Accepted Manuscript

Bioremediation of heavy metals by microbial process

Samakshi Verma, Arindam Kuila

PII: S2352-1864(18)30591-1
DOI: https://doi.org/10.1016/j.eti.2019.100369
Article number: 100369
Reference: ETI 100369

To appear in: Environmental Technology & Innovation

Received date : 15 December 2018

Revised date : 3 April 2019
Accepted date : 8 April 2019

Please cite this article as: S. Verma and A. Kuila, Bioremediation of heavy metals by microbial
process. Environmental Technology & Innovation (2019), https://doi.org/10.1016/j.eti.2019.100369

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 1 Bioremediation of heavy metals by microbial process

2 Samakshi Verma, Arindam Kuila*

3 Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan- 304022, India

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20 Corresponding Author

21 Tel: +91-1438-228302, Fax: +91-1438-228365

22 Email: arindammcb@gmail.com

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24 Abstract

25 Bioremediation is an inventive and optimistic technology which is applicable for the retrieval

26 and reduction of heavy metals in water and polluted lands. Microorganism plays an essential part

27 in bioremediation of heavy metals. By using genetic engineering, genetically modified organisms

28 can be generated which can likely reduce different types of poly cyclic hydrocarbons (PAHs).

29 Flavobacterium, Pseudomonas, Bacillus, Arthrobacter, Corynebacterium, Methosinus,

30 Rhodococcus, Mycobacterium, Stereum hirsutum, Nocardia, Methanogens, Aspergilus niger,

31 Pleurotus ostreatus, Rhizopus arrhizus, Azotobacter, Alcaligenes, Phormidium valderium,

32 Ganoderma applantus are some microbial species that help in bioremediation of heavy metals.

33 This review not only discussed about the importance of microbes for bioremediation of heavy

34 metals but also discussed about the challenges and limitations of native and engineered bacteria

35 for bioremediation. Significance of bioremediation with the help of genetically engineered

36 bacteria is in light because of its eco-friendly nature and minimum health hazards other than the

37 physio-chemical dependent strategies, which are less eco friendly and dangerous to life.

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39 Keywords: Bioremediation; Heavy metals; Microorganisms; Biosorption; Genetically

40 engineered bacteria; Poly cyclic hydrocarbons.

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47 1. Introduction

48 Today human needs and activities are increasing day by day because of the enhanced population

49 all over the world due to which our environment has been polluted with a huge amount of

50 hazardous contaminants from various sources (Raghunandan et al., 2014, 2018). There are some

51 consequences of industrialization which are affecting human health and they are also harmful for

52 every global region i.e. reduction of natural resources, an expansion in carbon emissions and

53 pollution (Ahuti, 2015). Industrialization has some limiting effects like it affects economic and

54 social transformation of human societies and it also requires hi-tech renovations (Mgbemene et

55 al., 2016). According to the industrial revolution, large scale utilization of contaminants results

56 in causing hazardous health problems. Industrial and technological modifications not only bring

57 their unacceptable partners with them but it also causes environmental pollution and degradation.

58 And because of these revolutions, there is accidental and intentional release of xenobiotic,

59 chemicals and toxic gases into the environment.

60 Environmental pollution is a persistent issue which will affect human health. Even though

61 several strategies have been utilized to record and tackle this issue but it remains a distressing

62 problem. Environment and humans both are affected by these hazards globally. A novel

63 technology should be designed in order to protect environment and humans from the

64 disadvantageous reactions of environmental pollution, and bioremediation is one of those

65 methods (Raghunandan et al., 2014, 2018). Bioremediation, which has emerged as a

66 comparatively economical process, can prove to be an effective tool to counter the ill-effects of

67 pollution and render the contaminated soil less polluted and free of toxic or recalcitrant

68 compounds (Singh and Gupta, 2016). Microbial population utilizes toxic heavy metals as a

69 source of nutrition in bioremediation. There are two sites at which bioremediation can be

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70 performed: on site contamination (in situ) or on contamination which is brought away from its

71 native place (ex situ). In situ bioremediation contains the therapy of heavy metals on the site at

72 which they are detected. Subsequently, there is an increased interest regarding microbial

73 bioremediation of contaminants amongst people as they aspire to remediate polluted

74 environments by discovering sustainable methods (Raghunandan et al., 2014, 2018; Kumar et al.,

75 2016).

76 The vast amount of inorganic and organic chemicals that are dumped onto the earth’s surface,

77 either deliberately through industrial processes or accidentally through spillage results their

78 accumulation beyond the permissible limits. Magnification of such heavy metals or chemicals

79 contaminated soil remnant with various organic modifications such as biosolids, MSW and

80 manure composts guides to enhance the physical properties and potency of the soils and also to

81 expand the bioavailability of nutrients for microbes (Jin et al., 2011). The conventional technique

82 of land filling has many negative-points; it is not only a costly affair but also requires monitoring

83 (Mgbeahuruike et al., 2018). A multifaceted point of view is utilized to detect the bioremediation

84 processes such as- system biology, plant-endophyte relationships and microbial diversity in

85 disruptive areas (Asha and Sandeep, 2013).

86 Few metals are beneficial for the human body in minimum amount such as nickel, copper, iron

87 and arsenic but are toxic (cytotoxic as well as mutagenic and carcinogenic in nature) at high

88 concentration (Valko et al., 2016). High density holding heavy metals are found to be hazardous

89 at minimum concentrations (Iram et al., 2013). Membrane filtration, ion exchange, adsorption,

90 coagulation, reduction or oxidation, electrochemical treatment, evaporation, and chemical

91 precipitation are some of the techniques which may remove heavy metals from contaminated soil

92 and water (Rajasulochana and Preethy, 2016). Heavy metal containing salts get easily dissolved

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 93 in wastewater hence they can’t purified by physical segregation strategies (Hussein et al., 2004).

94 Heavy metal contamination in soil leads to prominent changes in microbial population as there is

95 higher energy requirement under metal stress due to which microbial carbon usage is also

96 reduced (Xu et al., 2019). Heavy metals are found to be toxic at low concentrations so they can

97 harm living organisms followed by accumulation inside them as they are capable of entering the

98 food chain. Specific density of heavy metals is more than 5 g/cm3which causes unfavorable

99 effects on environment and living organisms (Jaishankar et al., 2014).

100 The functions of lungs, brain, liver, kidney, blood composition and other organs can be inhibited

101 and the energy levels can be decreased by the heavy metal toxicity. Some metals and their

102 compounds can cause cancer due to their repeatedly long term exposure (Jaishankar et al., 2014).

103 The background concentrations of some metals that are present naturally in the ecosystem can be

104 lower than the toxicity level of a few heavy metals. Hence, it is necessary to provide proper

105 prevention against the excessive contact with heavy metals (Toth et al., 2016).

106 2. Role of biotechnology in bioremediation

107 Biotechnology may be defined as the process which employs the exercise of engineering and

108 scientific principles to the creation of materials by utilizing biological agents to supply goods and

109 services to humans and environment (McHughen, 2016). The chemical compounds which

110 interact with the metal ions to make a stable, water soluble complex are known as the chelating

111 agents or chelants, sequestering agents or metal sequester. With the help of these chemicals soil

112 washing is performed, soil particles are purified by the selective transfer of pollutants from soil

113 towards solution (Ferraro et al., 2015). Different classes of compounds are utilized for soil

114 washing which involve chelating agents, surfactants, co-solvents, cyclodextrins, and organic

115 acids (Morillo and Villaverde, 2017). Natural organic acids having low molecular weight i.e.,

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116 formic acid, citric acid, oxalic acid, acetic acid etc. are the original products of root exudates,

117 microbial secretion, animal and plant remnants that are decomposed in soils (Hayat et al., 2017).

118 EDTA and NTA are two chelating agents which help in increasing the phytoextraction of soil

119 polluting heavy metals (Naghipour et al., 2016).

