Journal of Environmental Management 287 (2021) 112279

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Journal of Environmental Management

journal homepage: http://www.elsevier.com/locate/jenvman

Review

Chromium pollution and its bioremediation mechanisms in bacteria:

A review
Bhupendra Pushkar a, b, *, Pooja Sevak a, c, Sejal Parab a, Nikita Nilkanth a
a
Department of Biotechnology, University of Mumbai, Kalina, Santacruz (E), Mumbai, 400098, Maharashtra, India
b
Global Biotech Forum, Maharashtra, India
c
Society for Innovations in Biosciences, Maharashtra, India

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

Keywords: Environment pollution is at its peak and is creating havoc for living beings. Industrial wastes containing toxic
Bioremediation pollutants have contributed to a great extent in this disastrous environment pollution. Chromium (Cr3+/Cr6+) is
Chromium highly toxic and one of the most common environmental pollutants because of its extensive use in industries
Bacteria
especially tanneries. Lack of efficient treatment methods has resulted in extensive chromium pollution. Biore­
Mechanisms
mediation of chromium using bacteria is very thoughtful due to its eco-friendly and cost-effective outcome.
Reduction
Efflux Bacteria possess numerous mechanisms such as biosorption, reduction, efflux or bioaccumulation, naturally or
Etc acquired to counter the toxicity of chromium. This review focuses on the bacterial responses against chromium
toxicity and scope for their application in bioremediation. The differences and similarities between Gram
negative and positive bacteria against chromium are also highlighted. Further, the knowledge gap and future
prospects are also discussed in order to fill these gaps and overcome the problem associated with real-time
applicability of bacterial bioremediation.

set by various environment monitoring agencies (EPA and WHO) (Sayal

1. Introduction
Environmental pollution by heavy metals has devastating effects on
humans, plants and aquatic animals etc. (Management, 2018; Mathur
et al., 2016; Yin et al., 2019) and has disturbed the natural ecosystem to
a great extent (Dabir et al., 2019). The major cause of environmental
pollution is unplanned industrial and urban development which neglects
the importance of a healthy environment. Such activities have increased
the levels of heavy metals pollution massively which disturbs the
ecological balance (Wang et al., 2018; Abarikwu et al., 2017; Posthuma
et al., 2019). As per one report of WHO, more than 1.7 million children
under the age of 5 years die because of exposure to toxic pollutants
including heavy metal (Xu et al., 2018). Therefore, environmental
pollution with heavy metals is a sensitive issue and needs immediate
attention.
Various kinds of toxic heavy metals such as chromium (Cr), cadmium
(Cd), zinc (Zn), copper (Cu), nickel (Ni), lead (Pb), mercury (Hg) etc. are
often utilized in various industrial processes which ultimately find their
way to natural resources like land, soil, river, and sea (Jaishankar et al.,
2014). Various studies from all around the globe have reported the
presence of heavy metals in the environment above the stipulated values
et al., 2016; Barkouch et al., 2016; Rahman et al., 2014; Yoshinaga et al.,
2018; Fan et al., 2019a). With the increase in heavy metal pollution in
the environment, the chances of human exposure to these heavy metals
have also increased. Non-degradable nature of heavy metals makes it a
potential threat to the living being. Heavy metals tend to circulate
within the ecosystem and bioaccumulate at various trophic levels.
Heavy metals from untreated waste can leach to groundwater and can
enter irrigation water where they are easily absorbed by crops (Banerjee
et al., 2019a). Heavy metal upon entering living being via air, water or
food etc. can have lethal effects on them (Yaashikaa et al., 2019a; Xu
et al., 2018; Majumder et al., 2017).
Chromium is known for its widespread utility in various industries
like tanneries, chrome-plating, chemical industry, mining, steel etc. (Liu
et al., 2020a; Elahi et al., 2019). With the increased use of chromium in
industries, it has surfaced as a common environmental pollutant
(Pradhan et al., 2019). Cr6+ at ≥0.2 mg/mL and Cr3+ at ≥1.0 mg/mL
can damage DNA (Novotnik et al., 2016). Hence, Cr6+ is listed as Class A
human carcinogen by US Environmental Protection Agency (US-EPA,
1998). Considering the toxic effects of chromium on various living be­
ings, it has been studied widely to find a sustainable solution to manage
its pollution. A large number of bioremediation studies have also been

* Corresponding author. Department of Biotechnology, University of Mumbai, Mumbai, 400098, Maharashtra, India.
E-mail address: bpushkar@mu.ac.in (B. Pushkar).

https://doi.org/10.1016/j.jenvman.2021.112279
Received 21 November 2020; Received in revised form 10 February 2021; Accepted 25 February 2021
Available online 8 March 2021
0301-4797/© 2021 Elsevier Ltd. All rights reserved.
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

2. Physico-chemical properties of chromium

Abbreviations
Chromium is a transition element and belongs to VI-B group of the
Cr6+ Hexavalent Chromium periodic table. It’s ground electronic configuration is [Ar] 3d5 4s1
Cr3+ Trivalent Chromium (Mathur, Kalaji and Jajoo, 2015). It is a highly reactive element and
mg/L milligram per litre exists in nature in several oxidation states which ranges from − 2 to +6.
EPS Exopolysaccharide Out of these oxidation states only trivalent (3+) and hexavalent (6+)
LPS Lipopolysaccharide forms of chromium are predominant due to its stability in nature. But
ROS Reactive Oxygen Species their chemical properties contradict and thus impacts living cells in
DMRB Dissimilatory Metal Reducing Bacteria different ways (Bharagava and Mishra, 2018a,b,c). Tri and hexavalent
SOD Superoxide Dismutase ionic forms of chromium show difference in their physicochemical
CAT Catalase properties and biological reactivity (Sanjay et al., 2020). Trivalent
chromium (Cr3+) is less toxic and also acts as a micronutrient and is
required for growth and metabolic activities in some organisms (Ma
et al., 2019). However, Cr6+ is more toxic as compared to its counterpart
conducted which elaborate the potential of bioremediation processes for Cr3+ due to its high solubility and mobility in biological systems. Cr6+
chromium detoxification. rapidly permeates through the biological membrane and can readily
Progress in research of chromium remediation was analysed by interact with the biomolecules in the cell cytoplasm (Bharagava and
determining the number of studies published in the last 20 years (Fig. 1). Mishra, 2018a,b,c).
The search was performed for a different combination of keywords at Ionic state of the chromium is influenced by pH and redox condition
Scifinder portal. Progress in the research studies with time was analysed. of aqueous solution in which it is present. The solubility of Cr3+ depends
Studies on bioremediation increased from 2000 to 2014 while little on pH, as it is insoluble at neutral to alkaline pH (Ma et al., 2019). At pH
progress was seen after 2014. Same was observed for chromium biore­ below 5, Cr3+ predominates whereas, at higher pH, Cr6+ is more in
mediation studies. While the studies on the keywords such as chromium concentration (Kanagaraj and Elango, 2019). Cr6+ is a strong oxidizing
remediation and chromium bacteria were found to increase continu­ agent and is present in different oxyanionic forms i.e. hydro-chromate
ously with time. A large number of studies on bioremediation indicate its (HCrO−4 ), chromate(VI) and dichromate(VI) in the aqueous system. In
popularity in treatment process. A thorough analysis of the large infor­ acidic conditions, Cr6+ is present in form of dichromate (Cr2O2-7) while
mation available on the chromium bioremediation can help in designing in the alkaline condition it is mostly found as chromate (CrO− 4) (Srinath
the efficient treatment process for on-ground application. et al., 2002). The basic properties of different forms of chromium are
This review attempts to present different aspects of chromium presented in Table 1.
bioremediation with respect to microorganisms. Levels of chromium
pollution reported around the world was analysed exhaustively to un­
derstand its current status along with the possible sources of chromium
pollution. Microorganisms have chromium remediation properties and Table 1
special emphasis has been given to bacteria, which have a great po­ Physical properties of the different forms of chromium (WHO, 1996).
tential to bioremediate chromium. An effort has been made to discuss Properties Melting point Boiling point Solubility in water Density (g/
the bacterial chromium bioremediation mechanisms in detail. Also, the (◦ C) (◦ C) (g/litres) cm3)
gaps and bottleneck in the field of chromium bioremediation were Cr 1185 2672 Insoluble 7.14
analysed in order to build a bridge between available knowledge and CrCl3 1152 – Slightly soluble 2.76
challenges to be overcome. K2CrO4 968.3 – 790 2.73
Cr2O3 226 4000 Insoluble 5.21
CrO3 196 – 624 2.70

Fig. 1. Graphical presentation of the progress in the research paper published on different topics when searched with suitable keywords (Scifinder).

