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Innovative low-cost biosorption process of Cr6+ by Pseudomonas alcaliphila NEWG-2

Chromium is one of the heavy metal pollutants that causing risky health issues when discharged into the aquatic ecosystems. The current investigation focused on the bioremoval of Cr6+ depending on the bacterial sorption process by using Pseudomonas sp. NEWG-2 which was identified on the basis of morphological, cultural characteristics, 16S rRNA sequencing and phylogenetic analysis as Pseudomonas alcaliphila strain NEWG-2. It is clear from the FCCD experiments that the bacterium can grow normally and remove 96.60% of 200 mg/l of Cr6+ using yeast extract (5.6 g/l), glucose (4.9 g/l), pH (7) for 48 h incubation period. SEM and EDS analyses proved that the Cr6+ was biosorbed by P. alcaliphila NEWG-2. FTIR spectra indicated that the phenolic, carbonyl ester, acetyl, carboxylate, alkanes and carbonyl were the main groups involved in the chromium biosorption. Of the equilibrium isotherms models, the Langmuir model was more obedient, with a maximum uptake (qmax) of 10 mg/g (bacterial-alginate beads), than the Freundlich one. The findings reveal the efficiency of P. alcaliphila NEWG-2 in Cr6+ biosorption, with feasibility in the treatment of chromium-contaminated water as a green-technology tool. Interestingly, to the best of our knowledge, this is the first report on Cr6+ biosorption process by P. alcaliphila.


Results and discussion
The ecosystem's healthiness and balance are greatly governed by heavy metals pollution. Chromium discharged from the effluent of the tannery and/or metal industries is a major risk and disturbance factor, in this respect 25 . That is because of the high toxicity, e.g. carcinogenic and teratogenic features, especially in the form of the hexavalent state. The annual discharged quantity of chromium into the environment is approximately 1,70,000 tonnes 26 . The relatively expensive and ineffective traditional disposal procedures motivate interests towards safe and effective alternative strategies, mainly biological-based strategy. One of such profoundly driven approach in recent times is biosorption through microbial biomass and their products 2 . The biosorption of this metal could occur during the metabolic pathways of the microorganism, which are known to survive and tolerate heavy metals stress in the contaminated environment.
Comparing to the other microorganisms, bacteria have many merits, such as the small in size, the abundant distribution, the more resistant to harsh environmental conditions, the ability to be cultured under controlled conditions, and further, possess various mechanisms of bioleaching, including bioaccumulation, biomineralization, biotransformation and biosorption 22,27 . cr 6+ tolerability of the studied bacterium. From the start point of view, the present investigation was initiated to study the tolerability of Pseudomonas sp. to Cr 6+ ions, followed by optimization of the biosorption efficiency by modifying some nutritional and physical culturing conditions. Initially, K 2 Cr 2 O 7 was used for testing the tolerability of Pseudomonas sp. to different Cr 6+ ion concentrations in the batch cultured system, with running over wide ranges of initial Cr 6+ concentrations (50-250 mg/l based on Cr 6+ ion).
The biosorption capacity of Cr 6+ , the bacterial growth and the final cultural pH are illustrated in Fig. 1. The bacterial biosorption capacity initiated with 97.2% at 50 mg/l, and kept around the same percent with the increasing of the initial concentration of Cr 6+ and reached to saturated value at a concentration of 200 mg/l, with a removal efficiency of 96.1%. Higher concentrations (250 mg/l) decreased the tolerability of the bacterium down to 81%. Although the bacterium was able to grow and reduce Cr 6+ ion content in the fermentation medium, the growth and final cultural pH slightly reduced with the increment of Cr 6+ . There was an inverse relationship between bacterial growth and final cultural pH from one side and Cr 6+ concentrations, on the other side, this latter observation indicates the survivability and tolerability of the bacterial strain under the stress of Cr 6+ . Noticeably, Pseudomonas sp. was found to be able to reduce the yellow color of K 2 Cr 2 O 7 in the medium (Supplementary Fig. 1), indicating the ability of the bacterium to grow and neutralize Cr 6+ .