120 There is another technique to degrade pollutants from soil by using plants which is known as

121 phytoremediation. This technique utilizes the exploitation of higher plants for the purification of

122 soil from heavy metals. These plants act like metal accumulator (Table 1). They absorb the

123 essential as well as non-essential metal i.e. cadmium. The process of Cd aggregation has not

124 been explained yet. A system involved in the transportation of other essential micronutrients i.e.,

125 Zn2+ may have helped in the possible uptake of Cd in roots. Plants cannot differentiate among

126 these 2 ions because cadmium is analogue to zinc (Fontanili et al., 2016).

127 We can make use of biotechnology to engineer a single microorganism with all the needed

128 enzymes or the required degradative pathway for purpose of remediation (Dangi et al., 2019).

129 Two different groups of genes are responsible for degradative pathway of PCBs but owing to the

130 inhibitory effects of catecholic intermediates involved in both pathways these two groups of

131 genes are not found in same organism. The recombination between B.cepacia LB400 bph and P.

132 Pseudoalcaligenes KF707 genes shows simultaneous degradation of toluene and benzene and an

133 accelerated rate of breakdown of PCBs (Seeger et al., 2010).

134 In an enzyme Haloalkane dehalogenase, alanine was used to replace heavy amino acid groups

135 around the catalytic cavity and a variant was obtained that was much more efficient in causing

136 dechlorination of dichlorohexane (Dua et al., 2002). It is significant to identify microorganisms

137 that have the potential of biodegrading a specific pollutant and conventional techniques are time

138 consuming. The DNA probes can go a long way in this respect if one wants to identify a

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139 particular strain from a population. Such probes have been developed for genotypes having PCB

140 degrading ability (Dua et al., 2002).

141 The application of genetic engineering is used by biotechnology to enhance the cost and

142 productivity, which are the main factors in the upcoming universal utilization of microbes to

143 remove toxic substances from the ecosystem. Biological agents have proved their capacity for

144 remediation, however, their long term and large scale use needs the application of genetic tools

145 (Tylecote, 2019). Bacillus thuringensis has been used for enhanced bacterial biodegradation of

146 diesel oil (Mnif et al., 2017). Radioactive resistant bacteria (Deinococcus radiodurans) have

147 updated to assimilate and utilize ionic mercury and toluene from overly radioactive waste

148 (Manobala et al., 2018; Shukla et al., 2017).

149 Microbial cells produce nanostructures ranging from natural products, i.e. polymers and

150 magnetosomes, to protein constructs or engineered proteins, i.e. tailored metal particles and

151 (VLP) virus-like proteins (Park et al., 2016). Deinococcus radiodurans, a radioactive-resistant

152 organism is capable of tolerating radiation beyond the naturally occurring levels, so it is

153 significant in clean-up initiatives of radioactive waste which was funded by US Department of

154 Energy (DOE) (Manobala et al., 2018; Shukla et al., 2017).

155 3. Mechanism of action of microbial remediation

156 Microbes are ubiquitous in nature which is superior in heavy metal polluted sites hence they

157 play a crucial role in converting toxic heavy metals into non-toxic forms. Microbes help in

158 mineralizing some organic pollutants into end-products i.e. CO2 and H2O or to metabolic

159 intermediates which are being utilized as primary substrates for cell growth within the process

160 of bioremediation. Two-way defense system is maintained by microorganisms- i) producing

161 degradative enzymes for the target contaminants ii) opposition towards relevant heavy metals.

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162 There are various methods through which microorganisms are significant in restoring the

163 environment such as- binding, immobilization, oxidation, transformation and volatizing of

164 heavy metals. The process of bioremediation can be more efficient in specific areas by the

165 designer microbial approach, by perceiving the mechanism which regulates the activity and

166 growth of microbes in the polluted sites, their metabolic potential and their reaction towards

167 environmental modifications (Alvarez et al., 2017).

168 Bioaccumulation, bioleaching, biosorption, biotransformation and biomineralisation and

169 metal-microbe interactions are some mechanisms of bioremediation. Chemicals are needed for

170 the development and growth of microorganisms which are helpful in reducing heavy metals from

171 soil (Sikkema et al., 1995). Microorganisms are not only helpful in dissolving metals but also in

172 reduction and oxidation of transition metals. Cell membranes can be disrupted because of

173 contamination caused by some organic solvents, but sometimes defense mechanisms are

174 developed by the cells such as the formation of hydrophobic or solvent efflux pumps which act

175 as an outer cell-membrane-protective material (Dixit et al., 2015). It has been reported that

176 energy-dependent and plasmid-encoded metal efflux systems i.e. chemiosmotic ion/proton

177 pumps and ATPases are observed for the resistance of As, Cd and Cr in many bacteria (Ahemad,

178 2014).

179 4. Microbes dependent cleaning system

180 Modifications in the biologically encoded oxidation state can help in remediatin g environment

181 niches of sediments, soil and water altered with heavy metals. Bioremediation is the microbe-

182 mediated clearance or immobilization mechanism of contaminants such as- agrochemicals,

183 hydrocarbons and other organic toxicants but microorganisms are unable to convert inorganic

184 toxic compounds like heavy metals into harmless simpler forms and hence, they should be

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185 utilized according to their specificity towards contaminants. So, the mechanism of

186 bioremediation for heavy metals based upon the actively metabolizing microbes. There are

187 various microbes that are known to utilize varying amounts of heavy metals for their growth

188 and development as an essential micronutrients i.e., Fe3+ is essentially utilized by all bacteria

189 while Fe2+ is significant for anaerobic bacteria (Ahemad, 2014). By utilizing microorganisms,

190 rehabilitation and detoxification of polluted soil has emerged as the most effective, easy and

191 safest technology. Human activities like fuel and industrial processes, mining of ores, oil and

192 gas extraction, organic solvents, pesticides, pigments and plastics have released toxic products

193 in the environment which can be removed or detoxified by native soil microbes (Garbisu and

194 Alkorta, 2001).

195 4.1 Bioremediation by Adsorption

196 The extracellular polymeric substances are most important among all other reactive compounds

197 that are associated with bacterial cell walls as they show significant effects on metal adsorption

198 and acid-base properties (Guine et al., 2006). Extracellular polymeric substances (EPS) show a

199 great metal binding ability towards complex heavy metals via different methods, i.e. micro

200 precipitation of metals and proton exchange (Comte et al., 2008; Fang et al., 2010). The protons

201 were characterized and quantified and the metals were adsorbed on bacterial cells and EPS free

202 cells so that they can detect the relative significance of EPS molecules in metal degradation

203 according to the recent studies (Fang et al., 2011). The incomplete understanding of genetics and

204 genome level characteristics of the organisms which was utilized in the metabolic pathway and

205 their kinetics and in metal adsorption is the basic reason for hampering bioremediation research

206 and practice in the current scenario (Carter et al., 2006; Gan et al., 2009; Haritash and Kaushik,

207 2009; Kinya and Kimberly, 1996; Onwubuya et al., 2009).

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208 4.2 Bioremediation by Physio-bio-chemical mechanism

209 The higher affinity of a biosorbent towards metal ions (sorbate) to maintain equilibrium between

210 the two components is known as biosorption (Das et al., 2008). The degradation of Cd (II) and

211 Zn (II) is performed by ion exchange method through Saccharomyces cerevisiae (biosorbent)

212 (Chen and Wang, 2007; Talos et al., 2009). Heavy metals which are released through textile

213 wastewater are degraded by a promising sorbent i.e., Cunninghamella elegans (Tigini et al.,

214 2010). Energy is required for the cell metabolic cycle under heavy metal degradation process.

215 Immobilization is a technique which is utilized for reducing the mobilization of heavy metals

216 from polluted sites by modifying the chemical or physical state of the toxic metals. If any

217 chemical agent is mixed at heavy metal polluted site or precipitation of hydroxides is performed

218 at the contaminated site then it comes under the solidification treatment process (Karna et al.,

219 2017). The oxidation state or organic complex of heavy metals can be transformed into water-

220 soluble, less toxic and precipitated form as they can never be destroyed completely (Garbisu and

221 Alkorta, 2001).

222 Transformation of the toxic metal state and making it unavailable which is known as

223 detoxification and active efflux pumping of the toxic metal from cells are two basic methods that

224 are usually preferred for developing resistance in bacteria (Ma et al., 2016).

