Source of Cr(VI) in the aquatic ecosystem, its genotoxic effects and microbial removal from contaminated water

ABSTRACT Cr(VI) compounds have important industrial applications and are used in various sectors like tanning, chrome plating, anti-corrosion agents and wood preservation. The Cr(VI) contamination in the wastewater is generally due to several natural and anthropogenic sources. Anthropogenic activities like several industrial operations play a major role in the Cr(VI) contamination in the aquatic ecosystem. Cr(VI) well-known toxic metal ion and its exposure in humans causes several health issues. Cr(VI) enters the cells and gradually reduces into a lower oxidation state and generates oxidative stress in the cell which damages cell organelles. The Cr(VI) mediated genotoxicity has been described as damaging the DNA base pairing, sugar-phosphate backbone, histone modification and chromosomal damage. Water and wastewater must be treated to remove Cr(VI) due to its high toxicity. There are several, physiochemical methods used for Cr(VI) remediation but these approaches are expensive and produce hazardous sludge during the treatment process. Therefore, a suitable environmentally friendly and effective Cr(VI) removal approach is urgently needed. Microbial removal of Cr(VI) is considered an eco-friendly and cost-effective process. In this, the authors focused on sources, genotoxicity and microbial remediation approaches of Cr(VI). GRAPHICAL ABSTRACT


Introduction
Cr(VI) is a very toxic ionic form of chromium that originated from anthropogenic activity (1).It is rarely available on the Earth in metallic or elemental form.It is mostly found in the form of a chromium-containing compound or ionic form (2). Various types of chromium-containing compounds occur in nature such as sodium chromate, potassium dichromate, sodium dichromate and chromite (3).The chief and widely commercial source of chromium is chromite (FeCr 2 O 4 ) and it is considered a primary source of Chromium (4).Chromium is widely used for metallurgical applications, it covers about 67% of total chromium used for various industries (5).
EPA (Environmental Protection Agency) and ATSDR prepared a list of toxic substances based on their toxicity and exposure to human health.ATSDR substance priority list has been given 1st rank for most toxic heavy metals or substances.According to ATSDR ranking arsenic has 1st rank and is considered as 1st most toxic priority element.In this toxicity ranking, Cr(VI) has the 17th rank in ATSDR's substance priority list.Human beings and other living organisms are affected by chromium contamination in different ways such as skin contact, inhalation by airways, food contamination, etc (6).Various environmental protection agencies have suggested the acceptable limit of Cr(VI) as mentioned in Figure 1 (7).
Cr(VI) contamination in the environment comes from various anthropogenic activities.The Cr(VI) pollution in water is also caused by the oxidation of Cr(III) (8).The conversion of Cr(III) into Cr(VI) through many oxides such as manganese dioxide present in nature (9).These chromium contaminations cause several health problems in humans and other living organisms in various ways.Contamination in drinking water causes different types of cancer in the alimentary canal (10).It is a very reactive form of chromium species that easily crosses the cell membrane.The chromate ions have a similar structure to the phosphate and sulfate ions, hence they cross the cell membrane by sulfate transporter (11).Intracellular lethal Cr(VI) converts to a non-toxic lower oxidation state by chromate reductases.During the reduction process, free radicals generated cause DNA, protein and other cell organelles damage (12).Reduce Cr (III) can also form Cr (III)-DNA complex finally responsible for DNA mutation (13).
The Cr(VI) in water or food materials higher than the permissible limit shows several toxic effects.Considering its toxicity level, it is very necessary to eliminate the Cr (VI) level from contaminated water (14).There are several conventional approaches such as physical adsorption, oxidation and chemical reduction are used for Cr(VI) and other metal ion decontamination (15,16).There are several nanomaterials have been used for the removal of heavy metals from wastewater (17)(18)(19).Composite materials have also been reported as effective adsorbents for heavy metal removal (20)(21)(22)(23)(24)(25).Moreover, polymeric materials have also been reported as suitable adsorbents for heavy metals and dye removal (26)(27)(28)(29)(30).Some disadvantages of conventional methods are high operation costs, the lot of energy input required and the generation of toxic sludge (31).Hence, it is necessary to find an eco-friendly and inexpensive method for Cr(VI) remediation.Some ecofriendly methods such as biosorption, bioreduction, bioaccumulation and phytoremediation are considered the best option for this application (32).
Biosorption is generally performed by dead biomass of plants, bacteria, fungi and algae.In this process, Cr (VI) binds to the biosorbent by several functional groups like carboxyl, nitrogenous and hydroxyl on the biosorbent surface (33).The live microbes can intake heavy metals which is known as bioaccumulation (31,34).The Cr(VI) inside the cell reduces into the less toxic Cr(III) through several antioxidants and chromate reductases (14,35).Bacteria are considered a good option for Cr(VI) decontamination due to their short life span, high resistance against Cr(VI), ease of growth and wide availability (36).Singh and Mishra (8) isolated M. paraoxydans bacterial species from coal mining contaminated water.This bacterial species has a high Cr (VI) reduction property (99.96%) at 50 mg/L initial Cr (VI) ion concentration.Li et al (37) extracted microbial strain B. cereus 332 from contaminated soil and reported that B. cereus 332 was able to remove 99.9% Cr(VI) after incubation for 24 h.Plestenjak et al (38) isolated bacterial species affiliated with Mammaliicoccussciuritannery effluent and observed that it has 50% Cr(VI) removal efficiency.This review is focused on various sources of Cr(VI) contamination, health hazards including genotoxic effects and biological approaches for Cr(VI) detoxification.

