Microbial interactions with uranium: Towards a bioremediation approach

Uday Kumar Banala a,b, Nilamadhab Prasad Indradyumna Das a, Subba Rao Toleti b,c*

aRadiologicaland Environmental Safety Division, Indira Gandhi Centre for Atomic

Research, Kalpakkam-603102, India
bHomi Bhabha National Institute, Anushakti Nagar, Mumbai-400094, India
cWaterand Steam Chemistry Division, Chemistry Group, Bhabha Atomic Research Centre,
Kalpakkam-603102, India.

*Corresponding author:
Subba Rao Toleti,
Water and Steam Chemistry Division,
Chemistry Group, Bhabha Atomic Research Centre,
Kalpakkam-603102, India
Email: subbarao@igcar.gov.in, tsrmicro@gmail.com

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Contents:

1. Introduction

2. Uranium speciation and bioavailability

3. Chemical methods for uranium sorption

4. Microbial interactions with uranium

4.1. Bioaccumulation

4.2. Bioprecipitation

4.3. Bioreduction

4.3.1. Factors affecting the bioreduction process

4.3.2. Mechanism of Bioreduction

4.4. Biosorption

5. Conclusion

Declaration of competing interest

Acknowledgement

Funding

Figures and Tables

References

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Abstract

Rapid developments in industrialization and other anthropogenic activities resulted in the

release of elevated levels of contaminants to the environment. One such contaminant is

uranium, naturally present in rocks, minerals and the significant application of this

radioactive element in the nuclear industry and other processes resulted in its increased

distribution in nature. Most of the microorganisms that are present in terrestrial and aquatic

environments have developed survival strategies to grow in such toxic conditions and have

metabolic processes to sequester heavy metal toxicants such as uranium. Thus,

microorganisms can adsorb, accumulate, or precipitate uranium as a survival mechanism.

These microbial metabolic strategies can be employed for the bioremediation approach for

treating toxic pollutants. The purpose of this review is to discuss the plausible uranium-

microbial interaction mechanisms with a detailed description of bioaccumulation,

bioprecipitation, bioreduction and biosorption of uranium by various biological groups.

Comprehensive information about the uranium speciation and bioavailability was also

discussed which is essential for the in situ uranium bioremediation. Finally, a comparative

assessment of chemical methods versus biological procedures in the remediation of uranium

was explained.

Keywords: Uranium, Bioaccumulation, Bioprecipitation, Bioreduction, Biosorption,

Chemical methods

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Introduction

Uranium (U) is a naturally occurring very heavy radioactive metal. Earth crust contains

approximately 2.8 mg U/kg. In river water, the concentration of uranium is 0.01-6.6 µg/L,

while 30 µg/L and 3.32 µg/L are reported in groundwater and seawater, respectively

(Markich, 2002). The syngenetic and epigenetic deposition of impurities in carbonate rocks is

one among the primary source of uranium. It is present as a mineral of uraninite or

pitchblende which contains 50-80% of uranium and forms the primary mineral in

sedimentary carbonate rock formations such as calcareous sandstone and limestone

formations. Besides silicates, phosphates, carbonates and vanadates are also noted for the

presence of Uranium (Choppin et al., 2013; Francis, 1994). Natural uranium isotopes are
238U, 235U and 234U and their proportions are 99.27%, 0.72% and 0.005% respectively

(Sheppard et al., 2005). The growing necessity of uranium for nuclear power production

makes it metal in demand, extracting uranium from the aqueous medium is exorbitant.

Human activities such as mineral mining, nuclear weapon testing, and utilization of uranium-

containing phosphate fertilizers and natural leaching of uranium-bearing rocks have brought

the increased distribution of uranium in nature (Todorov, 2004). For instance, in the USA, a

few groundwater samples, soil and sediments have reported relatively high 11.7 g/L and 16

mg/g U, respectively. A nuclear waste storage site at Oak Ridge National Laboratory has 0.8

mg/g U (Kolhe et al., 2018). According to WHO guidelines, 30 µg/L is the maximum

permissible concentration of uranium in water, and daily open admission is 0.6 µg/kg of body

weight per day (WHO, 2011). In India, the occurrence of uranium in groundwater above the

WHO guideline value was reported, to be due to geogenic and anthropogenic factors like

groundwater overexploitation (Coyte et al., 2018). This is a severe environmental concern

because of its both radiological (235U) and chemical toxicity behaviour, which affects the

natural ecosystem.

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 Uranium exists in different oxidation states ranging from U (III) to U (VI) of which +4

and +6 are significant and essential. U (IV) is reduced form, non-mobile and less toxic, while

U (VI) is mobile, soluble and more toxic. The corresponding oxide of U (IV) is uranium

dioxide (UO2) and of U (VI) is uranium trioxide (UO3). Physical and chemical properties of

the soil matrices like pH, redox conditions, presence of ligands and others define the

oxidation state of U. Under oxidizing conditions and at pH ≤ 2.5, it exists as mobile U (VI)

wherein uranyl ion (UO22+) is the prevalent ion. In reducing conditions, insoluble and

immobile uraninite (UO2)[s] form predominates. At pH ≥ 6.5, hydroxyl complexes are

formed, and at much higher pH uranyl carbonate complexes prevail (Chopin et al., 2001).

Due to oxygenic conditions of soil surfaces and water, U (VI) is the dominant ion and

complexes of (bi) carbonate, citrate or phosphate are significant. Due to high solubility,

U(VI)-bicarbonate and U(VI)-citrate complexes are extremely mobile in the environment.

While U(VI)-phosphate complex is insoluble and leads to immobilization. Among the

commonly found ligands in nature, the order of affinity for complexation of UO2 is as follows

CO32->PO43->SO42->NO3->Cl- (Brugge, 2014). Uranium interactions with the biotic

metabolites and organic ligands have a significant role in uranium recycling in the ecosystem

(Haas et al., 1998).