Comparably, previous studies find out that some bacterial strains were tolerant up to 300 mg/l at pH 7.0 and temperature of 37 °C 28 . Nevertheless, the frequency of changes in metal uptake may be attributed to the metal's characteristics (e.g. the capacity for metal reductions, atomic weight, or ionic size ) and could be due to the characteristics of the bacterium, such as surface area, functional groups and structure 20 . In this connection, proteins and lipids may play a crucial role in the biosorption process. The capsule and slime layers of bacteria contain polysaccharides, acting as building blocks and/or barriers for heavy metal modulation. The exopolysaccharide is the self-defense of the bacterium against harsh conditions, e.g. pH, temperature, starvation and could play a crucial role in the biosorption process of metals. Likewise, Gupta and Diwan 2 reported that the leaching of heavy metal depending on microbial extracellular polymeric substances such as polysaccharides, uronic acid, humic substances and lipids. The species of Pseudomonas contain exopolysaccharides with anionic functional groups, which incorporated in biosorption of heavy metals 29,30 .
On the other hand, the lower tolerability of some bacteria could be attributed to the mutagenic effect of chromium ion towards bacterial cells 31 , further, Komori et al. 32 reported that mutation in Enterobacter cloacae as a result of DNA damage, leading to disruption of normal cell replication due to interactions with Cr 6+ . In some cases, the mutated cell may be more resistant to (Cr 6+ ) 33 . Herein, the decline in the tolerability of Pseudomonas sp. NEWG-2 at the higher concentration of chromium (250 ppm of Cr 6+ ) (Supplementary Fig. 1) could be due to a reduction in biosorption sites on the surface of bacteria 34 . However, it is worthy to note that the concentration applied in the present study is calculated based on Cr 6+ ion content in K 2 Cr 2 O 7 . Consequently, the initial concentration of Cr 6+ at 200 mg/l, has been chosen as the tolerable concentration for further investigation during the next trial.
Identification of Pseudomonas sp. strain NEWG-2. The microscopic characterization of the aerobic non-spore-forming, Gram-negative; Pseudomonas strain NEWG-2 showed straight rods of about 1.0 to 5.0 µm long and 0.5 to 1.0 µm wide with polar flagella.
The molecular identification using 16S rRNA gene sequence analysis was employed. The obtained 16S rRNA sequence of Pseudomonas sp. NEWG-2 was determined and the amplified fragment gave sequence with 1,500 bp ( Supplementary Fig. 2). The obtained 16S rRNA sequence was subjected to the BLAST search 35 on the GenBank database, and the most related sequences were obtained and compared with the present sequence.
The phylogenetic tree, generated by neighbor-joining analysis (Fig. 2), was constructed using MEGA5.0 software 36 . The isolate showed a high similarity of 99.20% with Pseudomonas alcaliphila. Accordingly, the bacterial isolate was identified as P. alcaliphila strain NEWG-2. The 16S rRNA sequence had been deposited in the DDBJ/EMBL-Bank/Gen Bank database under the accession number of MN025267.
Modeling the optimization of cr 6+ bioremoval process. The medium fermentation conditions of P.
alcaliphila NEWG-2 were subjected to a statistical modulating process based on response surface methodology, i.e. the face-centered central composite design (FCCD). The FCCD of the 30-experimental runs were performed to improve the removing efficacy of Cr 6+ ions by P. alcaliphila NEWG-2. The design of the matrix includes two nutritional variables, i.e. yeast extract (X 1 ) and glucose (X 2 ) and two physical variables, i.e. pH (X 3 ) and incubation time (X 4 ). The actual and coded levels of the variables as well as the investigated and predicted values of Cr 6+ ions removal percent of the 30 runs of the FCCD matrix are displayed in Table 1.
