Comparison of Rhodotorula sp. and Bacillus megaterium in the removal of cadmium ions from liquid effluents

2.1 Biosorbents: growth and inactivation

Both microorganisms were isolated within the microbiology laboratory of the Department of Environmental Engineering and Management, Iasi, Romania. Rhodotorula sp. was isolated from students’ hands, while B. megaterium was isolated from food products. Identification of microorganisms was made by microscopic observation of the appearance of colonies, cell shape, color and growth on specific media [21]. Rhodotorula sp. was grown in Sabouraud medium containing 20 g/l glucose and 10 g/l peptone in 1 l distilled water, for 72 h, at 28°C and 150 rpm. For the growth of B. megaterium, Lysogeny broth medium containing 10 g/l tryptone, 5 g/l yeast extract and 5 g/l NaCl in 1 l of distilled water was used. All chemicals were purchased from Sigma-Aldrich, Germany. The microorganism was grown for 72 h, at 30°C and 150 rpm. Afterwards, both microorganisms were centrifuged at 7000 rpm for 10 min (using a Hettich Mikro 220R centrifuge, Germany), inactivated in an autoclave (Autoclave Trade Raypa AH-21 Clase N, Spain) for 15 min at 121°C, and further dried at 60°C for 48 h. The dried biomass of Rhodotorula sp. and B. megaterium was crushed and sieved to 125–250 μm particle size and stored in a desiccator until further use.

2.2 Reagents and equipment

All chemicals used in this work were of analytical grade and no further purification was necessary. A stock solution of 1000 mg/l Cd(II) was obtained by dissolving 1.3722 g Cd(NO₃)₂.4H₂O (Riedel-de Haën, Germany) in 500 ml distilled water. The stock solution was diluted with distilled water based on the experiments employed. The pH of the solutions was adjusted by the addition H₂SO₄ 1 m and NaOH 1 m (Fisher Scientific, UK), the amount added being negligible (Multi-Parameter ProLab 2000 Meter, SI Analytics GmbH, Germany). Cd(II) concentration in solution was analyzed using an inductively coupled plasma spectrometer (Optima 8000 ICP-OES model, PerkinElmer, Inc., USA).

2.3 Biosorption studies

The biosorption of Cd(II) ions by Rhodotorula sp. and B. megaterium was studied in a batch system. Experiments were conducted in 200 ml Erlenmeyer flasks with 50 ml Cd(II) solution, in an incubator at 150 rpm. In the investigation concerning the pH influence, the following conditions were considered: working with 5 g/l Rhodotorula sp. and 3 g/l B. megaterium, at 25°C for 48 h, while maintaining constant a concentration of 50 mg/l Cd(II). The pH ranged from 3 to 7 to avoid a possible precipitation of cadmium ions. The biosorbent dosage varied from 1 g/l to 10 g/l, when investigating the biosorbent dosage influence, keeping a constant pH (pH 6 for Rhodotorula sp. and pH 4 for B. megaterium) and heavy metal concentration (50 mg/l). After the biosorbent dosage and optimum experimental pHs were established, the following step was to evaluate the influence of contact time (samples were taken at time intervals from 0 min to 48 h) for two different concentrations [50 mg/l and 100 mg/l Cd(II)]. Kinetic studies were conducted in 250 ml Erlenmeyer flasks with 100 ml Cd(II) solution. The influence of initial concentration of Cd(II) in solution on the biosorption process was assessed in the range between 25 mg/l and 300 mg/l. Temperature ranged from 25°C to 45°C. After incubation, samples were centrifuged at 10,000 rpm for 5 min, the supernatants were acidified (in 2%) with nitric acid (69%) (Fisher Scientific, UK) and further analyzed. Control experiments were also conducted for each investigation by considering the same procedure stated above, but in the absence of metal ions. All experiments were carried out in duplicate and the mean average values were used in further analysis. The experimental error was less than 5%.

The uptake of metal ions per gram of biosorbent was calculated using the following expression [Eq. (1)] [22]:

where q is the biosorption capacity (mg/g), C_i is the initial metal ion concentration in solution (mg/l), C_f is the concentration of metal ions in solution at the end of the biosorption process (mg/l), V is the volume of the solution (l) and m is the amount of biosorbent used in the experiment (g).

The removal efficiency (R, %) was calculated using Eq. (2):

2.4 Characterization of the biosorbents

Characterization of biosorbents is important in understanding the mechanism of heavy metals removal and consisted of the analysis of biomass surface for qualitative assessment of the main functional groups responsible for the metal binding. For this purpose, the point of zero charge (pH_pzc) was considered, along with FTIR spectroscopy and SEM-EDX.

