Bioresource Technology 326 (2021) 124750

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Role of microbial community and metal-binding proteins in

phytoremediation of heavy metals from industrial wastewater
Pooja Sharma, Ashutosh Kumar Pandey, Aswathy Udayan, Sunil Kumar *
CSIR-National Environmental and Engineering Research Institute (CSIR-NEERI), Nehru Marg, Nagpur 440 020, India

H I G H L I G H T S

• Industrial wastewater contains huge amount of toxic metals.

• Bioremediation of toxic heavy metals by microorganisms was thoroughly reviewed.
• Phytoremediation of metals assisted by microbes and MBPs was also reviewed.
• Phyto-bioremediation technique can be efficiently used for eco-restoration.
• Issues and suggested strategies were discussed along with future perspectives.

A R T I C L E I N F O A B S T R A C T

Keywords: This review illustrated the role of metal-binding proteins (MBPs) and microbial interaction in assisting the
Metal pollution phytoremediation of industrial wastewater polluted with heavy metals. MBPs are used to increase the accu­
Industrial wastewater mulation and tolerance of metals by microorganisms via binding protein synthesis. Microbes have various
Microorganism
protection mechanisms to heavy metals stress like compartmentalization, exclusion, complexity rendering, and
Hyperaccumulator plants
the synthesis of binding proteins. MBPs include phytochelatins, metallothioneins, Cd-binding peptides (CdBPs),
cysteines (gcgcpcgcg) (CP), and histidines (ghhphg)2 (HP). In comparison with other physico-chemical methods,
phytoremediation is an eco-friendly and safe method for the society. The present review concentrated on the
efficiency of phytoremediation strategies for the use of MBPs and microbe-assisted approaches.

1. Introduction
Pollution in the environment caused mainly by the pulp and paper,
tannery, and distillery industry contaminants has become a serious
concern for various aquatic and terrestrial ecosystems. Discharged
wastewater contains a variety of pollutants like metals (Ni, Fe, Zn, Cr,
Cd, As, Cu, Mn, and Mg), androgenic, mutagenic, and carcinogenic
compounds, which are toxic even after secondary treatment and has
become one of the grave concerns in India and other developing coun­
tries (Sharma et al., 2020a). Industrial wastewater leads to toxicity to
phytoplankton and zooplankton by the increased presence of factors like
total suspended solids (TSS), total dissolved solids (TDS), biochemical
oxygen demand (BOD), and chemical oxygen demand (COD) (Sharma
et al., 2020d). Furthermore, some metals like Cd, Cu, Pb, Hg, Cu, As, Ni
and Cr are declared as environmental pollutants by the Environmental
Protection Agency (EPA) (Oconnor et al., 2019; Selvi et al., 2019;
Jaskulak et al., 2019). Pollution of wastewater has been a significant risk
to the humans, and other living organisms (Ngo et al., 2020; Hoang
et al., 2020).
The role of MBPs in plant–microbe interaction increases the accu­
mulation and resistance or tolerance of heavy metals which has great
potential for the detoxification of heavy metals. Microbes and plants
during stress conditions adopt to various defence mechanisms, like
compartmentalization, making complexes, exclusion, and the secretion
of metal-binding proteins like phytochelatins (PCs) and metal­
lothioneins (MTs) (Pal and Rai, 2010; Gautam et al., 2012). Different
bacterial genera like Bacillus, Mycobacterium, Paenibacillus, Alcaligenes,
Acidovorax, Rhodococcus and Pseudomonas, are widely reported which
aids in phytoremediation. Microalgae have also been reported as an
excellent tool for the bioremediation of heavy metals in an environ­
mentally acceptable and feasible manner (Sreekumar et al., 2018; Leong
and Chang, 2020). The remaining microalgal biomass after

* Corresponding author.
E-mail address: s_kumar@neeri.res.in (S. Kumar).