2
B. Pushkar et al.
3. Sources of chromium pollution
Chromium enters into the ecosystem via natural or anthropogenic
sources and is recycled by the geochemical cycle to balance its levels in
the environment. An upsurge in chromium released by the anthropo­
genic activities has disturbed the normal geochemical cycle of chro­
mium and overburdened the ecosystem. Hence, increased chromium
concentration is noticed in soil, water resources, groundwater, sea, etc.
3.1. Natural sources of chromium pollution
Chromium is the twentieth most abundant element present in earth’s
crust. It is naturally present in combined form and the overall concen­
tration is 125 mg/kg (Kanagaraj and Elango, 2019). The natural mode of
chromium entry into the ecosystem is via weathering and rock leaching
of chromium from chromite mine and its other natural reservoirs (Zhang
et al., 2019). Ultramafic, basaltic and serpentinites rocks are natural
geological formations that store high concentrations of chromium and
are also the natural sources of chromium pollution. The average con­
centration of chromium in these rocks is 2400 mg/kg in ultramafic and
serpentinites and 200 mg/kg in basaltic. Under the natural geochemical
cycle, chromium leach from these rocks and move to various sites in the
ecosystem such as river, groundwater, soil etc. (Kanagaraj and Elango,
2019).
3.2. Anthropogenic chromium contamination sources
Various types of anthropogenic activities such as metallurgy, elec­
troplating, paint, pigment, tanneries, chemical production, dyeing, steel
production, and paper pulp are the major contributor of chromium to
the environment (Jaishankar et al., 2014). There are approximately
3000 tanneries across India mainly situated in Tamil Nadu, West Bengal,
Uttar Pradesh, Andhra Pradesh, Bihar, Gujarat, and Maharashtra
generating approximately 1,75,000 m3 wastewater per day. It consumes
a large volume of water and also generates a huge quantity of
chromium-containing wet waste. Tannery effluent contains chromium
in the range of 2000–5000 mg/L which is much higher than the stipu­
lated value of chromium in wastewater (Table 2) (Bharagava and Mis­
hra, 2018a,b,c). Both Cr3+ and Cr6+ are released in excess into the
environment, but the sources of these forms may vary. Cr3+ majorly
found in tannery waste and paint industries, however, Cr6+ is released
from mining, metallurgy, fossil fuel combustion, wood preservation and
cooling effluent (Kiliç et al., 2010).
Untreated effluent containing chromium from these industries in­
fluences various parameters of water resources like colour, TSS (Total
Suspended Solid), COD (Chemical Oxygen Demand) and BOD (Biolog­
ical Oxygen Demand). It also pollutes soils of agricultural field and water
used for irrigation which may channelize the entry of chromium in the
food chain (Yaashikaa et al., 2019a; Vijayaraj et al., 2018). Poultry feed
was also found to contaminated with chromium which is further accu­
mulated in body parts of the chicken, is another way of chromium entry
in the food chain (Ahmed et al., 2017).
Table 2
Permissible limit and toxic impact of Cr3+/Cr6+ (Banerjee et al., 2019a; WHO, 2017).
Agencies Bureau of Indian Standard United State Environmental Protection Agency
(BIS) (USEPA)
Fresh water 0.05 mg/L 0.011 mg/L
Drinking water 0.05 mg/L 0.1 mg/L
Industrial 2.0 mg/L – 2.0 mg/L
discharge
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Journal of Environmental Management 287 (2021) 112279
4. Chromium toxicity
4.1. Human
US Environmental Protection Agency (USEPA) designated Cr6+ as
one of the priority pollutant (Bharagava and Mishra, 2018a,b,c).
Mammalian cells are extremely sensitive to Cr6+ with LD50 of 0.15 mg/L
(Liu et al., 2015). It possesses mutagenic, genotoxic and carcinogenic
properties and therefore listed as Group A human carcinogen by Inter­
national Agency for Research on Cancer and the US Environmental
Protection Agency (Liu et al., 2015; Rahman and Singh, 2014; Huang
et al., 2017b). A study by Tiwari and De Maio has shown the increased
risk of cancer due to Cr3+/Cr6+ contaminated groundwater (Tiwari and
De Maio, 2017). A case study conducted on Sukinda Chromite Mine,
Odisha, India has revealed a correlation of Cr3+/Cr6+ contamination
with high to very high cancer risk in male as well as female. The cancer
risk increases with oral exposure of Cr6+ rather than dermal, due to less
contact time in the dermal pathway (Naz et al., 2016). Though, in
human, Cr6+ is usually absorbed via the respiratory tract, skin, and
digestive tract (Dong et al., 2018). Alimba et al. have reported chro­
mium (0.0052–52 mg/L) induced chromosomal DNA damage in
WIL2-NS cells which aggravate under folate deficiency (Alimba et al.,
2016). Upon absorption of Cr6+, it can alter the expression of genes by
interacting with cell biomolecules such as DNA, protein, lipid, etc.
(Karthik et al., 2017a). Cr6+ reduction generate various intermediates
such as pentavalent chromium (Cr5+), tetravalent chromium (Cr4+) or
Cr3+ end product. These intermediate can damage the DNA or
DNA-protein cross-links. Though, Cr3+ cannot cross cell membrane
easily but accumulate around the cell and alter the cell surface
morphology by damaging the cell membrane lipid which results in
disruption of cellular integrity and function. Cr6+ directly damage the
DNA while Cr3+ do this indirectly hence Cr3+ is less toxic then Cr6+
(Wang et al., 2017). Cr6+ also exhibits greater permeability in living
cells and tissues. Once inside the cells it acts as a strong oxidizing agent
by generating Reactive Oxygen Species (ROS) like superoxide [O2•-],
hydrogen peroxide [H2O2], and hydroxyl radical [•OH] during intra­
cellular reduction from Cr6+ to Cr3+, and contribute to its carcinogenic
effects (Karthik et al., 2017a; Banu et al., 2018). Wise et al. have
confirmed the chromosome damage upon Cr6+ exposure in cell lines.
Chronic exposure to Cr6+ also causes concentration-dependent aneu­
ploidy along with chromosomal instability which results in bladder
cancer (Wise et al., 2016). Even short term exposure to chromium is
found to generate stomach ulcers, irregular bowel movement and skin
irritation (Banerjee et al., 2019a).
Cr6+ is also reported to affect the pregnancy outcome and health for
subsequent next 2 generations. Cr6+ exposure can lead to spontaneous
abortion, stillbirth, preterm birth, and neonatal death. The neonate of
Cr6+ exposed woman may have respiratory issues, perinatal jaundice
and higher birth defects as Cr6+ accumulate in placenta tissue and
generate oxidative stress and high apoptosis. Also, sexual dimorphism is
reported to positively correlated with the level of Cr3+/Cr6+ exposure
(Banu et al., 2018).
Cr6+ concentration in air is strongly associated with incidences of
cancer in the respiratory system, buccal cavity and pharynx, prostate,
and stomach and also with mortality risk by lung, larynx, bladder,
kidney, testicular, bone, and thyroid cancer (Deng et al., 2019).
World Health Organization European Union Standards
(WHO) (EU)
– –
0.05 mg/L 0.05 mg/L
–
B. Pushkar et al.
Inhalation of Cr6+ initiates oxidative, genotoxic and proteotoxic stresses
which can cause lung cancer (Abreu et al., 2018). Cr6+ produces atypical
lesion in bronchial epithelial cells which may develop into malignant
neoplasm (Abreu et al., 2018). Cr6+ activate Gli2 in the bronchial
epithelial cell which proliferates the cell by inhibiting the autophagy
(Huang et al., 2017b). A study on human liver cell line (HepG2) confirms
the hepatotoxicity of chromium where Cr6+ generated oxidative stress
and induced caspase 3-dependent apoptosis with subsequent mito­
chondrial damage. Cr6+ affects the stress response pathway by inhibit­
ing the expression of heat shock protein namely Hsp72 and Hsp90a by
reducing the mRNA of Hsp72 and protein level of Hsp90a (Deng et al.,
2019). Besides this, Cr6+ translocate Nrf2 into the nucleus and activate
HO1, GCLC and SOD2 antioxidant via the Nrf-2 signalling pathway in
hepatocytes (Das et al., 2015). In an in vitro study, Cr3+/Cr6+ nano­
particles were reported to affect the platelet function by causing platelet
aggregation and decreased platelet membrane integrity and lysis
(Taterra et al., 2020). Like other heavy metals, chromium shares the
property of biomagnification upon entering in the food chain (Vijayaraj
et al., 2018) and is also reported to cause many disorders in human, like
eardrum perforation, ulceration and impaired foetal development
(Vijayaraj et al., 2018).
4.2. Aquatic animals
Chromium has a deleterious effect on aquatic organisms which show
varying response to different concentrations of chromium. Various re­
searchers have studied the effects of chromium on numerous aquatic
organisms and their reports indicates that aquatic animals are severely
affected via direct mortality or due to the sub-lethal effects induced by
chromium (Fernando et al., 2016; Arunkumar et al., 2000; Bozcaar­
mutlu and Arinç, 2007; Farag et al., 2006; Steinhagen et al., 2004). Fish
consumed by a large population around the globe has the potential for
bioaccumulation of heavy metals including chromium. Yin et al. (2019)
have reported the accumulation of Cr6+ in tissues of fish in order of head
> ventral muscle > dorsal muscle which are the edible parts of fish and
mostly consumed. Chromium also increases the total fat content in fish
(Yin et al., 2019).
Chromium accumulation in tissues depends on various factors that
vary with the type of species. Generally, chromium disrupts the enzy­
matic and metabolic pathways of aquatic animals. It also causes a
reduction in overall weight due to its neurotoxic effects that in turn
affects the food intake (Azmat et al., 2018; Fawad et al., 2016). Various
morphological and behavioural changes are provoked in aquatic ani­
mals which include irregular swimming, imbalance and abrupt move­
ments, decrease in food intake, etc. Increased mucus secretion is
reported in golden fish which is the immune response to the irritation
caused by chromium toxicity (Fawad et al., 2016). Another study on
Duttaphrynus melanostictus has revealed that chromium causes anemic
and leucopenic conditions and also contributes to genotoxicity. It also
exhibits a dose-dependant genotoxic effect in Asian toad which is widely
distributed. Finally, the mortality is induced by the various mechanisms
which is the collective action of chromium toxicity at different meta­
bolic levels (Fernando et al., 2016).
4.3. Microbial community
Microbial communities present at contaminated sites are directly
exposed to chromium pollution which has several detrimental effects on
their diversity. Adverse effects of chromium pollution on the microbial
community are highly dependent on the level of contamination and
other environmental factors such as pH, substrate concentration etc. (Pei
et al., 2018; Zheng et al., 2017). Chromium pollution decreases a viable
number of microbes present in the chromium polluted environment and
inhibits the growth of chromium sensitive microorganisms. However,
the chromium resistant microbes flourish in presence of chromium thus
changing the microbial diversity. Zheng et al. (2018) have reported that
4
Journal of Environmental Management 287 (2021) 112279
microorganisms such as Aphanocapsa delicatissima, Merismopedia punc­
tata, Merismopedia tenuissima, Microcystis aeruginosa, Diplochloris decus­
sata, Dictyosphaerium pulchellum, Actinocyclus normanii, Cyclotella
atomus, C. meneghiniana, Proteobacteria sp., Acidobacteria and Plancto­
mycetes reduce in number (Zheng et al., 2018), while Pei, Yu, Ji, Khan, &
Li (2018) have reported the increase in the number of microbes such as
Firmicutes and Bacteroidetes due chromium pollution. A study by Sathicq
& Gómez (2018) on the planktonic community have shown that the
relative abundance of chlorophytes and diatoms decreases, while that of
cyanobacteria increases in presence of chromium.
Change in microbial diversity of soil is one of the indicators of soil
quality. Hu et al. (2018) have reported that the chromium pollution
results in loss of richness of soil by decreasing the total nitrogen and
organic matter as well as the abundance of the fungal community (Hu
et al., 2018). Further, chromium is also reported to affect the metabolic
functions of the microbial community by inhibiting the metabolic
pathways and functional genes, especially those involved in the deni­
trification process (Thorgersen et al., 2017). It strongly affects nitrate
oxidizing bacteria followed by the ammonia-oxidizing bacteria and
denitrifying bacteria (Zheng et al., 2017). Anaerobic Ammonium
Oxidation (Anammox), an important reaction that converts nitrite and
ammonia to diatomic nitrogen and water is inhibited by chromium, thus
affecting the global nitrogen cycle (Yu et al., 2019a). Chromium at a
concentration of 5.0 mg/L reduces the heterotrophic ammonia and ni­
trate uptake rate, while at lower chromium concentrations there is
transient increase in the nitrate uptake rate (Zheng et al., 2017). Oxygen
uptake by microbes is also affected by chromium thus turning down the
whole respiration process (Zheng et al., 2018). A2O process is hampered
at high concentration of chromium (Sun et al., 2019) thus affecting the
microbial-based BOD and COD reduction of wastewater during
treatment.
4.4. Plants
Deleterious effects of chromium pollution on plants range from
biochemical changes to affecting the plant growth and crop yield.
Chromium polluted soil disturbs the overall growth of plants (Hu et al.,
2018). The increased concentration of chromium is reported to reduce
the growth of root and shoots in the brassica plant due to adverse effect
of chromium on auxin synthesis by the plant (Ghani et al., 2017).
Chromium reduces the total number of active reaction centres of
Photosynthesis System II (PS II) along with a decrease in the rate of
electron transport, changes in antenna size of PSII, heterogeneity of PS II
and damage of light-harvesting complex of PSII which result in impaired
photosynthesis process (Rai et al., 2004; Mathur et al., 2016).
The deleterious effect on plant development and yield is majorly
attributed by the oxidative stress generated by chromium. Moreover, the
harmful effects might be owing to the competitive absorption of chro­
mium instead of other divalent ions required for the normal develop­
ment of plant (Anjum et al., 2016). Chromium accumulation in various
parts of plants varies with different types of plants. In brown mustard,
chromium accumulates majorly in shoots while in tomato & pea it ac­
cumulates in roots (Pedreno et al., 1995). Active absorption of essential
mineral by the plant is affected in presence of chromium thus reducing
the mineral element composition in the plant where stems and branches
of plants show a drastic reduction in nutrient mineral elements. Brown
mustard and tomato growing in presence of 25–100 mg/L of chromium
pollution have shown a drastic decrease in its N, P, K Ca and Fe content
(Ghani et al., 2017; Pedreno et al., 1995). Chromium is antagonistic to
phosphorous absorption and mostly accumulate where phosphorous is
normally found in plants (Bishnoi, Dua, Gupta and Sawhney, 1993a,b;
Ghani et al., 2017; Pedreno et al., 1995). Further, chromium toxicity
also affects the germination of seedlings by affecting respiration as well
as the mobility of food reserve (Mathur, Kalaji and Jajoo, 2015). In pea
plant the number of pods per plant decreases with increasing concen­
tration of chromium and also the elongation of the embryonic axis is
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

