Kinetics of uranium sequestration by two natural

isolates from alkaline uranium mine waters

Abstract:

The present study aims at the isolation of bacterial strains that have potential in sequestering

uranium from alkaline wastewaters. The two bacterial strains, MPW02 and MPW03 isolated

from uranium mine pond waters were molecularly characterized and identified as Aeromonas

and Bacillus species. These two isolates studied for uranium sequestration from carbonate-

containing alkaline wastewaters. The pseudo-second-order model well described the kinetics

of uranium sequestrations by these isolates. Langmuir and Freundlich's models were fitted

well with the experimental data. The maximum loading capacity by the two isolates MPW02

and MPW03 were 105 and 30 mg U/ g dry biomass, respectively. Autoclaved biomass of

MPW03 cells was able to retain the uranium sequestration capacity. Whereas, dead MPW02

cells were not able to sequester uranium. Increase in carbonate-bicarbonate buffer

concentration also affected differently by two isolates. It indicates that these two isolates

adapt the different mechanisms of uranium sequestration. Further, FTIR analysis was used to

elucidate the role of functional groups in uranium sequestration. Overall, this study signifies

the potential application of these isolates for treating the alkaline uranium wastewaters.

Keywords: Uranyl carbonate, Uranium mine pond, Bioremediation, Kinetics

1. Introduction:
Uranium (U) contamination is a foremost environmental apprehension because of its

radiological and chemical toxicity. Although uranium is a ubiquitous element, anthropogenic

activities like nuclear energy production, nuclear weapon testing, and overexploitation of

groundwater and uncontrolled use of phosphate fertilizers put pressure on earth. Moreover,

natural disasters and geogenic activities also contribute to the elevated levels of

radionuclides/heavy metals in the environment (Coyte et al., 2018; Lloyd and Renshaw,

2005; Scholten and Timmermans, 1995; Skipperud et al., 2013). Uranium contamination and

its distribution in the environment primarily depend on the speciation and vice versa

(Markich, 2002). The most dominant oxidation states of uranium are U(VI) and U(IV). Under

oxidizing and at lower pH conditions soluble U(VI) and in reducing conditions insoluble

U(IV) state exists (Acharya, 2015; Konstantinou and Pashalidis, 2004; Sun et al., 2014).

Since U(VI) is highly soluble, it has more prospect to contaminate near-surface water and

groundwater (Schneider et al., 2017; Selvakumar et al., 2018; Song et al., 2019). Alkaline

wastes with uranium traces generate from uranium powered nuclear reactors and power

plants. For example, intermediate-level liquid waste (ILLW) and low-level liquid waste

(LLW) (pH 8-12) will contain 5-20 mM of residual uranium. Savannah River Site, Aiken; SC

is planning to dispose of 130 million litres of alkaline nuclear waste containing uranyl

carbonate (Duff et al., 2004; Kulkarni et al., 2013; Raj et al., 2006; Valsala et al., 2010). At

this alkaline pH, uranyl carbonate species such as [UO2(CO3)22-] and [UO2(CO3)34-] are more

prevalent and these are also highly soluble and stable. Although uranyl ion (UO 22+) is more

toxic than uranyl carbonate, the later may find its way through the groundwater from the

contamination site to other sites and may cause significant environmental threat.

There are several reports on biosorption, bioreduction, bioaccumulation and

biomineralization of uranium by various microorganisms (Gerber et al., 2018; Kolhe et al.,

2020; Li et al., 2017; Shukla et al., 2019; Zhang et al., 2018). All these studies were done at

acidic, neutral or near alkaline pH. However, very few reported the interaction of uranyl

carbonate with bacteria at alkaline pH (Chandwadkar et al., 2018; Kulkarni et al., 2016, 2013;

Nilgiriwala et al., 2008). These studies suggest that the presence of carbonates greatly

influence the uranium sequestration and is challenging to immobilization. Nevertheless,

uranyl carbonates are toxic pollutants and should be treated at par with uranyl ion from an

environmental perspective.

Depending on the mineralogical composition of the ore, either acid leaching or

alkaline leaching methods are used for uranium extraction. Tummalapalle uranium mine

(Andhra Pradesh, India) has been identified as one of the largest uranium reservoirs in India.

