et al., 2011). The conventional technique of land filling has many
negative-points; it is not only a costly affair but also requires monitoring (Mgbeahuruike, 2018). A multifaceted point
of view is utilized to detect the bioremediation processes such as — system biology, plant–endophyte relationships and
microbial diversity in disruptive areas (Asha and Sandeep, 2013).
Few metals are beneficial for the human body in minimum amount such as nickel, copper, iron and arsenic but are toxic
(cytotoxic as well as mutagenic and carcinogenic in nature) at high concentration (Valko et al., 2016). High density holding
heavy metals are found to be hazardous at minimum concentrations (Iram et al., 2013). Membrane filtration, ion exchange,
adsorption, coagulation, reduction or oxidation, electrochemical treatment, evaporation, and chemical precipitation are
some of the techniques which may remove heavy metals from contaminated soil and water (Rajasulochana and Preethy,
2016). Heavy metal containing salts get easily dissolved in wastewater hence they cannot purified by physical segregation
strategies (Hussein et al., 2004). Heavy metal contamination in soil leads to prominent changes in microbial population
as there is higher energy requirement under metal stress due to which microbial carbon usage is also reduced (Xu
et al., 2019). Heavy metals are found to be toxic at low concentrations so they can harm living organisms followed by
accumulation inside them as they are capable of entering the food chain. Specific density of heavy metals is more than 5
g/cm3 which causes unfavorable effects on environment and living organisms (Jaishankar et al., 2014).
The functions of lungs, brain, liver, kidney, blood composition and other organs can be inhibited and the energy levels
can be decreased by the heavy metal toxicity. Some metals and their compounds can cause cancer due to their repeatedly
long term exposure (Jaishankar et al., 2014). The background concentrations of some metals that are present naturally
in the ecosystem can be lower than the toxicity level of a few heavy metals. Hence, it is necessary to provide proper
prevention against the excessive contact with heavy metals (Toth et al., 2016).

2. Role of biotechnology in bioremediation

Biotechnology may be defined as the process which employs the exercise of engineering and scientific principles to the
creation of materials by utilizing biological agents to supply goods and services to humans and environment (McHughen,
2016). The chemical compounds which interact with the metal ions to make a stable, water soluble complex are known
as the chelating agents or chelants, sequestering agents or metal sequester. With the help of these chemicals soil washing
is performed, soil particles are purified by the selective transfer of pollutants from soil towards solution (Ferraro et al.,
 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369 3

Table 1
Metal hyper accumulator plant species.
Plant species Metals Amount of leaf (ppm) References
Aegiceras corniculatum Brominated diphenyl ethers (BDE-47) 37,000 Chen et al. (2015)
Spartina maritime As, Cu, Pb, Zn 5000 Mesa et al. (2015)
Eichhorina crassipes (water hyacinth) Heavy metals (Fe, Zn, Cd, Cu, B, and Cr) 40,000 Elias et al. (2014)
Phragmites australis PAHs – Di Gregorio et al. (2014)
Sparganium sp. Polychlorinated biphenyls – Di Gregorio et al. (2013)
Ipomea alpina Cu 12,300 Baker and Walker (1990)
Thlaspi caerulescens Zn : Cd 39,600 : 1800 Baker and Walker (1990);
Reeves and Brooks (1983)
Astragalus racemosus Se 14,900 Beath et al. (1937)

Table 2
Xenobiotics degrading microorganisms (Vidali, 2001).
Microbes Harmful chemicals References
Pseudomonas Anthracene, Benzene, Hydrocarbons, PCBs, Cybulski et al. (2003)
Bacillus spp. Halogenated hydrocarbons, Phenoxyacetate Cybulski et al. (2003)
Rhodococcus spp. Aromatics, Hydrocarbons Dean et al. (2002)
Azotobacter spp. Branched hydrocarbons benzene, Aromatics, cycloparaffins Dean et al. (2002);
Jogdand (1995)
Alcaligenes spp. Linear alkylbenzene sulfonates, PCBs polycyclic aromatics, Halogenated hydrocarbons, Kapley et al. (1999)
Mycobacterium spp. Hydrocarbons, Aromatics, Polycyclic hydrocarbons Park et al. (1998)
Arthrobacter spp. Benzene, long chain alkanes, Hydrocarbons, Phenoxyacetate, Cresol, Jogdand (1995)
Pentachlorophenol, Polycyclic aromatic, Phenol.
Corynebacterium Aromatics Jogdand (1995)
Flavobacterium Naphthalene, Aromatics, biphenyl Jogdand (1995)
Methanogens. Biphenyl, PCBs, Polycyclic aromatics Jogdand (1995)
Nocardia spp. Phenoxyacetate, Halogenated hydrocarbon diazinon Jogdand (1995)
Methosinus sp. PCBs, Formaldehyde Ijah and Antai (1988)