2.5 Biosorption modeling

For modeling the sorption process, Lagergren pseudo-first-order and Ho pseudo-second-order kinetic models and Langmuir and Freundlich isotherm models were initially applied. These models predict the parameters used for designing and modeling of the process and help in elucidating the mechanism involved in Cd(II) biosorption.

The pseudo-first-order model (Lagergren’s rate equation) describes the adsorption of the metallic ions from the liquid phase and is one of the most widely used equations. The linearized form of this expression is given by Eq. (3) [27]:

where q_e and q_t (mg/g) are the biosorption capacity at equilibrium and at time t (min), respectively, and k₁ (min⁻¹) is the rate constant of the pseudo-first order equation.

The pseudo-second-order kinetic model (Ho model) is related to the assumption that the rate-limiting step may be chemical sorption or chemisorption involving valence forces through sharing or exchange electrons between sorbent and sorbate and is expressed in linear form as Eq. (4) [28]:

where q_e and q_t (mg/g) are the biosorption capacity at equilibrium and at time t (min), respectively, and k₂ (g/mg min) is the rate constant of the pseudo-second order equation.

The Langmuir isotherm (1918) considers sorption as a chemical phenomenon and provides information on uptake capabilities that occur on a homogenous surface by monolayer sorption. It can be used for describing equilibrium conditions and to predict the performance of different biosorbents. The non-linear form of the Langmuir isotherm equation can be expressed as follows [Eq. (5)] [27], [29]:

where C_e (mg/l) is the equilibrium concentration of the metal in solution, q_m (mg/g) is the maximum sorption upon complete saturation at the biomass surface and K_L (l/g) is a constant related to the adsorption/desorption energy.

The Freundlich isotherm relationship is an exponential and an empirical equation; it provides information about the surface heterogeneity and the exponential distribution of active sites and their energies, and it is expressed by the following equation [Eq. (6)] [27], [29]:

where K_F (mg¹⁻ⁿ g⁻¹lⁿ) represents the sorption capacity when metal ion equilibrium concentration is equal to 1, and n represents the degree of dependence of sorption with equilibrium concentration.

In order to determine the biosorption efficiency as a function of the working conditions [pH, temperature, contact time, initial concentration of Cd(II) ions, biosorbent dosage] the biosorption process was modeled using a linear regression approach with Durbin-Watson statistics provided by the IBM SPSS Statistics software (version 23). Taking into consideration that two types of microorganisms were used, the same modeling approach was applied twice, resulting in two different models, one for each microorganism tested.

Further, the accuracy and precision of experiments were evaluated by ANOVA in order to ensure that the determined model predictions follow the experimental data and are statistically significant.

3.1 Influence of pH on biosorption process

The biosorption process of Cd(II) ions from aqueous solutions is influenced by different parameters and one of the most important is the solution pH. The sorption efficiencies and uptake capacities of Rhodotorula sp. and B. magaterium for Cd(II) ions removal tested for different pH values are shown in Figure 1. It can be observed that changing the pH values between 3 and 7 had an important effect on Cd(II) biosorption. In the case of Rhodotorula sp., the maximum removal efficiency was 65%, while the maximum uptake was 5.3 mg/g at pH=6, for an initial concentration of 50 mg/l Cd(II) ions and 5 g/l biosorbent dosage. For this biosorbent, the data showed an increase of Cd(II) uptake and removal efficiency at pH values from 3 to 5. The uptake capacity of Rhodotorula sp. increases from 2.7 mg/g to 5.3 mg/g, while the removal efficiency increases from 40% to 65%. In the case of B. megaterium, the optimum pH is 4, with a maximum efficiency of 97% and an uptake capacity of 16.2 mg/g (values obtained for 50 mg/l initial concentration and 3 g/l biosorbent dosage). For pH values between 3 and 4, the removal efficiency increased from 86% to 97%, and the uptake rose from 14.4 mg/g to 16.2 mg/g. For pH values higher than 4, the removal efficiency decreased at 72%, and the uptake diminished at 12.0 mg/g. It was not taken into account to maintain a constant pH during the experiments, therefore at the end of the process, it was noticed that the pH increased up to 8 for initial pH values higher than 5, which could be the reason for these results. The decrease of removal efficiencies at higher pH values may be caused by the formation of soluble hydroxylated complexes of Cd(II) ions and their ionized nature; these ions were converted into their hydroxide forms and got precipitated.