Although the bacterial growth and final culture pH showed marked variation along with the 30-experimental runs of the design matrix (Table 1), their modeling and analysis failed to have significant trend and did not reveal any reasonable relationships (data not shown) and did not have any effect on the bioremoval process, therefore these data were not subjected to additional evaluation or modeling. The subsequent modeling was continued only on Cr 6+ removal by the tested bacterium.
The variations in Cr 6+ removal ranged from 93.30 to 96.59%. The highest level of Cr 6+ removal percent, with a value of 96.59% (run No.8) has occurred at the center values of the tested independent variables. This observation indicates the accuracy of selected independent variables and their levels. In contrast, the minimum Cr 6+ removal percent (93.30%) was obtained in the run no. 30, at the level of 3 g/l for both yeast extract and glucose, pH 6 and incubation time for 24 h.

AnoVA and multiple regression analysis.
To explore the fitted model, the results were subjected to multiple regression analysis (Tables 2, 3). If the value of the determination coefficient (R 2 ) higher than 0.9, the regression model is described as very significant 37 . To be an adequate model, the R 2 value should not be less than   www.nature.com/scientificreports/ (0.75) 38 . However, the quadratic model was the best-fitted model, recording the highest values of R 2 (0.9860), adjusted R 2 (0.9730) and predicted R 2 (0.9539) for Cr 6+ removal percent using P. alcaliphila NEWG-2. Further, the summary of the fit statistics shows a high significance of the quadratic model (very low P-value < 0.0001), and non-significant lack of fit (F-value = 0.47 and P-value = 0.8541), with a small standard deviation (0.18). So, the quadratic model was selected for fitting the FCCD data of Cr 6+ removal by P. alcaliphila NEWG-2 ( Table 2). The data were subjected to ANOVA for further exploring the aptness of the various interaction effects of the quadratic model (Table 3). However, the experimental and predicted values of chromium removal are in a decent agreement, and the value of adjusted R 2 and predicted R 2 are high enough to indicate a high model significance. Predicted R 2 measures the model efficacy and significance in the prediction of new response values. The values of both predicted and adjusted R 2 should not be ˃ 20% of each other to be in decent agreement 39 . In the present study, the predicted R 2 is, realistically, in line with the adjusted R 2 value, explaining the high agreement between the experimental and the predicted values of Cr 6+ removal. The model variability (95.39%) is, thus, satisfactory enough for predicting the values of experimental variables, within the tested range.
Linear, mutual interactions or quadratic coefficient estimate with a negative value indicates that the effects of such variables are negative (antagonistic effect) on Cr 6+ removal percent by P. alcaliphila NEWG-2, that is to say,   (Table 3).  www.nature.com/scientificreports/ The Fisher's test (F-value = 75.73) and the very small value of probability (P-value less than 0.0001) obtained from ANOVA demonstrate that the model is very significant. Values of P ˃ 0.05 designate that the model term is significant 40 . When evaluating the prediction ability of the model in the Cr 6+ removal by P. alcaliphila NEWG-2, the model is considered accurate with a high cogency since the statics recorded high values of adjusted R 2 (0.973), F-value (75.73) and adequate precision (24.38), low values of standard deviation (0.18), coefficient of determination (0.19) and PRESS (1.53) and non-significance lack-of-fit (P-value = 0.8541 and F-value = 0.47) (Table 3).
Moreover, the significance of each parameter's coefficient was determined by the P-values and F-value, as a rule, the parameter is significant if the P-value < 0.05. Based on such rule, the significant coefficients are yeast extract (X 1 ), pH (X 3 ) and incubation time (X 4 ) as linear coefficients, X 1 X 3 , X 2 X 3 and X 3 X 4 as interaction coefficients and finally all the quadratic effect of the factors. On the other hand, the interactions between X 1 X 2 , X 1 X 4 and X 2 X 4 are not significant and not significantly contributed to the Cr 6+ removal by P. alcaliphila NEWG-2.