Both biochemical interactions and radiological effects of uranium isotopes and its

progenies pose toxicity to humans and other organisms. Toxicity by biochemical interactions

is more ubiquitous than radioactive. Generally, uranium decays by emitting an alpha particle,

which cannot penetrate the outer skin layer and does not cause any hazard when radiated

outside the body. However, the exposures from radionuclide can be both external and

internal. The radiation energy deposited inside the organism can cause DNA damage leading

to cancer (Mukherjee et al., 2012; Park and Jiao, 2014). In vivo the uranyl cation is the

dominant form and is known to damage the kidneys when ingested (act as a nephrotoxin)

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(Anke et al., 2009). Uranium has a high binding affinity towards biomolecules. When it

enters the organism, through the blood, it transports to other organs. Once it is in the blood, it

forms various complexes like uranyl bis- and tris-carbonate complexes and UO2-protein

complexes with human serum albumin, transferrin and other proteins. One known mechanism

of chemical toxicity of uranium is the disruption of the glycolysis pathway by displacing the

magnesium in the enzyme, hexokinase (Nomiyama and Foulkes, 1968). Depleted uranium

(DU) is also a health hazard; it arises as a by-product during the production of enriched

uranium. Studies have shown that DU also harms brain, kidney and bone (Briner, 2010).

Uranium contamination also has many ecological consequences, apart from health

issues to human beings. Microcosms are one of the suitable methods for studying the impact

of uranium contamination on microbial diversity, and one of such studies revealed

detrimental effects on microbial populations (Islam and Sar, 2011a). Metals are essentials for

the biological processes (not the uranium), but they can be toxic at higher concentration. The

microbes have developed various mechanisms to immobilize toxic metals or radionuclides to

survive in harsh environments. Few of the mechanism involves enzymatic precipitation,

biosorption, bioaccumulation and redistribution of toxic metals/radionuclides in a stable

mineral phase. Microbial interactions with the metal ions lead to change in concentration or

oxidation, which controls the mobility as well as bioavailability of that metal (Acharya,

2015). The above mentioned unique properties of microorganisms can be utilized to clean up

the contaminated sites.

"The process of utilizing organisms to eliminate or detoxify the pollutants from the

environment mainly the contaminated soil and water, which otherwise threaten to public

health, is known as bioremediation". Unlike in conventional methods (physical and

chemical), microbiological methods are eco-friendly and economical. Moreover, the

generation of other hazardous by-products is also less. High surface to volume ratio is also a

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definite property of microbes used in bioremediation, and indigenous bacteria are crucial in

the geochemical cycling of toxic elements. Several literature reports suggest that indigenous

microbes from toxic metal-contaminated sites can also have a higher tolerance to heavy

metals and other toxic substances (Islam and Sar, 2011b; Pollmann et al., 2006).

This review focuses on reporting comprehensive details on uranium concerning

biological interactions. It encompasses the speciation and bioavailability of uranium followed

by bioremediation processes by biological agents and their interactions. We also discuss

advantages, disadvantages and future applications of uranium bioremediation. Finally, a

comparative account of bioremediation with chemical methods is also presented for the

benefit of the readers.

1. Uranium Speciation and Bioavailability

Speciation is defined as "the process of identifying and quantifying the different defined

chemical species, forms, or phases present in a material or describes the concentration and

kinds of these species, forms, or phases present" (Markich, 2002) Bioavailability is the

amount of the active metal that is available from the total amount of metal available for

uptake by the living organisms in their immediate surroundings. Such speciation of metal was

determined by the environmental factors like pH, organic and inorganic ligands present in it.

For speciation evaluation in sediments, chemical extraction methods are widely employed.

Ambiguity in U speciation was observed while evaluating using non-standardized extraction

methods, source of U and composition of the sediment. The equilibrium speciation modelling

can be done by using Visual MINTEQ software (Gustafsson et al., 2009). Speciation is an

essential factor to consider for the bioremediation purpose. Since speciation ultimately

determines its solubility and mobility in the environment; it is a prerequisite to know about

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the speciation of uranium in the contaminated sites before proceeding with any remediation

procedure.

In freshwater, in oxic conditions at pH ≤ 5 free uranyl ion (UO22+) is the dominant

species, UO2CO3 at pH 5.5-6.0, from pH 6.0-7.5 the mixed uranyl-hydroxide-carbonate

species, UO2(OH)3CO3- and UO2(OH)3- is the main uranyl species at pH >7.5. The above U

speciations are at environmentally relevant conditions and increase in U concentration causes

transference in the percentage distribution of U (Konstantinou and Pashalidis, 2004). The

concentration of metal and pH are the two crucial factors that influence speciation. At

different pH and uranium concentrations, uranyl ion is present in various forms like UO 2OH+,

UO2(OH)2(aq),(UO2)2(OH)22+, (UO2)3(OH)5+, (UO2)4(OH)7+, (UO2)3(OH)7- and UO2(OH)3.

Carbonates and phosphates form stable complexes with uranium. When the carbonate

concentration increases, mono-, di- and tri-nuclear uranyl carbonate species, UO 2CO3 (aq),

UO2(CO3)22- and UO2(CO3)34- are dominant (Acharya, 2015). In seawater (pH ~8) at low U

concentration, i.e. 3 µg/L uranyl carbonate complexes are essential than the other

compounds. Speciation of uranium in different experimental media as predicted by Visual

MINTEQ software is summarized in Table 1.

Uranium has no biological role in organisms, and it is taken up by the cell mistakenly

for Ca (Chao and Lin-shiau, 1995). Toxicity studies on the freshwater mussel, Velesunio

angasi (Markich et al., 2000), unicellular micro-algae Chlorella sp.(Franklin et al., 2000),

and uptake studies on the pea, Pisum sativum (Ebbs et al., 1998) have shown that the pH and

the ligands were essential factors that contribute to the toxicity and uptake. These studies

suggested that as the pH increases the percentage proportion of uranyl ion (UO 22+) and

UO2OH+ decreases and has reduced toxicity and absorption of U. Similarly, with the

organic/inorganic ligands, uranyl complexes with carbonate, phosphates and humic

substances reduce the UO22+ and UO2OH+ activity. As UO22+ has higher binding affinity at

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the cell surface, there is significant bioavailability of UO22+ and UO2OH+ on the contrary to

the uranyl phosphate complexes (Markich et al., 2000). Hence before proceeding, the

bioremediation or protective approaches in aquatic or environmental systems, the speciation

and bioavailability of the metal have to be determined.