Figure 1:

Influence of pH on biosorption of Cd(II) ions by Rhodotorula sp. (biomass dosage 5 g/l, temperature 25°C, contact time 48 h, initial Cd(II) concentration 50 mg/l) and B. megaterium (biomass dosage 3 g/l, temperature 25°C, contact time 48 h, initial Cd(II) concentration 50 mg/l).

3.2 Influence of biosorbents dosage on biosorption process

Another parameter which has an important effect on the biosorption of heavy metals ions is the biosorbent dosage. The studies concerning the influence of biosorbents dose on Cd(II) ions removal by Rhodotorula sp. and B. megaterium showed that the biosorption capacity of Cd(II) decreased with increasing the biosorbent dosage from 1 g/l to 10 g/l (Figure 2). The maximum uptake of cadmium by Rhodotorula sp. was 13.8 mg/g at pH 6 and 33.1 mg/g at pH 4 for B. megaterium, respectively (considering 1 g/l biosorbent dosage). The maximum efficiency in the same conditions of pH was 65% for Rhodotorula sp. at 5 g/l biosorbent dosage and 97% for B. megaterium at 3 g/l biosorbent dosage. The increase in the percentage of the metal ion removal with increase in biosorbent dosage is due to the greater availability of the exchangeable sites or surface area at higher concentration of the biosorbent [30], [31]. Based on the results obtained up to now, further experiments were performed considering 5 g/l biosorbent dosage for Rhodotorula sp., and 3 g/l for B. megaterium.

Figure 2:

Influence of biosorbent dosage on biosorption of Cd(II) ions by Rhodotorula sp. (pH 6, temperature 25°C, contact time 48 h, initial Cd(II) concentration 50 mg/l) and B. megaterium (pH 4, temperature 25°C, contact time 48 h, initial Cd(II) concentration 50 mg/l).

3.3 Influence of contact time on biosorption process

For the optimization of the biosorption process, it is necessary to study the influence of contact time on Cd(II) ions removal from aqueous solution. The effect of contact time on Cd(II) removal efficiency and uptake capacity of Rhodotorula sp. and B. megaterium is shown in Figure 3. It was observed that the percentage of Cd(II) removal is improved with any increase in contact time, the maximum uptake and efficiency for Rhodotorula sp. being attained after 48 h, and for B. megaterium, in the first 10 min. After this time, it was found that the efficiency of the process did not show a considerable increase [for Rhodotorula sp., the maximum efficiency after 72 h of contact time for 50 mg/l initial concentration of Cd(II) ions in solution was 69%, data not shown, while after 48 h of contact time the removal efficiency was 65%, and 80% after 24 h, data not shown, and 79% after 20 min of contact time for B. megaterium]. Therefore, the optimum contact time was selected as 48h for Rhodotorula sp. and 20 min for B. megaterium in further experiments. Similar results were obtained by Liu et al. [16] for B. megaterium dead biomass, when a maximum biosorption efficiency of 95% for Au³⁺ ions removal from solution was attained in 30 min.

Figure 3:

Influence of contact time on biosorption of Cd(II) ions by: (A) Rhodotorula sp. (pH 6, temperature 25°C, biomass dosage 5 g/l) and (B) B. megaterium (pH 4, temperature 25°C, biomass dosage 3 g/l).

3.4 Influence of temperature on biosorption process

Temperature is one of the basic parameters for sorption processes due to the already demonstrated fact that the sorption processes tend to be exothermic and the sorption performances vary with temperature [29]. Biosorption is not considered a strongly exothermic process such as other physical adsorption processes, but the variation of temperature can improve its performance [22], [29]. Studies concerning the influence of temperature on Cd(II) biosorption process of by the selected microorganisms were carried out at four different temperatures (ranging from 25°C to 45°C) for 50 mg/l initial metal solution, and the results are shown in Figure 4. The increase of temperature resulted in the maximum biosorption efficiency and uptake for Rhodotorula sp. achieved at 30°C (85% and 14.2 mg/g), and at 35°C for B. megaterium (90% and 15.1 mg/g). With a higher increase in temperature, the efficiency of the process decreases for both microorganisms. This could be due to the deactivation of the biosorbent surface, while destructing some active sites on the biosorbent surface caused by bond ruptures or due to the weakness of biosorption forces between the active sites of the biosorbents and the metal ion species [32], [33]. The same behavior of heavy metals biosorption at different temperatures was obtained by Sulaymon et al. [32].