https://doi.org/10.1016/j.biortech.2021.124750
Received 25 November 2020; Received in revised form 15 January 2021; Accepted 15 January 2021
Available online 22 January 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
P. Sharma et al.
bioremediation can be successfully used to produce nutraceuticals and
other high-value metabolites (Udayan et al., 2017; Nidhin et al., 2020).
Strengthening metal solubility by improving the microbiota of the
rhizosphere of plants facilitates the synthesis of organic acids and
polysaccharides which is proved to be a competitive advantage in phy­
toremediation (Wang et al., 2017; Verma and Kuila, 2019).
Metals detoxification phytoremediation technology for treatment of
wastewater can be used in a cost-effective manner because metals are
considered as a distinctive category of pollutants (Cameselle and Gou­
veia, 2019). Cd, Pb, Ni, and Hg are the most common pollutants reported
worldwide (Oconnor et al., 2019). Native plant species used for phy­
toremediation are considered as an attractive tool for industrial waste­
water treatment (Ekperusi et al., 2019; Shah and Daverey, 2020). Plants
have the potential to tolerate complex pollutants from different
contaminated sites due to some special characters like biomass, reactive
oxygen species (ROS) enzymes, antioxidants activity, etc. Plant metal-
binding protein-like genes were isolated from various microbes and
plants like soybean, maize, tobacco, brassica, wheat, and rice and its
role in the deterioration of metal accumulation in a different part of
plants and microbes has been recognized till now (Duan et al., 2012).
Recently, Chauhan and Mathur (2020) reported the phytoremediation
efficiency of Helianthus annuus L. for the restoration of industrial site
polluted with heavy metals. Some other plants like Helichrysum italicum,
Zea mays, Erato polymnioides , Festuca arundinacea , Hibiscus cannabinus,
Paxillus involutus, Populus canescens, and Solanum lycopersicum, etc. are
suitable for phytoremediation as reported (Liu et al., 2020)
Plant-microbes’ interaction together with heavy metals uptake is
done through different biochemical processes including translocation,
chelation, immobilization, solubilization, precipitation, volatilization,
and complexation. For instance, various elemental pollutants come into
plants through a nutrient transport system. In phytodegradation or
phytotransformation, organic contaminants get metabolized inside the
plant cell by the use of specific enzymes. The enzymes responsible for
the degradation of anilines, pesticides, and the chlorinated compounds
are dehalogenase and nitroreductase which assist in the degradation of
the nitroaromatic compound. Metals are the elements that show the
highest degree of metallic activity, whereas non-metals are those ele­
ments that have no metallic behaviour. Metalloids are elements that are
intermediate between metals and non-metals. The concentration of
metals and metalloids in the industrial areas is high due to the contin­
uous anthropogenic activity (Man et al., 2020). These pollutants cause
severe diseases, like cancer, therefore, raising awareness of the health
hazards related to toxic chemicals present in wastewater is very much
needed, primarily about the absorption of metals in soil, water, and
edible plants from the contaminated site. Wastewater is highly
contaminated with harmful pollutants, such as Mn, Ni, Cd, Cu, As, Cr,
Pb, Zn, Ni, and Cr creating a problem for the use of the herb in cultivated
fields (Yu et al., 2012). Moreover, certain species like Cistus salvifolius,
Mentha suaveolens, Phytolacca americana, Digitalis purpurea, Agrostis
castellana, Hypochaeris radicata, Pteridium aquilinum, Pinus pinaster have
potential for the detoxification of metals and metalloids like Zn, W, Sb,
and As, which is feasible for wastewater treatment (Favas et al., 2014).
MBPs are compounds that are responsible for binding to metals like
Fe, Cr, Zn, As, Cd, Ni, and Pb, respectively. Naturally occurring heavy
metals binding proteins including PCs and MTs are rich in cysteine
residues. It is believed that the improved tolerance or resistance of
hyperaccumulating plants is due to the presence of an elevated chelating
molecule in the plant cells like MTs which are cysteine-rich proteins, and
PCs (cysteine- and glutathione-rich compounds). Phenolic compounds
can help in the sequestration of metals and defend the cells from harmful
proteins. The current study in molecular biotechnology like omics
analysis improves the capacity of phytoremediation of hazardous pol­
lutants by metagenomics, metatranscriptomics, and metabolomics
(Sarma et al., 2019). These pollutants are considered as critical to the
health of plants and animals.
Phytoremediation is the remediation of industrial contaminants that
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Bioresource Technology 326 (2021) 124750
is promising and considered as a low budget and green technology for
the elimination of hazardous pollutants. However, few efforts with the
help of plant and microbes’ interface will clean and detoxify from heavy
metals and organic pollutants at the structural, physiological, and mo­
lecular levels. The relationship between plant and microbes includes
various processes like biochemical, physiological, endogenous, genetic
as well as the secondary metabolite synthesis which help in wastewater
degradation and detoxification. The studies of metal-binding protein or
peptides using heavy metals resistance/tolerance or build-up by mi­
crobial cells attracted more interest recently. The metals concentration
in wastewater and plant parts depend on the industry raw material and
accumulation potential and biochemical response of a plant during
specific stress condition. The capability and response of the plant during
metal stress were measured by bioconcentration and translocation fac­
tor. For example, the Chenopodium album showed maximum accumula­
tion of Mn in leaves when grown in pulp and paper industry wastewater
(Sharma et al., 2020a). While Argemone Mexicana showed maximum
accumulation of Fe in root from sugarcane-molasses based distillery
waste. Moreover, Brassica napus hairy roots have the potential to remove
80% of Cr from tannery wastewater (Perotti et al., 2020).
This review documents the metals and other toxic pollutants present
in industrial wastewater after secondary treatment. It contained various