2. Chemical methods for uranium sequestration

The primary purpose of any remediation process is to ease the risks associated with

contaminant to the ecosystem. In the soils, heavy metals or radionuclides may be adsorbed or

complexed with soil particles, and subsequent reactions between them play a significant role

in their mobility. Thus, complexation leads to precipitation, the reduction immobilizes, and

oxidation results in movement, these interactions form the basis for remediation (Gavrilescu

et al., 2009). To recover uranium from the contaminated soils or water, various methods have

been extensively studied, from conventional methods to advanced technologies. Some of the

methods include chemical precipitation (Baker et al., 2019), membrane separation (Kolev et

al., 2013), solvent extraction (Wang and Zhuang, 2019), electrochemical purification (Wei et

al., 2016), reverse osmosis (Shen and Schäfer, 2014), ion exchange (Khawassek et al., 2018)

and adsorption (Sureshkumar et al., 2010). Of all these methods, adsorption and solvent

extraction are the most promising strategies for pollutant removal.

The solvent extraction process uses organophosphorus compounds, which are having

high affinity and good coordinating ability towards uranyl ion. Some of the widely reported

organophosphorus compounds for the abstraction of uranium are tributyl phosphate (TBP),

octyl(phenyl)-N,N-di-isobutyl carbamoyl methyl phosphine oxide (CMPO), trioctyl

phosphine oxide (TOPO), dihexyl-N,N-di-ethylcarbamoyl- methylphosphonate (CMP), di-2-

ethyl hexyl phosphoric acid (D2EHPA), monododecyl phosphoric acid (DDPA), dibutyl

butyl phosphonate (DBBP) and glycerophosphate (Wang and Zhuang, 2019). Among all the

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organophosphorus compounds studied, tributyl phosphate (TBP) is the commonly used and

has been commercialized for recovery of uranium from the spent nuclear fuels in the nuclear

industry. This TBP centered extraction process is known as Plutonium Uranium reduction

Extraction (PUREX) process (Lanham and Runion, 1949). Amide based extraction has also

been studied; it could be considered as a substitute for the organophosphorus compounds as it

is considered as green extractant (Manchanda and Pathak, 2004). Solvent extraction is

considered as a suitable method for uranium separation for its high separation factor, high

selectivity. However, this also has some disadvantages like solvent loss and generation of

secondary waste.

Solubilization using chelating agents like carbonates (Mason et al., 1997) and citrate

(Kantar and Honeyman, 2006) are used to recover uranium. Addition of carbonate or

bicarbonate increases the solubilization and leads to the mobility of the uranium ions from the

contaminated site. Sodium carbonate and bicarbonate are also used for solubilization and

subsequent precipitation of uranium from carbonate-containing ore materials (Suri et al.,

2014). Citric acid is also an active chelating agent in the mobilization of uranium-

contaminated soils. It is effectively applied in in-situ and ex-situ extraction of soils and

decontamination in the nuclear industry. One of the advantages of citrate in complexing

uranium is the biodegradability of U-citrate complex. Its biodegradation does not release the

uranium (Huang et al., 1998).

Adsorption is another widely studied, common and most efficient method for removal

of pollutants from the environment. It is based on the physicochemical interactions between

adsorbate and adsorbent. It is affected by the pH, metal ion concentration, competing ions,

nature of functional groups on adsorbents and others. The advantages of adsorption based

technology are low cost, excellent stability and ease of modification of functional groups for

higher adsorption capacity. For adsorption based remediation of pollutants various organic

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(resins, chitosan, cellulose, polypropylene, and others), inorganic (silica, carbon nanotubes,

graphene oxide) and hybrid (magnetic composites) adsorbents have been developed and

studied (Abdi et al., 2017; Anirudhan and Sreekumari, 2010; Calì et al., 2018; Christou et al.,

2019; Guo et al., 2018; Khawassek et al., 2018; Qian et al., 2018; Vivero-Escoto et al., 2013).

First-time Polypyrroles have been used to remove the uranium, and it showed a maximum

loading capacity of 87.72 mg U/g (Abdi et al., 2017). Polymers serve as good adsorbents for

removal of uranium as they provide excellent chemical stability and ample functional groups.

These can be used directly or could be chemically modified to enhance the complex

formation towards uranyl ion. Polyvinyl-pyrrolidone/chitosan blended nanofibrils have been

synthesized and studied for uranyl adsorption from aqueous solutions. This blended polymer

has the extreme uranium loading capacity of 167 mg U /g and can be used multiple times

without loss in efficiency (Christou et al., 2019). Some of the polymer adsorbents that are

used to remove uranium are collagen immobilized tannins (Sun et al., 2010), titanium loaded

collagen fibres (Cheng et al., 2011) and alkali-activated collagen fibres (Deng et al., 2018).

Nanotechnology is also playing a critical role in removing the pollutants from the

environment, including the uranium. Generally, a suitable adsorbent should have the

properties of maximum adsorption capacity and desorption cycle performance along with

high chemical and mechanical stability. Nanoparticles and carbon nanotubes can achieve

these properties. Recently, studies have reported the use of magnetic nanoparticles for

pollutant removal from the aqueous environment (Ngomsik et al., 2005; Yantasee et al.,

2007). However, bare nanoparticles and nanotubes will have low adsorption capacity and

selectivity towards the uranium. For example, a study by Das et al. (2010) has shown the

maximum loading capacity of 5 mg U/g of nanoparticles. Functionalized nanoparticles can

overcome this low loading capacity of nanoparticles. Therefore, nanoparticles are grafted

with the functional groups having a high affinity for uranyl ions. These modified sorbents

11
will be having high adsorption capacity and selectivity. In a recent study by Calì et al.,

(2018), functionalized magnetic nanoparticles with phosphate via a ligand exchange reaction

were used. These phosphate-Fe3O4 nanoparticles have an extremely high loading capacity

of ̴1700 mg U/g along with specificity and selectivity. Table 1 summarizes the adsorption

capacities of different chemical sorbents used for uranium sequestration.