225 In aerobic degradation method, electron acceptor is oxygen whereas organic contaminants get

226 oxidized with the reduction of electron acceptors by utilizing microbes in anaerobic mode of

227 degradation. By oxidizing the organic compound with Mn (IV) or Fe (III) as an electron

228 acceptor, microbes derive energy for their growth (Lovley and Phillips, 1988). The higher

229 availability of Fe (III) for microbial reduction is significant for stimulating anaerobic degradation

230 of organic contamination (Lovely et al., 1996; Spormann and Widdel, 2000). When metals are

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231 utilized as terminal electron acceptors then it is referred as dissimilatory metal reduction (Lovely,

232 2002). Reductive dechlorination is responsible for the biodegradation of chlorines or chlorinated

233 solvents (which act as electron acceptors in respiration) from contaminants. Geobaccter species

234 is utilized for biodegradation method as it is helpful in reduces the Uranium soluble state (U6+)

235 into its insoluble state (U4+). Thus, the microbes are also significant in reducing state of metals

236 and in modifying the metal solubility (Lovely et al., 1991).

237 Synthesis of binding protein and peptides, complex formation, compartmentalization and

238 exclusion are various defense systems that are helpful in reducing the stress generated by toxic

239 metals (Gomez Jimenez et al., 2011). The expression of phytochelatins and metal binding protein

240 and peptides (metallothionein) is required for studying the heavy metal accumulation by

241 microbes (Cobbett et al., 2002). Hormones and redox signaling process are mediated by

242 metallothionein transcription factors in context of toxic metal (Au, Cd, Ag, Co, Cu, Bi, Hg, Zn

243 and Ni) exposure (Abdelmigid, 2016). The production of metal-binding protein and expression

244 of smtA gene have been reported by Synechococcus sp. (cynobacterial strains) (Huckle et al.,

245 1993).

246 4.3 Bioremediation by Biosorption

247 Microbes are helpful in binding with metals from aqueous solution known as biosorbent under

248 the process of biosorption and it becomes mandatory that the physical nature of biosorbent,

249 kinetics of sorption, regeneration, maximum sorption capacity, and recovery of bound metals

250 should be analyzed to detect the stability of microbes as a biosorbent. There are certain criteria

251 which should be followed for biosorbent selection: (a) Less expensive and reusable biosorbent

252 should be utilized, (b) There should be quite and efficient separation from the solution, (c) The

253 kinetics of metals should be fast (Bae et al., 2001, 2002, 2003). For biosorption of metals from

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254 solutions, there are mainly three broad categories of biosorbent that are used: (1) exo

255 polysaccharides, (2) dead biomass and preparations, (3) living cultures. On comparison, dead

256 cells absorb metals more than the living cells. Filamentous fungi have high metal uptake capacity

257 like Aspergillus, Rhizopus, Mucor, Penicillium, Actinomycetes, and Streptomycetes.

258 4.4 Bioremediation by Bioaugmentation

259 Additionally, chemicals which were made to trigger the natural degradation can also be added,

260 such as vegetable oil, molasses, oxygen or permanganate. Presenting specialized microorganisms

261 at a site of contamination to basically “eat” is called bioaugmentation. The indigenous or

262 exogenous insertion of microbes to the polluted sites is frequently involved in this process. It is

263 an affordable, effective and fast remediation process, and it is finding favor among remediation

264 experts and site managers (EPA, 2006). To upgrade and encourage microbial growth and

265 development, bioaugmentation (microbes) or biostimulation (amendments) i.e. nutrients, air,

266 electron acceptors/donors and organic substrates, other compounds that can control and influence

267 investigation in their deficiency, are introduced. To investigate groundwater or surface water,

268 other solids, and soil biostimulation and bioaugmentation are two basic applications of

269 bioremediation that could be utilized (EPA, 2006).

270 5. Microbial Bioremediation

271 The type of micro-flora is a significant factor affecting bioremediation which depends on making

272 use of gene and metabolic diversity of microorganisms to render the pollutants less toxic

273 (Juwarkar et al., 2010). Bioremediation utilizes either indigenous strains or those derived from a

274 different site (Santos et al., 2011). The use of indigenous microbial strain eliminates the need of

275 monitoring (Asha and Sandeep, 2013). Energy and a carbon source are two basic requirements

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276 that are utilized for bioremediation (Vidali, 2001). Here is a list of heavy metals utilizing

277 microorganisms followed by Vidali, 2001 is given below-

278 ❖ Bacillus and Pseudomonas aeruginosa utilize Zinc, Copper (Gunasekaran et al., 2003;

279 Philip et al., 2002).

280 ❖ Zooglea spp. and Citrobacter spp. utilize Copper, Nickel, Urea, Cadmium, Cobalt, Nickel

281 (Sar and D'Souza, 2001).

282 ❖ Citrobacter spp. and Chlorella vulgaris utilize Cadmium, Lead, Uranium, Copper, Gold,

283 Lead, Nickel, Mercury, Uranium (Gunasekaran et al., 2003).

284 ❖ Aspergilus Niger utilizes Cadmium, Thorium, Uranium, Zinc (Gunasekaran et al., 2003).

285 ❖ Pleurotus ostreatus utilize Cadmium, Copper, and Zinc (Gunasekaran et al., 2003).

286 ❖ Rhizopus arrhizus utilize Cadmium, Calcium, Mercury, Lead, Phosphorous, Silver

287 (Favero et al., 1991; Gunasekaran et al., 2003).

288 ❖ Stereum hirsutum utilize Cadmium, Copper Cobalt, Nickel (Gabriel et al., 1994, 1996).

289 ❖ Phormidium valderium utilize Cadmium; Lead (Gabriel et al., 1994, 1996).

290 ❖ Ganoderma applantus utilize Copper, Lead, Mercury (Gabriel et al., 1994, 1996).

291 Autochthonous microbes are those microbes that naturally occupy the soil or water ecosystems

292 encountering separation, or by some other microbes which are originated from various

293 ecosystems can be utilized to perform bioremediation. Plenty of microbes can be utilized to

294 reduce xenobiotic compounds (Table 2), metals from ecosystems such as algae, bacteria, yeast

295 and fungi (Vieira and Volesky 2000; White et al., 1997). The removal of pollutants and cleaning

296 of waste products from water, soil, air, and raw materials from industries can be performed by

297 bioremediation. The less expensive examination of contaminated sites has become a difficult

298 task (Zeyaullah et al., 2009).

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299 Microorganisms which are going to be used may vary; they have to be selected very carefully

300 because these organisms can survive within a limited range of heavy metals based on their

301 toxicity (Dubey, 2004; Prescott, 2002). Various groups of microorganisms will be required for

302 effective mediation because several kinds of contaminants are to be captured within a

303 contaminated site (Watanabe et al., 2001). It has been reported that a strain of Pseudomonas

304 putida was the first patent that was registered in 1981, as a biological remediation agent which

305 was able to degrade petroleum (Glazer and Nikaido, 2007; Prescott, 2002).

306 The active members of microbial consortium are identified as the microorganisms like

307 Xanthofacter, Penicillium, Bacillus, Pseudomonas, Flavobacterium, Mycobacterium, and

308 Nitrosomonas which carry out biodegradation in different environments. These microbes can be

309 subcategorized into the following types:

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311 6. Genetic engineering of microbes involved for enhanced bioremediation

312 Microbial strains have the potential to remove a variety of several kinds of hydrocarbons through

313 genetic manipulation. A multi plasmid containing Pseudomonas strain is successfully produced

314 that can oxidize polyaromatic hydrocarbons, terpenic aromatic, and aliphatic hydrocarbons. An

315 alteration in the genetic material of microorganisms can be done by genetic engineering

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316 techniques known as recombinant DNA technology, which involve gene exchange between

317 microbes. Such microorganisms are known as genetically modified microorganisms (GMM) or

318 genetically engineered microorganisms (GEM). Genetically engineered microorganisms (GEMs)

319 are known to be effective in soil, activated sludge and groundwater bioremediation. Four

320 principal approaches are being considered in the development of GEM for bioremediation:

321 1) Bioaffinity bioreporter sensor applications in chemical sensing, end point analysis and toxicity

322 reduction; 2) Bioprocess development, monitoring and control; 3) Enhancement of affinity and

323 enzyme specificity; 4) Construction and pathway regulation.