types of carcinogenic, mutagenic, and endocrine-disrupting chemicals
(EDCs) like compounds among other contaminants, including heavy
metals. Molecular biology is considered as a new tool for the accelera­
tion to clean-up environmental pollution. Therefore, it is crucial to
investigate the phytoremediation process with microbiomes to illustrate
the stress tolerance pathways.
Interdisciplinary biotechnology studies may alleviate the dynamic
existence of environmental issues related to water, human health, and
agriculture to meet sustainable development goals (SDGs) (Baleta et al.,
2019). Phytoremediation with microbes is considered as one of the top
ten biotechnology opportunities in terms of preventing environmental
pollution (Eapen et al., 2007) (Fig. 1).
2. Industrial wastewater: main source of environmental
pollutants
Phytoremediation is an efficient way to clean up contaminated areas
and it has long-term suitability and environmental benefits. The key to
the sustainability of the society is industrialization but environmental
pollution is a major concern (Pandey et al., 2017). Its impact on the
development should be clear while thinking about the progress in the
ecosystem (Kassaye et al., 2017). Water is among the most valuable
natural resources and essential for all the forms of life, but different
activities, such as rapid population growth, urbanization, and industri­
alization ultimately pollute the environment every day. Moreover,
approximately 30,000 people are dying from water-related diseases
nearly every day (Howard et al., 1985). One of the fast-growing tradi­
tional industries with high environmental risks is the pulp and paper,
distillery, and tannery industry (Christopher et al., 2016; Sharma et al.,
2020a; Yadav et al., 2020). The estimated amount of wastewater dis­
charged by the Indian tannery, pulp and paper, and distillery industry
was about 25,000 kiloliters per day (KPD), 50,000 m3/day, and 5–10
million per day, along with high organic pollutant concentrations (Tare
et al., 2003). The primary issue for the environment and living organ­
isms is the release of heavy metals from industries.The huge amount of
wastewater discharged from industries like the pulp and paper industry,
distillery, and tanneries industry after secondary treatment can directly
affect the aquatic and terrestrial life (Mehmood et al., 2019). The leather
industry uses nearly 90% of Cr-based tanning agents to process raw skins
into leather. Treated wastewater from the pulp and paper, distillery, and
tannery industry have different properties, such as color, pH, TDS, TSS,
BOD, COD, total solids (TS), electric conductivity (EC), and heavy metals
and metalloid viz., Mg, Cu, Zn, Cd, Fe, Cr, As, Pb, and Ni which are above
the permissible limit (Table 1). In wastewater, the dark color was
P. Sharma et al.
Fig. 1. Plant and their mechanism with rhizospheric microbes and their accumulation process. PGPB- Plant growth promoting bacteria, HM-Heavy metals, MBPs-
Metal binding proteins, IAA- Indole-3- acetic acid.
reported to be exceptionally high, which may be a cause of complexity
and that can harm aquatic flora and fauna. The high toxicity is indicated
by the high BOD and COD ratio in industrial wastewater. The high
concentration of EC is related to the amount of ion and salt in contam­
inated water. The COD values are also heightened due to the release of
various extracts of wood and chemical compounds in wastewater used in
the pulping process (Muller et al., 2019).
Moreover, most of the tanneries (nearly 80%) are involved in the
process of chrome tanning, which releases ~2000–3200 tonnes of Cr
annually into the environment (Belay, 2010). It is well known that Cr
compounds have toxic, genotoxic, mutagenic, and carcinogenicity on
humans, animals, plants, and microbes. Cr present in many oxidation
states, ranging from − 2 to +6 but only types of trivalent (III) and hex­
avalent (VI) Cr are most prevalent and stable (Mishra and Bharagava,
2016). Udayan et al. (2020a); (2020b;) reported Cr biosorption using the
extracts of Calotropis gigantea (Udayan et al., 2020a; 2020b). Hazardous
metals discharged from the pulp and paper industries are polluted by
large areas of agricultural and freshwater environments (Chandra et al.,
2018). Selenastrum capricornutum, Chlorella vulgaris, Eisenia fetida,
Daphnia magna, Oryzias latipes and several other aquatic organisms’
growth and metabolism are highly affected by industrial wastewater
(Miyazaki et al., 2002). Phytotoxic and cytotoxicity assessment of pulp
and paper industry wastewater was conducted on Trigonella foenum-
graecum L Triticum aestivum, Aestivum sativa, Allium cepa, and Phaseolus
mungo (Haq et al., 2017; Sharma et al., 2020c). Researchers have
3
Bioresource Technology 326 (2021) 124750
recently been focusing on the environmental remediation of heavy
metals instead of chemical processes (Hu et al., 2019; Kumar and Tha­
kur, 2019; Sharma et al., 2020b).
3. Role of enzymes in phytoremediation
Phytoremediation via the native hyperaccumulator plants are widely
accepted for detoxification of metal-polluted wastewater as a cost-
effective sustainable technique (Rai et al., 2020). To solve such limita­
tions and its accelerated implementation, hyperaccumulators-based
phyto techniques processes could be improved by genetic engineering
(Kärenlampi et al., 2000). Besides, transgenic hyper-accumulators’
plants will also reduce the hazardous effects of environmental pollutants
on human health. Cytochrome P450 is composed of monooxygenases
that help in organic declination via hydroxylation and enhanced the
phytoremediation of metals (Rostami and Azhdarpoor, 2019). Cyto­
chrome P450 enters to plants in phase I like functionalization and
enhances the degradation process of organics, phase II conjugation with
glutathione and sugars. To speed up the rate of remediation of toxic
chemicals from fertile land, gene editing techniques are used (Takahashi
et al., 2001). For better performance of phytoremediation, it is impor­
tant to understand the gene engineering and molecular mechanism with
stress resistance against environmental pollutants. The advancement in
phytoremediation required for the detoxification of organic xenobiotics
via microbial consortia associated with below-ground plant biomass
P. Sharma et al. Bioresource Technology 326 (2021) 124750