3. Microbial interactions with uranium

Biological agents such as bacteria, fungi or plants interact with the uranium in a variety

of ways to protect and survive in the environment containing naturally abundant uranium or

contaminated sites (Merroun and Selenska-Pobell, 2008). A wide range of organisms has

been identified and characterized from mining and contaminated sites that can remove or

immobilize the uranium. These interactions of biological agents with uranium results in the

sequestration or complexation and thereby decrease the concentration of toxic metal in the

environment. The metal-microbe interactions include bioaccumulation, bioprecipitation,

bioreduction and biosorption. These methods are of great significance in terms of

bioremediation.

3.1. Bioaccumulation

Bioaccumulation is an energy-consuming process by which uranium is sequestered by

transporting inside the cell. Uranium has no biological role, and there are no transporter

proteins identified yet for its transport into the cell. Uranium may accumulate inside of the

cell due to increased permeability of the cell membrane when it is under heavy metal or other

stress condition (Suzuki and Banfield, 2019). Once it enters into the cell, it will be

sequestered by the internal phosphates as uranyl phosphate (Suzuki and Banfield, 2004;

VanEngelen et al., 2010). Accumulation of uranium was reported in a diversity of organisms

including bacteria, fungi and plants (Gerber et al., 2018; X. Li et al., 2016; Sasmaz et al.,

12
2016). Schematic illustration of the bioaccumulation process is presented in Figure 1. There

is no definite evidence available in open literature signifying bioaccumulation of uranium

could be a proper strategy for bioremediation of contaminated soil or water (Dummi

Mahadevan and Zhao, 2017).

3.2. Bioprecipitation

Bioprecipitation is the formation of insoluble uranium precipitate by complexation with

the inorganic ligands such as phosphates, through an enzymatic process. The microbial

phosphatases hydrolyze the organic phosphate substrate and release the inorganic phosphate,

which forms a metal-phosphate insoluble precipitate. This is also termed as biomineralization

(Renninger et al., 2004). In this process, the oxidation state of the uranium does not change as

in bioreduction. Bacillus and Rahnella spp. were tested positive for phosphatase activity and

precipitated 73% and 95% of U from 200 µM uranyl acetate and 10 mM glycerol-3-

phosphate, respectively. A negative phosphatase Arthrobactor sp. isolated from subsurface

soils at the US DOE Oak Ridge Field Research Center (ORFRC) did not precipitate the

uranium (Beazley et al., 2007). Shewanella putrefaciens immobilized uranium as

chernikovite [H2-(UO2)2(PO4)2.8H2O], on the cell surface at slightly acidic pH (Huang et al.,

2017). At neutral pH, Aeromonas hydrophila, Pantoea agglomerans, and Pseudomonas

rhodesiae strains have mineralized the uranium as insoluble hydroxyapatite [Ca5(PO4)3OH]

in aerobic and nitrate-reducing conditions (Shelobolina et al., 2009). Indigenous bacteria

present in uranium mine waters at Uranium Corporation of India Limited (UCIL), revealed an

exceptional uranium biomineralization property. This strain could remove 99% of soluble

U(VI) as crystalline uranyl phosphate species [(UO2(PO3)2, (UO2)3(PO4)2H2O, and

U2O(PO4)2] (Choudhary and Sar, 2011). Biomineralization by phytate was also demonstrated

in uranium-contaminated sediments of ORFRC (USA). Hydrolysis of phytate releases the

inorganic phosphate which precipitates uranium as ternary sorption complexes, U(VI)-

13
phytate precipitate and U(VI)-phosphate mineral. In a study by Beazley et al. (2011), nearly

complete phytate hydrolysis was noticed at pH 5.5. Initial sorption followed by

biomineralization was reported using B.cereus 12-2 sp. They suggested that the extracellular

amorphous U containing particles were transferred into the cell and converted into uramphite

intracellularly by phosphatase (Zhang et al., 2018). Bioprecipitation of uranium by isolates

Bacillus sphaericus JG-7B and Sphingomonas sp. S15-S1 at acidic conditions via acid

phosphatase was also reported (Merroun et al., 2011). Staphylococcus aureus biofilms have

also been studied recently for uranium sequestration in which, the acid phosphatase activity

of biofilms presented a new method for bioremediation (Shukla et al., 2019). The

phosphatase enzymes responsible for the activity were identified as PhoY and phytase in

Caulobacter crescentus (Yung and Jiao, 2014), PhoK (alkaline phosphatase) in

Sphingomonas sp. BSAR-1 (Nilgiriwala et al., 2008) and PhoN (acid phosphatase) in

Serratia sp. (Paterson-Beedle et al., 2012). Figure 2 provides a general schematic illustration

of bio precipitation mechanism.

Several recombinant strains such as E. coli with acid phosphatase genes (Basnakova et

al., 1998), P. veronii and P. rhodesiae with alkaline phosphatase genes (Powers et al., 2002),

and D. radiodurans (Appukuttan et al., 2006) were developed for biomineralization of

uranium. Recombinant E.coli DH5α bearing cloned phoN was able to sequester uranyl ion

(UO22+) from dilute aqueous flows as polycrystalline uranyl phosphate (HUO2PO4).

Genetically modified strains of P. veronii and P. rhodesiae with alkaline phosphatase genes

(phoA) accumulated sufficient PO43- which allowed the precipitation of uranium. D.

radiodurans was engineered with phoK gene from Sphingomonas sp. which encodes novel

alkaline phosphatase. This Deino-PhoK recombinant strain can precipitate >90% of uranium

from 1 mM solution at pH 9.0. The precipitated uranyl phosphate complex was identified as

chernikovite. Another recombinant D. radiodurans strain with phoN from Salmonella

14
enterica serovar Typhi, codes for a non-specific acid phosphatase, retained the

bioprecipitation ability even after 6 kGy of 60Co gamma irradiation.