Source of Cr(VI)
It is widely used in the chemical, metallurgical and leather industries (39).It is found in several forms like sodium dichromate (Na 2 Cr 2 O 7 ), ammonium dichromate ((NH 4 ) 2 Cr 2 O 7 ), potassium dichromate (K 2 Cr 2 O 7 ), etc (40).Chromium generally exists in nature in the form of Cr (III) (41).Anthropogenic activities such as tannery, chrome plating and coal mining are the major sources of Cr(VI) contamination (42).The major sources of Cr (VI) are described in Figure 2.

Natural sources
It generally originated from various types of human activity.However, many natural sources are responsible for the Cr(VI) in different types of environmental sources.Additionally, Cr(VI) was also reported in groundwater (0.05-0.073 mg/L) in many countries such as Mexico, Italy and California (43,44).The maximum part of natural chromium is available in ultramafic rocks.It is present in ultramafic rocks in the form of chromite, manesio chromite and other compounds (9).The external factors play a significant role in the conversion of Cr (III) to Cr(VI).The trivalent form of chromium converts into hexavalent ionic form in the presence of different types of oxides such as manganese oxides, ferric oxide (Fe + 3 ) and oxyhydroxides in groundwater and surface water.Manganese oxides have more capability than other oxides for the oxidation of Cr(III) (44,45).Equation (1) represents the oxidation of trivalent chromium into hexavalent chromium.
1)Moreover, hydrogen peroxide is a strong oxidizing agent which converts Cr(VI) into a less oxidation state.The rate of Cr(VI) oxidation by H 2 O 2 is very fast (46).Dissolved oxygen and atmospheric oxygen also tend to convert the trivalent form into the hexavalent form of chromium.Dissolved present in the water sources oxidized to Cr(III) but its significance is much less in the Cr(VI) contamination.Atmospheric oxygen has a major role in the generation of Cr(VI) from trivalent form (46,47). Gaseous oxygen species convert Cr (III) into Cr(VI) form during many natural processes such as forest burning (47).

Anthropogenic source
Anthropogenic activities are the source of Cr(VI) contamination in the groundwater and surface water.Various types of industrial processes generate Cr(VI) containing waste materials and release them into the environment as effluent (6).These waste materials are directly disposed of into the environment and are responsible for Cr(VI) contamination in water and soil (48).A few industrial operations are given below which generate Cr(VI) into their effluents.

Tanning process
Cr(VI) has a crucial role in the leather industry.Tanning is the process of conversion of raw animal skins and hides into usable leather (49).Chrome sulfate is widely used in the tanning process.It causes structural modification in collagen protein present in leather (50).Cr(VI) has a major role in the tanning process as a crosslinking agent.Chromium crosslinking occurs through; (a)two collagen molecules and one chromium ion attached by covalent bonding, and (b) protein molecules attached with chromium ions by hydrogen bonding (50,51).Cr (VI) containing industrial effluent directly discharges into water sources such as rivers and other sources.These chromium contaminations cause many toxicological effects in living organisms (52).