Table 1 remediation of metals which are present in Pseudomonas strains. More­

Physico-chemical characteristics and heavy metals contents of discharged over, Serratia sp and Trichosporon sp. 4S3 are potential bacterial strains
wastewater of pulp and paper, distillery, and tannery industry in India isolated from a Zn contaminated agricultural field with potential of
(Chowdhary et al., 2018; Bhargava and Mishra, 2018; Chandra et al., 2018). bioremediation capacity (Munoz et al., 2012; Jain et al., 2020; Ameen
Parameters Pulp and Distillery Tannery Permissible et al., 2020). Recently Singh, 2020 reported that cyanobacteria have
paper wastewater wastewater limit (EPA, great potential for metals tolerance and biosorption (Tables 2 & 3).
industry (mean) (mean) 2002)
wastewater
(mean) 3.1. Metal-binding proteins as a remediation tool
pH 8.1 ± 0.20 8.9 ± 0.3 7.8 ± 0.2 5–9
Color (Co-Pt) 2500 ± 125 Dark brown Turbid Dark brown For plants and animals, heavy metal ions are important as trace el­
Total solid (mg 616 ± 120 47,422 ± 11,028 ± –
ements, but their increasing amount in the environment becomes
L− 1 ) 336 805.2
Total 560 ± 13.25 13158.33 ± 3491.3 ± 30
extremely toxic. The MBPs in microorganisms and plants have excellent
dissolved 52.04 239.4 potential for the tolerance of metals in the contaminated side of indus­
solid (mg trial wastewater (Table 4). Comprehensive classes of gene families in
L− 1 ) heavy metals transport, including natural resistance-associated macro­
Total 56 ± 2.13 194 ± 23.5 35
phage proteins (Nramps), adenosine triphosphatases (ATPases), cation
suspended
solids (mg
L− 1 ) Table 2
Chemical 17999 ± 53200.00 ± 5248.00 ± 120 Relationship between plant and microbes for tolerance and detoxification of
oxygen 205.00 200 561.00
heavy metals from industrial wastewater.
demand (mg
L− 1 ) Heavy Plant species Microbes Plant microbe’s Reference
Biochemical 6000 ± 127 2656600 ± 345693.78 40 metals mechanism
oxygen 208.16 ± 46.28
Pb Brassica Pseudomonas Increased Fuloria et al.,
demand (mg
napus fluorescens biomass 2009
L− 1 )
Zn Brassica B. mucilaginosus Increased Rathore
Electrical 1762 ± 86 13158.33 ± 20348.38 ± 1000
juncea HKK-1 biomass and et al., 2019
conductivity 52.36 587.139
metal
(μS cm 1)
bioavailability
Total phenols 413 ± 18.23 2.44 ± 0.06 2.52 ± 0.03 0.50
Cd, Ni Lycopersicon Bacillus sp. RJ16, Biosorption Madhaiyan
(mg L− 1)
esculentum Methylobacterium considerable et al., 2007
Heavy metals (mg L− 1)
oryzae amount of Ni
Iron (Fe) 67.53 ± 2.00 148.46 ± 5.21 ± 0.52 2.00
and Cd
0.87
Ni Brassica Enterobacter Increased Kumar et al.,
Zinc (Zn) 13.90 ± 0.30 10.35 ± 1.79 ± 0.03 2.00
juncea aerogenes, biomass and 2008
011.21
metal uptake
Cupper (Cu) 2.15 ± 0.06 2.38 111111 1.23 ± 0.06 0.50
Cu Cajanus Proteus vulgaris Increased Rai et al.,
± 0.05
cajan KNP3 germination, 2020
Cadmium (Cd) 0.255 ± 0.10 – – 0.01
biomass, and
Manganese – 9.56 ± 0.65 1.02 ± 0.25 0.20
chlorophyll
(Mg)
Ni Cicer Pseudomonas sp. Increased Ahemad,
Chromium 2.30 ± 0.06 2.32 ± 0.07 7.65 ± 0.46
arietinum biomass and 2019.
(Cr)
decreased metal
Nickel (Ni) 3.30 ± 0.02 3.38 ± 0.21 0.36 ± 0.03 0.01
uptake
Cd Vigna mungo P. aeruginosa Increased Ma et al.,
MKRh3 biomass and 2011
plays an important role. The microbe’s metal resistance mechanisms rooting, and
arise from the active efflux pumping of the metals from enzymatic decreased
detoxification and transforming a harmful ion into a less toxic. The­ cadmium
uptake
lypteris palustris and Typha latifolia are used for the removal of Zn and Cu
Cd Solanum Serratia Production of Chen et al.,
via the bioaccumulation process (Hejna et al., 2020). Few other poten­ nigrum L. nematodiphila IAA, and 2010
tial plants are also reported which are useful for metals phytor­ LRE07, siderophores,
emediation like Typha latifolia, Thelypteris palustris, and Helianthus Ni Ricinus Pseudomonas sp. Increased Rai et al.,
annuus L, (Hejna et al., 2020). communis M6, biomass and 2020
IAA
The degradation of xenobiotics compounds via microbes is an
Cr Eichhornia P. diminuta, Increased metal Rai et al.,
important function related explicitly to the phytoremediation process. crassipes uptake; 2020
For example, fungal Trichoderma virens based glutathione transferase mechanism
(GST) has improved the potential of phytoremediation against recalci­ unknown
trant polyaromatic hydrocarbons (PAHs) like anthracene via heterolo­ As Alnus firma Bacillus Production of Babu et al.,
thuringiensis GDB- IAA, 2013
gous over-expression in tobacco plants (Dixit et al., 2011). The toxic 1 siderophores,
effects of environmental pollutants can be reduced by endophytic mi­ and
crobes, like Stenotrophomonas sp, Flavobacterium sp Pantoea sp, and solubilization of
Pseudomonas sp, (Mitter et al., 2019). Moreover, Pseudomonas sp and phosphate
Zn Rhizosphere Thlaspi Increased zinc Lodewyckx
Bacillus sp were the most efficient in microbe-assisted phytoremediation
bacteria caerulescens uptake et al., 2002
(Sharma et al., 2020b; Cherian et al., 2012). In Pseudomonas sp, some mechanism
genes like Nah, pah, and phn are enhanced by phytoremediation As Helianthus Pseudomonas Increased Moeinzadeh
(Abbasian et al., 2016). Phytoremediation is an economically feasible annuus fluorescens growth et al., 2010
technique with microorganisms which produces several enzymes that mechanism
Fe, Zn, Phragmites Bacillus sp Production of Sharma
help in the degradation of organics (Hussain et al., 2018). For example, Cr IAA, et al., 2020e
gene encoding, extra-chromosomal DNA, and enzymes enhance the siderophores,