In addition to bacteria, fungi are also reported to mineralize the uranium (Gadd and

Fomina, 2011). Several yeast strains have also been studied for phosphatase mediated

uranium biomineralization and reported that initial sorption of uranium to the cell surface and

subsequent precipitation (Lopez-Fernandez et al., 2018). In yeast strains, Cryptococcus

filicatus, Kluyveromyces lactis, and Pichia acaciae phosphatase mediated uranium

precipitation was reported when amended with glycerol-2-phosphate (G2P). This U-

phosphate bio-mineral complex was identified as ankoleite, chernikovite, bassetite, and

uramphite by XRD analysis (Liang et al., 2016). Biomineralization of uranium was reported

via uranyl phosphate formation in Saccharomyces cerevisiae when grown in high phosphate

containing media (Ohnuki et al., 2005). In filamentous fungi, Aspergillus niger and

Paecilomyces javanicus uranium biomineralization were observed when organic phosphorous

G2P was supplied externally. The formed bio-minerals were identified as potassium uranyl

phosphate hydrate, meta-ankoleite, uranyl phosphate hydrate, meta-ankoleite, uramphite, and

chernikovite (Liang et al., 2015).

Microorganisms were reported to precipitate uranium depending on speciation in

environmental conditions (Kulkarni et al., 2016; Neiss et al., 2007). However, such studies

are limited. In oversaturated conditions, uranium precipitates with calcite, whereas in under-

saturated condition uranyl complexes with calcium or carbonate, thereby increasing its

mobility (Catalano et al., 2006). In a natural environment where calcium and carbonates are

relatively high and at alkaline pH uranium is present as Ca2UO2(CO3)3 and UO2(CO3)34- and

this further increases the mobility (Bernhard et al., 2001; Zheng et al., 2003). Silicates also

play a pivotal role in biomineralization of uranium. The major problem to apply

biomineralization as a remediation strategy is an economic viability. The usage of organic

15
phosphate as a donor may limit its application as in situ radionuclide remediation technique.

Another problem with this technique is the precipitation of metal on the surface of the cell

may hinder the metabolism of the organism and may lead to reduced efficiency (Dummi

Mahadevan and Zhao, 2017).

3.3. Bioreduction

Reduction process of U(VI) to U(IV), makes insoluble precipitate of uranium and

removes it from groundwater. Many bacterial and fungal species are recognized to reduce

uranium. More than 25 species of prokaryotes have been identified which are involved in

uranium reduction. For example, iron-reducing bacteria (Geobacter uraniireducens and

Geobacter daltonii) and sulfate-reducing bacteria (SRB) (Desulfovibrio, Desulfobacterium,

and Desulfotomaculum genera) could able to reduce U(VI) to U(IV) (Akob et al., 2012).

These bacteria can use uranium as an electron donor in addition to iron and sulphate. Many

laboratory and field studies have reported the reduction and reduced bioavailability of

uranium. When the contaminated sites are augmented with external organic substances, these

organisms can reduce uranium and utilized in the long term (~2 years) experiment at

contaminated sites (up to 60 mg/L and 800 mg/kg solids) of the US Department of Energy at

Oak Ridge. When the groundwater and subsurface were conditioned with ethanol,

bioreduction was achieved to a sub-micromolar level. Conditioning with ethanol stimulated

the growth of denitrifying, iron-reducing, and sulphate-reducing bacteria which were low or

absent in controls (Istok et al., 2004). When sites were enriched with slow-release substrates

such as emulsified vegetable oil U(VI) reduction was noticed with Comamonadaceae,

Geobacteriaceae and Desulfobacterales (Gihring et al., 2011). Figure 3 describes the general

aspects of the bioreduction process.

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3.3.1. Factors affecting the bioreduction process

Many environmental factors affect the in situ bioreduction processes, and it is essential

to know uranium reducing microbial populations in a natural environment. Other

environmental factors such as salinity, temperature, redox potential, pH, organic substrates

and presence of other contaminants will also influence the population of uranium-reducing

microbes and bioreduction process. In anaerobic uranium reduction, Geobacter spp. are

dominant. While Desulfovibrio spp. are prevailing bacteria in a sulphate-reducing enrichment

and Clostridium spp., Ferribacterium spp., and Geothrix spp. are mainly present in an iron-

reducing environment (Boonchayaanant et al., 2009; Cardenas et al., 2008). Elevated pH and

presence of calcium or carbonates will decrease the uranium reduction process by forming

the calcium-uranyl-carbonate and uranyl-carbonate complexes, respectively. These uranyl

complexes are highly soluble and likely to decrease the reduction (Brooks et al., 2003; Fox et

al., 2013). Uranium bioreduction will be facile at pH 5.0 to 6.0 and in the absence of ligands

such as carbonates, phosphates and sulphates. Uranyl hydroxyl complexes are readily

reduced, which are the dominant species in that condition. At different environmental pH

conditions, quality and quantity of microbial populations will also change. At neutral, pH

Ralstonia and Dechloromonas spp., and an acidic pH Castellaniella and Burkholderia spp.

are widely studied (Spain and Krumholz, 2012). Presence of oxygen will affect the stability

of reduced uranium. Reports suggest that nearly all the bioreduced uranium is reoxidized over

a while (Komlos et al., 2008). Nitrates may also inhibit the uranium reduction process

because of its higher electric potential (Istok et al., 2004). Presence of sulphates increased the

U(VI) reduction rates by both D. desulfuricans and a D. vulgaris and Clostridium sp.

coculture (Spear et al., 2000). Some electron donors such as acetate, glucose, lactate, ethanol

and methanol, aromatic hydrocarbons like toluene, pyruvate, fumarate and emulsified

17
vegetable oils have been tested for bioreduction and each of which has its ability (Cardenas et

al., 2008; Gihring et al., 2011; Luo et al., 2007; Madden et al., 2009).

3.3.2. Mechanism of bioreduction

Pathway of uranium reduction, enzymes or genes responsible and the number of

electrons involved has not been well established yet. Generally, the reduction of U(VI) to

U(IV) needs two electrons. Though, literature reports suggest that a single electron reduction

system is the most likely mechanism of reduction. One electron reduction mechanism was

explained by using G. sulfurreducens in which U(VI) initially reduced to U(V) by one

electron and then to U(IV) by disproportionation (Lloyd and Renshaw, 2005). It has been

reported that bacterial pili act as electron conductors between cells to an electron acceptor

(Reguera et al., 2005). Microscopic studies revealed that the reduced U (IV) was

predominantly localized at periplasm and outside of the cells. This indicates that outer

membrane-bound enzymes are responsible for uranium reduction. Crude cell extracts, whole-

cell experiments and mutational analysis of D. vulgaris have suggested the role of

cytochrome c3 in U(IV) reduction.