Anti-corrosion coatings (chrome plating, spray coatings)
Anti-corrosion coating provides a barrier between metallic materials and their outer environment.It protects metallic materials against gradual damage due to the corrosion process (53).An ideal coating should be thin and less porous.It is the best corrosion inhibitor and provides less porous polyaniline film.The resulting Cr(VI) coating provides better protection for vehicles and other metallic materials (54).Various types of chromium-containing compounds such as ZnCrO 4 and SrCrO 4 used for anti-corrosion coating.These Cr(VI) containing compounds cause various types of Cr(VI) contamination in the water (55, 56).

Stainless steel
Cr(VI) is widely used in the steel industry.Stainless steel is a commercial product used in many sectors such as industrial equipment, vehicles, and household products (57).Various types of chromium species are used in the stainless steel industry such as (NH 4 ) 2 Cr 2 O 7 , K 2 Cr 2 O 7 , etc.These species were responsible for Cr(VI) contamination in wastewater (55).

Wood preservation
Chromated copper arsenate (CCA) is a pesticide containing Cr(VI), As(V) and Cu(II) widely used for wood preservation.It protects wood materials against various types of wood-decaying organisms such as termites (58).
When preserving sites of wood come in contact with the soil and water, the preserving chemical is gradually released into the environment (59).These Cr(VI) containing chemicals are responsible for soil and water contaminations.Cr(VI) in the soil slowly leaches and causes contamination in groundwater (60,61).As(V), Cr(VI) and Cu(II) contamination present in wastewater cause toxic effects on aquatic and terrestrial organisms.These metals accumulate in fishes and other organisms enter into the food chain and cause various types of carcinogenic effects in humans (58).Chromium trioxide is also used in wood preservation (14).Exposure to chromium contamination in a human occurs through skin contact with chromium-contaminated soil, inhalation by airways and ingestion of contaminated food (62).

Coal mines and coal washeries
The effluent generated from coal mines contains a large number of pollutants that cause various types of contamination in the environment (63).Wastewater generated from coal processing plants is also known as acid mine drainage.The wastewater of coal mines contains a huge number of heavy metals and other toxic substances that contaminate our surrounding environment (64).Cr(VI) is one of the major toxic contaminants that come in coal washery effluents.The coal washery effluent is directly discharged into the surface water sources which cause Cr(VI) contamination (65).This wastewater shows a broad toxicological profile in the living system (64, 65).

Genotoxicity of Cr(VI)
The genotoxicity of Cr(VI) is a complex mechanism and it can be explained by damage to DNA base pairs and its sugar-phosphate backbone.DNA damage may occur through direct or indirect mechanisms.In the direct mechanism, Cr(VI) interacts with the DNA molecules and forms a Cr(VI)-DNA complex.On the other hand, intracellular Cr(VI) ions generate oxidative stress in the form of ROS and carbon radicals.Cr(VI) widely exists in the form of chromate oxyanion (CrO 4 ) in the environment.CrO 4 ions show a similar structure to sulfate ions; hence, chromate ions enter the animal cell through its membrane transporters (66).Once, Cr(VI) enters the cell, it reduces to Cr(III) by ascorbate and glutathione (GSH) (67).It has been reported that cysteine amino acid residues also have a crucial role in the Cr (VI) conversion (68).The step-wise reduction of two electrons from Cr(VI) through ascorbate and generate Cr(IV).This reaction generally occurs in vivo, on the other hand, tissue culture systems contain a very low level of ascorbate.In this case, Cr(VI) reduces to Cr(V) by GSH and finally converts into Cr(III) (69).During Cr(VI) reduction, hydrogen peroxides and free radicals are generated as reaction intermediates which generate oxidative stress, responsible for damage to cellular proteins, DNA, RNA and lipids (66,70).Moreover, the intracellular oxidative stress can be decreased by adding ascorbate into the cell growth media but at this stage, the addition of ascorbate causes DNA double-strand breaks.The ascorbate was also responsible for the formation of ternary DNA adduct structure (71).The DNA adduct structure contains Cr(III) crosslink with glutathione or histidine, ascorbate and cysteine (72).The DNA repair of Cr(III) holding DNA adduct is very difficult and it is responsible for Cr(VI)-induced malignant cellular transformation (73).The interaction of Cr(VI) with animal cells and its fate in the intracellular environment including its toxicity is shown in Figure 3.