4
P. Sharma et al. Bioresource Technology 326 (2021) 124750

Table 3
List of the potential plants used for the remediation of heavy metals from the contaminated site.
Plant species Plant parts Metals Medium Metal accumulation Concentration of metal Reference
1
Alyssum heldreichii Leaves Ni Soil 11,80 1070–3280 mg kg Salihaj et al., 2016
Thlaspi caerulescens Shoots Cd Soil 263 19 mg kg1 Whiting et al., 2001
Sedum alfredii Leaves Cd Soil 2183 100 mM Cd Yang et al., 2004
Azolla pinnata Roots Cd Aquariums 740 3 mg L1 Rai, 2008
A. murale Leaves Ni Soil 4730–20,100 1070–3280 mg kg1 Salihaj et al., 2016
Eleocharis acicularis Shoots Zn Wastewater 11,200 NA Sakakibara et al., 2011
Euphorbia Cheiradenia Shoots Zn Soil 1873 2400 ppm Chehregani and Malayeri, 2007
E. cheiradenia Shoots Pb Soil 1138 13,500 ppm Chehregani and Malayeri, 2007
Corrigiola Telephiifolia Aerial parts As Soil 2110 3104 mg g1 Garcia-Salgado et al., 2012
Isatis pinnatiloba Aerial parts Ni Soil 1441 2000–3000 mg kg_1 Alitinozlu et al., 2012
Eleocharis acicularis Shoots Zn Wastewater 11,200 — Sakakibara, et al., 2011
Pteris cretica Aerial parts and roots As Compost 2200–3030 100 mg kg− 1 Wang et al., 2002
Alternanthera philoxeroide Root Mn Wastewater 77.20 46.37 mg L− 1 Sharma et al., 2020b
Eclipta alba L Root Mn Wastewater 65.37 46.37 mg L− 1 Sharma et al., 2020b