Bioreduction seems to be more attractive choice when compared to alternatives such as

biosorption or bioaccumulation. However, recent studies showed that when biogenic

uraninite was exposed to oxygen, it readily reoxidized to U(VI), making it a futile

bioremediation strategy. In the case of humic substances, it did not cause precipitation of

reduced uranium. Instead, it remained in the solution as U(VI)-humic complex (Gu et al.,

2005; Wan et al., 2011). Presence of organic ligands impairs the movement of the uranium

even in its reduced form. Clostridium sps reduced the citric acid associated with U(VI) as

U(VI)-citric acid complex, which remained in solution instead of precipitation. It increases

the mobility in the environment and is a potential for contaminating groundwater and other

18
sites (Francis and Dodge, 2008). The end products formed also are an essential factor during

the uranium bioreduction process. Thermobacterium ferrireducens could able to reduce the

uranium as ningyoite [CaU(PO4)2.H2O], a U(IV)-phosphate complex. It has been reported

that several U(IV) reduced products are being formed like biogenic uraninite, monomeric

U(IV). It is a well-known fact that the biogenic uraninites are more stable to reoxidation and

preferred for bioremediation rather than other forms (Bargar et al., 2008; Khijniak et al.,

2005; Lee et al., 2010; Renshaw et al., 2005).

3.4. Biosorption

Biosorption is well-defined as "the uptake of metal species by physicochemical

mechanisms and is independent of metabolic process" (Gadd, 2009). However, sometimes

pre-growth conditions can affect the efficiency of biosorption, and metabolic activity may

also affect the process, e.g. by triggering localized changes in pH or by releasing metal

complexing ligands (Gadd, 2004). All biological moieties like bacteria, fungi, algae, plant

and animal biomass as well as derived products such as chitosan have the capability of

biosorption. The chemical composition of the cell wall determines the capacity of the

biosorption. Specific functional groups present on the cell surface such as carboxyl, amine,

hydroxyl, phosphate and sulfhydryl groups interact with the metal and leads to the sorption of

metals (Fowle et al., 2000; Gorman-Lewis et al., 2005; Lütke et al., 2012). Gram-positive

bacterial cell wall consists of peptidoglycan with few teichoic acids. Whereas, Gram-negative

bacteria cell wall is more complicated due to its additional lipopolysaccharide (LPS)

membrane outside a thin peptidoglycan cell wall. Carboxyl groups in the peptidoglycan and

the phosphoryl groups in the teichoic acids are the most probable sites for the metal

complexing (Merroun et al., 2005). Langley and Beveridge (1999) depicted the role of

carboxylic groups in the binding of metal cations to O-side-chains of LPS and inferred that

metal was most likely to bind to phosphoryl groups in the core-lipid 'A' of LPS. The bacterial

19
extracellular polymeric substances (EPS) can also aid in sequestration of metal ions. For

example, P. aeruginosa, A.ferroxidans, Myxococcus xanthus and other strains isolated from

different habitats have exhibited the complexation of U within EPS (Beech and Cheung,

1995; Kazy et al., 2008). Several environmental bacterial isolates have been used to quantify

the uranium binding capacity. Schematic mechanism of biosorption on a typical bacterial cell

wall is represented in Figure 4.

Biosorption capacities and characteristics of different microbial types are presented in

Table 3 (bacteria), Table 4 (fungi) and Table5 (algae). From Table 3, it was observed that

actinomycetes named Brachybaterium sp. G1 which was isolated from the German salt dome

Gorleben had the maximum biosorption capacity of 971 mg U/g biomass. The process was

complete biosorption, and carboxylic functional groups played a significant role in the

interaction of uranium with the bacterial cells (Bader et al., 2018). Immobilized biomass was

used for sequestration of the uranium and proved to a viable option in various studies. For

instance, Geobacillus thermoleovorans subsp stromboliensis was immobilized onto an

Amberlite XAD-4 ion exchange resin and used for pre-concentration of uranium in the solid-

phase extractor (SPE) (Ozdemir and Kilinc, 2012). Synechococcus elongatus cells were

immobilized in polyacrylamide gels and used for the removal of uranium in a continuous

flow-through system. It has shown significant uranium binding capacity up to three

adsorption-desorption cycles (Acharya and Apte, 2013a). The multiwalled carbon nanotubes

(MWCNTs) were loaded with B. mojavensis and B. vallismortis and studied for pre-

concentration of uranium as SPE (Ozdemir et al., 2017; Özdemir et al., 2017a). The

biosorption capacities of B. mojavensis and B. vallismortis loaded MWCNTs were 25.8 and

23.6 mg U/g, respectively. They could be used in multiple adsorption-desorption cycles

without significant loss in biosorption efficiency. Similarly, in a recent study by Tong, (2017)

reported the immobilization of B. subtilis in alginate-chitosan microcapsules for uranium

20
removal. These alginate-chitosan microcapsules had good mechanical strength along with a

high loading capacity of 374.64 mg U/g biomass. This was the first report on sequestration of

uranium by alginate-chitosan microcapsules. The immobilization of biomass will offer the

feasibility of its application in bioremediation technology.

Unicellular yeast and filamentous fungi were studied for biosorption of uranium. Live

cells, dead cells and modified cells have been used to study the uranium biosorption capacity.

Uranyl adsorption by magnetically modified Rhodotorula glutinis was studied successfully

(Bai et al., 2012). Magnetic adsorbents have the advantage of the trouble-free separation of

compounds from solutions. Rhodotorula glutinis cells were also immobilized in Ca-alginate

beads and investigated for uranium removal capacity (Bai et al., 2014). The typical regular

lab strain ascomycetous yeast Saccharomyces cerevisiae has also been reported for uranyl

adsorption. Heat killed cells of Saccharomyces cerevisiae have shown the higher biosorption

capacity than the live cells (Lu et al., 2013; Wang et al., 2017). Recently, marine yeast

Yarrowia lipolytica has been explored for uranyl removal (Kolhe et al., 2020). Y. lipolytica

showed a biphasic uranium binding pattern with a maximum loading capacity of 37.5 mg U/

g dry weight of cells. In filamentous fungi, Trichoderma harzianum, Aphanocladium

spectabilis, Lentinus sajor- caju, Schizophyllum commune and others were found to be useful

in uranium biosorption with significant efficiency (Table 4).

