The kinetics of degradation of Fe (II) by isolated ferrobacteria was studied in beakers (Figure 1). The strains were grown in a mineral salt environment in well-defined proportions. The operation of biological degradation of Fe (II) in beakers was carried out according to the experimental parameters in particular pH, oxygen and temperature. The tests of the biological elimination of Fe (II) were carried out in water “doped” with iron at concentrations between 0.5 and 2 mg·L⁻¹, with a pH between 3.4 and 10 and a temperature ranging from 10˚C to 40˚C (Toyoda & Tebo, 2016).

For the measurement of the maximum wavelength, in a beaker, we put the solvent on we will do the “optical zero”. In another beaker we put the prepared 2 mg Fe (II) sulfate solution per litre. After determining absorbance of the solution at different wavelengths, we found that there is a wavelength at which the absorbance is maximum. Temperature and pH are continuously measured by the pH meter (multimeter). We used a DO meter HI 8043 oximeter to measure dissolved oxygen. It consists of a probe, a protective cap, an oxygen permeable teflon membrane and a cable. It also has an easy-to-read LCD screen. Dissolved oxygen is shown in mg·L⁻¹. The Fe (II) concentration is calculated by multiplying the obtained concentration by 10. Iron concentrations were evaluated according to the following relationship (Faye et al., 2019):

[ Fe 2 + ] total = ABS − 0.0577 0.1418 (1)

The treatment method relied on batch-operated fixed-bed beakers to remove Fe (II) from the doped water. The beakers were filled with coated jujube seeds with an average diameter of 8.08 mm, used as the filter material. For starting the

filter, the application of the method described by (Michalakos et al., 1997; Tekerlekopoulou et al., 2013) was used. The strain was taken from a Petri dish and added to the ascending filter with 10 mg/L of Fe²⁺ in the form of FeSO₄·H₂O (Tekerlekopoulou et al., 2006). The addition of nitrogen (20 g/L of NaNO₂) and phosphorus (10 g/L of KH₂PO₄), had been made to ensure the rapid growth of bacteria. The beakers were aerated at the top of the column opening using a MECAFER type air compressor (Q_G = 760 L/h). After a follow-up time of 10 days, the examination of the identification showed the presence of bacteria in convex form which could be of the genus Gallionella (G. ferruginea).

The column in batch mode is regularly aerated with an air compressor if the oxygen in the medium is less than 4 mg·L⁻¹. The start-up of the biological filter was carried out 2 months to obtain maximum rates of biological oxidation of iron (Tekerlekopoulou et al., 2008). To assess the kinetics of dissolved iron depletion, plastic beakers were perforated at the bottom as a sampling point with a syringe, allowing samples to be collected based on contact time. In batch operation, a solution was added with iron concentrations in the feed between 0.5 - 2 mg·L⁻¹ (Papadopoulos et al., 2019; Tekerlekopoulou et al., 2006). This concentration range covers concentrations found in groundwater sources in some regions of Senegal. Prepared solutions were stored at 4˚C to prevent growth of bacteria in the feeding medium (Pham & Waite, 2008; Tekerlekopoulou et al., 2006). The kinetics of the oxidation of Fe (II) by isolated ferrobacteria are presented in (Figure 1).

The determination of the physical characteristics of coated jujube seeds obeys the procedure described in my scientific work on: “Comparative study of two fillings (nuts of monkey bread and jujube) in a packed column: hydrodynamics and transfer of matter (Mamadou et al., 2018). Figure 2 shows the jujube seeds before and after the coating operation.

The physical parameters of the coated jujube seeds are shown in following Table 1.

To measure the amount of biomass in the three (3) beakers over time, we first determine the maximum wavelength from the release of a small amount of wash sludge in distilled water. The preparation of the solutions to be assayed follows the same protocol for the determination of Fe (II) by ortho-phenanthroline (Faye et al., 2019). Indeed, the principle will be to take the prepared solution and pass the monochromatic light at different wavelength, then build a graph of the absorbance as a function of the wavelength. The variation curve of the optical densities as a function of the wavelengths is described in Figure 3.

The peak of the graph (Figure 3) shows the maximum values of λ_max = 530 nm and O.D_max = 0.676. In the following experiments, we will set ourselves to the wavelength λ_max = 530 nm (adjust the device: UV-visible spectrophotometry) such that the absorbance (light absorbed) is maximal so that we will have a greater accuracy of optical density values.

Oxidation rates of Fe (II) depend strongly on the temperature of the reaction medium. Indeed, different temperatures have been set, from 10˚C to 60˚C to pH = 9.6, with the addition of airflow (generated by the MECAFER type compressor) at each temperature as it has been demonstrated in literature that dissolved oxygen affects the Fe (II) oxidation rate to Fe (III). The optimum temperature found for Fe (II) oxidation was 40˚C (Figure 4).

Over a temperature range of 10˚C to 40˚C, the oxidation rate increases. This phenomenon leads to the activation of the biological reaction in this temperature range. This evolution could be modeled by the Arrhenius equation in this region (Štembal et al., 2005):

( k k r e f ) T = A e − E a / R T (2)

where k is a magnitude of the evolution of the microbial system, E_a activation energy, A is a constant and T temperature.

From 40˚C, the biological oxidation rate of Fe (II) decreases. This decrease is explained by the thermal deactivation of the enzymatic system. Correlation equations could describe the evolution of oxidation rates as a function of temperature (Štembal et al., 2005). However, experimental data were insufficient to estimate

treatment efficiencies at elevated temperatures. Hence the importance of proceeding with pilot tests to find the limit temperature of thermal deactivation of biological oxidation in our pilot column.