324 • Pseudomonas aeruginosa (NRRLB-5472) and Pseudomonas putida (NRRLB-5473) are

325 two genetically modified strains which were firstly patent in 1981 by USA. These two

326 strains were constructed in early 70s (Chakrabarty, 1986) and include genes for camphor,

327 naphthalene and salicylate degradation which are highly toxic in nature. These strains are

328 efficient for toxin degradation.

329 • The PCB catabolic genes which were located on the chromosome of R.eutropha A5,

330 A.denitrificans JB1, and Achromobacter sp. LBS1C1 was transferred to the heavy metal

331 resistant strain R.eutropha CH34 via natural conjugation for the removal of

332 polychlorinated biphenyls (Valls et al., 2000).

333 • For heavy metals, Arabidopsis thaliana gene was introduced into Mesorhizobium huakuii

334 subsp. Rengei strain B3 for phytochelatin synthase (PCS; PCSAt), that further generated

335 the symbiosis between M.huakuii subsp. Rengei strain B3 and Astragalus sinicus

336 (Sriprang et al., 2003).

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337 • In the presence of bacteroid specific promoter (the nif H gene), genes were expressed to

338 accumulate Cd2+ and release phytochelatins.

339 • Heavy metal degradation using genetically engineered microorganisms has gained a great

340 interest as in chromium degradation from industrial wastewater Alcaligenes eutrophus

341 AE104 (pEBZ141) was used and simultaneous expression of the Hg transport system and

342 metallothionein for Hg2+ degradation from heavy metal and wastewater

343 Rhodopseudomonas palustris, the recombinant photosynthetic bacterium was

344 constructed.

345 Molecular techniques like genetic engineering or plasmid breeding can efficiently generate

346 microorganisms having favorable catalytic potential, which are capable in removing any

347 environmental pollutant. The evident potential of genetic engineering in bioremediation was

348 observed by several microbiologists and molecular biologists (Table 3).

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

350 o Although the genetic engineering has produced various strains and bacterial species to

351 degrade pollutants, there are lots of obstacles related with this too. One major problem

352 related in this respect is that the growing strains and microbial species are not the ones to

353 perform a bulk of bioremediation process.

354 o The use of stable isotope probing (SIP) and relative method in microbiology ecology

355 have revealed that Rhodococcus and Pseudomonas and typical fast growers widely

356 favored as hosts of biodegradation are less significant under certain natural condition

357 (Joutey et al., 2013). The major problem encountered in this successful status of

358 bioremediation is to captive the field condition for modified microorganisms (Joutey et

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359 al., 2013). “Suicidal genetically engineered microorganisms” (S-GEMS) have been

360 utilized to reduce prominent risks and obtain safer and efficient decontamination of

361 contaminated sites (Pandey et al., 2005).

362 o P.fluorescens HK44 is used in field release for bioremediation and it has been helpfully

363 supervised on optimum wide-ranging and limited ground conditions (Ripp et al., 2000).

364 Hence for pollution remediation, genetically engineered bacteria do not lack the threats

365 related with their release into the ecosystem.

366 o Major problem captured in bioremediation process relates to hostile field factors for the

367 engineered microorganisms. Some well characterized bacteria e.g., E. coli (Bondarenko

368 et al., 2008), B. subtilis (Ivask et al., 2011) and P. putida (Wu et al., 2006) are utilized in

369 field conditions emphasizing on ways for which the molecular significance are mainly

370 confined. Modification of engineered bacterial strains is required to satisfy the new

371 challenge is the significant feature of open biotechnological applications.

372 o It is the main concern to create genetically engineered bacteria with an appropriate level

373 of environmental certainty for field release in bioremediation. Within a harsh

374 environmental condition, the efforts that are made to analyze the performance of

375 engineered bacteria are endurance and capability of horizontal gene transfer which can

376 affect the native microflora of environment. Bacteria are specifically designed for in vitro

377 bioremediation to avoid the field requirements and critical conditions. It is not evident

378 whether there is any adverse effect on the natural microorganisms because of the

379 deliberate release of genetically engineered bacteria for bioremediation (Sayler and Ripp,

380 2000). Therefore, the survival of genetically engineered bacteria in harsh environment is

381 still a reason of immense worry.

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382 8. Conclusion and Future Aspects

383 Increased development in technologies and industrial manufacturing has caused some harmful

384 side effects i.e. soil health degradation and soil pollution. Utilization of microorganisms emerges

385 as time saver for bioremediation due to some intricacy involved in remediation of soil by

386 conventional methods. However, there is a limitation of bioremediation process because several

387 microorganisms are not capable of converting toxic heavy metals into their non-toxic forms and

388 hence results in causing inhibitory effects on microbial activity. So, in order to enhance the

389 biodegradation potential of microorganism, genetic engineering is being utilized. Biological

390 methods have higher specificity other than the chemical and physical methods, convenience

391 towards in situ methodologies (e.g. toxic chemical addition or ignoring high energy), and have

392 the potential for development by genetically engineered microbes (GEMs).

393 Undoubtedly, bioremediation is moving towards the process of giving a way to greener

394 pastures. Due to this reason of bioremediation, it is well organized and inexpensive way to

395 handle contaminated ground water and soil. Environmental biotechnology has an aim of

396 approaching and resolving these issues to allow the utilization of microbes in bioremediation and

397 because of which it becomes mandatory to favor the reactions of the indigenous microbes in

398 contaminated biotopes and to increase the degradative potential of microbes by using the process

399 of bioaugmentation or biostimulation. Bioremediation of pollutants becomes successful which

400 makes a change in our capacity to diminish wastes, strike industrial pollution, and enjoy a more

401 endurable future.

402 Subsequently, there should be focus on improving in situ bioremediation technologies with

403 the help of genetically modified microbes or GEMs and also the adaptability and applicability of

404 these GEMs in each and every unfavorable condition and in various heavy metal contaminated

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405 sites. Future studies should also consider the various doubts among public to undertake GEM for

406 bioremediation, and prove them harmless to the environment.

407

408 9. References

409 Abdelmigid, H.M., 2016. Expression analysis of Type 1 and 2 Metallothionein genes in

410 Rapeseed (Brassica napus L.) during short-term stress using sqRT-PCR analysis.

411 Ahemad, M., 2014. Remediation of metalliferous soils through the heavy metal resistant plant

412 growth promoting bacteria: paradigms and prospects. Arab. J. Chem.

413 Ahuti, S., 2015. Industrial growth and environmental degradation. Int. Educ. Res. J. 1, 5–7.

414 Alvarez, A., Saez, J.M., Costa, J.S.D., Colin, V.L., Fuentes, M.S., Cuozzo, S.A., Benimeli, C.S.,

415 Polti, M.A., Amoroso, M.J., 2017. Actinobacteria: current research and perspectives for

416 bioremediation of pesticides and heavy metals. Chemosphere. 166, 41-62.

417 Asha, L.P., Sandeep, R.S., 2013. Review on bioremediation- potential tool for removing

418 environmental pollution. Int. J. Basic Appl. Chem. Sci. ISSN, 2277- 2073.

419 Bae, W., Mehra, R.K., Mulchandani, A., Chen, W., 2001. Genetic engineering of Escherichia

420 coli for enhanced uptake and bioaccumulation of mercury. Appl. Environ. Microbiol. 67,

421 5335-5338.