Algae are photosynthetic, eukaryotic aquatic organisms. Biosorption of uranium by

different groups have also been reported (Table 5). Cystoseira indica, a brown algae biomass

was investigated for uranium biosorption. The maximum loading capacity of Ca-pretreated,

protonated and non-protonated Cystoseira indica was 454.5, 322.58 and 224.67 mg/g

respectively (Khani et al., 2006). Padina sp., a marine brown algae biomass, was studied for

removal of aqueous uranium solution, and it could load up to 377 mg /g (Khani, 2011).

21
 Biosorption is the preferred technique over other conventional physical and chemical

processes for treating the contaminants which are having low to medium level concentrations.

Since the binding of pollutant /toxic metal to cell wall is spontaneous and biosorbent can be

regenerated by removing the bound metal. The advantages of biosorption are low cost, high

efficiency, regeneration of the biosorbent and metal recovery. Though biosorption seems to

be a promising strategy for bioremediation of industrial effluents, there are some drawbacks

in using it as an industrial application. The major disadvantage with biosorption are 1) rapid

desorption, 2) presence of competing ions, and 3) saturation of binding sites. The efficiency

of biosorption will be decreased when complexing ligands such as humic, fulvic acids and

competing cations like Ca2+ and Mg2+ are present (Siegel and Bryan, 2003). Algae, bacteria

and fungi are potential biological agents in biosorption of the metals. Furthermore, the cell

wall composition is vital in determining the efficiency of metal ion binding.

4. Conclusion

Bioremediation is gaining more importance to treat heavy metals contaminant sites due

to its cost-effective, eco-friendly and superior efficiency over conventional methods. Various

methods such as biosorption, bioreduction and bioprecipitation have been tested by using a

different group of organisms. Many of such strains have shown the ability to minimize the

bioavailability of the pollutant under laboratory studies. Nevertheless, in terms of significant

scale field application, none of them has proved to be efficient over a long time. Since the

experimental conditions in the laboratory are more convenient for the bacteria/algae/fungi

than the actual environmental conditions. To overcome this limitation, the specific metabolic

activity of an organism and the environmental conditions has to be studied. Hence, more

studies have to be focused on the field application and fine-tuning the required parameters for

better sustainability and pollution-free environment.

22
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank Dr B. Venkatraman, Director SQ&RMG, IGCAR for support and

encouragement during the course of this study.

Funding

This work was funded by Department of Atomic Energy, Government of India.

23
Figure Legends

Fig. 1: Schematic representation of bioaccumulation of uranium

Fig. 2: Schematic representation of biomineralization of uranium

Fig. 3: Schematic representation of bioreduction of uranium

Fig. 4: Schematic representation of biosorption of uranium

24
Figure 1

25
Figure 2

26
Figure 3

27
Figure 4

28
 Table 1: Uranium speciation in different experimental media as predicted by Visual
MINTEQ software

Uranium Dominant
Conditions pH Ref.
concentration species
5 mM Ca & 2.5 mM
Ca2Uo2(CO3)3 aq
KHCO3
0 Mm Ca & 2.5 mM Carvajal et al.
87 µM Uranyl nitrate 7.3 UO2(CO3)2-2
KHCO3 (2012)
0 Mm Ca & 5 mM
UO2(CO3)3-4
KHCO3
LPM media (UO2)2CO3(OH)3- Lopez-
0.5-1 mM Uranyl
7 (UO2)4(OH)7+& Fernandez et
nitrate NaClO4
(UO2)3(OH)5+ al. (2018)
5 mM G3P & 10 mM (UO2)2(OH)5+&
6.8
MOPS (UO2)4(OH)7+
1 mM Uranyl Kulkarni et al.
5 mM G3P, 2.4 mM
carbonate (2016)
Ammonium Carbonate & 9 UO2(CO3)3-4
10 mM MOPS
UO22+& UO2-
1 mM Uranyl nitrate 100 mM Acetate buffer 5
Acetate+
(UO2)3(OH)5+&
7 Chandwadkar
(UO2)4(OH)7+
1 mM Uranyl et al. (2018)
20 mM MOPS Buffer (UO2)3(OH)7-,
carbonate
9 UO2(CO3)24-
&UO2(CO3)34-
UO22+,
03 - 05 (UO2)2(OH)22+&
100 mg/L U as UO2CO3 Huang et al.
14.56 mM Na2CO3
Uranyl nitrate (2017)
UO2(CO3)22-
05 - 08
&UO2(CO3)34-

29
Table 2: Adsorption performances of different chemical sorbents for uranium

Sorbent pH Maximum Ref.