The influence of pH on iron degradation was studied by varying this quantity by intervals of 0.5, from 7 to 10.5, while maintaining the other parameters constant (Figure 5) (Hesslein, 2005).

In these experiments, the maximum value of the oxidation rate of Fe (II) was observed at pH 9.5 (Figure 5). This value is close to the value obtained during tests in tubes for the elimination of iron present in water reconstituted by ferrobacteria isolated on a Petri dish (9.8) (Faye et al., 2019). We have tried to reproduce the works of whose optimum pH was 7.3. The results of our experiments are not very far from those described by these two previous authors. The results of (Brouwers et al., 2000) are similar to those of (Adams & Ghiorse, 1987).

Much research work in the biofiltration sector has shown that the biological oxidation yields of Fe (II) are strongly influenced by pH. Indeed, the effect of pH in the column can be modeled by the equation of (Zhang et al., 2002) under the same operating conditions:

[ k k r e f ] pH = k pH 1 + [ H + ] / K 1 + K 2 / [ H + ] (3)

k is the oxidation constant of Fe (II), k_ref = k at pH = 9.5; [O₂] = 6.5 mg·L⁻¹; T = 25˚C; K₁ = 3.05 × 10⁻⁸; K₂ = 2.46 × 10⁻⁸, k_pH = 2.82.

Thus, the increase in the oxidation conversion rate of Fe (II) to Fe (III) between a pH of 7.0 to 9.5 (Figure 5) is caused by the enzymatic activity of microorganisms which is pH dependent. Above pH = 9.5, the concentration of measured Fe (II) ions decreases with increasing pH due to the likely inhibition of bacteria. Although a decrease in Fe (II) could be explained by the decrease in yields at high pH (Teien et al., 2008), it is believed that this finding is unlikely since there is no formation of precipitate in the column. The absence of Fe (III) suggests that there is an imbalance with O₂ or that the precipitates formed as FeCO₃, Fe(OH)₂ or Fe(OH)₃ were supersaturated and that, therefore, Fe (II) remained under its control dissolved or reduced form. Additionally, if a precipitate is present, it would have been retained on the outer walls of the jujube nuts. Thus, the decrease in Fe (II) conversion yields above pH = 9.5 is most likely the result of the deactivation of the biological reaction, hence the inhibition of the bacterial population.

The influence of oxygen (O₂) on iron degradation was studied at different concentrations of dissolved oxygen, keeping the other parameters constant (Figure 6) (Zhang et al., 2002).

The modeling of the oxidation yields of Fe (II) shows a proportionality with the oxygen (O₂) contents, which shows that all the O₂ concentrations considered to be higher than the exact concentration required for the biofilter would cause the oxygen saturation of the bed (Duesterberg et al., 2008; Zhang et al., 2002). Experimental difficulties have been encountered with accurate control of O₂ for oxygen concentrations greater than about 8 mg·L⁻¹. The equation of (Zhang et al., 2002) was used to model the influence of dissolved oxygen on the oxidation rate of Fe (II):

( k k r e f ) O 2 = k O 2 [ O 2 ] (4)

[O₂] is the oxygen concentration = 8.05 mg·L⁻¹ [oxygen saturation medium at 8.05 mg·L⁻¹ for an air temperature of 25˚ C and an ionic strength of 0.05 mg·L⁻¹.

For a wavelength λ_max = 530 nm, measurements of the development of microorganisms were made. Within a few days after sowing, a fixed biofilm had developed on the media of the coated jujube seeds. Figure 7 below shows the evolution of microbial cells in the median filter (Zhu et al., 2010).

After a follow-up period of 39 days, the biofilm reaches the permanent phase, the biological oxidation of Fe (II) to Fe (III) becomes incomplete. However, if Fe (II) is added to the column during this period, the Fe (II) conversion efficiencies are the same as the biofilters that received Fe (II) at 2 months of operation. Thus, the oxidation kinetics of the Fe (II) was reasonably constant during the first 10 days of the stationary phase, compared with the results of (Mouchet, 1992) which indicated that the highest oxidative activity of Gallionella genus by Fe (II) was obtained in early stationary phase cultures after at least 2 months of culture. These batch tests will predict in subsequent experiments, on a column in continuous mode, which addition of the Fe (II) could be done in the first 10 days of the stationary phase of growth.

The biological oxidation of iron was to follow the enzymatic kinetics of Michaelis-Merten, with the corresponding velocity law:

dFe ( II ) [ X ] d t = − k [ Fe ( II ) ] K s + [ Fe ( II ) ] (5)

This modeling of bacterial growth is very close to the Monod model which is the oldest. The latter is an empirical model, which accounts for the exponential

phases of growth and slowdown, very close to Michaelis Merten’s law relating to enzymatic reactions. K_s is the half-rate constant at half the initial concentration, k is the maximum rate constant of Fe (II) (μmol Fe²⁺/mg cells. min), and [X] is the concentration of microorganisms in mg·L⁻¹.

The value of the constant k varies with the pH of the water, the temperature of the medium and the concentration of dissolved oxygen. The operating parameters were maintained constant [O₂] = 6.5 mg·L⁻¹, T = 25˚C and pH = 9.5. Fe (II) conversion yields increase with increasing Fe (II) contents (Figure 8) (Teien et al., 2008). The analysis of the experimental data by the nonlinear regression method gives values of K_s 17.02 mol·L⁻¹, and k 0.082 mol/(min·mg).