2015). Different classes of compounds are utilized for soil washing which involve chelating agents, surfactants, co-solvents,
cyclodextrins, and organic acids (Morillo and Villaverde, 2017). Natural organic acids having low molecular weight
i.e., formic acid, citric acid, oxalic acid, acetic acid etc. are the original products of root exudates, microbial secretion,
animal and plant remnants that are decomposed in soils (Hayat et al., 2017). EDTA and NTA are two chelating agents
which help in increasing the phytoextraction of soil polluting heavy metals (Naghipour et al., 2016).
There is another technique to degrade pollutants from soil by using plants which is known as phytoremediation. This
technique utilizes the exploitation of higher plants for the purification of soil from heavy metals. These plants act like
metal accumulator (Table 1). They absorb the essential as well as non-essential metal i.e. cadmium. The process of Cd
aggregation has not been explained yet. A system involved in the transportation of other essential micronutrients i.e., Zn2+
may have helped in the possible uptake of Cd in roots. Plants cannot differentiate among these 2 ions because cadmium
is analogue to zinc (Fontanili et al., 2016).
We can make use of biotechnology to engineer a single microorganism with all the needed enzymes or the required
degradative pathway for purpose of remediation (Dangi et al., 2019). Two different groups of genes are responsible for
degradative pathway of PCBs but owing to the inhibitory effects of catecholic intermediates involved in both pathways
these two groups of genes are not found in same organism. The recombination between B.cepacia LB400 bph and
P. Pseudoalcaligenes KF707 genes shows simultaneous degradation of toluene and benzene and an accelerated rate of
breakdown of PCBs (Seeger et al., 2010).
In an enzyme Haloalkane dehalogenase, alanine was used to replace heavy amino acid groups around the catalytic
cavity and a variant was obtained that was much more efficient in causing dechlorination of dichlorohexane (Dua
et al., 2002). It is significant to identify microorganisms that have the potential of biodegrading a specific pollutant and
conventional techniques are time consuming. The DNA probes can go a long way in this respect if one wants to identify
a particular strain from a population. Such probes have been developed for genotypes having PCB degrading ability (Dua
et al., 2002).
The application of genetic engineering is used by biotechnology to enhance the cost and productivity, which are the
main factors in the upcoming universal utilization of microbes to remove toxic substances from the ecosystem. Biological
agents have proved their capacity for remediation, however, their long term and large scale use needs the application of
genetic tools (Tylecote, 2019). Bacillus thuringensis has been used for enhanced bacterial biodegradation of diesel oil (Mnif
et al., 2017). Radioactive resistant bacteria (Deinococcus radiodurans) have updated to assimilate and utilize ionic mercury
and toluene from overly radioactive waste (Manobala et al., 2018; Shukla and Rao, 2017).
Microbial cells produce nanostructures ranging from natural products, i.e. polymers and magnetosomes, to protein
constructs or engineered proteins, i.e. tailored metal particles and (VLP) virus-like proteins (Park et al., 2016). Deinococcus
radiodurans, a radioactive-resistant organism is capable of tolerating radiation beyond the naturally occurring levels, so it
4 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369

Table 3
GEMs that are used in heavy metal bioremediation.
Microbes Modified gene expression Heavy metals References
Sphingomonas desiccabilis and Over expression of arsM gene Arsenic Liu et al. (2011)
Bacillus Idriensis strains
B. subtilis BR151 (pTOO24) Luminescent Cadmium sensors Cadmium Ivask et al. (2011)
Methylococcus capsulatus (Bath) CrR genes for Cr (VI) reductase activity Chromium(VI) Hasin et al. (2010)

Caulobacter crescentus RsaA-6His fusion protein Cadmium(II) Patel et al. (2010)