422 Bae, W., Mulchandani, A., Chen, W., 2002. Cell surface display of synthetic phytochelatins

423 using ice nucleation protein for enhanced heavy metal bioaccumulation. J. Inorg. Biochem.

424 88, 223-227.

425 Bae, W., Wu, C.H., Kostal, J., Mulchandani, A., Chen, W., 2003. Enhanced mercury biosorption

426 by bacterial cells with surface-displayed MerR. Appl. Environ. Microbiol. 69, 3176-3180.

19
427 Baker, A.J.M., Walker, P.L., 1990. Eco-physiology of metal uptake by tolerant plants. In heavy

428 metal tolerance in plants: Evolutionary Aspects, Eds: Shaw, A.J., CRC Press, Boca Raton,

429 FL; 155-177.

430 Beath, O.A., Eppsom, H.F., Gilbert, C.S., 1937. Selenium distribution in and seasonal variation

431 of vegetation type occurring on seleniferous soils. J. American. Pharm. Assoc. 26, 394-405.

432 Bondarenko, O., Rolova, T., Kahru, A., Ivask, A., 2008. Bioavailability of Cd, Zn and Hg in soil

433 to nine recombinant luminescent metal sensor bacteria. Sensors 8, 6899-6923.

434 Brim, H., McFarlan, S.C., Fredrickson, J.K., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J.,

435 2000. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed

436 waste environments. Nat. Biotechnol. 18, 85–90.

437 Carter, P., Cole, H., Burton, J., 2006. Bioremediation: Successes and Shortfalls. In: Proceedings

438 of Key International Conference and Exhibition for Spill Prevention, Preparedness, Response

439 and Restoration (Interspill), London, UK.

440 Chakrabarty, A.M., 1986. Genetic engineering and problems of environmental pollution.

441 BioTechnology 8, 515-530.

442 Chen, C., Wang, J.L., 2007. Characteristics of Zn2+ biosorption by Saccharomyces cerevisiae.

443 Biomed. Environ. Sci. 20, 478-482.

444 Chen, J., Zhou, H.C., Wang, C., Zhu, C.Q., Tam, N.F.Y., 2015. Short-term enhancement effect

445 of nitrogen addition on microbial degradation and plant uptake of polybrominated diphenyl

446 ethers (PBDEs) in contaminated mangrove soil. J. Hazard. Mater. 300, 84-92.

447 Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: Role in heavy metals

448 detoxification and homeostatis. Annu. Rev. Plant Biol. 53: 159-182.

20
449 Comte, S., Guibaud, G., Baudu, M., 2008. Biosorption properties of extracellular polymeric

450 substances (EPS) towards Cd, Cu and Pb for different pH values. J. Hazard. Mater. 151, 185-

451 193.

452 Cybulski, Z., Dzuirla, E., Kaczorek, E., Olszanowski, A., 2003. The influence of emulsifiers on

453 hydrocarbon biodegradation by Pseudomonadacea and Bacillacea strains. Spill Sci. Technol.

454 Bull. 8, 503-507.

455 Dangi, A. K., Sharma, B., Hill, R. T., & Shukla, P., 2019. Bioremediation through microbes:

456 systems biology and metabolic engineering approach. Critical reviews in

457 biotechnology. 39(1), 79-98.

458 Das, N., Vimala, R., Karthika, P., 2008. Biosorption of heavy metals—an overview. Indian J.

459 Biotechnol. 7, 159-169.

460 Dean, R.D., Moody, J., Cerniglia, C.E., 2002. Utilization of mixtures of polycyclic aromatic

461 hydrocarbons by bacteria isolated from contaminated sediment. Microbiol. Ecol. 41, 1-7.

462 Deng, X., Li, Q.B., Lu, Y.H., Sun, D.H., He, N., 2005. Genetic engineering of Escherichia coli

463 SE5000 and its potential for Ni2+ bioremediation. Proc. Biochem. 40, 425-430.

464 Deng, X., Yi, X.E., Liu, G., 2007. Cadmium removal from aqueous solution by gene modified

465 Escherichia coli JM109. J. Hazard. Mater. 139, 340-344.

466 Di Gregorio, S., Azaizeh, H., Lorenzi, R., 2013. Biostimulation of the autochthonous microbial

467 community for the depletion of polychlorinated biphenyls (PCBs) in contaminated

468 sediments. Environ. Sci. Poll. Res. 20(6), 3989-3999.

469 Di Gregorio, S., Gentini, A., Siracusa, G., Becarelli, S., Azaizeh, H., Lorenzi, R., 2014.

470 Phytomediated biostimulation of the autochthonous bacterial community for the acceleration

21
471 of the depletion of polycyclic aromatic hydrocarbons in contaminated sediments. BioMed.

472 Res. Int. 2014.

473 Dixit, R., Malaviya, D., Pandiyan, K., Singh, U., Sahu, A., Shukla, R., ... Paul, D., 2015.

474 Bioremediation of heavy metals from soil and aquatic environment: an overview of

475 principles and criteria of fundamental processes. Sustainability. 7(2), 2189-2212.

476 Dua, M., Singh, A., Sethunathan, N., Johri, A.K., 2002. Biotechnology and bioremediation:

477 successes and limitations. Appl. Microbiol. Biotechnol. 59, 143-152.

478 Dubey, R.C., 2004. A text book of Biotechnology, 3rd edition.Chand and Company Ltd., New

479 Delhi, India; 365-375.

480 Elias, S.H., Mohamed, M.A.K.E.T.A.B., Ankur, A.N., Muda, K., Hassan, M.A.H.M., Othman,

481 M.N., Chelliapan, S., 2014. Water hyacinth bioremediation for ceramic industry wastewater

482 treatment-application of rhizofiltration system. Sains Malaysiana. 43(9), 1397-1403.

483 EPA., 2006. Engineering issue: in situ and ex situ biodegradation technologies for remediation of

484 contaminated sites. 62, 6-15.

485 Fang, L., Wei, X., Cai, P., Huang, Q., Chen, H., Liang, W., Rong, X., 2011. Role of extracellular

486 polymeric substances in Cu(II) adsorption on Bacillus subtilis and Pseudomonas putida.

487 Bioresour. Technol. 102, 1137-1141.

488 Fang, L.C., Huang, Q.Y., Wei, X., Liang, W., Rong, X.M., Chen, W.L., Cai, P., 2010.

489 Microcalorimetric and potentiometric titration studies on the adsorption of copper by

490 extracellular polymeric substances (EPS), minerals and their composites. Bioresour. Technol.

491 101, 5774-5779.

492 Favero, N., Costa, P., Massimino, M.L., 1991. In vitro uptake of cadmium by basidiomycete

493 Pleurotus ostreatus. Biotechnol. Lett. 10, 701-704.

22
494 Ferraro, A., van Hullebusch, E.D., Huguenot, D., Fabbricino, M., Esposito, G., 2015.

495 Application of an electrochemical treatment for EDDS soil washing solution regeneration

496 and reuse in a multi-step soil washing process: Case of a Cu contaminated soil. J. Environ.

497 Manag. 163, 62-69.

498 Fontanili, L., Lancilli, C., Suzui, N., Dendena, B., Yin, Y. G., Ferri, A., ... & Sacchi, G. A., 2016.

499 Kinetic analysis of zinc/cadmium reciprocal competitions suggests a possible Zn-insensitive

500 pathway for root-to-shoot cadmium translocation in rice. Rice. 9(1), 16.

501 Freeman, J.L., Persans, M.W., Nieman, K., Salt, D.E., 2005. Nickel and cobalt resistance

502 engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel

503 hyperaccumulator plant Thlaspi goesingense. Appl. Environ. Microbiol. 71, 8627-8633.

504 Gabriel, J., Kofronova, O., Rychlovsky, P., Krenzelok, M., 1996. Accumulation and effect of

505 cadmium in the wood rotting basidiomycete, Daedalea quercina. Bull. Environ. Cont.