The coefficients of regression were used to generate the equation model. The value of Cr 6+ removal by P. alcaliphila strain NEWG-2 could be predicted by fitting data with the equation of the second-order polynomial in terms of the next regression model: where Y is the value of predicted chromium removal percent, X 1 = yeast extract concentration, X 2 = glucose concentration, X 3 = pH and X 4 = incubation time.

Model adequacy checking.
To confirm the adequacy of the model, some analytical statics were checked and depicted in Fig. 3. Plotting the normal probability of the data of the experimental residuals (Fig. 3A) shows that data points concentrated closely along the straight line, meaning that the residuals follow the normal distribution without linearity 41 . Where most of the observations gathered around the center and the values located away from the general mean and dwindled equally on both sides of the central peak. Extreme residual values on both sides are not desired. The residuals versus predicted values (Fig. 3B) was plotted, the residuals were found to be scattered randomly around the centerline and no specific patterns could be drawn, this, in turn, indicates that the residuals not correlated and distributed independently, consequently have constant variance, therefore, the model is adequately meeting the postulations of the study. Plotting the values of predicted versus actual data points (Fig. 3C) showed that the data points split equally along the 45° line. This can help detect if any value(s) cannot be easily predicted by the model. However, all data points are detectable, assuring the aptness of the model.
Generating of the three-dimensional (3D) surface plot. Following the previous assurance of the aptness of the model, the relationship between the interaction of each couple of the tested variables and the Cr 6+ removal was explored. For such purpose, the 3D-plots were created (Fig. 4) by drawing the two independent factors on X-and Y-axes against Z-axis (Cr 6+ removal percent), while the other two factors are kept at the central points.
The 3D-surface plot of the simultaneous effect of yeast extract and glucose on Cr 6+ removal was constructed (Fig. 4A). The maximum Cr 6+ removal was located around the central points, out of this range, a rather small percentage of chromium removal was noticed. The removal percent increased with the increment of glucose concentration, but the higher-level supports a fairly low percentage of Cr 6+ removal percent. The 3D-surface plot of Fig. 4B shows Cr 6+ removal efficacy as a function of yeast extract concentration and pH. The Cr 6+ removal percent maximized at pH and yeast extract around the midpoints of both. The same trend of Cr 6+ removal was observed for the other pair-wise combinations, i.e. yeast extract and incubation time (Fig. 4C), glucose and pH (Fig. 4D), glucose and incubation time (Fig. 4E) and pH and incubation time (Fig. 4F).
Experimental validation of the model. According to the model's Eq. (1), the highest theoretical value of Cr 6+ removal was calculated to be 96.33% and the predicted values of the tested variable were 5.6 g/l (yeast extract), 4.9 g/l (glucose), 7 (pH) and 48 h incubation period. Under these conditions, the Cr 6+ removal was experimentally verified and reached up to 96.60%. The experimental data verified a high grade of model precision, confirming the validation of the model under the levels of the factors used. It's obvious that the biosorption process of chromium has been influenced by tested variables levels.
These results are comparable with the previous findings on Bacillus REP02, grown on various yeast extract and dextrose levels using Box-Behnken design, the predicted (98.86%) and experimentally (99.0%) values of Cr 2+ removal were closely related 42 . Other findings reported that the maximum removal of Cr 2+ being 94%, was occurred at pH 6.0 by coated bacteria 6 . Whereas Abhirami et al. 10 reported 87.19% removal of Cr 6+ at pH 4.0.