DNA protein crosslinking and histone modification
The epigenetic profiling of Cr(VI) at both levels of histone modification and DNA methylation has been reported in several studies (74).The toxicity of Cr(VI) causes various changes in the histone such as decreased levels of H3K27Me3 and increased levels of H3K4Me3, H3K9Me2 and H3K9Me3 (75).Moreover, alteration in the gene expression of gene MLH1gene is also caused by Cr(VI).MLH1 is a mismatch repair gene and has an important role in DNA repair.The histone-methyltransferase G9a (HMTG) is associated with H3K9 demethylation and additionally, Cr(VI) is responsible for the expression of HMTG.The Cr(VI) toxicity is also increased the expression level of H3K9Me2 which responsible for the decreased expression level of the MLH1 gene.Lower expression of the HLH1 gene in the cell is responsible for limited DNA repair capacity (76).Furthermore, Cr(VI) is associated with MMR protein at the adducts region and shows toxicity.MMR protein is involved in the recruitment of the site for γ-H2AX foci.This focus is responsible for p53-mediated apoptosis and DNA denaturation (77).This study provides evidence of Cr (VI) mediated genotoxicity.Cr(VI) inhibits cell repair machinery or disrupts the components of the DNA repair mechanism and is responsible for introducing genotoxicity.Exposure to Cr(VI) can also interfere with epigenetic machinery and inhibit its function by histone deacetylation (78).

Chromosomal damage and DNA strand breaks
The intracellular Cr(VI) toxicity is associated with DNA strand scission and established as single-strand breaks (SSBs).The DNA strand breaks with exposure to Cr(VI) in the cultured cells of rodents and humans have been documented (79,80).The in vitro experiment model has been designed using soluble Cr(VI) as a single reductant and isolated plasmid DNA showed mixed results (81,82).It has been observed in this experiment that the production of SSBs by Cr(VI) reduction.Moreover, SSBs were produced by Cr-DNA interaction or due to ROS/carbon radicals.The exposure of Cr(VI) also activates the ATMdependent pathway and the recombination process is necessary for survival following Cr(VI) exposure (83).The conclusive research is linked to Cr(VI) reduction is responsible for the development of DNA DSBs (83).There are several investigations have reported that Cr (VI) exposure significantly surges the quantity of alkalilabile sites on DNA in vitro and in vivo (84,85).Moreover, Cr(VI) mediated DNA strand breaks have been investigated through single-cell gel electrophoresis (86,87).However, the mechanism of DNA strand breaks by reduction of Cr(VI) is unclear.However, one possibility is the generation of hydroxyl radicals in the Cr(VI) reduction process and subsequent attack on the base sugar.Cr(VI) has a high oxidation state and Cr(VI) may oxidize DNA through the abstraction of the C-1 or C-4 hydrogen of deoxyribose and is responsible for strand scission (88).The SSB formation in the liver and kidney of mice treated with Cr(VI) was observed and dimethylthiourea (hydroxyl radical scavenger) inhibited the SSB formation in the absence of Cr(VI) at a toxic level (79).In most cases, metaphase damage has been observed in the presence of both water-soluble and insoluble chromate ions (89).Finally, this evidence revealed genotoxicity which has the possibility of the development of human lung cancers by Cr(VI) exposure (90).