506 Toxicol. 57, 383-390.

507 Gabriel, J., Mokrejs, M., Bily, J., Rychlovsky, P., 1994. Accumulation of heavy metal by some

508 Woodrooting fungi. Folia Microbiologica. 39, 115- 118.

509 Gan, S., Lau, E.V., Ng, H.K., 2009. Remediation of soils contaminated with polycyclic aromatic

510 hydrocarbons (PAHs). J. Hazard. Mater. 172, 532-549.

511 Garbisu, C., Alkorta, I., 2001. Phytoextraction: A cost-effective plant-based technology for the

512 removal of metals from the environment. Bioresour. Technol. 77, 229-236.

513 Glazer, A.N., Nikaido, H., 2007. Microbial biotechnology: fundamentals of applied

514 microbiology, 2nd Edition, Cambridge University Press, Cambridge, New York; 510-528.

23
515 Gomez-Jimenez, T.R., Moliternib, E., Rodríguezb, L., Fernandezc, F.J., Villasenorc, J., 2011.

516 Feasibility of mixed enzymatic complexes to enhanced soil bioremediation processes.

517 Procedia Environ. Sci. 9, 54-59.

518 Guine, V., Spadini, L., Sarret, G., Muris, M., Delolme, C., Gaudet, J.P., Martins, J.M., 2006.

519 Zinc sorption to three gram-negative bacteria: Combined titration, modeling and EXAFS

520 study. Environ. Sci. Technol. 40, 1806-1813.

521 Gunasekaran, P., Muthukrishnan, J., Rajendran, P., 2003. Microbes in heavy metal remediation.

522 Ind. J. Exp. Biol. 41, 935-944.

523 Haritash, A.K., Kaushik, C.P., 2009. Biodegradation aspects of polycyclic aromatic

524 hydrocarbons (PAHs): a review. J. Hazard. Mater. 169, 1-15.

525 Hasin, A.A., Gurman, S.J., Murphy, L.M., Perry, A., Smith, T.J., Gardiner, P.E., 2010.

526 Remediation of chromium (VI) by a methane-oxidizing bacterium. Environ. Sci. Tech. 44,

527 400-405.

528 Hayat, S., Faraz, A., & Faizan, M., 2017. Root exudates: composition and impact on plant–

529 microbe interaction. Biofilms in Plant and Soil Health, 179.

530 Huckle, J.W., Morby, A.P., Turner, J.S., Robinson, N.J., 1993. Isolation of a prokaryotic

531 metallothionein locus and analysis of transcriptional control by trace metal ions. Mol.

532 Microbiol. 7, 177-187.

533 Hussein, H., Farag, S., Moawad, H., 2004. Isolation and characterization of Pseudomonas

534 resistant to heavy metals contaminants. Arab. J. Biotechnol. 7, 13-22.

535 Ijah, U.J.J., Antai, S.P., 1988. Degradation and mineralization of crude oil by bacteria. Nig. J.

536 Biotechnol. 5, 79-87.

24
537 Iram, S., Uzma, G., Rukh, S., Ara, T., 2013. Bioremediation of heavy metals using isolates of

538 filamentous fungus collected from polluted soil of Kasur, Pakistan. Int. Res. J. Biol. Sci. 2,

539 66-73.

540 Ivask, A., Dubourguier, H.C., Pollumaa, L., Kahru, A., 2011. Bioavailability of Cd in 110

541 polluted top soils to recombinant bioluminescent sensor bacteria: effect of soil particulate

542 matter. J. Soils Sediments. 11, 231-237.

543 Jaishankar, M., Tseten, T., Anbalagan, N., Mathew, B.B., Beeregowda, K.N., 2014. Toxicity,

544 mechanism and health effects of some heavy metals. Interdiscip. Toxicol. 7, 60-72.

545 Jin, H.P., Dane, L., Periyasamy, P., Girish, C., Nanthi, B., Jae-Woo, C., 2011. Role of organic

546 amendments on enhanced bioremediation of heavy metal (loid) contaminated soils. J.

547 Hazard. Mat. 185, 549-574.

548 Jogdand, S.N., 1995. Environmental biotechnology, 1st Edition, Himalaya Publishing House,

549 Bombay, India; 104-120.

550 Joutey, T.N., Bahafid, W., Sayel, H., Ghachtouli, E.I.N., 2013. Biodegradation: involved

551 microorganisms and genetically engineered microorganisms. Intech. 288-320.

552 Juwarkar, A.A., Singh, S.K., Mudhoo, A., 2010. A comprehensive overview of elements in

553 bioremediation. Reviews in Environmental Science and bio/technology, 9(3), 215-288.

554 Kang, S.H., Singh, S., Kim, J.Y., Lee, W., Mulchandani, A., Chen, W., 2007. Bacteria

555 metabolically engineered for enhanced phytochelatin production and cadmium accumulation.

556 Appl. Environ. Microbiol. 73, 6317-6320.

557 Kapley, A., Purohit, H.J., Chhatre, S., Shanker, R., Chakrabarti, T., 1999. Osmo tolerance and

558 hydrocarbon degradation by a genetically engineered microbial consortium. Bioresource

559 Technol. 67, 241-245.

25
560 Karna, R.R., Luxton, T., Bronstein, K.E., Hoponick Redmon, J., Scheckel, K.G., 2017. State of

561 the science review: Potential for beneficial use of waste by-products for in situ remediation

562 of metal-contaminated soil and sediment. Critical reviews in environmental science and

563 technology, 47(2), 65-129.

564 Kinya, K., Kimberly, L.D., 1996. Current use of bioremediation for TCE cleanup: results of a

565 survey. Remediat. J. 6, 1-14.

566 Kiyono, M., Pan-Hou, H., 2006. Genetic engineering of bacteria for environmental remediation

567 of mercury. J. Health Sci. 52, 199-204.

568 Kiyono, M., Sone, Y., Nakamura, R., Pan-Hou, H., Sakabe, K., 2009. The Mer E protein

569 encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli. FEBS Lett.

570 583, 1127-1131.

571 Kostal, J.R.Y., Wu, C.H., Mulchandani, A., Chen, W., 2004. Enhanced arsenic accumulation in

572 engineered bacterial cells expressing ArsR. Appl. Environ. Microbiol. 70, 4582-4587.

573 Kumar, A., Chanderman, A., Makolomakwa, M., Perumal, K., Singh, S., 2016. Microbial

574 production of phytases for combating environmental phosphate pollution and other diverse

575 applications. Crit. Rev. Environ. Sci. Technol. 46, 556–591. doi:

576 10.1080/10643389.2015.1131562

577 Liu, S., Zhang, F., Chen, J., Sun, G.X., 2011. Arsenic removal from contaminated soil via

578 biovolatilization by genetically engineered bacteria under laboratory conditions. J. Environ.

579 Sci. 23, 1544-1550.

580 Lopez, A., Lazaro, N., Morales, S. and Margues, A.M. 2002. Nickel biosorption by free and

581 immobilized cells of Pseudomonas fluorescens 4F39: a comparative study. Water Air Soil

582 Pollut. 135, 157-172.

26
583 Lovely, D.R., 2002. Dissimilatory metal reduction: from early life to bioremediation. ASM

584 News. 68, 231-237.

585 Lovley, D.R., Phillips, E.J.P., 1988. Novel mode of microbial energy metabolism: Organic

586 carbon oxidation to dissimilatory reduction of iron or manganese. Appl. Environ. Microbiol.

587 54, 1472-1480.

588 Lovley, D.R., Coates, J.D., Blunt-Harris, E.L., Philips, E.J.P., Woodward, J.C., 1996. Humic

589 substances as electron acceptors for microbial respiration. Nature 382, 445-448.

590 Lovley, D.R., Philips, E.J., Gorby, Y.A., Landa, E.R., 1991. Microbial reduction of uranium.

591 Nature 350, 413-416.

592 Ma, Y., Oliveira, R.S., Freitas, H., Zhang, C., 2016. Biochemical and molecular mechanisms of

593 plant-microbe-metal interactions: relevance for phytoremediation. Frontiers in plant

594 science, 7, 918.