Ranjithkumar and Mahalingam 28 stated that at pH values of 5.7 and 9, the Cr 6+ tolerability of Pseudomonas sp. was up to 400 mg/l. Another data stated the significant impact of pH on the Cr 6+ biosorption among the various variables tested using Box-Behnken design 43 . Whereas, the maximum removal of Cr 6+ by Cyanobacteria was 81.72% at pH 11.0 and initial concentration of 15 mg/l 21 . Additionally, Durga Devi et al. 44 investigated the biosorption of Cr 6+ by P. fluorescens, the maximum uptake was found to be 800 mg/l. Other factors such as www.nature.com/scientificreports/ temperature, pH, redox potential and presence of other metals, play a crucial role in the chelation of chromium by Bacillus spp. 26 . Importantly, the influence of medium pH values on the biosorption process could be explicated based on that the higher pH values cause the formation of metal hydroxide complexes, which decrease the concentration of chromium ions, thereby, causing a decrease in the equilibrium biosorption capacity 45 . While, a high electrostatic force of desirability at lower pH values, results in a high removal rate of chromium. Commonly, the influence of pH on the biosorption process could be due to the type of adsorbents and adsorbates 20 . Another study explored the disturbance of the bioremediation process of metals, where the building of bacterial exopolysaccharides (EPS) could be influenced by pH, in addition to other carbon sources available, temperature and the growth phase of the bacterium during which synthesis occurs 46 . Further, the strategies of heavy metal remediation through bacterial EPS must be focused on utilizing the non-neutral, negatively charged EPS to be incorporated as a suitable biosorbent. Otherwise, the bioremediation process could be influenced by nutritional factors, e.g. glucose or other carbon sources, whereby the monosaccharides form nucleotide diphosphate or monophosphate sugar, which is a crucial step in the synthesis of biosorbent agent such as EPS 47 .
In general, the microbial tolerance of heavy metal is attributed to a variety of detoxifying mechanisms, i.e. complexation by exopolysaccharides, binding with bacterial cell envelopes, metal reduction and/or metal efflux 5 . These mechanisms are sometimes encoded in plasmid genes facilitating the transfer of toxic metal resistance from one cell to another 48 . Additionally, the microbial metal tolerance can be categorized into (i) specific tolerance, which involves inducible mechanism (ii) nonspecific tolerance, which might be inducible or constitutive, some species of Pseudomonas fall under the nonspecific inducible resistance 49 . All these mechanisms may be applied as the suggested mode sorption process of Cr 6+ by the present P. alcaliphila NEWG-2 strain.   www.nature.com/scientificreports/ during the metabolic process compared to glucose that needs to be catabolized into pyruvate before entering the Krebs cycle pathway 51 . Commonly, the oxidation process of the organic compounds in synthetic media of chromium removal process might play a crucial role as an electron donor for the removal process, accumulating various types of functional groups on the surface of bacterial cells to enhance the Cr 6+ biosorption 50 . Furthermore, the accumulation of several anionic organic compounds inside the cell induces the formation of complexes with different metal cations including chromium 26 .
Another, the reduction process of chromium by bacteria could be occurred in aerobic or anaerobic conditions, in which the aerobic reduction of Cr 6+ is associated with a soluble protein fraction utilizing NADH or NADPH as an electron donor 52 . Whereas, in anaerobic reduction, Cr 6+ acts as the terminal electron acceptor through membrane-bound reductase activity 53 . The species of Pseudomonas aeruginosa, Pseudomonas fluorescens and Enterobacter cloacae showed to be tolerant of Cr 6+ under anaerobic conditions 33 .

Fourier transform infrared spectroscopy (FTIR).
The FTIR spectra of P. alcaliphila NEWG-2 dry biomass were investigated before and after Cr 6+ biosorption (Fig. 5). The difference in content could be returned to the interface of the metal ion with the bacterial cell wall functional groups that may include hydroxyl, carboxylate, phosphate and amino groups 54 . The ion-exchange method is the probable biosorption of metal ions on the surface of the cell. In the range of 400-4,000 cm −1 wave-number, the adsorption spectra were investigated 55 56 . FTIR spectra confirmed that the phenolic, carbonyl ester, acetyl, carboxylate, alkanes and carbonyl were the main groups involved in chromium biosorption. These results are conceding with the previous investigation, which showed that the pres- Scanning electron microscopy (SEM). SEM data that recorded the morphological variation in P. alcaliphila NEWG-2 before and after the biosorption process of Cr 6+ are depicted in Fig. 6. The scanning electron micrograph clearly illustrates the surface texture and morphology of P. alcaliphila NEWG-2 with a large magnification. The SEM analysis revealed also a piece of crucial information on the surface morphology of bacterium, with more extension. The graph indicates, obviously, the difference between the micrographs before and after adsorption of Cr 6+ ions.