diminished in presence of chromium (Bishnoi et al., 1993b). Also, it
inhibits the nodule formation in pea, thus affecting its nitrogen fixation
capability (Bishnoi et al., 1993a).
Chromium is reported to be phytotoxic to wheat, affecting its
metabolism with interveinal chlorosis in young leaves which turn to
necrosis at the later stage of growth. The concentration of reducing
sugars is also reported to increase in plants as chromium gets accumu­
lated in veins of the plant which hampers the export of reduced photo­
assimilated products to other parts of plants (Sharma et al., 1995).
Chromium damage the cell membrane in the plant by generating
malondialdehyde during lipid peroxidation (Anjum et al., 2016). In­
crease chromium stress decreases the protein content due to enhanced
protein degradation by proteases. However, increased proline concen­
tration under chromium stress is involved in the protection of thylakoid
membranes by quenching ROS (Anjum et al., 2016).
5. Chromium bioremediation
Pollution of natural resources by heavy metals is increasing at an
alarming rate which has a detrimental impact on living forms. Table 3
represent the chromium pollution reported in various studies which
confirms the severity of the issue. Numerous methods applied for
effective chromium remediation includes physical, chemical & biolog­
ical. With the advancement in technology, several combinations of
methods have been developed in order to achieve the goal of effective
remediation. Physical remediation methods such as landfilling, soil
washing, flushing, ultra-filtration, membrane filtration, and excavation
are conventional techniques for heavy metal remediation (Elahi and
Rehman, 2019; Sumiahadi and Acar, 2018). Whereas, chemical treat­
ment includes advanced oxidation, precipitation, solvent extraction, ion
exchange, adsorption, and chemical reduction. Perhaps, both physical &
chemical methods used for heavy metal remediation are not economi­
cally feasible and are deleterious to the environment (López et al., 2009;
Mwandira et al., 2017; Fan et al., 2019b). They often involve the gen­
eration of other hazardous chemicals which in turn causes pollution.
Also, the high cost involved and the problem of waste disposal
associated with these methods make these treatment methods unviable
(Vijayaraj et al., 2018; Elahi and Rehman, 2019).
On the other hand, biological methods of heavy metals remediation
are found to be more appropriate which involve the use of biological
mechanisms and processes. Biological means of waste treatment process
is termed as bioremediation which utilizes living organisms totally or
partially for treatment of pollutants to control the environment pollution
(Elahi and Rehman, 2019). Bioremediation technology offers several
advantages over other treatment processes like cost-effectiveness,
convenient to perform at large scale, efficiency, specificity, environ­
mental compatibility, etc. (Banerjee et al., 2019b; Huang et al., 2019a,
b). These attractive features of bioremediation process have attracted
the scientists around the world to study and develop bioremediation
process for remediation of heavy metals. Many studied have conducted
the chromium bioremediation in industrial effluent using bacteria iso­
lated from contaminated sites which confirms the potential in biore­
mediation process using bacteria (Faisal and Hasnain, 2004; Venkatesan
and Subramani, 2019; Raman et al., 2017; Elahi and Rehman, 2019).
6. Chromium resistant microbial diversity
Abundant studies have been conducted to explore the heavy metal
removal capability of bacteria (Fig. 1). A large number of bacteria
showing chromium bioremediation capacity have been isolated from
different habitat and identified (Table 4). Betaproteobacteria, Gammap­
roteobacteria, Firmicutes, and Alphaproteobacteria are major bacterial
communities found at chromium contaminated sites (He et al., 2016). A
study by Bansal, Coetzee, & Chirwa has shown the dominance of Gram
negative Pseudomonas aeruginosa, Serratia marescens, Alcaligenes faecal,
Klebsiella oxytoca in the chromium contaminated sludge sample and
their reduction properties under aerobic and anaerobic evironment
(Bansal et al., 2019). Various studies have supported the effectiveness of
bacterial consortia against chromium as compared to individual bacte­
ria. Different metabolic processes of bacteria present in consortia work
together to deal with the toxicity of chromium and hence give better
results. Thus, the metabolic versatility of bacteria complements each

Table 3
Cr3+/Cr6+ concentration reported at different water bodies above the standard.
Sr. Cr3+/Cr6+ reported area Country Concentration Any other heavy metal Source of contamination References
No. reported present

1 Xigu district, near Yellow river China 506.58 mg/kg in soil Copper and Zinc Industrial effluent Pei et al. (2018)
sample
2 Bandeirantes do Norte river Brazil 47.49 mg/kg in Other metal in limit tannery effluent Lunardelli et al.
sediment sample (2018b)
3 Clyde River catchment Scotland *971 mg/L Lead Naturally occurring ore minerals Bearcock et al. (2019)
4 Tarnaveni Romania *525.8 mg/kg total Lead and Manganese Chemical industry Mihaileanu et al.
chromium (2019)
5 Aosta Town Italy *0.165 mg/L Dolomite and calcite Superficial slag deposits by a steel Tiwari et al. (2019)
company
6 Birjand Iran *0.132 mg/L NM extraction of chromite mines Fallahzadeh et al.
andits drainage (2018)
7 Palar river India 0.060 mg/L Fluoride Tannery effluent Kanagaraj and Elango
andhydrogeochemical processes (2019)
8 Sukinda region India 3.022 mg/L Lead, cadmium, ferrous Chromite mining Mohapatra et al.
manganese, nickel (2018)
9 Mithi River India 593 mg/kg Arsenic, cadmium, Industries (U. Singare et al.,
mercury, lead 2012)
10 Tannery waste, Uttar Pradesh India 5.7 ± 0.2 mg/L Zinc, PCP, phenol Tannery industry (Bharagava and
Mishra, 2018a,b,c)
11 Jharia, Uttar Pradesh India 761 mg/L NM Tannery industry Banerjee et al. (2019b)
12 Chinnavarikkam, Vellore India 52.91 mg/kg NM Tannery effluent Karthik et al. (2017a)
13 Erode, Tamil Nadu India 490 mg/L BOD, COD leather tanneries Raman et al. (2017)
14 Shaanxi Province, surface water China 0.185 mg/L* TDS, NO−3 Loass and mudstone He and Li (2020)
15 Hazaribagh area, Soil sample Bangladesh 30,615 mg/kg NM Tannery industry Khan et al. (2020)
16 Dongxie channel of Changhua Taiwan 338.46 mg/kg NM Industries using chromium Tseng et al. (2019)
county, farmland soil
17 Vaniyambadi, Vellore, tannery India 5.46 mg/L Cadmium, copper, nickel Tannery effluent Yaashikaa et al.
effluent (2019b)

Note: *Highest reported and - not reported, NM-not mentioned.

5
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

Table 4
Efficacy of heavy metal remediation by bacteria.
Sr. Name of bacterial MIC Percentage of heavy metal removal/ Optimum pH for Optimum temperature for Reference
No. reduction removal removal

1 Cellulosimicrobium sp. 800 mg/ 99.33% at 50 mg/L 7 37 ◦ C (Bharagava and Mishra,

L And 62.28% at 300 mg/L 2018a,b,c)
2 Pseudomonas stutzeri 1900 27.47 mg/g of adsorbent 2 30 ◦ C Yaashikaa et al. (2019a)
mg/L
3 Bacillus cereus 2000 100% at 200 mg/L 7.5 37 ◦ C Banerjee et al. (2019b)
mg/L
4 Bacillus aerius S1 1820 2703.48 mg/g 8 37 ◦ C Elahi and Rehman (2019)
mg/L
5 Brevibacterium iodinum S2 1820 2600 mg/g 8 37 C
◦
Elahi and Rehman (2019)
mg/L
6 Citrobacter freundii NM (52%) 44.61 mg/L to 12.17 mg/L (effluent) (effluent) Vijayaraj et al. (2018)
7 Bacillus sp. M6 NM 45.9% of 200 mg/L NM 30 ◦ C Li et al. (2019)
8 Cellulosimicrobium funkei 250 mg/ 86.67% reduction at 200 mg/L 7 35 ◦ C Karthik et al. (2017a)
L
9 Stenotrophomonas maltophilia 400 mg/ 260 mg/L from 490 mg/L in effluent 7.3 NM Raman et al. (2017)
L
10 Microbacterium sp. Z5 1040 100% of 52, 104, 153 mg/L and 86% 9 NM Wang and Cui (2019)
mg/L of 520 mg/L
11 Enterobacter sp. SL 100 mg/ 91.10% of 100 mg/L 6 45 ◦ C Sun et al. (2020)
L
12 Bacillus licheniformis SR3 1000 95% of 100 mg/L 9 37 ◦ C Kumaresan Sarankumar
mg/L et al. (2020)
13 Shewanella sp. 65 mg/L 89% of 500 mg/L 7 37 C
◦
Kheirabadi et al. (2020)
14 Bacillus subtilis strain SZMC 100 mg/ 93.50% of 55 mg/L 5.02 35 ◦ C Liu et al. (2020a)
6179 J L
15 Morganella morganii 4600 92% of 4600 mg/L 7 37 ◦ C Princy et al. (2020)
mg/L
16 Halomonas sp. DK4 250 mg/ 81% of 100 mg/L 7 30 ◦ C Kalola and Desai (2020)
L

NM- Not mentioned.

other in heavy metal bioremediation (Ma et al., 2019). Besides being
efficient in heavy metal remediation, bacteria are often able to tolerate
other extreme environmental stress such as temperature, pH and
salinity, etc. which are often associated with polluted sites. Hence, the
multi-resistance character of bacteria can be explored to treat environ­
mental chromium pollution effectively. Karthik et al. (2017a), b have
isolated haloalkaliphilic chromium resistant Cellulosimicrobium funkei
AR8 from tannery industrial effluent which can be used for onsite
remediation of chromium. Various researchers have reported the chro­
mium reducing bacteria which can convert Cr6+ to Cr3+, which is a
non-toxic form. Physico-chemical properties and heavy metal content at
the polluted site have a direct effect on bacterial efficiency of heavy
metal remediation (Banerjee et al., 2019b). The microbial diversity at
contaminated sites plays an important role in heavy metal remediation
(Wang and Cui, 2019). A diverse group of bacteria resistant to chromium
have been reported in various studies and few of recently identified
chromium resistant bacteria are presented in Table 4.
7. Chromium detoxification mechanisms in microorganisms
Various researchers have isolated different types of bacteria pos­
sessing the chromium bioremediation capacity and also studied the
mechanism of its interaction with chromium (Zhu et al., 2019; Zhou
et al., 2017; Baldiris et al., 2018). Chromium resistant bacteria are re­
ported to exhibit an array of mechanisms to overcome the stress induced
by chromium in order to ensure their survival. These mechanisms
include biosorption, bioaccumulation, biotransformation, efflux, enzy­
matic reduction, non-enzymatic reduction, precipitation, cytosolic
binding, biofilm formation, etc. (Table 5) (Banerjee et al., 2019b).
Chromium resistant bacteria may use single or a combination of several
mechanisms to deal with the chromium toxicity (Bharagava and Mishra,
2018a,b,c).