Microbial mediated Cr(VI) detoxification
The Cr(VI) contamination in the wastewater is removed through several conventional methods and these methods are expensive, generate secondary pollutants, and have low Cr(VI) remediation efficiency and high demand for energy input (91).Microbial-based Cr(VI) decontamination is considered a safe and cost-effective process (92).Microorganisms are suitable for heavy metal removal due to their widespread in the environment and some microbes are highly resistant to the Cr (VI) (93,94).There are several researchers have reported their findings on Cr(VI) reducing microbes (95).A few Cr (VI) reducing microbes are shown in Table 1.
Bacteria can remediate Cr(VI) from the contaminated site.The bacterial cells produce reductase enzymes which reduce and convert lethal chromium to the nontoxic form of chromium.The surface molecules on the microbial cells bind to the Cr(VI) ions (105).Cr(VI) bioremediation involves several biochemical reactions outside and intracellular regions of bacterial cells.The Cr(VI) enters the microbial cell and reduces into Cr(III) by several reductase enzymes and electronic shuttles of the cell (106).Finally, Cr(VI) accumulates inside the bacterial cell.The overall mechanism of Cr(VI) detoxification is shown in Figure 4.
There are two main Cr(VI) resistant mechanisms of the microbes involved in Cr(VI) detoxification.One mechanism prevents the accumulation of Cr(VI) inside the cells.Another mechanism is affected in the presence of an enzymatic system that involves the conversion of Cr(VI) and promotes bioaccumulation (13).The gene expressed in the bacterial cell, especially ChrA.ChrAexpels Cr(VI) and inhibits bioaccumulation (107).The ChrB and CopZ genes involved in the decrease meant the intracellular Cr(VI) by pumping Cr(VI) ions outside of the bacterial cell.The Chr-promoter of the bacterial cell is induced by Cr(VI) but is not affected by Cr(III) and sulfate (108).
The microbes have a high diversity of genes involved in the Cr(VI) detoxification but sometimes these genes give negative responses (109).

Aerobic and anaerobic mechanism of Cr(VI) reduction
The microbial-mediated Cr(VI) detoxification can be done by anaerobic and aerobic bacterial genera.In the anaerobic condition, glutathione, vitamins, etc., are required for Cr(VI) detoxification because they donate electrons that convert Cr(VI) to non-lethal Cr(III).The anaerobic reaction requires NADH/NADPH as cofactors for reductase enzymes.Cr(VI) ions reduce into other chromium ions of lower oxidation (13).In the aerobic process, soluble Cr(VI) is reduced into insoluble Cr(III) ions by soluble chromate reductase (110) as shown in Figure 5.
In the oxygen-deficient environment, Cr(VI) has the role of a terminal electron acceptor in the microbial respiratory system which resides on the bacteria membrane.In a sufficient oxygen environment, Cr(VI) reduces into its lower oxidation state such as Cr(V), Cr (IV) and finally reduced into less toxic Cr(III).It has been reported in some microbial genera that cytochrome-b actively participate in the transport of electron and it occurs under anaerobic condition (111,112).It has also been reported in several findings that cytochrome electron shuttle-based mechanisms are involved in extracellular electron transfer (113).

Factors affecting detoxification
The temperature is one of the most important parameters for bacterial growth and Cr(VI) remediation.Moreover, a higher temperature is required for the growth of thermophiles compared to mesospheric.Moreover, the maximum reduction of Cr(VI) occurs at physiological temperature but varies from microbe to microbe (107).It has been reported that Cr(VI) at an initial concentration of 40 mg/L was completely reduced by C. saccharolyticus supplemented with glucose at 70°C in 12 h.The Cr-reduced Cr(III) was immobilized on the bacterial surface in the form of precipitation (114).Mesophilic bacteria such as B. dabaoshanensis and facultative anaerobic bacteria perform the Cr(VI) reduction process at a mesothermal temperature (113,114).These mesophilic bacteria have more Cr(VI) tolerant properties than the thermophile (115).Moreover, bacterial growth at lower temperatures is inhibited because the fluidity of the cell membrane reduces at a lower temperature which affects the membrane transport system of the cell and the entry of Cr(VI) ions (116).The protein synthesis mechanism of bacteria affects high temperatures and also changes membrane structure which leads to the inhibition of chromate reductase (117).The researchers have reported that microorganisms have stronger chromate removal efficiency at a slightly higher temperature (35-37°C) (118).
Solution pH has also a crucial role in the Cr(VI) remediation.Solution pH determines the proton content and oxidation state of chromium (119).pH affects the Cr(VI) biosorption on the bacterial surface and the activity of chromate reductase.The maximum reduction of Cr(VI) has been reported at lower pH in previous research and it was decreased with increasing solution pH in the alkaline condition.Cr(VI) reduction also affects very acidic pH, because microbial growth and activity of chromate have been affected at very acidic pH (120).Electron donors such as glucose and fructose in the reaction medium enhance the Cr(VI) reduction process (121).Moreover, the initial Cr(VI) dosage in the microbial growth medium also influences the Cr(VI) remediation efficiency.The microorganism can be tolerant of heavy metal concentration at a certain limit and thereafter, start decreasing Cr(VI) removal with increasing heavy metal dosage in the growth medium (14)

