Where qeq and qt represent the amount of uranium adsorbed (mg/g) at equilibrium and

at time t respectively. k1 is the equilibrium rate constant of pseudo-first-order sorption (min -1).

The linearized form of the pseudo-first-order reaction can be obtained by rearranging the

equation (3)

ln(qe – qt) = lnqe – k1t (4)

Where, qe is equilibrium uranium uptake (mg/g), and qt is the amount of uranium

adsorbed at time t. k1 is the first-order rate constant and can be determined from the slope of

the plot of ln(qe – qt) vs time t.

The equation corresponding to the pseudo-second-order kinetic model is as follows:

dqt/dt = k2(qeq – qt)2 (5)

The linearized form of the pseudo-second-order kinetic equation can be expressed as

(t/qt) = (1/k2qe2) + (1/qe)t (6)

Where k2 is the rate constant of pseudo-second-order biosorption (g/mg min). The

value of k2 and qe can be obtained experimentally from the slope and intercept of the plot t/qt

vs time t, respectively.

The equilibrium data of the uranium sequestration onto the bacterial cells were fitted

with the Langmuir and Freundlich isotherms equations (Volesky and Schiewer, 2002)

Langmuir isotherm is represented by the following equation:

Ce /Qe = 1/qmax kL × 1/Ce + 1/qmax (7)

Where, Qe and Ce is the equilibrium metal uptake capacity (mg/g) and residual metal

concentration at equilibrium (mg/L), respectively. qmax represents the maximum metal uptake

(mg/g), and kL is the affinity constant (mg/L). The affinity constant and qmax were calculated

from the slope of the linear plot of Ce versus Ce /Qe.

The linear form of Freundlich isotherm equation used is:

log q = log k + 1/n log Ce (8)

 The constant k is the measure of adsorption capacity, and n is the intensity of

adsorption. The plot of log q versus log Ce was used to calculate the value of k and Ce.

2.5. FT-IR analysis

For FTIR analysis, bacterial cells treated with or without uranium were recovered by

centrifugation, washed with distilled water and lyophilized. The metal-free control and metal

loaded bacterial cells were prepared as KBr pellets. The infrared spectra were recorded in the

range of 400-4000 cm-1.

3. Results and discussion

3.1. Identification of bacterial isolates

The two bacterial strains MPW02 and MPW03 were isolated from Tummalapalle

uranium mine pond water using low nutrient R2A medium. Phylogenetic affiliation of the

isolated bacterial strains was assigned based on 16s rRNA gene analysis. The gene sequence

analysis indicated that the isolates MPW02 and MPW03 belong to Aeromonas and Bacillus

sp. The obtained gene sequences were deposited in NCBI with accession numbers MT477807

and MT477808.

3.2. Uranium sequestration study

The bacterial isolates MPW02 and MPW03 were studied for the uranium

sequestration ability. The pH of the uranium solution is an essential factor which determines

the sequestration capacity. Uranium sequestration by these two isolates was done at alkaline

pH range (9.2) and in the presence of carbonates (1 mM). It was observed that sequestration

was strongly affected by the pH and carbonates. Fig. 1 describes the effect of contact time of

different initial uranium concentrations on uranium sequestration by two isolates, MPW02

and MPW03. The results indicated that the uranium sequestration was increased as initial

uranium concentration increases by both the isolates. The isolate MPW02 has the maximum

uranium sequestration capacity of 112 mg U/ g dry biomass at 75 mg/L of initial uranium

concentration within 8 h of contact time. Whereas the isolate MPW03 has a maximum

capacity of 36 mg U/ g dry biomass at 50 mg/L of initial uranium concentration within 4 h of

contact time. Fig. 1 also describes the effect of contact time on sequestration by two isolates.

It can be observed that the sequestration capacity increased for 0-4 h and then reached

equilibrium after 6-8 h. It can also be observed that the isolate MPW02 has a better

sequestration capacity over the other isolate, MPW03. The uranium concentration in

controlled samples remains unchanged over the experimental incubation period indicating no

adsorption or precipitation onto the tubes. The contact time for all the experiments was

chosen as 8 h.

Fig. 2 shows the sequestration percentage (R%) and loading capacity (Q) of the

isolates at different initial uranium concentrations. It can be sen that MPW02 has a better

sequestration capacity at higher uranium concentrations while MPW03 had the highest

sequestration at lower uranium concentrations (10 mg/L). The percentage sequestration of

uranium by isolate MPW03 has remained almost unchanged (~ 80%) with the increase in

initial uranium concentration from 5 to 75 mg/L. Further increase in initial uranium

concentration from 75 to 100 mg/L caused a decrease in sequestration percentage to 55%.