diffusion facilitator (CDF) family, and zinc transporters (ZIP) proteins

Table 4
like IRT, CdBPs, ZRT, HP, CP, and cation anti-porters’ family which help
List of plants and microbes’ metal-binding protein during the phytoremediation
in the metal transportation (Zhang et al., 2017; Khalid et al., 2021).
of metals in industrial wastewater.
Alternatively, these mechanisms are used in engineering of bacteria and
Name Example Metals Reference
plants for industrial wastewater restoration. Over-expression of metal-
Fungal 1 Neurospora crass MT Zn, Cd Cobine et al., binding proteins is usually used to enhance the accumulation and
2004 tolerance efficiency, and possible mechanisms regulating toxic metals
Fungal 2 Candida glabrata MT2 Cu, Cr García-
Santamarina,
are compartmentalization; active efflux; decreased cell membrane
and Thiele, 2015 transport; cell wall metal binding and, chelation. Novel metal-binding
Fungal 3 Candida glabrata MT2 As, Ni Mejare and proteins have a higher affinity, high metal-binding strength, and selec­
Bülow, 2001 tivity than nonmetal-binding proteins. The accumulation of various
Plant MTs Type 4 Triticum aestivum MT Zn, Ba, Fe Mierek-Adamska
metals by plants are classified as (1) Zn/Cd/Pb; (2) Ni accumulators and,
et al., 2018
Phytochelatins and Schizosaccharomyces Zn Cobbett and (3) Cu/Co. Plant root characteristics like high absorption, and root
other non- pombe Goldsbrough, density are appropriate for remediation (Tangahu et al., 2011). LamB
proteinaceous MT- 2002. and OmpA, are the outer membrane protein that is anchored to the
like polypeptides peptidoglycan sheet, were anchored to the metal-binding proteins. In
Plasma membrane of NRAMPs1 Fe2+, Zn2+ Avalos, 2004
1–12 repeats, the Neurospora crassa MT was expressed as a fusion in the
plant root Mn 2+,
Cd2+, periplasm of E with the maltose-binding protein (Fig. 2).
Cu2+, &
Pb2+
Bacteria, fungi, MTPs or CDFs1 Mn2+, Mejare and 3.2. Phytochelatins and metallothioneins
animal & plant Fe2+, Zn2+ Bülow, 2001
Arabidopsis root ZIP (ZRT&IRT) Cd, Ni, Mn, Mejare and
Zn & Fe Bülow, 2001 The most important peptides involved in metal accumulation and
tolerance are PCs and MTs. MTs are cysteine-rich proteins that is low
molecular weight (6–7 k Da) and found in prokaryotes and plants. PCs
are related to a family of peptides were first reported in Schizosacchar­
omyces pombe exposed to Cd as cadmium-binding compounds known as

Fig. 2. Metals accumulation in plants root and their detoxification through metals binding proteins like phytochelatins dependent pathway.