595 Manobala, T., Shukla, S., Rao, T.S., Kumar, M.D., 2018. A New Uranium Bioremediation

596 Approach using Radio-tolerant Deinocoocus radiodurans Biofilm. bioRxiv. 503896.

597 McHughen, A., 2016. A critical assessment of regulatory triggers for products of biotechnology:

598 Product vs. process. GM crops & food. 7(3-4), 125-158.

599 Mesa, J., Rodriguez-Llorente, J.D., Pajuelo, E., Piedras, J.M.B., Caviedes, M.A., Redondo-

600 Gomez, S., Mateos-Naranjo, E., 2015. Moving closer towards restoration of contaminated

601 estuaries: bioaugmentation with autochthonous rhizobacteria improves metal

602 rhizoaccumulation in native Spartina maritima. J. Hazard. Mater. 300:263–271.

603 doi:10.1016/j.jhazmat.2015.07.006

604 Mgbeahuruike, L.U., 2018. An investigation into soil pollution and remediation of selected

605 polluted sites around the globe (Doctoral dissertation, Manchester Metropolitan University).

27
606 Mgbemene, C.A., Nnaji, C.C., Nwozor, C., 2016. Industrialization and its backlash: focus on

607 climate change and its consequences. J. Environ. Sci. Technol. 9, 301–316. doi:

608 10.3923/jest.2016.301.316

609 Mnif, I., Sahnoun, R., Ellouz-Chaabouni, S., Ghribi, D., 2017. Application of bacterial

610 biosurfactants for enhanced removal and biodegradation of diesel oil in soil using a newly

611 isolated consortium. Pro. Saf. Environ. Protec. 109, 72-81.

612 Morillo, E., & Villaverde, J., 2017. Advanced technologies for the remediation of pesticide-

613 contaminated soils. Sci. Total Environ. 586, 576-597.

614 Murtaza, I., Dutt, A., Ali, A., 2002. Biomolecular engineering of Esherichia coli

615 organomercuriallyase gene and its expression. Ind. J. Biotech. 1, 117-120.

616 Naghipour, D., Gharibi, H., Taghavi, K., & Jaafari, J., 2016. Influence of EDTA and NTA on

617 heavy metal extraction from sandy-loam contaminated soils. J. Environ. Chem. Engine. 4(3),

618 3512-3518.

619 Ng, S.P., Davis, B., Polombo, E.A., Bhave, M., 2009. Tn 5051 like mer containing transposon

620 identified in a heavy metal tolerant strain Achromobacter sp. AO22. BMC Res. Notes. 7, 2-

621 38.

622 Onwubuya, K., Cundy, A., Puschenreiter, M., Kumpiene, J., Bone, B., 2009. Developing

623 decision support tools for the selection of “gentle” remediation approaches. Sci. Total

624 Environ. 407, 6132–6142.

625 Pandey, G., Paul, D., Jain, R.K., 2005. Conceptualizing “suicidal genetically engineered

626 microorganisms” for bioremediation applications. Biochem. Biophy. Res. Commun. 327,

627 637-639.

28
628 Park, A.J., Cha, D.K., Holsen, T.M., 1998. Enhancing solubilization of sparingly soluble organic

629 compounds by biosurfactants produced by Nocardia erythropolis. Water Environ. Res. 70,

630 351-355.

631 Park, T.J., Lee, K.G., Lee, S.Y., 2016. Advances in microbial biosynthesis of metal

632 nanoparticles. Appl. Microbiol. Biotechnol. 100(2), 521-534.

633 Patel, J., Zhang, Q., Michael, R., McKay, L., Vincent, R., Xu, Z., 2010. Genetic engineering of

634 Caulobacter crescentus for removal of cadmium from water. Appl. Biochem. Biotechnol.

635 160, 232-243.

636 Philip, L., Iyengar, L., Venkobacher, L., 2000. Site of interaction of copper on Bacillus

637 polymyxa. Water Air Soil Pollut. 119, 11-21.

638 Prescott, L.M., Harley, J.P., Klein, D.A., 2002. Microbiology, 5 Edition, McGraw-Hill, New

639 York.

640 Raghunandan, K., Kumar, A., Kumar, S., Permaul, K., and Singh, S., 2018. Production of gellan

641 gum, an exopolysaccharide, from biodiesel-derived waste glycerol by Sphingomonas spp.

642 3Biotech 8:71. doi: 10.1007/s13205-018-1096-3

643 Raghunandan, K., McHunu, S., Kumar, A., Kumar, K.S., Govender, A., Permaul, K., et al.,

644 2014. Biodegradation of glycerol using bacterial isolates from soil under aerobic conditions.

645 J. Environ. Sci. Heal. A Tox. Hazard. Subst. Environ. Eng. 49, 85–92. doi:

646 10.1080/10934529.2013.824733

647 Rajasulochana, P., Preethy, V., 2016. Comparison on efficiency of various techniques in

648 treatment of waste and sewage water–A comprehensive review. Res. Eff. Technol. 2(4), 175-

649 184.

29
650 Reeves, R.D., Brooks, R.R., 1983. European species of Thlaspi L. (Cruciferae) as indicators of

651 nickel and zinc. J. Geochem. Explor. 18, 275-283.

652 Ripp, S., Nivens, D.E., Ahn, Y., Werner, C., Jarrell, J., Easter, J., Burlage, R., Sayler, G.S., 2000.

653 Controlled field release of a bioluminescent genetically engineered microorganism for

654 bioremediation process monitoring and control. Environ. Sci. Tech. 34, 846-853.

655 Santos, H.F., Carmo, F.L., Paes, J.E.S., Rosado, A.S., Peixoto, R.S., 2011. Bioremediation of

656 mangroves impacted by petroleum. Water, Air and Soil Pollut. 216, 329-350.

657 Sar, P., D'Souza, S.F., 2001. Biosorptive uranium uptake by Pseudomonas strain:

658 characterization and equilibrium studies. J. Chemic. Technol. Biotech. 76, 1286-1294.

659 Sasaki, Y., Minakawa, T., Miyazaki, A., Silver, S., Kusano, T., 2005. Functional dissection of a

660 mercuric ion transporter Mer C from Acidithiobacillus ferrooxidans. Biosci. Biotech.

661 Biochem. 69, 1394-1402.

662 Sayler, G.S., Ripp, S., 2000. Field applications of genetically engineered microorganisms for

663 bioremediation processes. Curr. Opin. Biotechnol. 11, 286-289.

664 Seeger, M., Hernandez, M., Mendez, V., Ponce, B., Cordova, M., Gonzalez, M., 2010. Bacterial

665 degradation and bioremediation of chlorinated herbicides and biphenyls. J. Soil. Sci. Plant

666 Nut. 10, 320–332.

667 Shukla, S.K., Rao, T.S., 2017. The first recorded incidence of Deinococcus radiodurans R1

668 biofilm formation and its implications in heavy metals bioremediation. bioRxiv. 234781.

669 Sikkema, J., de Bont, J.A., Poolman, B., 1995. Mechanisms of membrane toxicity of

670 hydrocarbons. Microbiol. Rev. 59, 201-222.

671 Singh, S., Gupta, V.K., 2016. Biodegradation and bioremediation of pollutants: perspectives

672 strategies and applications. Int. J. Pharm. Biol. Sci. 10(1). 53.

30
673 Singh, S., Kang, S.H., Mulchandani, A., Chen, W., 2008. Bioremediation: environmental

674 cleanup through pathway engineering. Curr. Opin. Biotechnol. 19, 437-444.

675 Spormann, A.M., Widdel, F., 2000. Metabolism of alkylbenzenes, alkanes, and other

676 hydrocarbons in anaerobic bacteria. Biodegradation 11, 85-105.