JS4022/p723–6H
Pseudomonas strain K-62 MerE protein encoded by transposon Tn21 Mercury Kiyono et al. (2009)

Achromobacter sp AO22 Mercury reductase expressing mer gene Mercury Ng et al. (2009)
E. coli strain Metallothionein Arsenic Singh et al. (2008)
E. coli strain AsIII S-adenosylmethionine Arsenic Yuan et al. (2008)
methyltransferase gene
Pseudomonas fluorescens OS8; MerR/CadC/ZntR/Pmer/PcadA/PzntA Cadmium, Lead, Mercury, Zinc, Bondarenko et al. (2008)
Escherichia coliMC1061;
Bacillus subtilisBR151;
Staphylococcus aureus RN4220
E .coli strain PCS gene expression (SpPCS) Cd2+ Kang et al. (2007)
E. coli JM109 Cadmium transport system and Cadmium Deng et al. (2007)
metallothionein
P. putida 06909 Expression of metal binding peptide EC20 Cadmium Wu et al. (2006)
Pseudomonas K-62 Expression of mercury transport system and Mercury Kiyono and Pan-Hou (2006)
Organomurcuriallyase
E. coli SE5000 Nickel transport system and metallothionein Nickel Deng et al. (2005)
E. coli JM109 Hg2+ transporter and metallothionein Mercury Zhao et al. (2005)
E .coli strain Over expression of Serin acetyl transferase Nickel and cobalt Freeman et al. (2005)

Acidithiobacillus ferrooxidans
E. coli
Escherichia coli and Moraxella sp.
Mesorhizobium huakuii B3
E. coli strain
P. fluorescens 4F39
Deinococcus radiodurans
Mercury ion transporter gene expression Mercury Sasaki et al. (2005)
Metalloregulatory protein ArsR (over Arsenic Kostal et al. (2004)
expressing ELP153AR)
Expressing EC20 Mercury and Cadmium Bae et al. (2003)
Phytochelatin synthase (PCS) Cd2+ Sriprang et al. (2003)
gene expression
Organomurcuriallyase Mercury Murtaza et al. (2002)
gene expression
Nickel transport system Nickel Lopez et al. (2002)
Hg (II) resistance gene (merA) Mercury (Radioactive waste Brim et al. (2000)
from nuclear weapons)

is significant in clean-up initiatives of radioactive waste which was funded by US Department of Energy (DOE) (Manobala
et al., 2018; Shukla and Rao, 2017).

3. Mechanism of action of microbial remediation

Microbes are ubiquitous in nature which is superior in heavy metal polluted sites hence they play a crucial role
in converting toxic heavy metals into non-toxic forms. Microbes help in mineralizing some organic pollutants into
end-products i.e. CO2 and H2 O or to metabolic intermediates which are being utilized as primary substrates for cell
growth within the process of bioremediation. Two-way defense system is maintained by microorganisms- (i) producing
degradative enzymes for the target contaminants (ii) opposition towards relevant heavy metals. There are various methods
through which microorganisms are significant in restoring the environment such as — binding, immobilization, oxidation,
transformation and volatizing of heavy metals. The process of bioremediation can be more efficient in specific areas by
the designer microbial approach, by perceiving the mechanism which regulates the activity and growth of microbes in
the polluted sites, their metabolic potential and their reaction towards environmental modifications (Alvarez et al., 2017).
Bioaccumulation, bioleaching, biosorption, biotransformation and biomineralisation and metal-microbe interactions are
some mechanisms of bioremediation. Chemicals are needed for the development and growth of microorganisms which
are helpful in reducing heavy metals from soil (Sikkema et al., 1995). Microorganisms are not only helpful in dissolving
metals but also in reduction and oxidation of transition metals. Cell membranes can be disrupted because of contamination
caused by some organic solvents, but sometimes defense mechanisms are developed by the cells such as the formation
of hydrophobic or solvent efflux pumps which act as an outer cell-membrane-protective material (Dixit et al., 2015). It
has been reported that energy-dependent and plasmid-encoded metal efflux systems i.e. chemiosmotic ion/proton pumps
and ATPases are observed for the resistance of As, Cd and Cr in many bacteria (Ahemad, 2014).