Table 5
Different mechanisms of microorganisms.
Sr. No. Name of bacterial Mechanism involved Taxonomy of bacteria Reference

1 Azotobacter beijreinckii MTCC 2641 Exopolysachharide(EPS) secretion Azotobacteraceae Chug et al. (2016)
2 Bacillus strain TCL EPS secretion, Cr6+ reduction, and efflux Bacillaceae Banerjee et al. (2019b)
3 Cellulosimicrobium funkei I) extracellular reduction and II) intracellular reduction Promicromonosporaceae Karthik et al. (2017a)
4 Stenotrophomonas maltophilia Cr6+ reduction Xanthomonadaceae Baldiris et al. (2018)
5 Shewanella oneidensis Efflux and reduction Shewanellaceae Baaziz et al. (2017)
6 Lysinibacillus sphaericus OT4b.31 Efflux Bacillaceae Shaw and Dussan (2018)
7 Pannonibacter phragmitetus BB Reduction, efflux, Reactive Oxygen Species Rhodobacteraceae Chai et al. (2019)
detoxification
8 Sinorhizobium sp. SAR1 Biosorption and biotransformation Rhizobiaceae Jobby et al. (2019)
9 Aeromonas hydrophila ATCC 7966 Cr6+ reduction Aeromonadaceae (Huang et al., 2019a, b)
10 Pseudomonas brenneri Cr6+ reduction, accumulation, Cr3+ adsorption of LPS Pseudomonadaceae Banerjee et al. (2019a)
11 Sphingopyxis macrogoltabida SUK2 Biosorption (Cr6+ and Cr3+), Cr6+ reduction Sphingomonadaceae Prabhakaran et al. (2019)
12 Bacillus sp. CRB-1 Cr6+ reduction, efflux Bacillaceae Zhu et al. (2019)
13 Corynebacterium paurometabolum Biosorption and bioreduction Corynebacteriaceae Prabhakaran and Subramanian
(2017)
14 Pseudomonas alcaliphila strain NEWG-2 Biosorption Pseudomonadaceae El-Naggar et al. (2020)

6
B. Pushkar et al.
7.1. Cell surface interaction
The cell surface is the first site of interaction for various molecules
present in surrounding of the cell. It also serves as the first line of
defence against the outer environment and it also transmits signals in­
side the cell. Bacterial cell envelop contains various molecules such as
anionic lipopolysaccharides (LPS), phospholipid and membrane pro­
teins. In Gram-Negative bacteria, a thin layer of peptidoglycan is situ­
ated under the LPS layer and these two layers play an important role in
interaction with heavy metals on the cell surface. However, Gram-
positive bacteria possess only a thick layer of peptidoglycan on the
cell membrane (Fang et al., 2018). LPS in gram negative bacteria acts as
a metal chelator which facilitates the binding of chromium with the cell
surface. Heavy metals such as chromium are reported to induce the
enhanced secretion of lipopolysaccharide at outer cell surface thus
facilitating its binding with cell surface (Kiliç et al., 2010).
Cell surface also contains various functional groups such as carboxyl,
alkanes, amide, amines, hydroxyl, phosphonate, sulfonate, carbonyl,
imidazole, and phosphodiester which are part of larger molecules such
as peptidoglycan and lipopolysaccharide. These functional groups are
reported to be actively involved in chromium interaction on the cell
surface and its adsorption. Changes in the composition of cell surface
molecule occur in bacteria upon exposure to chromium (Karthik et al.,
2017a). Sohini Banerjee et al. (2019c) have observed a shift in FTIR
peaks which were corresponding to amino, carboxyl, nitrogen and ox­
ygen of peptide due to their binding with chromium. Chromium in­
teracts with bacterial cell surface via functional groups which are mostly
C- and O- based. Cell surface functional groups such as N–H, –CONH–
and C–NH2 do not interact with chromium at alkaline pH (pH 9) as these
groups are electrostatically neutral at alkaline pH, while –COOH and
–OH groups are negatively charged at this pH and hence can electro­
statically interact with chromium. Though, Cr3+ interacts with proton­
ated functional groups present on the bacterial cell surface via
electrostatic interaction (Li et al., 2019). Also, maximum interaction of
Cr6+ ions occurs at acidic pH because protonation of cell surface occurs
at this pH by the deposition of H+ ions which helps in the electrostatic
attraction between Cr6+ (as HCrO−4 ) and the charged cell surface. With
an increase in pH, the HCrO4− starts transforming to CrO2− 4 and Cr2O7
2−
ions and the protonation of the surface of adsorbent is disabled with the
availability of OH− ions, which causes an increment in the electrostatic
force between negatively charged particles and adsorbent surface. This
reduces the adsorption of Cr6+ at alkaline pH (Yaashikaa et al., 2019a).
Like charges on the bacterial surface tend to neutralize in presence of
chromium thus inducing the clumping of cells which is one of the re­
sponses and a self-defence mechanism against chromium toxicity (Kar­
thik et al., 2017a). Cr6+ can also form insoluble Cr3+ which is colloid
chromium hydroxide and is absorbed on the surface of the bacterial cell,
which leads to a change in total protein composition of the cell surface
(Asatiani et al., 2004). It is reported that topological protrusions appear
on the cell surface after the interaction of chromium (520 mg/L) with
bacteria, which may be due to the absorption of Cr3+ and subsequent
changes in the protein composition of the cell surface (Wang and Cui,
2019). Cell surface also acts as Cr6+ reduction site in certain bacteria like
A. oxydans leading to the formation of Cr5+ complex on its cell surface.
Cr5+ reduced from C6+ can penetrate the cell and also anion-transport of
(CrO4)2- occurs via cell surface (Asatiani et al., 2004). Sulfate trans­
porters present on cell surface help in the transport of chromate (CrO2− 4 )
due to its similar structure. Thus chromium competitively inhibits sul­
fate uptake, which is compensated by increasing the uptake of
cysteine-containing molecules by cell (Gang et al., 2019). Overall, the
bacterial cell surface plays a very important role in chromium resistance
and chromium remediation.
A wide group of gram negative bacteria are reported to be resistant to
chromium (Table 5) whereas Bacillus sp. predominates among gram
positive chromium resistant bacteria (Table 4) (Bansal et al., 2019;
Purwanti et al., 2017; Shaw and Dussan, 2018). A microbial study on
7
Journal of Environmental Management 287 (2021) 112279
chromate mine seepage water also showed the dominance of Gram
negative bacteria (Dey and Paul, 2010). Though the Gram negative and
positive bacteria are different but chromium resistant gene is shared
within and across the two types of bacteria. This may be due to the
horizontal gene transfer among bacterial communities and selection of
chromium resistant bacteria over a period of time (Abou-Shanab et al.,
2007). Patra et al. have studied the chromate reductase gene of Gram
positive bacterial isolate namely Arthrobacter aurescens strain MM10,
Bacillus atrophaeus strain MM20, and Rhodococcus erythropolis strain
MM30 which have shown >99% sequence similarity with available
Gram negative E. coli and Shigella spp. (Patra et al., 2010).
A comparative study between E. coli and Staphylococcus epidermidis
by Quiton et al. (2018) have shown higher biosorption potential of Gram
negative E. coli bacteria due to negative charge of cell wall (LPS)
structure (Quiton et al., 2018). However, the Gram positive bacteria
achieve chromium biosorption largely due to the presence of high
content of anionic polymers in cell wall majorly comprising of pepti­
doglycan teichoic or teichuronic acids. The cell surface ligands such as
phosphoryl, SO2− 3 , RNH2 and R2NH, carbonyl (COO ) groups of Gram
−
positive bacteria possess high affinity for transition metal cations
including chromium (Şahin and Öztürk, 2005). While Prabhakaran and
Subramanian in 2017 have revealed that the hydroxyl group is also
involved in chromium biosorption in Gram positive bacteria at pH 1 to 4
(Prabhakaran and Subramanian, 2017).
Gram negative bacteria can efficiently reduce Cr6+ extracellularly as
compared to Gram positive bacteria due to presence of lipopolysac­
charides, lipoproteins and phospholipids in the outer membrane (Batool
et al., 2017; Faisal and Hasnain, 2004b; Faisal, M., & Hasnain, 2001).
According to Shaw and Dussan, efflux pumps and regulators of
Gram-negative and Gram-positive bacteria can be clustered in lineage I
and II. Amino acid alignment study of these clusters revealed the pres­
ence of different amino acid signatures and conserved regions in each
lineage. Gram negative efflux pumps and regulators are grouped in
lineage I and Gram positive in lineage II (Shaw and Dussan, 2018). The
similarities and difference in the response of Gram negative and positive
bacteria to Cr3+/Cr6+ is schematically represented in Fig. 2.
7.2. Exopolysaccharide (EPS)
Bacteria secrete exopolysaccharide (EPS) as a protective mechanism
against extreme conditions present in the surrounding environment.
Exopolysaccharide is majorly composed of proteins & carbohydrates,
which accounts for 75–90% of its total composition. The other minor
components of EPS include nucleic acids, lipids, lipoproteins, glyco­
proteins, lipopolysaccharides, phospholipid, uronic acid and humic acid
(Vu et al., 2009; Chug et al., 2016). EPS derived from different sources
show variations in chemical composition and are highly complex in
nature. Metal chelation by EPS is one of its property that makes it useful
in chromium remediation. The chelation property of EPS is attributed by
various functional groups such as carboxyl, phosphoryl, sulfhydryl,
phenolic and hydroxyl present on its macromolecules (Chug et al., 2016;
Saba et al., 2019).
EPS can be divided into three types according to the multiple-layer
structure model: soluble EPS (S-EPS), loosely bound EPS (L-EPS) and
tightly bound ESP (T-EPS). The protein and polysaccharide present in
the EPS act as an electron donor during the Cr6+ reduction by the
extracellular enzyme as shown in Fig. 3. EPS immobilize the Cr3+ after
reduction via its negatively charged functional groups such as carboxyl
and hydroxyl (Yu et al., 2019b). The nitrogen and oxygen groups of the
LB-EPS and T-EPS transfer their electron to biosorbed Cr6+ and reduce it
to Cr3+ (Wang et al., 2014). L-EPS layer is reported to contribute to
non-enzymatic chromium reduction activity as well as Cr3+ immobili­
zation (Banerjee et al., 2019b). FTIR analysis of chromium interaction
with cell surface suggests the involvement of –OH groups of carbohy­
drate and –NH of primary amine of membrane proteins in
non-enzymatic chromium reduction (Srinath et al., 2002).
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

Fig. 2. A comparative analysis of the chromium response by Gram negative and Gram positive bacteria.