Tummalapalle mine is a carbonate-rich dolostone type and alkaline pressure leaching process

is being used to extract the uranium from the ore. It is the first of its kind in India to use

alkaline pressure leaching and is considered to be more economical when carbonate content

is high (Suri et al., 2014). Hence, mine tailing and waste residuals will be rich in carbonates

and highly alkaline, and the dominant uranium species will be uranyl carbonate. Further,

groundwater is also buffered with carbonates. These uranyl carbonates are difficult to

sequester biologically as they are highly stable and interfere with the metal-microbe

interactions (Carvajal et al., 2012). In this scenario, the remediation or stabilization of these

uranyl carbonate species from the contaminated sites is an essential aspect of current

research.

In this present paper, we aimed at uranium sequestration from carbonate-rich alkaline

wastewater. Indigenous isolates from alkaline uranium mine pond water were selected for the

study. Their uranium sequestration properties were studied on a temporal scale with respect

to different initial uranium concentration, carbonate concentration and different pH as well.

Further, uranium sequestration by the isolates was assessed in terms of equilibrium and

isotherm kinetic model studies.

2. Materials and methods

2.1. Bacteria and culture conditions

The bacterial strains used in the present study were isolated from the Tummalapalle

uranium mine pond waters. Microscopic, biochemical and 16s rDNA sequences were used to

characterize the bacterial strains. Stock cultures of bacterial strains were maintained in

glycerol stocks. For maintenance and routine lab experiments, R2A media was used.

2.2. Analysis of uranium by Arsenazo III

Uranyl carbonate stock was prepared by a combination of sodium carbonate and stock

solution of uranyl nitrate. The uranyl carbonate species were confirmed by the presence of

specific peaks at 434, 448 and 464 nm which are characteristic of uranyl, [U(VI)] dicarbonate

species, [UO2(CO3)2]2- (Acharya et al., 2009; Francis et al., 2000).

Uranium was estimated by standard arsenazo (III) method (Golmohammadi et al.,

2012). To 1 mL of sample 125 μL of 2.5% DTPA, 62.5 μL of 10% tartaric acid and 62.5 μL

of 0.25% arsenazo-III reagent were added and mixed gently. After 5 min, the absorbance of

the pink-violet complex was measured at 651 nm in 96 well plates using Tecan multimode

reader and analyzed by Magellan software.

2.3. Uranium sequestration

For uranium sequestration experiments, biomass was harvested from an overnight

grown culture in R2A medium by centrifugation and washed twice with sterile saline. Cells

were resuspended at a concentration of OD ~ 1.0 (600 nm) in the carbonate-bicarbonate

buffer (1 mM) at pH 9.2 in polypropylene tubes. Then, uranyl carbonate was added to the cell

suspensions. These tubes were kept at continuous stirring at ambient room temperature. Each
set of experiment was performed in triplicates. At a specified time, aliquots of samples were

withdrawn and centrifuged to separate the cell biomass. Uranium in the supernatant was

estimated by arsenazo (III) method as described in section 2.2. Also, negative control without

bacterial cell biomass was prepared in each batch of an experiment.

Removal percentage (R %) and removal capacity [Q (mg/g)] were calculated by

following equations (1) and (2), respectively:

R = (C0-Ce)/C0 × 100% (1)

Q = (C0-Ce) × V / W (2)

Where, C0 and Ce are initial and final concentrations of uranium (mg/L), respectively.

V is the volume of the solution (mL), and W is the dry bacteria weight (g).

Effect of pH was studied by adjusting the initial pH of the carbonate-bicarbonate

buffer (pH 9.2, 9.9 and 10.6). Effect of biomass dosage (OD ~ 0.5, 1.0 and 1.5) and

carbonate-bicarbonate buffer (10, 20, 30, 40 mM) was investigated. Different concentrations

of uranium (1-100 mg/L) were used to study the effect of initial concentration on

sequestration. Further, uranium sequestration was also investigated by dead bacterial

biomass.

2.4. Kinetic and equilibrium studies

To study the mechanism of uranium sequestration and potential rate-limiting steps,

widely used pseudo-first-order and pseudo-second-order kinetic equations were employed to

fit the experimental data. The equation corresponding to the pseudo-first-order kinetic is

expressed as follows (Lagergren, 1898):

dqt/dt = k1(qeq – qt) (3)