4. Microbes dependent cleaning system

Modifications in the biologically encoded oxidation state can help in remediating environment niches of sediments, soil
and water altered with heavy metals. Bioremediation is the microbe-mediated clearance or immobilization mechanism
 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369 5

of contaminants such as — agrochemicals, hydrocarbons and other organic toxicants but microorganisms are unable to
convert inorganic toxic compounds like heavy metals into harmless simpler forms and hence, they should be utilized
according to their specificity towards contaminants. So, the mechanism of bioremediation for heavy metals based upon
the actively metabolizing microbes. There are various microbes that are known to utilize varying amounts of heavy metals
for their growth and development as an essential micronutrients i.e., Fe3+ is essentially utilized by all bacteria while
Fe2+ is significant for anaerobic bacteria (Ahemad, 2014). By utilizing microorganisms, rehabilitation and detoxification
of polluted soil has emerged as the most effective, easy and safest technology. Human activities like fuel and industrial
processes, mining of ores, oil and gas extraction, organic solvents, pesticides, pigments and plastics have released toxic
products in the environment which can be removed or detoxified by native soil microbes (Garbisu and Alkorta, 2001).

4.1. Bioremediation by adsorption

The extracellular polymeric substances are most important among all other reactive compounds that are associated
with bacterial cell walls as they show significant effects on metal adsorption and acid–base properties (Guine et al., 2006).
Extracellular polymeric substances (EPS) show a great metal binding ability towards complex heavy metals via different
methods, i.e. micro precipitation of metals and proton exchange (Comte et al., 2008; Fang et al., 2010). The protons
were characterized and quantified and the metals were adsorbed on bacterial cells and EPS free cells so that they can
detect the relative significance of EPS molecules in metal degradation according to the recent studies (Fang et al., 2011).
The incomplete understanding of genetics and genome level characteristics of the organisms which was utilized in the
metabolic pathway and their kinetics and in metal adsorption is the basic reason for hampering bioremediation research
and practice in the current scenario (Carter et al., 2006; Gan et al., 2009; Haritash and Kaushik, 2009; Kinya and Kimberly,
1996; Onwubuya et al., 2009).

4.2. Bioremediation by physio-bio-chemical mechanism

The higher affinity of a biosorbent towards metal ions (sorbate) to maintain equilibrium between the two components
is known as biosorption (Das et al., 2008). The degradation of Cd (II) and Zn (II) is performed by ion exchange method
through Saccharomyces cerevisiae (biosorbent) (Chen and Wang, 2007; Talos et al., 2009). Heavy metals which are released
through textile wastewater are degraded by a promising sorbent i.e., Cunninghamella elegans (Tigini et al., 2010). Energy
is required for the cell metabolic cycle under heavy metal degradation process.
Immobilization is a technique which is utilized for reducing the mobilization of heavy metals from polluted sites by
modifying the chemical or physical state of the toxic metals. If any chemical agent is mixed at heavy metal polluted site or
precipitation of hydroxides is performed at the contaminated site then it comes under the solidification treatment process
(Karna et al., 2017). The oxidation state or organic complex of heavy metals can be transformed into water-soluble, less
toxic and precipitated form as they can never be destroyed completely (Garbisu and Alkorta, 2001).
Transformation of the toxic metal state and making it unavailable which is known as detoxification and active efflux
pumping of the toxic metal from cells are two basic methods that are usually preferred for developing resistance in
bacteria (Ma et al., 2016).
In aerobic degradation method, electron acceptor is oxygen whereas organic contaminants get oxidized with the
reduction of electron acceptors by utilizing microbes in anaerobic mode of degradation. By oxidizing the organic
compound with Mn (IV) or Fe (III) as an electron acceptor, microbes derive energy for their growth (Lovley and Phillips,
1988). The higher availability of Fe (III) for microbial reduction is significant for stimulating anaerobic degradation of
organic contamination (Lovley et al., 1996; Spormann and Widdel, 2000). When metals are utilized as terminal electron
acceptors then it is referred as dissimilatory metal reduction (Lovely, 2002). Reductive dechlorination is responsible for
the biodegradation of chlorines or chlorinated solvents (which act as electron acceptors in respiration) from contaminants.
Geobaccter species is utilized for biodegradation method as it is helpful in reduces the Uranium soluble state (U6+) into
its insoluble state (U4+). Thus, the microbes are also significant in reducing state of metals and in modifying the metal
solubility (Lovley et al., 1991).
Synthesis of binding protein and peptides, complex formation, compartmentalization and exclusion are various defense
systems that are helpful in reducing the stress generated by toxic metals (Gomez-Jimenez et al., 2011). The expression
of phytochelatins and metal binding protein and peptides (metallothionein) is required for studying the heavy metal
accumulation by microbes (Cobbett and Goldsbrough, 2002). Hormones and redox signaling process are mediated by
metallothionein transcription factors in context of toxic metal (Au, Cd, Ag, Co, Cu, Bi, Hg, Zn and Ni) exposure (Abdelmigid,
2016). The production of metal-binding protein and expression of smtA gene have been reported by Synechococcus sp.
(cynobacterial strains) (Huckle et al., 1993).