Electron dispersive spectroscopy (EDS). One useful tool, for analysis of elemental content and/or chemical characterization of sorbents, is the EDS 57 . Herein, the EDS analysis was studied to investigate how well the Cr 6+ attached to the cell wall of P. alcaliphila NEWG-2 biomass. Figure 7 depicts the presence of additional Cr 6+ peak after the biosorption process by P. alcaliphila NEWG-2 and these data confirmed the ability of this bacteria to remove Cr 6+ ions from aqueous effluents.
isotherms of cr 6+ biosorption by immobilized cells. Biosorption equilibrium isotherm modeling of alginate beads alone or immobilized with P. alcaliphila NEWG-2 was studied for Cr 6+ . Data of initial and residual concentrations of Cr 6+ after 4 h of direct contact at 25 °C, as well as, the summary of constants for both Langmuir and Freundlich's constants are presented in Table 4. Initially, the lower the value of b constant of the Langmuir model and the higher the values of both K f and n constants of the Freundlich model, indicate directly the higher affinity of the biomass. The amount of metal adsorbed at the equilibrium (q e ) versus residual concentration (C e ) sorption isotherm relationship was mathematically expressed and plotted (Fig. 8). The value of q max of bacterial-alginate beads was 10 mg/g compared with only 7.405 mg/g for alginate beads. This, in turn, reflecting the critical role of the immobilized cells in the biosorption process. The linear regressions of data for the Langmuir and Freundlich isotherms for Cr 6+ biosorption using immobilized P. alcaliphila NEWG-2 cells were plotted in Fig. 9. The equilibrium parameter (R L ) or the dimensionless constant separation factor is essentially used to express the Langmuir www.nature.com/scientificreports/ isotherm, which indicates the shape of the isotherm adsorption for the adsorbents and adsorbates. In this respect, R L has various types, i.e. favorable (0 < R L < 1), linear (R L = 1) and, unfavorable (R L > 1) the present value (0.01002) at different initial concentrations of Cr 6+ for the Langmuir model indicates a favourable adsorption process. The coefficients of determination (R 2 ) of both Langmuir and Freundlich are close to 1.0, i.e. 0.999 and 0.918, respectively, therefore, adequately, describing the experimental data of Cr 6+ biosorption. The higher the R 2 the more robustness of the model. Langmuir model was found to be more efficient in this respect. Likewise, the biosorption process in alginate beads only (Fig. 9) was apt to Langmuir model than Freundlich, in which the R 2 is 0.9225 and 0.7443, respectively. Briefly, the immobilization of the bacterial cells made a difference in the biosorption process and such process is more obey to Langmuir model than Freundlich's one.  www.nature.com/scientificreports/   www.nature.com/scientificreports/ The current data are inconsistent with the findings of Rezaei 58 , in which the equilibrium isotherm models of the sorption process of Cr 6+ by Spirulina sp. preciously followed Freundlich than Langmuir. Various equilibrium models for fitting the experimental data for examining the correlation between solid and liquid phase concentrations of Cr 6+ at equilibrium had been investigated 21 . In common, the two major models of kinetic study, i.e. Langmuir and Freundlich isotherms, were the major models 59 , in which, these mathematical models can fit the data reasonably well, and hardly reflect the sorption mechanism.

Materials and methods
Bacterium and growth conditions. Pseudomonas sp. was isolated, 2 years ago, from the soil and identified at the genus level by the Department of Microbiology, Soils, Water and Environment Research Institute, Agricultural Research Center (Affiliation ID: 60019332), Giza, Egypt. The isolate was kindly provided to the present study.