Fig. 3. Interaction of Cr3+/Cr6+ with different bacterial EPS.

The S-EPS can bind to Cr6+/Cr3+ after releasing from cells. While T- content of EPS under Cr6+/Cr3+ stress. And the production of L-EPS and
EPS remain associated on cell and accumulate Cr6+/Cr3+ on the cell T-EPS increases under low chromium pollution (Li et al., 2020). In Ba­
surface (Johnson et al., 2020). Bacillus subtilis increases the protein cillus sp. Strain MRP-3, T-EPS plays a major role in the adsorption of

8
B. Pushkar et al.
chromium as compared to LB-EPS, where functional groups of T-EPS are
involved in Cr6+/Cr3+ binding (Shao et al., 2019). Pseudochrobactrum
saccharolyticum LY10 is reported to increase T-EPS production with
increasing Cr6+ concentration around its cells to mitigate Cr6+ toxicity
by accumulating Cr6+ in EPS (Long et al., 2019). Also, the nitrogenous
component of S-EPS is reported to increase in microorganism in pres­
ence of Cr3+/Cr6+ in activated sludge as a cell response mechanisms to
heavy metal (Liu et al., 2020b). Further, the EPS producing bacterial
cells flocculate in presence of chromium, which acts as a defence
mechanism against chromium toxicity (Srinath et al., 2002). Thus, EPS
helps bacteria in various ways to remediate chromium. Future research
focusing on the enhancement of EPS production by bacterial cells can
help in further improving the process of bioremediation of chromium
and other heavy metals.
7.3. Biosorption
Biosorption is a physicochemical interaction between bacteria and
heavy metals which leads to adsorption of heavy metals on the bacterial
surface (Mullen et al., 1989). It is a non-specific response of bacteria
against toxic heavy metals and involves the binding of chromium with
the active functional groups present on the cell surface (Morillo Pérez
et al., 2008). Biosorption process is passive in nature and depends on
physicochemical properties of the cell wall as well as the surrounding
environment. Bacterial efficiency to biosorp heavy metals varies with
different types of bacteria and also depends on their cell wall composi­
tion (Bharagava and Mishra, 2018a,b,c).
Biosorption comprises of two phases. The first phase of biosorption is
passive physical adsorption and takes place by complexation, ion ex­
change, coordination, adsorption, chelation and precipitation on the
bacterial cell surface. These physical adsorption processes of biosorption
may act synergistically or independent of each other, which depends
upon the type of bacteria and its surrounding environment. Also, it is
independent of bacterial metabolism and hence can be achieved by
living as well as non-living bacterial cells (Jarosławiecka and
Piotrowska-Seget, 2014). The second phase of biosorption is bio­
accumulation which is a slower process and involves metabolism
dependent active chromium transport into the bacterial cell. Bio­
accumulated chromium further gets sequestered intracellularly
following the routes like localization into the particular organelles,
binding to metallothionein, accumulation as a particulate heavy metal,
extracellular precipitation or formation of the complex (Srinath et al.,
2002; Bharagava and Mishra, 2018a,b,c; Elahi and Rehman, 2019).
Chromium is biosorbed either in form of Cr6+ ions or in its reduced form
as Cr3+ ion (Ma et al., 2019).
C. freundii is reported to biosorb chromium through extracellular
polysaccharide substances which leads to alteration in cell morphology
and increase in its cell size (Vijayaraj et al., 2018). P. alcaliphila strain
NEWG-2 has phenolic, carbonyl, ester, acetyl, carboxylate, alkanes and
carbonyl functional groups which interact with Cr3+/Cr6+. Cr–O and
Cr–N complexes found on P. alcaliphila strain NEWG-2 show a major role
of hydroxyl, carboxylate, phosphate and amino groups in biosorption
(El-Naggar et al., 2020). The live as well dead biomass of Kocuria sp.
ASB107 biosorb 40% and 54.1% of chromium at pH 4.5 (Akbarpour
Nesheli et al., 2018). Similarly, a study by Srinath et al. (2002) has re­
ported the increase in biosorption efficiency of dead biomass of Bacillus
circulans and Bacillus megaterium as compared to the live cell, which may
be due to lack of chromium toxicity to dead cells or the absence of active
efflux mechanism to transport out the intracellular chromium (Srinath
et al., 2002). Biosorption capacity of the broken cell wall is higher due to
the increased surface area of cell wall which provides more binding sites
for the heavy metal (Srinath et al., 2002).
Various functional groups such as amines, carboxyl, phosphate, and
hydroxyl groups present on the cell surface are involved in chromium
biosorption via protonic exchange (H+) (Raman et al., 2017). At lower
pH Cr6+ exist as HCrO− 4 and CrO2−7 (Kalola and Desai, 2020) and there is
9
Journal of Environmental Management 287 (2021) 112279
an increase in protonation of carboxyl and amino groups present on the
cell wall, which results in an electrostatic attraction between anionic
Cr3+/Cr6+ and cell surface. Therefore, lower pH help in higher bio­
sorption of chromium. But with the increase in pH, the negative charges
on cell wall increases due to deprotonation of the functional groups,
which causes repulsion between negatively charged Cr3+/Cr6+ and cell
surface, thus decreasing the overall biosorption. Also, the metals pre­
cipitate at higher pH which reduces the affinity and solubility of metal
ions thus decreasing the biosorption process (Jobby et al., 2019; Kalola
and Desai, 2020; Akbarpour Nesheli et al., 2018). At higher pH, bio­
sorption process is also hampered due to dual competition between
CrO2−4 and OH where OH predominate. Akbarpour Nesheli et al. have
− −
reported the improvement in biosorption upon immobilization of
Kocuria sp. ASB107 cells by cross-linking techniques (Akbarpour Nesheli
et al., 2018). Halomonas sp. DK4 possess hydroxyl, carboxyl, amide, and
phosphate groups which are reported to be involved in chromium
binding and can biosorb 63% of chromium at pH 6, beyond this pH the
removal efficiency decreases (Kalola and Desai, 2020). In contract a
study by Kurniawan et al. has shown the maximum chromium bio­
sorption by Gram positive isolate Azotobacter s8 (10.53%) and B. subtilis
(5.68%) while Gram negative P. putida could not perform any chromium
removal (Kurniawan et al., 2019).
Some studied have reported the maximum chromium biosorption at
alkaline pH. Rizvi et al. reported 96% of chromium biosorption in
B. subtilis at pH ranging from 8 to 9. With substantial decline in bio­
sorption at pH 5 which is in contrast to studies reported by Kalola, and
desai, 2020 and Jobby et al. (2019) (Rizvi et al., 2020). C. funkei can
biosorb chromium and also possess other chromium removal mecha­
nisms such as reduction and bioaccumulation. Optimum pH for bio­
sorption of chromium by C. funkei is 7. Chromium removal is
metabolism dependent in C. funkei and pH 7 supports the maximum
growth and active metabolism. Therefore, maximum chromium removal
occurs at pH 7 (Karthik et al., 2017b).
Based on available biosorption studies using different bacteria at the
varying condition, it can be inferred that biosorption efficiency varies
among different types of bacteria and is affected by pH, temperature,
initial Cr concentration and biomass used etc. Also, the cell wall
composition of bacteria affects the biosorption of chromium. The cell
wall composition of bacteria changes with the type of growth medium,
and growth conditions. Therefore, the same bacteria isolated from
different site have shown dissimilar biosorption results (Kurniawan
et al., 2019; Rizvi et al., 2020; El-Naggar et al., 2020). Considering the
research evidence, biosorption is found to be an effective chromium
bioremediation method. Further research for enhancing the biosorption
potential of microorganisms will add immense value to the heavy metal
bioremediation process.
7.4. Chromium reduction
Another very important and widely studied mechanism of chromium
detoxification in bacteria is the reduction of highly toxic and mobile
Cr6+ to less toxic and insoluble Cr3+ by chromium reductase enzyme or
by the non-enzymatic process. Most of the chromium resistant bacteria
exhibits chromium reductase activity. Bacterial species that are reported
to possess chromium reductase activity are Cellulosimicrobium sp.,
Ochrobactrum sp., Bacillus sp. JDM-2-1, Staphylococcus capitis, Cellulosi­
microbium funkei, Pseudomonas putida, Lysinibacillus fusiformis ZC1,
Achromobacter xylosoxidans SHB 204, Pediococcus pentosaceus, Provi­
dencia sp. (Bharagava and Mishra, 2018a,b,c; Thacker and Madamwar,
2005; Zahoor and Rehman, 2009; Karthik et al., 2017a; Biotechnol and
Stanford, 2003; He et al., 2011; Tadishetty Hanumanth Rao et al., 2017;
Ilias et al., 2011a, b; Thacker et al., 2006).
There are two types of chromium reductases such as membrane-
associated reductase and intracellular reductase depending on the
location of chromium reduction (Table 6). Various studies have reported
the presence of chromium reductase activity in the cell membrane, cell
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