4.3. Bioremediation by biosorption

Microbes are helpful in binding with metals from aqueous solution known as biosorbent under the process of
biosorption and it becomes mandatory that the physical nature of biosorbent, kinetics of sorption, regeneration, maximum
sorption capacity, and recovery of bound metals should be analyzed to detect the stability of microbes as a biosorbent.
6 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369

There are certain criteria which should be followed for biosorbent selection: (a) Less expensive and reusable biosorbent
should be utilized, (b) There should be quite and efficient separation from the solution, (c) The kinetics of metals should
be fast (Bae et al., 2001, 2002, 2003). For biosorption of metals from solutions, there are mainly three broad categories of
biosorbent that are used: (1) exo polysaccharides, (2) dead biomass and preparations, (3) living cultures. On comparison,
dead cells absorb metals more than the living cells. Filamentous fungi have high metal uptake capacity like Aspergillus,
Rhizopus, Mucor, Penicillium, Actinomycetes, and Streptomycetes.

4.4. Bioremediation by bioaugmentation

Additionally, chemicals which were made to trigger the natural degradation can also be added, such as vegetable oil,
molasses, oxygen or permanganate. Presenting specialized microorganisms at a site of contamination to basically ‘‘eat’’ is
called bioaugmentation. The indigenous or exogenous insertion of microbes to the polluted sites is frequently involved
in this process. It is an affordable, effective and fast remediation process, and it is finding favor among remediation
experts and site managers (EPA, 2006). To upgrade and encourage microbial growth and development, bioaugmentation
(microbes) or biostimulation (amendments) i.e. nutrients, air, electron acceptors/donors and organic substrates, other
compounds that can control and influence investigation in their deficiency, are introduced. To investigate groundwater
or surface water, other solids, and soil biostimulation and bioaugmentation are two basic applications of bioremediation
that could be utilized (EPA, 2006).

5. Microbial bioremediation

The type of micro-flora is a significant factor affecting bioremediation which depends on making use of gene and
metabolic diversity of microorganisms to render the pollutants less toxic (Juwarkar et al., 2010). Bioremediation utilizes
either indigenous strains or those derived from a different site (Santos et al., 2011). The use of indigenous microbial strain
eliminates the need of monitoring (Asha and Sandeep, 2013). Energy and a carbon source are two basic requirements that
are utilized for bioremediation (Vidali, 2001). Here is a list of heavy metals utilizing microorganisms followed by Vidali
(2001) is given below-

• Bacillus and Pseudomonas aeruginosa utilize Zinc, Copper (Gunasekaran et al., 2003; Philip et al., 2000).
• Zooglea spp. and Citrobacter spp. utilize Copper, Nickel, Urea, Cadmium, Cobalt, Nickel (Sar and D’Souza, 2001).
• Citrobacter spp. and Chlorella vulgaris utilize Cadmium, Lead, Uranium, Copper, Gold, Lead, Nickel, Mercury, Uranium
(Gunasekaran et al., 2003).
• Aspergilus Niger utilizes Cadmium, Thorium, Uranium, Zinc (Gunasekaran et al., 2003).
• Pleurotus ostreatus utilize Cadmium, Copper, and Zinc (Gunasekaran et al., 2003).
• Rhizopus arrhizus utilize Cadmium, Calcium, Mercury, Lead, Phosphorous, Silver (Favero et al., 1991; Gunasekaran
et al., 2003).
• Stereum hirsutum utilize Cadmium, Copper Cobalt, Nickel (Gabriel et al., 1994, 1996).
• Phormidium valderium utilize Cadmium; Lead (Gabriel et al., 1994, 1996).
• Ganoderma applantus utilize Copper, Lead, Mercury (Gabriel et al., 1994, 1996).