The fermentation medium consisting of (g/l) glucose (5), yeast extract (5) and MgSO 4 ·7H 2 O (0.2) and pH 7.2. For the maintenance, the bacterial strain was cultured on slants of the same medium supported with 15 g agar and incubated at 25 ± 1 °C for 48 h, the autoclavation was carried out at 121 °C for 20 min. the bacterium was sub-cultured periodically and preserved at 4 °C.
For inoculum preparation, the bacterium was grown on the previous broth medium under shaking at 100 rpm and 25 ± 1 °C for 48 h, the bacterial count was adjusted to obtain 10 8 ml −1 CFU, 5% (v/v) inoculum was used to inject 50 ml of fermentation medium Erlenmeyer flasks.
Bacterial tolerance to cr 6+ . The potentiality level of Pseudomonas sp. NEWG-2 to survive in Cr 6+ was investigated. The preceding fermentation medium was supplemented with various concentrations of K 2 Cr 2 O 7 to cover a range from 50 to 250 ppm of Cr 6+ , and incubated for 48 h. Then the bacterial tolerability was evaluated in terms of growth reduction, Cr 6+ removal after fermentation and a reduction in the final cultural pH.

Molecular identification.
Pseudomonas sp. NEWG-2 was molecularly identified using 16S rRNA sequencing. DNA extraction for the bacterial sample was performed using the Thermo Gene JET Genomic DNA Purification Kit (#K0721), followed by the PCR reaction and sequencing according to the method of El-Naggar Statistical optimization of cr 6+ biosorption. The matrix of FCCD was performed for statistically modeling of the medium conditions for the maximization of the bioremoval process of Cr 6+ by P. alcaliphila NEWG-2. Two nutritional (yeast extract and glucose) and two physical (pH and incubation time) factors were the independent factors used for constructing the design matrix. Each of the four factors was investigated at three levels during the fermentation ( Table 1). The FCCD was arranged to include eight axial points, sixteen factorial points, six center points and to estimate the pure error 62 . Following the laboratory experimentation of the design, the fermented medium was centrifuged (5,000 rpm for 20 min), the biosorption process was evaluated.
evaluation of the biosorption process. The efficacy of bacterial isolate to survive in the Cr 6+ -containing medium was evaluated, measuring the bacterial growth (OD at 610 nm) and the ability to modify the fermentation medium pH (using pH-meter with a glass electrode (HI 9,321 microprocessor pH-meter). Finally, the residual Cr 6+ was determined in the supernatant after fermentation. The concentration was recorded on a Buck Scientific Accusys 211 series, Atomic Absorption Spectrophotometer, USA by an air/acetylene flame system 63 .
Data modeling. The obtained data were then fitted to the next second-order polynomial quadratic model: where; Y is the Cr 6+ removal percent, X i , and X j are independent variables; β 0 model constant, β i , is linear coefficients; β ii is the quadratic coefficients and β ij , is the interaction coefficients. To evaluate the equation model, laboratory validation was performed to ensure the fitness of the theoretically calculated value of each factor. ftiR spectroscopy. Before and after Cr 6+ removal, the cells of P. alcaliphila NEWG-2 were analyzed using FTIR spectroscopy to detect the functional groups present in the cells. With KBr pellets, the bacterial cells were implemented. The FTIR spectra of P. alcaliphila NEWG-2 were estimated in the range from 400 to 4,000 cm −1 (Thermo Fisher Nicolet IS10, "USA spectrophotometer) at Spectral Analyses Unit, Mansoura University, Egypt 3 .

SeM investigation.
To evaluate the removal of the Cu 2+ and to examine the surface of bacterial cells, dry cells of P. alcaliphila NEWG-2 (before and after removal of the Cr 6+ ) were gold-coated and examined at various magnifications using SEM (JEOL TEM-2100) attached to a CCD camera at an accelerating voltage of 200 kV at Central Laboratory, Electron Microscope Unit, Mansoura University, Egypt 3 .