Table 6 reduction.
List of bacteria having chromium reductase activity in different parts. Chromium reduction can occur in aerobic as well as in anaerobic
Sr. Name of bacteria Chromium reductase activity Reference conditions. The anaerobic reduction process is associated with the
No. attributed by membrane-bound reductase proteins/enzymes which are generally
1 S. aureus sub sp. Aureus Culture supernatant and cell (Ilias et al., involved in the electron transport chain, where Cr6+ acts as a terminal
debris (not in cell extract 2011a, b) electron acceptor. Under anaerobic condition, the electrons produced by
supernatant) ubiquinone passes to cytochrome c via cytochrome b and finally, the
2 P. pentosaceus ATCC Culture supernatant and cell (Ilias et al., reduced cytochrome c get oxidized to reduce Cr6+ extracellularly (Gang
25745 NC_009641.1 debris (not in cell extract 2011a, b)
supernatant)
et al., 2019). The reduced form Cr3+ either remains attached to func­
3 Bacillus strain TCL EPS and Cell membrane Banerjee et al. tional groups like amine, carboxyl, hydroxyl, sulfhydryl, and phosphate
(2019b) on the cell surface or is released in the surrounding. The overall Cr6+
4 Bacillus sp. M6 Cell envelop and cell Li et al. reduction under the anaerobic condition with glucose as a carbon source
cytoplasm (2019)
was reported as given below (eqs. (1)–(3)). The Cr6+ is converted to
5 Stenotrophomonas Soluble fraction of the cell Baldiris et al.
maltophilia (2018) insoluble chromium(III)hydroxide in aqueous solution. Glucose can be
6 Aeromonas hydrophila Intracellular and (Huang et al., replaced by other strong electron donors which facilitates the reduction
ATCC 7966 extracellular 2019a, b) process (Singh et al., 2011).
7 Oceanobacillus Cr6+ reduction by cell Zeng et al.
oncorhynchi W4 envelop (2019) (1)
(2)
cytoplasm and cell culture supernatant supporting the presence of (3)
membrane-associated & intracellular reductase (Ilias et al., 2011a, b).
Recent studies have also reported the constitutive expression of chro­ (Singh et al., 2011).
mium reductase with localization of chromium reductase at the mem­ Intracellular reductase is located in the cytoplasm which reduces the
brane (90%) and EPS (80%) fractions in Bacillus strain TCL (Sohini intracellular Cr6+ to Cr3+ in the cytoplasm and is encoded by chromate
Banerjee et al., 2019). Membrane-associated reductase involves sulfate reductase gene such as ChrR or YieF etc. (Fig. 5) (Sanjay et al., 2018).
transport channels present on the bacterial surface which transports The soluble form of reductase is constitutively produced in E. coli FACU
Cr6+ into the cytoplasm and during the process of transport, it also re­ strains but can also be induced (Mohamed et al., 2020). Aeromonas
duces the Cr6+ to Cr3+ at cell envelope (Li et al., 2019). hydrophila ATCC 7966 reduces chromium extracellularly using the
Steps involved in intracellular chromium reduction include. metal-reducing and respiratory pathway as well as intracellularly
(Huang et al., 2019a, b). Similarly, Desulfovibrio and Desulfuromonas
1. Chromium biosorption on the cell surface - Cr6+ in the sur­ reduce Cr6+ intracellularly by the enzyme in presence of NADH and
rounding medium is adsorbed on the cell surface of bacteria via the extracellularly by cytochrome. EPS also contributes to extracellular Cr6+
functional groups like alkane, amines, amide, etc. reduction with help of carboxyl and hydroxyl functional groups (Zhang
2. Cr6þ transport - Cr6+ exists in tetrahedral CrO2− et al., 2020). In contrast, Geobacter sulfurreducens PCA like dissimilatory
4 ionic form which is
analog of physiologically important anions such as SO2− 2− metal reducing bacteria (DMRB) have outer membrane cytochromes
4 and PO4
2− 2−
and is transported via SO4 and PO4 channel (Wang et al., 2017). which play a critical role in the extracellular Cr6+ reduction (Gong et al.,
3. Cr6þ reduction - The intracellular Cr6+ is reduced to insoluble Cr3+ 2018). Also, the cell envelope of Oceanobacillus oncorhynchi W4 reduced
by cytosolic molecules. 82.9% of Cr6+ as compared to only 11.1% reduction intracellularly. Cell
4. Cr3þ bioaccumulation - The reduced Cr3+ is accumulated in the envelope has major reduction properties in Oceanobacillus oncorhynchi
cytosol (Karthik et al., 2017a). W4 (Zeng et al., 2019) which is in contrast to C. funkei and
M. thermautotrophicus which perform reduction majorly in the cell
Reducing compounds produced by bacteria during carbon oxidation cytoplasm (Karthik et al., 2017a; Singh et al., 2015). The permeable cells
are utilized as an electron donor for chromium reduction instead of of Bacillus sp. M6 can perform reduction more efficiently as compared to
making building blocks of the cell (Karthik et al., 2017a). Parameswari the intact cells (Li et al., 2019). Whereas Oceanobacillus oncorhynchi W4
et al. (2009) reported that the presence of high chromium concentra­ show no difference in chromium reduction upon increase permeability
tions prevents the multiplication of bacteria and increases the reduction as major reduction occurs at cell envelope (Zeng et al., 2019).
of chromium. Bacterial energy is diverted to chromium reduction and its Cheng at al. have reported Cr6+ reduction in Shewanella oneidensis
detoxification, which leads to a decrease in bacterial biomass in pres­ MR-1 by bidirectional extracellular electron transfer (EET) where elec­
ence of chromium. Ikegami et al. have shown that bacteria in logarith­ trons are transferred from cell interior to outer membrane with the help
mic phase can perform Cr6+ reduction significantly as compared to of various redox proteins such as CymA (the electron-transfer hub),
bacteria in any other growth phase most probably due to the presence of MtrA (periplasmic c-type cytochrome), MtrB (β-barrel protein), MtrC
the high number of the active cell with maximum enzymatic activity and OmcA (two outer membrane c-type cytochromes) which are also
(Ikegami et al., 2020). Chromium reduction time increases with an in­ involved in the metal reduction and respiratory pathway. S. oneidensis
crease in chromium concentration which may be due to saturation of the MR-1 also secretes flavins to mediate reduction (Cheng et al., 2020).
enzyme (Jeyasingh and Philip, 2005). MtrC and OmcA are extracellular terminal reductase that transfers
Chromium exists as chromate (CrO2− electrons to Cr6+ during the reduction process in the metal respiratory
4 ) in aqueous conditions with pH
range from 6.5 to 9.0. Change in pH affects the ionic state of reductase pathway in S. oneidensis MR-1 (Belchik et al., 2011).
enzyme which in turn affects chromium reduction. Pseudomonas sp. Aerobic reduction usually occurs in the cytoplasm. Soluble enzymes
MAI4 can reduce chromium most efficiently at pH 7 (Wani et al., 2019). like flavoprotein, dehydrogenase, NADH-dependant nitroreductase,
E. coli FACU strains showed maximum growth and reductase enzyme azoreductase are involved in aerobic Cr6+ reduction (Chai et al., 2019;
activity at pH 7 and fluctuation in pH from 7 affected the reduction Dong et al., 2018). Shewanella oneidensis MR-1 is reported to use NADH
(Mohamed et al., 2020). Thus the chromium reduction rate decreases or endogenous electron reservoir for chromium reduction under aerobic
with either a decrease or increase in the optimum pH value of reductase condition, while under anaerobic condition, the electrons are fetched
enzyme. Besides pH, cell membrane permeability is reported to influ­ from the electron transport system involving membrane-associated cy­
ence the Cr6+ reduction rate in certain bacteria with intracellular tochrome as discussed earlier (Gang et al., 2019). The supply of external
electron donors is reported to enhance chromium reduction. Various

10
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

carbon sources like lactose, glucose, sodium formate, sodium acetate, alkaline pH. However, N-based groups such as while N–H, –CONH– and
sodium lactate, trisodium citrate, glycerol etc. are also found to be good C–NH2 cannot interact because of their electroneutrality which is in
electron donors for reduction (Li et al., 2019). Li et al. (2019) found agreement with a study by Zeng et al. (2019) on Oceanobacillus onco­
glycerol as an excellent electron donor for chromium reduction in Ba­ rhynchi W4 (Li et al., 2019). Cr3+ precipitate on the cell surface after
cillus sp. M6 (Li et al., 2019). Oceanobacillus oncorhynchi W4 show reduction on cell envelope Oceanobacillus oncorhynchi W4 via C– – O,
enhanced reduction in presence of glycerine followed by NADH, glucose C–OH and C–O–C groups as observed in Bacillus sp. M6 (Zeng et al.,
and lactate (Zeng et al., 2019). However, lactate act as a remarkable 2019).
electron donor for a reduction under anaerobic condition (Huang et al., Chromium reduction process promotes the synthesis of GSH, GSSG
2019a, b). ChrT protein reduces Cr6+ using NAD(P)H with propensity in and NPSH (Non-protein Sulfhydryl) in order to maintain a reduced
order NADPH > NADH > non-NAD(P)H (Gu et al., 2020). While E. coli environment in the cell (Gu et al., 2020). Pseudomonas brassicacearum
FACU showed an enhanced reduction in presence of NADH (Mohamed LZ-4 has potential to simultaneously bioremediate naphthalene and
et al., 2020). The presence of Fe2+ and Fe3+ positively influence the Cr6+ chromium. Also, the presence of naphthalene as a sole source of carbon
reduction in aerobic as well as anaerobic conditions by Gram negative has tremendously increased its Cr6+ reduction ability (from 25% to
Pseudomonas aeruginosa, Serratia marescens, Alcaligenes faecal, Klebsiella 96.2%) due to the induction of NahG in presence of naphthalene. H.
oxytoca (Bansal et al., 2019). Anthraquinone-2,6-disulfonate (AQDS) or Huang et al. reported that naphthalene catabolic gene NahG has Cr6+
humic acid enhances the Cr6+ reduction in Aeromonas hydrophila ATCC reduction ability as NahG deleted mutant showed 52% decrease in
7966. While the MtrC deleted mutant does not respond to AQDS dosing reduction activity. NahG encodes for salicylate hydroxylase (an oxido­
shows the role of the respiratory pathway in metal reduction (Huang reductase) with FAD as a cofactor, this FAD may be used to fetch elec­
et al., 2019a, b). Thus, enzyme activity of chromium reductase is posi­ tron from NADH for Cr6+ reduction (Huang et al., 2017a).
tively influenced by NADPH, carbon source, K2Cr2O7 and K2HPO4 and
negatively influenced by nitrogen source, NaCl and temperature and Ni
7.5. Efflux mechanism
(Banerjee et al., 2019b; Ma et al., 2019; Raman et al., 2017). Sulfur act as
an electron donor in heterotrophic Cr6+ reducers such as Desulfovibrio
Chromium when present inside the cell cytoplasm can damage vital
and Desulfuromonas (Zhang et al., 2020). As shown in Fig. 4 the reduced
biomolecules which are crucial for cell survival. Therefore certain bac­
sulfite pool in cell cytoplasm can also reduce the Cr6+ but with
teria have developed an active efflux pump as defensive machinery of
increasing chromium toxicity the sulfite pool decreases in the cell which
cells, which acts by extruding toxic chromium ions out of the cell into
also decreases Cr6+ reduction. Thus, the sulfur assimilation pathway is
the periplasm or in the surrounding. Generally, the efflux mechanism is
also useful for chromium detoxification in bacteria (Thorgersen et al.,
employed for various bacterial cell functions like maintaining cell ho­
2017).
meostasis, developing resistance to antibiotics, heavy metals & salts
The medium composition can alter the Cr6+ reduction product. As
which help them to survive under extreme conditions (Pal et al., 2019;
seen in Aeromonas hydrophila ATCC 7966 that simple salt medium leads
Canovas et al., 2003). Efflux system is one of the mechanisms that
to the generation of Cr3+ precipitate which accumulates on cell surface
majorly contributes to chromium resistance in bacteria and is reported
while a shift in medium to nutrient-rich composition has led to a gen­
to be encoded by ChrA gene (Dong et al., 2018). Deletion of ChrA gene
eration of the soluble organo-Cr3+ complex such as lactate-Cr3+, yeast
affects the survival of bacteria in presence of chromium due to the
extract-Cr3+, ketoglutarate-Cr3+, histidine-Cr3+. These organo-Cr3+
inactivation of efflux mechanism (Baaziz et al., 2017). Active efflux
complexes are much stable and do not readily undergo reoxidation as
pumps are reported to transport tetracycline along with chromium out
compared to than aqueous Cr3+ or Cr(OH)3 (Huang et al., 2019a, b). The
of the cell, thus many chromium resistant bacteria are also
reduced product is accumulated on cell membrane than supernatant and
antibiotic-resistant (Pal et al., 2019). Chromium resistant bacteria Ba­
cytoplasm in Pseudomonas sp. MAI4 after reduction in the cytoplasm by
cillus strain TCL is reported to effectively efflux ethidium bromide (EtBr)
reductase (Wani et al., 2019). Whereas Bacillus sp. M6 reduces Cr6+ in
and chromium, thus minimizing the intracellular damage by chromium
the cell cytoplasm (68.8%) and cell envelope (62.8%) and accumulate
(Banerjee et al., 2019b; Pal et al., 2019). Hence, the efflux of chromium
the reduced product in the cytoplasm as well as on cell envelope. The
works in cooperation with the efflux of other toxic molecules. Chromium
membrane-associated sulfate channel reduces the Cr6+ in Bacillus sp. M6
efflux is an energy-dependent chemiosmotic homeostasis process that is
while transporting it onto the cell envelope (Li et al., 2019). B. subtilis
driven by transmembrane potential, thus it is a concentration-dependent
MNU16 also accumulate Cr3+ in the cell cytoplasm after reduction
response (Shaw and Dussan, 2018). It is suggested to be associated with
(Upadhyay et al., 2017). Geobacter sulfurreducens PCA accumulates Cr3+
electron transport chain where efflux of chromium inhibits the electron
on cell wall-bound via carboxylate functional groups and also inside the
transport chain by fetching the electrons from electron transport chain
cell. Beside this, a small portion of reduced product Cr3+ also present in
to aid the expulsion of chromium out of the cell (Fig. 6). ChrA and ChrB
solution as after reduction by Geobacter sulfurreducens PCA. Cr3+ on the
genes are reported to be involved in Cr3+/Cr6+ transport by playing a
cell surface and in solution remained in organo-Cr3+ (Gong et al., 2018).
variety of roles such as generating membrane potential, electron transfer
Reduced Cr3+ bind to the cell by coordinating with C-based and O-
and transmembrane proton gradient. Cr6+ enters the cell via sulfate ion
based in Bacillus sp. M6, while no N-based groups are reported to
channel and is transported out using ChrA transmembrane protein. ChrA
involved in chromium binding. The negatively charged –COOH and –OH
protein generates transmembrane electrochemical proton gradient using
(upon dehydrogenation) can interact to Cr3+ via electrostatic forces at
H+ to efflux chromium out of the cell. ChrAT and ChrA cloned bacteria