Autochthonous microbes are those microbes that naturally occupy the soil or water ecosystems encountering separation,
or by some other microbes which are originated from various ecosystems can be utilized to perform bioremediation.
Plenty of microbes can be utilized to reduce xenobiotic compounds (Table 2), metals from ecosystems such as algae,
bacteria, yeast and fungi (Vieira and Volesky, 2000; White et al., 1997). The removal of pollutants and cleaning of waste
products from water, soil, air, and raw materials from industries can be performed by bioremediation. The less expensive
examination of contaminated sites has become a difficult task (Zeyaullah et al., 2009).
Microorganisms which are going to be used may vary; they have to be selected very carefully because these organisms
can survive within a limited range of heavy metals based on their toxicity (Dubey, 2004; Prescott et al., 2002). Various
groups of microorganisms will be required for effective mediation because several kinds of contaminants are to be
captured within a contaminated site (Watanabe et al., 2001). It has been reported that a strain of Pseudomonas putida
was the first patent that was registered in 1981, as a biological remediation agent which was able to degrade petroleum
(Glazer and Nikaido, 2007; Prescott et al., 2002).
The active members of microbial consortium are identified as the microorganisms like Xanthofacter, Penicillium, Bacillus,
Pseudomonas, Flavobacterium, Mycobacterium, and Nitrosomonas which carry out biodegradation in different environments.
These microbes can be subcategorized into the following types:
 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369 7

6. Genetic engineering of microbes involved for enhanced bioremediation

Microbial strains have the potential to remove a variety of several kinds of hydrocarbons through genetic manipulation.
A multi plasmid containing Pseudomonas strain is successfully produced that can oxidize polyaromatic hydrocarbons,
terpenic aromatic, and aliphatic hydrocarbons. An alteration in the genetic material of microorganisms can be done
by genetic engineering techniques known as recombinant DNA technology, which involve gene exchange between
microbes. Such microorganisms are known as genetically modified microorganisms (GMM) or genetically engineered
microorganisms (GEM). Genetically engineered microorganisms (GEMs) are known to be effective in soil, activated
sludge and groundwater bioremediation. Four principal approaches are being considered in the development of GEM
for bioremediation: (1) Bioaffinity bioreporter sensor applications in chemical sensing, end point analysis and toxicity
reduction; (2) Bioprocess development, monitoring and control; (3) Enhancement of affinity and enzyme specificity; (4)
Construction and pathway regulation.

• Pseudomonas aeruginosa (NRRLB-5472) and Pseudomonas putida (NRRLB-5473) are two genetically modified strains
which were firstly patent in 1981 by USA. These two strains were constructed in early 70 s (Chakrabarty, 1986) and
include genes for camphor, naphthalene and salicylate degradation which are highly toxic in nature. These strains
are efficient for toxin degradation.
• The PCB catabolic genes which were located on the chromosome of R.eutropha A5, A.denitrificans JB1, and Achro-
mobacter sp. LBS1C1 was transferred to the heavy metal resistant strain R.eutropha CH34 via natural conjugation for
the removal of polychlorinated biphenyls (Valls et al., 2000).
• For heavy metals, Arabidopsis thaliana gene was introduced into Mesorhizobium huakuii subsp. Rengei strain B3 for
phytochelatin synthase (PCS; PCSAt), that further generated the symbiosis between M.huakuii subsp. Rengei strain
B3 and Astragalus sinicus (Sriprang et al., 2003).
• In the presence of bacteroid specific promoter (the nif H gene), genes were expressed to accumulate Cd2 + and release
phytochelatins.
• Heavy metal degradation using genetically engineered microorganisms has gained a great interest as in chromium
degradation from industrial wastewater Alcaligenes eutrophus AE104 (pEBZ141) was used and simultaneous ex-
pression of the Hg transport system and metallothionein for Hg2+ degradation from heavy metal and wastewater
Rhodopseudomonas palustris, the recombinant photosynthetic bacterium was constructed.

Molecular techniques like genetic engineering or plasmid breeding can efficiently generate microorganisms having
favorable catalytic potential, which are capable in removing any environmental pollutant. The evident potential of genetic
engineering in bioremediation was observed by several microbiologists and molecular biologists (Table 3).