Technology challenges and future prospects of microbial mediated Cr(VI) remediation
The microbial Cr(VI) detoxification represents a promising technique for Cr(VI) remediation in terms of high Cr(VI) removal capacity and eco-friendly approach.Microbial remediation has several advantages such as an eco-friendly and cost-effective approach.However, microbial remediation methods also have some limitations such as the requirement of media, providing a suitable environment for the growth of the microbes and most cases microbial Cr(VI) remediation applied at laboratory stages not employed at large-scale (125).Moreover, Cr(VI) interaction with eukaryotic microbes is still unclear and additional research is required in this field (125).
In a critical analysis of the literature, some research fields require further investigation.Genetic engineering as genome editing can also provide the best approaches to develop Cr(VI) remediation tools for water treatment (126).Moreover, the immobilization of bacteria on solid supports provides long-lasting Cr (VI) treatment techniques.The economic analysis of microbial-based Cr(VI) detoxification (including media costs, energy consumption, labor, etc.) should be used.It is an important study to provide a simple cost assessment of microbial-based Cr(VI) remediation (127).Moreover, life cycle assessment (LCA) measures the environmental impacts of a process at different life stages, such as applying microbes to detoxify Cr (VI) and disposing of it at the end.As a result, mitigation strategies can be adopted for the environmental impact (128).To ensure sustainability, LCA studies are recommended for future studies on microbial-based Cr(VI) remediation [129].

Conclusion
Cr(VI) contamination is becoming a serious problem to the ecosystem and a major health risk to the biota.Additionally, the detrimental contamination of Cr(VI) in the water by natural and anthropogenic activities are serious concern for food security and safety.Hence, there is a need to find a long-lasting Cr(VI) removal technique for its removal from the environment.This requires a deep understanding of Cr(VI) detoxification from contaminated water.In this review, we discussed the source of Cr(VI) contamination, the genotoxicity of Cr(VI) and its microbial-mediated detoxification.Further, we also highlighted the important mechanisms involved in the Cr(VI) detoxification.Based on this review, we conclude that microbes have a higher tendency of Cr(VI) remediation and can be considered as an alternative to conventional water treatment methods.This review can be also beneficial to researchers and the construction of microbial-based water treatment systems.

Figure 1 .
Figure 1.The permissible limit of Cr(VI) approved by WHO, USEPA, CPCB and EPA.

Figure 2 .
Figure 2. Major sources of Cr(VI) in the environment.

Figure 3 .
Figure 3. Entry and toxicity of Cr(VI) in the animal cell.

Figure 4 .
Figure 4.The entry of Cr(VI) into the bacterial cell and intracellular response.
Duran et al  (122)  anaerobic granular consortium showed higher Cr (VI) biosorption at a lower Cr(VI) level in the solution.Chen et al(123) isolated Asperillusflavus CR500 from wastewater and analyzed the Cr(VI) bioreduction properties of isolated bacteria.Authors observed that Cr(VI) reduction by Asperillusflavus CR500 declined with rising Cr(VI) concentration.Incubation time is another important aspect that affects microbial remediation efficiency.Singh et al(8) reported Cr(VI) remediation by isolated bacterial strain from wastewater and observed the effection of incubation time on Cr(VI) remediation.Some other factors such as inoculum dosage, agitation rate and type of growth media also affect the Cr(VI) reduction of efficiency(48,124).