EDS evaluation.
Energy-dispersive X-ray analysis was performed, employing JEOL TEM-2100 connected to a CCD camera at an accelerating voltage of 200 kV at Central Laboratory, Electron Microscope Unit, Mansoura University, Egypt 3 .
Bacterial immobilization. Immobilization of P. alcaliphila NEWG-2 cells was performed in 4% sodium alginate in distilled water, prepared by constant stirring at 60 °C for 30 min 64 . Following cooling, the sterile sodium alginate gel was supplied with bacterial cells (10 5 CFU/ml from 48 h old culture) with stirring for 5 min at room temperature. The beads with a diameter of 1.5 ± 0.2 mm were generated by adding drop-wise of the alginate alone or alginate-bacterial biomass combination in a cold sterile solution of CaCl 2 (2.5%) through a 3-ml syringe with gentle stirring at room temperature to make spheres beads, then, by washing, three times with distilled sterilized water, the trace of CaCl 2 was removed and finally, stored at 4 °C overnight in sterilized distilled water for stabilization and hardness. For biosorption procedure, glass separating funnel (Simax) was packed with the alginate-bacterial beads or alginate beads without bacteria. Initial concentrations ranging from 200 to 400 ppm of Cr 6+ ion solution were added and left at 25 °C under shaking (100 rpm). Samples of each concentration from both treatments were collected after 4 h from the separating funnel effluent. The collected fractions were analyzed for the residual ions with the Atomic Absorption Spectrophotometer (Buck scientific 210 VGP, Inc.). equilibrium isotherms. The biosorption equilibrium uptake capacity in the absence or presence of the bacterial cells in sodium alginate beads at each concentration of Cr 6+ was calculated according to mass balance on the ions expressed using the next equation: where M is the biomass dry weight (g), C e is the concentration of Cr 6+ (mg/l) at equilibrium, C 0 is the original Cr 6+ concentration (mg/l), V is the sample volume (liter), and q e is the biomass biosorption equilibrium ions uptake capacity (mg/g). Freundlich's and Langmuir isotherms were used to characterize the equilibrium between adsorbed ions by the bacterial cells and ions in the solution in this study. The equation of Langmuir isotherm empirical model based on the sorption on the surface is as follows: www.nature.com/scientificreports/ Then after arrangement, the next equation is generated; The b and q max values (the adsorption equilibrium constants) can be obtained from the intercepts and the slopes of the linear plots; respectively, where experimental data of C e /q e as the function of C e . freundlich isotherm model. The equation of Freundlich isotherm empirical model based on sorption on a heterogeneous surface is as follows: K and 1/n: an experimental constant, n represents the concentration effect on the adsorption efficiency and reflects the adsorption strength. K means the adsorption capacity of the adsorbent. The equation can be linearized in the following logarithmic form: These values K f and n can be obtained from the intercepts and the slopes of the linear plots; respectively, where experimental data of ln q e as the function of ln C e .

conclusions
The present investigation concluded the optimization process for biosorption of Cr 6+ using statistical modeling; FCCD. The maximum removal of Cr 6+ (96.60%) by P. alcaliphila strain NEWG-2 was achieved in a medium containing yeast extract (5.6 g/l) and glucose (4.9 g/l) with an initial pH (7) and fermented for 48 h. The immobilized cells showed also efficacy in the biosorption process of Cr 6+ following Langmuir and Freundlich isotherms model. The equilibrium data were compliant to Langmuir isotherm model compared to the other one. Finally, the current new green tool technology; P. alcaliphila NEW-2 is recommended to be the biosorption of chromium from the contaminated water as a safe and cost-effective strategy.
Received: 8 April 2020; Accepted: 30 July 2020 (4) q e = q max C e b 1 + C e b (5) C e q e = 1 q max b + C e q max (6) q e = K f (C e ) n (7) ln q e = ln K f + 1 n ln C e