7. Problems that are faced by genetically engineered microbes in bioremediation

◦ Although the genetic engineering has produced various strains and bacterial species to degrade pollutants, there
are lots of obstacles related with this too. One major problem related in this respect is that the growing strains and
microbial species are not the ones to perform a bulk of bioremediation process.
8 S. Verma and A. Kuila / Environmental Technology & Innovation 14 (2019) 100369

◦ The use of stable isotope probing (SIP) and relative method in microbiology ecology have revealed that Rhodococcus
and Pseudomonas and typical fast growers widely favored as hosts of biodegradation are less significant under
certain natural condition (Joutey et al., 2013). The major problem encountered in this successful status of biore-
mediation is to captive the field condition for modified microorganisms (Joutey et al., 2013). ‘‘Suicidal genetically
engineered microorganisms’’ (S-GEMS) have been utilized to reduce prominent risks and obtain safer and efficient
decontamination of contaminated sites (Pandey et al., 2005).
◦ P.fluorescens HK44 is used in field release for bioremediation and it has been helpfully supervised on optimum wide-
ranging and limited ground conditions (Ripp et al., 2000). Hence for pollution remediation, genetically engineered
bacteria do not lack the threats related with their release into the ecosystem.
◦ Major problem captured in bioremediation process relates to hostile field factors for the engineered microorgan-
isms. Some well characterized bacteria e.g., E. coli (Bondarenko et al., 2008), B. subtilis (Ivask et al., 2011) and P.
putida (Wu et al., 2006) are utilized in field conditions emphasizing on ways for which the molecular significance
are mainly confined. Modification of engineered bacterial strains is required to satisfy the new challenge is the
significant feature of open biotechnological applications.
◦ It is the main concern to create genetically engineered bacteria with an appropriate level of environmental certainty
for field release in bioremediation. Within a harsh environmental condition, the efforts that are made to analyze
the performance of engineered bacteria are endurance and capability of horizontal gene transfer which can affect
the native microflora of environment. Bacteria are specifically designed for in vitro bioremediation to avoid the
field requirements and critical conditions. It is not evident whether there is any adverse effect on the natural
microorganisms because of the deliberate release of genetically engineered bacteria for bioremediation (Sayler and
Ripp, 2000). Therefore, the survival of genetically engineered bacteria in harsh environment is still a reason of
immense worry.

8. Conclusion and future aspects

Increased development in technologies and industrial manufacturing has caused some harmful side effects i.e. soil
health degradation and soil pollution. Utilization of microorganisms emerges as time saver for bioremediation due to
some intricacy involved in remediation of soil by conventional methods. However, there is a limitation of bioremediation
process because several microorganisms are not capable of converting toxic heavy metals into their non-toxic forms and
hence results in causing inhibitory effects on microbial activity. So, in order to enhance the biodegradation potential of
microorganism, genetic engineering is being utilized. Biological methods have higher specificity other than the chemical
and physical methods, convenience towards in situ methodologies (e.g. toxic chemical addition or ignoring high energy),
and have the potential for development by genetically engineered microbes (GEMs).
Undoubtedly, bioremediation is moving towards the process of giving a way to greener pastures. Due to this reason of
bioremediation, it is well organized and inexpensive way to handle contaminated ground water and soil. Environmental
biotechnology has an aim of approaching and resolving these issues to allow the utilization of microbes in bioremediation
and because of which it becomes mandatory to favor the reactions of the indigenous microbes in contaminated biotopes
and to increase the degradative potential of microbes by using the process of bioaugmentation or biostimulation.
Bioremediation of pollutants becomes successful which makes a change in our capacity to diminish wastes, strike
industrial pollution, and enjoy a more endurable future.
Subsequently, there should be focus on improving in situ bioremediation technologies with the help of genetically
modified microbes or GEMs and also the adaptability and applicability of these GEMs in each and every unfavorable
condition and in various heavy metal contaminated sites. Future studies should also consider the various doubts among
public to undertake GEM for bioremediation, and prove them harmless to the environment.

Acknowledgement

Authors are thankful to Prof. Aditya Shastri, Vice Chancellor of Banasthali Vidyapith for provide Bioinformatics facility
to complete the article.

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