The isolate MPW03 had the sequestration capacity of up to 75% when treated with 10 mg/L

initial uranium concentration. However, the sequestration capacity of isolate MPW03 had

decreased when initial uranium concentration increased from 10 to 100 mg/L.

Effect of pH on the uranium sequestration was tested by the two isolates in the range

of pH 9.2 to 10.6 and presented in Fig. 3. The results indicated that uranium sequestration

strongly affected by the change in the pH. The maximum sequestration was observed at pH

9.2 and 9.9, and the lowest sequestration was observed at pH 10.6 by both the isolates. The

observed decreased in the uranium sequestration can be explained by uranium speciation at

higher pH. Effect of carbonate buffer concentration on uranium sequestration was

investigated, and results are presented in Fig. 4. Carbonate concentration strongly affected the

sequestration by isolate MPW02. Increase in the carbonate-bicarbonate buffer from 1 to 30

mM caused the sequestration to decrease to ~ 5% as it was evident from the previous

literature (Kulkarni et al., 2016; Nilgiriwala et al., 2008) that the presence of an excess of

carbonates and at higher pH decreases the uranium sequestration ability. The negatively

charged uranyl carbonate species will be dominated at the higher pH and/or in the presence of

carbonates which will have repulsive nature preventing the sequestration of uranium by

bacterial cell surface. Further, change in pH also influences the functional groups on the

bacterial surfaces, which might affect the sequestration capacity (Imam et al., 2019). But in

contrary, the isolate MPW03 had a little influence on the sequestration of uranium by

carbonates. From Fig. 4, it was clear that even at 30 mM of carbonate-bicarbonate buffer

concentration able to retain the sequestration capacity of ~ 60%. It was almost similar to the

capacity at 1mM carbonate-bicarbonate buffer concentration. From this, it can be concluded

that both the strains had a different mechanism of uranium sequestration.

Fig. 5 shows the effect of biomass concentration on uranium sequestration was

studied with varying amount of biomass (0.5, 1.0, 1.5, 2.0 OD at 600). The increase in

biomass did not affect uranium sequestration by MPW02. In contrast, an increase in

sequestration was observed with the increase in cell concentration by MPW03. Further to

investigate the mechanism of uranium sequestration by these two isolates live and dead

biomass was used. The isolate MPW03 was able to retain the sequestration capacity

indicating the biosorption to be the dominant mechanism of uranium sequestration (Fig. 6).

When the uranium sequestration by live and dead cells of MPW02 was compared, a

noticeable decrease in uranium sequestration was observed depicting the metabolic activity

might be playing the role (Gerber et al., 2018).

3.3. Kinetic modelling

 To investigate the mechanism of uranium sequestration, most commonly used kinetic

models such as pseudo-first-order and pseudo-second-order were chosen. Experimental data

was used to fit these kinetic models. The regression coefficients (R2) calculated indicated that

experimental data well agree with the pseudo-second-order than the pseudo-first-order kinetic

model. Thus the two isolates followed the pseudo-second-order kinetic model. The calculated

qeq values from the pseudo-second-order model well agreed with the experimental values

(Table 1). The better fit of the pseudo-second-order kinetic model indicates that the rate-

limiting step in the uranium sequestration by the isolates depends on the chemical reaction of

uranium ions and the functional groups on the cell surfaces (Ho et al., 2000; Kavitha and

Namasivayam, 2007)

3.4. Isotherm studies

In this study, Langmuir and Freundlich isotherms models were used to study the

uranium binding capacity of two test isolates. The results of the respective models were

presented in Table 2. The experimental data produced good correlation values for Langmuir

and Freundlich isotherm models. For Langmuir isotherm model, the correlation coefficient

values are 0.94 and 0.99 for MPW02 and MPW03, respectively. This indicates the

acceptability of the model for uranium sequestration by these two isolates. From Langmuir

equation maximum loading capacity of MPW02 and MPW03 was calculated and is found to

be 105 and 30 mg U/g dry biomass, respectively. The calculated values are also in good

agreement with the experimental values. Langmuir constants for isolates MPW02 and

MPW03 are 0.28 and 0.16, respectively, which is the measure of the affinity of the binding

sites.

The characteristics of Langmuir adsorption isotherm can be expressed separation

factor (RL) as described by Hall et al. (1966), which was expressed by the following equation:

RL = 1 / (1+ kL C0) (9)