5
P. Sharma et al.
cadystins (Inouhe, 2005). PCs, contain cysteine-rich sulfhydryl groups,
and are small polypeptides with a basic consist (g-GluCys) n Gly, where
n 1⁄4 2–111 which bind and sequester heavy metal ions (Mirza et al.,
2014). PCs are enzymatically synthesized proteins which is involved in
the detoxification of metals that are measured in plants grown at high
concentrations of heavy metals, but very few studies have investigated
plant reaction even at lower metal concentrations which are environ­
mentally significant. Cysteine-rich MTs (20–30%), molecular weight
(w7 kDa), are thermally strong but have stable metal tolerance
comprising two cysteine-rich binding domains α and β. In-plants, there
are four MT genes, for example, class MT1 in the root area, class
MT2 genes in leaves, class MT3 in the ripe fruit, and PEC/pec MT genes
in the seeds (Huang et al., 2011). The leaves and roots of the host plant
shows excess metal ions which are tolerant to both Cu and Zn obtained
by the activation of the class MT4 gene (Rodríguez-Llorente et al., 2010).
During metal binding, the absorption of metals occurs and translocate
into the root and shoot from the rhizosphere zone, there are several
intracellular methods as well as cytosolic approaches utilized for the
detoxification of metals through PCs (Polak et al., 2014). It encourages
the over-expression of natural chelators like PCs, MTs, and organic
acids, not only the penetration of metal ions via plant cells, as well as the
translocation using xylem. To improve the tolerance and uptake of E. coli
cells, MBPs and MTs are mainly utilized. MT biosynthesis is regulated
through different stimuli like hormones, cytotoxic agents, and metals.
Advanced technologies in understanding the regulation of PCs biosyn­
thesis and expression of the MT gene and the potential functions of PCs
and MTs in heavy metal homeostasis and detoxifying are discussed.
Recently, Balzano et al., 2020 reported that the microalgae PCs and MTs
are highly potential for bioremediation of metals. PCs-mediated metals
tolerance against lead exposure reported in Ceratophyllum demersum L.
(Mishra et al., 2006).
3.3. Signaling molecule and secondary metabolites in phytoremediation
Plant and microorganism interact through various chemical mole­
cules called signaling molecules, and they have the potential to play a
crucial role for both plants and microbes for sustainable growth and
fitness. Plant-associated with microbes has an important impact on plant
growth, tolerance to the toxicity of heavy metals as well as disease
resistance. However, the surrounding condition of plants and microor­
ganisms is responsible for their signaling molecules. In the interaction
between plants and microbes, the signaling of growth regulators like
ethylene (ETH), abscisic acid (ABA), salicylic acid (SA), and jasmonic
acid (JA), as well as several others play a significant role. In some plant
microbe interaction like fungal network, symbiotic interaction and
production of flavonoids are recognized as the primary signaling
element. Plant growth-promoting rhizobacteria (PGPR) are free-living
which could exchange root exudate chemical structure and thus
release numerous signaling molecules like Nod factor, Myc factor, the
volatile organic compound (VOCs), molecular associated microbes’
pattern (MAMPs), and exopolysaccharides. MAMPs reveal new rham­
nolipid MAMPs released from Pseudomonas aeruginosa defended against
grapevine plant pathogens. The MAMPs signaling molecule produced
from PGPR like Pseudomonas fluorescens and Chryseo bacterium balusti­
num, help in metabolism of Papaver somniferum (Bonilla et al., 2014).
Also, signaling molecules synthesized by rhizobia (Nod factors) and
AMF (Myc factor) helps to regulate the profiling of the root system and
regulate the lateral root system and nodule formation, thus promoting
the process of symbiotic evolution infections or nodule organogenesis
(Maillet et al., 2011). The environment and bacterial community pro­
vide a cell-to-cell signal called quorum sensing (QS) in this process
formation of a film called biofilm occurs.
The QS processes are induced by plant-microbes communication,
sporulation, competence, biofilm formation, and antibiotics. N-acyl-L-
homoserine lactones (AHLs) are an important part of the QS molecule
network (Wang et al., 2010). Research on importance of microbes and
6
Bioresource Technology 326 (2021) 124750
plant signaling compounds is required to study their roles to mediate
this inter-kingdom interaction in the rhizosphere, which may build an
innovative practice to boost the phytoremediation process. The bacterial
siderophores are catecholate produced by enterobactin, and some are
carboxylates like rhizobactin and hydroxamates, i.e., ferrioxamine B. The
hydroxamates are the most common siderophores molecule produced by
fungi which belong to the family ferrichrome. Microbial community is
attached with mineral surface and action for the formation of a micro­
environment condition is helpful that protects microorganism from
different environmental stress condition. The microbial siderophores
has an essential role in the weathering and land formation in nature.
These are also promoting the mineral dissolution of the insoluble phase.
The synthesis of siderophores molecules by the way of sufficient quan­
tities of biocontrol agents restricts the supply of Fe3+ to the pathogen
and may contribute to the induction of host resistance to the pathogen.
Fe3+ is transformed into Fe2+ in the cell, which has a low affinity for the
siderophores cell which improves the growth of microorganisms.
4. Uptake toxic heavy metals via transport, translocation, and
transformation
Improved translocation of the absorbed metals from industrial
wastewater to a plant shoot is significant for hyperaccumulating plants.
The transformation of organic compounds is divided into three stages,
the first is chemical changes by oxidation, reduction, and hydrolysis, the
second is the conjugation of sugars, and amino acids of foreign xeno­
biotics and the third is sequestration (Rai et al., 2020). Uptake, trans­
location, and detoxifying of metals by spinach grown on a waste site are
polluted with metals (Eissa and Negim, 2018; Yan et al., 2020; Nze­
diegwu et al., 2020). Glutathione is released by glutathione synthetase
(GSHS), alpha-glutamylcysteine synthetase (γ-ECS), and glutathione S
transferase (GST). Two ATPs used for this molecular process led to
organic environmental toxins which are becoming detoxified (Guo et al.,
2008; Hussain et al., 2018). Unfortunately, there seems to be no natural
transporter inside plant cells for organic environmental pollutants.
Removal of metals via various physico-chemical and biological methods
have been proposed, which is considered as a challenging task in terms