Figure 4:

Influence of temperature on biosorption of Cd(II) ions by Rhodotorula sp. (pH 6, biomass dosage 5 g/l, contact time 48 h, initial Cd(II) concentration 50 mg/l) and B. megaterium (pH 4, biomass dosage 3 g/l, contact time 2 h, initial Cd(II) concentration 50 mg/l).

3.5 Influence of initial concentration of Cd(II) ions in solution on biosorption process

The effect of initial concentration on the removal of Cd(II) ions by the selected biosorbents was studied and the results are given in Figure 5. The analysis of results reveals that the efficiency of Cd(II) removal decreased exponentially with the increase in initial concentration of ions. During the experiments, the capacity of biosorbents for Cd(II) removal was tested for seven different initial concentrations (ranging from 25 mg/l to 300 mg/l). For both types of biosorbents, it was observed that for initial concentrations between 25 mg/l and 100 mg/l, the biosorption capacity increased while biosorption efficiency decreased (from 4.3 mg/g [86%] to 11.2 mg/g [57%] for Rhodotorula sp., and from 7.1 mg/g [86%] to 23.8 mg/g [48%] for B. megaterium). For an initial Cd(II) concentration of 300 mg/l, the biosorption capacity decreased to 6.2 mg/g (11%) for Rhodotorula sp. and to 8.1 mg/g (8%) for B. megaterium. The increase of biosorption capacity with increase of initial metal ion concentration indicates that surface saturation of biosorbents is dependent on the initial metal ion concentrations [34]. Similar results concerning the influence of initial ions concentration on the biosorption process were obtained by Ezzouhri et al. [35] for lead removal by Penicillium sp.

Figure 5:

Influence of initial concentration on biosorption of Cd(II) ions by Rhodotorula sp. (pH 6, biomass dosage 5 g/l, contact time 48 h, temperature 25°C) and B. megaterium (pH 4, biomass dosage 3 g/l, contact time 2 h, temperature 25°C); Langmuir and Freundlich isotherms modeling.

3.6 Biosorption modeling

3.6.4 Analysis of variance

The analysis of variance (ANOVA) was used as a statistical technique to determine the variation among and between groups. This analysis can be applied for comparing three or more groups or variables for statistical significance [19], [40].

The ANOVA Table 7 indicates how well the regression equation fits the data. The value of the Sig. (probability value, p_model>F=0.00), which is lower than 0.05, shows that both models are statistically significant for the prediction of the outcome variable. Also, a high F-value (20.22 for B. megaterium and 39.38 for Rhodotorula sp.) indicates that using the models is better than guessing the mean.

Table 7:

ANOVA statistics.

Model	Sum of squares (SS)	df	Mean square (MS)	F-values	Sig.(p>F)
1
Regression	10,225.72	5	2045.14	20.21	0.000
Residual	3135.99	31	101.16
Total	13,361.71	36
2
Regression	21,004.99	5	4200.99	39.38	0.000
Residual	5440.43	51	106.67
Total	26,445.42	56

1. df, Degree of freedom; p, probability.

The normal probability plot of the residuals (Figure 6) shows that the residuals are normally distributed, the relationship being approximatively linear, indicating that in the linear regression analysis, there is no tendency in the error term and showing that both equations perform satisfactorily.

Figure 6:

Normal probability plot of regression standardized residual for the dependent variable: (A) Rhodotorula sp. and (B) Bacillus megaterium.

3.7 Biosorbents characterization

3.7.1 Point of zero charge (pH_pzc)

For biosorbents characterization, the value of point of zero charge was initially determined. This value puts into evidence the different surface functional groups with different acid and basic characteristics. This analysis gives information about the possible electrostatic interactions between biosorbents and chemical species of metal. The pH_pzc was determined using the pH drift method which was tested as indicated by several authors [23], [24]. The results obtained are presented in Figure 7. The black straight line is used to observe the acidic or basic character of the material surface, and represents the plot of initial pH vs. final pH, for equal values. The intersection of experimental points with the black line represents the point of zero charge (pH_pzc). If the experimental points are above the black line, the surface of the biosorbent is negatively charged, while if the points are under the black line, the surface of biosorbent is positively charged. Based on the results obtained, it can be concluded that the surface of the studied biosorbents is negatively charged (pH_pzc for Rhodotorula sp. is 4.8 and for B. megaterium is 0.9) and during the biosorption process, the biosorbent surface attracted the metal ions with positive charges [23], [31].

Figure 7:

Representation of the pH_pzc of (A) Rhodotorula sp. and (B) Bacillus megaterium using the pH drift method.