677 Sriprang, R., Hayashi, M., Ono, H., Takagi, M., Hirata, K., Murooka, Y., 2003. Enhanced

678 accumulation of Cd2+ by a Mesorhizobium sp. transformed with a gene from Arabidopsis

679 thaliana coding for phytochelatin synthase. Appl. Environ. Microbiol. 69, 1791-1796.

680 Talos, K., Pager, C., Tonk, S., Majdik, C., Kocsis, B., Kilar, F., Pernyeszi, T., 2009. Cadmium

681 biosorption on native Saccharomyces cerevisiae cells in aqueous suspension. Acta Univ.

682 Sapientiae Agric. Environ. 1, 20-30.

683 Tigini, V., Prigione, V., Giansanti, P., Mangiavillano, A., Pannocchia, A., Varese, G.C., 2010.

684 Fungal biosorption, an innovative treatment for the decolourisation and detoxification of

685 textile effluents. Water 2, 550-565.

686 Toth, G., Hermann, T., Da Silva, M. R., Montanarella, L., 2016. Heavy metals in agricultural

687 soils of the European Union with implications for food safety. Environ. Int. 88, 299-309.

688 Tylecote, A., 2019. Biotechnology as a new techno-economic paradigm that will help drive the

689 world economy and mitigate climate change. Res. Pol. 48(4), 858-868.

690 Valko, M., Jomova, K., Rhodes, C. J., Kuča, K., Musilek, K., 2016. Redox-and non-redox-

691 metal-induced formation of free radicals and their role in human disease. Arch.

692 toxicol. 90(1), 1-37.

693 Valls, M., Atrian, S., de-Lorenzo, V., Fernandez, L.A., 2000. Engineering a mouse

694 metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy

695 metals in soil. Nat. Biotechnol. 18, 661-665.

31
696 Vidali, M., 2001. Bioremediation. An overview. Pure Appl. Chem. 73(7), 1163–1172.

697 Vieira, R., Volesky, B., 2000. Biosorption: a solution to pollution? Internat. Microbiol. 3, 17-24.

698 Watanabe, K., Kodoma, Y., Stutsubo, K., Harayama, S., 2001. Molecular characterization of

699 bacterial populations in petroleum contaminated ground water discharge from undergoing

700 crude oil storage cavities. Appl. Environ. Microbiol. 66, 4803-4809.

701 White, C., Sayer, J.A., Gadd, G.M., 1997. Microbial solubilization and immobilization of toxic

702 metals: key biochemical processes for treatment of contamination. FEMS Microbiol. Rev.

703 20, 503-516.

704 Wu, C.H., Wood, T.K., Mulchandani, A., Chen, W., 2006. Engineering plant-microbe symbiosis

705 for rhizoremediation of heavy metals. Appl. Environ. Microbiol. 72, 1129-1134.

706 Xu, Y., Seshadri, B., Bolan, N., Sarkar, B., Ok, Y.S., Zhang, W., ... Dong, Z., 2019. Microbial

707 functional diversity and carbon use feedback in soils as affected by heavy metals. Environ.

708 Int. 125, 478-488.

709 Yuan, C.G., Lu, X.F., Qin, J., Rosen, B.P., Le, X.C., 2008. Volatile arsenic species released from

710 Escherichia coli expressing the AsIII S-adenosyl methionine methyltransferase gene.

711 Environ. Sci. Technol. 42, 3201-3206.

712 Zeyaullah, M.D., Atif, M.D., Islam, B., Azza, S., Abdelkafel, S.P., ElSaady, M.A., Ali, A., 2009.

713 Bioremediation: a tool for environmental cleaning. Afr. J. Microbiol. Res. 3, 310-314.

714 Zhao, X.W., Zhou, M.H., Li, Q.B., Lu, Y.H., He, N., Sun, D.H., Deng, X., 2005. Simultaneous

715 mercury bioaccumulation and cell propagation by genetically engineered Escherichia coli.

716 Proc. Biochem. 40, 1611-1616.

717
718
719

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720
721
722 Table 1: Metal hyper accumulator plant species.
Plant species Metals Amount of leaf References
(ppm)
Aegiceras Brominated 37,000 Chen et al., 2015
corniculatum diphenyl
ethers (BDE-
47)
Spartina maritime As, Cu, Pb, Zn 5000 Mesa et al., 2015

Eichhorina crassipes Heavy metals 40,000 Elias et al., 2014

(water (Fe, Zn,
hyacinth) Cd, Cu, B, and
Cr)
Phragmites australis PAHs - Gregorio et al., 2014

Sparganium sp. Polychlorinated - Gregorio et al., 2013

biphenyls
Ipomea alpina Cu 12,300 Baker and Walker, 1990
Thlaspi caerulescens Zn : Cd 39,600 : 1,800 Baker and Walker, 1990;
Reeves and Brooks, 1983

Astragalus Se 14,900 Beath et al., 1937

racemosus

723
724
725
726
727

33
728 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, Dean et al., 2002;
cycloparaffins Jogdand, 1995
Alcaligenes spp. Linear alkylbenzene sulfonates, PCBs polycyclic Kapley et al., 1999
aromatics, Halogenated hydrocarbons,
Mycobacterium spp. Hydrocarbons, Aromatics, Polycyclic hydrocarbons Park et al., 1998

Arthrobacter spp. Benzene, long chain alkanes, Hydrocarbons, Jogdand, 1995

Phenoxyacetate, Cresol, 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
729
730
731
732
733
734
735
736
737
738
739
740
741

34
742 Table 3. GEMs that are used in heavy metal bioremediation.
Microbes Modified gene expression Heavy metals References
Sphingomonas Over expression of arsM gene Arsenic Liu et al., 2011
desiccabilis and
Bacillus Idriensis
strains
B. subtilis BR151 Luminescent Cadmium sensors Cadmium Ivask et al., 2011
(pTOO24)
Methylococcus CrR genes for Cr (VI) reductase Chromium(VI) Hasin et al., 2010
capsulatus (Bath) activity

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

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

Achromobacter sp Mercury reductase expressing Mercury Ng et al., 2009

AO22 mer gene
E. coli strain Metallothionein Arsenic Singh et al., 2008
E. coli strain AsIII S-adenosylmethionine Arsenic Yuan et al.., 2008
methyltransferase gene
Pseudomonas MerR/CadC/ZntR/Pmer/PcadA/ Cadmium, Lead, Bondarenko et al.,
fluorescens OS8; PzntA Mercury, Zinc, 2008
Escherichia
coliMC1061;
Bacillus
subtilisBR151;
Staphylococcus

35
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 Cadmium Wu et al., 2006
peptide EC20

Pseudomonas K-62 Expression of mercury transport Mercury Kiyono and Pan-

system and Hou, 2006
Organomurcuriallyase
E. coli SE5000 Nickel transport system and Nickel Deng et al., 2005
metallothionein
E. coli JM109 Hg2+ transporter and Mercury Zhao et al., 2005
metallothionein
E .coli strain Over expression of Serin acetyl Nickel and Freeman et al.,
transferase cobalt 2005

Acidithiobacillus Mercury ion transporter gene Mercury Sasaki et al., 2005

ferrooxidans expression
E. coli Metalloregulatory protein ArsR Arsenic Kostal et al., 2004
(over expressing ELP153AR)

Escherichia coli and Expressing EC20 Mercury and Bae et al., 2003
Moraxella sp. Cadmium
Mesorhizobium Phytochelatin synthase (PCS) Cd2+ Sriprang et al.,
huakuii B3 gene expression 2003

E. coli strain Organomurcuriallyase Mercury Murtaza et al.,

gene expression 2002

P. fluorescens 4F39 Nickel transport system Nickel Lopez et al., 2002

36
 Deinococcus Hg (II) resistance gene (merA) Mercury Brim et al., 2000
radiodurans (Radioactive
waste from
nuclear weapons)
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769

37
770
771
772
773 Highlights

774 ➢ Bioremediation of heavy metals using microbes

775 ➢ Bioremediation using genetically engineered microbes

776 ➢ Biosorption and bioaugmentation of toxic heavy metals

777

778

779

780

781

38