of cost and technological difficulty (Sheoran et al., 2016).
Consequently, plant membranes that produce ATPase and proton
pump H+ are ubiquitin with electrochemical potential that energize the
absorption of nutrients. The mechanism of membrane transport depends
on the direction and electric charge of transport. In the outermost sur­
face of plasma membrane (pm), several plant transporters are present,
which help in the uptake of nutrients and cell efflux. Those are the su­
perior transporter category because they can also be activated for
functional characterization in the outer cell, like Xenopus oocytes. Root
surfaces have vast surface areas and strong chemical receptors, which
have evolved primarily to adsorb elemental nutrients. Plant transport
mechanisms for pollutants are immobile within their infancy, with two
related subfamilies of zinc transporters proteins implicated in Zn (II) and
Fe (II) accumulation being present in the excellent process. Some che­
lators molecules like ethylenediaminetetraacetic acid (EDTA) often help
in the production of auxin in the plant as regulators of growing plants,
while increasing the level of plant growth, translocation of nutrients and
accumulation of metals in plants (Naghipour et al., 2016).
Moreover, metals break the nucleic acids, DNA oxidation, DNA
protein crosslink, chromosomal aberrations, plant metabolism, gene
expression regulation, and DNA checkpoint effect on apoptosis. In
plants, different metals, such as Zn and Cd have more mobility when
compared to Cu and Pb. Metals are bound to the root cell wall during the
transfer from the soil and the concentration of heavy metals in root is
increased. Metal mobility from the root to the shoot is facilitated by
metal chelating agents, including citric acid, uric acid, malic acid,
organic acid, benzoic acid, amino acid, phenol, lactic acid, and thiols,
respectively. The plant absorbs metal from various sources in root cells
to the cation exchangeability of the xylem cell. Once heavy metal ion
P. Sharma et al.
enters plant root, it can either be deposited in the root or transferred to
the shoot and first highest from the start to the end of the xylem vessel
which is mainly deposited in the vacuole of the leaf. Besides, anions like
NO3 must be deposited in vacuoles for the balancing of significant and
acts as a carrier of growth and cell expression. Plastids as well as the
mitochondrial membrane performs a vital role in the organic and inor­
ganic molecule transport in a plant. Plastid is essential to transport
inorganic ions at this membrane, and nutrient assimilation and some
internal gene products are reported, but its transport pathway is little
known. Heavy metal is an essential nutrient for the growth and devel­
opment of plants. The membrane proteins represent a different class of
plant metal carriers and can act at tissues. Mesophyll and epidermal cells
are the most transportable of the heavy metal family. ATPase is a heavy
metal-associated transporter (HMAT) related to the transfer of free
metal ions from the cytoplasm into the plasma membrane. ATP-binding
cassette (ABC) transporters helps in the translocation of hormone-
like auxin, a successful developmental stimulate and abscisic acid (ABA)
involved in environmental stress tolerance. The comparison between the
bioconcentration factor and translocation factor is an essential param­
eter for analyzing the absorption of metals from wastewater to root and
root to shoot. The bioconcentration factor and translocation values were
less than one (<1.0), which is unacceptable for phytoremediation (Fitz
and Wenzel, 2002). As hyperaccumulator plants can effectively
concentrate metallic elements, such as Ni and Co into their cells showed
in Berkheya coddii from South Africa (Rue et al., 2020) (Fig. 3 & Table 4).
5. Research needs and future directions
Phyto-bioremediation is a socially accepted technique for accumu­
lation, tolerance, and detoxifying of the metals pollution emanating
from industrial contaminated sites. Phytoremediation is an
environment-friendly approach for the reclamation of the polluted area
without disturbing soil fertility, microbial community, and biodiversity
(Xiao et al., 2019). Future research on the combination of microbes-
plant interaction for detoxification and resistance of heavy metals
Fig. 3. Microbes interaction with metals and their biochemical process during metal detoxification at the contaminated site.
7
Bioresource Technology 326 (2021) 124750
from the discharged site is very much required. There is no technology
for the detoxification of wastewater contaminants for the prevention of
groundwater and river pollution. Although the treatment of wastewater
pollutants is widely studied, extremely limited experimental research
work has been carried out concerning metal-binding proteins and
plant–microbe commination for the mobility of heavy metals. The
communication of plant and microbe’s role is to act as decontaminators
from hazardous pollutants by improving phytoremediation and pro­
tecting the food chain in the ecosystem by decreasing the different
pollutants in agricultural land. The long-term beneficial effect of plant
microbe’s interaction with metal-binding proteins might result in
improved plant adaptability leading to sustainable development. The
cause of short-term interaction between plants and microorganisms is a
beneficial result for improved plant health. The present review focussed
on industrial wastewater pollution that influences the microbial com­
munity function and structure to build up a novel strategy for improving
plant performance, identifying the signalling molecule and MBPs assis­
ted phytoremediation technology.
6. Conclusion
This study explored the advantages and possible strategies of mi­
crobes and metal-binding proteins for sustainable phytoremediation of
metal-contaminated wastewater. Heavy metals and metalloids contam­
inants seriously cause health problems to a considerable number of
people worldwide requiring a novel technology for the detoxification of
industrial wastewater by employing phyto-bioremediation technology.
Hence, these techniques can be used as a field-scale green detoxification
technology for eco-restoration of the polluted sites. This review will
provide beneficial insights to support the potential development of a
successful and economically feasible heavy metals detoxification tech­
nology based on microbes and MBPs.
P. Sharma et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors acknowledge the support extended by the Director,
CSIR-NEERI towords facilating the work to perform this review. The
authors gratefully acknowledge all the anonymous reviewers and Ex­
ecutive Editor-in-Chief for their valuable comments and suggestion on
earlier version of this paper.
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