loading capacity
(mg/g)
Fe3O4 NPs 7.0 5 Das et al. (2010)
Phosphated- Fe3O4 NPs 7.0 1690 Calì et al. (2018)
Tributyl PO4-C nanotubes 5.0 166.7 Mishra et al. (2016)
Amidoxime-SiO2@ Fe3O4 NPs 5.0 105 Zhao et al. (2014)
Graphene/ Fe3O4 NPs 5.5 69.5 Zong et al. (2013)
Amidoxime (AO) modified multiwalled 5.0 67.9 Wu et al. (2018)
carbon nanotubes (MWCNT)
Chitosan modified multiwalled carbon 5.0 41 Chen et al. (2013)
nanotube
Chitosan-tripolyphosphate beads 5.0 236.9 Sureshkumar et al.
(2010)
Amidoxime-grafted multiwalled carbon 4.5 145 Wang et al. (2014)
nanotubes
phosphoryl multiwalled carbon nanotubes 5.0 194.17 Guo et al. (2018)
(PS-MWCNTs)
Amidoxime modified chitosan/ bentonite 8.0 49.09 Anirudhan et al.
composite (2019)
Polyvinylpyrrolidone/Chitosan blended 6.0 167 Christou et al.
nanofibers (2019)
Fe3O4 into calcium alginate coated 6.0 392.69 Yi et al. (2018)
chitosan HCl hydrogel beads
α-aminophosphonate functionalized 5.0 245 Imam et al. (2018)
chitosan
MMSNs-PP 3.5 37.5 D. Li et al. (2016);
Li et al. (2017)
MMSNs-PPI 9.6 133.3 D. Li et al. (2016);
Li et al. (2017)
TBP-SBA-15 5 197.8 Xue et al. (2017)
MSPh-III 8.3 185.2 Vivero-Escoto et
al. (2013)
Amberjet 1200 H 1.2 133 Khawassek et al.
(2018)
Succinic acid impregnated amberlite 4.5- 12.33 Metilda et al.
XAD-4 8.0 (2005)
Gel-amide 7.0 28.98 Venkatesan et al.
(2004)
Polypyrrole 5.0 87.72 Abdi et al. (2017)
Anionic resin Dowex A 3.9 79 Ladeira and
Gonçalves (2007)
Zeolite X- PAN --- 90 Akyil and Eral
(2005)
GO-COOH/UiO-66 8 1097 Yang et al. (2017)
salicylaldoxime/polydopamine modified 5 1049 Qian et al. (2018)
reduced graphene oxide

30
Table 3: Uranium biosorption capacities of different bacteria

Name of organism Biomass Uranium pH Loading Ref.

quantity concentra capacity
(mg) tion (mg/g)
Halobacterium --- 0.04 mM 6.0 9.3 Bader et al.
(2018, 2017)
Bacillus sp. (dwc-2) 0.5 g/L 10 mg/L 3.0 3.03 Li et al. (2014)
Bacillus mojavencis (carbon 20 10 mg/L 5.0 25.8 Özdemir et al.
nanotubes loaded) (2017b)
Bacillus vallismortis (carbon 50 mg 10 mg/L 5.0 23.6 Ozdemir et al.
nanotubes loaded) (2017)
Bacillus subtilis (alginate- --- 150 mg/L 6.0 376.64 Tong (2017)
chitosan microcapsules)
Geobacillus Thermoleovorans 250 4.20 mM 5.0 11 Ozdemir and
subsp. Stromboliensis Kilinc (2012)
(immobilized AmberliteXAD-4
ion exchange resin)
Streptomyces levoris 15 mg 0.1 mM 3.5 9.04 Tsuruta (2004)
Streptomyces sporoverrucosus 100 mg 0.042 mM 3.0 3.0 Li et al. (2016)
dwc-3
Amycolatopsis sp. K47 0.001 0.168 mM 4.0 38.8 Celik et al.
(2018)
Brachybacterium sp. G1 --- 0.04 mM 6.0 971 Bader et al.
(2018)
Citrobacter freudii --- 0.084-0.63 --- 48 Xie et al.
mM (2008)
Pseudomonas aeruginosa CSU 30-50 0.4 mM 2.4 100 Strandberg et
al. (1981)
Myxococcus xanthus 20 1 mM 4.5 28.56 Gonzälez-
Muñoz et al.
(1997)
Synechococcus elongatus --- 0.1 mM 7.8 53.5 Acharya et al.
(2009)
Synechococcus elongatus --- 1 mM 7.8 72.5 Acharya and
(immobilized) Apte (2013a)
Anabaena torulosa --- 0.1 mM 7.8 77.35 Acharya et al.
(2012);
Acharya and
Apte (2013b)
Microcystis aeruginosa --- 0.168 mM 7.0 44 Li et al. (2004)

31
Table 4: Uranium biosorption capacities of different fungi

Name of organism Biomass Uranium pH Loading Ref.

quantity concentration capacity
(mg/g)
Saccharomyces --- 0.0042 mM 5.5 29.8 Lu et al.
cerevisiae (heat (2013)
killed)
Saccharomyces 25-200 mg 1 mg/L 5.0 94.9-4.3 Wang et al.
cerevisiae (heat (2017)
killed)
Saccharomyces 25-200 mg 1 mg/L 5.0 3.2-1.9 Wang et al.
cerevisiae (Live) (2017)
Saccharomyces 1 mg/mL 60 mg/L 6.0 47.4 Bai et al.
cerevisiae(PAO-B (2016)
modified)
Rhodotorulla glutinis 5.15 mg 0.420 mM 6.0 235 Bai et al.
(magnetically (2012)
modified)
Rhodotorulla glutinis 5 mg 0.153 mM 6.0 17.3 Bai et al.
(immobilized) (2014)
Rhodotorulla glutinis 4 mg 0.588 mM 6.0 98.4 Bai et al.
(raw) (2010)
Trichoderma 0.420 mg 4.20 mM 4.5 196 Akhtar et al.
harzianum (2009)
Aphanocladium 40 mg 100 mg/L 3.5 161.5 Gargarello et
spectabilis al. (2008)
Acremonium 40 mg 100 mg/L 3.5 162.1 Gargarello et
minutisporum al. (2008)
Lentinus sajor- caju --- 0.840 mM 4.5 182 Bayramoǧlu
(Alkali treated) et al. (2006)
Schizophyllum 300 mg 0.0021-0.5 5.0-6.0 280 (Günther et
commune mM al., 2014)
Fusarium sp. #ZZF51 100 mg 0.21 mM 4.0 16 Yang et al.
2012)

32
Table 5: Uranium biosorption capacities of different algae

Name of Biomass Uranium pH Loading Ref.

organism quantity concentration capacity
(mM) (mg/g)
Chlorella --- 0.1 4.4 14 Vogel et al.
vulgaris (2010)
(live)
Chlorella --- 0.1 4.4 28 Vogel et al.
vulgaris (2010)
(dead)
Cystoseria --- 1.47 4.0 455 Khani et al.
indica (2006)
(calcium
pretreated)
Cystoseria --- 0.210-4.20 4.0 198 Khani et al.
indica (2008)
Catenella 50 mg 0.420 4.5 303 Bhat et al.
repens (2008)
Cladophora 12 g/L 5.0 152 Bağda et al.
hutchinsiae (2017)
Padina sp --- 0.210-4.20 4.0 377 Khani (2011)

33
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