3.7.2 FTIR spectroscopy

The FTIR spectroscopy provides information about the functional groups present on the surface of the biosorbents. According to Wang and Chen [41], the functional groups related to the biosorption are: hydroxyl, carboxyl, amino, ester, sulfhydryl, carbonyl and phosphate. Also, Michalak et al. [5] and Chojnacka et al. [42] mentioned that the participation in metal ion binding of these functional groups depends on the initial pH values: for values between 2 and 5 carboxyl groups are participating, for pH=5–9, carboxyl and phosphate groups are involved in the process, and for pH between 9 and 12, the carboxyl, phosphate and hydroxyl (or amine) groups. For Rhodotorula sp. biomass, the FTIR spectroscopy (Figure 8A) indicates around 3425 cm⁻¹ and 3394 cm⁻¹, the existence of O-H and amine groups. The carboxylic groups are observed at 2923.87 cm⁻¹, 1739.66 cm⁻¹ and 1747.38 cm⁻¹, and the absorption from 2854.44 cm⁻¹ can be attributed to the stretching vibration of the C-H group. A vibration at 2360.70 cm⁻¹ can be attributed to phosphine groups and the vibrations at 1654.80 cm⁻¹, 1546.80 cm⁻¹ and 1539.68 cm⁻¹ are specific for amides. The C-O-C polysaccharides groups are indicated around of 1080 cm⁻¹ [43], [44]. The FTIR analysis of the B. megaterium before and after Cd(II) biosorption (Figure 8B) showed that on the surface of the biosorbents are present the following functional groups: hydroxyl group or amine (3394.47 cm⁻¹ and 3433.09 cm⁻¹), carboxylic (peaks at 2923.87 cm⁻¹, 2931.58 cm⁻¹, 1404.07 cm⁻¹ and 1400.22 cm⁻¹), amide (1654.80 cm⁻¹ and 1542.94 cm⁻¹), phosphoryl (1234.35 cm⁻¹ and 1230.49 cm⁻¹), phosphines (peaks at 2356.84 cm⁻¹ and 2360.70 cm⁻¹), CH primarily from lipids (2854.44 cm⁻¹) and C-O-C polysaccharides (1060.77 cm⁻¹ and 1080.06 cm⁻¹) [42], [44], [45].

Figure 8:

Fourier transform infrared (FTIR) analysis for (A) Rhodotorula sp. and (B) Bacillus megaterium before and after biosorption.

3.7.3 SEM-EDX

To determine the textural characteristics of the Rhodotorula sp. and B. megaterium before and after the Cd(II) biosorption process, a morphological analysis of the biosorbents was performed. SEM images of the biosorbents before and after metal uptake at 3000× magnification and tension of 5 kV are shown in Figure 9. The comparison of SEM images of the biosorbents shows that during the biosorption process of Cd(II), the cell-surface morphology of the biosorbents suffers significant changes. The Rhodotorula sp. cells before exposure were smooth, round and had certain dimensions, but after cadmium ions biosorption, the cells were destroyed, lost their shape and became flat. In the case of B. megaterium cells after biosorption, the form was approximately the same but the cells length was shorter. These changes were probably caused by precipitated cadmium ions around the cell surface, linked with their functional groups and the ion exchange process between biosorbents and Cd(II) ions [46], [47]. Similar changes of the biosorbent structure were observed after cadmium removal by green algae, Ulva lactuca, as reported by Ghoneim et al. [46].

Figure 9:

Scanning electron microscopy (SEM) analysis of biosorbents before and after biosorption of Cd(II) ions: Rhodotorula sp. (A) before, (B) after; Bacillus megaterium (C) before, (D) after.

The EDX analysis was performed in the same time with SEM analysis for the biosorbents after biosorption (Figure 10). This analysis indicates the presence of cadmium ions and provides information about the concentration and distribution of elements in sorbents biomass. The proportions of principal elements that are in the composition of Rhodotorula sp. after biosorption are: C (65.8%), O (31.8%), P (1.3%), Cd (0.4%), K (0.4%), S (0.3%) and Mg (0.1%). In the case of B. megaterium, the composition includes C (68.3%), O (25%), P (2.5%), Cd (2.5%), Ca (0.3%), K (0.3%), Na (0.3%) and Mg (0.1%).

Figure 10:

Energy dispersive X-ray microanalysis (EDX) analysis after biosorption of Cd(II) ions by: (A) Rhodotorula sp. and (B) Bacillus megaterium.