Fig. 4. The pathway shows the steps of sulfur assimilation and how the chromium gets reduced by oxidizing the reduced sulfur pool (Thorgersen et al., 2017).

11
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

Fig. 5. Intra and extracellular Cr6+ reduction in bacteria by and accumulation of reduced product.

Fig. 6. ChrA efflux pump. Hydrolysis of bound ATP to ADP led to conformational changes in the metal-binding domain that transport the Cr6+ out of the cell. Source
of image (Shaw and Dussan, 2018).

have more resistance toward chromium then ChrT engineered bacteria.
Thus, showing the importance of ChrA based chromium efflux mecha­
nism in bacteria and transport of Cr3+ out of the cell via ChrA protein
after reduction (Gu et al., 2020).
Oxyanions such as sulfate and molybdate and inhibitors like vali­
nomycin and CN− can markedly hamper the Cr3+/Cr6+ efflux by ChrA
and ChrAB however NADPH significantly enhances the efflux (He et al.,
2018). Sulfate and molybdate act as an inhibitor of Chr efflux due to the
identical structure and size with chromate and competes with chromate
for ChrA protein, thus decreasing the overall chromium efflux. Valino­
mycine specifically allow K+ ion entrance and exit through trans­
membrane. Valinimycien inhibitory effect on efflux shows that chrome
efflux is a chemiosmotic process driven by the membrane potential
(Shaw and Dussan, 2018). On other hand, CN− is a respiratory inhibitor,

12
B. Pushkar et al. Journal of Environmental Management 287 (2021) 112279

which blocks the transfer of electrons from cytochrome aa3 to oxygen. Chromium resistant genes transferred amongst bacteria possess a special
Thus, the inhibitory effect of CN− on efflux shows the involvement of the function that facilitates the survival of bacteria in presence of chro­
respiratory pathway in chromium efflux. Contrary, NADPH enhances mium. Genes involved in chromium resistance are located on chromo­
the efflux of Cr6+/Cr3+ out of the cell by donating its electron and also somal DNA and/or on plasmid DNA. Several Bacillus sp. are reported to
promotes Cr6+ reduction. The H+ from NADPH generate transmembrane possess chromium resistant genes that are located on plasmid as well as
electrochemical proton gradient to promote the Cr3+/Cr6+ efflux by genomic DNA (Banerjee et al., 2019b; Aguilar-Barajas et al., 2008).
ChrA protein. Efflux of chromium by ChrA and ChrAB genes concur­ Many genes are reported to be involved in chromium detoxification
rently involve a variety of mechanisms, such as membrane potential which protects the bacterial cell from damage caused by chromium as
driving force, respiratory chain electron transfer, and transmembrane mentioned in Table 7. These genes include chrA, chrB, chrR, chrC, YieF,
proton concentration gradient (He et al., 2018). Thus, bacteria use efflux ChrT, etc. ChrA protein from Pseudomonas aeruginosa and Cupriavidus
mechanism for its protection by removing chromium from cells using metallidurans was first reported to be involved in chromium efflux pro­
transmembrane channels and preventing the intracellular components cess. ChrA gene is present on chromosomal as well as on plasmid DNA
from oxidative damage. and the proteins encoded by it belongs to chromate ion transporter
(CHR) family and is involved in chromium transport (Baaziz et al., 2017;
7.6. Stress enzyme Gu et al., 2020). ChrB acts as a modulator for ChrA protein expression
and is important for conferring resistance to chromium. ChrB is induc­
Heavy metals are known to create oxidative stress in cells by ible in nature, which is stimulated by chromium and it positively reg­
generating reactive oxygen species (ROS) and thus damaging the ulates the expression of ChrA. It acts as a metal sensitive transcription
cellular components. Heavy metal resistant bacteria produces a range of regulator of Chr operon which binds to repressor in apo form to release
enzymes that neutralizes the oxidative stress and protects the cellular the operator when bound to chromium (Shaw and Dussan, 2018; Branco
components from oxidative damage (Lunardelli et al., 2018a; Joshi and Morais, 2013). It is also suggested that ChrB has a role in the
et al., 2018). When Cr6+ enters the cell through sulfate channels, it is reduction of Cr6+ to Cr3+ before transporting Cr6+ from the cell via
reduced during the transport process. Cr6+ reduction under aerobic ChrA. So, ChrB protein majorly performs two functions, firstly it confers
conditions produces Cr5+ and Cr4+ which are short-lived intermediates bacteria with chromium resistance and secondly it acts as a regulator of
and generates ROS in the process. ROS thus generated, creates oxidative Chr operon (He et al., 2018). Both ChrA and ChrB are expressed by the
stress in the cell which further stimulates the activation of antioxidant bacteria when grown in presence of high chromium concentration and
defence mechanism in the cell. Antioxidant defence mechanism uses are reported to work better in coalition for conferring resistance to
antioxidant compounds and enzymes such as superoxide dismutase, chromium when compared to their individual capacity (Dong et al.,
glutathione transferase, peroxidase, catalase, and thiol-containing 2018; Branco et al., 2008).
molecules which converts the biologically oxidative species into harm­ Gene product of YieF and ChrR genes are involved in transferring the
less compounds (Elahi and Rehman, 2019). electrons to Cr6+ with simultaneous generation of ROS through two
A comparative study by Elahi and Rehman (2019) using Bacillus mechanisms, namely Class I (tight) and Class II (semi-tight) respectively
aerius S1 and Brevibacterium iodinum S2 have indicated that the expres­ (Baldiris et al., 2018). Oxidative stress in form of ROS can cause damage
sion of antioxidant enzyme against chromium stress varies with different to genetic material and affect DNA replication, translation, etc. Bacteria
bacterial types. For example, Bacillus aerius S1 activates a higher level of overcome the chromium induced oxidative damage to genetic material
antioxidant molecules as compared to Brevibacterium iodinum S2,
whereas both bacteria produces higher levels of peroxidase enzyme
Table 7
(Elahi and Rehman, 2019). In contrast, Escherichia coli produce super­
Genes involved in the heavy metal remediation.
oxide dismutase (SOD) and catalase while Shewanella oneidensis MR-1
generates a high concentration of thioredoxins and glutaredoxins. Sr. Name of Name of gene Role of the gene Reference
No. bacterial
Oxidative stress caused by the chromium also changes the expression of
certain genes that helps in combating oxidative stress (Thorgersen et al., 1 Bacillus sp. SFC chrA- Cr6+ extrusion or Ontañon et al.
500-1 E Chromate ion efflux (2018a)
2017).
transporter
Kiliç et al. (2010) have reported that reduced form of glutathione (CHR) family
(GSH) plays an important role in Cr6+ detoxification, where it interacts 2 Shewanella chrA- Cr6+ extrusion or Baaziz et al.
with chromate and produces Cr6+ redox-cycling. This helps in mini­ oneidensis MR1 Chromate ion efflux (2017)
mizing the oxidative stress-induced DNA damage and also leads to the transporter
(CHR) family
development of glutathione-Cr3+-DNA adducts (Kiliç et al., 2010). In 3 Serratia sp.S2 ChrB a positive He et al. (2018)
presence C6+, intracellular concentration of glutathione and NPSH in­ regulatory role in
creases to maintain the reduction state in the cytoplasm. The C6+ ChrA gene
reduction process also promotes the synthesis of Glutathione and NPSH regulation
4 Pseudomonas ChrR Cytosolic Cr6+ Ackerley et al.
to counter the ROS and improve reduction ability (Gu et al., 2020).
putida reduction (2004b)
Further, Ma et al. (2019) have reported that Cu2+ which is the cofactors 5 Escherichia coli YieF Cytosolic Cr6+ Ackerley et al.
for antioxidant enzymes such as superoxide dismutase (SOD), catalase reduction (2004b)
(CAT) and chromium reductase is elevated in the cell upon the exposure 6 Cupriavidus chrC Protection from Aguilar-Barajas
to chromium. Cu2+ is also involved in electrons shuttle among the metallidurans ROS et al. (2008)
7 Pannonibacter LuxI/LuxR Quorum sensing Chai et al. (2019)
protein subunit which facilitates the Cr6+ detoxification (Ma et al., phragmitetus BB and TraI/TraR
2019). Thus, various oxidative stress neutralising enzymes help the 8 Serratia sp. S2 nfsB Nitroreductase Dong et al.
bacterial cell to protect itself from the oxidative stress generated by (2018)
chromium. 9 Escherichia coli nfsA Nitroreductase Ackerley et al.
(2004a)
10 Bacillus subtilis nfrA Chromium Zheng et al.
8. Genes and proteins involved in chromium detoxification reduction nitro/ (2015)
flavinreductas
Bacterial communities develop heavy metal resistance by horizontal 11 Serratia sp. S2 ChrT Chromium Alhebshi and
gene transfer, which is mostly intra species but sometimes it can also reduction El-bestawy
FMN-reductase (2018)
occur at interspecies level (Srinath et al., 2002; Mergeay et al., 2003).

13
B. Pushkar et al.
by activating Rec system and SOS repair system (Thorgersen et al.,
2017). ChrR and YieF genes are reported in Pseudomonas putida and
E. coli where they perform chromium reduction in the cell cytoplasm.
The products of these genes belong to Class I chromate reductases. Both
these gene use a different mechanism for reduction of chromium. YieF
directly reduces the Cr6+ to Cr3+ via four-electron transfer reaction
whereas ChrR performs chromium reduction by combining one or
two-electron transfer steps (Thatoi et al., 2014; Ackerley et al., 2004b).
Therefore, YeiF one-step reduction process generates less ROS as
compared to multi-step ChrR based chromate reduction (Baaziz et al.,
2017; Cheung and Gu, 2007). ChrR gene is also reported to reducte and
detoxify chromate in association with ChrA which further enhances the
chromium resistance properties of ChrA and helping it to confer strong
chromium resistance in bacteria (Aguilar-Barajas et al., 2008). However,
class II chromate reductase requires NAD(P)H as an electron donor and
generally has nitroreductase activity (Baaziz et al., 2017). ChrT protein
is reported in Serratia sp. S2 which belong to FMN-reductase family and
also involved in the reduction of Cr6+ to Cr3+ (Alhebshi and El-bestawy,
2018; Gu et al., 2020).
Various other genes such as NfsA, NfrA, NemA and LpDH are also
reported for their catalytic reduction reaction of Cr6+ to Cr3+ (Baaziz
et al., 2017; Ackerley et al., 2004a; Ontañon et al., 2018b). Where NfrA
belongs to nitro/flavoreductase family and is reported to possess a broad
electron accepting capability which is used for reduction reactions
(Zheng et al., 2015; Hasin et al., 2010). NfsA protein aid LpDH-catalysed
Fig. 7. Data available on the different protein involved in the chromium detoxification (UniProt). Schematic representation of the activity of important proteins
responsible for chromium detoxification in microorganisms.
14
Journal of Environmental Management 287 (2021) 112279
chromate reduction with less ROS generation and it is a two electrons
reducer of quinones and nitro compounds. Reduction occurs by its
four-electron reduced dimer which transfers three electrons in one step
to Cr6+ to produce Cr3+ with simultaneous transfer of remaining elec­
tron to other strong electronegative molecules such as molecular oxygen
which in turn generate ROS which is similar to YieF reduction activity
(Ackerley et al., 2004a). Ma et al. (2019) have reported that the ex­
pressions of NfsA and NemA genes significantly increase upon exposure
to Cr6+ along with many other genes (Ma et al., 2019).
Analysis of the data available on the chromium resistant genes and
protein revealed that ChrA is mostly studied and reported protein in the
UniProt database. This indicates that the efflux mechanism is reported in
the maximum number of chromium resistant microorganisms and also
studied very well. The other majorly reported proteins are ChrR and
ChrB as shown in Fig. 7. While the other proteins involved in chromium
remediation such as ChrC and Yie are very less reported and need further
exploration. The mechanism of action of important proteins against
chromium is represented in Fig. 7 along with protein structure.
Changes occurring in the surrounding environment are sensed by the
bacterial cells, and they respond to it by fine-tuning their transcription
and translation process. Presence of chromium in the surrounding
environment leads to the upregulation or downregulation of certain
proteins. It also induces qualitative as well as quantitative variation in
protein metabolism of the bacterial cell. The proteins that help bacterial
cells in alleviating the chromium toxicity belong to various classes such
B. Pushkar et al.
as envelop stress response protein, proteins involved in energy meta­
bolism, oxidative stress protein, redox enzymes, quorum sensing pro­
teins, DNA repair protein, transcription and translation regulators, etc.
(Ontañon et al., 2018b; Kiliç et al., 2010; Cheng et al., 2009). Proteins
involved in various metabolic processes such as nitrogen metabolism,
cell membrane integrity, fatty acid metabolism, LPS metabolism, tran­
scription and translation process etc. also alters their expressions to
adjust the overall metabolism in the bacterial cell (Shah and Damare,
2020; Kiliç et al., 2010). Further, membrane proteins also play an
important role in the trafficking of chromium in and out of the cell.
OmpA is an outer membrane protein and is involved in LPS trafficking.
Cr6+ is speculated to LPS bind before it enters the cell (Kiliç et al., 2010).
Asatiani et al. (2004) have reported the up-regulation of cell wall pro­
teins, mostly of 40–55 kDa molecular weight in Arthrobacter oxydans
upon exposure to chromium in the surrounding medium (Asatiani et al.,
2004). Elahi and Rehman (2019) have reported the hike in the
non-protein thiols molecules upon chromium exposure (Elahi and
Rehman, 2019). The whole bacterial molecular system is rearranged in
response to chromium which ensures the minimum damage to bacteria
and its survival. The overall mechanisms observed in various bacteria
against chromium are schematically represented in Fig. 8. All these
mechanisms are discussed in detail in the previous section.
9. Gaps and bottleneck in chromium bioremediation
Though the chromium bioremediation using bacteria is subject of
research for a very long time and many progresses have been done in this
field but still, there are many questions unanswered especially when it
comes to bacterial molecular response towards respective heavy metal
and the possible utility of these molecular responses for environmental
chromium bioremediation. A study by Rivera et al. on P. aeruginosa
PA01 has shown the involvement of as oprE (encoding outer mem­
brane), rmlA (cell wall LPS) and ftsK (cytoplasm) in chromium resis­
tance. The role these genes are not studied in details. OprE is actively
15
Journal of Environmental Management 287 (2021) 112279
involved in chromium efflux mechanisms as OPrE mutant strains does
not restore efflux mechanisms even upon expression of ChrA gene
(Rivera et al., 2008). Fig. 7 shows that ChrR gene is majorly studied to
understand the chromium reductase mechanism while other genes such
as ChrT and YieF are very less explored though these genes have high
chromium reduction ability (Gu et al., 2020). Further, in contrast to the
diversity of chromium resistant Gram negative bacteria, very less group
of Gram positive bacteria are reported for chromium bioremediation.
Also, there is a gap in knowledge of the difference in chromium resis­
tance mechanism between Gram negative and positive bacteria (Shaw
and Dussan, 2018).
Following are the major bottlenecks in the bioremediation treatment
process.
1. One of the major bottlenecks in the bioremediation process during
treatment is the sensitivity of microorganisms towards other pol­
lutants and environmental stress present at the treatment site. Major
research on bioremediation is limited to the isolation of chromium
resistant microorganisms and testing their bioremediation efficacy at
lab scale under controlled condition. Bioremediation capacity of the
bacteria may be affected by various factors of the polluted sites.
Hence, future research can be focused on the application of chro­
mium remediating bacteria for on-ground remediation of chromium.
2. Industries that are releasing chromium pollutants are highly unor­
ganised and there is no check on waste treatment and disposal.
Mixing of different industrial waste further complicate the biore­
mediation process. The bacterial consortium could be developed for
the remediation of chromium along with other pollutants that are
released by various industries.
3. Genes responsible for chromium remediation may be present in
genomic or plasmid DNA. Bacteria may lose its chromium resistant
gene if the bacteria losses their plasmid. In such a case, the biore­
mediation capability of bacteria will be lost. More research is needed
Fig. 8. A schematic representation of the
different mechanisms initiated by bacteria
against chromium. (a) efflux of the chro­
mium out of the cell, (b) reduction of Cr6+ to
Cr3+ (b1) intracellular reduction of Cr6+,
(b2) Cr6+reduction by cytochrome in the
cytoplasm, (b3) Cr6+reduction by cell
envelop, (b4) chromium reduction by c-type
cytochrome under anaerobic condition, (c)
biosorption (c1) of Cr6+ and (c2) Cr3+, (d)
chromium entrapment by EPS, (e) bio­
accumulation, (f) complexation of chromium
by MT, and (g) activation of SOS response to
counter the DNA damage caused by
chromium.
B. Pushkar et al.
for devising the methods for maintaining a high copy number of the
plasmids bearing the chromium remediation genes.
4. Heavy metals are non-degradable and the remediation process
should focus on their extraction from the polluted sites. Future
research should be conducted on the development of a process which
can help in the removal of bacteria from the polluted sites, once they
accumulate the heavy metals. Bacteria could be immobilised on
various substrates which can help in the removal of bacteria from the
polluted sites.
10. Conclusion and future perspective
The present review highlights the issue of chromium pollution faced
throughout the world and the need for immediate actions. Chromium
bioremediation method offers several advantages over other conven­
tional methods. Bacterial chromium bioremediation mechanisms were
discussed in detail which confirms the utility of bacteria for purpose of
chromium remediation. A comprehensive analysis of the literature
available suggests that huge study has been conducted on bioremedia­
tion which is majorly limited to lab scale.
This review pointed out the major gaps in the bacterial bioremedi­
ation process and suggests that 1) Different molecular mechanisms of
chromium remediation in bacteria should be explored further to clearly
understand the bacterial response, as various mechanisms work paral­
lelly to remediate the pollutants, 2) There is need to promote more field
bioremediation studies rather than just bacterial isolation and lab-scale
treatment assays. Very few studies have reported on-site waste treat­
ment using bioremediation (Gao et al., 2018; Sheng et al., 2013), 3) The
bacterial bioremediation can be combined with other techniques such as
phytoremediation, immobilizations which can support the growth of
bacteria and also help in achieving maximum bioremediation.
Author’s contribution
Dr. Bhupendra Psuhkar: Conceptualization, Validation, and Super­
vision, Pooja Sevak: Conceptualization, Formal analysis, Investigation,
Writing – original draft and Visualization, Sejal Parab and Nikita Nil­
kanth: Data Collection and Compilation. Nikita Nilkanth: Data Collec­
tion and Compilation. I would like to declare that the descriptions are
accurate and agreed by all authors.
Declaration of competing interest
The authors declare no competing financial interests or personal
relationships with respect to the work reported in this paper.
Acknowledgement
The authors are thankful to the unknown reviewers for giving their
valuable comments and suggestions to improve the manuscript.
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